Mechanistic Studies On Anti-Liver Fibrosis Of Oroxylin A Down-regulates LDH-A To Block HSC Aerobic Glycolysis-Dependent Contraction

BackgroundLiver fibrosis is a compensatory process for chronic liver injury such as viral hepatitis,alcoholic liver,fatty liver.In fibrogenic liver,the quiescent HSCs undergo transdifferentiation into myofibroblasts with proliferative and migratory properties,and subsequently secrete massive extracellular matrix molecules,accumulating in liver parenchyma and promoting the pathogenesis of hepatic fibrosis.The enhanced contractility of hepatic stellate cells increases the resistance of sinusoidal blood flow,thus exacerbating the capillarization and remodeling of hepatic sinusoids,In addition,the contractile force generated by HSCs aggravates scar contracture mediating hepatic fibrosis.Cell contraction is a highly energy-consuming process.New evidence suggests that aerobic glycolysis is a faster and shorter energy production pathway when HSC is activated and can be used to meet the high requirements of rapid proliferation.Therefore,revealing and targeting the role of aerobic glycolysis in controlling of HSC contraction may become a new strategy to prevent liver fibrosis.There are great basic and clinical studies revealed that traditional Chinese medicine has a definite curative effect on liver fibrosis.Our previous study showed that oroxylin A as one of the important active components of Scutellaria baicalensis has a definite curative effect on anti-hepatic fibrosis.Previous studies have found that oroxylin A ameliorated hepatic fibrosis via inhibiting angiogenesis of LSEC in liver;and oroxylin A reduced liver fibrosis associated with induction of HSC autophagy.We hold that the underlying mechanisms of the anti-fibrotic effects of oroxylin A are not fully understood.Here,we explore to clarify the effects of oroxylin A on hepatic fibrosis and to elucidate the regulation of HSC contraction by oroxylin A.Our findings made oroxylin A as a candidate anti-hepatic fibrosis drug and provide novel insights for the study of the underlying mechanism of oroxylin A on liver fibrosis.Methods1.In vitro studiesHSC-LX2 cell line was used in this study.Collagen lattice contraction and skeleton staining showed the ability of HSC contraction;Western Blot and immunofluorescence were used to detect the level of phosphorylated of MLC2.Glucose uptake,glucose consumption,lactate production,extracellular acidification rate kit were performed to detect glycolysis flux and level of glycolytic product.Real-Time PCR and Western Blot were used to detect levels of gene and protein of glycolytic rate-limiting enzymes:hexokinase 2,phosphofructokinase 1 and M2 pyruvate kinase;Activity of glycolytic rate-limiting enzyme were measured using ELISA assay kits.Overexpression plasmid and siRNA transfection were used to validate the effect of LDH-A2.In vivo studiesThirty male ICR mice were randomly divided into 5 groups(n=6):normal control group,CCl4 model group,CCl4+oroxylin A treatment+adenovirus empty vector,CCl4+oroxylin A treatment+LDH-A plasmid adenovirus.HE,Masson,Sirius red staining of liver tissue showed liver structure and collagen deposition,Immunohistochemistry staining of a-SMA showed HSC activation in liver tissue.Scanning electron microscope was used to observe hepatic sinusoidal capillary.Levels of serum alanine aminotransferase,aspartate aminotransferast,total bilirubin and indirect bilirubin were determined by commercial kits,and the expression of serum hyaluronic acid,laminin and Ⅲ procollagen were detected by ELISA assay kits.Real-time PCR were used to detect the levels of target gene.Western blot and Immunofluorescence double staining were used to detect the levels of protein in liver tissue.Clinical liver samples(n=5)were randomly selected from Nanjing second Hospital of Nanjing University of Chinese Medicine.Real-time PCR was used to investigate the expression of LDH-A gene in the livers of patients with hepatic fibrosis and normal patients.ResultsOroxylin A inhibited HSC contraction associating with inhibition of HSC skeleton formation and MLC2 phosphorylation.Oroxylin A reduced aerobic glycolysis capacity in HSC.Oroxylin A inhibited expression of genes and protein of aerobic glycolysis rate-limiting enzymes HK2,PFK1 and PKM2,and also decreased enzymes activity Glycolysis inhibitor 2-DG confirmed that oroxylin A blocked aerobic glycolysis leading to inhibition of HSC contraction.Further exploration found that oroxylin A has a significant regulatory effect on LDH-A(potential target of aerobic glycolysis).We found that a higher gene expression of LDH-A in livers of patients with liver fibrosis than normal patients.While oroxylin A downregulated gene and protein expression,and enzyme activity of LDH-A in HSC.Next,LDH-A pharmacological inhibitor,LDH-A siRNA and LDH-A overexpression plasmid verified that oroxylin A played a key role in blocking HSC glycolysis and inhibiting HSC contraction by down-regulating LDH-A.Finally,the in vivo adenovirus packaging LDH-A plasmid verified that effects of oroxylin A in anti-hepatic fibrosis through the regulation of LDH-A.Oroxylin A reduced serum liver injury and liver fibrosis index which were upregulated during liver fibrosis.Oroxylin A alleviated the disorder of liver structure and collagen deposition during liver fibrosis.Oroxylin A also inhibited HSC activation and restored the fenestrae of liver sinusoidal endothelial cells in liver fibrosis.However,the intervention of oroxylin A on hepatic fibrosis was blocked by LDH-A overexpression.In addition,oroxylin A blocked the elevated HSC contraction and HSC aerobic glycolysis during fibrosis.But overexpression of LDH-A diminished oroxylin A improvement effects during liver fibrogenesis.In vivo and in vitro results showed that down-regulation of LDH-A and inhibition of aerobic glycolysis-dependent contraction of HSC were the basis of oroxylin A’s anti-hepatic fibrosis effect.ConclusionIn conclusion,our current work linked the aerobic glycolysis pathway to the contractile phenotype of HSCs,and uncovered that oroxylin A blocked glycolysis-dependent HSC contraction and reduced hepatic fibrosis through downregulation of LDH-A.We suggested LDH-A as a promising target for disruption of HSC metabolism and treatment of liver fibrosis.Our studies provided an experimental evidence for oroxylin A regulating LDH-A for the improvement of liver fibrosis.