Berberine Inhibits NLRP3 Inflammasome Activation And Subsequent Pyroptosis In Nonalcoholic Steatohepatitis Cellular Model Through ROS/TXNIP Axis

Objective:To investigate the effects of Berberine?BBR?on methionine-choline-deficient?MCD?medium and palmitic acid?PA?-induced nonalcoholic steatohepatitis?NASH?.The therapeutic effect of NLRP3 inflammatory corpuscle activation and subsequent pyroptosis in the model provides a further theoretical basis for the clinical treatment of NASH with berberine.Methods:?1?Cells:Mouse normal liver cell line AML12 cells were purchased from ATCC.?2?Experimental group:blank control group,MCD+LPS group,MCD+LPS+BBR?10?M,20?M?group,PA group,PA+BBR?10?M,20?M?group,MCD+LPS+NAC?10?M?group,MCD+LPS+BBR?20?M?+H₂O₂?200?M?group,PA+NAC?10?M?group,PA+BBR?20?M?+H₂O₂?200?M?group.?3?Nile red staining was used to detect the changes of lipid deposition in MCD+LPS and PA model groups,and the effect of BBR dosing group on lipid deposition in NASH model.?4?Cells were labeled with a DCFH-DA fluorescent probe,and the levels of cellular reactive oxygen species?ROS?in each group were detected by flow cytometry.?5?The lipid peroxidation?MDA?kit was used to detect the level of cellular lipid oxidation in each group.?6?Real-time PCR was used to detect the mRNA expression level of inflammatory factor TNF-?in each group.?7?The caspase-1 activity level of each group was measured using a caspase-1 activity assay kit.?8?Western blot was used to detect the expression of inflammatory factors,inflammatory corpuscles and cell-related proteins in various groups,including TNF-?,NF-?B p65,p-NF-?B p65,NLRP3,TXNIP,GSDMD,GSDMD-N.?9?Statistical analysis:Statistical analysis data were expressed using GraphPad Prism 7,mean±standard deviation data.Comparisons between groups were performed using one-way ANOVA,and p<0.05 indicates statistically significant differences.Result:?1?Nile red staining showed that compared with the blank control group,the MCD+LPS model group and the PA model group showed a significant increase in lipid deposition?red fluorescence increased significantly?compared with the blank control group;Compared with the BBR dosing group,the lipid deposition was observed to decrease in a concentration-dependent manner,and the lipid deposition?red fluorescence was significantly reduced?was significantly reduced with increasing concentration.The results of flow cytometry showed the same trend.?2?The results of flow cytometry showed that compared with the blank control group,the levels of ROS in the MCD+LPS model group and the PA model group were significantly higher.Compared with the two model groups,the BBR dosing group could significantly down-regulate the ROS level.In a concentration-dependent manner,ROS levels were significantly down-regulated with increasing concentrations;and the NAC-dosing group also down-regulated ROS levels;whereas the H₂O₂ group compared with the BBR-dosing group reversed the down-regulated ROS levels.?3?The results of MDA kit showed that the lipid peroxidation level of MCD+LPS model group and PA model group was significantly higher than that of the blank control group;compared with the two model groups,the BBR dosing group could be significantly down-regulated.Lipid peroxidation levels.?4?The results of Real-time PCR showed that compared with the blank control group,the mRNA expression of TNF-?was significantly increased in the MCD+LPS model group and the PA model group,while the BBR administration group was able to down-regulate the mRNA expression level of TNF-?.?5?The results of caspase-1 activity assay showed that the activity level of caspase-1 was significantly increased in the MCD+LPS model group and the PA model group.The BBR plus drug group could down-regulate the activity of caspase-1 and was similar to the NAC dosing group.The H₂O₂group reversed the down-regulated caspase-1 activity level.?6?Western blot analysis showed that compared with the blank control group,the expression levels of inflammation-related p-NF-?B p65 and TNF-?protein in MCD+LPS model group and PA model group were significantly increased,and BBR plus drug group could be used.Compared with the blank control group,the expression levels of NLRP3,TXNIP and caspase-1 proteins in the inflammatory corpuscles of the MCD+LPS model group and the PA model group were significantly increased,and the BBR dosing group could reduce it,and the down-regulation effect and NAC.The drug-added group was similar,and the H₂O₂ group could reverse the decreased protein level.Compared with the blank control group,the expression of GSDMD-N protein in the MCD+LPS model group and the PA model group was significantly increased.BBR plus drug group It can be lowered,and the down-regulation effect is similar to that of the NAC dosing group,while the H₂O₂ group can reverse the decreased protein expression level.Conclusion:?1?Berberine can effectively down-regulate the lipid deposition and lipid peroxidation level in NASH model induced by MCD+LPS and PA.?2?Berberine can effectively down-regulate the expression of NF-?B/TNF-?axis in the NASH model and effectively inhibit the inflammatory effects in the NASH model.?3?Berberine inhibits the production of ROS in the NASH model,and down-regulates the expression of TXNIP/NLRP3-related proteins by ROS,thereby inhibiting the activation of caspase-1 and ultimately inhibiting the pyroptosis in NASH.