| Objective:Liver fibrosis is the common sequel of chronic liver diseases and is associated withhigh morbidity and mortality in affected patients.As the perfect way to preventprogression to liver fibrosishas not been discovered, it is important to establish aneffective and safe antifibrotic therapy. Rho-kinase has pleiotropic functions includingthe regulation of cellular contraction, motility,morphology, polarity, cell division, andgene expression. Pharmacological analyses have revealed that Rho-kinase is involved ina wide range of diseases such as vasospasm, pulmonary hypertension, nerve injury, andglaucoma, and is therefore considered to be a potential therapeutictarget.Gan-fu-kang(GFK) Decoction is a traditional Chinese medicine developed bypathophysiology department of Da Lian medical university. The aim of the presentstudy was to investigate its intervention of the RHO signaling pathway in hepaticfibrosis.Methods:In vivo:All healthy Sprague-Dawley rats were randomly divided into hepatic fibrosismodel group, control group and three treatment groups.All groups except the controlgroup were subcutaneously injected with10%CCL4(0.5ml/kg)2times a week for12week. The rats in the3treatment groups including a high-dose group(3125mg/kg/day),a medium-dosegroup (312.5mg/kg/day), and a low-dose group(31.25mg/kg/day) were daily gavaged with GFK Decoction, and the rats in the modeland normal control groups were given saline vehicle.The three treatment groups weretreated with GFK from the9th weeek to the th20week.The control group and modelgroup were put to death and obtained the liver tissues at the end of12th week.The threetreatment groups were put to death at the end of the20th week. At the time of killing, hepatectomy was performed and liver tissue samples were stored at-70C. Liver tissueswere stained with hematoxylin and eosin (HE) to observe liver pathologicstructures.The mRNA Expression of RHOA,ROCK,FAK,vinclin in liver tissue wasobserved by semi-quantitation reverse transcriptase polymerase chainreaction(RT-PCR).The immunohistochemistry and western blot were employed to vaulethe protein expression of ROCKI.In vitro: Rat HSC-T6cells were cultured in dulbecco’s modified Eaglemedium(DEMN) supplemented with10%foetal bovine serum,100units ml penicillinG and100mg ml streptomycin at37oC under5%CO295%O2.The cells wererandomly divided into three groups：control group(contains10%normal rats serum),acetaldehyde group(contains10%normal rats serum and acetaldehyde200umol/L),GFK group(contains10%medium-does GFKtreated-rats and acetaldehyde200umol/L)。The mRNA Expression of RHOA,ROCK,FAK,vinclin in liver tissue andHSC cells was observed by semi-quantitation reverse transcriptase polymerase chainreaction(RT-PCR).Western blot were employed to vaule the protein expression ofROCKI.Results:（1）The affect of GFK GFK Decoction on the rat liver tissue:changes of livertissue:abserved by HE stain the liver of control rats had no appreciable alterations,and inthe model group more fibrous tissues formed and extended into the hepatic lobules toseparate them incompletely and thick intralobular septa were evident. While in the3treatment groups, especially in the medium-dose GFK group, the pathological changesin liver were rather milder, showing less fibrous tissue proliferation.The gene levels ofRHOA,ROCKI,FAK,vinclin were markedly increased in the model group comparedwith the control group (P <0.05). Compared with the model group, the gene level ofRHOA,ROCKI,FAK and vinclin were significantly decreased in the3treatment groups,especially in the medium-does GFK group.Expressions of protein level:detected byimmunohistochemical and western blot shows that the expression of ROCKI proteinincreased evidently in model group.and the lower express in the three treatedgroups,especially on the medium-does GFK group(P<0.05).（2）The affect of GFK GFK Decoction on the HSC-T6: the gene levels ofRHOA,ROCKI,FAK,vinclin were markedly increased in the acetaldehyde groupcompared with the control group,and the expression lever of this genes are significantlylower in the medium-does GFK group compared with the model group..Expressions of protein level detected by western blot shows that the expression of ROCKI proteinincreased evidently in model group.and the lower express in the medium-does GFKgroup(P<0.05).Conclution：1RHO signal pathway maybe stimulate HSC proliferation，migrationand conglutination to promoting liver fibrosis2GFK have the effects of liver protection, anti-liver fibrosis,and maybe it associatedwith... |
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