Molecular Mechanism Research On The Anti-liver Fibrosis Of Dihydroartemisinin Through Regulating Autophagy And Senescence Of Hepatic Stellate Cells

Hepatic fibrosis(HF)is a common pathological process for all kinds of chronic liver diseases to develop cirrhosis and even hepatocarcinoma(HCC).The essence of hepatic fibrosis is the excessive deposition of extracellular matrix(ECM),and activated hepatic stellate cell(HSC)is the most important cell source of ECM.Therefore,the removal of activated HSC is considered to be the main strategy to treat hepatic fibrosis.Interestingly,cell senescence refers to the irreversible separation of cells from normal division into a relatively stable state of proliferation stagnation.Autophagy mainly consists of two layers of membranes encapsulating senescent or damaged organelles,misfolded protein,pathogenic microorganisms to form autolysosome,and contents of packages are then degraded by various proteolytic enzymes in lysosomes to provide material and energy sources for cell itself.Recent studies have shown that autophagy can regulate the process of cell senescence,and autophagy has become a potential breakthrough point in the study of aging and anti-aging.If appropriate drugs are used to regulate autophagy and senescence in HSCs,it will be able to block HSC overproliferation,inhibit HSC activation,reduce ECM deposition,and alleviate pathological changes of hepatic fibrosis.Importantly,our previous studies showed that dihydro artemisinin(DHA)can significantly improve the pathological damage of hepatic fibrosis,activate HSC autophagy,and induce HSC senescence.Base on this,we innovatively put forward a scientific hypothesis that DHA may play an anti-hepatic fibrosis role by regulating autophagy and senescence signaling network.We will focus on this scientific hypothesis to reveal the potential molecular mechanism of DHA in anti-hepatic fibrosisFirst of all,we confirmed that DHA can improve the pathological damage of hepatic fibrosis both in vivo and in vitro.According to the literatures,a classical rat model of hepatic fibrosis induced by carbon tetrachloride(CCl4)was established.After successful establishment,different doses of DHA were injected intraperitoneally for 4 weeks.Interestingly,ELISA assays showed that DHA can significantly reduce the serum levels of liver injury markers including ALT,AST,ALT,LDH and TBIL and liver fibrosis markers including LN,HA,IV-C,PC-Ⅲ and HYP.Moreover,histopathological examination showed that DHA can alleviate the pathological changes of hepatic fibrosis and also can reduce the excessive deposition of collagen Furthermore,western blot,real-time PCR and immunofluorescence assays showed that DHA can significantly decrease the levels of hepatic fibrosis markers including a-SMA,collagen 1,fibronectin,TGF-βR1 and PDGF-RP.Besides,primary HSCs were isolated from normal rat liver,and PDGF-BB was used to activate HSC in vitro.Then different doses of DHA were added to intervene activate HSCs.Attractively,DHA can down-regulate the expression levels of HSC activation markers including a-SMA,collagen 1,fibronectin,desmin,TGF-βR1 and PDGF-RB in a dose-dependent manner.Overall,these results suggest that DHA can improve the pathological damage of hepatic fibrosis both in vivo and in vitroNext,we determined whether DHA plays an anti-hepatic fibrosis role by regulating autophagy and senescence pathway network.The liver fibrosis model was established according to the above methods,and DHA was given by the way of therapeutic administration.At the same time,primary HSCs were isolated from normal rat livers and were activated by PDGF-BB,and then were treated with different doses of DHA.Interestingly,DHA can induce HSC senescence in vitro and in vivo,and this effect is related to GATA6 up-regulation.GATA6 siRNA can impair DHA-induced HSC senescence,whereas GATA6 plasmid can enhance the senescence of HSCs induced by DHA.Importantly,activation of autophagy may be a potential mechanism of DHA-induced senescence and GATA6 accumulation in HSCs.Blocking autophagy can impair the effect of DHA-induced HSC senescence and GATA6 accumulation.Molecular mechanism studies revealed that DHA can promote degradation of the autophagic substrate p62 by activating autophagy,and induce the dissociation of p62 and GATA6,leading to the accumulation of GATA6,and then triggering the occurrence of cell senescence.Autophagy inhibitors 3_MA,CQ,Bafilomycin A1 or p62 plasmid could block the degradation of p62,thereby impairing DHA-induced HSC senescence and GATA6 accumulation.Taken together,these data show that DHA plays an anti-hepatic fibrosis role by regulating autophagy and senescence pathway networkIt is well known that the typical characteristics of senescent cells are permanent cell cycle arrest and senescence-associated secretory phenotype(SASP).Cell senescence can block the proliferation of damaged cells or cancer cells,and can also secrete a variety of inflammatory related factors to affect the function of surrounding normal cells by SASP.Then,we determined whether DHA could inhibit production and release of inflammatory factors by activating autophagy in HSCs.Both in vivo and in vitro experiments were carried out in accordance with the above description.Interestingly,DHA can inhibit the production and release of inflammatory factors both in vivo and in vitro,and autophagy activation may be related to the effect.Blocking autophagy can impair the anti-inflammatory effect of DHA,whereas up-regulating autophagy can enhance the anti-inflammatory effect of DHA.Molecular mechanism studies showed that DHA may trigger ROS accumulation,induce JNK1/2 upregulation,promote autophagy activation,and then play an anti-inflammatory role in HSCs.ROS scavengers including NAC,GSH,Na Py,Cat or JNK1/2 specific inhibitors SP600125 and JNK1/2 siRNA all can attenuate the activation of autophagy and anti-inflammatory effects induced by DHA.Collectively,these findings indicate that DHA could inhibit production and release of inflammatory factors by activating autophagy in HSCs.Finally,we explored whether DHA can induce ferroptosis in HSCs through activation of autophagy.Both in vivo and in vitro experiments were carried out in accordance with the above description.Interestingly,DHA can induce HSC ferroptosis both in vivo and in vitro,and this effect is related to autophagy activation.Blocking autophagy can impair DHA-induced HSC ferroptosis,whereas promoting autophagy can enhance DHA-induced HSC ferroptosis Importantly,NCOA4 upregulation may mediate DHA-induced HSC ferroptosis.NCOA4 can direct autophagy to recognize and degrade FTH1,and then increased the level of iron in HSCs.NCOA4 siRNA can attenuate DHA-induced HSC ferroptosis,whereas NCOA4 plasmid can enhance DHA-induced HSC ferroptosis.Altogether,these results suggest that DHA can induce ferroptosis in HSCs through activation of autophagy.In summary,the present study reveal the key role of autophagy and senescence signaling network in hepatic fibrosis at the level,and further elucidate the molecular mechanism of DHA in anti-hepatic fibrosis.Our study may provide reliable evidence for the development of DHA as a clinical drug for anti-hepatic fibrosis.