Determination Of Pyroptosis Regulatory Pathway Of HSC-LX2 Induced By AngⅡ-ROS And Intervention Effect Of AngⅡ-ROS On Albizia Julibrissin Durazz.-Tribulus Terrestris L.drug Pair

Objective Current evidence suggests that liver fibrosis is reversible even at late stages.Pyroptosis is reportedly regulated by classical and non-classical pathways and is also involved in the activation of the human hepatic stellate cell line LX2.This study sought to identify regulatory pathways that pyroptosis of HSC during AngⅡ-ROS induced HSC activation,and to explore the intervention effect of Albizia julibrissin Durazz.-Tribulus terrestris L.drug pair on it,so as to provide new insights for the treatment of liver fibrosis.Methods 1.To establish the activation model of HSC-LX2 cells induced by AngⅡ.The HSCLX2 was used for all tests,and an optical inverted microscope was used to track the morphological changes in the cells.Cell activity and proliferation were identified using CCK8.DCFH-DA identified ROS production.To find the LDH that was released by injured cells,an LDH assay kit was employed.The amounts of IL-18 and IL-1β were measured using ELISA.Western blotting and RT-PCR:AngⅡ activated the protein expression and mRNA levels of αSMA,NLRP3,Caspase-1,-4,-5,ASC and GSDMD-N in HSC-LX2 cells.2.An AngⅡ induced HSC-LX2 cell activation model was established.After different concentrations of NAC and NSA intervention,cell viability was detected by CCK8.DCFH-DA detects ROS generation.LDH assay kits were used to detect LDH released by damaged cells,and ELISA was used to quantify IL-18 and IL-1β levels.Western blotting and Real-time PCR detected protein expression and mRNA levels of α-SMA,ASC,NLRP3,Caspase-1,-4,-5,and GSDMD-N.3.In the study of network pharmacology,drug components and drug and disease-related targets were obtained through online databases TCMSP,TCMID,DrugBank,PubChem,SwissADME,SwissTargetPrediction,GeneCards,OMIM and CNKI,and related networks were constructed.GO and KEGG analysis were performed and preliminarily verified by molecular docking.4.An AngⅡ induced HSC-LX2 cell activation model was established.After intervention with different concentrations of Albizia julibrissin Durazz.-Tribulus terrestris L.drug pair medicated serum,cell proliferation and activity were detected by CCK8.DCFH-DA detects ROS generation.LDH assay kits were used to detect LDH released by damaged cells,and ELISA was used to quantify IL-18 and IL-1β levels.Western blotting and Real-time PCR detected protein expression and mRNA levels of α-SMA,NLRP3,Caspase-1,and GSDMD-N.Results 1.Compared with the blank group,after Ang Ⅱ stimulation,HSC-LX2 cell viability(P<0.05),intracellular ROS content(P<0.01),LDH content(P<0.01),IL-18 and IL-1β content were up-regulated(P<0.05).The protein and mRNA levels of α-SMA,NLRP3,ASC,Caspase1,-4,-5,GSDMD-N were considerably enhanced(P<0.05,P<0.01).2.HSC-LX2 was induced by AngⅡ,and then treated with NAC and NSA.Compared with AngⅡ,after NAC and NSA treatment,HSC-LX2 cell viability decreased(P<0.01),intracellular ROS content(P<0.01),LDH content(P<0.01),IL-18 and IL-1β content decreased(P<0.05).At the same time,α-SMA,NLRP3,ASC,Caspase-1,-4,-5 and GSDMD-N had considerably lower levels of mRNA and protein expression(P<0.05,P<0.01).3.From the results of network pharmacology,it can be shown that NLRP3 and Caspase-1 are used in the therapy of liver fibrosis.In the enrichment analysis of KEGG,the NOD-like receptor signaling pathway was at the forefront.Through molecular docking technology,it was found that the active ingredients of Albizia julibrissin Durazz.-Tribulus terrestris L.drug pair,machaerinicacid lactone and(-)-syringaresinol,had strong docking activity with NLRP3 and CASP1.4.In vitro experiments showed that the drug pair could inhibit the activated HSC-LX2 and significantly inhibit the expression of inflammatory factors such as ROS,IL-18 and IL-1β(P<0.05).At the same time,under the influence of AngⅡ,HSC-LX2 cells significantly increased their expression of NLRP3,Caspase-1,α-SMA,and GSDMD.while the expression was significantly decreased under the action of drug pair(P<0.05).Conclusion In the above studies,AngⅡ-ROS can cause pyroptosis in HSC-LX2 cells.Unlike LPS-induced pyroptosis,AngⅡ-induced Caspase-4 and-5 activation is related to its upregulation of ROS content.The intervention effect of Albizia julibrissin Durazz.-Tribulus terrestris L.drug pair on pyroptosis is related to its classical pathway of inhibiting pyroptosis,which is consistent with the prediction of network pharmacology.Its mechanism needs further study and verification.