Objective To investigate the expression of squamous cell heat shock protein 53（CRNN） in esophageal squamous cell carcinoma（ESCC）， and toevaluate its impact on the biological behavior of ESCC cells Eca9706. Methods Immunohistochemical method was used to detect the expression of CRNN protein in 93 ESCC tissues and 101 normal esophageal epithelial tissues adjacent to cancer，and the associations of CRNN expression levels with the clinical pathological characteristics and survival prognosis of ESCC patients were analyzed. Receiver operating characteristic（ROC） curve was used to analyze the predictive performance of CRNN expression level on ESCC. The Eca9706 cells were divided into control group and CRNN group （overexpression of CRNN）. Cell counting kit-8（CCK-8） assay was used to detect the proliferation activities of Eca9706 cells in two groups； Transwell chamber assay was used to detect the numbers of migration cells of Eca9706 cells in two groups； plate clone formation assay was used to assess the numbers of clone formation of Eca9706 cells in two groups； flow cytometry was used to detect the apoptotic rates of Eca9706 cells in two groups. Results Compared with adjacent normal esophageal epithelial tissue， the expression intensity of CRNN protein in ESCC tissue was significantly decreased （χ²=23.476， P<0.001）. The downregulation of CRNN protein expression in ESCC patients was associated with tumor location（χ²=5.353， P=0.021） and histological grade （χ²=4.434， P=0.035）， but not with age（χ²=0.102， P=0.750）， gender（χ²=0.050， P=0.822），tumor stage （χ²=0.047， P=0.828） or lymph node metastasis （χ²=0.553， P=0.457）. Survival analysis showed that ESCC patients in high expression of CRNN protein group had better prognosis than those in low expression of CRNN protein group （P=0.013）. Univariable Cox proportional hazards regression analysis showed the associations between overall survival rate in ESCC patients and the expression level of CRNN protein ［hazard ratio （HR）=0.198，95% confidence interval （CI）： 0.047-0.842，P=0.028］ and tumor stage（HR=2.479，95%CI：1.247-4.929，P=0.010）. Multivariable Cox regression analysis showed that the expression level of CRNN protein （HR=0.213，95%CI： 0.050-0.895， P=0.035） and tumor stage （HR=2.391， 95%CI： 1.198-4.772， P=0.013） were independent factors for the prognosis of ESCC. Compared with control group，the proliferation activity of cells in CRNN group was significantly decreased （P=0.004）， the number of clone formation was decreased （P=0.002）， the number of migration cells was decreased （P=0.002）， and the apoptotic rate was significantly increased （P=0.006）. Conclusion Low expression level of CRNN protein suggests poor prognosis for the ESCC patients. Overexpression of CRNN may inhibit the proliferation，migration and invasion abilities of ESCC cells，and promote their apoptosis.

Objective To investigate the effects of human glycolipid transfer protein （GLTP） on proliferation， migration and invasion of pancreatic cancer （PC） cells， and to elucidate their mechanisms. Methods The difference in the expression levels of GLTP proteins between PC tissue and normal pancreas tissue was analyzed by University of Alabama at Birmingham Cancer Data Analysis Platform（UALCAN） Database， as well as the difference in the expression levels of GLTP protein between PC tissue and normal pancreas tissue of the PC patients with different clinicopathological characteristics. The PANC-1 cells were cultured in vitro anddivided into control group （transfected with pFLAG-CMV4 plasmid） and GLTP-overexpression（GLTP-OE） group （transfected with pFLAG-GLTP plasmid）. The stably GLTP transfected cells were established using the antibiotic screening method. Knock-down experiments were performed using non-specific siRNA transfected PANC-1 cells as control group and si-GLTP transfected PANC-1 cells as si-GLTP group. Western blotting method was used to detect the expression of GLTP protein in the cells in various groups， cell counting kit-8 （CCK-8） method was used to detect the proliferation activities of PANC-1 cells， clone formation assay was used to detect the number of clone formation， and Transwell chamber assay were used to detect the numbers of migration and invasion cells in various groups. Transcriptomics sequencing analyses were conducted to assess the possible mechanism of GLTP in the PANC-1 cells. Western blotting method was employed to detect the expression levels of phosphatidylinositol 3-kinase （PI3K）， phosphorylated PI3K （p-PI3K）， protein kinase B （Akt）， and phosphorylated Akt （p-Akt） proteins in the PANC-1 cells in various groups； Real-time fluorescence quantitative PCR （RT-qPCR） was used to assess the expression levels of amphiregulin （AREG） and kinase insertion domain receptor （KDR） mRNA in the cells in various groups. The mice were randomly divided into control group （injected with pFLAG-GMV4 transfected PANC-1 cells） and experimental group （injected with pFLAG-GLTP stably transfected PANC-1 cells）， and the subcutaneously transplanted tumor models were prepared； the volumes and weights of the transplanted tumors of the mice in two groups were measured. Results UALCAN database analysis showed that the expression level of GLTP protein in PC tissue was lower than that in normal pancreas tissue （P<0.01）， and there were statistically significant differences in the GLTP protein expression between PC tissue and normal pancreas tissue of the PC patients with different cancer stages （P<0.05）， tumor grades （P<0.05）， ages （P<0.001）， and genders （P<0.05）. Compared with control group， the proliferation activity （P<0.01） and the number of clone formation （P<0.001） of the cells in GLTP-OE group were decreased， while the numbers of migration cells （P<0.001） and invasion cells （P<0.01） were decreased. In the knock-down experiment， compared with control group， the proliferation activity （P<0.01） and the number of clone formation （P<0.05） of the cells in GLTP-OE group were increased， while the numbers of migration cells （P<0.001） and invasion cells （P<0.001） were increased. Compared with control group， the tumor weight and volume of the mice in experimental group were decreased （P<0.01）， following the injection of tumor cells for a period of four weeks. In the over-expression experiment， compared with control group， the expression levels of p-PI3K （P<0.01）， p-Akt-S473 （P<0.01）， and p-Akt-T308 （P<0.05） proteins in the cells in GLTP-OE group were decreased； the expression levels of AREG （P<0.01） and KDR （P<0.01） mRNA were decreased. In the knock-down experiment， compared with control group， the expression levels of p-PI3K （P<0.01）， p-Akt-S473 （P<0.01）， and p-Akt-T308 （P<0.01） in the cells in si-GLPT group were increased， and the expression levels of AREG （P<0.01） and KDR （P<0.05） mRNA were increased. Conclusion Low expression levels of GLTP in PC tissue are present. The over-expression of GLTP can inhibit the proliferation， migration and invasion of PANC-1 cells， as well as the growth of subcutaneously transplanted tumors in the nude mice； its possible mechanism may be related to decreasing the activity of the PI3K/Akt signaling pathway.

Objective To investigate the protective effect of prunetin on the neurons in the rats with cerebral ischemia reperfusion injury （CIRI）， and to clarify its possible mechanisms. Methods Thirty-six SD rats were randomly divided into sham operation group， model group， low dose of prunetin group （3.5 mg·kg^-1）， medium dose of prunetin group （7.0 mg·kg^-1）， high dose of prunetin group （14.0 mg·kg^-1）， and positive drug edaravone （Eda） group （n=6）. Zealonga method was used to evaluate the neurological function damage of the rats in various groups； open field experiment was used to evaluate the autonomous motor function； Triphenyltetrazolium chlorde （TTC） staining was used to evaluate the areas of cerebral infarction of the rats in various groups； HE staining and Nissl staining were used to observe the pathomorphology of brain tissue of the rats in various groups. Additionally，twenty-one SD rats were randomly divided into sham operation group， model group， prunetin group， c-Jun N-terminal kinase （JNK） inhibitor group， p38 inhibitor group， JNK inhibitor+prunetin group， and p38 inhibitor+prunetin group （n=3）. TUNEL staining was used to detect the positive rates of apoptosis of neurons of the rats in various groups； Western blotting method was used to detect the expression levels of apoptosis-related proteins and JNK/p38 signaling pathway-related proteins in brain tissue of cerebral infarction side of the rats in various groups. Results Compared with sham operation group， the neurological deficit score of rats in model group was significantly increased （P<0.001）， the total motor distance was shortened （P<0.001）， and the ratio of cerebral infarction area was increased （P<0.001）. In sham group，the neuronal structure in the rat brain tissue was clear and well-organized， with an abundance of Nissl bodies and no apparent pathological changes observed. Compared with model group， the neurological deficit scores of the rats in medium and high doses of prunetin groups were decreased （P<0.05）， total motor distances of rats were increased （P<0.05）， and the cerebral infarction areas of rats were decreased（P<0.05）； the neurons showed disarrayed arrangement， cytoplasmic condensation， nuclear consolidation， and lysing and deletion of Nissl bodies were decreased. Compared with sham operation group， the positive rate of apoptosis of neurons in model group was significantly increased （P<0.001）， the expression level of B-cell lymphoma-2（Bcl-2）， Bcl-2-associated X protein（Bax） and cleaved Caspase-3 proteins in brain tissue of the rats were significantly increased （P<0.05 or P<0.01）. Compared with model group， the positive rats of apoptosis of neurons of the rats in prunetin group were decreased （P<0.05）， the expression level of Bcl-2 protein in brain tissue of the rats was increased （P<0.001）， and the expression levels of Bax and cleaved Caspase-3 proteins were significantly decreased（P<0.05）. Compared with inhibitor groups， the positive rates of apoptosis of neurons in inhibitor+prunetin groups were decreased （P<0.01）， and the expression levels of p-JNK and p-p38 proteins in brain tissue of the rats as well as the ratios of p-JNK/JNK and p-p38/p38 were decreased （P<0.05）. Conclusion Prunetin has the effect of reducing the neurological function damage， decreasing the area of cerebral infarction， reducing the pathological damage， and inhibiting neuronal apoptosis in the rats， and its mechanism may be related to inhibiting neuronal apoptosis through regulating the JNK/p38 signaling pathway.

Objective To investigate the effect of securinine （SEC） on apoptosis of the human colon cancer cell line SW620， and to elucidate its possible mechanism. Methods The nude mice with subcutaneously transplanted tumor were divided into control group （n=6）， oxaliplatin （OXA） group （n=7）， and SEC group（n=7）. The volume and mass of subcutaneous tumors in the nude mice were measured in various groups， and the tumor inhibitory rates in various groups were calculated. The SW620 cells were treated with different doses （5-120 μmol·L^-1） of SEC for 12， 24， 48， and 72 h， respectively. Cell counting kit-8 （CCK-8） assay was used to assess the survival rates of cells in various groups， and the optimal doses of SEC were confirmed. The SW620 cells were divided into control group， 20 μmol·L^-1 SEC group， 40 μmol·L^-1 SEC group， and 40 μmol·L^-1 OXA group. TUNEL staining method and flow cytometry were used to detect the apoptotic rates of cells in various groups. JC-1 staining was used to detect the mitochondrial membrane potentials of cells in various groups， and 2'，7'-dichlorodi-hydrofluorescin diacetate （DCFH-DA） fluorescence staining and flow cytometry were used to measure the reactive oxygen species （ROS） levels in the cells in various groups. Western blotting method was used to detect the expression levels of cytochrome C， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， c-Jun N-terminal kinase （JNK）， phosphorylated JNK （p-JNK）， mitogen-activated protein kinase p38， phosphorylated p38 （p-p38）， extracellular signal-regulated kinase （ERK） and phosphorylated ERK （p-ERK） proteins in the cells in various groups. Results Compared with control group， the volume and mass of subcutaneously transplanted tumors in the nude mice in SEC group were significantly decreased （P<0.05 or P<0.01 or P<0.001）； the inhibitory rates of tumor in SEC group and OXA group were 20.42% and 6.50%. The CCK-8 assay results showed that compared with 0 μmol·L^-1 SEC， when the SEC dose exceeded 20 μmol·L^-1， the survival rates of SW620 cells were significantly decreased （P<0.001）. The optimal condition for subsequent experiments was set as doses of 20 μmol·L^-1 SEC and 40 μmol·L^-1 SEC， and duration of 24 h. The TUNEL results showed that compared with control group， the apoptotic rates of cells in 20 and 40 μmol·L^-1 SEC groups were significantly increased （P<0.05 or P<0.001）. The results of flow cytometry showed that compared with control group， the apoptotic rate in 40 μmol·L^-1 SEC group was significantly increased （P<0.001）. The JC-1 staining results showed that compared with control group， the mitochondrial membrane potentials of cells in 20 and 40 μmol·L^-1 SEC groups were significantly decreased （P<0.001）. Compared with control group， the levels of ROS detected by DCFH-DA fluorescence staining in the cells of 20 and 40 μmol·L^-1 SEC groups and 40 μmol·L^-1 OXA group were significantly increased （P<0.001）， while the level of ROS detected by flow cytometry in 40 μmol·L^-1 SEC group was significantly increased （P<0.05）. Compared with control group， the expression levels of Bcl-2 protein in the cells in 20 and 40 μmol·L^-1 SEC groups and 40 μmol·L^-1 OXA group were decreased （P<0.01）， while the expression levels of cytochrome C and Bax proteins were increased （P<0.001）.Compared with control group， the ratios of p-JNK/JNK， p-p38/p38 and p-ERK/ERK in 20 and 40 μmol·L^-1 SEC groups were significantly increased （P<0.05 or P<0.01 or P<0.001）. Conclusion SEC can inhibit the proliferation of SW620 cells， increase the cellular ROS levels， reduce the mitochondrial membrane potential， and induce the mitochondrial apoptosis； its mechanism may be related to the regulation of the mitogen-activated protein kinase（MAPK） signaling pathway.

Objective To investigate the influence of 17β-estradiol （17β-E2） in the proliferation and differentiation capabilities of primary cultured hippocampal neural stem cells （NSCs），and to clarify its potential mechanism. Methods The NSCs were isolated from the hippocampal tissue of SD rats within 24 h of birth，and divided into control group and 17β-E2 group. Immunofluorescence method was used to detect the expressions of Nestin and 5-ethynyl-2'-deoxyuridine （EdU） in NSCs in two groups， and the proliferation rates of NSCs were calculated. Western blotting method was used to detect the expression level of Nestin protein. Flow cytometry was used to analyze the percentages of NSCs at different cell cycles. Immunofluorescence method was used to identify the expressions of markers for neurons βⅢ-tubulin and astrocyte marker glial fibrillary acidic protein （GFAP） in the NSCs after differentiation， and the relative ratio of neurons to astrocytes was calculated. Western blotting method was used to detect the expression levels of βⅢ-tubulin and GFAP proteins as well as phosphatidylinositol 3-kinase （PI3K）， protein kinase B （Akt）， phosphorylated Akt （p-Akt）， glycogen synthase kinase-3 beta （GSK-3β）， phosphorylated GSK-3β （p-GSK-3β）， and beta-catenin （β-catenin） proteins related to PI3K/Akt/GSK-3β pathway. Results Immunofluorescence assay revealed positive Nestin expression in NSCs in two groups； compared with control group，the proliferation rate of NSCs in 17β-E2 group was increased （P<0.01）. Compared with control group， the expression level of Nestin protein in the NSCs in 17β-E2 group was increased （P<0.05），and the percentage of NSCs in S phase was increased （P<0.01）. Compared with control group，the relative ratio of neurons to astrocytes in 17β-E2 group was significantly increased （P<0.05）. After differentiation，compared with control group，the expression level of βⅢ-tubulin protein in the NSCs in 17β-E2 group was increased （P<0.05）， and the expression level of GFAP protein was decreased （P<0.01）， while the expression levels of Akt， p-Akt， β-catenin， and p-GSK-3β proteins were increased （P<0.05 or P<0.01）， and the expression level of GSK-3β protein was decreased （P<0.05）. Conclusion 17β-E2 can promote the proliferation of NSCs and facilitate their differentiation towards neurons，and its mechanism may be related to the PI3K/Akt/GSK-3β signaling pathway.

Objective To investigate the effect of bone marrow mesenchymal stem cells （BMSCs） on the proliferation and collagen expression levels of L929 cells， and to clarify its related mechanism. Methods The BMSCs were extracted from the 4-week-old C57BL/6 mice. The phenotypes of BMSCs were identified by immunofluorescence staining. The L929 cells were divided into control group （L929 cells）， co-culture group （L929 cells and BMSCs）， inhibitor of Janus kinase（JAK） WP1066 group （WP1066-treated L929 cells and BMSCs）， and dimethyl sulfoxide （DMSO） group （DMSO-treated L929 cells and BMSCs）. Cell counting kit-8 （CCK-8） assay was used to detect the proliferation activities of the L929 cells in various groups at different time points； Western blotting method was used to detect the expression levels of type Ⅰ collagen （ColⅠ） and type Ⅲ collagen （ColⅢ） in the L929 cells in various groups； immunofluorescence staining was used to detect the expressions of ColⅠ and ColⅢ proteins in the L929 cells in various groups. Results The fluorescence assay results of surface antigen（SA） showed that the surface markers CD29+， CD45-， CD90+ and CD105+ were found in the BMSCs. The CCK-8 assay results showed that compared with control group， the proliferation activities of the L929 cells in co-culture group and DMSO group were significantly increased（P<0.01）； compared with co-culture group， the proliferation activity of the L929 cells in WP1066 group was significantly decreased （P<0.01）. The Western blotting method results showed that compared with control group， the expression levels of ColⅠ and ColⅢ proteins in the L929 cells in co-culture group and DMSO group were significantly increased （P<0.01）； compared with co-culture group， the expression levels of ColⅠ and ColⅢ proteins in the L929 cells in WP1066 group were significantly decreased （P<0.01）； compared with DMSO group， the expression levels of ColⅠ and ColⅢ proteins in the L929 cells in WP1066 group were significantly decreased （P<0.01）. The immunofluorescence staining results showed that compared with control group， the fluorescence intensities of ColⅠ and ColⅢ proteins in the L929 cells in co-culture group and DMSO group were significantly increased（P<0.01）； compared with co-culture group， the fluorescence intensities of ColⅠ and ColⅢ in the L929 cells in WP1066 group were significantly decreased（P<0.01）； compared with DMSO group， the fluorescence intensities of ColⅠ and ColⅢ in the L929 cells in WP1066 group were significantly decreased （P<0.01）. Conclusion MSCs can promote the proliferation and collagen production of the L929 cells of the mice through the JAK2/signal transducer and activator of transcription 3（STAT3） signaling pathway.

Objective To investigate the effect of type 2 taste receptor （T2R）38 activation on ferroptosis of human airway epithelium NuLi-1 cells induced by cigarette smoke exposure， and to clarify its possible mechanism. Methods The human airway epithelial NuLi-1 cells were divided into control group （without any treatment）， cigarette smoke extract （CSE） group （treated with 5% CSE for 24 h） and CSE+T2R38 specific agonist phenylthiocarbamide （PTC） group（CSE+PTC group） （treated with 5% CSE and 1 mmol·L^-1 PTC for 24 h）. The expression levels of T2R38 mRNA and protein in NuLi-1 cells in various groups were determined by real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting methods. The cell viabilities in various groups were determined by cell counting kit-8 （CCK-8） assay. The activities of inducible nitric oxide synthase （iNOS）， endothelial nitric oxide synthase （eNOS）， and superoxide dismutase （SOD） in the cells in various groups were measured by kits. DAX-J2 red fluorescence probe was used to determine the levels of nitric oxide（NO） in the cells in various groups. The reactive oxygen species（ROS） levels in the cells in various groups were detected by fluorescent probe kit. The levels of malondialdehyde （MDA）， Fe²⁺， and reduced glutathione （GSH） in the cells in various groups were determined by enzyme-linked immunosorbent assay （ELISA） method. Western blotting method was used to determine the expression levels of nuclear factor erythroid 2-related factor 2 （Nrf2） and glutathione peroxidases 4 （GPx4） proteins in the cells in various groups. Results Compared with control group， the expression levels of T2R38 mRNA and protein in NuLi-1 cells in CSE group were increased （P<0.05）. Compared with control group， the viability of NuLi-1 cells in CSE group was decreased （P<0.05）， the activities of iNOS and SOD in cells in CSE group were increased （P<0.05）， the levels of NO and ROS were increased （P<0.05）， the levels of MDA and Fe²⁺ were increased（P<0.05）， and the GSH level and the expression levels of Nrf2 and GPx4 proteins were decreased. Compared with CSE group， the viability of NuLi-1 cells in CSE+PTC group was increased（P<0.05）， the activity of SOD and the GSH level in the cells were increased（P<0.05）， the activity of iNOS in cells was decreased （P<0.05）， the levels of NO and ROS in cells were decreased （P<0.05）， the levels of MDA and Fe²⁺ were decreased （P<0.05）， and the expression levels of Nrf2 and GPx4 proteins were increased （P<0.05）. There was no significant difference in eNOS activity among control group， CSE group， and CSE+PTC group （P>0.05）. Conclusion Activation of bitter taste receptor T2R38 can inhibit ferroptosis in human airway epithelium NuLi-1 cells induced by cigarette smoke exposure， and its mechanism may be related to the reduction of iNOS activity in the cells.

Objective To explore the correlation between IκB kinase-interacting protein （IKBIP） expression in tumor cells within cervical cancer tissues （TCCCT） and the clinical pathological characteristics and prognosis of patients， as well as its impact on the biological behaviors of cervical cancer （CC） cells HeLa and SiHa， and to elucidate its potential mechanism. Methods GENT2 and Kaplan-Meier plotter databases were utilized to analyze the differential expression of IKBIP mRNA in CC and normal cervical tissues， as well as its correlation with the clinical prognosis of CC patients. The Hallmark reference gene set was chosen for pathway enrichment analysis using the Gene Set Enrichment Analysis（GSEA） software. Immunohistochemistry method was used to detect the IKBIP protein expression in TCCCT and epithelial cells in normal cervical tissue （ECNCT）， and analyze the correlations between its expression level and clinicopathological characteristics and prognosis of CC patients. Furthermore， univariate and multivariate Cox regression analyses were performed to identify the risk factors impacting the prognosis of CC patients. The stably transfected cells of CC （HeLa cells and SiHa cells） with IKBIP knockdown were established for the experiment， which were divided into sh-NC group and sh-IKBIP group. The expression of IKBIP protein in various groups was assessed using Western blotting method. The cell proliferation activity and the percentage of 5-ethynyl-2'-deoxyuridine （EdU） positive cells were measured using cell counting kit-8 （CCK-8） and EdU methods， while Transwell chamber assay was employed to determine the numbers of migration and invasion cells in various groups. Additionally， the expression levels of E-cadherin， N-cadherin， and Snail proteins in the cells in various groups were analyzed by Western blotting method. Results The GENT2 database analysis revealed that the expression level of IKBIP mRNA in CC tissue was higher than that in normal cervical tissue （P<0.001）. The GSEA enrichment analysis identified the epithelial-mesenchymal transition （EMT） pathway as the top-ranked pathway in IKBIP high-expression group. The immunohistochemistry results indicated the positive expression rate of IKBIP protein in TCCCT was higher than that in ECNCT （50.5% vs 8.0%）， and its over-expression was associated with FIGO stage （2018） and differentiation grade of tumor （P<0.05）. The univariate and multivariate Cox regression analyses suggested that lymph node metastasis （LNM） and high expression of IKBIP were the risk factors affecting the overall survival （OS） and progression-free survival （PFS） of CC patients （P<0.05）. The Western blotting method results showed that compared with sh-NC group， the expression level of IKBIP protein in the cells in sh-IKBIP group was decreased （P<0.05）. The CCK-8 and EdU assay results showed that compared with sh-NC group， the proliferation activity and the percentage of EdU positive cells in sh-IKBIP group were decreased （P<0.05）. The Transwell chamber assay results showed that compared with sh-NC group， the numbers of migration and invasion cells in sh-IKBIP group were significantly decreased （P<0.05）. Additionally， the Western blotting method results indicated that compared with sh-NC group， the expression level of E-cadherin protein in sh-IKBIP group in CC cells was increased （P<0.05）， while the expression levels of N-cadherin and Snail protein were decreased （P<0.05）. Conclusion The expression of IKBIP protein is significantly up-regulated in HeLa and SiHa cells derived from CC， and it is closely correlated with poor prognosis in CC patients. Suppression of IKBIP protein expression can effectively inhibit the proliferation， invasion and migration capabilities as well as EMT process of CC cells.

Objective To investigate the protective effect of a composite hydrogel against hydrogen peroxide（H?O?）-induced oxidative stress injury in the cardiomyocytes， and to clarify its possible mechanism. Methods The mice were subcutaneously injected with 100 μL of hydrogel. After normal feeding for 1， 14， and 28 d， the mice were sacrificed. Tissue samples were collected and subjected to HE staining to observe the histocompatibity of the hydrogel. The primary cardiomyocytes isolated from 1-day-old SD rats were used to establish an oxidative stress injury model. The primary cardiomyocyties were divided into control， H₂O₂ and H₂O₂+Hydrogel groups. The primary cardiomyocytes in control group were cultured normally， the primary cardiomyocytes in H₂O₂ group were treated with 200 μmol·L^-1 H₂O₂ for 24 h， and the primary cardiomyocytes in H₂O₂+Hydrogel group were incubated with 1 g·L^-1 composite hydrogel and 200 μmol·L^-1 H₂O₂ for 24 h. The viabilities of cardiomyocytes in various groups were assessed by cell counting kit-8（CCK-8） method. Dihydroethidium （DHE） and 2'，7'-dichlorodihydrofluorescein diacetate （DCFH-DA） staining were used to assess the reactive oxygen species（ROS） levels in the cells. The expressions of filamentous actin（F-actin） in the cells in various groups were detected by phalloidin fluorescence staining； the expressions of connexin 43 （Cx43） and cardiac troponin T （cTnT） proteins in the cardiomyocytes in various groups were detected by immunofluorescence method. The apoptotic rates of cardiomyocytes in various groups were assessed with TUNEL staining method. The expression levels of apoptosis-related proteins B-cell lymphoma-2（Bcl-2）and Bcl-2-associated X protein （Bax） in the cardiomyocytes in various groups were assessed by Western blotting method. Results The HE staining results showed that the inflammatory cells around the implanted hydrogel were less infiltrated， and the inflammatory reaction of subcutaneous implantation was less. Compared with control group， the viability of cardiomyocytes in H₂O₂ group was significantly decreased （P<0.05）， the level of ROS in the cells was increased（P<0.05）， the expression levels of Cx43， cTnT and F-actin proteins in the cells were decreased （P<0.001）， the apoptosis rate of cardiomyocytes were decreased （P<0.01）， the expression level of Bcl-2 protein in the cells was increased （P<0.001）， and the expression level of Bax protein was decreased（P<0.01）. Compared with H₂O₂ group， the viability of cardiomyocytes was significantly increased （P<0.05）， the level of ROS in the cells was decreased （P<0.01）， the expression levels of cTnT， Cx43 and F-actin proteins were increased （P<0.01），the apoptotic rate of cardiomyocytes were significantly decreased （P<0.001）， the expression level of Bcl-2 protein in the cells were decreased （P<0.01），and the expression level of Bax protein was increased （P<0.01）. Conclusion Hydrogel may promote the expression of cardiomyocyte-related proteins by scavenging ROS in the environment and inhibit the cardiomyocyte apoptosis to achieve the protective effect on the cardiomyocytes under oxidative stress.

Objective To prepare a novel ginseng polypeptide thermosensitive hydrogel， and to investigate its repair effect on heat-induced skin injury in the rats and explore the underlying mechanisms. Methods Thermosensitive hydrogels were formulated using Pluronic F127 and β-sodium glycerophosphate（β-GP）， and their phase transition temperatures， spatial structures， elemental compositions， and water retention capacities were evaluated. The rat models of heat-induced skin injury were established and the model rats were divided into PBS group， Gel group， and ginseng polypeptide gel （GP-Gel） group. After 11 d of treatment， the morphological changes of wound and collagen deposition in the wound of the rats in various groups were observed by HE and Masson staining. Immunohistochemistry was used to detect the expression levels of α-smooth muscle actin （α-SMA）， connective tissue growth factor （CTGF）， basic fibroblast growth factor （bFGF）， proliferating cell nuclear antigen （PCNA）， cell proliferation marker Ki67， epidermal growth factor （EGF）， CD31， vascular endothelial growth factor （VEGF）， P50 and P65 proteins in the skin wound tissue of the rats in various groups. Western blotting method was used to detect the expression levels of Toll-like receptor 4 （TLR4） in the skin wound tissue of the rats in various groups. ELISA method was used to measure the levels of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， interleukin-6 （IL-6）， interleukin-15 （IL-15）， and interleukin-10 （IL-10） in the serum of the rats in various groups. Results Compared with PBS and Gel groups， the wound area of the rats in GP-Gel group was reduced（P<0.01）， the expression levels of PCNA， Ki67， EGF， CD31， VEGF， α-SMA， and CTGF proteins in the skin wound tissue were increased （P<0.05 or P<0.01）， and the expression levels of P65 and TLR4 proteins were decreased （P<0.01）； the level of anti-inflammatory factor IL-10 in serum was increased （P<0.01）， while the levels of pro-inflammatory factors TNF-α， IL-1β， IL-6 and IL-15 were decreased （P<0.05 or P<0.01）. Conclusion The ginseng polypeptide thermosensitive hydrogel promotes the repair of heat-induced skin injury by enhancing cell proliferation， collagen synthesis， angiogenesis， and reducing inflammatory responses.

Objective To investigate the inhibitory effect of astragaloside Ⅳ（AS-Ⅳ） on cisplatin（CDDP）-induced liver injury in the mice，and to elucidate its possible mechanism. Methods Forty male C57BL/6 mice with body weights of 18-22 g were randomly divided into control group，model group，AS-Ⅳ group and adenosine 5'-monophosphate-activated protein kinase （AMPK） inhibitor （Compound C）+ AS-Ⅳ group. The mice in control group and model group were gavaged with the same volume of normal saline，and the drug was administered continuously for 9 d. The mice in AS-Ⅳ group and Compound C+AS-Ⅳ group were given AS-Ⅳ aqueous solution（150 mg·kg^-1·d^-1）， respectively. On the 6th day of experiment，the mice in Compound C+AS-Ⅳ group were intraperitoneally injected with Compound C （20 mg·kg^-1）， and on the 7th day，except for control group，the mice in other groups were intraperitoneally injected with 20 mg·kg^-1 CDDP to establish the mouse liver injury models，and the mice were sacrificed 48 h later. Serum and liver tissues were collected，and the levels of aspartate aminotransferase （AST） and alanine aminotransferase （ALT） in the serum of the mice，as well as the activities of superoxide dismutase（SOD） and catalase （CAT） and the levels of malondialdehyde（MDA） in the liver tissue of the mice in various groups were detected by kits. The pathomorphology of liver tissue of the mice in various groups were detected by HE staining. The expression levels of glutathione peroxidase 4 （GPX4）， ferritin heavy chain 1 （FTH1） and ferroptosis inhibitory protein 1 （FSP1） proteins in liver tissue of the mice in various groups were detected by immunohistochemical staining， and the expression levels of nuclear factor-E2-related factor 2 （Nrf2）， heme oxygenase-1 （HO-1） and AMPK proteins in liver tissue of the mice in various groups were detected by Western blotting method. Results Compared with control group， the levels of AST and ALT in serum of the mice in model group were increased （P<0.01）， the activities of SOD and CAT in the liver tissue were significantly decreased （P<0.01）， and the MDA level was increased （P<0.01）； compared with model group，the levels of AST and ALT in serum of the mice in AS-Ⅳ group were decreased （P<0.01）， the MDA level in the liver tissue was decreased （P<0.01）， and the activities of SOD and CAT were increased （P<0.01）； compared with AS-Ⅳ group （P<0.01）， the levels of AST and ALT in serum of the mice in Compound C+AS-Ⅳ group were increased （P<0.01）， the level of MDA in liver tissue was increased （P<0.05）， and the activities SOD and CAT were decreased（P<0.01）. The HE staining results showed that compared with control group， the liver damage degree of the mice in model group was enhanced，the hepatocyte arrangement was disordered， and some hepatocyte edema were increased； compared with model group， the liver morphology of the mice in AS-Ⅳ group returned to normal； compared with AS-Ⅳ group， the hepatocyte arrangement of the mice in Compound C+AS-Ⅳ group was disordered and the edges were blurred. The immunohistochemistry results showed that compared with control group， the expression levels of GPX4， FTH1 and FSP1 proteins in liver tissue of the mice in model group were decreased （P<0.05）； compared with model group， the expression levels of GPX4， FTH1 and FSP1 proteins in liver tissue of the mice in AS-Ⅳ group were increased （P<0.05）； compared with AS-Ⅳ group， the expression levels of GPX4， FTH1 and FSP1 proteins in liver tissue of the mice in Compound C+AS-Ⅳ group were decreased （P<0.05 or P<0.01）. The Western blotting results showed that compared with control group，the expression levels of Nrf2， HO-1 and AMPK proteins in liver tissue of the mice in model group were decreased （P<0.01）； compared with model group，the expression levels of Nrf2， HO-1 and AMPK proteins in liver tissue of the mice in AS-Ⅳ group were increased （P<0.01）； compared with AS-Ⅳ group，the expression levels of Nrf2， HO-1 and AMPK proteins in liver tissue of the mice in Compound C+AS-Ⅳ group were decreased （P<0.01）. Conclusion AS-Ⅳ can alleviate the CDDP-induced liver injury，and its mechanism may be related to the regulation of AMPK/Nrf2/HO-1 signal pathway and ferroptosis by AS-Ⅳ.

Objective To investigate the expression of protein kinase D2 （PRKD2） in bladder cancer （BLCA） tissue using bioinformatics analysis method and its effect on the prognosis of BLCA patients， and to clarify the role of PRKD2 in the occurrence and development of BLCA. Methods The data from 9 normal bladder samples， 19 BLCA paracancerous samples， and 407 BLCA tumor samples were downloaded from the UCSC Cancer Genome Database. The Mann-Whitney U test was applied to analyze the difference in expression of PRKD2 mRNA in BLCA tumor and normal bladder tissues， and the Human Protein Atlas （HPA） database was used for proteomic validation. DESeq2 package in R software was applied to screen the differentially expressed genes （DEGs） in BLCA tissue in PRKD2 low- and high-expression groups. The co-expression heatmaps of PRKD2 were plotted using the ggplot2 package， Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） were used for functional annotation analysis and pathway enrichment analysis of DEGs， and Gene Set Enrichment Analysis （GSEA） was used to obtain the gene sets that were significantly enriched for DEGs. The BLCA samples were divided into low- and high-expression groups according to the expression level of PRKD2， and the correlations between PRKD2 expression and immune cell infiltration in the BLCA patients were analyzed with GSVA package. The relationship between PRKD2 and prognosis of BLCA patients was further analyzed using the survival package and the survminer package. The PRKD2 gene mutations in BLCA tissue were analyzed using the cBioPortal database. The cystitis， bladder polyp and BLCA tissues were collected， and the expression levels of interleukin-17F （IL-17F） protein in BLCA and control tissues were detected using immunohistochemical staining technique. Results PRKD2 was highly expressed in a variety of malignant tumors， and the expression levels of PRKD2 mRNA and protein in BLCA tissue were significantly increased compared with those in normal bladder tissue （P<0.05）. Single gene differential analysis of PRKD2 yielded a total of 1 058 DEGs， of which a total of 29 genes were up-regulated and 1 029 were down-regulated. The results of GO functional enrichment analysis showed that DEGs were mainly enriched in the biological process （BP）， such as chemical stimuli involved in sensory perception， Cajal body， and endopeptidase inhibitor activity. The results of KEGG pathway analysis showed that DEGs were mainly enriched in the pathway of Staphylococcus aureus infection and the pathway of maturity onset diabetes of the young. GSEA analysis showed that DEGs were mainly enriched in the Notch signaling pathway， the retinoic acid-inducible gene-Ⅰ（RIG-Ⅰ）-like receptor signaling pathway， the cytoplasmic DNA screening pathway， the base excision repair signaling pathway， natural killer （NK） cell-mediated cytotoxicity signaling pathway and T cell receptor signaling pathway. The results of immune infiltration analysis indicated that the expression of PRKD2 was positively correlated with five types of cells， such as activated dendritic cells （aDC）， NK CD56dim cells and central memory T cells （Tcm） （P<0.05）， and negatively correlated with three types of immune cells， including macrophages， effector memory T cells （Tem） and plasmacytoid dendritic cells （pDC） （P<0.05）.The clinical characteristic subgroup analysis results showed that the expression levels of PRKD2 mRNA in BLCA patients who were over 70 years old and developed lymphovascular invasion were decreased （P<0.05）； the overall survival （OS）， disease-specific survival （DSS） and progression-free interval （PFI） in the BLCA patients with PRKD2 high expression were significantly longer than those with PRKD2 low expression （P<0.05）. The univariate and multivariate Cox analyses indicated that distant metastasis， primary therapy outcome and clinicopathologic stage were the important factors affecting BLCA prognosis. About 9% patients had PRKD2 gene mutations， including missense mutation， gene amplification， mRNA low or high expression， and multi-motif mutation. The immunohistochemistry results showed that the expression level of IL-17F protein in BLCA tissue was significantly higher than that in cystitis tissue （P<0.05）. Conclusion The expression level of PRKD2 in BLCA tissue is obviously increased， which could up-regulate the expression of IL-17F protein， and the decrease of PRKD2 protein expression may be a potential factor for the poor prognosis of BLCA patients.

Objective To investigate the expression of chondroitin sulfate proteoglycan 4 pseudogene 12 （CSPG4P12） in the small cell lung cancer （SCLC） tissue， its relationship with immune infiltration， and its effect on cell biological functions， and to clarify its effect in the occurrence and development of SCLC. Methods The E-GEOD-60052 cohort was obtained by searching the ArrayExpress database for SCLC. The R language Bioconductor package was used to complete data filtering standardization， and 63 samples of SCLC tumor tissues and 7 samples of normal tissues were obtained. The Mann-Whitney U test was used to analyze the difference in CSPG4P12 expression levels between two groups. Pearson correlation analysis was used to evaluate the associations between CSPG4P12 expression levels and 47 immune checkpoint genes. The ESTIMATE algorithm and CIBERSORT algorithm were used to evaluate the correlations between CSPG4P12 expression and tumor immune cell infiltration. A case-control study was used to analyze the clinical data. A total of 230 patients with SCLC were selected as case group， and 230 healthy subjects were selected as control group. The genotyping of CSPG4P12 rs2880765， rs6496932 and rs8040855 was performed using TaqMan-MGB fluorescent probe labeling method. Odds ratio （OR） and 95% confidence interval （CI） were calculated by unconditional Logistic regression model to analyze the association between polymorphic genetic variation of CSPG4P12 gene and the risk of SCLC. The SCLC DMS114 cells were transfected with pUC-57 plasmid （control group） and CSPG4P12 over-expression plasmid （OV-CSPG4P12 group）， respectively. The efficiencies of CSPG4P12 over-expression in two groups were verified by real-time fluorescence quantitative PCR（RT-qPCR） method. Cell counting kit-8 （CCK-8） method was used to detect the cell proliferation activities of cells in two groups. Transwell chamber assay was used to detect the numbers of migration and invasion cells in two groups， respectively. Hoechst 33342 fluorescence staining was used to observe the cell apoptosis in two groups. Results The ArrayExpress database E-GEOD-60052 cohort analysis showed that the expression level of CSPG4P12 mRNA in SCLC tissue was decreased compared with normal tissue （P<0.001）. The expression of CSPG4P12 had positive correlations with the immune checkpoint genes including leukocyte associated immunoglobulin like receptor 1 （LAIR1） （r=0.47， P<0.001）， tumor necrosis factor（TNF） receptor superfamily member 9 （TNFRSF9） （r=0.38， P<0.01）， and TNF superfamily member 9 （TNFSF9） （r=0.44， P<0.001）. The ESTIMATE algorithm results showed that the matrix score， immune score and ESTIMATE composite score of the patients in CSPG4P12 low expression group were lower than those in CSPG4P12 high expression group （P<0.01）. The CIBERSORT algorithm results showed that compared with CSPG4P12 high expression group， the infiltration of M0 macrophages in CSPG4P12 low expression group was increased （P<0.05） and the infiltration of mast cells resting was decreased （P<0.05）. The CSPG4P12 expression level had positive correlations with infiltration of mast cells resting （r=0.35， P=0.03） and mononuclear cell infiltration （r=0.34， P=0.034）. In case-control studies， compared with AA genotype， CSPG4P12 rs2880765 AT and TT genotype carriers had a higher risk of SCLC （OR=1.68， 95%CI=1.15-2.45， P<0.01）. The stratified analysis showed that genetic variation of rs2880765 A>T increased the risk of SCLC in the male， younger age group（≤60 years） and smoking subgroups （males： OR=1.86， 95%CI=1.18-2.93， P<0.01； ≤60 years： OR=1.73， 95%CI=1.11-2.68， P<0.01； smoking： OR=2.76， 95%CI=1.49-5.13， P=0.001）. The cell biology experiment showed that compared with control group， the proliferation abilities of the cells in OV-CSPG4P12 group were significantly decreased at 48 and 72 h （P<0.01）， while the number of migration cells at 24 h was significantly decreased （P<0.01）， the number of apoptotic cells at 24 h was increased （P<0.05） and the number of invasion cells at 48 h was significantly decreased （P<0.01）. Conclusion CSPG4P12 is lowly expressed in SCLC tumor tissue， which is associated with immune infiltration. The genetic variation of CSPG4P12 rs2880765 A>T can increase the risk of SCLC， and its over-expression can inhibit cell proliferation， migration and invasion， and promote apoptosis.

Objective To investigate the induction effect of different concentrations of glucose on the polarization of Raw264.7 macrophages in vitro. Methods The Raw264.7 cells cultured in DMEM medium were divided into control group （5.5 mmol·L^-1 glucose）， different high-glucose groups （15.0， 25.0， 35.0， and 45.0 mmol·L^-1 glucose）， and positive control group ［lipopolysaccharide（LPS）］. The cells were cultured for 3， 6， and 9 h， respectively. Cell morphology was observed in various groups. Cell counting kit-8 （CCK-8） method was used to assess the survival rates of cells in various groups. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， and interleukin-10 （IL-10） mRNA in cells in various groups. Enzyme-linked immunosorbent assay （ELISA） was used to measure the levels of IL-6， TNF-α， and IL-10 in the cell supernatants in various groups. Flow cytometry was used to determine the percentages of M1 and M2 macrophage markers， CD86+ and CD163+ cells， in various groups. Results In control group， the Raw264.7 cells adhered to the culture dish， with a predominantly round morphology. The cells in 35.0 mmol·L^-1 high-glucose group and positive control group showed elongated shapes and pseudopodium formation， indicating inflammatory changes. Compared with control group， the survival rates in different high-glucose groups at 6， 12， 24， and 48 h after treatment were increased （P<0.05）. Compared with control group， after 3 h of treatment， the expression levels of IL-6 and IL-10 mRNA in the cells in 35.0 mmol·L^-1 high-glucose group were increased （P<0.05 or P<0.01）， while no significant changes were observed in IL-6， TNF-α， and IL-10 levels in the cell supernatants （P>0.05）； after 6 h of treatment， the expression level of TNF-α mRNA in the cells in 35.0 mmol·L^-1 high-glucose group was increased （P<0.001）， and the levels of IL-6， TNF-α， and IL-10 in the cell supernatants were significantly increased （P<0.05 or P<0.001）； after 3 h of treatment， the percentages of macrophage markers CD86+ cells and CD163+ cells in 35.0 mmol·L^-1 high-glucose group were significantly increased （P<0.01 or P<0.001）. Conclusion A certain high concentration of glucose may induce the polarization of Raw264.7 macrophages towards the M1 subtype in vitro.

Objective To investigate the effect of up-regulation of microRNA-31 （miRNA-31） on the osteogenic differentiation of dental pulp stem cells （DPSCs）， and to elucidate its possible mechanism. Methods The DPSCs in logarithmic growth phase were divided into control group （no treatment）， NC group （transfected with random sequence control）， Agomir group （transfected with miR-31 mimic agomiR-31）， and combination group （transfected with miR-31 mimic agomiR-31 and added with XAV939）. After 48 h of transfection， real-time fluorescence quantitative PCR （RT-qPCR） was used to detect the expression levels of miR-31 in the DPSCs in various groups. MTT assay was used to detect the proliferation abilities of the DPSCs in various groups. Alizarin red staining was used to detect calcium deposition in the DPSCs in various groups. Alkaline phosphatase （ALP） staining was used to detect the degree of osteogenic differentiation of the DPSCs in various groups. Western blotting method was used to detect the expression levels of proteins related to the wingless-type MMTV integration site family（Wnt）/β-catenin signaling pathway in the DPSCs in various groups. Results There were no significant differences in the miR-31 expression level， the cell proliferation abilities at 24， 48 and 72 h， the ratio of calcified region， and the ALP ability between control group and NC group （P>0.05）. Compared with control group and NC group， the expression level of miR-31， the cell proliferation abilities at 24， 48 and 72 h， the ratio of calcified region， and the ALP activity in the DPSCs in Agomir group were increased （P<0.05）. Compared with Agomir group， the expression level of miR-31， the cell proliferation abilities at 24， 48 and 72 h， the ratio of calcified region， and the ALP activity in the DPSCs in combination group were decreased （P<0.05）. There were no significant difference in the expression levels of glycogen synthase kinase 3β （GSK-3β）， β-catenin and Runt-associated transcription factor 2 （Runx2） in the DPSCs between control group and NC group （P>0.05）. Compared with control group and NC group， the expression level of GSK-3β protein in the DPSCs in Agomir group was decreased （P<0.05）， and the expression levels of β-catenin and Runx2 proteins in the DPSCs were increased （P<0.05）. Compared with Agomir group， the expression level of GSK-3β protein in the DPSCs in combination group was increased （P<0.05）， while the expression levels of β-catenin and Runx2 proteins were decreased （P<0.05）. Conclusion Up-regulation of miR-31 can promote the proliferation and osteogenic differentiation of DPSCs， and its mechanism may be related to the activation of Wnt/β-catenin signaling pathway.

Objective To investigate the effects of Juglone on the apoptosis of osteosarcoma（OS） cells （U2OS and MG63 cells） through the cysteinyl aspartate specific proteinase-3（Caspase-3）/gasdermin E（GSDME）-mediated pyroptosis pathway. Methods The U2OS and MG63 cells were cultured in vitro and divided into control group， different concentrations （5， 10 and 20 μmol·L^-1） of Juglone groups and Caspase-3 inhibitor Z-DEVD-FMK group （10 μmol·L^-1 Juglone + 30 μmol·L^-1 Z-DEVD-FMK）. The survival rates of cells in various groups were assessed by cell counting kit-8（CCK-8） assay， and the apoptotic rates were detected by flow cytometry. Lactate dehydrogenase （LDH） release assay was used to measure the release rates of LDH from the cells. Western blotting method was used to detect the expression levels of apoptosis-related proteins including B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein（Bax）， cleaved-Caspase-3 and poly（ADP-ribose） polymerase（PARP）and pyroptosis-related proteins including GSDME full form（GSDME-F） and GSDME N-terminal（GSDME-N）. The levels of interleukin-1β （IL-1β） and interleukin-18 （IL-18） in the cell supernataut in various groups were measured by enzyme-linked immunosorbent assay（ELISA） method. Results Compared with control group， the survival rates of cells in 5， 10， and 20 μmol·L^-1 Juglone groups were significantly decreased （P<0.05 or P<0.01）， and the 50% inhibitory concentration （IC₅₀） values of U2OS cells and MG63 cells were 8.4 and 10.2 μmol·L^-1， respectively. Compared with control group，the apoptotic rates and LDH release rates of U2OS and MG63 cells in 5 and 10 μmol·L^-1 Juglone groups were significantly increased （P<0.05 or P<0.01）. Compared with control group， the expression levels of Bax， cleaved-Caspase-3， and cleaved-PARP proteins in 5 and 10 μmol·L^-1 Juglone groups were significantly increased （P<0.01）， while the expression levels of Bcl-2 protein were significantly decreased （P<0.01）. Compared with control group， the levels of IL-1β and IL-18 in cell supernatant in 5 and 10 μmol·L^-1 Juglone groups were increased（P<0.01）. Compared with control group， the expression levels of cleaved-Caspase-3 and GSDME-N proteins in 5 and 10 μmol·L^-1 Juglone groups were significantly increased （P<0.01）， while there was no difference in the expression level of GSDME-F protein （P>0.05）. Compared with 10 μmol·L^-1 Juglone group， the expression levels of cleaved-Caspase-3 and GSDME-N in Z-DEVD-FMK group were significantly decreased（P<0.01），while there was no difference in the expression level of GSDME-F protein （P>0.05）. Conclusion Juglone can induce the apoptosis of U2OS and MG63 cells and cause the Caspase-3/GSDME-mediated pyroptosis.

Objective To investigate the protective effect of quercetin against 5-fluorouracil （5-FU）- induced damage in the human immortalized keratinocytes （HACAT）， and to elucidate its possible mechanism. Methods The HACAT cells were divided into control group （normal cultured cells）， 5-FU group （treated with 7.5 mg·L^-1 5-FU for 24 h）， and low， medium， and high doses of quercetin groups （HACAT cells treated with 25， 50， and 75 μmol·L^-1 quercetin combined with 7.5 mg·L^-1 5-FU for 24 h）. Cell counting kit-8（CCK-8） assay was used to detect the survival rates of HACAT cells treated with different doses（0，10，25，50，75 and 100 μmol·L^-1） of quercetin in various groups. The fluorescent probe of reactive oxygen species（ROS） was used to detect ROS levels in the HACAT cells in various groups. Annexin Ⅴ-FITC/PI double staining was used to detect the apoptosis of HACAT cells in various groups. Western blotting method was used to detect the expression levels of B-cell lymphoma-2（Bcl-2）， Bcl-2-associated X protein （Bax）， Cleaved Caspase-3， cyclooxygenase-2 （COX-2）， interleukin-1β （IL-1β）， and interleukin-6（IL-6） in the HACAT cells in various groups. Results The CCK-8 assay results showed that compared with 0 μmol·L^-1 quercetin group， the survival rates of HACAT cells in 10， 25， 50 and 75 μmol·L^-1 quercetin groups showed no significant differences （P>0.05）， while the survival rates of HACAT cells in 100 μmol·L^-1 quercetin group was significantly decreased （P<0.05）. Compared with 5-FU group， the survival rates of the HACAT cells in low， medium and high doses of quercetin group were significantly increased （P<0.05）. Compared with 5-FU group， the ROS levels in low， medium， and high doses of quercetin groups were significantly decreased （P<0.05）. Annexin Ⅴ-FITC/PI double staining assay showed that compared with 5-FU group， the apoptotic rates in low， medium and high doses of quercetin groups were significantly decreased （P<0.05）. The Western blotting results showed that compared with 5-FU group， the expression levels of Bcl-2 protein in medium and high doses of quercetin groups were significantly increased （P<0.05）， the expression levels of Bax and Cleaved Caspase-3 proteins in low， medium and high doses of quercetin groups were significantly decreased （P<0.05）， the expression levels of COX-2 protein in medium and high doses of quercetin groups were significantly decreased （P<0.05）， and the expression levels of IL-1β and IL-6 proteins in medium dose of quercetin group were significantly decreasd （P<0.05）. Conclusion Quercetin has protective effect on 5-FU-induced damage in the HACAT cells， and its mechanism may be related to the reduction of the expression of ROS and inflammatory factor COX-2 which attenuate the apoptosis.

Objective To screen out the differentially expressed genes （DEGs） in multiple primary lung cancers （MPLCs） using bioinformatics methods， and to analyze their biological functions and their influence in the prognosis of lung adenocarcinoma. Methods Single-cell transcriptome sequencing data （GSE200972） was downloaded from the Gene Expression Omnibus （GEO） database. After preliminary data processing with R software， the Seurat R package was used for data processing， cell clustering， and annotation. The clusterProfiler R package was used for Gene Set Enrichment Analysis （GSEA）. The STRING database and Cytoscape software were employed to construct the protein-protein interaction （PPI） network and to screen out the key genes （Hub genes）. The gene expression levels in the lung adenocarcinoma database were analyzed using Gene Expression Profiling Interactive Analysis （GEPIA） database. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the gene expression in tumor tissue of A549 xenograft mice and lung tissue of normal mice. Kaplan-Meier Plotter was used for prognosis analysis. Results Seven cell types were identified from cell clustering analysis，which were epithelial cells， endothelial cells， fibroblasts， T cells and natural killer （NK） cells， B cells， myeloid cells， and mast cells. A total of 14 605 DEGs were screened out between tumor epithelial cells and normal epithelial cells. The GSEA results revealed four activated pathways in tumor samples ［myelocytomatosis oncogene （MYC） pathway， P53 pathway， oxidative phosphorylation pathway and glycolysis pathway］ and one inhibited pathway ［tumor necrosis factor-α （TNF-α） and nuclear factor kappa B （NF-κB） pathway］. The Hub genes identified from PPI network included CXC motif chemokine ligand 8 （CXCL8）， glyceraldehyde-3-phosphate dehydrogenase （GAPDH）， CXC motif chemokine receptor 4 （CXCR4）， kirsten rat sarcoma viral proto-oncogene （KRAS）， CXC motif chemokine ligand 1 （CXCL1）， C-C motif chemokine ligand 2 （CCL2）， mucin 1 （MUC1）， and secreted phosphoprotein 1 （SPP1）. The GEPIA database analysis and animal experiments showed that the expression levels of SPP1 mRNA in non-small cell lung cancer tissue were increased compared with normal lung tissue （P<0.01）. The Kaplan-Meier survival analysis indicated that the patients with high expression level of SPP1 had shorter overall survival （OS） than those with low expression level （P<0.01）. Conclusion There are activation of oncogene-related pathways and activation of tumor suppressor pathway antagonizing tumor progression in MPLCs. Moreover， elevated expression of SPP1 in non-small cell lung cancer may indicate a relatively poor prognosis.

Objective To analyze the relationship between the expression level of calumenin （CALU） and the prognosis of hepatocellular carcinoma （HCC） patients by bioinformatics tools and establish the prognostic prediction nomogram， and to clarify its possible mechanism. Methods The data of 374 HCC tissue samples were downloaded from The Cancer Genome Atlas （TCGA） database and the data of 160 normal tissue samples were down loaded from Genotype-Tissue Expression（GTEx） database. Paired sample t-test was used to analyze the difference in CALU expression between the HCC tissue samples and the paired adjacent normal tissue samples. Human Protein Atlas （HPA） database was used to verify the results. DESeq2 package was used to screen the differentially expressed genes （DEGs） between CALU low expression group and CALU high expression group in the HCC tissue samples. R package pROC was used to analyze the receiver operating characteristic（ROC） curve. Univariate and multivariate Cox regression analyses were used to confirm the prognosis value of CALU in the HCC patients with different clinicopathological characteristics， and ggplot2 package was used to construct the forest plot. R packages rms and survival were used to construct the nomogram and its calibration curve， and the diagnostic value of CALU in distinguishing HCC tissue from normal tissue was analyzed. The data from Kaplan-Meier Plotter database were used to further verify the relationship between CALU and the prognosis of HCC patients. The gene transcriptional expression data of 216 HCC samples obtained from GSE14520 dataset were used to verify the prediction accuracy of the nomogram. Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analyses were used to determine the function and enrichment pathways of the DEGs， and Gene Set Enrichment Analysis （GSEA） was used to obtain the significantly enriched gene sets of the DEGs. Single-cell sequencing data of 10 HCC tissue samples and 8 adjacent normal tissue samples obtained from GSE149614 dataset were used to verify the relationship between CALU and the prognosis of HCC patients and its mechanism. Results Compared with normal tissue， the expression level of CALU mRNA in HCC tissue was significantly increased （P<0.001）， and the expression level of CALU protein in HCC samples was also increased. A total of 928 DEGs were identified between CALU low expression group and CALU high expression group in the HCC samples， including 784 upregulated DEGs and 144 downregulated DEGs. The ROC analysis results indicated that CALU showed high diagnostic value in distinguishing cancer tissue from adjacent non-cancer tissue with an area under curve（AUC） of 0.839. Kaplan-Meier survival analysis showed that the survival rate of HCC patients in CALU high expression group was significantly lower than that in CALU group low expression（P<0.001）. Univariate and multivariate Cox regression analyses results demonstrated that high expression of CALU was an independent risk factor of the prognosis in HCC patients， and a prognosis prediction nomogram was constructed. The applicability of nomogram on the prognosis of HCC was verified by GSE14520 dataset. The GO enrichment analysis results showed that DEGs were mainly enriched in pathways related to the oxidative stress， ferroptosis and cuproptosis （P<0.05）. The KEGG enrichment analysis results showed that DEGs were mainly enriched in the pathways related to extracellular matrix（ECM） receptor interaction， linoleic acid metabolism and neuroactive ligand receptor interaction （P<0.05）. The GSEA results showed that high expression of CALU may promote the G₁-S phase transition of the cell cycle， ubiquitination protein polymerization and HCC progression， while low expression of CALU may activate oxidative stress， ferroptosis and cuproptosis in HCC cells. Single-cell sequencing analysis results showed that the expression level of CALU mRNA was significantly increased in HCC cells with advanced tumor stages. HCC_CALU_High cell subset was mainly related to ubiquitination， p53 and cell cycle （P<0.01）， and HCC_CALU_Low cell subset was mainly related to oxidative stress， ferroptosis， and histone binding （P<0.01）. Conclusion The high expression of CALU may be related to the poor prognosis of HCC patients. The constructed nomogram of HCC prognosis shows favourable effect in predicting the survival rate of the HCC patients. The up-regulation of CALU may promote HCC progression by regulating the G₁-S phase of the cell cycle and ubiquitination of protein， while down-regulation of CALU may inhibit HCC progression by inducing oxidative stress， ferroptosis and cuproptosis in cells.

Objective To investigate the pathways involved in bisphenol S （BPS） and bisphenol F （BPF） induced male reproductive injury by bioinformatics methods and experimental verification. Methods Bioinformatics analysis was conducted to screen the genes related to male reproductive system diseases associated with BPF and BPS from the Comparative Toxicogenomics Database （CTD）. Functional enrichment using Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis were performed to predict potential signaling pathways and key genes. Cell counting kit-8（CCK-8） method was used to assess the cell viability in various groups treated with different concentrations of BPS and BPF （1×10^-3， 1×10^-2， 1×10^-1， 1×10⁰，1×10¹， and 1×10² μmol·L^-1）. TM3 cells were divided into control group （0.1% DMSO）， different doses of BPS groups， and different doses of BPF groups. The cells were treated with 20， 40， and 80 μmol·L^-1 of BPS and BPF for 72 h， respectively. Real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting method were used to detect the expression levels of key genes mRNA and proteins in various groups. Results The bioinformatics analysis results revealed that 507 and 447 male systemic disease genes related to BPS and BPF were screened by CTD， respectively. The GO enrichment analysis results showed that the selected genes were primarily enriched in biological processes （BP） such as reproductive system development and reproductive structure development. The KEGG pathway analysis results indicated that these genes were significantly enriched in signaling pathways such as phosphatidylinositol 3-kinase/protein kinase B （PI3K/AKT）， hypoxia-inducible factor-1（HIF-1）， and cellular senescence（P<0.001）. The CCK-8 method results showed that compared with control group， the cell viabilities in 1×10² μmol·L^-1 BPF and BPS groups were significantly decreased （P<0.05）， while the viabilities of TM3 cells in other groups had no significant changes （P>0.05）. After BPS treatment， compared with control group， the expression levels of PI3K， AKT， hypoxia-inducible factor-1α （HIF-1α）， and CREB-binding protein （CBP） mRNA in low， medium， and high doses of BPS groups were decreased （P<0.05）， the expression levels of PI3K protein were decreased （P<0.05）， the expression levels of B-cell lymphoma-2-associated X protein （Bax） protein were increased （P<0.05）， and the expression levels of serine protease inhibitor clade B， member 10 （SERPINB10） mRNA were increased （P<0.01）； the expression levels of Bax and intraflagellar transport 80 homolog （IFT80） mRNA in the cells in medium and high doses of BPS groups were increased （P<0.05）； the expression levels of B-cell lymphoma-2 （Bcl-2） mRNA and protein in low and high doses of BPS groups were decreased （P<0.05）； the expression levels of additional sex combs like 2 （ASXL2） mRNA in low and medium doses of BPS groups were decreased （P<0.01）. After BPF treatment， compared with control group， the expression levels of Bcl-2， HIF-1α， and structural maintenance of chromosomes protein 1B （SMC1B） mRNA in low， medium， and high doses of BPF groups were decreased （P<0.05）， and the expression levels of IFT80 mRNA （P<0.01） and Bax protein （P<0.01） were increased； the expression levels of PI3K， AKT， and ring finger protein 130 （RNF130） mRNA in low and high doses of BPF groups were decreased （P<0.05）； the expression level of CBP mRNA in medium dose of BPF group was decreased （P<0.05）， while the expression level of RNF130 mRNA was increased （P<0.05）； the expression levels of PI3K and Bcl-2 proteins in high dose of BPF group were decreased （P<0.05）. Conclusion BPF and BPS may cause cell cytotoxicity and impair male reproductive health through PI3K/AKT and HIF-1 signaling pathways. RNF130 and SMC1B may be important targets for their induction of male reproductive toxicity.

Objective To investigate the mutations of TP53 and KRAS genes in the patients with six common types of digestive system tumors， including colorectal cancer（COAD）， cholangiocarcinoma（CHOL）， gallbladder cancer（GBC）， liver hepatocellular carcinoma（LIHC），stomach adenocarcinoma（STAD）， and pancreatic adenocarcinoma（PAAD）， and to analyze the relationships between TP53 and KRAS gene mutations and clinical pathological characteristics， tumor mutation burden（TMB）， and microsatellite instability （MSI） of the patients. Methods The pathological paraffin or biopsy samples of 112 patients from January 2022 to December 2023 diagnosed with six types of tumors based on imaging and pathology were collected. Hybrid capture-based gene sequencing technology was used to detect TP53 and KRAS gene mutations in the patients with different types of tumors； mutation landscapes of common digestive system tumor samples were constructed. The patients were divided into high and low TMB groups according to the TMB levels. The mutation statuses of TP53 and KRAS genes in the patients with different types of digestive system tumors were compared， and the TP53 and KRAS gene mutations in the patients with different clinicopathological characteristics were examined. Results A total of 276 mutations were detected in the 112 samples， with the highest mutation rate in TP53 gene （67%），followed by KARS gene （34%）. TP53 gene mutation was most prominent in COAD， followed by LIHC， while KRAS gene mutation was most significant in PAAD. TP53 gene mutation mainly occurred in exons 5-8， while the KRAS gene mutation primarily occurred in exon 2. There was no statistically significant difference in TP53 gene mutation rate among the six types of digestive system tumors （P>0.05）， while the KRAS gene mutation rate showed statistically significant difference （P<0.05）. The mutation rates of TP53 and KRAS gene co-mutation also showed statistically significant difference among the six types of tumors （P<0.05）. There were statistically significant differences in TP53 and KRAS gene mutation rates between the patients with high TMB and low TMB （P<0.05）， while there were no statistically significant differences in TP53 and KRAS mutation rates between the patients with different sex， age， tumor size， differentiation degree， TNM stage， lymph node and/or distant metastasis and MSI （P>0.05）. Conclusion The mutation rates of TP53 and KRAS genes are higher in common digestive system tumors， which are related to tumor types and TMB.

Objective To explore the causal association between insulin-like growth factor-1（IGF-1） and colorectal cancer（CRC） based on two sample Mendelian randomization（MR） analysis. Methods A bidirectional two sample MR analysis was conducted based on publicly aggregated data from the IEU OpenGWAS project. The inverse variance weighted（IVW） method was used as the main analysis model to assess the causal relationship between IGF-1 and CRC. Additional analyses were performed using weighted median （WM）， MR-Egger regression， weighted mode estimator （WME）， and simple mode （SM） methods. Sensitivity analysis was performed to assess the robustness of the results. Results A total of 386 single nucleotide polymorphisms（SNPs）were selected as instrumental variables （IVs） with IGF-1 as the exposure factor. The MR analysis results revealed a positive causal association between IGF-1 and the risk of CRC ［odds ratio （OR）=1.178， 95% confidence interval （CI）： 1.092-1.272）］（P<0.001）， and the association remained significant after adjusting for height ［OR（95%CI）=1.214（1.111， 1.327）］（P<0.001）. Cochran’s Q-test showed heterogeneity among the IVs（P<0.05）， while the horizontal pleiotropy of IV was not detected by the MR-Egger regression（P>0.05）. The leave-one-out analysis showed that the MR results were robust. Reverse MR analysis indicated no reverse causal relationship between IGF-1 and CRC ［OR（95%CI）：1.017（0.997， 1.037）］（P=0.103）. Conclusion There is a causal relationship between IGF-1 level and CRC， and elevated IGF-1 level could be a risk factor for CRC.

Objective To discuss the expression of tripartite motif-containing protein 24 （TRIM24） in the clear cell renal cell carcinoma （ccRCC） tissue， and to clarify its relationships with the clinicopathological features and prognosis of the ccRCC patients. Methods The cancer and paracancer normal tissues were collected from 90 ccRCC patients who had not undergone preoperative radiotherapy or chemotherapy. Tissue microarray and immunohistochemistry were used to detect the expression levels of TRIM24 protein in ccRCC tissue. The differences in TRIM24 protein expression between ccRCC and paracancer normal tissues were analyzed. The score of TRIM24 protein expression and the average value were calculated， and based on the average value， the patients were classified into TRIM24 protein low-expression and TRIM24 protein high-expression groups. The associations between the TRIM24 protein expression and different clinicopathological features of the patients were analyzed， and the relationship between the TRIM24 protein expression and the prognosis of the patients was analyzed. Results The immunohistochemistry results showed that the TRIM24 protein was expressed in both the nucleus and cytoplasm of the ccRCC tumor cells， and there were significant differences in the TRIM24 protein expression level in ccRCC tissue when compared with paracancer normal tissue （P<0.05）. The TRIM24 protein expression in the nucleus of ccRCC tissue was associated with the patient’s age， gender， and tumor size （P<0.05）. The Kaplan-Meier survival analysis results showed that the overall survivals of the patients with high TRIM24 protein expression in the cytoplasm of ccRCC tissue， older age， and higher pathological grade were shorter than those with low TRIM24 protein expression， younger age， and lower pathological grade （P<0.05）. The multivariate Cox regression analysis results showed that the prognosis of the patients with high TRIM24 protein expression in the cytoplasm and higher pathological grade were poorer compared with the patients with low TRIM24 protein expression and lower pathological grade （P<0.05）. Conclusion The ccRCC patients with high TRIM24 expression in the cytoplasm of ccRCC tumor tissue and higher pathological grade have the lower postoperative survival rates and poorer prognosis.

The patients with skeletal class Ⅱ malocclusion are often accompanied by structural and functional abnormalities of the upper airway， and obstructive sleep apnea hypopnea syndrome （OSAHS） may occur in severe cases. The morphologic and fluid dynamic changes of the upper airway in one patient with skeletal class Ⅱ malocclusion accompanied by OSAHS after maxillomandibular advancement （MMA） surgery were observed.The patient was a 27-year-old male with skeletal class Ⅱ malocclusion and moderate OSAHS. Combined orthodontic and orthognathic treatment was conducted， involving the anterior movement of the maxilla and mandible. After surgery， the patient’s facial and intraoral features showed a satisfactory occlusion relationship with normal overbite， overjet and canine-molar relationships. There was also a significant improvement in the upper airway flow field. Two years after surgery， the cross-sectional area of the nasopharyngeal airway increased by 10.76% with a 55.36% reduction in pressure. The oropharyngeal airway showed a 108.25% increase in cross-sectional area with a 98.14% pressure reduction. The hypopharyngeal airway exhibited a 97.51% increase in cross-sectional area with a 351.03% pressure reduction. The laryngopharyngeal airway demonstrated a 27.54% increase in cross-sectional area with a 95.62% pressure reduction. The apnea-hypopnea index （AHI） decreased by 55.45%， achieving the treatment goal. Morphological measurements combined with fluid dynamic analysis can comprehensively evaluate the condition of the upper airway. The approximate value of the critical closing pressure（Pcrit） obtained from computational fluid dynamics （CFD） analysis may serve as a simple quantitative indicator for assessing the upper airway condition.

Cornelia de Lange syndrome is a rare congenital malformation disease， and its typical features include growth restriction， mental retardation， craniofacial abnormality and hirsutism. This study reported 2 cases of CdLS patients， summarized their clinical manifestations and gene mutation characteristics， and the relevant literatures were reviewed. Patient 1， a 5-year-old girl， was admitted to the hospital due to growth retardation. Physical examination revealed hirsutism， monobrow， small and sparse teeth， hemangiomas （approximately 2 cm×2 cm） on the chest and back， delayed language development， and intellectual disability. The height was 98 cm ［≤-2 standard deviation（SD）］， the weight was 15 kg （-2SD--1SD）， the head circumference was 46 cm （-3SD--2SD）. Brain magnetic resonance imaging（MRI） plain scan showed slightly enlarged left lateral ventricle and bilateral lateral ventricle triangles， slightly thickened bilateral maxillary sinus and ethmoid sinus mucosa. Echocardiography revealed mild regurgitation of the mitral and tricuspid valves. Patient 2， a 1-month-old girl， was admitted to the hospital due to postpartum shortness of breath. The physical examination highlighted hirsutism， short nose， soft cleft palate， dysphagia， positive three-concave sign， audible throat sound， small hands， palm penetration in the left hand， short fifth finger of the right hand， limited right hip abduction， foot varus， and a white spot at the bottom of the right eye. Ultrasonography at 1 month showed mild regurgitation of the tricuspid valve and an open foramen ovale. Brain MRI at 2 d showed a few patchy low-signal shadows in the longitudinal fissure cistern and tentorium， a small amount of subarachnoid hemorrhage， and a small amount of fluid in the bilateral maxillary sinus， ethmoid sinus， and middle ear mastoid. No obvious structural or numerical abnormalities were observed in the chromosome karyotypes. Whole-exome sequencing detected a heterozygous variation of c.6653_6655del in the NIPBL gene in patient 1 and a heterozygous variation of c.337C>T in the NIPBL gene in patient 2. These variations were not found in their parents. The study revealed that NIPBL gene variation could be the genetic cause of the two patients with CdLS. The identification of the variation c.337C>T may expand the variation spectrum of the NIPBL gene， providing valuable insights into the pathogenic genetic basis of CdLS.

Skeletal class Ⅱ malocclusion is characterized by maxillary protrusion， mandibular retrognathia， or a combination of both， and often accompanied by vertical dimensional discrepancies； treatment is complex， and combined orthodontic-orthognathic surgery is needed for the adult patients. Clear aligner therapy has gradually been applied in complex orthodontic cases. However， limited cases have been reported domestically and internationally regarding the application of clear aligner therapy combined with orthognathic surgery. This article presented a case of a patient with skeletal class Ⅱ high-angle malocclusion treated with the combined therapy and analyzed the clinical efficacy of the treatment appraoch to provide reference for the clinical practice. Extraction of impacted wisdom teeth 18， 28， 38， and 48， as well as orthodontic teeth 15， 25， 34， and 44， was performed in stages before orthodontic treatment. Clear aligner therapy was used for preoperative orthodontics. In sagittal plane， a super-complete class Ⅱ canine and molar relationship and a 13-14 mm overjet of the anterior teeth were established. The maxillary and mandibular arch morphology was matched horizontally. The orthognathic surgery included maxillary LeFort Ⅰ osteotomy， bilateral sagittal split ramus osteotomy （BSSRO） and chinplasty. Fine occlusal adjustment was conducted after operation. After treatment， the skeletal relationship between upper and lower jaw was corrected to normal； subspinale-nasion-supramental angle （ANB） was improved from 12.3° to 4.7°； the patient established the class Ⅰ canine and molar relationship， with normal overjet and overbite； root parallelism was good and there was no obvious root resorption； the facial soft tissue profile was significantly improved， and nasion-subnasale-pogonion angle（N-Sn-Pg） was improved from 143.9° to 162.8°. The curative effect was stable 1 year after operation. Clear aligner therapy can efficiently complete combined orthodontic and orthodontic surgery in the complex cases. Compared with the fixed appliance， it is more beneficial to the patients’ need for beauty and the maintenance of periodontal health.

Objective To establish a method of dual nueleic acid test strips for rapid detection of Bacillus cereuscytK and nhe toxin genes based on polymerase chain reaction（PCR）and colloidal gold technique，and to evaluate its specificity， sensitivity， repeatability and stability. Methods Bacillus cereus DNA was extracted by boiling method. Specific primers were designed with Bacillus cereus cytK and nhe as the target genes. Clonal transformation was used to identify the PCR products. The optimal labeling amounts of colloidal gold-labeled streptavidin， quality control line （C line）， cytK detection line （T₁） and nhe detection line （T₂） were determined. The nucleic acid test strips were assembled and its specificity，sensitivity，reproducibility and stability were evaluated. Results The DNA concentration of Bacillus cereus was 248 mg·L^-1，and the purities were 1.8-2.0. After cloning and plasmid sequencing，the similarities between the PCR products and the sequences of cytK and nhe registered in the GenBank database were 100%. Under the condition of pH 7.0， the optimal amount of streptavidin labeling per 200 μL of colloidal gold solution was 6.0 μL； the optimal marking amount was 2.00 g·L^-1 for the quality control line （C line）， 0.550 g·L^-1 for cytK gene detection line （T₁） and 0.2 g·L^-1 for nhe gene detection line （T₂）. In the specificity test， positive result on the test strips was seen only for Bacillus cereus，and no cross-reactivity was observed for Staphylococcus aureus， Escherichia coli， Pseudomonas aeruginosa and Bacillus subtilis， which were consistent with the electrophoresis results. Sensitivity assay showed that even when DNA concentration was reduced to 10^-2 mg·L^-1， three bands （C line， T₁ line and T₂ line） could be observed， and the detection limit of the test strip was one-tenth of agarose gel electrophoresis （10^-1 mg·L^-1）. The nucleic acid test strips were verified by different operators in different laboratories， and the results were consistent. The stability of the test strips was verified at the 6th，9th and 12th months， and the results showed good stability. Conclusion The dual nucleic acid test strip method established in this study can simutaneously detect the cytK and nhe toxin genes of Bacillus cereus with high sensitivity and specificity，achieving short-term visual detection.

The Hedgehog （HH） signaling pathway is involved in various developmental processes of vertebrates and invertebrates，including embryogenesis，stem cell renewal，tissue regeneration，the occurrence and development of tumor and chemotherapy resistance. Overactivation and abnormal expression of key components in the HH signaling pathway are associated with the development of various malignant tumors. Phosphorylation plays an important role in regulating the activity and function of proteins in normal cells， and the disorder of phosphorylation-dephosphorylation cascade is closely related to tumorigenesis. The abnormal activation of this signaling pathway is also related to protein phosphorylation，while its complex underlying mechanism needs to be further explored. This article reviewed the research progress of the HH signaling pathway and protein phosphorylation modification in malignant tumors， focusing on the regulatory effect of phosphorylation modification on the activity of HH signaling pathway and its effect on the occurrence and development of malignant tumors，in order to provide a theoretical basis for the in-depth development and clinical application of targeted drugs for phosphatase/dephosphatase.

Protein phosphatase 2A（PP2A） is one of the major serine-threonine protein phosphatases in mammalian cells which plays an important role in regulating biological activities such as cellular mitosis and protein dephosphorylation. PP2A acts as a tumor suppressor and has been demonstrated to be genetically altered or functionally inactivated in a variety of solid cancers and leukemias， and its activity is inhibited， leading to subsequent proliferation of tumor cells. Clinical studies have shown that endogenous inhibitors such as SET， cancerous inhibitor of PP2A（CIP2A） and protein phosphatase methylesterase-1（PME-1） may reduce PP2A activity， which could be important indicators of tumor progression or recurrence. On the other hand， PP2A-activating drugs （e.g.FTY720） can restore the tumor-suppressing activity of PP2A by altering the structure of the inhibitor SET， thus effectively inhibiting tumor development. Therefore， PP2A and its inhibitors may serve as potential therapeutic targets in clinic. This article provides a comprehensive review on the mechanism of action of PP2A and its inhibitors in the pathogenesis of malignant tumor as well as their applications in oncotherapy， aiming to provide new directions for the treatment of malignant tumors.

Functional gastrointestinal disease （FGID） and inflammatory bowel disease （IBD） are two common gastrointestinal diseases in clinical practice. FGID refers to non-structural changes in gastrointestinal function， with irritable bowel syndrome （IBS） and functional dyspepsia （FD） being the common types. On the other hand， IBD is a group of chronic inflammatory bowel diseases with well-defined pathological features， mainly including Crohn’s disease （CD） and ulcerative colitis （UC）. Over the past decades， the incidence rates of both FGID and IBD have increased with years， accompanied by high recurrence rates， and the clinical outcomes remain unsatisfactory， which seriously affect the physical and mental health of patients. Further research has revealed that neuropsychiatric abnormalities， including mental and psychological disorders， play important roles in the occurrence and development of FGID and IBD. The comorbidity of gastrointestinal and psychiatric diseases shares a common pathophysiological basis， that is， the abnormalities in the bidirectional communication between the gut and the central nervous system， and the theory of brain-gut axis has emerged as a research hotspot in this field. Better therapeutic outcomes can be achieved by screening and identifying key brain regions closely related to FGID and IBD through quantifiable assessments， and implementing combined pharmacotherapy on both the digestive system and the nervous system based on the theory of brain-gut axis. Current neuroimaging studies have provided the preliminary results for understanding the changes in the nervous system in FGID and IBD patients， but there is still a lack of systematic evaluation of the application of neuroimaging studies based on the brain-gut axis in gastrointestinal diseases. This paper reviewed the changes in brain structure and function associated with FGID and IBD， and the neuroimaging-based analysis of the brain-gut axis theory in the occurrence and development of these diseases， in order to provide theoretical basis for future personalized precision medicine of FGID and IBD based on the brain-gut axis.

Sutureless scleral fixation of posterior chamber intraocular lens （SSF-PCIOL） is a technique mainly employed for the implantation and fixation of intraocular lens （IOL） when there is insufficient support from the crystalline lens capsule. In terms of clinical effects， SSF-PCIOL offers the advantages of improved visual quality and enhanced IOL stability in the patients. The surgical technique for SSF-PCIOL has evolved from the transvitreal scleral fixation approach， to the fibrin glue assisted approach， and then to the transconjunctival approach. The types of IOL available for clinical use have also shifted from traditional three-piece designs to specialized IOL designed for scleral fixation. SSF-PCIOL can be carried out in combination with other ophthalmic procedures and advanced auxiliary equipment to increase the precision of surgical procedures and the objectivity of outcome assessments while minimizing surgical trauma in the patients and ensuring surgical outcomes. This review discusses the evolution of SSF-PCIOL techniques， the characteristics of available IOL types， the synergistic application of combined procedures and auxiliary equipment， and the comprehensive evaluation of clinical outcomes， with the aim of providing clinical evidence for further refinement of the technique and offering references for surgical options for the patients with insufficient crystalline lens capsule support.