Protective Effect And Part Of Mechanisms Of Prunella Vulgaris Sulfated Polysaccharide On Liver Fibrosis In Rats

Aim:Prunella vulgaris sulfated polysaccharide（PVSP）,the main bioactive compound of PV that possesses anti-inflammatory,antioxidant,immunoregulatory and anti-tumor activities.However,the antifibrotic effects of PVSP remains poorly understood.The present study was designed to the effect of Prunella vulgaris sulfated polysaccharide（PVSP）on TGF-β1-activated HSC-T6 cell and CCl₄-induced liver fibrosis,and to explore related mechanism.Methods:HSC-T6 cells were stimulated by TGF-β1and treated with different doses of PVSP（50,100,200μg/ml）,the HSC-T6 proliferation were detected by CCK8.The expression levels ofα-smooth muscle actin（α-SMA）and collagen type I（Col-I）mRNA were detected by qRT-PCR.The production ofα-SMA and Col-I protein was measured by Western blot.The liver fibrosis model was established by intraperitoneal injection with 40%CCl₄.The rats were randomly divided into five groups:a nomal control group,a positive control group（colchicines 0.25 mg/kg）,a high-PVSP group（PVSP 300 mg/kg）,a low-PVSP group（100 mg/kg）and a model group.Serum levels of ALT（aspartate aminotransferase）,AST(aspartate aminotransferase,HA（hyaluronic acid）,LN（laminin）,PC-III（procollagen-III-peptide）and C-IV（type IV collagen）were examined by ELISA（enzyme linked immunosorbent assay）in dfferent groups.Hematoxylin and eosin（HE）staining and sinus red staining were used to examine the degree of hepatic fibrosis.RT-PCR was used to detect the expression ofα-SMA,Col-I and TGF-β₁ mRNA in liver tissues of rats,and immunohistochemistry Staining was detected the content ofα-SMA,Col-I and TGF-β₁ protein in liver tissues.Moreover,the levels of Smad3,Smad2/3 and Smad7in liver tissues were examined by Wertern blot.Results:In vitro,PVSP could inhibit the proliferation of HSC-T6 induced by TGF-β₁.Compared with the TGF-β₁ group,PVSP significantly reduced mRNA and protein expression ofα-SMA and Col-I in HSC-T6 cells（P<0.05 or P<0.01）.In vivo,in the model group,serum levels of ALT,AST,HA,LN,PC-III and C-IV increased obviously compare with the normal group（P<0.05 or P<0.01）.Compared with the model group,the levels of ALT,AST,HA,LN,PC-III and C-IV significantly deccreased in the PVSP groups（P<0.05）,and the high-PVSP group had a better effect（P<0.05）.The pathological results showed that PVSP could collagen deposition and the degree of hepatic fibrosis.The expression ofα-SMA,Col-I and TGF-β₁ mRNA and protein in liver tissues rats increased obviously compare with the normal group.The expression of Smad3 and Smad2/3 increased in model group compare with the normal group（P<0.05 or P<0.01）,while the content of Smad7decreased（P<0.05）.Compared with the model group,the levels of Smad3 and Smad2/3 were significantly reduced（P<0.05）,and the expression of Smad7decreased（P<0.05）.Conclusion:PVSP treatment could inhibit TGF-β1-activated HSCs.PVSP treatment also could alleviate CCl₄-induced liver fibrosis,and its mechanism may be through regulating the TGF-β₁/Smads signal pathway,thereby inhibiting proliferation and activation of HSCs,downregulating the formation of ECM,finally could reverse the development of hepatic fibrisis.This study provided a basis for using PVSP to treat hepatic fibrosis and may encourage further experimental and clinical trials for developing new drugs to treat this disease.