Effects Of Carvedilol On Autophag And Apoptosis Of Human Hepatic Stellate Cells

Background and AimsLiver fibrosis is a histopathological change caused by excessive deposition of extracellular matrix(ECM)in the interstitial tissue of liver tissue caused by various acute or chronic liver damage,and is a common link in the progression of chronic liver disease to cirrhosis.Controlling this process is of great significance in preventing the occurrence and progression of cirrhosis.Liver fibrosis is a complex pathological process involving multiple cytokines and signaling pathways,and the exact mechanism has not been fully elucidated.The characteristics of activation,proliferation,migration,secretion of hepatic stellate cell(HSC)located in the space of Disse are the main processes in the occurrence and development of liver fibrosis.Non-Selective Beta Blocker(NSBB),the stone of cirrhotic portal hypertension drug treatment,can reduce cardiac output through βt blockade,and contract visceral blood vessels through β2 receptor blockade reducing portal blood flow,thereby reducing portal pressure.Propranolol is the first NSBB to prevent hemorrhage of esophagogastric varices in cirrhosis.Carvedilol is a third-generation NSBB with strong non-selective beta blockade and alphal receptor blockade.Based on the traditional NSBB effect,the alphal receptor blockade of carvedilol makes it not only reduce cardiac output and blood flow in portal system,but reduce intrahepatic vascular resistance.Therefore,carvedilol has a stronger effect on reducing portal pressure than propranolol.Whether carvedilol has the effect of attenuating liver fibrosis,there have been several studies in the past.Previous studies have suggested that carvedilol could anti-mouse liver fibrosis,inhibited rat HSC proliferation,fibrosis,and HSC activation.Moreover,activation and proliferation of HSC play a dominant role in liver fibrosis,so inhibition of activation and proliferation of HSC are beneficial to reversing liver fibrosis.At the same time,autophagy and apoptosis play an important role in the activation of hepatic stellate cells.Autophagy is an evolutionarily conserved process by which cytoplasmic materials,including damaged proteins and organelles,are sequestered for lysosome-dependent degradation.Thus,the autophagic machinery is responsible for the maintenance of cellular homeostasis.When the lysosomal pathway is activated,cells begin to consume their own components.This process is one of self-protection mechanism of cell,which allowing cell to produce nutrients,energy to maintain their functions,and eliminates excess or damaged organelles,invasive microorganisms when they are undernourished.What’s more,both autophagy and apoptosis all play an important role in the activation of HSC.Increasing the apoptosis of HSC or inhibiting autophagy can significantly reduce their viability and activation.The aim of this study was to investigate the effects of carvedilol on autophagy and apoptosis in human HSC and to explore its potential mechanism for inhibiting liver fibrosis.Methods1.The HSC cell line LX-2 was used for in vitro study.2.Western blot was used to verify the effect of carvedilol on the expression of autophagy upstream markers(LC3B-I,LC3B-Ⅱ,Beclinl,Atg5).3.Transmission Electron Microscope was used to observe the morphology of autophagic vacuoles in LX-2 under the treatment of carvedilol.4.Application of autophagy inducers and inhibitors was used to verify the effect of carvedilol on autophagic flow via cell immunofluorescence staining and Western blot.5.Application of autophagy inducers and inhibitors was used to verify the effect of carvedilol on autophagosome and lysosome colocalization in LX-2 via cellular immunofluorescence staining.6.Application of autophagy inducers and inhibitors was used to verify the effect of carvedilol on LX-2 lysosomal pH via cell immunofluorescence staining and Western blot.7.Western blot,flow cytometry and CCK-8 experiments were used to confirm the effect of carvedilol on LX-2 viability and proliferation.8.Western blot was used to confirm the effect of carvedilol on LX-2 apoptosis-related signaling pathway.9.Application of autophagy inducers and inhibitors was used to investigate the relationship between autophagy and apoptosis in LX-2 under the treatment of carvedilol via Western blot and flow cytometry.Results:1.Carvedilol increased the conversion of LC3B-I to LC3B-II in LX-2,but did not affect the expression of Beclinl and Atg5.2.Transmission Electron Microscopy showed that the number of autophagosomes in the carvedilol group was significantly higher than that in the control group.3.Compared with the carvedilol group,carvedilol combined with autophagy early inhibitors in the 3-MA group significantly reduced the number of autophagosomes in the LX-2,while carvedilol combined with the autophagy late inhibitor chloroquine(CQ)group did not significantly reduce the number of autophagosomes compared to the carvedilol group.4.Carvedilol increased the expression of P62,a downstream marker of autophagy in LX-2.After transfected the double-labeled adenovirus GFP-RFP-LC3B,was then performed by immunofluorescence staining,the results showed that the application of carvedilol can increase the number of autophagosomes in LX-2 but the number of autolysosomes.5.After transfected the double-labeled adenovirus GFP-RFP-LC3B,LX-2 was then performed by immunofluorescence staining,the results showed that carvedilol did not affect the fusion of autophagosomes with lysosomes.6.Carvedilol increased the lysosomal pH in LX-2.7.Carvedilol inhibited the proliferation of LX-2 in a time-and dose-dependent manner,IC50=25.10μgM for 12 hours and IC50=18.16μM for 24 hours.8.Carvedilol increased the levels of cleaved-caspase8,cleaved-caspase3,cleaved-PARP,and BAX and decreased the expression of Bcl-2 in LX-2.9.The autophagy-associated protein in HSC changed after 6 hours of carvedilol treatment,but the apoptosis-related protein in HSC changed until 12 hours after treatment.Conclusions:Carvedilol could inhibit the autophagy of human HSC and promote the apoptosis of human HSC.The inhibitory effect of carvedilol on autophagy is earlier than its effect on apoptosis.