Objective To investigate the protective effect of astragaloside Ⅳ（ASⅣ） on hypoxia-induced vascular injury in the mice， and to elucidate its possible mechanism. Methods Sixty male healthy Kunming mice were randomly divided into control group， hypoxia group，low， medium and high doses （40，80， and 120 mg·kg^－1）of ASⅣ groups，with 12 mice in each group. The mice in hypoxia group and different doses of ASⅣ groups were fed in hypoxia chamber （oxygen concentration 10%） for 4 consecutive weeks. The mice in low， medium and high doses of ASⅣ groups were intragastricly administered on the second day of hypoxia，lasted for 4 weeks. The vascular tension of mice in various groups was measured by in vitro vasclarring experment. The levels of interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in serum of the mice in various groups were determined by enzyme linked immunosorbent assay（ELISA） method.The rates of periostin （POSTN） positive cells in aorta tissue of the mice in various groups were detected by immunohistochemistry method. The expression levels of POSTN，intercellular adhesion molecule-1 （ICAM-1），vascular cell adhesion molecule-1 （VCAM-1） and phosphorylated nuclear factor κB（p-NF-κB） p65 proteins in aorta tissue of the mice in various groups were detected by Western blotting method. Results Compared with control group，the maximum diastolic rate of acetylcholine（Ach） of the mice in hypoxia group was significantly decreased （P<0.01）； compared with hypoxia group， the maximum diastolic rate of Ach of the mice in low dose of ASⅣ group had no significant difference（P>0.05），but the maximum diastolic rates of Ach of the mice in medium and high doses of ASⅣ groups were significantly increased （P<0.01）. Compared with control group，the levels of IL-6 and TNF-α in serum of the mice in hypoxia group were significantly increased （P<0.01）； compared with hypoxia group，the levels of IL-6 and TNF-α in serum of the mice in low， medium， and high doses of ASⅣ groups were significantly decreased （P<0.05 or P<0.01）. The rates of POSTN positive cells in aorta tissue of the mice in control group was extremely low； compared with control group，the rate of POSTN positive cells in aorta tissue of the mice in hypoxia group was significantly increased （P<0.01）； compared with hypoxia group， the rate of POSTN positive cells in aorta tissue of the mice in low dose of ASⅣ group had not significant difference（P>0.05），but the rates of POSTN positive cells in aorta tissue of the mice in medium and high doses of ASⅣ groups were significantly decreased （P<0.01）. Compared with control group， the expression levels of POSTN，ICAM-1，VCAM-1，and p-NF-κB p65 proteins in aorta tissue of the mice in hypoxia group were significantly increased（P<0.01）； compared with hypoxia group， the expression levels of POSTN，ICAM-1，VCAM-1，and p-NF-κB p65 proteins in aorta tissue of the mice in low dose of ASⅣ group had no significant difference（P>0.05），but the expression levels of POSTN，ICAM-1，VCAM-1，and p-NF-κB p65 proteins in aorta tissue of the mice in medium and high doses of ASⅣ groups were significantly decreased （P<0.01）. Conclusion ASⅣ can ameliorate the endothelial dysfunction and inflammation in the hypoxia-induced mice，and it mechanism may be related to regulation of POSTN/NF-κB signaling pathway.

Objective： To construct the prokaryotic expression plasmid of transcription factor EB（TFEB） serine 114 site mutant based on gene splicing by overlap extension PCR （SOE PCR）technique， and to induce the expression and purification in vitro. Methods The mutant primers were designed according to the principle of SOE PCR technique. First，the pGEX-6p-1-TFEB plasmid was used as the template， outer primers F and R and mutation primers Fn and Rn were used for the first step PCR amplification to obtain product 1 and product 2 containing mutation sites. Secondly， overlap splicing of product 1 and product 2 was performed by annealing and extension of the second step PCR. Lastly， using the spliced DNA fragment as a template， the third step PCR amplification was performed to obtain the target DNA containing the mutation sites by using outer primers F and R. Then the target DNA fragments were cloned into the pGEX-6p-1 vector， which were identified by enzyme digestion with BamH Ⅰ and Sal Ⅰ. The mutation results were verified by DNA sequencing. The expressions of TFEB and its mutant recombinant proteins were induced in Escherichia coli（E. coli） with different concentrations of isopropyl-beta-D-thiogalactopyranoside（IPTG） and different conditions. The purified proteins were purified by Glutathione-Sepharose 4B agarose beads， and the protein concentrations and molecular weights of the purified products were detected by SDS-PAGE gel electrophoresis. Results Two DNA fragments containing mutation sites were obtained by the first step of PCR amplification， and a large number of complete DNA fragments containing mutation sites were obtained after the annealing and extension of the second step of PCR and the amplification of the third step of PCR. The double-enzyme digestion identification results showed that the size of the ligated DNA fragment was the same as that of the target fragment. The DNA sequencing results showed that the 114-position serine （TCT） of TFEB was successfully mutated to alanine （GCT）. The soluble protein expression of TFEB and its mutants were successfully obtained under the condition of 0.5 mmol·L^－1 IPTG and overnight at 16 ℃.The results of SDS-PAGE gel electrophoresis showed that the purified protein concentration was higher and the molecular size was correct. Conclusion The site-directed mutagenesis of serine 114 of TFEB is successfully achieved by SOE PCR technique， and TFEB and its mutant genes are successfully expressed in E. coli.

Objective To investigate the effect of arctigenin on the metabolites in serum of the cerebral ischemia-reperfusion injury model rats， and to clarify the molecular mechanism of arctigenin for protection of cerebral ischemia-reperfusion injury. Methods A total of 18 SPF SD rats were randomly divided into sham operation group， model group and arctigenin group， with six rats in each group. The rats in arctigenin group were administered with 100 mg·kg^－1 arctigenin by gavage，and the rats in sham operation group and model group were administrated with saline at the same volume lasted for 14 d， then the cerebral ischemia-reperfusion rat models were established by improved line embolization. After 24 h of reperfusion， the neurological deficit scores of the rats in various groups were assessed， HE staining was used to observe the pathomorphology of ischemic penumbra of the rats in variuos groups， and metabolomics technique was used to detect the metabolite levels in serum of the rats in various groups. Results Compared with sham operation group， the score of neurological deficit of the rats in model group was significantly increased （P<0.05）， the morphology of brain tissue was significantly changed and a small amount of glialcell hyperplasia was seen，the levels of 14 kinds of serum metabolites including 3，3'，4'，5-tetrahydroxysilbene and docosahexaenoic acid （DHA） and so on were significantly decreased （P<0.05）， and the levels of 12 kinds of serum metabolites including carnosine and so on were significantly increased （P<0.05）； compared with model group， the score of neurological deficit of the rats in arctigenin group was significantly decreased （P<0.05）， neuronal injury was improved， the levels of 14 kinds of serum metabolites including 3，3'，4'，5-tetrahydroxysilbene and DHA and so on were significantly increased （P<0.05）， and the levels of 12 kinds of serum metabolites including carnosine and so on were significantly decreased （P<0.05）. Conclusion Arctigenin may play an anti-cerebral ischemia-reperfusion role by regulating the metabolites such as DHA， 3，3'，4'，5-tetrahydroxysilbene and carnosine involved in the amino acid metabolism pathway such as histidine metabolism and arginine and proline metabolism.

Objective To investigate the three-dimensional structure of oligoribonuclease from Pseudomonas aeruginosa （P.aeruginosa） Orn （PaOrn），and to verify the active sites of PaOrn，and explore the effect of PaOrn on the intracellular Bis-（3'-5'） cyclic diguanylic acid（c-di-GMP） levels and virulence of P. aeruginosa. Methods A fusion protein with a histidine （His） tag and a glutathione （GST） tag was obtained by constructing the recombinant plasmid pET 32M3C-PaOrn that was expressed by the Escherichia coli（E. coli） BL21.The protein was purified with multistage purifiation method； after Ni affinity chromatography， GST affinity chromatography， and anion exchange chromatography， and finally yielding the PaOrn target protein with high purity and high homogeneity were obtained.The crystals were screened with commercial crystallization reagents and sitting drops， and they were further optimized according to the crystal state. The quality of crystal was verified by X-ray diffraction method， and resolution above 3 ? was used for structure resolution.The activity of PaOrn degradation 5'-phosphoguanylyl-（3'，5'）-guanosine（pGpG） was verified by using high resolution mass spectrometry Q TOF， and the GMP product was used as an indicator to verify the activity. The hydrolytic activities of the PaOrn and the mutant PaOrn proteins （Orn^D11A， Orn^E13A， Orn^D111A， Orn^H157A， and Orn^D162A） against the 10 nt RNA was verified by using Urea-PAGE， and the degradation bands were used as analytical indicators for the normal activity. Results The target protein PaOrn was obtained with purity up to 95% after prokaryotic expression and multistage purification. Good quality single crystals were grown at Wizard Ⅰ # 12［1.0 mmol·L^－1（NH₄） ₂HPO₄， 0.1 mmol·L^－1 Imidazole］ pH 8.0， the resolution of crystal PaOrn was 1.85 ?，and the resolutions of PaOrn-GMP complex and PaOrn^D11A-pGpG complex were 1.90 ? and 1.70 ?， respectively.The Urea-PAGE results showed that PaOrn hydrolyzed the substrate pGpG to GMP. The urea polyacrylamide gel electrophoresis results revealed that PaOrn could degrade up to 10 nt of RNA， and after mutation of the active sites of PaOrn （Asp11， Glu13， Asp111， His157， and Asp162），the activity of PaOrn was lost，and failed to degrade the substrate pGpG and RNA（10 nt）. Conclusion PaOrn is involved in the regulation of intracellular c-di-GMP and RNA levels through degradation of oligonucleotides，and affects transcription initiation and the multiple cellular biological behaviors.

Objective To explore the effects of Parnassia palustris Linn extracts on Helicobacter pylori （Hp） eradication and gastric mucosa， and to clarify its antibacterial and anti-inflammatory mechanisms in vitro and in vivo. Methods The six-week old male Kunming mice were randomly divided into blank control group， normal saline group， Hp group， triple group， quadruple group， Parnassia palustris Linn group， triple + Parnassia palustris Linn group， and quadruple + Parnassia palustris Linn group. The minimal inhibitory concentration （MIC） of Parnassia palustris Linn extracts on Hp SS1 standard strains in vitro and the expression levels of Hp colonization- and adhesion-related gene mRNA were detected by real-time fluorescence quantitative （RT-qPCR） method. Animal experiments were used to evaluate the eradication rate of Hp by different methods， and enzyme linked immunosorbent assay（ELISA） method was used to detect the serum levels of inflammatory factors such as interleukin-2 （IL-2）， interleukin-8 （IL-8） and tumor necrosis factor alpha （TNF-α） of the mice in various groups. Rapid urease test （RUT） was used to detect the infection rate of Hp in stomach tissue， HE staining and transmission electron microscope were used to detect the pathomorphology and ultrastructures of stomach tissue. Results The MIC of Parnassia palustris Linn was 10 g·L^－1.The RT-qPCR results showed that compared with Hp group，the expression levels of Bab A， Cag A and Vac A mRNA in Parnassia palustris Linn group were decreased （P<0.05 or P<0.01）.The immunohistochemistry results showed that the part of gastric mucosa had erosion and ulcers，obvious bleeding and conggestion，and positive Hp infection of the mice in Hp group； the symptoms of gastric mucosal bleeding and ulcers in Parnassia palustris Linn group were improved， and Hp infection was mostly negative.The Rut results showed that compared with Hp group，the infection rate of Hp of the mice in Parnassia palustris Linn group was decreased（P<0.05），and the infection rates of the mice in triple group and quadruple group were decreased（P<0.05）；compared with Parnassia palustris Linn group，the eradication rates of Hp of the mice in triple group and quadruple group had no significant differences（P>0.05）； compared with triple group or quadruple group， the eradication rates of Hp of the mice in triple+Parnassia palustris Linn group and quadruple+Parnassia palustric Linn group were increased（P<0.01）. The gastric mucosa was apparently repaired， and the structures of mitochondria and microvilli of gastric mucosal epithelial cells were intact of the mice in triple+Parnassia palustris Linn group and quadruple+Parnassia palustris Linn group.Compared with Hp group，the levels of IL-2 in serum of the mice in triple， quadruple，Parnassia palustris Linn， triple+Parnassia palustris Linn and quadruple+Parnassia palustris Linn groups were increased（P<0.05）， and the levels of serum IL-8 and TNF-α were decreased（P<0.05 or P<0.01）. Conclusion Parnassia palustris Linn extracts can effectively eradicate Hp and protect gastric mucosa， and its mechanism is related to decreasing the levels of IL-2，IL-8 and TNF-α and protecting the integrity of structures of gastric mucosa subcellular organelle after Hp infection.

Objective： To investigate the protective effect of ultra-filtration extract from Angelica Sinensis Radix and Hedysari Radix （UFE-AH） on the injury of human umbilical vein endothelial cells （HUVECs） induced by X-ray，and to clarify its possible mechanism. Methods The HUVECs were cultured in vitro and irradiated with different doses （0，2，4，6，8 and 10 Gy） of X-ray； the optimal radiation dose （6 Gy） was selected. The HUVECs were intervened with different concentrations （0，50，100，200，400，600，800 and 1 000 μg·L^－1） of UFE-AH， and the optimal proliferation promoting concentrations （100，200 and 400 μg·L^－1） were selected.The experiment was divided into blank group， model group （6 Gy X-ray）， low dose of UFE-AH group （6 Gy X-ray + 100 μg·L^－1 UFE-AH）， medium dose of UFE-AH group （6 Gy X-ray+200 μg·L^－1 UFE-AH） and high dose of UFE-AH group （6 Gy X-ray + 400 μg·L^－1 UFE-AH）.The survival rates of cells in various groups were measured by CCK-8 assay， the apoptotic rates of cells in various groups were measured by flow cytometry，the ultrastructures were observed by transmission electron microscope， and the expression levels of phosphatidylinositol 3-kinase （PI3K）， protein kinase B （Akt）， phosphorylated Akt（p-Akt），vascular endothelial growth factor （VEGF），B-cell lymphoma-2（Bcl-2），Bcl-2 associated X protein （Bax） and cysteinyl aspartate specific proteinase-3（caspase-3） proteins in the cells in various groups were measured by Western blotting method. Results The CCK-8 assay results showed that compared with 0 Gy X-ray，the inhibitory rates of HUVECs irradiated by X-ray were increased with the increase of radiation dose when the indiation dose was greater than 2 Gy at 48 and 72 h after radiation （P<0.05 or P<0.01）， and the increasing of inhibition rates at 6 Gy X-ray and above were more significant（P<0.01）. Compared with 0 μg·L^－1 UFE-AH， the survival rates of cells after treated with 100，200，400 and 600 μg·L^－1UFE-AH were increased（P<0.05 or P<0.01），especially treated with 100，200 and 400 μg·L^－1 UFE-AH for 24，48 and 72 h （P<0.01）. Compared with blank group，the survival rate of the cells in model group was decreased（P<0.01）；compared with model group， the cell survival rates in medium and high doses of UFE-AH groups were significantly increased（P<0.01）. Compared with blank group，the apoptotic rate of the cells in model group was significantly increased（P<0.01）；compared with model group， the apoptotic rates of the cells in different doses of UFE-AH groups were significantly decreased（P<0.01）.The transmission electron microscope results showed that the cell membrane in blank group was intact and the cytoplasm was more uniform， the mitochondria were oval； no obvious swelling was observed， and a small amount of lysosomes were observed in the cells；compared with blank group， the cells in model group had severe swelling， multiple damage of the cell membrane， loose cytoplasm and many vacuolar areas， mildly irregular nuclei， the significantly reduced mitochondria， mild or moderate swelling， shallower and uneven matrix， local breakage and shortening of a small part of the cristae of mitochondria， and a large amount of autolysosomes （ASS） in the cells； compared with model group， the cell swelling in low， medium， and high doses of UFE-AH groups was alleviated，intracellular organelle vacuoles were gradually reduced， mitochondrial swelling was alleviated， the mitochondrial number was increased， and a certain amount of ASS were observed， but they still did not return to normal. Compared with blank group，the expression levels of PI3K， p-Akt， Bcl-2 and VEGF proteins in the cells in model group were significantly decreased（P<0.01）， and the expression levels of Bax and caspase-3 proteins and the Bax/Bcl-2 ratio were significantly increased（P<0.01）； compared with model group， the expression levels of PI3K， p-Akt， Bcl-2 and VEGF proteins in the cells in medium and high doses of UFE-AH groups were significantly increased（P<0.01），and the expression levels of Bax and caspase-3 proteins and the Bax/Bcl-2 ratio were significantly decreased（P<0.01）. Conclusion UFE-AH at a certain dose has a protective effect on the X-ray-induced injury of HUVECs， and its mechanism may be related to the effect of UFE-AH on the PI3K， Akt， p-Akt， VEGF， Bcl-2， Bax and caspase-3 protein expressions in the HUVECs.

Objective To investigate the effect of lipopolysaccharide （LPS） of Porphyromonas gingivalis （P.g） on the expression levels of ferroptosis-related factors in the macrophages， and to clarify the influence of ferroptosis-related factors in the pathogenesis of periodontitis. Methods The mouse mononuclear/macrophage line RAW264.7 cells were cultured and divided into experimental group and control group. The cells in experimental group were treated with 10 mg·L^－1P.g-LPS for 6， 12， and 24 h， respectively； the cells in control group were not treated. Real-time fluorescence quantitative PCR （RT-qPCR） method was performed to detect the expression levels of long chain acyl-CoA synthetase 4 （ACSL4）， glutathione peroxidase 4 （GPX4）， and transferrin receptor 1 （TfR1） mRNA in the RAW264.7 cells in two groups. The levels of reactive oxygen species （ROS） in the RAW264.7 cells in two groups were detected by 2'，7'-dichlorodihydrofluorescein diacetate（DCFH-DA） fluorescence probe method. The levels of malondialdehyde （MDA） in the RAW264.7 cells in two groups were detected by thiobarbituric acid （TBA） method and the levels of ferrous ion （Fe²⁺） in the RAW264.7 cells in two groups were detected by Ferrousion fluorescent probe （FeRhonox-1） method. The expression levels of ACSL4， GPX4， and TfR1 proteins in the RAW264.7 cells in two groups were detected by Western blotting method. Results Compared with control group， the expression levels of GPX4 mRNA in the RAW264.7 cells in experimental group after treated for 6， 12， and 24 h were decreased （P<0.05）， the expression levels of ACSL4 and TfR1 mRNA in the RAW264.7 cells in experimental group after treated for 12 and 24 h were increased （P<0.05）； the expression levels of ACSL4 and TfR1 proteins in the RAW264.7 cells in experimental group after treated for 12 and 24 h were increased （P<0.05）；the expression levels of GPX4 protein in the RAW264.7 cells in experimental group after treated for 12 and 24 h were decreased （P<0.05）.Compared with control group，the levels of ROS， MDA， and Fe²⁺ in the RAW264.7 cells in experimental group were significantly increased （P<0.05）， and showed an increasing trend with time prolongation. Conclusion The expression levels of ferroptosis-related factors ACSL4 and TfR1 are increased and the expression level of GPX4 is decreased in a time-dependent manner induced by P.g-LPS， suggesting that the changes of ferroptosis-related factors may be related to the pathogenesis of periodontitis.

Objective To investigate the therapeutic effects of Astragali Radix on the intestinal inflammation， intestinal barrier and intestinal flora in the rats with rhubarb-induced diarrhea，and to elucidate the possible mechanisms. Methods Fifty rats were randomly divided into control group， model group， positive control group （2.468 g·kg^－1 Shenlingbaizhu Powder）， low dose（1.35 g·kg^－1） of Astragali Radix group， and high dose（2.70 g·kg^－1） of Astragali Radix group，with 10 rats in each group. The rat diarrhea models were replicated by rhubarb gavage in combination with diet indiscipline.The body weights， fecal water contents and diarrhea scores of the rats in various groups were detected； HE staining was used to observe the pathomorphology of colon tissue of the rats in various groups， and Western blotting method was used to detect the expression levels of protease activated receptor-2（PAR-2），phosphorylated p38 （p-p38）， myosin light chain kinase （MLCK）， phosphorylated myosin light chain （p-MLC）， zonula occludens-1（ZO-1），Occludin，Toll-like receptor 4 （TLR4），tumor necrosis factor receptor-associated factor 6（TRAF6）， myeloid differentiation factor 88 （MyD88）， and nuclear factor-κB （NF-κB） proteins in colon tissue of the rats in various groups；the levels of motilin （MTL）， gastrin （GAS）， tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， interleukin-6 （IL-6），interleukin-10 （IL-10），malondialdehyde （MDA），and secretory immunoglobulin A （SIgA） and the activities of superoxide dismutase （SOD） in serum of the rats in various groups were measured by enzyme linked immunosorbent assay（ELISA） method；16S r DNA gene sequencing was performed to analyze the intestinal flora of the rats in various groups. Results Compared with control group，the body weight of the rats in model group was significantly decreased（P<0.01）， the fecal water content and diarrhea score were significantly increased （P<0.01），and the levels of MTL and GAS in serum were significantly increased （P<0.01）； compared with model group，the body weights of the rats in positive control and low and high doses of Astragali Radix groups were significantly increased（P<0.01）， the fecal water contents and diarrhea scores were significantly decreased （P<0.01），and the levels of MTL and GAS in serum of the rats in were significantly decreased （P<0.01）. The HE staining results showed that compared with control group， the colonic epithelial mucosa of the rats in model group was damaged， with a large number of infiltrated inflammatory cells and disordered glands. Compared with model group， the mucosal structures of the epithelium in positive control and high dose of Astragali Radix groups were more regular， with less inflammatory cell infiltration and without obvious glandular disorder； the mucosal structure of the colonic epithelium in low dose of Astragali Radix group had slight mucosal damage， with partial inflammatory cell infiltration and more neatly arranged glands.The Western blotting results showed that compared with control group，the expression levels of PAR-2，MLCK，TLR4，TRAF6，MyD88，NF-κB，p-p38 and p-MLC proteins in colon tissue of the rats in model group were significantly increased （P<0.01），and the expression levels of ZO-1 and Occludin proteins in colon tissue of the rats in model group were significantly decreased （P<0.01）； compared with model group，the expression levels of PAR-2， MLCK， TLR4， TRAF6， MyD88， NF-κB，p-p38 and p-MLC proteins in colon tissue of the rats in positive control group，low dose of Astragali Radix group and high dose of Astragali Radix group were significantly decreased （P<0.05 or P<0.01）， and the expression levels of ZO-1 and Occludin proteins in colon tissue of the rats in positive control group， low dose of Astragali Radix group and high dose of Astragali Radix group were significantly increased （P<0.05 or P<0.01）.Compared with control group，the levels of TNF-α， IL-1β， IL-6 and MDA in serum of the rats in model group were significantly increased （P<0.01），the levels of IL-10， and SIgA in serum and the SOD activity of the rats were significantly decreased（P<0.01）； compared with model group， the levels of TNF-α， IL-1β， IL-6 and MDA in serum of the rats in positive control group， low dose of Astragali Radix group and high dose of Astragali Radix group were significantly decreased （P<0.01）， and the levels of IL-10 and SIgA in serum and the SOD activities were significantly increased （P<0.01）.At the level of phylum，the intestinal flora detection results showed that compared with control group， the abundance of Proteobacteria and Verrucomicrobia in model group was significantly increased （P<0.01）； compared with model group， the abundance of Proteobacteria and Verrucomicrobia in low and high doses of Astragali Radix groups was significantly decreased （P<0.01）， and the abundance of Epsilonbacteraeota in intestinal flora in high dose of Astragali Radix group was significantly increased （P<0.01）. Conclusion Astragali Radix has a therapeutic effect in the rhubarb-induced diarrhea model rats， and its mechanism may be related to inhibiting intestinal inflammation through TLR4/TRAF6/NF-κB signaling pathway， thereby repairing the intestinal barrier and restoring the composition of intestinal flora.

Objective To investigate the improvement effect of Phellinus igniarius acidic polysaccharide（PIP） on liver fibrosis in the mice induced by bile duct ligation （BDL）， and to clarify its possible mechanism. Methods A total of sixty 4-week-old male C57/BL6 mice were randomly divided into control group， model group， positive drug group，and PIP group，with 15 mice in each group. The mice in control group only received the passed surgical line without ligation around the common bile duct， and the bile duct of mice in model group， postive drug group and PIP group were ligated. After administration for 3 weeks， the blood was taken from eyeball 12 h after the last administration， and the mice were sacrificed. The body weights and liver weights of mice were weighed， and the liver index was calculated. Microplate method was used to determine the levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in serum of the mice in various goups； the levels of type Ⅲ procollagen （PC Ⅲ） and cellagen type Ⅳ（Col Ⅳ） in serum were determined by double antibody sandwich method. HE staining and Sirius red staining were used to observe the pathomorphology and fibrosis degrees of liver tissue of the mice in various groups； the activities of superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px） and the levels of malondialdehyde （MDA）and glutathione （GSH） in liver tissue of the mice in various groups were determined. The expression levels of nuclear factor-E2 related factor （Nrf2）， heme oxygenase-1 （HO-1） and quinone oxidoreductase 1 （NQO1） proteins in liver tissue of the mice in various groups were detected by Western blotting method. Results Compared with control group，the body weight of the mice in model group had no significant difference（P>0.05），and the liver weight and liver index were increased（P<0.05）；compared with model group， the body weight of the mice in PIP group had no significant change（P>0.05）， and the liver weight and liver index were decreased（P<0.05）.The HE staining results showed no abnormalities in liver tissue of the mice in control group；the liver lobular structure was destroyed and focal liquefied hepatic necrosis and more inflammatory cell infiltration were seen in liver tissue of the mice in model group；compared with model group， the necrosis degrees of liver tissue of the mice in positive drug group and PIP group were significantly reduced， and the infiltration of inflammatory cells was reduced. The Sirius red staining results showed that compared with control group，the content of collagen fibers in liver tissue of the mice in model group was significantly increased；compared with model group，the content of collagen fibers in liver tissue of the mice in PIP group was significantly decreased.Compared with control group，the ALT and AST levels in serum of the mice in model group were increased（P<0.01），PC Ⅲ and Col Ⅳ levels were increased（P<0.05）， the activities of SOD and GSH-Px were significantly decreased（P<0.01），and the levels of MDA and GSH were significantly increased（P<0.05 or P<0.01）； compared with model group， the levels of ALT and AST in serum of the mice in positive drug group and PIP group were significantly decreased （P<0.01）， the levels of PC Ⅲ and Col Ⅳ were significantly decreased （P<0.05 or P<0.01）， the activities of SOD and GSH-Px in liver tissue of the mice were increased （P<0.05）， and the levels of GSH and MDA were decreased （P<0.05）.The Western blotting results showed that compared with control group，the expression levels of Nrf2，HO-1，and NQO1 proteins in liver tissue of the mice in model group were decreased （P<0.05）；compared with model group， the expression levels of Nrf2，HO-1，and NQO1 proteins in liver tissue of the mice in PIP group were significantly increased （P<0.05 or P<0.01）. Conclusion PIP can improve the liver fibrosis in the mice caused by BDL， and its mechanism may be related to reducing the level of liver oxidative stress and regulating the expression levels of Nrf2， HO-1 and NQO1 proteins in Nrf2 / HO-1 signaling pathway.

Objective To investigate the protective effect of ginsenoside Rg1 on cardiac injury in the rats with severe acute pancreatitis （SAP）， and to elucidate the related molecular mechanisms. Methods The SD rats were randomly divided into control group， model group and Rg1 treatment group，with 6 rats in each group. The abdominal cavity of the rats in control group was closed after laparotomy； the rats in model group were retrogradely injected with 5% sodium taurocholate （0.15 μL·kg^－1） into the biliopancreatic duct to induce SAP after laparotomy； the rats in Rg1 treatment group were injected with Rg1 （4 mg·kg^－1） via the tail vein 30 min before operation of preparing the SAP model； the rats in control group and model group were injected with normal saline 30 min before operation. Enzyme linked immunosorbent assay（ELISA） method was used to detect the levels of serum tumor necrosis factor-α （TNF-α）， endotoxin， endothelin 1 （ET-1）， interleukin-1β （IL-1β） and the activities of serum creatine kinase-MB （CK-MB）， cardiac troponin I （cTnI） and mylase of rats in various groups. Ultrasound imaging system was used to detect the heart rate（HR），left ventricular end-systolic diameter （LVDs） and left ventricular end-diastolic diameter （LVDd） of rats in various groups and calculate the fractional shortening （FS）.Western blotting method was used to detect the expression levels of NADPH oxidase （NOX） 2 and NOX4， phosphorylated p38（p-p38），phosphorylated extracellular regulated protein kinase 1/2（p-ERK1/2） and phosphorylated c-Jun N-terminal kinase（p-JNK） proteins in myocardium tissue of rats in various groups. Results Compared with control group， the levels of TNF-α， endotoxin，IL-1β and ET-1 in serum of the rats in model group were significantly increased （P<0.05）； the activities of CK-MB，cTnI，and amylase were significantly increased （P<0.05）. Compared with model group， the levels of TNF-α， endotoxin，IL-1β and ET-1 in serum of the rats in Rg1 treatment group were decreased （P<0.05），and the activities of CK-MB，cTnI，and amylase were decreased （P<0.05）. Compared with control group， the HR and LVDs of the rats in model group were significantly increased （P<0.05），the LVDd had no significant difference（P>0.05），and the FS was significantly decreased（P<0.05）； compared with model group， the LVDs of the rats in Rg1 treatment group were significantly decreased （P<0.05），the LVDd had no significant difference（P>0.05），and the FS was significantly increased （P<0.05）.The Western blotting results showed that compared with control group， the expression levels of NOX2，NOX4，p-p38， p-ERK1/2 and p-JNK proteins in myocardium tissue of the rats in model group were significantly increased （P<0.05 or P<0.01）； compared with model group， the expression levels of NOX2，NOX4，p-p38， p-ERK1/2 and p-JNK proteins in myocardium tissue of the rats in Rg1 treatment group were significantly decreased （P<0.05）. Conclusion Rg1 has a protective effect on cardiac injury in the SAP rats，and its mechanism may be related to reducing myocardial tissue oxidative stress by inhibiring MAPK signaling pathway.

Objective To investigate the inhibitory effect of fermented red ginseng total saponins （FRGTS） on the epithelial-mesenchymal transition（EMT） of renal tubular epithelial cells induced by high glucose， and to clarify its mechanisms. Methods The rat renal tubular epithelial cells NRK-52E were divided into normal control group （5.5 mmol·L^－1 D-glucose）， high glucose group（30.0 mmol·L^－1 D-glucose）， silent information regulator 1 （SIRT1） inhibitor EX527 group （30.0 mmol·L^－1 D-glucose+10 μmol·L^－1 EX527）， FRGTS group （30.0 mmol·L^－1 D-glucose+25 mg·L^－1 FRGTS） and EX527+FRGTS group （30.0 mmol·L^－1 D-glucose+10 μmol·L^－1 EX527+25 mg·L^－1FRGTS）. Immunofluorescence method was used to detect the expression levels of E-cadherin and α-smooth muscle actin （α-SMA） protein in the cells in various groups. Real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the expression levels of E-cadherin， α-SMA and SIRT1 mRNA， ELISA method was used to detect the levels of collagen type Ⅰ （ColⅠ） in culture supernatant， and Western blotting method was used to detect the expression levels of SIRT1， transforming growth factor-β1 （TGF-β1） and Smad3 proteins in the cells in various groups. Results After 48 h of high glucose culture， compared with normal control group， the expression levels of E-cadherin mRNA and protein in the NRK-52E cells in high glucose group were decreased （P<0.01）， the expression levels of α-SMA mRNA and protein were increased （P<0.01）， the level of ColⅠ in culture supernatant was increased（P<0.01），the expression levels of SIRT1 mRNA and protein were significantly decreased （P<0.01），and the expression levels of TGF-β1 and Smad3 proteins were increased（P<0.01）. Compared with high glucose group， the expression level of E-cadherin mRNA in the NRK-52E cells in FRGTS group was increased（P<0.01）， the expression level of α-SMA mRNA was decreased （P<0.05）， the level of ColⅠ in culture supernatant was decreased（P<0.05），the expression levels of SIRT1 mRNA and protein were increased （P<0.05 or P<0.01），and the expression levels of TGF-β1 and Smad3 proteins were decreased （P<0.05 or P<0.01）. Compared with FRGTS group，the expression level of E-cadherin mRNA in the cells in EX527+FRGTS group was significantly decreased（P<0.01），the expression level of α-SMA mRNA was increased（P<0.05），the level of ColⅠ in culture supernatant was increased（P<0.05），the expression levels of SIRT1 mRNA and protein were significantly deceased（P<0.01），and the expression levels of TGF-β1 and Smad3 were significantly increased（P<0.01）. Conclusion FRGTS can upregulate the expression of SIRT1 and then inhibits TGF-β1/Smad signaling pathway， which effectively inhibits the EMT of renal tubular epithelial cells.

Objective To investigate the promotion effect of Toll-like receptor 4（TLR4） adapters， myeloid differentiation factor 88（MyD88） and Toll/interleukin-1（IL-1） receptor（TIR） domain containing adaptor protein inducing interferon-β（TRIF） after gene knowout in M2 polarization of macrophages induced by lactate， and to elucidate the regulation mechanism of immune responses by lactate. Methods The murine macrophage cell line Raw264.7 were cultured in vitro， CRISPR/Cas9 gene editing system was constructed by lentivirus method， MyD88 and TRIF genes were knocked out（Myd88-KO group and TRIF-KO group），and the untransfection Raw264.7 cells were used as control group. The knockout effects of gene knowout were detected by real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting methods. The experiment was divided into untreated Raw264.7 cell group， Raw264.7 + lactate group， MyD88-KO group， MyD88-KO + lactate group， TRIF-KO group and TRIF-KO+lactate group.The indexes were determined after 15 mmol·L^－1 lactate were induction for 24 h.The expression levels of M2 polarization phenotype molecules macrophage mannose receptor CD206 and arginase 1 （Arg1） mRNA in the cells in various groups were detected by RT-qPCR method， the morphology of cells in various groups was observed under light microscope， and the levels of tumor necrosis factor-α （TNF-α）， interferon-γ （INF-γ） and interleukin-10 （IL-10） in the cell supernatant in various groups were detected by enzyme linked immunosorbent assay（ELISA） method. The expression levels of nuclear factor-κB （NF-κB） signaling pathway-related proteins were detected by Western blotting method. The molecular docking simulation of lactate with TLR4， MyD88 and TRIF were carried out by Autodock software，and the binding was observed. Results After targeted gene was knocked out using CRISPR/Cas9 technique，compared with control group， the expression levels of MyD88 and TRIF mRNA and proteins in the cells in MyD88-KO group and TRIF-KO group were significantly reduced （P<0.05 or P<0.01）. After 24 h of lactate treatment， compared with untreated Raw264.7 cell group， the expression levels of markers of M2 polarization CD206 and Arg1 mRNA in the cells in Raw264.7+lactate group were significantly increased （P<0.05）； compared with MyD88-KO group， the expression levels of CD206 and Arg1 mRNA in the cells in MyD88-KO+lactate group were increased（P<0.05）； compared with TRIF-KO group， the expression levels of CD206 and Arg1 mRNA in the cells in TRIF-KO+lactate group were decreased （P<0.05）. The cells in Raw264.7+lactate group showed M2-polarized morphology， such as increased volume， obvious protruding pseudopods and increased proportion of conical cells； the cells in MyD88-KO+lactate group also showed M2-polarized morphological changes；the cells in TRIF-KO+lactate group showed no significant morphological changes. The ELISA results showed that compared with untreated Raw264.7 cell group，the TNF-α level in the cells in Raw264.7+lactate group was significantly decreased （P<0.05）， and the differences in INF-γ and IL-10 levels were not statistically significant （P>0.05）； compared with MyD88-KO group，the level of TNF-α in the cells in MyD88-KO+lactate group was significantly decreased （P<0.05）， the difference in INF-γ level was not statistically significant （P>0.05）， and the IL-10 level was significantly increased （P<0.05）；compared with TRIF-KO group，the TNF-α level in the cells in TRIF-KO+lactate group was significantly increased （P<0.05）， and the differences in the INF-γ and IL-10 levels were not statistically significant （P>0.05）.The Western blotting results showed that compared with untreated Raw264.7 cell group，the expression levels of TLR4 protein in the cells in Raw264.7+lactate group， MyD88-KO group， MyD88-KO+lactate group， TRIF-KO group and TRIF-KO+lactate group had no statistically significant differences（P>0.05）； the expression levels of nuclear factor-κB inhibitor protein-α（IκB-α） and p65 protein in the cells in Raw264.7+lactate group were significantly decreased （P<0.05）； compared with MyD88-KO group， the expression levels of IκB-α and p65 proteins in the cells in MyD88-KO+lactate group were decreased （P<0.05）； compared with TRIF-KO group， the expression levels of IκB-α and p65 proteins in the cells in TRIF-KO+lactate group had no statistically significant differences （P>0.05）.The number of hydrogen bonds formed by molecular docking of lactate with TLR4， Myd88 and TRIF was 2， 4 and 4， and the binding energies were －2.72， －3.24 and－3.66. Conclusion Lactate can inhibit the activity of IκB-α，and further inhibit NF-κB and thus promote M2 polarization through regulation of TRIF after treatment in the macrophages.

Objective To investigate the changes of silencing information regulator 1 （SIRT1） in ligation-induced kidney injury in the chronic periodontitis rats， and to clarify the effect of SIRT1 on kidney injury in the chronic periodontitis rats. Methods Twenty rats were randomly divided into control group and periodontitis group，with 10 rats in each group.The periodontitis model was established by ligation of necks of bilateral maxillary first molars. The gingival color and shape of the rats in two groups were observed， and the gingival bleeding index （BI）， periodontal probing depth （PD） and tooth mobility （TM） were measured and recorded. Hematoxylin-eosin（HE） staining was used to observe the pathomorphology of periodontium of the rats in two groups； micro-computed tomography （Micro-CT） was used to evaluate the alveolar bone resorption of the rats in two groups. HE and PAS staining were used to observe the pathomorphology of kidney tissue of the rats in two groups. Biochemical kits were used to assess kidney function indicators and detect the oxidative stress levels of the rats in two groups； Western blotting method was used to detect the expression levels of tumor necrosis factor-α （TNF-α） and nuclear factor kappa B （NF-κB） p65 protein in kidney tissue of the rats in two groups； real-time fluorescence quantitative PCR（RT-qPCR） and immunohistochemical method were used to detect the expression levels of SIRT1 and peroxisome-proliferator-activated receptor γ coactivator 1α （PGC-1α） mRNA and proteins in kidney tissue of the rats in two groups. Results The clinical indicators of peridontium tissue detection results showed that compared with control group， swollen gums， soft texture， deep periodontal pocket， bleeding gums and loosening of tooth of the rats in periodontitis group were found.The HE staining and Micro-CT results of peridontium tissue showed that compared with control group， attachment loss and alveolar bone absorption of the rats occurred in periodontitis group.The HE and PAS staining results of kidney tissue showed that compared with control group， the renal cystic space was dilated， the basement membrane of the glomerular was slightly thickened， the epithelial cells of the renal tubules were exfoliated， and the brush edge was destroyed in periodontitis group.The biochemical detection results showed that there was no significant difference in renal function indexes of the rats in two groups （P>0.05）； compared with control group，the levels of total antioxidative status （TAS），managanse superoxide dismutase （Mn-SOD） and glutathione （GSH） in kidney tissue of the rats in periodontitis group were decreased（P<0.01）， while the level of malondialdehyde （MDA） was increased （P<0.01）.The Western blotting results showed that compared with control group， the expression levels of TNF-α and NF-κB p65 proteins in kidney tissue of the rats in periodontitis group were increased （P<0.05 or P<0.01）.The RT-qPCR and immunohistochemistry results showed that compared with control group， the expression levels of SIRT1 and PGC-1α mRNA and proteins in kidney tissue of the rats in periodontitis group were decreased （P<0.05 or P<0.01）. Conclusion In the rat model of chronic periodontitis， the expression of SIRT1 in kidney tissue is down-regulated and the level of oxidative stress is increased， which aggravats the kidney injury of chronic periodontitis rats.

Objective To explore the characteristics of antioxidant stress of muscle tissue after aging based on the nuclear factor E2-related factor 2（Nrf2） dependent antioxidant pathway， and to clarify the effect of the dynamic balance between reactive oxygen species （ROS） production and antioxidation in the age-related sarcopenia. Methods Twenty four C57BL/6J （C57） male mice were randomly divided into 3-month-old group （3 M group）， 12-month-old group （12 M group） and 22-month-old group （22 M group）. The soleus muscle tissue of mice was taken， and the skeletal muscle mass index （SMI） was calculated. The pathomorphology of muscle tissue of the mice in various groups was observed by HE staining. The ROS levels of mice in various groups were detected by 2'，7'-dichlorodihydrofluorescein diacetate（DCFH-DA） fluorescence probe method， the mRNA expression levels of eroxisome proliferators-activated receptor-γ coactivator-1α （PGC-1α）， Nrf2 and antioxidant stress genes in muscle tissue of the mice in various groups were detected by real-time fluorescence quantitative（RT-qPCR） method，and the protein expression levels of Nrf2 in muscle tissue of the mice in various groups were detected by Western blotting method. Results Compared with 3 M group， the cross-sectional area of soleus muscle fibers of the mice in 12 M group was increased， the number of nuclei in some muscle fibers was increased， and some muscle fibers showed denaturation changes；the SMI value was decreased， but the difference was not statistically significant （P>0.05）；the level of ROS was increased （P<0.05），the expression level of Nrf2 mRNA had no significant difference（P>0.05）， the expression level of PGC-1α mRNA was significantly increased （P<0.01），the expression levels of Nqol， Cat， Gclc， Sod1 and Sod2 mRNA were significantly increased （P<0.01）， and the expression level of Nrf2 protein was significantly decreased （P<0.01）. Compared with 12 M group， the white muscle fibers of soleus muscle of the mice in 22 M group were increased， the morphology was irregular， the number of nuclei of muscle fibers was increased and the phenomenon of nuclear translocation occurred， the gap between muscle cells was increased， the number of cells was decreased， and the SMI value was decreased， but the difference was not statistically significant （P>0.05）； the level of ROS in muscle tissue was significantly increased （P<0.01）， the expression levels of Nrf2 mRNA was significantly increased （P<0.01）， the expression levels of Nqol， Cat， Sod1 and Sod2 mRNA were significantly increased （P<0.05 or P<0.01）， while the expression level of Gclc mRNA was decreased （P<0.05）， and the expression level of Nrf2 protein was significantly increased （P<0.01）. Conclusion The soleus muscle of aging mice shows sarcopenia phenotype. The soleus muscle with oxidative metabolism as the main has certain characteristics of antioxidant stress ability， which is closely related to the enhancement of Nrf2 dependent antioxidant pathway.

Objective： To investigate the effect of Schisandra chinensis polysaccharide （SCP） on the proliferation and apoptosis of human bladder cancer T24 cells， and to elucidate its possible mechanisms. Methods The human bladder cancer T24 cells were cultured in vitro and divided into control group and 50， 100， and 200 mg·L^－1 SCP groups. After 24－48 h of intervention with different doses of SCP， the inhibitory rates of proliferation of T24 cells in various groups were detected by MTT method， the apoptosis of T24 cells in various groups was detected by Hoechst 33258 fluorescence staining method，the apoptotic rates of T24 cells in various groups were detected by Annexin Ⅴ-FITC/propidium iodide（PI） double staining method， the percentages of T24 cells in different cell cycles and the apoptotic rates of T24 cells in various groups were detected by flow cytometry， and the expression levels of phosphatase and tensin homolog deleted on chromosome ten（PTEN）， phosphorylated phosphatidylinositol-3 kinase （p-PI3K），protein kinase B （Akt） and phosphorylated Akt （p-Akt） proteins in the T24 cells in various groups were detected by Western blotting method. Results Compared with control group， the inhibitory rates of proliferation of T24 cells in 50，100，and 200 mg·L^－1 SCP groups were significantly increased（P<0.05 or P<0.01）； the apoptotic rates of T24 cells were significantly increased （P<0.05 or P<0.01）；the percentages of T24 cells in 50，100 and 200 mg·L^－1 SCP groups in G₀/G₁ phase were significantly increased （P<0.05 or P<0.01）， the percentages of T24 cells in 100 and 200 mg·L^－1 SCP groups in S phase and G₂/M phase were significantly decreased （P<0.01）； the expression levels of PTEN protein in the T24 cells in 50，100，and 200 mg·L^－1 SCP groups were significantly increased （P<0.05 or P<0.01）， the expression levels of p-PI3K protein and the ratios of p-Akt/Akt in the T24 cells were significantly decreased （P<0.05 or P<0.01）. Conclusion SCP can inhibit the proliferation of T24 cells and promote their apoptosis，and its mechanism may be related to the regulation of expressions of PTEN and PI3K/Akt signaling pathway-related proteins.

Objective To establish a method for rapid detection of three toxin genes of Bacillus cereus nhe， cytK and entFM based on multiplex PCR，and to evaluate its efficiency. Methods The DNA of Bacillus cereus was extracted by boiling method. The specific toxin genes of Bacillus cereus nhe， cytK and entFM were used as target genes， and the specific primers were designed by NCBI primer-BLAST 5.0 software to optimize the reaction system. The triple PCR detection system was established and the specificity， sensitivity and stability were detected. Results The Bacillus cereus DNA was extracted by boiling method with a concentration of 227 mg·L^－1 and a purity of 1.50－1.60. The pathogenic genes of three toxins of Bacillus cereus were detected simultaneously by multiplex PCR system. When the annealing temperature was 56 ℃， the amplification fragments of Bacillus cereus gene primer nhe499， gene primer cytK191 and gene primer entFM363 were 499， 191 and 363 bp， respectively. The bands of three toxin genes of Bacillus cereus amplified by PCR system were clear and bright， and there were no non-specific bands， and no amplification with Staphylococcus aureus， Escherichia coli and Pseudomonas aeruginosa， indicating that the primers of nhe499， cytK191 and entFM363 of Bacillus cereus genes had strong specificities， the three primers did not interfere with each other in the same reaction system， and the lowest detection limit of multiplex PCR system was 10^－1 mg·L^－1. The stability test was consistent with the self-made preparation of multiple PCR system every 6 months. Conclusion The established multiplex PCR detection system has high sensitivity and specificity for three toxin genes of Bacillus cereus nhe499， cytK191 and entFM363， the detection results are accurate and stable， and the detection system has good stability， which is suitable for rapid detection of three different pathogenic toxin genes of Bacillus cereus.

Objective To investigate the effects of ovariectomy on the blood glucose， adipose tissue content and liver lipid deposition in the normal or hyperinsulinemia mice. Methods Twenty 6-week-old female loss of skeletal muscle-specific insulin-like growth factor-1 receptor function（MKR） hyperinsulinemia mice were randomly divided into MKR sham operation group （MKR group） and MKR ovariectomy group （MKR OVX group），with 10 mice in each group；twenty wild-type （WT） FVB/N mice were randomly divided into WT sham operation group（WT group） and WT ovariectomy group（WT OVX group），with 10 mice in each group. After two weeks of recovery feeding， the body weights and blood glucose levels of the mice in various groups were monitored. Glucose tolerance abilities and insulin sensitivities of the mice in various groups were detected by glucose tolerance test（GTT） and insulin tolerance test（ITT）. The mice were sacrificed 12 weeks after surgery，and the weights of liver and adipose of the mice in various groups were weighed.The pathomorphology of liver and gonadal adipose tissue of the mice in various groups were observed by HE staining，glycogen staining and oil red O staining. The expression levels of lipid metabolization-related gene mRNA in gonadal adipose tissue of the mice in various groups were detected by real-time fluorescence quantitative PCR（RT-qPCR） method. Western blotting method was used to detect the expression levels of hormone sensitive lipase（HSL） in gonadal adipose tissue of the mice in various groups. Results The body weight of the mice in MKR group was significantly decreased compared with WT group （P<0.05）； compared with MKR group， the body weight of the mice in MKR OVX group was significantly increased （P<0.05）， but compared with WT and WT OVX groups，the body weight of the mice in MKR OVX group was decreased （P<0.05）. In MKR OVX group， the blood glucose of the mice was gradually increased， and severe glucose intolerance and insulin resistance appeared. Compared with WT group， liver steatosis was found and glycogen was decreased in WT OVX group （P<0.05）， while the liver steatosis degree was increased and glycogen was decreased significantly in MKR OVX group.Compared with WT group，the gonadal adipose index and mesenteric adipose index of the mice in WT OVX group were increased（P<0.05）；compared with MKR group，the gonadal adipose index and perirenal adipose index of the mice in MKR OVX group was increased（P<0.05）.The HE staining results showed that the adipocytes in MKR group were significantly shunk compared with WT group， and the adipocytes were enlarged in WT OVX group and MKR OVX group，the adipocytes in MKR OVX group were still shunk compared with WT group and WT OVX group. Compared with WT group， the expression levels of HSL mRNA and protein in gonadal adipose tissue of the mice in MKR group were increased （P<0.05）， while the expression levels of HSL mRNA and protein in gonadal adipose tissue of the mice in WT OVX group and MKR OVX group were decreased（P<0.05）. Conclusion Ovariectomy can lead to dysglycemia， adipose tissue mass increasing and lipid deposition in the liver in the mice. High endogenous insulin levels and insulin resistance can further aggravate the dysglycemia caused by ovariectomy.

Objective To investigate the effect of overexpression of Toll-like receptor 4 （TLR4） gene by lentivirus on autophagy of the gastric cancer cells，and to clarify its mechanism. Methods The gastric cancer cells BGC-823 and SGC-7901 at logarithmic growth phrase were divided into non-infection group，negative control virus group and over-expression TLR4 group； they were infected with lentivirus-free， empty vector and lentivirus that overexpress TLR4， separately. Western blotting method was used to detect the expression levels of TLR4 protein in gastric epithelial cells GES-1 and gastric cancer cells BGC-823， SGC-7901， HGC-27 and MGC-803.CCK-8 assay was used to detect the proliferation activities of cells in various groups，real-time fluorescence quantitative PCR （RT-qPCR） method was performed to determine the expression levels of TLR4，Beclin1 and microtubule-associated protein light chain 3（LC3） mRNA，the expression levels of phosphatidylinositol-3 kinase/protein kinase B （PI3K/Akt）， mammalian target of rapamycin （mTOR）， phosphorylated PI3K/phosphorylated Akt （p-PI3K/p-Akt） and phosphorylated mTOR （p-mTOR） signaling pathway-related proteins and autophagy-related proteins （LC3， Beclin1 and p62） were detected by Western blotting method. Results The expression levels of TLR4 protein in BGC-823 cells， HGC-27 cells and MGC-803 cells were higher than that in gastric epithelial cells GES-1.Compared with non-infection and negative control virus groups，the TLR4 protein expression levels in BGC-823 and SGC-7901 cells in over-expression TLR4 group were significantly increased（P<0.05）.Compared with non-infection control and negative control virus groups，the proliferation activities of BGC-823 cells and SGC-7901 cells in over-expression TLR4 group were significantly increased（P<0.05 or P<0.01）.Compared with negative control group，the expression levels of TLR4 mRNA in the BGC-823 cells and SGC-7901 cells in over-expression TLR4 group were significantly increased（P<0.01），the expression level of Beclin1 mRNA in BGC-823 cells was decreased（P<0.05），and the expression levels of LC3 and Beclin1 mRNA in SGC-7901 cells were decreased（P<0.05）.Compared with non-infection group and negative control group，the expression levels of LC3 and Beclin1 proteins in the BGC-823 cells and SGC-7901 cells in over-expression TLR4 group were significantly decreased（P<0.05），while the expression levels of p62， p-PI3K、p-Akt and p-mTOR proteins were increaed（P<0.05）. Conclusion The over-expression of TLR4 gene can significantly inhibit autophagy in gastric cancer cells via regulating the PI3K/Akt/mTOR signaling pathway.

Objective To investigate the improvement effects of oil and ethanol extract of Atractylodes rhizome on the dextran sodium sulfate （DSS）-induced ulcerative colitis （UC） in the mice， and to provide the experimental basis for the clinical application of Atractylodes rhizome. Methods Forty male BALB/c mice were randomly divided into normal group， model group， Atractylodes rhizome oil （0.185 g·kg^－1） group， Atractylodes rhizome ethanol extract （1.107 g·kg^－1） group and sulfasalazine （0.250 g·kg^－1） group， with 8 mice in each group.The UC mouse model was established by freely drinking 3.5% DSS solution. The body weights and disease activity index （DAI） scores of the mice in various groups were detected. After dissecting the mice， the colon lengthes were detected and the spleen coefficient of mice in various groups were calculated.The myeloperoxidase（MPO） activities in colon tissue of the mice in various groups were detected by MPO kit， the pathomorphology of colon tissue of the mice in various groups was observed by HE staining， the expression levels of the tumor neosis factor-α（TNF-α），interleukin-1β（IL-1β） and interleukin-6（IL-6） mRNA in colon tissue of the mice in various groups were detected by real-time fluorescence quantitative PCR （RT-qPCR） method，the positive expressions of inflammatory factors in colon tissue of the mice in various groups were detected by immunohistochemical staining，and alcian blue-periodic acid-Schiff（AB-PAS） staining was used to observe the number of goblet cells in colon tissue of the mice in various groups. Results Compared with normal group， the body weights of the mice in model group on the 7th and 8th days were decreased （P<0.05）， the DAI scores on the 4th－8th days were increased （P<0.05）， the colon lengthes were decreased （P<0.05）， the spleen coefficients were increased （P<0.05）， the MPO activities in colon tissue were increased （P<0.05）， the colon tissue was seriously damaged， the expression levels of TNF-α，IL-1β and IL-6 mRNA on the 4th－8th days were increased（P<0.05）， the positive expressions of inflammatory factors were increased， and the number of goblet cells was decreased. Compared with model group， the body weights of the mice in Atractylodes rhizome oil group， Atractylodes rhizome ethanol extract group and sulfasalazine group on the 7th and 8th days were increased，but the difference was not statistically significant（P>0.05）， the DAI scores on the 6th－8th days were decreased （P<0.05）， the colon lengthes were increased （P<0.05）， the spleen coefficients were decreased （P<0.05）， the MPO activities in colon tissue were decreased （P<0.05）， the injury degrees of colon tissue were alleviated，the expression levels of TNF-α，IL-1β and IL-6 mRNA in colon tissue were decreased（P<0.05）， the positive expressions of inflammatory factors were decreased， and the number of goblet cells was increased. Compared with Atractylodes rhizome oil group， the MPO activity in colon tissue of Atractylodes rhizome ethanol extract group was decreased， but the difference was not statistically significant（P>0.05）； the expression levels of TNF-α，IL-1β and IL-6 mRNA were decreased（P<0.05）， and the number of goblet cells was increased. Conclusion The oil and ethanol extract of Atractylodes rhizome have improvement effects in the UC model mice. At the same dose， the ethanol extract of Atractylodes rhizome can improve UC in the mice better.

Objective To investigate the efficency of Toll-like receptor 3 （TLR3） activation in the human umbilical cord mesenchymal stem cells（hUC-MSCs） transplantation for the treatment of lupus nephritis （LN）， and to preliminariy clarify its mechanism. Methods The hUC-MSCs were cultured and identified， and treated with 10 mg·L^－1 polyinosinic acid. A total of 45 MRL/Lpr female mice were randomly divided into model group， hUC-MSCs group， and TLR3+hUC-MSCs group，and another 15 C57BL/6C female mice were selected as control group；the mice were the injected with hUC-MSCs or polyinosinic acid-treated hUC-MSCs via tail vein. After administration， the levels of 24 h urine protein of the mice were measured every two weeks； the blood sample and kidney tissue were collected after 8 weeks，and the levels of anti-double stranded DNA（anti-dsDNA） antibody in serum and the levels of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， interleukin-6（IL-6）， and interleukin-17（IL-17） in kidney tissue of the mice in various groups were detected by enzyme linked immunosorbent assay（ELISA） method. HE staining and PAS staining were used to observe the pathomorphology of kidney tissue； immunofluorescence staining was used to detect the deposition of IgG and complement C3 in kidney tissue. Western blotting method was used to detect the expression levels of protein kinase B （Akt）， mammalian target of rapamycin （mTOR） and autophagy-related proteins in kidney tissue of the mice in various groups. Results The hUC-MSCs had typical mesenchymal stem cell characteristics. Compared with control group， the level of anti-dsDNA antibody in serum of the mice in model group was significantly increased （P<0.05），the levels of TNF-α， IL-1β， IL-6 and IL-17 in kidney tissue were significantly increased （P<0.05）， and there were glomerular mesangial cell proliferation， renal tubular atrophy and necrosis， and inflammatory cell infiltration in kidney tissue； IgG and C3 deposition in glomerulus was obvious （P<0.05）， and the expression levels of Akt and mTOR proteins in kidney tissue were significantly increased （P<0.05），while the expression levels of microtubule-assaciated protein 1 light chain 3 Ⅱ（LC3Ⅱ） and Beclin1 proteins were significantly decreased （P<0.05）. Compared with model group， the level of 24 h urine protein of the mice in TLR3+hUC-MSCs group was significantly reduced at 4 weeks of administration（P<0.05）， and the levels of 24 h urine protein of the mice in hUC-MSCs group and TLR3+hUC-MSCs group were significantly decreased at 6 and 8 weeks of administration（P<0.05）；and the levels of anti-dsDNA antibody in serum of the mice in hUC-MSCs group and TLR3+hUC-MSCs group were significantly decreased （P<0.05）；the levels of TNF-α， IL-1β， IL-6 and IL-17 in kidney tissue were significantly decreased （P<0.05）， the lesion of kidney tissue was improved to a certain extent， the depositions of IgG and C3 in kidney tissue were reduced （P<0.05）， and the improvement effect in TLR3+hUC-MSCs group was more significant； the expression levels of Akt and mTOR proteins in kidney tissue of the mice in hUC-MSCs group and TLR3+hUC-MSCs group were significantly decreased （P<0.05）， and the expression levels of LC3Ⅱ and Beclin1 proteins were significantly increased （P<0.05）. Compared with hUC-MSCs group， the expression levels of LC3Ⅱ and Beclin1 proteins in kidney tissue of the mice in TLR3+hUC-MSCs group were significantly increased （P<0.05）. Conclusion TLR3 can enhance the effect of hUC-MSCs transplantation in the treatment of LN in the mice， and inhibit kidney tissue inflammation and regulate the level of autophagy.

Objective： To explore the effect of pituitary adenylate cyclase activating polypeptide（PACAP）27 on the cyclophosphamide-induced testicular injury in the rats， and to clarify its possible mechanism. Methods Sixty SD rats were randomly divided into control group， model group， PACAP27 group and L-carnitine group， with 15 rats in each group.Except control group，the rats in other groups were treated with cyclophosphamide to establish the models and administrited with drugs. On the 3rd day after the administration， the weights of rats and the testis were weighed， and the testis index was calculated；the levels of serum follicle-stimulating hormone （FSH）， luteinizing hormone （LH） and testosterone （T） of the rats in various groups were measured by radioimmunoassay， HE staining was used to observe the pathomorphology of testis tissue of the rats in various groups； the activities of superoxide dismutase （SOD）， catalase （CAT） and the levels of malondialdehyde （MDA） in testis tissue of the rats in various groups were detected by enzyme linked immunosorbent assay（ELISA） method， TUNEL staining was used to detect the apoptotic rates of testicular tissue of the rats in various groups， and Western blotting method was used to detect the expression levels of mitochondrial apoptosis pathway-related proteins.The testicular tissue mitochondria were extracted， the opening degrees of the mitochondrial permeability transition pore （mPTP） of the rats in various groups were detected with mitochondrionl isolation reagent， and JC-1 fluorescent staining was used to detect the mitochondrial membrane potential. Results Compared with control group， the body weight， testis weight and testis index of the rats in model group were decreased （P<0.05）， the levels of FSH， LH and T in serum were decreased （P<0.05）， the activities of SOD and CAT in testis tissue were decreased （P<0.05）， the level of MDA was increased （P<0.05）， the testis tissue had obvious pathological damage， and the apoptotic rate was increased （P<0.05）， the expression levels of cytochrome C（Cyt C），B-cell lymphoma-2 associated X protein（Bax），cysteinyl aspartate specific proteinase-3（caspase-3） and cysteinyl aspartate specific proteinase-9（caspase-9） proteins were increased （P<0.05）， the expression level of B-cell lymphoma-2（Bcl-2） protein was decreased （P<0.05）， the opening degree of mPTP was enhanced （P<0.05）， and the mitochondrial membrane potential was decreased （P<0.05）. Compared with model group， the testis weights and testis indexes of the rats in PACAP27 group and L-carnitine group were increased （P<0.05）， the levels of FSH， LH and T in serum were increased （P<0.05）， the activities of SOD and CAT were increased （P<0.05）， the levels of MDA were decreased （P<0.05）， the pathological damages of testis tissue were improved， the apoptotic rates were decreased （P<0.05）， the expression levels of Cyt C， Bax， caspase-3 and caspase-9 proteins were decreased （P<0.05）， the expression levels of Bcl-2 protein were increased （P<0.05），the mPTP opening degrees were decreased（P<0.05），and the mitochondrial membrane potentials were increased （P<0.05）. Conclusion PACAP27 may alleviate cyclophosphamide-induced testicular injury in the rats by inhibiting mitochondria-dependent apoptosis pathway.

Objective： To investigate the effects of different concentrations of traditional Chinese medicine indirubin derivative E804 on the proliferation， apoptosis and differentiation of non-small cell lung cancer （NSCLC） A549 cells， and to clarify its possible mechanism. Methods The NSCLC A549 cells were cultured in vitro and divided into control group （0 μmol·L^－1 E804） and different concentrations（2.5， 5.0 and 10.0 μmol·L^－1） of E804 groups. MTT assay was used to detect the proliferation rates of cells in various groups； cell scratch assay was used to detect cell migration abilities in various groups； soft agar cloning assay was performed to observe cell differentiation and colony formation abilities in various groups； flow cytometry was used to detect apoptotic rates of cells in various groups； Western blotting method was used to detect the expression levels of apoptosis-related proteins and Janus kinase 1 （JAK1）/signal transducer and activator of transcription 3 （STAT3） signaling pathway-related proteins in the cells in various groups. Results The MTT assay results showed that compared with control group，the proliferation rates of A549 cells in 2.5， 5.0 and 10.0 μmol·L^－1 E804 groups were significantly decreased（P<0.05 or P<0.01） in a time- and concentration-dependent manner； the cell scratch and soft agar cloning methods results showed that compared with control group， the migration distance and the number of colony formation of the cells in 2.5， 5.0 and 10.0 μmol·L^－1 E804 groups were decreased（P<0.05 or P<0.01）. The flow cytometry results showed that compared with control group， the apoptotic rates in 2.5， 5.0 and 10.0 μmol·L^－1 E804 groups were increased（P<0.05 or P<0.01）.The Western blotting method results showed that compared with control group， the expression levels of B-cell lymphoma-2（Bcl-2），phosphorylated JAK1（p-JAK1） and phosphorylated STAT3（p-STAT3） proteins in the cells in 2.5， 5.0 and 10.0 μmol·L^－1 E804 groups were decreased（P<0.05 or P<0.01）， while the expression levels of Bcl-2 related X protein（Bax）， cleaved cysteinyl aspartate specific proteinase-3（cleaved caspase-3） and cleaved cysteinyl aspartate specific proteinase-9（cleaved caspase-9）proteins were increased（P<0.05 or P<0.01）. Conclusion E804 can inhibit the proliferation， migration and differentiation of A549 cells in vitro， and induce their apoptosis， and its mechanism may be related to the down-regulation of expressions of JAK1/STAT3 signaling pathway-related proteins.

Objective To investigate the pregnancy outcomes of fresh embryo transfer in the same infertile patients receiving the GnRH antagonist （GnRH-ant） program and the GnRH agonist （GnRH-a） program for ovulation， and to provide basis for the selection of ovulation induction program. Methods Using retrospectively research method， the clinical data and laboratory examination data of 74 patients （148 cycles in total） received GnRH-a program and GnRH-ant program treatment for ovulation within 2 years were collected. The effects of ovulation promotion， embryo development and clinical outcome of fresh cycle transplantation were analyzed. Results The ovulation induction detection results showed that compared with GnRH-a group， the total dose and time of gonadotropin （Gn） use， and endometrial thickness of the patients in GnRH-ant group were decreased （P<0.05 or P<0.01）； the level of luteinizing hormone （LH） on the human chorionic gonadotropin （HCG） day was significantly increased （P<0.01）；there were no significant differences in the number of oocytes retrieved and the levels of progesterone（P） and estradiol（E2） on the HCG day （P>0.05）. The embryo status detection results showed that compared with GnRH-a group，the 2 pronucleus（2PN） rate， abnormal fertilization rate，2PN cleavage rate， available blastocyst formation rate and high quality blastocyst formation rate in GnRH-ant group were increased， but there were no significant differences （P>0.05），while the good embryo rate and available embryo rate in GnRH-ant group were significantly increased （P<0.05）.In clinical pregnancy outcome， there were no significant differences in the number of embryos transferred， ectopic pregnancy rate， abortion rate， implantation rate and clinical pregnancy rate in two groups （P>0.05）. Conclusion GnRH-ant program could be a more suitable plan for ovulation due to the higher good embryo rate， avaliable embryo rate， lower Gn dose and shorter Gn using time compared with GnRH-a program.

Objective To screen the specific genes of cystic fibrosis （CF） and predict the target genes， and to study its mechanism. Methods The datasets（GSE71799，GSE24206，GSE98925 and GSE69764） including CF samples and normal samples were downloaded from the Gene Expression Omnibus（GEO）database and divided into CF group and control group. The R software limma package was used to screen the differentially expressed genes（DEGs） in CF group and control group. Gene Ontology（GO） and Kyoto Encyclopedia of Genes and Genomes（KEGG）were used to analyze the function and pathway enrichment of DEGs， Gene Set Enrichment Analysis（GSEA）was used to obtain the significantly enriched gene sets of DEGs， and STRING database was used to establish the protein-protein interaction（PPI）network. The PPI network was analyzed and visualized by Cytoscape software， and the hub genes were screened. Results A total of 429 DEGs were obtained and screened， including 105 DEGs that were overexpressed in CF group and 324 DEGs that were overexpressed in control group（|log2（FC）|>1，P<0.05）. The GO enrichment analysis results showed that DEGs were mainly enriched in the neutrophil mediated immunity， chemokine activity and cell adhesion molecule binding and so on（P<0.05）. The results of KEGG pathway analysis demonstrated that DEGs were mainly enriched in interleukin-17（IL-17） signaling pathway（P<0.05）. The results of GSEA demonstrated that DEGs were mainly enriched in signaling pathway translation， ribosomal RNA（rRNA） processing and mitochondrial translation and so on. Through STRING database and Cytoscape software， 35 hub genes in the PPI network were screened out， including matrix metallopeptidase 9（MMP9）， C-X-C motif chemokine ligand 2（CXCL2） and C-X-C motif chemokine ligand 3（CXCL3）. In addition， CXCL2 and CXCL3 were associated with DNA methylation. Conclusion The disorder of neutrophil-mediated immunity-related genes and IL-17 signaling pathway-related genes may play an important role in the occurrence and development of CF. CXCL2 and CXCL3 genes may become the biomarkers for DNA methylation therapy of CF.

Objective To investigate the factors associated with late spontaneous abortion after pregnancy in the patients assisted by assisted reproductive technology （ART） ， and to illuminate the risk factors for late spontaneous abortion. Methods Using retrospective research method， the clinical data of 310 pregnant patients underwent ART were collected； according to the different pregnancy outcomes of patients， they were divided into term birth group （248 cases） and late abortion group （62 cases）. The general situation， main causes and related factors of infertility， clinical situation and pregnancy outcomes of the patients in two groups were recorded， and the factors with P<0.05 in single factor regression analysis were included in the multivariate Logistic regression equation to further analyze the risk factors of late abortion. Results The differences in number of previous late miscarriages or preterm births， previous history of cervical LEEP or conization， previous history of hysteroscopic electrodesection， history of polycystic ovary syndrome（PCOS）， body mass index （BMI）， cycle type， endometrial thickness，whether twin pregnancies and ovulation disorder of the patients between two groups were statistically significant （P<0.05）.The factors with P<0.05 were included in the multivariate Logistic regression equation，BMI（OR=1.194，95%CI：1.088－1.311， P<0.01），previous history of late abortion or premature delivery（OR=5.673，95%CI：1.189－27.069， P=0.029）， history of cervical LEEP or conization（OR=5.113， 95%CI：1.025－25.496， P=0.047） and twin pregnancy（OR=5.129，95%CI：2.377－11.067，P<0.01） were the risk factors of late spontaneous abortion. Frozen thawed embryo transfer （FET） cycle was the protective factor of late abortion compared with fresh transplantation cycle （OR=0.422， 95% CI：0.219－0.814，P=0.010）. Conclusion Previous history of premature or late abortion， cervical surgery，high BMI and multiple pregnancy are the risk factors of late spontaneous abortion after ART-assisted pregnancy of the patients， while FET is the protective factor of late spontaneous abortion.

Objective To investigate the effects of continuous infusion of dexmedetomidine on the perioperative stress response， lactate（Lac） clearance rate and postoperative rehabilitation of the patients with peritoneal cancer undergoing selective cytoreductive surgery（CRS） combined with hyperthermic intraperitoneal chemotherapy （HIPEC）. Methods The clinical data of 60 patients with peritoneal cancer underwent CRS combined with HIPEC under general anesthesia were collected.After screening，4 cases of patients were eliminated； the patients were divided into control group （general anesthesia）（27 patients） and dexmedetomidine group（29 patients）. All patients were subjected to general anesthesia，and the patients in dexmedetomidine group recieved dexmedetomidine at the same time. Intraoperative invasive arterial blood pressure （ABP） and central venous pressure （CVP） were monitored， cardiac index （CI） was measured， rate pressure product （RPP） were calculated， and intraoperative fluid replacement was guided by stroke volume variation （SVV）. Mean arterial pressure （MAP）， heart rate（HR）， RPP，CVP and CI were recorded before surgery （T0）， before the start of thermoperfusion chemotherapy （T1）， after thermoperfusion chemotherapy （T2）， and after surgery （T3），and the levels of Lac， base excess （BE），blood glucose （Glu） and cortisol in blood were calculated and Lac dearance rate was calculated. The postoperative complications within 3 d after surgery of the patients between two groups were recorded. Results There were no statistically significant differences in circulatory indicators such as MAP， HR，RPP， CVP and CI between two groups at T0 （P>0.05）.At T1，compared with control group，RPP of the patients in dexmedetomidine group was decreased（P<0.05）. At T2， compared with control group， HR and CVP of the patients in dexmedetomidine group were significantly decreased （P<0.05）， and RPP was decreased （P<0.05）.At T3，compared with control group，HR，MAP and CVP of the patients in dexmedetomidine group were significantly decreased （P<0.05），RPP was decreased （P<0.05）；there was no significant difference in CI between two groups （P>0.05）. At T0， there were no significant differences in the levels of Lac， BE， Glu and cortisol in blood of the patients between two groups （P>0.05）. At T1 and T2， compared with control group，the Lac， Glu and cortisol levels in blood of the patients in dexmedetomidine group were decreased （P<0.05）， and BE negative value was decreased （P<0.05）. At 12 h after surgery（T4）， 24 h after surgery（T5）， 48 h after surgery（T6） and 72 h after surgery（T7）， compared with control group，the Lac levels of the patients in dexmedetomidine group were decreased （P<0.05）， and the Lac clearance rates were significantly increased （P<0.05）；and the levels of alanine aminotrasferase（ALT） and aspartate aminotransferase（AST） were significantly decreased at T5 and T7 （P<0.05）.Compared with control group， the incidence cases of postoperative hypotension， nausea and vomiting， abdominal distention， chills and sleep disorders of the patients in dexmedetomidine group were significantly reduced at 3 d after surgery（P<0.05）. Conclusion Intraoperative dexmedetomidine continuous infusion can reduce the perioperative stress response and Lac levels， reduce the incidence of postoperative complications， and promote postoperative recovery in the patients with peritoneal cancer.

Objective： To investigate the expressions of Apelin and its receptor angtiotensin Ⅱ type 1 receptor associated protein （APJ） in placenta tissue of the patients with gestational diabetes mellitus （GDM），and to explore its effect on the insulin resistance（IR）， proliferation and invasion of trophoblast cells. Methods The placenta tissue of patients with GDM （GDM group，30 cases） and pregnant women with normal glucose tolerance （NGT） （NGT group，30 cases） were collected. Immunohistochemistry was used to detect the rates of positive cells of Apelin and APJ in placenta tissue of the subjects in two groups； Western blotting method was used to detect the expression levels of Apelin and APJ proteins in placenta tissue； real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the expression levels of Apelin， APJ and adenosine monophosphate-activated protein kinase （AMPK）mRNA； Pearson correlation analysis method was used to analyze the correlation between the expression levels of Apelin and APJ mRNA and the expression level of AMPK mRNA. The trophoblast cells HTR-8/SVneo （HTR-8） were cultured in vitro and divided into control group， Apelin group， high glucose group（HG group）， HG+Apelin group，and HG+Apelin+anti-APJ group.The expression levels of Apelin， APJ， insulin receptor substrate 1 （IRS1）， insulin receptor substrate 2 （IRS2），and glucose transporter 4 （GLUT4） proteins and the phosphorylated levels of phosphatidylinositol 3-kinase （PI3K）（p-PI3K/PI3K） and AMPK （p-AMPK/AMPK） in the cells in various groups were detected by Western blotting method； 5-bromo-2-deoxyuracil（EdU）method was used to detect EdU positive expression rates in the cells in various groups； the number of invasion cells in various groups was detected by Transwell chamber assay. Results Apelin and APJ were widely expressed in placental villus tissue； compared with NGT group， the expression levels of Apelin and APJ mRNA and protein in placenta tissue of the patients in GDM group were significantly decreased （P<0.05）. The correlation analysis results showed that the expression levels of Apelin and APJ mRNA in placenta tissue of the patients in GDM group was positively correlated with the expression level of AMPK mRNA （R²=0.148， P<0.05；R²=0.275， P<0.05）.The Western blotting results showed that compared with control group， the expression levels of Apelin， APJ， IRS1， IRS2，and GLUT4 proteins and the ratios of p-PI3K/PI3K and p-AMPK/AMPK in Apelin group were significantly increased （P<0.05）， the expression levels of Apelin，APJ，IRS1，IRS2，and GLUT4 proteins and the ratios of p-PI3K/PI3K and p-AMPK/AMPK in HG group， HG+Apelin group and HG+Apelin+anti-APJ group were significantly decreased （P<0.05）； compared with HG group， the expression levels of IRS1， IRS2， and GLUT4 proteins and the ratios of p-PI3K/PI3K and p-AMPK/AMPK in HG+Apelin group were significantly increased（P<0.05）， and the differences in the indicators mentioned above in HG+Apelin+anti-APJ group were not statistically significant （P>0.05）.The EdU and Transwell assay results showed compared with control group， the EdU positive expression rate and the number of invasion cells in Apelin group were significantly increased （P<0.05）， while those in HG group， HG+Apelin group and HG+Apelin+anti-APJ group were significantly decreased （P<0.05）； compared with HG group， the EdU positve expression rate and the number of invasion cells in HG+Apelin group were significantly increased （P<0.05）， while the differences between HG group and HG+Apelin+anti-APJ group were not statistically significant （P>0.05）. Conclusion The expressions of Apelin and APJ in placenta tissue of the GDM patients are decreased， and exogenous administration of Apelin can improve IR of trophoblast cells and promote the abilities of proliferation and invasion of trophoblast cells under HG environment，and its mechanism may be related to the up-regulation of AMPK signaling pathway proteins by Apelin.

Objective： To investigate the expression of long non-coding RNA gastric carcinoma high expression transcript 1 （lncRNA GHET1） in placenta tissue of the preeclampsia （PE） patients and its effects on the proliferation， cycle progression and invasion ability of trophoblast cells， and to clarify their related mechanisms. Methods Thirty pregnant and lying-in women were divided into normal group（n=15） and PE group （n=15）. The pathomorphology of placenta tissue of the subjects in two groups were observed by HE staining.The HTR-8 cells were cultured in vitro， and transfected with over-expression lncRNA GHET1 and control sequence plasmids， and the cells were divided into control group （conventional culture）， GHET1 group （trasfected with over-expressed lncRNA GHET1 plasmid） and negative control group （transfected with negative control sequence plasmid）.Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of lncRNA GHET1 mRNA in placenta tissue of the subjects in two groups and HTR-8 cells in various groups. The proliferation rates of HTR-8 cells in various groups were measured by CCK-8 method. The percentages of HTR-8 cells at different cell cycles in various groups were detected by flow cytometry. The invasion abilities of HTR-8 cells in various groups were detected by Transwell chamber method. Western blotting method was used to detect the expression levels of cellular-myelocytomatosis viral oncogene（c-Myc）， cyclinD1 and epithelial mesenchymal transformation （EMT）-associated protein E-cadherin，and Vimentin and the expression levels of Wnt/β-catenin signaling pathway-associated proteins and ratio of phosphorylated glycogen synthase kinase 3β （p-GSK3β）/glycogen synthase kinase 3β（GSK3β） and β-catenin proteins in the HTR-8 cells in various groups. Results Compared with normal group， the villi of placenta tissue of the patients in PE group were dysplasia and the amount was decreased， the vascular distribution in villi and interstitium was disorder， the vascular wall showed fibrinoid necrosis， and the calcified area and syncytial nodule on the villi were increased. Compared with normal group， the expression level of lncRNA GHET1 mRNA in placenta tissue of the patients in PE group was significantly decreased （P<0.05）. Compared with control group， the lncRNA GHET1 mRNA expression level in the HTR-8 cells in GHET1 group was significantly increased（P<0.05）， while there was no significant difference in the indicators mentioned above in negative control group （P>0.05）. Compared with control group， the proliferation rate， the percentage of cells at S phase，and the number of invasion cells of HTR-8 cells in GHET1 group were significantly increased （P<0.05）， while there were no significant differences in the indicators mentioned above in negative control group（P>0.05）. Compared with control group， the expression levels of c-Myc， cyclinD1， Vimentin and β-catenin proteins and ratio of p-GSK3β/GSK3β in the HTR-8 cells in GHET1 group were significantly increased （P<0.05）， while the expression level of E-cadherin was significantly decreased （P<0.05）； there were no significant differences in the indicators mentioned above in negative control group （P>0.05）. Conclusion Over-expression of lncRNA GHET1 in placenta tissue of the PE patients may promote trophoblast cell proliferation， cell cycle progression， and cell invasion through Wnt/β-catenin signaling pathway， and play a role in improving the progression of PE.

Objective To establish a method for rapid detection of Bacillus cereus entFM toxin gene based on nucleic acid test strip technology. Methods The Bacillus cereus DNA was extracted by boiling method， the Bacillus cereus entFM toxin gene was used as the target gene，the specific primers were designed using NCBI primer-BLAST 5.0， and the PCR products were identified by cloning and transformation；the nucleic acid test strips were established， and the specificity， sensitivity and stability of nucleic acid test strips were evaluated. Results The concentration of Bacillus cereus DNA was 300 mg?L^－1， and the purity was about 1.60. The positive bands of PCR products were 100% similar to entFM registered in GenBank database after gel cutting recovery， clone transformation and sequencing comparison. At the condition of pH value was 7.0， 3.3 μg streptavidin was added to each 100 μL colloidal gold solution for labeling， the quality control line concentration was 1.8 g?L^－1， the detection line concentration was 1 g?L^－1， and both the quality control line and the detection line on the nitro cellulose membrane could react with the PCR products to produce the clear red bands. The nucleic acid test strips were assembled according to the optimum conditions， with 6 μL of PCR product and 100 μL of sample developing solution， and the results could be observed after 10 min of detection. The specificity of the nucleic acid test strips was consistent with the electrophoresis results， only Bacillus cereus was a positive result， and there was no cross-reaction with Staphylococcus aureus， Pseudomonas aeruginosa， Escherichia coli and Salmonella， and they were negative results.The sensitivity test results showed that nucleic acid test strips could accurately detect when the DNA mass concentration droped to 10^－3 mg?L^－1 at the same time. The results of ordinary PCR electrophoresis showed that when the DNA mass concentration droped to 10^－1 mg?L^－1 at the same time， the target band appeared.The sensitivity of nucleic acid test strips was 100 times higher than that of ordinary PCR； for stability testing， nucleic acid test strips had the same stability in the 6th， 9th and 12th months. Conclusion The established nucleic acid test strips have high sensitivity， strong specificity and good stability for the detection of Bacillus cereus entFM toxin gene， which is suitable for rapid identification of Bacillus cereus entFM toxin gene.

Objective To construct the lentiviral expression vector of T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain-green fluorescent protein （TIGIT-GFP） and establish a cell line stably expressing TIGIT-GFP， and to explore the expression of TIGIT-GFP fusion protein in stable expression cell line. Methods The TIGIT plasmid and lentiviral vector plenti-GFP were doubly digested by endonucleases EcoR Ⅰ and Not Ⅰ. The inserted gene and vector in agarose gel were recovered and ligated to construct the recombinant plasmid pLenti-TIGIT-GFP. The human embryonic kidney HEK293T cells transfected with sequenced recombinant plasmid were used as experimental group， and the HEK293T cells transfected with pCMV6-TIGIT were used as control group. After 48 h of transfection， the expression of GFP in the cell membrane was detected under fluorescence microscope. The cells in experimental group were cultured and screened with puromycin for two weeks； the screened cells were selected and expanded，and the expression of TIGIT-GFP protein in cell membrance was examined by fluorescence microscope. Western blotting method was used to determine the TIGIT-GFP protein expression in monoclonal cells. Results The enzyme digestion results showed that TIGIT gene was inserted into the expression vector pLenti-GFP，and the DNA sequencing showed no mutation.The GFP in the cell membrane in experimental group was found. The Western blotting results showed a specific band of TIGIT-GFP in cell lysis buffer in experimental group. Conclusion The lentiviral expression vector pLenti-TIGIT-GFP is successfully established， the cell line stably expression TIGIT-GFP fusion protein is constructed，and the TIGIT-GFP protein is mainly expressed in the cell membrane of the stable cell line.