Study On The Molecular Mechanism Of Fraxetin Against Carbon Tetrachloride-induced Liver Fibrosis In Rats

Objective:Liver fibrosis is a kind of repair response which is answerable for the long-term invasion of various damage factors in our body.If no treatment as early as possible,liver fibrosis can progress to an irreversible stage of cirrhosis,even hepatoma which finally lead to death.Fraxetin,a coumarin monomer compound extracted from cortex cinerea,has many pharmacological activities such as antibacteria,antioxidant and anti-tumor.Recent studies have shown that fraxetin has a good anti-fibrosis effect,but the related mechanisms were not clear.Therefore,this study aims to further clarify the anti-hepatic fibrosis effect of fraxetin,and clarify the molecular mechanisms.Methods:48 male SD rats were divided into four groups at random which were named as normal group,model group,fraxetin 25 mg/kg and 50 mg/kg group.Except normal groups,the experimental model of liver fibrosis was founded by carbon tetrachloride?CCl₄?.Simultaneously,the rats of fraxetin groups were treated with fraxetin?25 mg/kg or 50 mg/kg?.Normal groups received equal volumes of saline and peanut oil.After 8weeks,all rats were killed,and then the liver tissue and serum were collected.The subsequent operations as follows:?1?the liver function makers were tested by a fully automatic biochemical analyser,and the fibrotic makers were tested by radioimmunoassay.?2?HE and masson stainings were performed for the examination of inflammation injury and collagen deposition.?3?The staining of immunohistochemistry and immunofluorescence were performed for inspecting the expression of?-SMA and the key proteins of MAPKs and Bcl-2/Bax pathways.?4?The key proteins of NF-?B/I?B?,MAPKs,Bcl-2/Bax and?-SMA were tested by Western blot.?5?The mRNA levels of inflammatory factors and HSCs activation makers were tested by Real-time PCR.Results:1.Fraxetin enable to alleviate the survival status and liver index of fibrotic rats.2.The serological results showed that fraxetin could effectively decrease liver function makers?AST,ALT,TBIL?and fibrotic makers?HA,LN,?-C,P?NP?.3.The results of HE and masson's stainings indicated that fraxetin could reduce inflammation and collagen deposition in fibrotic liver.4.The results of immunohistochemical and fluorescence staining were shown as follow:after fraxetin treatment,?1?the expression of p-p38 MAPK,p-JNK and p-ERK were decreased in fibrotic liver.?2?the ratio of Bcl-2/Bax was down-regulated.?3?the expression of?-SMA was inhibited in fibrotic liver.5.The results of Western blot were shown as follow:?1?fraxetin could inhibit NF-?B nuclear transportation through blocking the phosphorylation of I?B?in cytoplasm.?2?fraxetin could inhibit the phosphorylation of p38 MAPK,JNK and ERK in fibrotic liver.?3?fraxetin could up-regulate the ratio of Bcl-2/Bax,and decrease the expression of Cleaved Casepase-3.?4?fraxetin could inhibit the expression of?-SMA in the fibrotic liver.6.The results of Real-time PCR showed that fraxetin could decrease the mRNA levels of inflammatory factors?TNF-?,IL-6,IL-1?,COX-2,TGF-??,HSCs activation makers??-SMA,Vimentin,Desmin?and collagen regulator?Colla?,Colla?,TIMP-1?and inversely MMP-1 were increased in fibrotic liver.Conclusion:This study revealed that fraxetin possessed therapeutic potential for preventing liver fibrosis induced by CCl₄ in SD rats.The anti-fibrotic activity of fraxetin is closely linked to its capacity to restrain NF-?B/I?B?and MAPKs inflammatory responses in conjuction with Bcl-2/Bax apoptotic signaling pathway.These hepato-protective properties make fraxetin a promising therapeutic drug candidate for treating liver fibrosis.