消黄方对胆汁淤积性肝纤维化大鼠肝脏库普弗细胞激活的影响

Effect of "Xiaohuang Prescription" on Kupffer Cell Activation in Rats with Cholestasis-induced Liver Fibrosis

目的:检测消黄方对胆管结扎大鼠肝组织库普弗细胞(KCs)激活的干预作用。方法:将Wistar大鼠随机分为假手术组和胆管结扎组(BDL),BDL大鼠在造模后2周末再随机分为消黄方组和模型组。造模第3周起消黄方组予灌胃消黄方煎出液(1.73 g/kg),每日1次,至5周末;假手术组与模型组灌胃等量蒸馏水。实验结束留取肝组织样本及血清,以免疫印迹和免疫组化及实时定量PCR法检测KCs标志物CD68及炎症因子TNF-α表达。结果:免疫组化结果显示,假手术组肝组织CD68表达极低,几乎呈阴性表达;模型组肝组织CD68阳染表达显著升高,主要分布在肝窦及增生的胆管周围;消黄方组CD68表达显著下降。实时定量PCR结果和免疫印迹结果与上述结果基本一致,PCR结果显示TNF-αmRNA表达在假手术组较低,模型组TNF-αmRNA表达有所升高,但并不明显;消黄方对TNF-α降低作用不明显。ELISA法检测显示,假手术组血清TNF-α含量相对较低,模型组大鼠TNF-α含量显著升高,而消黄方组显著降低。结论:消黄方可显著抑制肝脏KCs激活和降低血清TNF-α含量,但对肝脏TNF-α表达无作用,提示TNF-α多器官来源性及消黄方的多靶点治疗作用,而TNF-α来源于何器官有待进一步研究。

Objective： To observe the inhibiting effect of ＂Xiaohuang Prescription＂ on Kupffer Cells （KCs） activation in rats with cholestasis-induced liver fibrosis. Methods： Wistar rats were randomized into sham operation group and bile duct ligation （BDL） group. After 2 weeks, the rats in BDL group were divided into ＂Xiaohuang Prescription＂ group and model group. At the 3rd week, the rats in ＂Xiaohuang Prescription＂ group were treated with ＂Xiaohuang Decoction＂ by intragastric administration at a dose of 0. 173 g/100 g once a day, till the end of 5th week; the rats in sham operation group and model group were treated with equal amount of distilled water. The sample of hepatic tissue and blood serum were kept, and the CD68, a specific maker of KC and expression of TNF-a were detected by western-blotting and real-time quantitative PCR. Results： The result of immunohistochemistry showed that the mRNA expression of CD68 was almost negative in sham operation group; the expression of CD68-positive staining was increased in model group, which appeared not only in hepatic sinusoids, but also in proliferative bile duct; the expression of CD68 in ＂Xiaohuang Prescription＂ group was decreased markedly. Real-time quantitative PCR results and western blot analysis were consistent with above results. The result of PCR showed that the expression of TNF-a mRNA was lower in sham operation group, and higher in model group, but without marked differences; ＂Xiaohuang Prescription＂ had insignificant effect on reduction of TNF-a. The results of ELISA showed that the content of TNF-a was lower in sham operation group, but higher in model group. ＂ Xiaohuang Prescription＂ lowered the content of TNF-a significantly. Conclusion： ＂ Xiaohuang Prescription＂ can inhibit KC activation and reduce content of TNF-a in serum, but without any effect on expression of TNF-a in liver. It is indicated that TNF-a derives from the multiple organs, and it is worthy, to have further study on the source of TNF-a.