Improvement And Mechanism Of Phillyrin On Liver Fibrosis

Liver fibrosis（LF）is a common reaction to chronic liver tissue damage and inflammation caused by hepatotropic virus infection,abnormal lipid metabolism,cholestasis,chronic alcoholism,and autoimmune disease.It is a vital pathological link in the development of cirrhosis.According to the China Business Intelligence Big Health Data,in 2015,the number of patients with fatty liver disease in China exceeded 300 million.And it is estimated that the number of patients with hepatitis B virus and hepatitis C virus or chronic fatty liver disease is expected to reach 447 million by 2020.Effective treatment of liver fibrosis is a major way to prevent the disease from chronic hepatitis progress to cirrhosis or liver cancer.However,there are no effective drugs for the prevention and treatment of liver fibrosis around the world.It is urgent to find a safe and effective medicine.Forsythia is a traditional Chinese medicine for reducing swelling,heat-clearing and detoxifying,which can reduce inflammation,regulate blood pressure,lower blood lipids,and inhibit pathogenic microorganisms.Phillyrin（PHI）is a natural small molecule compound derived from forsythia and PHI can ameliorate lung injury,kidney injury and brain injury.It has a wide range of pharmacological effects such as anti-inflammatory and anti-oxidation.In view of the anti-inflammatory and antioxidant pharmacological effects,PHI may have protective effect on liver fibrosis.However,the effect and mechanism of PHI on liver fibrosis is not clear.In this experiment,liver fibrosis in mice was induced by carbon tetrachloride（CCl₄）or alpha naphthylisothiocyanate（ANIT）to evaluate the anti-hepatic fibrosis effect of PHI.To further evaluate the mechanism of PHI,hepatic stellate cells and RAW264.7 cells were used.Methods1.Establishment of liver fibrosis model and PHI interventionMale C57BL/6 mice were administered with 10%CCl₄ in olive oil or 0.05%ANIT diet for 4 weeks to establish animal models of liver fibrosis.Simultaneously,the mice were intraperitoneally given PHI and normal saline.Finally,the anti-fibrotic pharmacological effects of PHI were evaluated by methods such as liver morphology analysis,liver function index detection,histopathological analysis,hydroxyproline content determination,RT-PCR and Western blot.2.MTT assayL02,HSC-T6,LX-2,and RAW264.7 cells were incubated with 0.1μM-100μM PHI for24h,48h,and 72h;RAW264.7 and HSC-T6 cells were stimulated with LPS as well as PHI treatment,the cell proliferation was measured by MTT assay.3.Transwell assayRAW264.7 and HSC-T6 cells were stimulated with LPS as well as PHI treatment.To analyze cell migration ability,Transwell test was used.4.Cytokine assayRAW264.7 and HSC-T6 cells were stimulated with LPS as well as PHI treatment,then the concentration of inflammatory factors was detected by ELISA.5.ROS testHSC-T6 cells were stimulated with LPS as well as PHI treatment,then the intracellular ROS was detected by Flow cytometry.6.Detection of protein expression level and nuclear translocationRAW264.7 cells were stimulated with LPS and cells were given PHI treatment.Immunofluorescence was used to analyze the nuclear translocation of NF-κB p65.LX-2 cells were stimulated with TGF-β₁ or TNF-αas well as PHI treatment.The differences in expression ofα-SMA,nuclear translocation of NF-κB p65 and Smad_2/3/3 were identified by using Immunofluorescence.7.Detection of protein expression and phosphorylationRAW264.7 and HSC-T6 cells were stimulated with LPS as well as PHI treatment,then Western blot analysis were performed to determine NF-κB p65 phosphorylation andα-SMA protein expression.LX-2 cells were stimulated with TGF-β₁ or TNF-αas well as PHI treatment.Western blot analysis was used to detectα-SMA protein expression,phosphorylation of NF-κB p65and Smad_2/3.Results1.In mice with liver fibrosis,PHI showed excellent protective effect against CCl₄ or ANIT-induced liver injury and fibrosis.The degree of liver damage,collagen content,hydroxyproline content,the level of inflammation and fibrosis gene expression were reduced.Moreover,the phosphorylation level of NF-κB p65 and Smad_2/3 in liver tissue of mice in the PHI group was also significantly reduced compared to the model group.2.0.1μM-30μM PHI had no effect on the proliferation of L02,HSC-T6,LX-2,RAW264.7 cells at 24h,48h,and 72h.And PHI markedly suppressed the proliferation,migration of RAW264.7 and HSC-T6 cells induced by LPS.3.PHI can dramatically decrease the ROS level by LPS-induced HSC-T6 cells,and suppress the secretion of inflammatory factors and the phosphorylation of NF-κB p65 in macrophages and hepatic stellate cells.4.PHI can obviously suppressα-SMA protein expression and Smad_2/3/3 protein phosphorylation in hepatic stellate cells.ConclusionPHI has a significant protective effect against liver fibrosis,and its mechanism is related to inhibit the inflammatory response of macrophages and the activation of hepatic stellate cells through inhibition of NF-κB signal pathway and TGF-β₁/Smad_2/3 signal pathway.