“有故无陨”法则下基于线粒体能量代谢途径的制首乌肝毒性机制研究

Mechanism of hepatotoxicity of prepared Polygonum multiflorum based on mitochondrial energy metabolism pathway in “You Gu Wu Yun”

目的:以中医证候为基础,研究制首乌致肝损伤的发生与中医肾虚证候的相关性,探讨制首乌致肝损伤的作用机制以及其与中医证候的关联性。方法:对不同肾虚证候模型大鼠灌胃给予制首乌药液,制备含药血清,流式细胞仪和MTT检测制首乌含药血清作用不同时间诱导L02肝细胞凋亡的情况;建立不同肾虚证候的整体动物模型,提取各组大鼠肝细胞线粒体,用分光光度计检测肝线粒体琥珀酸脱氢酶(SDH)、线粒体呼吸链复合物I活性、Na^+-K^+-ATP酶含量、Ca^2+-Mg^2+-ATP酶,使用紫外分光光度计和多功能酶标仪检测细胞色素氧化酶(COX)活性以及PTP开放程度。结果:在体外试验中,与空白组相比,肾阳虚24 g/kg制首乌组15%含药血清对L02肝细胞增殖具有明显的抑制作用;整体动物实验中,用制首乌治疗肾阳虚证候时,大鼠的肝功能出现明显损伤;与空白组比较,制首乌12 g/kg组SDH活性显著降低;线粒体呼吸链复合物I活性明显下降;Na^+-K^+-ATP酶、Ca^2+-Mg^2+-ATP酶活性显著降低;肾阳虚大鼠给药后PTP通透性较肾阴虚显著增大,COX活力显著降低。而制首乌6.12 g/kg治疗肾阴虚证候时,各项指标较空白组无统计学差异,说明大鼠肝功能无明显损伤。结论:制首乌引起的肝脏损害程度与中医肾虚证有关,其中制首乌治疗肾阳虚证候时其肝毒性明显强于对证治疗肾阴虚证候。

Objective: To study the relationship between liver injury caused by Polygonum multiflorum and Shenxu in TCM, and to explore the mechanism of liver injury caused by Polygonum multiflorum and its correlation with TCM syndromes. Methods: Rats with different TCM Shenxu syndrome were treated with Polygonum multiflorum by gavage to prepare the serum containing drugs. The apoptosis of L02 hepatocytes was detected by flow cytometry and MTT at different time after the treatment. Animal models of different TCM Shenxu syndrome were established. The mitochondria were extracted from rat hepatocytes in each group. The activity of mitochondrial respiratory chain complex I, Na^+-K^+-ATP enzyme, Ca^2+-Mg^2+-ATP enzyme and succinate dehydrogenase(SDH),) in hepatic mitochondria were detected by spectrophotometer. The activity of cytochrome oxidase(COX) and the opening degree of PTP were detected by UV spectrophotometer and multifunctional enzyme labeling instrument. Results: In vitro, compared with the control group, 15% serum containing 24 g/kg prepared Polygonum multiflorum significantly inhibited the proliferation of L02 hepatocytes. In vivo, the liver functions of the rats were obviously damaged in Shenxu treated by Polygonum multiflorum groups. Compared with the control group, the activity of SDH in 12 g/kg prepared Polygonum multiflorum group was significantly decreased, the activity of mitochondrial respiratory chain complex I, Na^+-K^+-ATP enzyme and Ca^2+-Mg^2+-ATP enzyme were significantly decreased. Compared with Shenyinxu model group, the permeability of PTP in Shenyangxu model group was significantly increased, the activity of COX was significantly decreased. In TCM Shenyinxu group, no significant difference was observed between the control group and Polygonum multiflorum groups at doses of 6 g/kg and 12 g/kg, which suggested that there was no obvious damage on liver function. Conclusion: Liver damage caused by Polygonum multiflorum is related to Shenxu syndrome in TCM. The treatment of Polygonum multiflorum in Shenyangxu group shows stronger liver toxicity than that in Shenyinxu group.