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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 detect the effect of Xiaohuang decoction on the activation of Kupffer cells (KCs) in the livers of rats with bile duct ligation. Methods: Wistar rats were randomly divided into sham-operation group and biliary ligation group (BDL). BDL rats were randomly divided into Xiahuang Fang group and model group at the end of 2 weeks after model establishment. From the third week after the model was established, the rats in Xiahuang prescription group were treated by Xiaowanfang decoction (1.73 g / kg) once daily until the end of 5 weeks. The rats in sham operation group and model group were given the same amount of distilled water. At the end of the experiment, liver samples and serum were collected, and the expression of KCs marker CD68 and inflammatory cytokines TNF-α were detected by Western blot, immunohistochemistry and real-time quantitative PCR. Results: The results of immunohistochemistry showed that the expression of CD68 in the liver tissue of the sham-operation group was extremely low and almost negative. The expression of CD68 in the liver tissue of model group was significantly increased, mainly distributed in the sinusoidal and hyperplastic bile duct. CD68 expression decreased significantly. Real-time quantitative PCR results and Western blotting results were consistent with the above results, PCR results showed that TNF-αmRNA expression in the sham-operated group was lower, the model group TNF-αmRNA expression increased, but not obvious; Xiaohuang on TNF- α reduce the effect is not obvious. The results of ELISA showed that the level of TNF-α in serum of sham operation group was relatively low, the content of TNF-α in model group was significantly increased, while the content of TNF-α in Xiaohuang group was significantly decreased. Conclusion: Xiaohuang Recipe can significantly inhibit the activation of KCs in the liver and decrease the level of serum TNF-α, but has no effect on the expression of TNF-α in the liver, suggesting the multi-organ origin of TNF-α and the multi-target treatment of Xiaohuang Decoction. -α derived from what organs to be further studied.