Objective: To investigate the effects of histone deacetylation enzyme 3 (HDAC3) on the quantity, developmental phenotypes and cytokine production function of the invariant nature killer T cells (iNKT), and to determine the regulatory effects of HDAC3 on the development and function of the iNKT cells. Methods: The T cell-specific hdac3 gene knockout (HDAC3KO) mice and their wild type normal control (WT) mice were used, and 3-5 mice were selected from every kind of mice in experiment. The lymphocytes from the thymus, spleen, lymph nodes and liver of the HDAC3KO and WT mice were separated, and the effects of HDAC3 on the number and developmental phenotypes of the iNKT cells were detected by cell surface antibody staining and flow cytometry methods. The experiment was repeated at least three times. The HDAC3KO mice and their homologous B6.SJL mice were used in the mixed hematopoietic chimerism experiment, and 4-6 mice were selected from every kind of mice. The bone marrow cells from HDAC3KO and B6.SJL mice were extracted, and after mixed together they were injected into the γ-ray irradiated B6.SJL mice via tail vein to prepare the bone marrow mixed chimeric models. Eight weeks later,the production and development of iNKT cells in the thymus from different bone marrows were detected by flow cytometry. The experiment was repeated for three times. The HDAC3KO and WT mice (3-5 mice from every kind of mice) were selected and introperitoneally injected by iNKT cell specific stimulator α-Galcer; 4 h later, the spleen lymphocytes and serum were collected,and the cytokine levels in the iNKT cells after treated with HDAC3 were detected by intracellular staining, flow cytometry and ELISA methods. The experiment was repeated at least 3 times. Results: Compared with the WT mice, the ratios and the number of iNKT cells in the thymus and peripheral immune organs of the HDAC3KO mice were significantly decreased(P<0.05);the ratios of CD44-NK1.1- and CD44+NK1.1-cells in the iNKT cells in thymus of the HDAC3KO mice were significantly increased(P<0.05); and the ratios of CD44+NK1.1+, CD122+, CD69+ and DX5+ cells were significantly decreased(P<0.05).The experiment of mixed bone marrow chimerism showed that the number of iNKT cells from the bone marrow cells of the HDAC3KO mice was significantly decreased compared with the bone marrow cells of the B6.SJL mice(P<0.05),and the ratio of CD44+NK1.1+ cells in the iNKT cells from the bone marrow cells of the HDAC3KO mice was significantly lower than that of the iNKT cells from the B6.SJL mice (P<0.05).The results of intracellular staining and ELISA showed that the levels of IFN-γ and IL-4 in the iNKT cells and serum of the HDAC3KO mice were significantly decreased compared with the WT mice(P<0.05). Conclusion: HDAC3 deficiency results in the decrease of the number of iNKT cells and the blocking of development and maturation of iNKT cells by an endogenous pathway.In addition, HDAC3 deficiency results in the significant reduction of cytokine production in the iNKT cells. All the results indicate that HDAC3 plays an important role in the regulation of iNKT cell development and function.

Objective: To investigate the effect of honokiol(HNK) on the inflammatory response in lung tissue of the asthma mice, and to clarify the intervention effect and its related mechanism. Methods: Twenty healthy female C57BL/6J mice were randomly divided into control group, model group, dexamethasone(DXM) group and HNK group,with 5 mice in each group.The asthma mouse model was established by intraabdominal injection of ovalbumin(OVA) combined with aluminum hydroxide[Al(OH)₃] suspension and OVA nasal stimulation.Thirty min before stimulation,the mice in DXM group and HNK group were treated with intraabdominal injection of the DXM solution and HNK solution, and the mice in model group were treated with the same amount of normal saline. After the mice were killed,the pathological changes of lung tissue were observed and evaluated by HE staining; the bronchoalveolar lavage fluid(BALF) was collected and the infiltration degree of inflammatory cells was observed by Diff-quik staining.The levels of IL-4,IL-6 and IL-17 in serum of the mice in various groups were detected by ELISA assay, and the expression levels of malondialdehyde(MDA) and the activities of superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) in lung homogenate of the mice in various groups were detected by colorimetric method;the expression levels of p-JNK,nuclear factor-κB(NF-κB),Caspase-3,Bcl-2 and γH2Ax proteins in lung tissue of the mice in various groups were detected by Western blotting method.The immunofluorescence intensities of γH2Ax in lung tissue of the mice in various groups were measured. Results: Compared with control group, the number of airway epithelial exfoliation and necrosis cells of the mice in model group was increased obviously, the airway wall was thickened and the airway side inflammation cell infiltration degree was increased obviously;compared with model group,the performances mentioned above of the mice in DXM group and HNK group were alleviated;the pathological changes of lung tissue of the mice in HNK group were similar to DXM group.Compared with control group,the leves of IL-4, IL-6 and IL-17 in serum of the mice in model group were increased(P<0.05);compared with model group, the levels of IL-4,IL-6 and IL-17 of the mice in DXM group and HNK group were decreased(P<0.05);there were no significant differences in the IL-4, IL-6 and IL-17 levels in serum of the mice between HNK group and DXM group(P>0.05).Compared with control group,the level of MDA in lung homogenate of the mice in model group was increased(P<0.05),but the activities of SOD and GSH-Px were decreased(P<0.05);compared with model group,the levels of MDA of the mice in DXM group and HNK group were decreased(P<0.05),but the activities of SOD and GSH-Px were increased(P<0.05);there were no significant differences in the MDA levels and the activities of SOD and GSH-Px of the mice between HNK group and DXM group(P>0.05).Compared with control group, the expression levels of p-JNK,NF-κB,Caspase-3 and γH2Ax proteins in lung tissue of the mice in model group were increased(P<0.05),but the expression level of Bcl-2 protein was decreased(P<0.05); compared with model group, the expression levels of p-JNK,NF-κB,Caspase-3 and γH2Ax of the mice in DXM group and HNK group were decreased(P<0.05),but the expression levels of Bcl-2 protein were increased(P<0.05);there were no significant differences in the expression levels of p-JNK, NF-κB, Caspase-3, γH2Ax and Bcl-2 proteins in lung tissue of the mice between HNK group and DXM group(P>0.05).Compared with control group,the immunofluorescence intensity of γH2Ax in lung tissue of the mice in model group was significantly enhanced;compared with model group,the immunofluorescence intensities of γH2Ax in lung tissue of the mice in DXM group and HNK group were relatively weak,while the immunofluorescence intensity of γH2Ax in HNK group was similar to that in DXM group. Conclusion: HNK can inhibit the lung tissue injury and inflammation response in the asthma mice to a certain extent, and its mechanism may be related to its inhibition of oxidative stress and DNA damage.

Objective: To observe the intervention effect of electro-acupuncture in the rats with neurogenic bladder caused by T10 spinal cord transection (SCT), and to clarify its mechanism. Methods: Forty-eight female SD rats were randomly divided into sham operation group (12 rats) and model group (36 rats). The T10 SCT models were induced by surgery,and the models were evaluated by Basso Beattie Bresnahan(BBB) score and urodynamics indexes. On the 15th day after surgery, the successful replication model rats were randomly divided into model control group (12 rats),electro-acupuncture group (12 rats) and electro-acupuncture control group (12 rats). The rats in electro-acupuncture group were treated with electro-acupuncture at "Dazhui"(DU14) and "Ciliao"(BL32); the rats in electro-acupuncture control group were treated with electro-acupuncture at the 1 cm acupuncture point next to the "Dazhui"(DU14) and "Ciliao"(BL32). The electro-acupuncture used dilatational wave (10 Hz/9 s, 50 Hz/5 s) for 20 min, and the intensity was suitable for regular contraction and twitching at the acupuncture point, once a day for a week in a row. After the last electro-acupuncture intervention, the urodynamic test was performed in the rats in each group, and the expression levels of Wnt-1 and β-catenin mRNA and protein in spinal cord tissue of the rats in various groups were detected by RT-PCR and immunofluorescence methods. Results: Compared with sham operation group, the bladder base pressures and maximum pressures of the rats in model control group, electro-acupuncture control group and electro-acupuncture group were increased(P<0.05 or P<0.01), and the bladder maximum capacities and compliance were decreased(P<0.01); the pressure of bladder leak point of the rats in model control group and electro-acupuncture control group were increased (P<0.01). Compared with model control group, the bladder base pressure, maximum pressure and leak point pressure of the rats in electro-acupuncture group were increased(P<0.01), and the bladder maximum capacity and compliance were increased (P<0.01).Compared with electro-acupuncture control group, the bladder basic pressure, maximum pressure and leak point pressure of the rats in electro-acupuncture group were decreased(P<0.05 or P<0.01), and the bladder maximum capacity and compliance were increased (P<0.05 or P<0.01). Compared with sham operation group, the expression levels of Wnt-1 and β-catenin mRNA and proteins in spinal cord tissue of the rats in model control group, electro-acupuncture control group and electro-acupuncture group were significantly increased (P<0.01). Compared with model control group, the expression levels of wnt-1 and β-catenin mRNA and proteins in spinal cord tissue of the rats in electro-acupuncture group were significantly increased (P<0.01). Compared with electro-acupuncture control group, the expression levels of Wnt-1 and β-catenin mRNA and proteins in the spinal cord tissues of the rats in electro-acupuncture group were significantly increased (P<0.01). Conclusion: electro-acupuncture on "Dazhui" (DU14) and "Ciliao" (BL32) has a significant improvement effect on the urodynamics of the rats with neurogenic bladder after T10 SCT, and its mechanism is related to regulating the activation of Wnt/β-catenin signal pathway.

Objective: To observe the regulatory effects of sericin on the insulin receptor(IR), insulin receptor substrate-1(IRS-1), phosphatidylinositol-3-kinase(PI3K) and protein kinase B(Akt) in the insulin PI3K/Akt signaling pathway in the rats of type 2 diabetes mellitus,and to discuss the mechanism of sericin in lowering the blood glucose. Methods: Thirty-six SPF male SD rats were randomly divided into normal group, model group and experimental group; there were 12 rats in each group.The rats in model group and experimental group were given with high-fat, high-sugar feeding and intraperitoneal injection of streptozotocin (STZ) of 35 mg·kg^-1·d^-1 for 2 d to establish the type 2 diabetic rat models, and the standard for successfully establishing model was the fasting blood glucose ≥ 11.1 mmol·L^-1 after injecting STZ for 72 h. After successfully establishing the diabetes models,the rats in experimental group were given sericin(2.4 mg·kg^-1·d^-1) by gavage, and the rats in normal group and model group were lavaged with the equal volume normal saline; lasted for 35 d. The glucose oxidase method was used to detect the fasting blood glucose of the rats in various groups.Immunohistochemical staining and Western blotting methods were used to detect the expressions of IR, IRS-1, PI3K and Akt proteins in liver tissue of the rats in various groups and periodic acid-schiff staining was used to determine the contents of liver glycogen of the rats in various groups. Results: The blood glucose level of the rats in model group was significantly higher than that in normal group(P<0.05), and the blood glucose level of the rats in experimental group was obviously lower than that in model group(P<0.05).The expressions of IR, IRS-1,PI3K and Akt proteins were tan granules located in the rat liver slices in normal and experimental groups,and some of positive proteins were diffuse;the expression amounts of IR,IRS-1,PI3K and Akt proteins in liver tissue of the rats in model group were significantly decreased,and they were tan granules;the IR, IRS-1 and Akt proteins were mainly located in the membrane and cytoplasm of hepatocytes, while the PI3K protein was mainly located in the cytoplasm of hepatocytes.The staining of liver glycogen of the rats in various groups was red or purple granules; the staining of liver glycogen of the rats in model group was relative light,and the area was small. Compared with normal group, the expression levels of IR,IRS-1,PI3K and Akt proteins and the content of liver glycogen in liver tissue of the rats in model group were decreased significantly(P<0.05);compared with model group, the expression levels of IR,IRS-1,PI3K,and Akt proteins and the content of liver glycogen in liver tissue of the rats in experimental group were increased significantly(P<0.05). Conclusion: Sericin can enhance the transduction effect of liver insulin PI3K/Akt signaling pathway by up-regulating the expressions of IR, IRS-1, PI3K and Akt in the liver tissue of the rats of type 2 diabetes mellitus, thus reduce the blood glucose level.

Objective: To explore the effect of Oxaliplatin on the proliferation and apoptosis of human tongue squamous cell carcinoma CAL27 cells, and to clarify its underlying mechanism. Methods: The CAL27 cells were treated with different concentrations of Oxaliplatin (0,10, 20, 40 and 80 mg·L^-1) and used as control group and 10, 20, 40 and 80 mg·L^-1 Oxaliplatin groups. The morphology of CAL27 cells in various groups were observed by crystal violet staining. The proliferation rates of the CAL27 cells in various groups were detected by CCK-8 assay. The percentages of CAL27 cells in different cell cycles and the apoptotic rates in various groups were detected by flow cytometry. The expression levels of cyclin-dependent kinase 2(CDK2)and E-type cyclin(cyclin E) mRNA in the CAL27 cells in various groups were analyzed by Real-time fluorescence quantitative PCR. The expression levels of anti-apoptotic protein survivin and pro-apoptotic protein Bax in the CAL27 cells in various groups were determined by Western blotting method. Results: Compared with control group, the CAL27 cells in 40 mg·L^-1 Oxaliplatin group wrinkled; the cells in 80 mg·L^-1 Oxaliplatin group lost the former morphology, and the intercellular connections of cells disapeared. Compared with control group, the proliferation rates of CAL27 cells in 10, 20, 40 and 80 mg·L^-1 Oxaliplatin groups were significantiy decreased (P<0.05 or P<0.01).Compared with control group, the percentage of CAL27 cells in G₀/G₁ phase in 40 mg·L^-1 Oxaliplatin group was significantly increased (P<0.01), and the expression levels of CDK2 and cyclin E mRNA were significantly decreased (P<0.05).Compared with control group, the apoptptic rates of CAL27 cells in 40 and 80 mg·L^-1 Oxaliplatin groups were markedly increased (P<0.01). Compared with control group, the expression levels of survivin protein in CAL27 cells in 40 and 80 mg·L^-1 Oxaliplatin groups were significantly decreased (P<0.01), and the expression levels of Bax protein were obviously increased (P<0.05 or P<0.01). Conclusion: Oxaliplatin can inhibit the proliferation and induce the apoptosis of CAL27 cells, and its mechanism may be related to arresting G₀/G₁ phase, up-regulating the expression level of survivin and down-regulating the expression level of Bax.

Objective: To observe the DNA damage of HT22 mouse hippocampal neuronal cells induced by inhaled anesthetic sevoflurane, and to clarify the possible mechanism of neurotoxicity induced by sevoflurane. Methods: The HT22 mouse hippocampal neuronal cells were randomly divided into blank control group, 2% sevoflurane groups (2%Sevo 6 h group, 2%Sevo 12 h group, 2%Sevo 24 h group), 4% sevoflurane groups (4%Sevo 6 h group, 4%Sevo 12 h group, 4%Sevo 24 h group) and 8% sevoflurane groups (8%Sevo 6 h group,8%Sevo 12 h group, 8%Sevo 24 h group).The survival rates and the mortalities of HT22 mouse hippocampal neuronal cells in various groups were measured by MTT method and LDH methods,respectively.According to the experiment results, the cells were divided into normal saline group, normal saline +4%Sevo 12 h group, normal saline+8%Sevo 12 h group, NAC group, NAC+4%Sevo 12 h group,and NAC+8%Sevo 12 h group.MTT method and LDH method were used to detect the survival rates and the mortalities of HT22 mouse hippocampal neuronal cells in various groups after NAC pretreatment; single cell gel electrophoresis was used to detect the DNA double-strand breaks in the HT22 mouse hippocampal neuronal cells in various groups; Western blotting method was used to detect the expression amounts of 8-OHdG, ATM, p-ATM, and γ-H2AX in the HT22 mouse hippocampal neuronal cells in various groups. DCFH-DA method was used to detect the levels of reactive oxygen species (ROS) in the HT22 mouse hippocampal neuronal cells in various groups. Results: Compared with blank control group, there were no significant differences in the survival rates and the mortalities of HT22 mouse hippocampal neuronal cells in 2% Sevo groups (P>0.05); the survival rates of HT22 mouse hippocampal neuronal cells in 4%Sevo groups and 8%Sevo groups were significantly decreased (P<0.01), and the mortalities were significantly increased (P<0.01). Compared with 4% Sevo groups, the survival rates of HT22 mouse hippocampal neuronal cells in 8% Sevo groups at the same time were decreased (P<0.05), and the mortalities were increased (P<0.05).Compared with 4% Sevo 6 h group, the survival rates of HT22 mouse hippocampal neuronal cells in 4% Sevo 12 h group and 4% Sevo 24 h group were decreased (P<0.05), and the mortalities were increased (P<0.05). The survival rate of HT22 mouse hippocampal neuronal cells in 4% Sevo 24 h group was lower than that in 4% Sevo 12 h group (P<0.05), and the mortality was higher (P<0.05).Compared with 8%Sevo 6 h group,the survival rates of HT22 mouse hippocampal neuronal cells in 8% Sevo 12 h group and 8% Sevo 24 h group were decreased (P<0.05),and the mortalities were increased (P<0.05); the survival rate of HT22 mouse hippocampal neuronal cells in 8%Sevo 24 h group was lower than that in 8%Sevo 12 h group (P<0.05), and the mortality was higher (P<0.05). Compared with normal saline group, the amounts of DNA double-strand breaks in the HT22 mouse hippocampal neuronal cells in normal saline + 4% Sevo 12 h group and normal saline + 8% Sevo 12 h group were increassed, the expression amounts of DNA damage-related proteins 8-OHdG, ATM, p-ATM, and γ-H2AX were increased, and the ROS levels were increased (P<0.05). Compared with normal saline + 4% Sevo 12 h group, the survival rate of hippocampal neurons of the HT22 mice in NAC + 4% Sevo 12 h group was increased (P<0.01), the mortality was reduced (P<0.01), the amount of DNA double-strand breaks in the hippocampal neuronal cells was reduced, the expression amount of DNA damage-related proteins 8-OHdG, ATM, p-ATM, γ-H2AX were decreased, and the ROS level in the hippocampal neuronal cells was significantly reduced (P<0.05). Compared with normal saline + 8% Sevo 12 h group, the survival rate of HT22 mouse hippocampal neuronal cells in NAC + 8% Sevo 12 h group was increased (P<0.01), the mortality was decreased (P<0.01), and the amount of DNA double-strand breaks in the hippocampal neuronal cells was reduced, the expression amounts of DNA damage-related proteins 8-OHdG, ATM, p-ATM,and γ-H2AX were decreased, and the ROS level in hippocampal neuronal cells was significantly reduced (P<0.05). Conclusion: Sevoflurane could cause the death of HT22 mouse hippocampal neuronal cells by inducing DNA damage,and its mechanism may be related to the intracellular ROS accumulation induced by sevoflurane.

Objective: To investigate the inhibitory effect of polysaccharides from panax ginseng fruit in the human tongue squamous cell carcinoma CAL27 cells,and to elucidate its potential mechanism. Methods: Water soluble ginseng berry polysaccharide (WGBP) was extracted by water extraction and alcohol precipitation method, and its monosaccharide components were analyzed by high performance liquid chromatography (HPLC). The human tongue squamous cell carcinoma CAL27 cells were divided into control group and different concentrations (1,2 and 5 g·L^-1) of WGBP groups. After culture for 24 h, CCK-8 method was used to detect the proliferation rates of the CAL27 cells in various groups, flow cytometry was used to detect the cell cycle and apoptotic rates of the CAL27 cells in various groups,and Western blotting method was used to detect the expression amounts of apoptosis-related proteins in the CAL27 cells in various groups. Results: WGBP was mainly composed of galactose and galacturonic acid, followed by arabinose, glucose, rhamnose and mannose. Compared with control group, the proliferation rates of CAL27 cells in 1,2 and 5 g·L^-1 WGBP groups were increased (P<0.05 or P<0.01) in a concentration-dependent manner.The flow cytometry results showed that compared with control group, the percentages of CAL27 cells in S phase in 2 and 5 g·L^-1 WGBP groups were decreased (P<0.05 or P<0.01), and the percentages of CAL27 cells in G₂/M phase were increased (P<0.05 or P<0.01) in a concentration-dependent manner.The Western blotting results showed that compared with control group, the apoptotic rates of CAL27 cells in 1,2 and 5 g·L^-1 WGBP groups were increased (P<0.05) in a concentration-dependent manner. Compared with control group, the expression amounts of pro-Caspase-3 and pro-PARP proteins in the CAL27 cells in 1,2 and 5 g·L^-1 WGBP groups were increased. Conclusion: WGBP can induce the apoptosis and inhibit the proliferation of human tongue squamous cell carcinoma CAL27 cells by inducing the G₂/M phase arrest and Caspase-3 activation.

Objective: To investigate the concentration distribution of gentiopicrin in the main organs and tissues of the rats before and after the preparation of gentian wine,and to provide the scientific basis for further discussion on the correlation between the processing methods and the efficacy of gentian. Methods: Sixty rats were randomly divided into gentian raw products group and gentian wine products group with 5 rats per group at each time point.The rats were given gentian raw products and gentian wine products (0.63 g·kg^-1) intragastrically and killed at 6 different time points (15, 30, 60, 120, 240 and 360min).The mass concentrations of gentiopicrin in heart,liver, spleen,lung,kidney and brain tissues of the rats in varrious groups were measured by high-performance liquid chromatography-mass spectrometry(HPLC-MS) method at different time points. Results: The linear relationship of gentiopicrin in the range of 50-20000 μg·L^-1 was good (r ≥ 0.996 9).The relative standard deviation (RSD) of precision and stability of this method was less than 15%.The recovery rate was 77.4%-87.2%,and RSD was less than 15%,which was in accordance with the technical guidelines for the study of non-clinical pharmacokinetics of chemical drugs. The results of HPLC-MS method showed that gentiopicrin was widely distributed in the rats,including heart,liver, spleen,lung,kidney and brain tissues and so on.At the point of 30 min,the gentiopicrin concentrations from gentian raw products and wine products reached the peak in heart tissue,and reached the peak values in liver spleen and lung and kidney tissues at 60 min.Compared with the gentian raw products,the area under curve (AUC_{0→360 min}) of gentiopicrin in the gentian wine products in liver,lung and kidney tissues were significantly increased (P<0.01),the AUC_{0→360 min} in brain tissue was increased (P<0.01),but the AUC_{0→360 min} in heart and spleen tissues were slightly decreased(P<0.05). Conclusion: Gentian can promote the absorption and utilization of gentiopicrin in liver,kidney and other organs,and change the concentration distribution of gentiopicrin in the main organs and tissues of the rats by processing with wine.

Objective: To observe whether magnesium-L-threonate (MLT) can enhance the antiviral effect of entecavir (ETV) in the HBV-infected mice, and to explore its mechanism. Methods: The recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome was injected into the C57BL/6 mice by tail vein to establish the HBV persistent infection mouse models. After successful modeling, the mice were divided into model group, MLT group (1.2 g·kg^-1 magnesium-L-threonate capsules), ETV group (75 μg·kg^-1 entecavir tablets), and combined treatment group (1.2 g·kg^-1 magnesium-L-threonate caspules+75 μg·kg^-1 entecavir tablets) (n=8), and another 8 normal mice uninfected with HBV were selected as control group. Then all the mice were intragastrically administered for 4 weeks. The serum levels of HBsAg and HBeAg were detected by ELISA method, the copied of HBV DNA in liver tissue and serum of the mice were detected by quantitative real-time PCR. The peripheral blood CD8+ T cells were isolated, and the contents of Mg²⁺ in serum and CD8+ T cells were detected. The expression levels of PD-1, NKG2D, CD95 and CD38 in the CD8+ T cells were detected by flow cytometry. Results: Compared with control group, the contents of Mg²⁺ in serum and CD8+ T cells and the expression levels of surface activation molecules NKG2D and CD38 in the CD8+ T cells in model group were significantly decreased (P<0.05), while the expression levels of surface inhibitory molecules PD-1 and CD95 in the CD8+ T cells were significantly increased (P<0.05). Compared with model group, the expression levels of serum HBsAg and HBeAg, and the copied of HBV DNA in liver tissue and serum of the mice in various administration groups were significantly decreased (P<0.05), and the improvement degree of the indexes in combined treatment group was superior to other administration groups. Compared with model group, the contents of Mg²⁺ and the expression levels of NKG2D and CD38 in MLT group and combined treatment group were significantly increased (P<0.05), the expression levels of PD-1 and CD95 were significantly decreased (P<0.05), but there were no significant differences in ETV group (P>0.05). Conclusion: MLT can enhance the antiviral effect of ETV in the HBV-infected mice, and the mechanism may be that MLT can cause the increase of Mg²⁺ contents in the CD8+ T cells to improve the activity of CD8+ T cells, and thereby HBV can be removed more effectively.

Objective: To investigate the effects of LincRNA-p21 knockdown on the proliferation, migration and invasion of gastric cancer cells, and to elucidate their mechanisms. Methods: The expression levels of LincRNA-p21 mRNA in the gastric cancer tissue, adjacent tissue, three types of gastric cancer cells (MGC-803, MKN-45 and SGC-790 cells) and normal gastric mucosal epithelial cells GES-1 were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The MGC-803 cells were used as the subjects and were divided into sh-NC group, sh-LincRNA-p21 group and AG490+sh-LincRNA-p21 group. The MGC-803 cells in sh-NC group were infected with sh-NC by lentivirus; the MGC-803 cells in sh-LincRNA-p21 group were infected with sh-LincRNA-p21 by lentivirus; the MGC-803 cells in AG490+sh-LincRNA-p21 group were infected with sh-LincRNA-p21 by lentivirus, and then treated with 10 μg·L^-1 AG490. The percentages of 5-ethynyl-2'-deoxyuridine (EdU) incorporation of the MGC-803 cells in various groups were detected by EdU incorporation assay. The viabilities of MGC-803 cells in various groups were measured by CCK-8 assay. The number of invasion and migration of MGC-803 cells in various groups was detected by Transwell assay. The expression levels of p-JAK1, p-STAT3 and p-STAT5 proeteins in the MGC-803 cells in sh-NC group and sh-LincRNA-p21 group were detected by Western blotting method. The MGC-803 cells from sh-NC group and sh-LincRNA-p21 group were subcutaneouly transplanted into the neck of the BALB/c nude mice, then the volumes and weights of the tumors of the mice were measured. Results: Compared with adjacent tissue, the expression level of LincRNA-p21 mRNA in gastric cancer tissue was significantly decreased (P<0.01); compared with the GES-1 cells, the expression levels of LincRNA-p21 mRNA in the MGC-803, MKN-45 and SGC-790 cells were significantly decreased (P<0.01).Compared with sh-NC group, the percentage of EdU incorporation, the cell viability, the number of migration and invasion cells, the expression levels of p-JAK1, p-STAT3 and p-STAT5 proteins in the MGC-803 cells in sh-LincRNA-p21 group were significantly increased (P<0.05 or P<0.01).Compared with sh-LincRNA-p21 group, the cell viability and the number of migration and invasion of the MGC-803 cells in AG490+sh-LincRNA-p21 group were significantly decreased (P<0.05 or P<0.01).The results of the tumorigenesis of nude mice showed that compared with sh-NC group, the volume and weight of the tumor of the mice in sh-LincRNA-p21 group were significantly increased (P<0.05 or P<0.01). Conclusion: LincRNA-p21 knockdown can significantly promote the growth and metastasis of gastric cancer cells, and this promotion may be related to promoting the activity of JAK-STAT signaling pathway.

Objective: To investigate the effects of montelukast (Mon) on the proliferation, apoptosis and secretion of inflammatory factors of the airway smooth muscle cells of the asthmatic rats, and to elucidate the molecular mechanism of airway remodeling and inflammatory response in the asthmatic rats. Methods: The acute asthma rat models were established by ovalbumin sensitization and stimulation.The primary cultured airway smooth muscle cells at the fifth generation were carried out for the experiment.The experiment was divided into control group (normal airway smooth muscle cells), model group (asthmatic model rat airway smooth muscle cells) and treatment group (asthmatic model rat airway smooth muscle cells +Mon intervention). The proliferation activities of the cells in various groups were detected by MTT method, the apoptotic rates of the cells in various groups were measured by flow cytometry, and the levels of interleukin-6(IL-6) and transforming growth factor-β1(TGF-β1) in cell supernatant in various groups were tested by ELISA method.The expression levels of proliferating cell nuclear antigen(PCNA), CyclinD1,B-cell lymphoma-2(Bcl-2), Bcl-2 associated X protein(Bax), Toll-like receptor-4(TLR4) and nuclear factor-κB (NF-κB) p65 proteins in the cells in various groups were examined by Western blotting method, and the expression levels of TLR4 and NF-κB p65 mRNA in the cells in various groups were detected by RT-PCR method. Results: Compared with control group, the proliferation activities of the cells in model group and treatment group were increased significantly(P<0.05), the apoptotic rates were decreased significantly(P<0.05), and the levels of IL-6 and TGF-β1 were obviously increased(P<0.05);the expression levels of PCNA, CyclinD1, Bcl-2, TLR4 and NF-κB p65 proteins and the expression levels of TLR4 and NF-κB mRNA were remarkably increased(P<0.05),but the expression levels of Bax protein was distinctly decreased(P<0.05).Compared with model group, the proliferation activity of the cells in treatment group was decreased (P<0.05), the apoptotic rate was increased (P<0.05), and the levels of IL-6 and TGF-β1 were clearly decreased(P<0.05);the expression levels of PCNA, CyclinD1, Bcl-2, TLR4 and NF-κB p65 proteins and the expression levels of TLR4 and NF-κB mRNA were remarkably decreased(P<0.05), but the expression levels of Bax protein was obviously increased (P<0.05). Conclusion: Mon can inhibit the proliferation of airway smooth muscle cells, promote the apoptosis and alleviate the airway inflammation in the asthmatic rats,and its mechanism may be related to inhibition of TLR4/NF-κB signaling pathway.

Objective: To investigate the effect of Prader-Willi/Angelman syndrome region protein 2(NIPA2) on the high glucose-induced osteoblast apoptosis, and to elucidate its mechanism; Methods: The osteoblasts MC3T3-E1 were treated with 26 nmol·L^-1 high glucose for 24 h; the cells were divided into HG+si-con group (transfected with si-con), HG+si-NIPA2 group (transfected with si-NIPA2), HG+si-NIPA2+DMSO group (transfected with si-NIPA2 and treated with DMSO), HG+si-NIPA2+AG490 (transfected with si-NIPA2 and treated with AG490),at the same time control group was set up. After the MC3T3-E1 cells were transfected by liposome method, they were treated with 26 nmol·L^-1 high glucose again.The qRT-PCR method was used to detect the expression level of NIPA2 mRNA in theMC3T3-E1 cells;Western blotting method was used to determine the expression levels of NIPA2, P21, Cleaved caspase-3, p-JAK2 and p-STAT3 proteins in the MC3T3-E1 cells; MTT method was used to detect the cell proliferation activities, and flow cytometry was used to measure the apoptotic rates. Results: Compared with control group, the mRNA and protein expression levels of NIPA2 in the MC3T3-E1 cells in HG group were significantly decreased (P<0.01). After knocking down the NIPA2, compared with HG+si-con group, the expression level of NIPA2 protein in HG+si-NIPA2 group was significantly decreased (P<0.01), the expression levels of P21 and Cleaved caspase-3 proteins were significantly increased (P<0.01), the proliferation activities of MC3T3-E1 cells were significantly decreased at 48 and 72 h (P<0.01), the apoptotic rate was significantly increased (P<0.01), and the expression levels of JAK/STAT signaling pathway-related proteins p-JAK2 and p-STAT3 were significantly increased (P<0.01).Compared with HG+si-NIPA2+DMSO group, the expression levels of NIPA2 protein in HG+si-NIPA2+AG490 group was significantly increased (P<0.01), the expression levels of P21 and Cleaved caspase-3 proteins were significantly decreased (P<0.01), the cell proliferation activities were significantly increased at 48 and 72 h (P<0.01), and the apoptotic rate was significantly decreased (P<0.01). Conclusion: NIPA2 can regulate the proliferation and apoptosis of the osteoblasts induced by high glucose, and its regulatory mechanism is related to the JAK/STAT signaling pathway.

Objective: To explore the effects of miR-125b on the proliferation and migration of cardiac fibroblasts via Toll-like receptor-4/nuclear factor-κB (TLR4/NF-κB) signaling pathway, and to clarify the role and mechanism of miR-125b in myocardial fibrosis. Methods: The HEH2 cells in logarithmic growth phase were randomly divided into blank control group, negative control group and miR-125b inhibitors group.The HEH2 cells in miR-125b inhibitors group were transfected with miR-125b inhibitors,and the HEH2 cells in negative control group were transfected with negative control inhibitors,and the HEH2 cells in blank control group were not transfected.The levels of miR-125b in the cells in various groups were determined by reverse transcription-polymerase chain reaction (RT-PCR) method.The proliferation activities of HEH2 cells were determined by MTTmethod.The Transwell chamber was used to measure the migration abilities of HEH2 cells;the expression levels of type Ⅰ collagen (Col Ⅰ), type Ⅲ collagen (Col Ⅲ), α-smooth muscle actin (α-SMA), TLR4 and NF-κB proteins in the HEH2 cells were determined by Western blotting method. Results: Compared with blank control group and negative control group, the level of miR-125b in the HEH2 cells in miR-125b inhibitors group was decreased (P<0.05), the proliferation activity and the number of migration cells were decreased (P<0.05), and the expression levels of Col Ⅰ, Col Ⅲ, α-SMA, TLR4, and NF-κB proteins were decreased (P<0.05).There were no significant differences in the miR-125b levels,the proliferation activities,the number of migration cells,the expression levels of Coll Ⅰ, Coll Ⅲ, α-SMA, TLR4, and NF-κB proteins in the HEH2 cells between blank control group and negative control group(P>0.05). Conclusion: Down-regulation of miR-125b level can inhibit the proliferation and migration of the cardiac fibroblasts, and its mechanism may be related to the TLR4/NF-κB signaling pathway.

Objective: To perform the function experiment in the pathogenic candidate gene TP53 via cell experiment,and to investigate the effects of TP53 G199X and V157fs point mutations on the protein expression, proliferation, migration and invasion of the ovarian cancer A2780 cells. Methods: The ovarian cancer A2780 cells without endogenous TP53 gene expression were selected and divided into wild-type group, G199X group, V157fs group and empty vector group. The TP53 wild-type plasmid was constructed, and the two point mutation plasmids of G199X and V157fs were constructed by using the point mutation kit. The wild-type, mutant and empty vector plasmids were transfected into the A2780 cells by lipofection. Western blotting method was used to detect the the expressions of P53 protein in the A2780 cells in various goups. The proliferation activities of A2780 cells in various groups were detected by CCK-8 assay. The rates of scratch healing of A2780 cells in various groups were detected by scratch assay. Transwell assay was used to examine the number of invasion cells of A2780 cells in various groups. Results: After transfection of A2780 cells, the P53 protein didn't express in the cells in G199X group, V157fs group and empty vector group. Compared with wild-type group, the proliferation activities of the cells in G199X group,V157fs group and empty vector group were significantly increased(P<0.01);the scratch healing rates of the cells in G199X group and V157fs group were increased (P<0.05 or P<0.01); the number of invasion cells in G199X group,V157fs group and empty vector group was increased(P<0.01). Conclusion: Two point mutations of TP53 G199X and V157fs can stop the expression of P53 protein in the A2780 cells and promote the proliferation, migration and invasion of ovarian cancer A2780 cells.

Objective: To establish the acute liver ischemia-reperfusion models of rats pre-treated with curcumin, and to investigate the protective effect of curcumin on the liver of the rats after acute ischemia-reperfusion. Methods: Ten SD rats were randomly assigned to sham operation group(n=3),solvent control group(n=3) and curcumin group(n=4).The rats in curcumin group and solvent control group were pre-treated with curcumin and 1% carboxymethyl cellulose (CMC) respectively by intraperitoneal injection before acute liver ischemia-reperfusion. The rats in sham operation group did not receive acute liver ischemia-reperfusion. Ultra performance liqwid chromatography-mass specteum(UPLC-MS) was used to determine the levels of serum curcumin of the rats in various. Bile duct cannulation and flurescence analysis were used to measure the bile flows of the rats in various groups. The malondialdehyde (MDA) kit was used to detect the MDA levels in liver tissue of the rats in various groups. Results: The serum curcumin level of the rats 1 h after introperitoneal injection of 200 mg·kg^-1 curcumin reached to(0.17±0.05)mg·L^-1, but decreased to below the detection level at 3 h after injection. The MDA level in liver tissue of the rats in curcumin group (9.18mmol·g^-1±1.78mmol·g^-1) was lower than those in solvent control group (527.54mmol·g^-1±237.38mmol·g^-1) and sham operation group (162.73mmol·g^-1±90.50mmol·g^-1).The bile flow of the rats in curcumin group recovered to approximately 40% of basal flow after ischemia-reperfusion, and had no significant difference compared with solvent control group (P>0.05).The bile flow of the rats in sham operation group kept in the basal flow level. Conclusion: Curcumin could be readily detected in the blood of the rats after introperitoneal injection; it is metabolized rapidly in vivo, and its level could decrease to below the detection level at 3 h after administration. Pre-treatment with curcumin can protect the liver by decreasing the MDA level in liver tissue of the rats after liver ischemia-reperfusion. However the treatment has the limited effect on bile flow restoration.

Objective: To investigate the effects of rhein on the proliferation, migration and invasion of the non-small cell lung cancer (NSCLC) A549 cells, and to elucidate the mechanisms. Methods: The NSCLC A549 cells were divided into control group and low, medium and high doses of rhein groups. The cells in each group were cultured for 24, 48 and 72 h in the culture medium without rheic and the culture medium containing 5, 10 and 20 μmol·L^-1 rhein. MTT method was used to analyze the proliferation rates of A549 cells in various groups. Flow cytometry was used to detect the apoptotic rates and the percentages of A549 cells at different cell cycles. Western blotting method was used to detect the expression levels of cysteine protease -3(Caspase-3), cysteine protease -9(Caspase-9), cytochrome C(Cyt-C) and apoptosis-induced factor (AIF) protines in the A549 cells in various groups. Scratch test and Transwell assay were used to detect the abilities of migration and invasion of A549 cells. Results: After treated with rhein for 24 h, compared with control group, the proliferation rates of the A549 cells in low, medium and high doeses of rhein groups were significantly decreased(P<0.05), the migration distances of cells were significantly reduced, and the number of invasion cells was significantly reduced; the apoptotic rates of the A519 cells in medium and high doses of rhein groups were significantly increased(P<0.05);the percentage of A519 cells in G₁ phase in high dose of rhein group was significantly reduced(P<0.05). After treated with rhein for 48 h, compared with control group, the expression levels of Caspase-3, Caspase-9, Cyt-C and AIF proteins in medium and high doses of rhein groups were significantly increased (P<0.05). Conclusion: Rhein can inhibit the proliferation, migration and invasion abilities of A549 cells and its mechanism may be related to increasing the expressions of Caspase-3, Caspase-9, Cyt-C and AIF proteins.

Objective: To investigate the effect of lipopolysaccharide (LPS) on the expressions of epithelial-mesenchymal transition (EMT) markers and β-catenin in the breast cancer MDA-MB-231 cells, and to clarify its possible mechanism. Methods: The breast cancer MDA-MB-231 cells were divided into control group and different concentrations (5, 10, 20, and 40 mg·L^-1) of LPS groups. Inverted microscope was used to observe the morphology of MDA-MB-231 cells in various groups. Immunofluorescence test was used to detect the β-catenin expression and location in the MDA-MB-231 cells in various groups. Real-time quantitative PCR(RT-qPCR) and Western blotting methods were used to detect the expression levels of the EMT markers E-cadherin, Vimentin and β-catenin mRNA and proteins in the MDA-MB-231 cells in various groups. Results: The morphology of MDA-MB-231 cells in control group was epithelial phenotype, and the morphology of MDA-MB-231 cells in different concentrations of LPS groups were the phenotype of mesenchymal cells. The results of immunofluorescence staining showed that the expression of β-catenin was mainly located in the nucleus. Compared with control group, the expression levels of Vimentin and β-catenin mRNA and proteins in the MDA-MB-231 cells in different concentrations of LPS groups were increased (P<0.05 or P<0.01), especially in 20 mg·L^-1 LPS group. Compared with control group, the expression levels of E-cadherin mRNA and proteins in the MDA-MB-231 cells in different concentrations of LPS groups were decreased (P<0.05 or P<0.01), especially in 20 mg·L^-1 LPS group. Conclusion: LPS could promote the EMT, invasion and metastasis of the breast cancer MDA-MB-231 cells by down-regulating the E-cadherin expression and up-regulating the Vimentin expression, and its mechanism may be related to Wnt/β-catenin signaling pathway.

Objective: To knock down the Toll-like receptor 2 (TLR2) gene in the colorectal cancer cells with lentiviral RNA interference technology, to explore its effect on the colorectal cancer cell proliferation, and to clarify its mechanism. Methods: The colorectal cancer HCT116 cells and HT29 cells in logarithmic phase were divided into non-infection control group, negative control group and gene knockdown group(TLR2-RNAi group); the cells were transfected with lentivirus-free, lentivirus-RNAi and lentivirus-TLR2-RNAi, respectively. The expression levels of TLR2 protein in the cells in various groups were detected by Western blotting method after stable transfection.The cell proliferation activities and the percentages of cells in different cell cycles were detected by CCK-8 method and flow cytometry. Western blotting method was used to detect the expression levels of PI3K/Akt and nuclear factor-κB(NF-κB) signaling pathway-related proteins and cell cycle-associated proteins cyclin D1 and cyclin D3 proteins in the cells in various groups. Results: Compared with non-infection control group and negative control group, the expression levels of TLR2 protein in the HCT116 cells and the HT29 cells in TLR2-RNAi group were decreased significantly (P<0.01). Compared with non-infection control group and negative control group,the cell proliferation activities in the HCT116 cells and the HT29 cells in TLR2-RNAi group were significantly decreased (P<0.05); the percentages of cells in S phase and G₂ phase were decreased significantly (P<0.05), and the percentages of cells in G₁ phase were increased significantly (P<0.05). Compared with non-infection control group and negative control group, the expression levels of PI3K, p-Akt, p-NF-κB, cyclin D1 and cyclin D3 proteins in the HCT116 cells and HT29 cells in TLR2-RNAi group were significantly decreased(P<0.01). Conclusion: Knockdown of TLR2 gene can inhibit the proliferation of colorectal cancer cells by regulating the expressions of PI3K/Akt and NF-κB cell signaling pathway proteins and cell cycle-associated proteins.

Objective: To investigate the effects of tripartitemotif containing 29(TRIM29)-siRNA transfection on the cell cycle and apoptosis of glioma U87MG cells,and to elucidate the role of TRIM29 in the occurrence and development of glioma. Methods: The U87MG cells cultured in vitro were divided into control group (untransfected), NC-siRNA group (transfected with negative control NC-siRNA), and TRIM29-siRNA group (transfected with TRIM29-siRNA). The expression levels of TRIM29 mRNA in the U87MG cells in three groups were detected by real-time fluorescent quantitative PCR,the percentages of U87MG cells in different cell cycles and the apoptotic rates in three groups were detected by flow cytometry,and the expressions levels of TRIM29, CyclinD1, p21, Bcl-2 and Bax proteins in the U87MG cells in three groups were detected Western blotting method. Results: There were significant differences in the expression levels of TRIM29, CyclinD1, P21, Bcl-2, Bax proteins, the expression levels of TRIM29 mRNA,and the percentages of U87MG cells in G₀/G₁, S, G₂/M phases and the apoptotic rates among three groups (P<0.05).Compared with control group, the expression levels of TRIM29 mRNA and TRIM29, CyclinD1, Bcl-2 proteins, the percentages of U87MG cells in G₀/G₁ phase, S phase,and G₂/M phase, and the apoptotic rates in the U87MG cells in NC-siRNA group had no significant differences (P>0.05).Compared with control group or NC-siRNA group,the expression levels of TRIM29 mRNA and TRIM29, CyclinD1,Bcl-2 proteins in the U87MG cells in TRIM29-siRNA group were decreased significantly(P<0.05); the percentage of U87MG cells in G₀/G₁ phase, the apoptotic rate of U87MG cells and the expression levels of p21 and Bax proteins were increased significantly (P<0.05). Conclusion: TRIM29-siRNA transfection can induce the cell cycle arrest and apoptosis in the U87MG cells and its mechanism may be related to down-regulation of Cyclin D1, Bcl-2 protein expressions and up-regulation of p21 and Bax protein expressions.

Objective: To investigate the protective effect of carnosine on the vascular cognitive impairment(VCI) of the rats, and to elucidate its mechanism. Methods: Fifty SD rats were randomly divided into sham operation group, model group (bilateral common carotid artery occlusion for 10 min→reperfusion for 10 min→occlusion for 10 min) and different doses of carnosine group (bilateral common carotid artery occlusion for 10 min→reperfusion for 10 min→occlusion for 10 minutes + 100, 300,and 900 mg·kg^-1·d^-1 carnosine);there were 10 rats in each group. The carnosine was administered by gavage once a day from 21 d before modeling to 12 d after modeling. Morris water maze test was carried out on the 7th day after operation to measure the learning and memory ability of the rats. The hippocampus tissue of the rats was obtained on the 12th day; the levels of carnosine and reduced glutathione(GSH) in hippocampus tissue of the rats in various groups were detected by high performance liquid chromatography (HPLC),the expression of glial fibrillary acidic protein(GFAP) in CA1 area of hippocampus of the rats in various groups was detected by immunohistochemistry method.The expression levels of phosphate nuclear factor-kappa B p65(p-NF-κB p65) protein in hippocampus tissue of the rats in various groups were determined by Western blotting method. The levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in hippocampus tissue of the rats in various groups were detected by ELISA method. Results: Compared with sham operation group, the pyramidal cells in the CA1 area of hippocampus of the rats in model group were partially absent, the escape latency was significantly prolonged (P<0.01), and the time staying in the platform quadrant was significantly shortened (P<0.01);the levels of carnosine and GSH in hippocampus tissue were decreased (P<0.01), the number of GFAP positive cells was increased, the expression level of p-NF-κB p65 protein was increased (P<0.01),and the levels of IL-1β and TNF-α were increased (P<0.01).Compared with model group, the pyramidal cells in CA1 area of hippocampus of the rats in different doses of carnosine groups were arranged in order, the escape latency was significantly shortened (P<0.01), and the time staying in the platform quadrant was significantly increased (P<0.01);the levels of carnosine and GSH in hippocampus tissue of the rats were increased (P<0.05 or P<0.01), the number of GFAP positive cells was decreased, the expression levels of p-NF-κB p65 protein were decreased(P<0.01),and the levels of IL-1β and TNF-α were decreased (P<0.01). Conclusion: Carnosine has the protective effect in the rats with VCI,and its mechanism may be related to the improvement of antioxidant capacity,inhibition of NF-κB pathway activation, thus inhibiting the abnormal activation of astrocytes and reducing inflammation.

Objective: To investigate the influence of galectin-3 expression suppression in the apoptosis of human gastric cancer MGC-803 cells, and to provide a potential target for gene targeting therapy. Methods: The human gastric cancer MGC-803 cells were selected and divided into siRNA interference group, blank control group and negative control group. The MGC-803 cells in siRNA interference group were transfected with the siRNA which targeted galectin-3, the MGC-803 cells in blank control group were added only transfection reagent, while the MGC-803 cells in negative control group were transfected with negative siRNA. Flow cytometry and propidium iodide (PI) staining method were used to detect the apoptosis.Western blotting method was applied to determine the expressions of galectin-3,Bcl-2 and Bax proteins. Results: Compared with negative control group and blank control group, the expression level of galectin-3 protein in siRNA interference group was significantly decreased (P<0.05). The PI staining results showed that the number of apoptotic cells in siRNA interference group was increased significantly in which nuclear pyknosis, chromatin concentration, crescent shape and apoptotic bodies were found; meanwhile only a small amount of apoptotic cells in negative control group. The flow cytometry and Western blotting results showed that compared with negative control group and blank control group, the apoptotic rate of the MGC-803 cells in siRNA interference group was significantly increased(P<0.05), the expression level of Bcl-2 protein was decreased significantly (P<0.05),and the expression level of Bax protein had no significant difference(P>0.05). Conclusion: To suppress galectin-3 expression can promote the apoptosis of MGC-803 cells, suggesting that galectin-3 might play an oncogene role in the gastric cancer cells and has the possibility of becoming a target for targeting therapy.

Objective: To observe the effect of immunostimulatory oligodeoxynucleotide (CpG ODN) BW006 on the expressions of Runt-related transcription factor 2(Runx2) and Osterix in periodontal tissue of the rats with experimental tooth movement, and to explore its effect on the periodontal remodeling of the rats with orthodontic tooth movement. Methods: The experimental tooth movement models were established in the 36 7-week-old male Wistar rats. The left maxilla was used as experimental group (local injection of CpG ODN BW006) and the right maxilla as control group (local injection of PBS), once every 3 d, 40 μL each time. Twelve rats were randomly selected at 3, 7 and 14 d after stress, and the maxillary tissue containing the first molars was prepared. HE staining and immunohistochemical staining were performed to observe the morphological changes of periodontal tissue, measure the tooth movement distance and detect the expressions of Runx2 and Osterix. Results: The HE staining results showed that the bone resorption on the pressure side of the rats in experimental group was lower than that in control group, and a large number of osteoblasts were seen on the tension side of the rats in experimental group; the alveolar bone fullness and height of the rats in experimental group were significantly higher than those in control group. The distances of tooth movement in experimental group were (0.347±0.007) and (0.443±0.016)mm, while they were (0.585±0.012) and (0.975±0.084)mm in control group at 7 and 14 d; there were significantly differences between two groups(P<0.01). The immunohistochemical staining results showed that the positive expression levels of Runx2 and Osterix in the periodontal tissue of the rats in experimental group were significantly higher than those in control group at 3 and 7 d (P<0.05 or P<0.01), and reached a peak on the 7th day; the positive expression levels of Runx2 and Osterix in experimental group were decreased slightly on the 14th day, but there were still significant differences compared with control group (P<0.05 or P<0.01). Conclusion: CpG ODN BW006 can regulate the periodontal tissue remodeling by promoting the expressions of Runx2 and Osterix during tooth movement.

Objective: To study the anti-aging effect of Agaricus blazei polyssaccharide-A (ABP-A) in the aging model mice induced by D-galactose (D-Gal), and to explore the anti-aging mechanism of Agaricus blazei polysaccharide(ABP). Methods: The crude polysaccharide from Agaricus blazei Murill(ABM) was extracted by water extraction and alcohol precipitation, and the fractions were analyzed by DEAE-cellulose ion exchange column chromatography;ABP-Awas obtained.A total of 48 male ICR mice were randomly divided into control group, model group, positive drug group(Piracetam)and ABP-A group(n=12). Morris water maze, dark avoidance and platform jumping behavior experiments were performed at 70 d after administration. The superoxide dismutase (SOD) activitives, the malondialdehyde (MDA) levels,the total antioxidant capacities (T-AOC), the catalase (CAT) activitives, and the reactive oxygen species (ROS) levels in serum of the mice in various groups were detected. Western blotting method was used to detect the expression levels of Nrf2, Keap1 and HO-1 proteins in the brain tissue of the mice in various groups. Results: The total sugar content of ABP was 75.1%. The ABP-A was graded and gained by DEAE-cellulose ion exchange column chromatography.The behavioral experiment results showed that compared with model group, the dark avoidance latency of the mice in ABP-A group was significantly lengthened (P<0.05), the number of errors was decreased significantly (P<0.05); the latency of step down was significantly prolonged (P<0.05), and the number of errors was decreased significantly (P<0.05); the positioning latency of the mice in ABP-A group was significantly shortened(P<0.05), and the number of entering to the platform was increased significantly (P<0.05). Compared with model group, the activities of serum SOD and CAT of the mice in ABP-A group were increased(P<0.05),the T-AOC was increased(P<0.05), the levels of MDA and ROS were decreased(P<0.05).The Western blotting results showed that compared with model group, the expression level of HO-1 protein in the brain tissue of the mice in ABP-A group was increased (P<0.05), and the expression levels of Nrf2 and Keap1 proteins were decreased (P<0.05 or P<0.01). Conclusion: ABP can improve the learning and memory ability of the aging model mice, and its mechanism may be related to regulating Keap1/Nrf2/ARE oxidative stress pathway related factors Nrf2, Keap1, and HO-1 with Nrf2 as the core to perform the anti-aging effect.

Objective: To investigate the effects of lead exposure during pregnancy and lactation periods on the learning and memory abilities and the expressions of c-fos and parvalbumin(PV) in hippocampus tissue of the offspring rats, and to clarify the possible mechanism of lead on the memory function impairment during nervous system development. Methods: Eight female Wistar rats during pregnancy period were randomly divided into control group and low,medium and high doses of lead-exposed groups. The rats in low,medium and high doses of lead-exposed groups were given deionized water containing 0.05%, 0.10%, and 0.20% lead acetate, and the rats in control group were given deionized water. Ten days after the pups were born, Morris water maze was used to measure the learning and memory abilities of the offspring rats and atomic absorption spectrometry was used to measure the blood and hippocampal lead concentrations of the offspring rats.Biochemical method was used to determine the nitric oxide (NO) levels and nitric oxide synthase (NOS) activities in hippocampus tissue of the offspring rats in various groups. The expressions of c-fos and PV proteins in hippocampus tissue of the offspring rats in various groups were detected by Western blotting method.Pearson correlation analysis was used to analyze the correlations between the expression levels of c-fos and PV proteins in hippocampus tisue of the offspring rats and the learning indexes,the NO level,and the NOS activity in hippocampus tissue. Results: In the positioning navigation test, compared with control group, the average escape latencies and the swimming distances of the offspring rats in medium and high doses lead-exposed groups were increased(P<0.05); in the space exploration experiment, the time of staying in the target quadrant and the number of crossing platform of the offspring rats in medium and high doses of lead-exposed groups were reduced(P<0.05).The blood lead and hippocampal lead concentrations of the offspring rats in various groups were statistically different (F=176.44,P<0.01;F=37.37,P<0.01); compared with control group, the blood lead and hippocampal lead concentrations of the offspring rats in low,medium and high doses of lead-exposed groups were significantly increased(P<0.05). There were statistical differences in the NO levels and the NOS activities in the hippocampus tissue of the offspring rats in various groups(F=4.105,P<0.05;F=3.443,P<0.05). Compared with control group, the NO level in hippocampus tissue of the offspring rats in high dose of lead-exposed group was significantly reduced(P<0.05), and the NOS activity was decreased (P<0.05).Compared with control group,the expression levels of c-fos protein in hippocampus tissue of the offspring rats in different doses of lead-exposed groups were decreased(P<0.05), and the expression levels of PV protein in hippocampus tissue in medium and high doses of lead-exposed groups were increased (P<0.05).The results of Pearson correlation analysis showed that the expression level of c-fos protein in hippocampus tissue of the offspring rats was positively correlated with the learning ability indexes of the water maze test and the NO level and the NOS activity in the hippocampus tissue (P<0.01); the expression level of the PV protein in hippocampus tissue of the offspring rats was negatively correlated with the learning ability indexes of the water maze test and the NO level and the NOS activity in the hippocampus tissue (P<0.01). Conclusion: Lead exposure during pregnancy and lactation periods can impair the learning and memory abilities of the offsprings rats, and the mechanism is related to the decrease of c-fos protein expression level and the increase of PV protein expression level in the hippocampus tissue.

Objective: To investigate the effects of hepatitis B virus (HBV) on the T lymphocytes and their subsets in different alanine aminetransferase(ALT) states,and to elucidate the immunological mechanism of ALT-based antiviral therapy for hepatitis B. Methods: A total of 363 patients with chronic hepatitis B were selected as the subjects. According to the ALT abnormalities, the patients were divided into ALT normal group (131 cases),normal ≤ ALT<2 times of upper limit group (110 cases),and ALT ≥ 2 times of upper limit group (122 cases).The patients in ALT ≥ 2 times of upper limit group were given entecavir and followed up for 24 weeks.The hepatitis B antigen antibody parameters were measured by chemiluminescence immunoassay analyzer,the liver function parameters were measured by automatic biochemical analyzer,the HBV loads were measured by quantitative PCR analyzer,the classification of T lymphocytes was detected by flow cytometry, and the levels of serum interleukin-2(IL-2),interferon-γ(IFN-γ),interleukin-4(IL-4) and interleukin-10(IL-10) of the patients were detected by enzyme-linked immunosorbent assay(ELISA) method.The influence of different HBV loads in the immunological indexes in various groups and the changes of virological and immunological indexes of the patients before and after antiviral therapy were detected. Results: In ALT normal group, there were no significant differences in the total number of T lymphocytes,the number of Th/i lymphocytes,the number of Ts/c lymphocytes and the serum IL-2,IFN-γ,IL-4,IL-10 levels of the patients with different loads of HBV(P>0.05).In ALT ≥ 2 times of upper limit group, with the increase of virus load,the total number of T lymphocytes and the number of Th/i lymphocytes were decreased(P<0.05),and the number of Ts/c lymphocytes was increased(P<0.05); the cytokines IL-2 and IFN-γ levels were increased(P<0.05),and the cytokines IL-4 and IL-10 levels were decreased(P<0.05).Compared with before treatment,24 weeks atfer entecavir treatment, the patient's HBV DNA was decreased significantly (P<0.05),the total number of T lymphocytes and the number of Th/i lymphocytes were increased(P<0.05), the number of Ts/c lymphocytes was decreased(P<0.05); the cytokines IL-2 and IFN-γ levels were decreased(P<0.05),and the cytokines IL-4 and IL-10 levels were increased(P<0.05). Conclusion: The influence of HBV on immune function is different in different ALT states. Therefore, antiviral therapy for the hepatitis B patients with ALT ≥ 2 times of upper limit can significantly change the immunological state of the patients.

Objective: To synthesize a light-curable polyurethane(PU) adhesive, and to explore its effects on the water absorption values,the water solubility values,the rates of double blond conversion and the micro-shear bond strength of three commercial resin adhesives. Methods: A polyether PU prepolymer was synthesized by solution polymerization using polyether triol, isophorone diisocyanate,and 2-hydroxyethyl methacrylate as raw materials. The structure of the product was characterized by Fourier transform infrared spectroscopy(FT-IR), and a diluent, a solvent and a photoinitiator were used to prepare a light-curable PU adhesive.The commercial adhesives Single Bond Universal (SBU), Single Bond 2 (SB2) and Clearfil SE (SE) were used as control groups;the SBU,SB2 and SE added with 70%PU adhesives(SBU-70% PU, SB2-70% PU and SE-70% PU) were used as experimental groups. According to the YY 1042-2011 standard, the water absorption values,the water solubility values of the adhesives in experimental groups and control groups were measured.The rates of double bond conversion of the adhesives in various groups were measured by FT-IR. The micro-shear bond strengths of adhesives in various groups were measured using universal testing machine. Results: The FT-IR results showed a characteristic absorption peak of a photo-curable group (C=C double bond) at 1 638 cm^-1, consistent with the expected product structure.The water absorption values of adhensives in experimental groups(SBU-70%PU group,SB2-70%PU group and SE-70%PU group) were significantly lower than those in the corresponding control groups (t=15.479,P<0.05;t=19.703,P<0.05; t=11.795,P<0.05);the solubility values of the adhesives in SBU-70%PU group and SB2-70%PU group were significantly lower than those in the corresponding control groups(t=29.845, P<0.05;t=28.862,P<0.05);the rates of double bond conversion of adhesives in experimental groups were significantly higher than those in the corresponding control groups (t=-11.612,P<0.05;t=7.213, P<0.05;t=-15.031,P<0.05); the micro-shear bond strength of the adhesive in SB2-70%PU group was significantly higher than that in the corresponding control group (t=-2.515,P<0.05). Conclusion: After successfully synthesizing light-curing PU adhesive, and adding 70% PU to the three commercial adhesives, it can significantly reduce the water absorption values and the solubility values of the adhesives, increase the rates of double bood conversion, and it has no significant effect on the micro-shear bond strength, so that the overall properties of the adhesive is effectively improved.

Objective: To investigate the expressions of M1 macrophage-related factors induced nitric oxide synthase(iNOS) and signal transducers and activators of transcription 1(STAT1) in gingiva tissue of the patients with severe chronic periodontitis, and to elucidate the possible role of M1 macrophages in the occurrence and development of chronic periodontitis. Methods: The gingiva tissue of 15 patients with severe chronic periodontitis was selected as experimental group, and 15 cases of healthy gingiva tissue of the patients requiring removal of the third molars were used as control group.The expression levels of iNOS and STAT1 mRNA in the gingiva tissues of the patients in two groups were detected by RT-PCR.The expression levels of iNOS and STAT1 proteins in the gingiva tissues of the patients in two groups were detected by Western blotting method. Results: Compared with control group,the expression levels of iNOS and STAT1 mRNA in the gingiva tissue of the patients in experimental group were significantly increased (P<0.01);compared with control group,the expression levels of iNOS and STAT1 proteins in the gingiva tissue of the patients in experiment group were also significantly increased (P<0.01). Conclusion: M1 macrophage-mediated immune response is enhanced in severe chronic periodontitis, suggesting that M1 macrophages are involved in the inflammatory response and tissue damage of periodontal tissue.

Objective: To investigate the clinical characteristics of the acute leukemia (AL) patients with complex karyotypes, and to elucidate their relationships with prognosis. Methods: A total of 33 AL patients with complex karyotypes were selected as the subjects (complex karyotype group), and 43 cases were randomly selected from 248 non-complex karyotype AL patients diagnosed at same time as the controls(non-complex karyotype group). The clinical and laboratory data of the patients in two groups were collected and the clinical characteristics and their relationships with prognosis were analyzed. Results: There were no significant differences in gender, age, hematological indexes before treatment, proportion of bone marrow primordial cells and extramedullary infiltration between the AL patients with complex karyotypes and non-complex karyotypes at same time (P>0.05); the constituent ratio of complex karyotypes AL in the AL patients was 11.7% (33/281).The complete remission (CR) rates of the AL patients in complex karyotype group and non-complex karyotype group were 59.4% (19/32) and 85.0% (34/40),there was significant difference between two groups (P=0.014); the CR rates of the acute myeloid leukemia (AML)patients in complex karyotype group and non-complex karyotype group were 47.6% (10/21) and 79.3% (23/29), there was significant difference between two groups (P=0.020).The CR rates of complex karyotypes AL patients with high karyotype complexity and low karyotype complexity were 41.2% (7/17) and 80.0% (12/15), there was significant difference between two groups(P=0.036).The Kaplan-Meier survival analysis showed that the median recurrence-free survival (RFS) and median overall survival (OS) of the patients in complex karyotype group and non-complex karyotype group were 162 and 462 d(P=0.002),256 and 769 d(P<0.01), and there were significant differences between two groups;the median OS of the AML and acute lymphoblastic leukemia(ALL) patients in complex karyotype group and non-complex karyotype group were 125 and 741 d (P<0.01), 293 and 939 d (P=0.008), and there were significant differences between two groups. Conclusion: The constituent ratio of complex karyotypes in the AL patients is relatively lower, and there are no significant differences in the general clinical characteristics, but the curative effect and prognosis are poor. The higher the chromosome complexity degree of the AL patients with complex karyotypes, the worse the curative effect.

Objective: To investigate the distribution of single nucleotide polymorphism (SNPs)of selenoprotein P(Sepp1)rs7579 site in the patients with papillary thyriod carcinoma(PTC) and the healthy controls and the interaction with non-genetic factors in the Inner Mongolia region,and to clarify the correlation of Sepp1 and the risk of PTC. Methods: The genotypic and allelic frequencies of Sepp1 from 138 patients with PTC and 140 healthy controls in the Inner Mongolia region were determined by allele-specific polymerase chain reaction(AS-PCR).Stratification analysis was conducted according to the different clinical characteristics(age,gender,tumor size and presence or absence of lymph node metastasis),and the association between the Sepp1 gene polymorphism with PTC incidence risk was analyzed in combination with non-genetic factors such as smoking,drinking,body mass index(BMI),iodized salt intake and mental state etc by multiple Logistic regression analysis.Pearson correlation analysis was used to analyze the correlations between the expression levels of c-fos and PV proteins in hipocampus tissue of the offspring rats and the learning ability indexes,the NO content,and the NOS activity in hipocampus tissue. Results: High BMI,iodized salt intake,bad mental state and big working pressure increased the risk of PTC.The differences in the frequencies of GG,GA,AA genotypes and G,A alleles of Sepp1 (rs7579) between the PTC patients and the healthy controls were not significant(χ²=0.622,P>0.05;χ²=0.673,P>0.05).But the stratification analysis results showed that there were significant differences in the genotypic frequencies of GG,GA and AA between the PTC patients and the controls in ≥ 50 years old group(χ²=7.717,P=0.028),and the frequency of AA genotype of the PTC patients was significantly lower than that of the controls(P<0.05).The relative risk of PTC incidence in the subjects with allele A (AA or GA) was lower than those with genotype GG (OR=0.481,95%CI:0.267-0.866). Conclusion: BMI, iodized salt intake,mental state and working pressure are the main influencing factors of PTC;allele A may be the protective factor of PTC.

Objective: To investigate the marginal microleakage of the new self-adhesive flow resin Constic and other three cavity filling materials in the in vitro experiments of permanent molars, and to provide the references for the selection of clinical Ⅴ -type cavity filling materials. Methods: A total of 40 fresh and extrated teeth including 20 premolars and 20 third molars were collected,and the V-type cavities were prepared on the buccal surface; the teeth were randomly divided into 4 groups(n=10),and the cavities were filled by self-adhesive flow resin Constic(Constic group),self-adhesive flow resin Dyad Flow(Dyad Flow group),flow resin Filtek Z350XT(Filtek Z350XT group) and Z350XT nano-resin(Z350 group).Five teeth were selected from each group and received cold and heat cycles for 100 times,and another five simples were used for 500 times.After thermal test,all teeth were stained with 0.1% Rhodamine-B solution. The microleakage was observed under confocal laser scanning microscope,the microleakage degree was evaluated,and the depth of microleakage was detected. Results: The marginal microleakage in different degrees was found in the materials in various groups.There was no significant difference in the microleak degree of the same meterial after 100 times and 500 times of cold and heat cycles(P>0.05).Compared with Constic group, the microleakage depths in the side of mouth and the side of gum in Dyad Flow group, Filtek Z350XT group and Z350XT group were increased(P<0.05);the microleakage depths in the side of mouth and the side of gum in Dyad Flow group and Filtek Z350XT group were lower than those in Z350XT group (P<0.05). Conclusion: Constic has low microleakage and high edge tightness and is more effective than Dyad Flow,Filtek Z350XT and Z350XT.

Objective: To analyze the treatment of a patient with recurrent and refractory small cell lung cancer(SCLC) intolerable further chemotherapy who gained significant efficacy by anlotinib combined with thoracic radiotherapy and to clarify the efficacy and safety of anlotinib combined with radiotherapy in the SCLC patients, and to provide the reference for the selection of treatment program of these patients. Methods: The clinical data of one patient with advanced recurrent and refractory SCLC who could not tolerate further chemotherapy were collected, and the related literatures were reviewed to analyze the efficacy and safety of anlotinib combined with thoracic radiotherapy. Results: The patient was a 70-year-old woman who had a long histroy of hypertention and diabetes,and presented with cough and shortness of breath.Based on the chest CT,biopsy and pleural effusion cytology,the patient was definitely diagnosed as extensive stage of SCLC(pleural metastasis) and then underwent a series of treatments. After three lines of chemotherapy regimens, the patient was unable to tolerate the continued chemotherapy due to the repeated reduce of neutrophil and platelet, and the disease progressed rapidly. Dyspnea aggravated,and the patient was unable to lie. Anlotinib combined with thoracic radiotherapy was initiated.Before radiotherapy,the PET-CT results demonstrated the hypermetabolic lesions including primary tumor located in upper lobe of left lung and bilateral hilar and mediastinal lymph nodes,bilateral pleural effusion.The patient was administrated orally with anlotinib 12 mg once per day for 3 d after thoracic radiotherapy of 6 G/3 f, the patient's wheeze was improved obviously and she could lie flat.However,she complained of dizziness with an increased blood pressure of 168/80 mmHg.Considering that it was related to anlotinib,and the patient's dyspnea was obviously relieved at present to ensure the safe implementation of radiotherapy,so anlotinib was suspended.After irradiation with 38 Gy/19 f,the patient's dyspnea disappeared completely;the primary tumor and metastatic lymph nodes shrank significantly. The CT results showed the intrathoracic lesions were shrank 40 d after radiotherapy;then the patient continued to system therapy. Conclusion: For the patients with SCLC unable to tolerate chemotherapy or progressed after multiple lines of chemotherapy, anlotinib combined with thoracic radiotherapy is promising.In the future,large-scaled clinical trials should be initiated to testify the efficacy and safety of anlotinib combined with thoracic radiotherapy.

Objective: To analyze the clinical features of gingival enlargement in the diabetic patients caused by nifedipine, and to provide the evidences for the diagnosis and treatment of drug-induced gingival enlargements with systemic diseases. Methods: The clinical materials of one diabetic patient with nifedipine-induced gingival enlargement were collected and the relative literatures were reviewed to summarize the etiology, clinical characteristics and treatment methods of the drug-induced gingival enlargement with system disease. Results: The patient, male, aged 67 years old, admitted hospital because of recurrence after 2 years of resection of gingival swelling. The patient had a history of diabetes and hypertension,as well as the use of nifedipine to control hypertension. Oral examination revealed poor oral hygiene, dark red gums,and bleeding during probing. The swelling of the gingiva exceeded 2/3 of the tooth, and there were varying degrees of mobility in many teeth. The clinical diagnosis was drug-induced gingival enlargement. First the blood pressure and blood glucose were controlled to the clinically operable range, oral hygiene education was performed,the periodontal basic therapy was done to eliminate inflammation, and gum swelling was significantly reduced. Gingival resection and gingivalplasty were used to restore the normal shape of the gums at the sites where the hyperplasia was still apparent after basic treatment. The plaque was strictly controlled throughout the procedure, no antihypertensive drugs were replaced, and there was no relapse within one year. Conclusion: The etiology of drug-induced gingival enlargement with systemic diseases is complex and easy to relapse. Initial periodontal therapy should be performed under the condition of controlling the systemic diseases. Surgery can be used if necessary,and long-term effective plaque control is a powerful measure to prevent the occurrence and recurrence of drug-induced gingival enlargement.

Objective: To explore the pathogenesis,clinical manifestations,diagnosis and treatment of the patient with catathrenia,to improve the clinician's understanding of the disease, and to provide the evidences for its diagnosis and treatments. Methods: The clinical data of a patient with catathrenia were retrospectively analyzed and the diagnosis and treatment were summarized;the relevent literatures were reviewed,and the clinical characteristics and diagnosis and treatment methods were explored. Results: The patient was a 28 years old woman with intermittent catathrenia for 3 years and went to hospital. She was healthy in the past,had no bad habits, and had regular work and rest. There were no abnormal signs on physical examination. The relevant examinations including electrocardiogram,chest radiograph,CT scan of the brain,fibrolaryngoscope and polysomnography(PSG) were performed. The PSG results showed exhalation prolongation after deep inhalation,followed by brief deep inhalation and exhalation. The respiratory rhythm slowed down,the arousal index and respiratory event index increased,and there were no oxygen saturation decreases. According to the patient's clinical manifestations and the PSG manifestations,the patient was diagnosed as catathrenia,and the possibility of central sleep apnea(CSA)was excluded. The application of continuous positive airway pressure(CPAP)was effective,and the condition was stable during the follow-up of two years. Conclusion: Catathrenia is relatively rare,and its pathogenesis,diagnosis and treatment are not clear at present. The patient's clinical manifestations and PSG manifestations are used clinically as a basis for diagnosis. The application of CPAP has a better prognosis.

Objective: To analyze the process of diagnosis and treatment of the patient with novel coronavirus pneumonia(COVID-19) in pregnancy, and to provid the refercnce for the diagnosis and treatment of COVID-19 in pregnancy. Methods: The general materials,COVID-19 patient contact history,physical examination,auxilliary examination,diagnosis methods and treatment methods of a COVID-19 patient in pregnancy, who recovered and was discharged, with the gastrointestinal symptoms as the first manifestations were retrospectively analyzed;the quick and correct diagnosis and treatment methods for COVID-19 in pregnancy were explored. Results: The female patient,aged 38 years old,was hospitalized because of "menelipsis 30⁺² weeks,diarrhea and nausea for 3 d".The acute gastrointestinal symptoms such as diarrhea and nausea were presented without obvious inducement.Because the patient had the contact history of the COVID-19 patients in Wuhan city,pharyngeal swab nucleic acid test and lung CT scan were performed in the patient.The result of pharyngeal swab nucleic acid test was positive,and the CT results showed that the inferior lobe of left lung and the upper and lower lobes of right lung had maltipre speckle shadows.The clinical diagnosis was COVID-19.In the treatment process,the maternal vital signs and the fetal state in utero were monitored strictly.The patient received combined treatment of Chinese and Western medicine.After treatment,the examination indexes of the pregnant woman were improved,and the results of sputum and pharyngeal swab nucleic acid test were negative for consecutive two times;the lung inflammation was improved;the fetal heart monitoring was normal;the time of patient in hospital was 15 d;the patient recovered safely and was discharged. Conclusion: The gastrointestinal symptoms such as diarrhea, nausea, and vomiting may be the first manifestations of the COVID-19 patients.CT examination plays an important role in screening the patients with suspected COVID-19, and it is safe to be used in pregnancy. Early and sufficient Chinese medicine combined with antiviral treatment for non-heavy COVID-19 is effective.