肝组织中NF-κB、TGF-β1及其Ⅰ型受体mRNA和HSC在肝纤维化中的改变及护肝片对其的影响

目的探讨中、晚期纤维化大鼠肝组织中肝星状细胞(HSC)的活化与增殖、核转录因子-κB(NF-κB)及转化生长因子-β1(TGF-β1)及其Ⅰ型受体(TβRⅠ)表达的改变及护肝片对其的影响。方法采用12.5%CCl4诱导的大鼠肝纤维化模型,自造模之日起,大鼠分组灌胃给药(护肝片921mg/kg)或溶媒,每日一次,直至8或13周末,分别处死动物,取左叶肝组织石蜡包埋,制作组织芯片。免疫组化S-P法检测大鼠肝组织α-平滑肌肌动蛋白(-αSMA)和NF-κB p65蛋白的表达,原位杂交检测TGF-β1及TβRⅠmRNA的表达;并用MetaMorph图像分析系统计数-αSMA阳性细胞数,对NF-κB p65蛋白、TGF-β1及TβRⅠmRNA的表达量进行定量分析。结果 1.模型复制8周和13周,模型组的肝损伤及其纤维化分级均明显高于正常组(P<0.01),护肝片组的肝损伤及其纤维化分级均轻于模型组。2.模型复制8周和13周,模型组活化的HSC(即-αSMA阳性细胞)数量较正常组明显增多,NF-κB p65蛋白、TGF-β1及TβRⅠmRNA的表达均较正常组明显增强(P<0.01);3.护肝片显著抑制8、13周纤维化肝组织HSC的活化与增殖和NF-κB p65蛋白、TGF-β1及TβRⅠmRNA的表达(P<0.01)。结论抑制HSC的活化与增殖和NF-κB p65蛋白与TGF-β1及TβRⅠmRNA的表达可能是护肝片抗肝纤维化作用的靶点之一。

Altered protein expression of NF-κB and mRNA expression of TGF-β1 and its type Ⅰ receptor and HSCs of hepatic tissue in the middle and late stage of rat hepatic fibrosis and the effect of liver-aid tablets on them

Objective To study the effect of Liver-aid tablets on the activation and proliferation of hepatic stellate cells（HSCs） and protein expression of NfkappaB（NF-κB） and mRNA expression of transforming growth factor beta-1（TGF-β1） and its typeⅠreceptor（TβRⅠ） in the middle and late stage of rat hepatic fibrosis.Methods SD rats were divided into 3 groups： normal control,model control and group of Liver-aid tablets.CCl4（12.5%,4ml·kg-1）was injected subcutaneously in model rats twice a week for 8 and 13 weeks.The drug（921mg·kg-1 in the group of Liver-aid tablets,but 0.5% CMC-Na in control groups）was given intragastrically once a day for 8 and 13 weeks.The rats were killed at the end of 8th and 13th week respectively,the left liver tissue was then used to make tissue microarrays（TMA）..IHC S-P method was used to detect the protein expression of α-smooth muscle actin（α-SMA） and NF-κB p65,while the mRNA expression of TGF-β1 and TRβⅠwas detected by ISH.α-SMA positive cells were counted in three random 250μm×250μm areas under light microscop（×200）.The expression of NF-κB protein,TGF-β1 and TβRⅠmRNA were quantified by MetaMorph imaging analysis system.Results 1.Afeter 8 and 13 weeks,the degrees of liver injury and fibrosis grades in the model group were higher than those in the normal group（P0.01）,which was ameliorated remarkably by Liver-aid tablets.2.The number of activated HSCs in fibrotic model liver tissue was more than that in the normal group after 8 and 13 weeks（P0.01）.The protein expression of NF-κB,and the mRNA expression of TGF-β1 and TβRⅠin the model group were stronger than those in the normal group after 8 and 13 weeks（P0.01）.3.Liver-aid tablets inhibited not only the activation and proliferation of HSCs,but also the protein expression of NF-κB and mRNA expression of TGF-β1 and TβRⅠof fibrotic liver tissue after 8 and 13 weeks（P0.01）.Conclusion Liver-aid tablets have anti-fibrosis effects on CCl4-induced rat liver fibrosis,which might be performed through the pathway of inhibiting the activation and proliferation of HSCs,the protein expression of NF-κB,and the mRNA expression of TGF-β1 and TβRⅠ.