Objective:To investigate the effect of oleanolic acid(OA)on inflammatory injury of pancreatic acinar cells in-duced by lipopolysaccharide(LPS)by regulating stromal cell-derived factor-1(SDF-1)/CXC chemokine receptor 4(CXCR4)signal axis.Methods:Rat pancreatic acinar cells AR42J were treated with 5,10,20 and 40 μmol/L OA,and the cell viability was detected using CCK-8 method to determine the optimal dosage for subsequent experimental OA treatment;AR42J cells were randomly divided into control group(NC),LPS group(10 mg/L LPS),OA low-dose group(OA-L group,10 mg/L LPS+5 μmol/L OA),OA medium dose group(OA-M group,10 mg/L LPS+10 μmol/L OA),OA high-dose group(OA-H group,10 mg/L LPS+20 μmol/L OA),Rr-SDF-1 activator(SDF-1)group(10 mg/L LPS+20 ng/ml Rr-SDF-1)and OA-H+Rr-SDF-1 group(10 mg/L LPS+20 μmol/L OA+20 ng/ml Rr-SDF-1).CCK-8 method and EdU staining were applied to detect proliferation of AR42J cells;flow cytometry was applied to detect apoptosis of AR42J cells;activity of superoxide dismutase(SOD)and content of malondialdehyde(MDA)in cell supernatant were detected by spectrophotometry;ELISA was applied to detect levels of IL-6 and TNF-α in cell supernatant;Western blot was applied to detect expressions of CyclinD1,Bcl-2 associated X protein(Bax),SDF-1 and CXCR4 proteins in cells.Results:5,10 and 20 μmol/L OA were selected as the low,medium and high doses for subsequent treatment of AR42J cells;compared with NC group,A450 value,EdU positive cell rate,SOD activity,and CyclinD1 protein expression in LPS group decreased,while apoptosis rate,MDA content,IL-6,TNF-α levels,Bax,SDF-1 and CXCR4 protein expressions increased(P<0.05);compared with LPS group,A450 value,EdU positive cell rate,SOD activity,and CyclinD1 protein expression in OA-L group,OA-M group and OA-H group increased,while apoptosis rate,MDA content,IL-6,TNF-α levels,Bax,SDF-1 and CXCR4 protein expressions reduced,the change trends of corre-sponding indicators in Rr-SDF-1 group was opposite to the above(P<0.05);compared with OA-H group,A450 value,EdU positive cell rate,SOD activity,and CyclinD1 protein expression in OA-H+Rr-SDF-1 group decreased,while apoptosis rate,MDA content,IL-6,TNF-α levels,Bax,SDF-1 and CXCR4 protein expressions increased(P<0.05).Conclusion:OA may alleviate LPS induced inflam-matory damage to pancreatic acinar cells in rats by inhibiting the SDF-1/CXCR4 pathway.

Objective:To investigate the effect of curcumin on Th17/Treg cell immune balance in acne mice by regulating the JAK2/STAT3 signaling pathway.Methods:An acne mouse model was established by injecting propionibacterium suspension.The suc-cessfully established mice were randomly separated into the acne group,low(curcumin-L,50 mg/kg curcumin),medium(curcumin-M,100 mg/kg curcumin),high dose(curcumin-H,150 mg/kg curcumin)curcumin groups,and curcumin-H+JAK2/STAT3 pathway activator-olivelin group(150 mg/kg curcumin+1.0 mg/kg colivelin),with healthy mice as the control group,after intervention,the mice were scored for skin lesions;blood was taken from the orbit,ELISA and flow cytometry were applied to detect serum levels of IL-6 and IL-10,and the ratios of Th17 and Treg cells;HE was applied to detect pathological changes in auricle tissue;Western blot was applied to detect the expressions of p-JAK2,p-STAT3,JAK2,STAT3,Treg transcription factor forked transcription factor 3(FOXP3)and Th17 transcription factor retinoic acid related solitary receptor(RORγt)proteins in auricle tissue.Results:Compared with the control group,the pathological damage in the acne group was severe,the skin lesion and pathological scores,IL-6 level,Th17 cells ratio,p-JAK2/JAK2,p-STAT3/STAT3,and RORγt expression were obviously increased,the IL-10 level,Treg cells ra-tio,and FOXP3 expression were obviously reduced(P<0.05);compared with the acne group,the skin lesion and pathological scores,IL-6 level,Th17 cells ratio,p-JAK2/JAK2,p-STAT3/STAT3 and RORγt expression in curcumin-L,curcumin-M,and curcumin-H groups were obviously reduced,the IL-10 level,Treg cells ratio,and FOXP3 expression were obviously increased(P<0.05);com-pared with the curcumin-H group,the skin lesion and pathological scores,IL-6 level,Th17 cells ratio,p-JAK2/JAK2,p-STAT3/STAT3,and RORγt expression in curcumin-H+colivelin group were obviously increased,the IL-10 level,Treg cells ratio,and FOXP3 expression were obviously reduced(P<0.05).Conclusion:Curcumin can improve the immune balance of Th17/Treg cells in acne mice,which may be related to the inhibition of the JAK2/STAT3 signaling pathway.

Objective:To investigate the impact of minocycline on myocardial injury in diabetes cardiomyopathy(DCM)rats by regulating 5'-AMP activated protein kinase(AMPK)/sirtuin 1(SIRT1)/peroxisome proliferator activator receptor gamma coactiva-tor 1α(PGC-1α)signal pathway.Methods:SD rats were fed with high-fat diet for 4 weeks,and then a single intraperitoneal injection of 35 mg/kg streptozotocin was used to induce the DCM model.They were randomly grouped into model group,low-dose minocycline(20 mg/kg)group,high-dose minocycline(40 mg/kg)group,high-dose minocycline+Dorsomorphin(AMPK inhibitor,0.2 mg/kg)group,with 12 rats in each group,another 12 normal rats were fed with normal feed for 4 weeks,and then were given a single intraper-itoneal injection of the same dose of citric acid buffer,which was set as a sham operation group,after the intervention with minocy-cline and Dorsomorphin,and the fasting blood glucose(FBG),total cholesterol(TC)and triglyceride(TG)were measured;left ven-tricular ejection fraction(LVEF)and left ventricular short axis shortening(FS)were measured by ultrasound;HE staining and Mas-son staining were applied to detect the pathological morphology of myocardial tissue of rats in each group;the levels of serum and myo-cardial tissue inflammation IL-6,IL-18,and myocardial tissue oxidative stress indicators-superoxide dismutase(SOD)and malondial-dehyde(MDA)were measured with the kit;Western blot was applied to detect the expression of AMPK/SIRT1/PGC-1α pathway pro-tein in myocardial tissue of rats in each group.Results:Compared with the sham operation group,the myocardial tissue in the model group was seriously damaged,the levels of FBG,TC and TG,the cross-sectional area of myocardial cells and the proportion of myocar-dial fiber area,the levels of IL-6 and IL-18 in serum and myocardial tissue,and the level of MDA in myocardial tissue were obviously increased(P<0.05),the levels of LVEF,FS and SOD in myocardial tissue,and the protein expression of p-AMPK/AMPK,SIRT1 and PGC-1α were obviously decreased(P<0.05);compared with the model group,the myocardial tissue damage of rats in the low-dose and high-dose minocycline groups were reduced,the levels of FBG,TC and TG,the cross-sectional area of myocardial cells and the proportion of myocardial fiber area,the levels of IL-6 and IL-18 in serum and myocardial tissue,and the level of MDA in myocardial tissue were decreased(P<0.05),the LVEF,the levels of FS and SOD in myocardial tissue,and the protein expression of p-AMPK/AMPK,SIRT1 and PGC-1α were increased(P<0.05);compared with the low-dose minocycline group,the myocardial tissue damage in the high-dose minocycline group was further reduced,the levels of FBG,TC and TG,the cross-sectional area of myocardial cells and the proportion of myocardial fiber area,the levels of IL-6 and IL-18 in serum and myocardial tissue,and the level of MDA in myo-cardial tissue were further decreased(P<0.05),the levels of LVEF,FS and SOD in myocardial tissue,and the protein expression of p-AMPK/AMPK,SIRT1 and PGC-1α were further increased(P<0.05);compared with the high-dose minocycline group,the myocar-dial tissue damage of rats in the high-dose minocycline+Dorsomorphin group increased,the levels of FBG,TC and TG,the cross-sec-tional area of myocardial cells and the proportion of myocardial fiber area,the levels of IL-6 and IL-18 in serum and myocardial tis-sue,and the level of MDA in myocardial tissue were increased(P<0.05),the LVEF,the levels of FS and SOD in myocardial tissue,and the protein expression of p-AMPK/AMPK,SIRT1 and PGC-1α were decreased(P<0.05).Conclusion:Minocycline can reduce oxidative stress and inflammation in DCM rats by activating AMPK/SIRT1/PGC-1α signal,and improve glycolipid metabolism,thereby reducing myocardial injury and repairing cardiac function in rats.

Objective:To investigate the impact and mechanism of polydatin(PD)on lipopolysaccharide(LPS)induced in-flammatory damage in pancreatic acinar cells.Methods:Rat pancreatic exocrine cells AR42J were cultured in vitro,LPS treated cells were used to construct a cell inflammatory injury model,and co-cultured with 0,12.5,25,50,100 and 200 μg/L PD,CCK-8 method was applied to detect cell proliferation activity;AR42J cells were grouped into blank group(CT group),inflammatory injury model group(M group),PD group(100 μg/L),and PD+WZ811 group[stromal cell derived factor 1(SDF-1)/CXC chemokine receptor 4(CXCR4)pathway inhibitor](100 μg/L PD+1 μmol/L WZ811),dinitrophenylhydrazine method was applied to detect the leakage rate of lactate dehydrogenase(LDH)in each group of cells,flow cytometry was applied to detect cell apoptosis rate,ELISA kit was ap-plied to determine the levels of IL-1β and TNF-α in cell supernatant,thiobarbituric acid method were applied to determine the content of malondialdehyde(MDA),Xanthine oxidation method was applied to measure superoxide dismutase(SOD)activity,immunofluo-rescence staining was applied to detect the expressions of SDF-1 and CXCR4 proteins.Results:Compared with 0 μg/L group,100 μg/L and 200 μg/L PD obviously increased cell proliferation activity;compared with the CT group,the leakage rate of LDH,apoptosis rate,and the contents of IL-1β,TNF-α and MDA of cells in the M group increased,the SOD activity and expressions of SDF-1 and CXCR4 proteins decreased(P<0.05);compared with the M group,the leakage rate of LDH,apoptosis rate,and the contents of IL-1β,TNF-α and MDA of cells in the PD group decreased,the SOD activity and expressions of SDF-1 and CXCR4 proteins increased(P<0.05);compared with the PD group,the leakage rate of LDH,apoptosis rate,and the contents of IL-1β,TNF-α,and MDA of cells in the PD+WZ811 group increased,the SOD activity and expression of SDF-1 and CXCR4 proteins decreased(P<0.05).Conclusion:The protective effect of PD on LPS-induced inflammatory damage in AR42J cells may be related to the activation of the SDF-1/CXCR4 signaling pathway.

Objective:To construct a prognostic model related to cellular senescence in gastric adenocarcinoma by bioinforma-tics tools and analyze the immune microenvironment.Methods:Gene expression data and corresponding clinical information of gastric adenocarcinoma patients were downloaded from TCGA database.Cellular senescence-related genes(CSRGs)were downloaded from CellAge database.The limma package was used to screen for differentially expressed CSRGs in gastric adenocarcinoma tissues and the survival package was used to screen for CSRGs affecting the prognosis of gastric adenocarcinoma patients in R software.The LASSO regression analysis was used to construct a risk model for gastric adenocarcinoma and calculate the risk score for each patient.The Kaplan-Meier survival analysis was used to compare the overall survival(OS)of patients in high and low risk groups and the validity of prognostic models to predict 5-year survival of patients was analyzed by receiver operator characteristic curve(ROC).Univariate and multivariate Cox regression analyses were used to screen for independent prognostic factors in patients with gastric adenocarcinoma.Gene set enrichment analysis(GSEA)was used to predict potential signaling pathways in patients of high and low risk groups.Based on ESTIMATE and single sample gene set enrichment analysis(ssGSEA)algorithms to analyze the differences in tumor immune micro-environment between high and low risk groups of patients.Results:Sixteen prognostically relevant differentially expressed CSRGs were screened.The prognosis model of the genes containing AGT,EZH2,NOX4 and ZFP36 were constructed by LASSO regression analy-sis,and the patients were divided into two groups of high and low risk according to the median risk score.Survival analysis showed that patients in the high-risk group had significantly lower OS than those in the low-risk group.ROC curve analysis proved the accuracy of the prognostic model to predict the 5-year survival of patients(AUC=0.703).Then multifactorial Cox regression analysis suggested that age and risk score were independent prognostic factors for patients with gastric adenocarcinoma.GSEA enrichment analysis demonstrated that the high-risk group was associated with signaling pathways such as epithelial mesenchymal transition,hypoxia,and angiogenesis,while the low-risk group was associated with signaling pathways such as G2M checkpoint,DNA repair,and peroxisome.Further analy-sis showed that patients in the high-risk group had a higher abundance of immune cell infiltration and higher levels of immune check-point-related gene expression.Conclusion:In this study,a new prognostic model related to cellular senescence in gastric adenocarci-noma was constructed,in which patients in the high-risk group had higher immunoreactivity.

Objective:To explore the therapeutic effect and mechanism of Baicalin(BG)on chronic migraine(CM)rats.Methods:CM rat model was established by intermittent subcutaneous injection of nitroglycerin.Rats were randomly grouped into con-trol group,chronic migraine group(CM group),low-dose and high-dose Baicalin groups(BG-L group,BG-H group),Baicalin+MIP-1α recombinant protein group(BG+MIP-1α group)and CCR1 inhibitor ZK811752 group(CCR1-ZK group),with 10 rats in each group.General behavior of rats were observed and scored;von Frey fiber optic pain gauge was applied to measure the mechanical pain threshold of rats;Nissl staining was applied to observe the neuronal structure of ventrolateral periaqueductal gray(vlPAG);ELISA was applied to detect levels of TNF-α and IL-1β;immunohistochemical method was applied to detect expressions of pain related pro-teins c-Fos and CGRP;Western blot was applied to detect expressions of MIP-1α and CCR1 proteins.Results:Compared with Control group,the body mass,mechanical pain threshold,and the number of Nissl bodies in vlPAG area of rats in CM group were obviously reduced,the general behavioral score,levels of TNF-α,IL-1β,and expressions of c-Fos,CGRP,MIP-1α and CCR1 were obviously increased(P<0.05);compared with CM group,the body mass,mechanical pain threshold,the number of Nissl bodies in vlPAG area of rats in BG-L group,BG-H group and CCR1-ZK group were obviously increased,the general behavioral score,levels of TNF-α,IL-1β,and expressions of c-Fos,CGRP,MIP-1α and CCR1 were obviously reduced(P<0.05);compared with BG-H group,the body mass,mechanical pain threshold,and the number of Nissl bodies in vlPAG area of rats in BG+MIP-1α group were obviously reduced,general behavioral score,levels of TNF-α,IL-1β,and expressions of c-Fos,CGRP,MIP-1α and CCR1 were obviously increased(P<0.05).Conclusion:Baicalin has certain therapeutic effect on chronic migraine rats,and its mechanism may be related to inhibiting the activation of MIP-1α/CCR1 signaling pathway.

Objective:To prepare rabbit polyclonal antibody against human FAM21 and analyze antibody specificity.Methods:Using the plasmid encoding human FAM21 full-length gene as a template,the nucleotide sequence of its 2 431～3 006 base was amplified by PCR and connected to the pGEX-6p-1 prokaryotic expression vector to construct pGEX-6P-1-FAM21 recombinant plasmid expressing the 811～1 002 amino acid fragment of FAM21.The recombinant plasmid was transformed into BL21(DE3)compe-tent Escherichia coli and was expressed inductively,and the protein was purified using GST fusion protein purification magnetic beads.The purified GST fusion protein was used as an antigen to immunize New Zealand rabbits,and the collected antiserum was purified by an agarose column containing GST protein.The specificity of antibody was detected by Western blot and immunofluorescence assay in stable FAM21 knockdown HeLa cells.Results:The pGEX-6p-1-FAM21 prokaryotic expression plasmid was successfully constructed and induced to express in BL21(DE3)competent Escherichia coli.The purified GST fusion protein had a molecular weight of approxi-mately 50 kD,and the purified antibody titer from immunized New Zealand rabbits was greater than 1∶128 000,with high specificity.Conclusion:The pGEX-6p-1-FAM21 prokaryotic expression plasmid is successfully constructed,and the rabbit polyclonal antibody against human FAM21 is prepared for Western blot and immunofluorescence assay.

T cell response plays an important role in anti-viral infection and anti-tumor immunity,and antigen-specific T cell detection is essential for study of T cell response.This article reviews progress of antigen-specific T cell detection technology,including enzyme-linked immunospot(ELISPOT)assay,intracellular cytokine staining(ICS)assay and activation-induced labeling(AIM)assay,which based on detection of cytokine secretion or activation phenotypes of specific T cells after stimulation and reactivation with antigen in vitro.Another class of methods include Tetramer technology based on known epitopes-human leukocyte antigen(HLA)restriction and recently developed single-cell transcriptomes and T-cell antigen receptor(TCR)sequencing technology.Application of the above methods has advanced our understanding of antigen specific T cell response:Strength and duration of the response,subpop-ulation information,epitopes and their associated HLA-restriction,TCR cloning information and transcriptome characteristic.

After ischemic stroke,intracranial cells experience stress due to ischemic and hypoxic injury,leading to a series of aseptic immune response processes.The oxidative stress process in microglias triggers the activation of the NLRP3 inflammasome,which promotes the release of inflammatory factors such as IL-1β and IL-18,contributing to the inflammatory reaction caused by isch-emic stroke.In addition,NLRP3 inflammasome is involved in the polarization,pyroptosis and autophagy of microglias,regulating the prognosis of ischemic stroke.This review summarizes the specific mechanisms of NLRP3 inflammasome in regulating microglial status and its involvement in ischemia-reperfusion injury.It also discusses the associated treatment strategies,identifies the current research focus and blanks,and provides some guidance and ideas for future research.

Tumor microenvironment(TME)is the living environment of tumor cells.The immune cells contained in TME are reprogrammed into tumor-promoting states due to various factors.Inhibiting this reprogramming effect of TME is an innovative strategy for treating tumors.As an essential factor in spindle assembly,TPX2 is considered to be a gene that promotes cancer cell proliferation and plays a role in cell response to replication stress.Highly expressed TPX2 in tumor cells promotes tumor growth through a variety of pathways,and these pathways can promote the tumor-promoting activity of immune cells in TME.Therefore,TPX2 may be a regulatory gene of TME.This paper discusses the possible mechanism of TPX2 involved in the regulation of TME by discussing the pathway medi-ated by TPX2 and the regulation of immune cells infiltrated in TME,and provides ideas for subsequent research directions.