委陵菜酸调节Keap-1/Nrf2和NLRP3炎症小体信号通路对乙醇诱导GES-1细胞损伤的保护作用及机制研究OA

AIM:To investigate the protective ef-fect and mechanisms of tormentic acid(TA)on GES-1 cells induced by alcohol.METHODS:The GES-1 cell injury model induced alcohol was established.The GES-1 cells were divided into control group,model group,TA(12.5 μg/mL)group,Nrf2 inhibitor group(ML385,10 μmol/L)group and NLRP3 in-hibitor(MCC950,8 μmol/L)group.After adding the corresponding drugs to the drug intervention group cells and culturing them for 22 hours,cells were treated with 7%alcohol except for the control group and cultured for another 2 hours,and then the cell supernatant and cells were collected,re-spectively.MTT assay was used to test cell viability;Cell scratch and Transwell were utilized to detect cell migration;AnnexinV FITC/PI double staining was used to test cell apoptosis;Immunofluores-cences were used to detect MMP and ROS level in alcohol induced GES-1 cells;Colorimetric and ELISA methods were used to test the levels of LDH,IL-4,IL-1β,IL-6,IL-10,IL-18,NO and TNF-α in the GES-1 cell supernatants;Colorimetric method was utilized to detect the levels of CAT,GSH,MDA,MPO,SOD,T-AOC in alcohol induced GES-1 cells,and cy-tochrome C in the cytosol and mitochondria of GES-1 cells;Immunofluorescence was utilized to detect the co-localization of NLRP3,ASC and caspase-1;The changes in intracellular ultrastructure were ob-served under an electron microscope;Real-time PCR was used to detect the mRNA expression levels of Apaf-1,ASC,Bcl-2,Bcl-xl,Bad,Bax,caspase-1,COX-1,COX-2,GCLC,HO-1,iNOS,Keap-1,NEK7,NL-RP3,NQO1,Nrf2,PGE2 and TXNIP in alcohol in-duced GES-1 cells;Western blot was utilized to test the protein expression levels of Apaf-1,ASC,Bcl-2,Bcl-xl,Bad,Bax,caspase-1,cleaved-caspase-3,cleaved-caspase-9,PARP-1,cleaved-PARP-1,GCLC,HO-1,Keap-1,NEK7,NLRP3,NQO1,nuclear Nrf2,pro-caspase-1,pro-caspase-3,pro-caspase-9,pro-IL-18,pro-IL-1β,total Nrf2 and TXNIP in GES-1 cells.RESULTS:Compared with the model group,TA prominently promote alcohol induced GES-1 cell mi-gration,depressed cell apoptosis and LDH release,substantially reduced intracellular ROS level,IL-1β,IL-6,IL-18,NO,TNF-α levels in the GES-1 cell super-natants,MDA,MPO levels in alcohol induced GES-1 cells and cytochrome C content in cytosol(P<0.01),substantially elevated mitochondrial membrane po-tential,IL-4,IL-10 levels in the GES-1 cell super-natants,CAT,GSH,SOD,T-AOC levels in alcohol in-duced GES-1 cells and cytochrome C content in the mitochondria(P<0.01).It dramatically down-regu-lated the ASC,Apaf-1,Bad,Bax,caspase-1,COX-2,iNOS,Keap-1,NEK7,NLRP3,TXNIP mRNA and ASC,Apaf-1,Bad,Bax,caspase-1,cleaved-caspase-3,cleaved-caspase-9,cleaved-PARP-1,Keap-1,NEK7,NLRP3,pro-caspase-1,pro-IL-1β,pro-IL-18,TXNIP protein expressions in alcohol induced GES-1 cells,restrained co-localization of NLRP3,ASC and cas-pase-1 in alcohol induced GES-1 cells;The structure of the nucleus and nucleolus was intact,chromatin was uniform,and mitochondrial swelling was re-duced;TA up-regulated the COX-1,Bcl-2,Bcl-xl,GCLC,HO-1,NQO1,Nrf2,PGE2 mRNA and Bcl-2,Bcl-xl,GCLC,HO-1,NQO1,total Nrf2,nuclear Nrf2,pro-caspase-3,pro-caspase-9 protein expressions and Bcl-2/Bax,Bcl-xl/Bad ratios in the alcohol in-duced GES-1 cells(P<0.01).CONCLUSION:TA has a significant protective effect on alcohol damaged GES-1 cells,and its mechanism is closely related to activating the Keap-1/Nrf2 pathway,reducing ox-idative stress,inhibiting the activation of the NLRP3 inflammasome pathway,alleviating inflammatory response,and thereby suppressing the activation of the mitochondrial apoptosis pathway.