基于NF-κB信号通路探讨蒙药七味清肝散抗肝纤维化作用的研究

Study on anti-hepatic fibrosis effect of Mongolian medicine Qiwei Qinggan powder based on NF-κB signaling pathway

目的探讨蒙药七味清肝散(QGS-7)经NF-κB信号通路发挥抗肝纤维化作用。方法体内实验取60只雄性Wistar大鼠随机分为空白组,四氯化碳(CCl4)模型组,QGS-7低、中、高剂量(135、270、405 mg·kg^(-1)·d^(-1))组。空白组每日灌胃0.5%CMC-Na溶液,模型组和QGS-7组大鼠灌胃50%CCl4花生油溶液造肝纤维模型,每周2次,同时各给药组每日予相应剂量的QGS-7灌胃8周。取材后采用HE和Masson染色法观察肝组织病理学改变。q-PCR法和Western blot法检测纤维化标志物α-平滑肌动蛋白(α-SMA)、胶原蛋白Ⅰ(collagenⅠ)和NF-κB信号通路相关因子的m RNA和蛋白表达量。体外实验中大鼠灌胃给予QGS-71350 mg·kg^(-1)·d^(-1),7 d后取血制备含药血清,将HSC-T6细胞分为空白组,脂多糖(LPS)模型组,QGS-7含药血清低、中、高浓度组。Annexin V-FITC和PI双染法检测细胞凋亡情况;q-PCR法和Western blot法检测α-SMA、collagenⅠ及NF-κB信号通路相关因子的m RNA和蛋白表达量。结果体内实验中,与空白组相比,CCl4模型组组织中成纤维细胞增多,细胞排列混乱,胶原沉积明显,QGS-7各剂量组均有不同程度改善。与空白组相比,CCl4模型组肝组织中α-SMA、collagenⅠ、NF-κBp65蛋白和m RNA表达量显著增加(P<0.05),TLR4、p-TAK1、p-IKKα/IKKα、p-IKKβ/IKKβ、p-NF-κBp65/NF-κBp65蛋白和m RNA表达量显著增加(P<0.05);与模型组相比,QGS-7各剂量组上述因子的蛋白和m RNA表达量均显著降低(P<0.05)。体外实验中,空白组和LPS模型组凋亡率均较低,与模型组相比,QGS-7含药血清各浓度组凋亡率显著增加(P<0.01),α-SMA、collagenⅠ和NF-κB信号通路相关因子的m RNA和蛋白表达量与体内实验表达趋势一致。结论QGS-7具有良好的抗肝纤维化作用,作用机制可能与下调NF-κB信号通路的关键靶点有关。

AIM To investigate the anti-hepatie fibrosis effeet of Qiwei Qinggan powder(QCS-7)through NF-κB signaling pathway in rivo and in ritro.METHODS In rivo experiment,sisty male Wistar rats were randomly divided into 5 groups:blank group,CCl4 model group,QGS-7 low,medium,high dose groups.The blank group was gavaged with O.5%CMCNa solution daily,and the model group and each dose groups were gavaged with 50%CCl4 peanut oil solution twice a week,while each dose groups were gavaged with corresponding dose of QGS-7(135,270,405 mg·kg^(-1)·d^(-1))for 8 weeks.The histopathological changes of liver were observed by HE and Masson staining.q-PCR and Western blot were used to detect the mRNA and protein expression of fibrosis markers of alpha smooth muscle actin(α-SMA),collagen Iand NF-κB signaling pathway-related factors.In vitro experiment,rats were gavage with QGS-71350 mg·kg^(-1)·d^(-1),and blood was taken after 7 d to prepare drug-containing serum,which was classified into the blank group,the LPS model group,and the QGS-7 drugcontaining serum low-,medium-and high-concentration groups using HSC-T6 cells.Detection of apoptosis by double staining with Annexin V-FITC and PI.The mRNA and protein expression ofα-SMA,collagen Iand NF-κB signaling pathway-related factors were detected by q-PCR and Western blot.RESULTS In vivo experiment,HE and Masson results showed that fibroblasts were increased of the tissues in the CCl4 model group,with disorganized cell arrangement and significant collagen deposition compared with the blank group.Compared with the blank group,α-SMA,collagen I and NF-κBp65 protein and mRNA expressions of the liver tissues were significantly increased in the CCl4 model group(P<0.05),and TLR4,p-TAK1,p-IKKa/IKKα,p-IKKB/IKKβ,p-NF-κBp65/NF-κBp65 protein and mRNA expression were significantly increased(P<0.05).Compared with the model group,the protein and mRNA expression of the above factors were significantly decreased in each dose group of QGS-7(P<0.05).In vitro experiments,the apoptosis rate was lower in both the blank group and the LPS model group.Compared with the model group,the apoptosis rate was significantly increased in each concentration of the QGS-7-containing serum group(P<0.01),and the mRNA and protein expression of theα-SMA,collagen I,and the NF-κB signalling pathway related to factors were consistent with the trend in vivo experimental expression.CONCLUSION QGS-7 has good anti-fibrotic effects,and the mechanism may through down-regulation of key targets of NF-κB signaling pathway to exert anti-fibrotic effects.