Oroxylin A Inhibits Liver Fibrosis Through Modulating Autophagy Of Hepatic Stellate Cells

Liver fibrosis is a reversible pathophysiological process occurring in all kinds of chrnonic liver diseases,and its end-stage cirrhosis is responsible for high morbidity and mortality worldwide.The transdifferentiation of hepatic stellate cells(HSCs)into contractile,matrix-producing myofibroblasts(MFBs)is a central event in liver fibrosis.Therefore,intervention of HSC biology,including inhibition of the fibro genic function and induction of apoptosis,necroptosis,pyroptosis and cellular senescence,are proposed therapeutic approaches to reverse liver fibrosis.Oroxylin A.a natural monoflavonoid isolated from Scutellariae radix,shows a wide range of pharmacological activities such as anti-inflammatory,anti-angiogenesis,anti-oxidant,and anti-tumor properties.However,the effects of Oroxylin A on liver fibrosis remain poorly understood.In the present study,we sought to determine the effects of Oroxylin A on carbon tetrachloride(CCl4)-induced liver fibrosis,and to further examine the molecular mechanisms.In the previous studies,we found that Oroxylin A had no obvious toxicity to normal hepatocytes,but obviously inhibited the expression of HSC activation marker a-SMA.Interestingly,Oroxylin A significantly increased the expression of autophagic marker LC3-Ⅱ.and reduced ROS levels,IL-1β expression,and mTOR phosphorylation.Based on the above results,we speculated that Oroxylin A may affect ROS production to regulate PI3K-AKT-mTOR pathway,promote HSC autophagy,and in turn inhibit HSC activation and improve the inflammatory microenvironment in liver fibrosis.This study will carry out experiments to validate this hypothesis.Firstly,we determined the effects of Oroxylin A on liver fibrosis and inflammatory microenvironment in vivo and in vitro,which can provide a basis for further exploring its mechanism of anti-hepatic fibrosis.We found that treatment with Oroxylin A markedly decreased the level of liver injury markers including alkaline phosphatase(ALP),aspartate ami-notransferase(AST),and alanine aminotransferase(ALT)in a dose-dependent manner.Moreover,Oroxylin A treatment remarkably inhibited extracellular matrix(ECM)deposition,and significantly down-regulated the mRNA and protein expression of liver fibrosis markers including al(Ⅰ)collagen,fibronectin,α-SMA,PDGF-βR,and TGF-β1R in CCl4-induced murine model of liver fibrosis.Interestingly,Oroxylin A might protect the liver against CC14-induced injury by suppressing inflammation.Immunofluorescent assay showed that the expression of pro-inflammatory cytokines including nuclear factor-KB(NF-kB).tumor necrosis factor a(TNF-α).IL-6 and IL-8 were down-regulated by treatment with Oroxylin A in a dose-dependent manner.Moreover,the levels of IL-1 β,IL-4,IL-10,and IL-18 in liver and serum were determined by ELISA assays.As expected,Oroxylin A not only inhibited the expression of proinflammatory factors,but also promoted the production of anti-inflammatory factors.Further experiments demonstrated that the elevated expression of NF-kB,NLRP3,TNF-a,IL-1 β,IL-6 and IL-8 in fibrotic liver was abolished by treatment with Oroxylin A.Consistently,experimental results in vitro showed that Oroxylin A treatment reduced the mRNA and protein expression of HSC activation markers a-SMA,desmin,a 1(Ⅰ)collagen,fibronectin,TGF-(3,and TNF-a,in a dose dependent manner.Moreover,the immunoblot analysis,ELISA assay,and immunofluorescence demonstrated that Oroxylin A also reduced the expression of inflammatory cytokines including NLRP3,cleaved-IL-1β,pro-IL-1β,pro-IL-4,pro-IL-18,and IL-1β in a dose-dependent manner in vitro.Overall,these results indicate that Oroxylin A alleviates liver fibrosis and inflammation.Secondly,we explored the correlation between the anti-fibrotic effects of Oroxylin A and the regulation of HSC autophagy.Attractively,Oroxylin A treatment markedly up-regulated the expression of autophagy makers,LC3-Ⅱ,Atg3,Atg4,Atg5,Beclinl/Atg6,Atg7,Atg9,Atg12,and Atg14,and apparently reduced the expression of autophagy substrate p62 in CCl4-induced murine model of liver fibrosis.Autophagy inhibitor chloroquine(CQ)blocked the promoting effect of Oroxylin A on autophagy,and thus impaired the inhibitory effect of Oroxylin A on liver fibrosis markers and inflammatory factors,indicating that blocking autophagy in vivo abolished the inhibitory effects of Oroxylin A on hepatic fibrosis and inflammatory microenvironment.Consistently,results in vitro showed that Oroxylin A treatment promoted the protein expression of autophagy marker LC3-II in activated HSCs.Moreover,treatment with Oroxylin A markedly up-regulated the protein expression of Atg3,Atg5,Beclinl/Atg6,Atgl2,and Atg14 in a dose-dependent manner in activated HSCs.Next,three important methods were used to determine autophagic flux in Oroxylin A-treated HSCs.First of all,western blot analysis revealed that Oroxylin A combined with CQ increased the protein expression of LC3-Ⅱ compared with Oroxylin A-treated HSCs.Secondly,immunofluorescent staining of autophagy substrate p62 was performed on Oroxylin A-treated HSCs,using p62 specific antibody.As expected,Oroxylin A treatment remarkably reduced the expression of autophagy substrate p62 in a dose-dependent manner.Thirdly,typical structures of autophagy were observed by the transmission electron microscopy(TEM)in vehicle-treated or Oroxylin A-treated HSCs.Interestingly,vehicle-treated HSCs displayed a certain number of autophagic vesicles in the cytoplasm.In contrast,treatment with Oroxylin A resulted in a remarkable increase in autophagic vacuoles,representing an intensified autophagy.Importantly,inhibition of autophagy by specific inhibitor 3-methyladenine(3-MA)completely abolished Oroxylin A-induced anti-fibrosis effect,indicating that activation of autophagy was required for Oroxylin A to alleviate liver fibrosis.Overall,these results showed that Oroxylin A inhibited liver fibrosis and improved the inflammatory microenvironmental by promoting autophagy of HSCs.Thirdly,it was clarified that inhibition of the PI3K-AKT-mTOR signaling pathway could promote HSC autophagy,thereby improving liver fibrosis and inhibiting the formation and release of inflammatory factors.The results showed that the ratio of p-PI3K/PI3K,p-AKT/AKT,p-mTOR/mTOR in activated HSC was reduced by Oroxylin A.Then we used the mTOR plasmid combined with Oroxylin A for reverse validation.It was found that mTOR plasmid significantly weakened the Oroxylin A-induced p-mTOR/mTOR inhibition.Moreover,mTOR plasmid significantly impaired the promoting effect of Oroxylin A on autophagy markers LC3-Ⅱ,Atg3,Atg5-Atg12,Atg6/Beclin 1,Atg9,Atg12,Atg14 and inhibition of p62.Furthermore,mTOR overexpression significantly damaged Oroxylin A-induced HSC inhibition.Importantly,mTOR overexpression also significantly impaired the inhibitory effects of Oroxylin A on the expression of proinflammatory cytokines,including TNF-a,IFN-γ,IL-1β,IL-6,IL-8,NF-kB,NLRP3,Cleaved-IL-1β,and Pro-IL-1β.At the same time,mTOR overexpression also significantly impaired the promotion effects of Oroxylin A on the expression of anti-inflammatory cytokine IL-10.Thus,it can be seen that Oroxylin A may promote HSC autophagy by inhibiting the PI3K-AKT-mTOR signaling pathway,and thus play a role in inhibiting HSC activation and inflammation.Fourthly,we aimed to determine whether Oroxylin A inhibited the PI3K-AKT-mTOR signaling pathway by influencing ROS generation.The results showed that the level of ROS in activated HSCs was reduced by Oroxylin A in both dose-dependent and time-dependent manners.Moreover,Oroxylin A significantly increased the GSH/GSSG ratio in activated HSC s.In addition,we found that inhibition of ROS accumulation may be related to promoting HSC autophagy.Interestingly,it was found that autophagy inhibitor 3-MA can significantly weaken Oroxylin A-induced inhibitory effect on ROS generation and promoting effect of GSH/GSSG ratio.Then we combinated of the GSH inhibitor BSO and Oroxylin A together to treat activated HSC for reverse validation.The results showed that BSO significantly damaged Oroxylin A-induced ROS generation,p-PI3K/PI3K,p-AKT/AKT,p-mTOR/mTOR inhibition,and autophagy activation.In addition,BSO also impaired the inhibitory effect of Oroxylin A on HSC activation markers,including a-SMA,al(I)collagen,Fibronectin,Desmin,TGF-β1R,and PDGF-βR.Importantly,BSO also significantly weakened the inhibitory effects of Oroxylin A on pro-inflammatory factors,including TNF-α,IFN-γ,IL-1β,IL-6,IL-8,IL-18,NLRP3,NF-KB,Pro-IL-1β,and Cleaved-IL-1β.Moreover,BSO also significantly impaired the promotion effects of Oroxylin A on the expression of anti-inflammatory cytokine IL-10.Altogether,these data showed that Oroxylin A inhibited PI3K-AKT-mTOR signaling pathway by influencing ROS generation.In summary,this study for the first time revealed that Oroxylin A relieved liver fibrosis and inflammation through regulating ROS-PI3K-AKT-mTOR-autophagy circuit.Given that effective anti-fibrosis drugs are lacking clinically,Oroxylin A is expected to be developed as a new anti-fibrosis drug in the future.