邓奕辉 李银 谭琥 覃弘宇 李定祥

摘要：目的 比较解毒化瘀方药物血清与药物血浆对凝血酶合并缺氧诱导PC12细胞损伤蛋白酶活化受体（PAR）-1、细胞外信号调节激酶（ERK）1/2表达的差异。方法 灌胃给药制备大鼠药物血清和药物血浆。采用三气培养箱和无糖DMEM培养液造成细胞缺氧模拟缺血，并同时加入150 U/mL凝血酶，建立体外缺氧合并凝血酶诱导的PC12细胞损伤模型。实验分为空白组、模型组、药物血清组及药物血浆组。实时荧光定量PCR检测细胞PAR-1、ERK1/2 mRNA表达，免疫荧光法检测细胞PAR-1、ERK1/2蛋白表达。结果 与空白组比较，模型组PAR-1、ERK1/2 mRNA和蛋白表达增强（P<0.05）；与模型组比较，药物血清组和药物血浆组PAR-1、ERK1/2 mRNA和蛋白表达下降（P<0.05，P<0.01）；药物血浆组PAR-1、ERK1/2表达较药物血清组下降（P<0.05）。结论 解毒化瘀方药物血浆抗缺氧合并凝血酶诱导PC12细胞损伤作用优于其药物血清。

关键词：解毒化瘀方；药物血浆；药物血清；PC12细胞；大鼠

DOI：10.3969/j.issn.1005-5304.2016.06.019

中图分类号：R285.5 文献标识码：A 文章编号：1005-5304（2016）06-0073-04

Abstract： Objective To compare the differences between Jiedu Huayu Prescription containing serum and plasma on PAR-1， ERK1/2 expression of the PC12 cell injury model. Methods Rat serum containing and plasma models was prepared through gavage. Three gas incubators and sugar-free DMEM medium simulated ischemia caused by hypoxia were used， and 150 U/mL of thrombin was added， with a purpose to establish the thrombin-induced hypoxic PC12 cell injury model. The experiment was divided as control group， model group， serum containing group and plasma containing group. Real-time fluorescent quantitative PCR was used to detect the expression levels of PAR-1 and ERK1/2 mRNA in each group； immunofluorescence was used to detect the protein expressions of PAR-1 and ERK1/2. Results Compared with the control group， the expressions of PAR-1 and ERK1/2 mRNA and protein in model group were enhanced （P<0.05）； compared with the model group， the expressions of PAR-1 and ERK1/2 mRNA and protein in serum containing group and plasma containing group decreased （P<0.05， P<0.01）； the expressions of PAR-1 and ERK1/2 mRNA and protein in plasma containing group decreased more significantly than serum containing group （P<0.05）. Conclusion Jiedu Huayu Prescription containing plasma was superior to its containing serum against the injury of PC12 cell induced by hypoxic and thrombin.

Key words： Jiedu Huayu Prescription； plasma containing medicine； serum containing medicine； PC12 cells； rats

中药血清药理学是指将中药或中药复方经口给动物灌服一定时间后采集动物血液、分离血清，用此含有药物成分血清进行体外实验的一种实验技术[1]。然而，近年来不少学者发现，中药复方或单味药药物血清与血浆在成分和作用上存在差异，且在大多数情况下，中药药物血浆较药物血清具有优势。

近年来，急性缺血性中风的中医病机理论取得重大突破，其中王永炎院士提出的“毒损脑络”病机假说得到了广泛的研究[2-6]。本课题组认为，缺血性中风主要病机瘀血阻滞、毒损脑络的形成与凝血酶毒性相关，化瘀解毒法是该病的有效治法。解毒化瘀方为基于“瘀毒”理论的中药复方，由黄连、黄芩、黄柏、栀子等8味中药组成。而凝血酶正是通过与蛋白酶活化受体（protease-activated receptor，PAR）结合发挥对神经细胞的毒性作用，同时，细胞外信号调节激酶（ERK）1/2系统在凝血酶诱导的脑耐受中发挥重要作用。……