Objective To evaluate the impact and clinical significance of NOD-like receptor pyrin domain-containing protein 3(NLRP3)in the activation of lipopolysaccharide(LPS)-induced BV2 microglia cells.Methods shRNA plasmids were devised for BV2 microglia transfection,and the most effective transfection segment was identified via RT-PCR and Western blot assays.NLRP3 expression in the cell line was detected by Western blotting,while light microscopy was used to observe morpho-logical alterations in BV2 cells transfected with NLRP3-shRNA.Furthermore,enzyme-linked immunosorbent assay(ELISA)was used to quantify levels of inflammatory cytokines IL-18,IL-1β,TNF-α and NO in cell supernatants.Immunofluorescence staining was used to observe the expression and localization of microglial activation markers iNOS,Arg-1,Iba1,and NLRP3.Results NLRP3 was highly expressed in LPS-induced BV2 cells.The Western blot result showed that the mRNA expression level was the lowest and transfection was the least effective in NLRP3-mus-727 group.Microscopic examination revealed a round or short spindle-shaped morphology in BV2 cells transfected with shNLRP3,which was akin to resting state cells in the blank control group.ELISA showed that pro-inflammatory mediators IL-18,IL-1β,TNF-α and NO levels were decreased in BV2 cells trans-fected with shNLRP3(all P＜0.05).Immunofluorescence staining indicated a relative decrease in iNOS and Iba1 expression,with an upregulation of Arg-1 in BV2 cells transfected with shNLRP3.Conclusion NLRP3 is highly expressed in LPS-induced BV2 cells.Inhibition of NLRP3 expression can suppress the inflammatory response of BV2 cells induced by LPS,promoting their polarization towards the M2 phenotype.

Objective To investigate the effects of peroxisome proliferator activated receptor-gamma(PPARγ)gene silen-cing in human bone marrow stromal cells(HS-5)on hematopoietic function in bone marrow-suppressed mice,and to explore the potential mechanisms involved.Methods A bone marrow-suppressed mouse model was established by whole-body X-ray irradi-ation.Two hours after modeling,the mice were randomly divided into three groups:experimental group(intravenous injection of PPARγ RNAi-interfered HS-5 cells through the tail vein),control group(intravenous injection of PPARγ RNAi-uninterfered HS-5 cells through the tail vein),and blank group(intravenous injection of an equal amount of saline through the tail vein),with 5 mice in each group.Peripheral blood routine tests were performed before,24 hours after,1 week after,and 2 weeks after radio-therapy.In vitro osteogenic and adipogenic induction was performed in cells,and the cells were divided into experimental group(PPARγ RNAi-interfered HS-5 cells),control group(PPARγ-uninterfered HS-5 cells),and blank group(HS-5 cells without os-teogenic/adipogenic induction).Osteogenic/adipogenic staining was observed.The effects of PPARγ gene-silenced HS-5 cells on mouse bone marrow hematopoietic stem cells(HSCs)were detected by CCK-8 proliferation assay.The groups included experi-mental group(PPARγ RNAi-interfered HS-5 cells were co-cultured with mouse HSCs after 3 days of osteogenic induction dif-ferentiation),positive control group(HS-5 cells treated with 50 μmol/L PPARγ inhibitor were co-cultured with mouse HSCs af-ter 3 days of osteogenic induction differentiation),negative control group(PPARγ RNAi-uninterfered HS-5 cells were co-cul-tured with mouse HSCs after 3 days of osteogenic induction differentiation),and blank group(Mouse HSCs were cultured alone without co-culturing with HS-5 cells).Results After radiotherapy,the hematological parameters of mice in each group showed a decreasing trend initially,and then increased.One week after radiotherapy,there were significant differences in platelet and white blood cell levels among the three groups(experimental group＞control group＞blank group,all P＜0.05).Two weeks after radiotherapy,there were significant differences in the percentage of adipocyte vacuole area among the three groups(experi-mental group＜control group＜blank group,all P＜0.05).Pearson correlation analysis showed a negative correlation between hematological parameters and PPARγ expression levels(all P＜0.05),as well as a negative correlation between hematological parameters and the percentage of adipocyte vacuole area(all P＜0.05).After in vitro osteogenic/adipogenic induction differenti-ation,compared to the control group,the experimental group showed a significantly lower proportion of orange-red cells and a significantly higher proportion of red calcium nodules.After 3 days of osteogenic induction differentiation,the experimental group,positive control group,and negative control group of human bone marrow stromal cells were co-cultured with mouse HSCs,while HSCs were solely cultured in the blank group.The results showed that after 24 h,48 h and 72 h of co-culture,the A values of mouse HSC cells in the experimental group and positive control group were higher than those in the negative control group and blank group(all P＜0.05).Conclusion Silencing of the PPARγ gene in HS-5 cells implanted into bone marrow-sup-pressed mice contributes to enhanced hematopoietic function in mice.After interference and silencing of the PPARγ gene,the os-teogenic differentiation ability of HS-5 cells is enhanced,while the adipogenic differentiation ability is weakened.Furthermore,osteogenic-induced HS-5 cells can further enhance the proliferation capacity of mouse HSCs.

Objective To investigate the effect of circFOXK2 on the proliferation,migration and invasion of lung cancer cells and its possible mechanism.Methods The cancer tissues and adjacent tissues of 45 patients with lung cancer were collect-ed,and the expression of circFOXK2 and miR-409-3p in the tissues were detected by qRT-PCR.Lung cancer cell A549 and H1299 was cultured in vitro,and the dual-luciferase reporter assay was used to verify the regulatory relationship between circ-FOXK2 and miR-409-3p.The CCK-8,clone formation and Transwell assays were used to detect cell proliferation,migration and invasion.Western blot was used to detect the protein expression of E-cadherin and N-cadherin in cells.Results The expression of circFOXK2 was significantly higher(P＜0.05),but the expression of miR-409-3p was significantly lower(P＜0.05)in lung cancer tissues.circFOXK2 could target miR-409-3p and negatively regulate its expression in lung cancer cells.The proliferation,migration and invasion were reduced in circFOXK2(all P＜0.05),N-cadherin level was decreased(P＜0.05),but E-cadherin level was increased(P＜0.05)in circFOXK2 knockdown or miR-409-3p overexpressed A549 and H1299 cells.The inhibitory effects of circFOXK2 knockdown on the proliferation,migration and invasion of lung cancer cells could be reversed by miR-409-3p inhibitor.Conclusion The circFOXK2 was upregulated in lung cancer,which promoted the proliferation,migration and inva-sion of lung cancer cells by targeting miR-409-3p.

Objective To study the influencing factors of overall survival(OS)and cancer-specific survival(CSS)in patients with hepatocellular carcinoma and pulmonary metastasis and establish nomograms to predict survival.Methods The study pop-ulation consisted of 2242 cases with a first primary hepatocellular carcinoma who presented with pulmonary metastasis at the time of diagnosis in the Surveillance,Epidemiology,and End Results(SEER)database of the National Cancer Institute from 2010 to 2016.The influencing factors of OS and CSS were evaluated by using multivariable Cox proportional hazards regression mod-els.Nomograms predicting 1-year OS and CSS were constructed.Data analysis and construction of nomograms were performed with Cox proportional hazards regression models,the Kaplan-Meier curves(log-rank test)and C-index.Results The 1-year OS and CSS rates in the cohort were 10.5％(95％CI:8.7％-12.7％)and 11.8％(95％CI:9.8％-14.2％),respectively.In multi-variable survival analysis,insurance status,small tumor,tumor stage 1-2,negative AFP,chemotherapy treatment,and surgical treatment were associated with OS.Sex,insurance status,tumor staging,AFP status,chemotherapy and surgery treatment were incorporated into the nomogram for CSS prediction.The bootstrap-corrected concordance indexes(C-indexes)predicted by nomo-gram were 0.72(95％CI:0.70-0.74)and 0.71(95％CI:0.69-0.73),which could be used to predict OS and CSS.The models were internally validated and shown to have good calibration.Conclusion The nomograms are established based on the associat-ed factors,which shows good performance in predicting survival in patients with hepatocellular carcinoma and pulmonary metas-tasis.

Objective To investigate the effects of puerarin on myocardial ischemia-reperfusion injury and its mecha-nism.Methods Molecular docking and dynamics simulation were utilized to predict the binding potential of puerarin and SIRT1.A myocardial ischemia-reperfusion model was established in SD rats by ligating the anterior descending branch of the left coronary artery.The protective effect of puerarin on myocardial injury was observed,and the therapeutic effect of puerarin was compared after inhibition of SIRT1 expression.The infarct volume was detected using 2,3,5-triphenyltetrazolium chloride(TTC)staining.The apoptosis rate and SIRT1 expression of cardiomyocytes were detected by using TUNEL combined with im-munofluorescence.Transmission electron microscope was used to observe the myocardial ultrastructure.Western blot was per-formed to detect the expression of ferroptosis-related proteins.Results Molecular docking studies confirmed the formation of stable complexes between puerarin and SIRT1.Puerarin treatment significantly increased myocardial ischemia-reperfusion injury through upregulation of SIRT1,SLC7A11 and GPX4 expression,and downregulation of IREB2 expression in rats.The protec-tive effect of puerarin on myocardium was abolished once SIRT1 protein expression was inhibited.Conclusion Molecular doc-king and molecular dynamics simulation techniques can accurately predict the interaction of puerarin,and the main target SIRT1.Puerarin inhibits ferroptosis by activating SIRT1 pathway,thereby alleviating myocardial ischemia-reperfusion injury.

Objective To investigate the effect of cholecystokinin octapeptide(CCK-8)on glutamate transporter 1(GLT-1)expression in hippocampal astrocytes induced by glutamate(Glu).Methods The mouse hippocampal astrocytes were isolated and the toxicity of CCK-8 at different concentrations on the mouse hippocampal astrocytes was detected.The cells were divided into control group,Glu group,Glu+0.1 μmol/L CCK-8 group,Glu+0.5 μmol/L CCK-8 group and Glu+1.0 μmol/L CCK-8 group.MTT assay was used to detect cell proliferation.Flow cytometry was used to detect cell apoptosis.Biochemical kit was used to detect Glu content in the extracellular supernatant,and qRT-PCR was used to detect the mRNA expression of GLT-1 and glutamate/aspartate transporter(GLAST).The protein expressions of Caspase-3,Bcl-2,GLT-1 and GLAST were detected by Western blotting,and the expression of TNF-α in the cell supernatant was detected by ELISA.Results CCK-8 at different concentrations had no significant effect on the proliferation of mouse hippocampal astrocytes.Compared with the control group,the cell proliferation ability and the expression levels of Bcl-2 protein,GLT-1 and GLAST mRNA and protein in Glu group were significantly decreased(all P＜0.01),the apoptosis rate,extracellular Glu content,Caspase-3 protein expression level in cells and TNF-α level in cell supernatant were significantly increased(all P＜0.01);Compared with the Glu group,the cell proliferation a-bility and the expression levels of Bcl-2 protein,GLT-1 and GLAST mRNA and protein in the Glu+0.5 μmol/L CCK-8 group and Glu+1.0 μmol/L CCK-8 group were significantly increased(all P＜0.05),the apoptosis rate,extracellular Glu content,Caspase-3 protein expression level in cells and TNF-α level in cell supernatant were significantly decreased(all P＜0.01).Con-clusion CCK-8 can inhibit Glu-induced inflammatory response of astrocytes,promote the expression of GLT-1,reduce the con-centration of extracellular Glu,promote cell proliferation and inhibit apoptosis.

Objective To explore the value of endoscopic ultrasonography combined with linked color imaging/blue laser imaging technology combined with magnifying endoscopy(LCI/BLI-ME)in determining the depth of infiltration in early gastric cancer after eradication of Helicobacter pylori(H.pylori),as well as the influencing factors affecting the accuracy of the judg-ment,so as to explore its clinical application value.Methods Clinical data of 91 patients with early gastric cancer after H.pylori eradication were collected from October 2017 to June 2023 in Wuhan No.1 Hospital.Based on the pathological diag-nosis,the endoscopic manifestations of gastric mucosa and early gastric cancer after eradication of H.pylori were summa-rized.The accuracy of endoscopic ultrasonography combined with LCI/BLI-ME in determining the infiltrating depth of early gastric cancer and the related factors affecting the accuracy were evaluated.Sensitivity,specificity,and accuracy were used to in-dicate the effectiveness of endoscopic diagnosis.Chi-square test was used to compare the difference between endoscopic diagnosis and pathological diagnosis.Logistic regression analysis was used to determine the factors affecting the diagnostic accuracy of en-doscopy.Results The overall accuracy of endoscopic ultrasonography combined with LCI/BLI-ME in determining the infiltra-tion depth of early gastric cancer after H.pylorieradication was 77.08％,and the accuracies of uT1a and uT1b stages were 82.86％and 61.53％,respectively.There were 12 under-diagnosed cases(17.14％)and 10 over-diagnosed cases(38.46％).The sensitivity,specificity,positive predictive value and negative predictive value of endoscopic ultrasonography combined with LCI/BLI-ME in the diagnosis of mucosal lesions were 85.29％,57.14％,82.86％and 61.5％,respectively.The results of univariate and multivariate logistic regression analysis showed that the maximum diameter of the lesion and the degree of tissue differentia-tion were the factors affecting the diagnostic accuracy,but not the location and shape of the lesion.The accuracy of endoscopy in judging the infiltration depth of early gastric cancer was decreased with the increase of lesion or the reduction of tissue differen-tiation degree.Conclusion Endoscopic ultrasonography combined with LCI/BLI-ME has good clinical application value in deter-mining the infiltration depth of early gastric cancer T1a after H.pylori eradication.The lesion size and the degree of tissue dif-ferentiation affect the accuracy of the judgment.

Objective To observe the effect of sirtuin 1(SIRT1)on rat myocardial fibrosis induced by pressure overload and the proliferation of cardiac fibroblasts induced by angiotensin Ⅱ(Ang Ⅱ),and to explore the molecular mechanisms.Methods The pressure overload-induced myocardial fibrosis was established by abdominal aorta constriction(AAC)procedure in vi-vo.After treatment with SIRT1 activator,the myocardial interstitial fibrosis and the collagen volume fraction were evaluated by Masson's trichrome staining.The protein expressions of TGF-β1/Smads were determined by immunohistochemical analy-sis.After in vitro intervention of Ang Ⅱ or Ang Ⅱ with SIRT1 activator,the fibroblasts proliferation was detected by MTT as-say.The mRNA and protein expressions of collagen Ⅰ/Ⅲ(Col1α1/3α1),SIRT1 and TGF-β1/Smads in myocardial tissue and fi-broblasts were evaluated by qRT-PCR and Western blotting.Results Compared with the sham operation group,myocardial in-terstitial fibrosis was significantly observed in the pressure overload model group,myocardial collagen volume fraction was in-creased,expressions of Col1α1/3α1 and TGF-β1/Smads were significantly increased,and SIRT1 expression was decreased.After the intervention of SRT1720,SIRT1 activator could improve the myocardial interstitial fibrosis induced by pressure overload,downregulate the expressions of Col1α1/3α1 and TGF-β1/Smads,and upregulate the expression of SIRT1.Meanwhile,correla-tion analysis showed that the protein expression of SIRT1 was negatively correlated with the expression of TGF-β1.In addition,SRT1720 also inhibited Ang Ⅱ-induced fibroblast proliferation and increased expression of Col1α1/3α1 and TGF-β1.Conclusion Activation of SIRT1 inhibits pressure overload-induced myocardial fibrosis and Ang Ⅱ-induced fibroblasts proliferation via regu-lation of the TGF-β1/Smads signaling pathway.

Objective To investigate the associations between preconception dietary patterns(DPs)among Chinese women of childbearing age and neonatal birth weight.Methods The subjects selected for the questionnaire survey and follow-up were women of childbearing age who underwent prenatal eugenic examination at Jiang'an Maternal and Child Health Hospi-tal.Dietary intake information was collected using a semi-quantitative food frequency questionnaire,dietary patterns were extrac-ted by principal component analysis,and the relationship between DPs and birth weight was analyzed by modified Poisson re-gression or linear regression models.Results The final analysis of 221 maternal and infant pairs showed that women who fol-lowed the"nuts-poultry"pattern,one of the four dietary patterns,had a lower risk of delivering large for gestational age(LGA)infants(RR:0.25;95％CI:0.08-0.79),which was more pronounced in those who delivered male infants(RR:0.14;95％CI:0.03-0.72).Conclusion The risk of having LGA newborn is decreased in woman who takes a preconception dietary pattern characterized by nuts and poultry,which is more pronounced in those delivering male infants.Females of childbearing age should maintain good dietary habits before conception to ensure proper growth and development of the fetus and reduce the risk of ad-verse birth outcomes.

Objective To explore the association between serum trace element levels in early pregnancy and gestational dia-betes mellitus(GDM),and the mediating effect of bile acid metabolism changes in this association.Methods A nested case-con-trol study was designed based on the Guangxi Zhuang Birth Cohort.A total of 248 pregnant women(case group=124,control group=124)were included from June 2015 to July 2019 in Nanning city.The concentrations of 8 trace elements and 31 bile acids in serum were measured in early pregnancy.Conditional logistic regression and BKMR models were used to analyze the associa-tion and combined effect between trace elements and GDM risk,respectively.Orthogonal partial least squares-discriminant anal-ysis(OPLS-DA)was used to screen potential bile acid biomarkers associated with GDM,and then conditional logistic regression was used to determine the association between specific bile acid levels and GDM risk.Multiple linear regression was used to e-valuate the association of serum trace element concentrations with differential bile acid metabolites.Mediation analysis was used to evaluate the mediating role of bile acids in the relationship between trace element exposure and GDM.Results After adjus-ting for confounding factors,serum vanadium(V)was found to be positively associated with the risk of GDM,while chromium(Cr),manganese(Mn),zinc(Zn),selenium(Se)and molybdenum(Mo)were negatively correlated with the risk of GDM(all P＜0.05).The OPLS-DA model and conditional Logistic regression analysis showed that taurocholic acid(TCA),glycochenodeoxy-cholic acid 3-sulfate(GCDCA-3S),glycochenodeoxycholic acid-3-O-β-glucuronide(GCDCA-3Gln),glycoursodeoxycholic acid-3-sulfate(GUDCA-3S),taurodeoxycholic acid-3-sulfate(TDCA-3S),and chenodeoxycholic acid(CDCA)might be potential bile acid metabolic markers of GDM(all P＜0.05).The concentrations of multiple trace elements were also significantly correlated with the levels of specific bile acids(all P＜0.05).Mediation analysis showed that GCDCA-3Gln and TCA mediated the associa-tion between serum Zn and Se and GDM risk,respectively(all P＜0.05).Conclusion Serum trace elements such as V and Cr are significantly associated with the risk of GDM in early pregnancy,and changes in bile acid metabolism may precede the occur-rence of GDM.It is suggested that the effect of trace elements on the metabolism of bile acids,especially conjugated bile acids,may be one of the mechanisms affecting the risk of GDM.