Penicilfuranone A Exerts Anti-fibrotic Effects By Endoplasmic Reticulum And Mitochondria Mediated Apoptosis Pathways In Activated Hepatic Stellate Cells

Background: Liver fibrosis is an intractable disease results from chronic damage and the excessive accumulation of synthetic extracellular matrix,which is characterised by a higher incidence.New targeted therapy has always been the focus in the prevention and reversal of liver fibrosis research.Since the activation of hepatic stellate cells(HSCs)plays a significant role in promoting the development of fibrosis,general anti-hepatic fibrosis treatment protocols are based on inducing apoptosis of activated HSCs or depressing the activation of HSCs.Endoplasmic Reticulum Stress was thought to be a crosstalk of signaling conduction in the pathogenesis of fibrosis of a variety of organs.For Discussing its molecular mechanism,for the prevention and therapy of fibrosis,perhaps can provide us with new targets.Previous studies have shown that Penicilfuranone A has obvious anti-fibrotic effects,but the precise mechanism of signaling pathway is unknown.Objective: To investigate whether PFA is able to exhibit the resistance of liver fibrosis by mediating the apoptosis of TGF-?1-induced activated HSCs,and find the medicinal target by exploring its molecular mechanism.Methods: We used the exogenous TGF-?1-induced HSCs as a cell model in vitro,the cell viability of normal hepatocytes and two activated HSCs were assessed by MTT cytotoxicity assay.Hoechst 33258 fluorescent staining,flow cytometry Annexin-V/PI and DNA Ladder was utilized to observe the morphological and biochemical changes of the cells after PFA treatment.The protein and mRNA levels of ERS-mediated PERK/eIF2?/ATF4,ATF6 and IRE1?/XBP1 signaling pathway related molecules were assessed by the western blot,PCR and PAGE silver staining,and detected the expression of IRE1?/ASK1 pathway related protein.In order to investigate the relationship between ER and mitochondrial apoptosis pathway,we assessed the expression of Bcl-2 family-related proteins and cytoplasm Cyt-c protein,next we detected the protein's expression of ERS-mediated caspase12 signaling pathway molecules,as well as the expression of cell apoptosis marker protein caspase3 and PARP.Results: The results of cytotoxicity test showed that PFA did not impact normal hepatocytes but rather significantly decreased the activity of HSCs in 10 ?M-40 ?M concentration ranges.Observing the cell morphological changes and biochemical characteristics,after different concentrations of PFA treated,suggested that the activated HSCs appeared nuclear agglutination,fragmentation,phosphatidylserine exposure to the outer plasma membrane,DNA fragments,with caspase3,PARP protein expression increasing,confirmed the emergence of apoptosis.Detection of protein and mRNA expression found that PFA did not activate the PERK/eIF2?/ATF4 pathway,ATF6 pathway and IRE1?/XBP1 pathway;PFA activated the ASK1/JNK/p38 signaling pathway by activating IRE1?.The regulation of BCL-2 family members increased the expression of cytoplasm cyt-c,while PFA could increase the expression of caspase12,but not calpain 1.caspase12 and Cyt-c were both activated caspase9,and the caspase cascade reaction was finally initiated to induce the apoptosis of activated HSCs.Conclusion: PFA has capability to trigger the apoptosis of TGF-?1 activated HSCs.The particular molecular mechanism as follows: PFA regulates three apoptosis pathways mediated by IRE1? in endoplasmic reticulum pathway: p38/CHOP,JNK/Bim,caspase12,and then associated with mitochondria apoptosis pathway,both signaling pathway together result in the apoptosis of activated HSCs.And IRE1? may be the target of PFA againsts hepatic fibrosis.