| Objective:Liver fibrosis is the repair reaction of various chronic liver injuries in the body,and reversible.If chronic liver injuries continue to exist and develop,liver fibrosis can deteriorate into liver cirrhosis.Ginkgo biloba extract(GBE)is extracted from dry ginkgo biloba leaveswhich has a variety of pharmacological effects such as antioxidant,free radical scavenging,anti-platelet aggregation and so on.In recent years,studies have found that GBE has a good activity in anti-liver fibrosis.This paper mainly explored the inhibitory effect of GBE on CCl4 induced liver fibrosis in rats and the molecular mechanism of inhibition by animal experiments.Methods:48 healthy male Sprague--Dawley(SD)rats were randomly divided into four groups:Normal control group(Control group),Gastric perfusion model group(Model group),GBE high dose treatment group(High dose group)and GBE low dose treatment group(Low dose group),12 rats in each group.Except for the control group,the rats in the other three groups were subjected to intragastric administration of 50%CCl4/peanut oil(1mL·kg-1)twice a week to establish liver fibrosis model.During the CCl4 exposures,the rats in high dose group and low dose group were also given GBE by intraperitoneal injection once per day at doses of 30mg/kg and 15mg/kg body weight,respectively.After 8 weeks,the full automatic biochemical analyzer was used to measure the liver function indexes of total bilirubin(TBIL),aspartic acid transaminase(AST)and alanine aminotransferase(ALT).Meanwhile Chemiluminescence detection was used to measure the liver fibrosis indexes of hyaluronic acid(HA),type IV collagen(IV-C),laminin(LN),and type III procollagen(PIIINP).In addition,the morphology of liver was observed and the liver samples were collected.HE staining and Masson staining were used to examine the pathological changes of liver tissues;Western blot technique was used to detect the changes of expression levels of nuclear protein(NF-?B),cytoplasmic proteins(NF-?B and I?B?)and total proteins(p38MAPK?p-p38MAPK?Bcl-2?Bax and Caspase-3)in liver tissues;The effects of GBE on the expression of p38MAPK,NF-?B,Bcl-2,and Bax in liver tissues of rats were investigated by immunostaining;HSCs activation factors(Collal?Colla2?MMP-1?TIMP-1?SMA and Desmin)and inflammation related factors(COX-2?IL-1??IL-6?TNF-a and TGF-?)were studied by semi-quantitative RT-PCR and Real-time PCR detection to investigate the effects of GBE on HSCs and inflammationResults:1.After 8 weeks of intragastric administration of 50%CCl4/peanut oil(1mL·kg-1),compared with the Control group,the body weightof rats in the Model group was significantly decreased(P<0.05),and the liver weight was significantly increased(P<0.05).Compared with the Model group,the body weightof rats in the Low dose group was significantly increased(P<0.05),and the liver weighthad no significant difference(P>0.05),the body weightof rats in the High dose group was increased significantly(P<0.05),liver weight was significantly reduced(P<0.05).Compared with the Low dose group,the body weightof rats in the High dose group was significantly increased(P<0.05),and the liver weight was significantly reduced(P<0.05).There was no significant difference between the High dose group and the Control group(P>0.05).2.The calculation results of liver index(liver weight/body weight)showed that compared with the Control group,the liver index in the Model group was significantly increased(P<0.05).Compared with the Model group,the liver indexes in the Low dose group and High dose groupwere significantly lower(P<0.05).Compared with the Low dose group,the High does groupsignificantly reduced the liver index(P<0.05).And the liver index of the High dose group was significantly higher than that ofthe Control group(P<0.05).3.The results of liver function indexes(TBIL/AST/ALT)showed that,compared with the Control group,the liver function indexes of the Model group were significantly increased(P<0.05).Compared with the Model group,GBE administration(the High dose group and the Low dose group)could significantly reduce the liver function indexes(P<0.05).Compared with the Low dose group,the liver function indexes in the High dose group were significantly lower(P<0.05)4.The results of liver fibrosis indexes(HA/IV-C/LN/PIIINP)showed that,compared with the Control group,the liver fibrosis indexes of the Model group were significantly increased(P<0.05).Compared with the Model group,GBE administration(the High dose group and the Low dose group)could significantly reduce the liver fibrosis indexes(P<0.05).Compared with the Low dose group,the liver fibrosis indexes in the High dose group were significantly lower(P<0.05)5.The pathological examination showed that collagen deposition in the Model group was significantly increased compared with that in other groups and the degree of liver fibrosis was significantly increased(P<0.05).After GBE administration(in High dose group and Low dose group),collagen deposition was significantly decreased and liver fibrosis improved obviously.In addition,the degree of liver fibrosis was significantly improved with the increase of GBE dose(P<0.05),but the recovery level from liver fibrosis of GBE administration groups still did not return to the level of the Control group6.The results of Western Blot experiment showed that GBE supplement could effectively reduce the protein levels of p38MAPK and p-p38MAPK(P<0.05),which indicated that GBE can inhibit the p38MAPK signal in rats with liver fibrosis.GBE administration could effectively reduce the nuclear protein(NF-?B)and increase the cytoplasmic protein(NF-?B?I?B?)levels(P<0.05),which showed that GBE can regulate the NF-?B/I?B? signal in rats with liver fibrosis.GBE administration could effectively increase the level of Bcl-2 protein(P<0.05),while reducing the levels of Bax and Caspase-3 protein(P<0.05),which indicated that GBE can regulate Bcl-2/Bax signaling in rats with liver fibrosis7.The results of immunohistochemical parallel experiment showed that GBE administration could effectively reduce the protein levels of p38MAPK?Bax and NF-?B(P<0.05),while increasing the Bcl-2 protein level(P<0.05),which is consistent with the results of Blot Western.This indicated that GBE can regulate p38MAPK signaling pathway,NF-?B/I?B? signaling pathway and Bcl-2/Bax signaling pathway in rats with liver fibrosis8.RNA levels of HSCs activation markers(Collal?Colla2?MMP-1?TIMP-1?a-SMA and Desmin)showed that GBE administration could effectively reduce the RNA levels of HSCs activating factors in rats with liver fibrosis(P<0.05),which indicated that GBE can inhibit the activation of HSCs in the process of liver fibrosis9.RNA levels of inflammation related factors(Cox-2?IL-1??IL-6?TNF-a and TGF-?)showed that GBE administration could effectively reduce the RNA levels of inflammation related factors in rats with liver fibrosis(P<0.05),indicating that GBE can inhibit the inflammatory reaction in the process of liver fibrosisConclusions:GBE can effectively inhibit the liver fibrosis induced by CCl4 in rats.The underlying mechanisms may include:GBE inhibits p38MAPK signaling pathway and regulates NF-?B/I?B? signaling pathway to effectively inhibit inflammation and HSCs activation.In addition,GBE can also inhibit the Bcl-2/Bax signaling pathway,which can inhibit the hepatocyte apoptosis.GBE could effectively inhibit liver fibrosis in rats by influencing these three signaling pathways... |
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