Oroxylin A Inhibits YAP/HIF-1? Signaling For Anti-angiogenesis In Liver Fibrosis

Objective:Liver fibrosis is a common pathological process of many chronic liver diseases,and also a self-repair response to the tissue after chronic liver injury.Liver fibrosis is reversible,however,continuing development without treatment will eventually lead to liver cirrhosis even liver cancer.Therefore,it is very important to postpone or reverse the pathological process of liver fibrosis to reduce the mortality of liver cirrhosis and liver cancer.The pathogenesis of liver fibrosis is more complex,angiogenesis as the main feature of injury-repair response,is the process of forming new blood vessels on the basis of existing blood vessels,which closely participate in the development of liver sinusoidal remodeling and fibrosis.At present,many studies have focused on targeting of LSEC capillarization to inhibit the pathological angiogenesis in the liver and delay the progression of liver fibrosis.However,the existing anti-fibrosis drugs are not enough effective,and have many adverse reactions.It is urgent to develop new and effective anti-fibrosis drugs.Oroxylin A is a kind of flavonoid extracted from herbs,which has extensive biological activities,including anti-tumor,antibacterial,anti-inflammatory,antiviral,neuroprotective and so on.Oroxylin A can promote the apoptosis of tumor cells and inhibit angiogenesis.It has been found that oroxylin A can inhibit the angiogenesis of tumor in nude mice,but the effect of oroxylin A on angiogenesis in liver fibrosis has not been reported.In this paper,the role of oroxylin A in the angiogenesis of liver fibrosis and its underlying mechanism will be preliminarily investigated in vivo and in vitro.Methods:It was mainly carried out through three parts of the experiment.1)The first part:Mice were intraperitoneal injected with CCl4 4 weeks to induce liver fibrosis,after which additional 4 weeks oroxylin A(40 mg/kg)was treated with continuing CCl4.Liver tissue sections were stained with HE,Masson and Sirius red;Immunofluorescence staining was used to observe the angiogenesis index,CD31;Screening the appropriate oroxylin A concentration by MTT;Western blotting and immunofluorescence staining were used to detect the protein expression of Ang-2,VEGF-A,HIF-la in LSEC.2)The second part:Screened suitable concentration of HIF-la inhibitor ACF by MTT;Western blotting investigated the effect of ACF and oroxylin on the protein expression of VEGF-A,CD31;Matrigel angiogenesis assay analysed the effect of ACF and oroxylin A on the formation of blood vessels;Mice were intraperitoneal injected with CCl4 4 weeks to induce liver fibrosis,after which mice were given HIF-1? overexpression plasmid and oroxylin A(40 mg/kg),liver tissue sections were stained with HE,Masson and Sirius red;Serum VEGF-A,Ang-2,LN,IV-C,HA level and ALT,AST and ALP activity were determined;Dual immunofluorescence staining was performed to detect angiogenesis index CD31 and HIF-la;Isolated mice primary LSEC and detected HIF-1?,VEGF-A protein expression.3)The third part:Transfected LSEC with YAP siRNA or plasmid respectively,transfection efficiency was determined,after successfully transfection,treated LSEC with hypoxic mimic CoCl2,Western blotting was performed to detect VEGF-A and Ang-2 protein level;Protein expression HIF-1? in cytoplasm and nuclear were detected;Immunofluorescence staining was used to observe the expression of HIF-1? and VEGF-A;Treated LSEC with different concentrations oroxylin A,phosphorylation and nuclear translocation of YAP was investigated by Western blotting;Treated LSEC with YAP siRNA or plasmid combined oroxylin A,protein levels of VEGF-A,Ang-2 and HIF-la was explored by Western blotting;VEGF-A was investigated by immunofluorescence staining.Results:1.Masson and Sirius red staining showed that oroxylin A can significantly reduce the liver collagen formation;HE staining showed that oroxylin A can improve the pathological structure in fibrotic liver;Immunofluorescence staining demonstrated that the expression of angiogenesis index CD31 was reduced effectively by oroxylin A;and the positive region for CD31 staining was also found positive-HIF-la-staining strongly,which were highly coincidence;20,30 and 40?M oroxylin A were identified by MTT assay;Western blotting analyses indicated that oroxylin A could dose-dependently reduced the high expression of HIF-1?,VEGF-A and Ang-2 induced by hypoxic mimic CoCl2,and immunofluorescence staining was similar;In addition,Matrigel angiogenesis assay also confirmed that oroxylin A could significantly inhibit LSEC angiogenesis.2.Western blotting analyses exhibited that the effect of ACF and oroxylin A were similar,both can decrease the protein levels of CD31,VEGF-A;Matrigel experiments verified the anti-angiogenic effect of ACF;Masson staining and Sirius red staining showed that HIF-1?overexpression weakened the inhibitory effect of oroxylin A on collagen expression;HE staining demonstrated that the improvement of oroxylin A on pathological liver structure was partially offset by HIF-la plasmid;Oroxylin A reduced the high expression of serum VEGF-A,Ang-2,LN,IV-C,HA level and ALT,AST and ALP activity induced by CCl4,but this effect was abrogated by HIF-la overexpression plasmid;Dual immunofluorescence staining showed that the suppression of oroxylin A on regulating HIF-la was located in LSEC,which was confirmed by isolated mouse primary LSEC.3.Western blotting analyses exhibited that YAP siRNA can reduced the increased levels of HIF-1? and Ang-2,VEGF-A induced by hypoxia;Immunofluorescence staining showed that YAP siRNA decreased the expression of HIF-la and VEGF-A,YAP overexpression plasmid group showed the opposite effect;Western blotting analyses demonstrated that hypoxia could decrease the phosphorylation level of YAP and promote the nuclear translocation of YAP,oroxylin A treatment can significantly raise p-YAP level,decrease the expression of YAP in the nucleus;Oroxylin A combined with YAP plasmid counteract the inhibition of oroxylin A on pro-angiogenic factor;It is observed that both YAP siRNA and oroxylin A can significantly reduce the VEGF-A expression under hypoxic conditions by immunofluorescence staining,combination of them indicated stronger resistance to VEGF-A expression,however,combined use of YAP plasmid and oroxylin A could partially abolish the suppression effect of oroxylin A on VEGF-A.Conclusions:1.Oroxylin A can protect the liver injury induced by CCl4 and significantly reduce the collagen deposition in the fibrotic liver.2.Oroxylin A may regulate pathological angiogenesis in fibrotic liver through HIF-1?-dependent pathway.3.The anti-angiogenesis effect of oroxylin A on LSEC may be related to the regulation of YAP/HIF-la signaling pathway.The results of this study support experimental evidence for anti-angiogenesis in liver fibrosis by oroxylin A,and provide a new research perspective for the development of anti-fibrosis drugs.