Study On The Mechanism Of Berberine On Regulation Of Hepatic Stellate Cell Autophagy And Liver Fibrosis

ObjectiveLiver fibrosis?LF?is considered as to the imbalance between generation and degradation of extracellular matrix?ECM?,leading to excessive deposition of ECM.Activated hepatic stellate cell?HSC?is the main source cells producing ECM,and autophagy upregulation is an important player in HSC activation.Therefore,inhibiting autophagy in activated HSC has become a pivotal therapeutic target in LF.Berberine,an isoquinoline alkaloid,is widely applied to dysentery and bacterial gastroenteritis.Berberine,as an autophagy regulator,can inhibit autophagy of various cells and disease progression.It is reported that berberine can effectively improve LF,however the specific molecular targets and mechanisms are lacking,which has affected the application and development of berberine in anti-LF treatment.This study aimed to explore the potential molecular mechanism of anti-LF of berberine both in vivo and in vitro,to provide a reliable experimental and theoretical basis for developing berberine as a clinical drug candidate against LF.MethodsA mouse model of LF was induced by carbon tetrachloride?CCl₄?injection,and berberine was administered intragastricly.After 38 days,the serum levels of ALT and AST in mice were measured.The liver was detected,and the liver pathological changes were observed by HE and Sirius red staining.Content of Hydroxyproline in livers of the mice was detected,and the levels of?-SMA and COL1A1 were determined using RT-qPCR,Western blot and tissue immunofluorescence.In vivo experiments,LX-2 was treated by PDGF-BB or berberine.HSC activation markers?-SMA and COL1A1,autophagy-related molecules p62,LC3-I,LC3-II,Atg5,Becn1,Atg7,and apoptotic molecules PARP,p53,Bcl-2,Bax were detected using RT-qPCR,Western blot and cellular immunofluorescence.Autophagosomes were evaluated under electron microscope.The autophagy levels and flux of cells were assayed by infecting Ad-GFP-LC3B and autophagy dual-labeled lentivirus mRFP-GFP-LC3 in LX-2 cells.Apoptosis and cell cycle were measured by flow cytometry and TUNEL assays,cell lipid droplet contents were evaluated using oil red O assay.Cell proliferation was determined using CCK-8,clone formation and EdU assays.Results?.Berberine can reduce collagen deposition,inflammatory cell infiltration,?-SMA and COL1A1,liver hydroxyproline Glycine,and serum AST and ALT concentrations in livers of LF mice.The above results indicated that berberine can effectively improve CCl₄-induced LF in mice.?.Berberine can decreased?-SMA and COL1A1 expression as well as promote lipid droplets accumulation in LX-2 cells.Berberine can reduce the cell viability and the number of cell clones and EdU incorporation.These results indicated that berberine can inhibit the activation and proliferation of HSC.?.Berberine can promote the expression of p62 in LX-2 cells,and reduce LC3-II and ATG5 protein expression,the number of LC3 fluorescent spots,and autophagosomes.Knockdown Atg5 using siRNA in LX-2 cell can up-regulate p62expression in LX-2 cells and reduce LC3-II protein expression,the number of LC3spots,and the expression of?-SMA and COL1A1 and inhibit autophagy flow.Overexpression Atg5 in LX-2 cell can reverse the effects of berberine.Berberine can inhibit HSC autophagy by down-regulating Atg5 expression.?.Berberine can up-regulate p53,Bax,cleaved PARP and down-regulate Bcl-2expression in LX-2 cells.Annexin V and TUNEL staining-positive cells increased in berberine-treated LX-2 cells in a dose-dependent manner.Overexpressed Atg5 in berberine-treated LX-2 cells can obviously reverse the pro-apoptotic effect of berberine.The above results indicated that berberine can suppress HSC autophagy and induce HSC apoptosis via down-regulating Atg5 expression.ConclusionBerberine possess an anti-fibrotic effect,which inhibits HSC activation and proliferation.Berberine suppresses autophagy and induces HSC apoptosis via down-regulating the expression of Atg5.It provides a reliable theoretical basis for development of berberine as a clinical drug candidate against LF.