摘要
目的:观察天麻钩藤饮含药血清对1-甲基-4-苯基-吡啶离子(1-methyl-4-phenyl pyridinium,MPP^+)诱导的PC12细胞凋亡的影响,并且探讨其是否通过c-Jun氨基末端激酶(JNK)信号通路发挥神经保护作用。方法:将32只SD大鼠随机分为空白组、天麻钩藤饮低、中、高剂量组,每组8只。天麻钩藤饮按照5.7,11.4,22.8 g·kg-1灌胃,空白组灌胃等体积生理盐水,采血制备空白和含药血清。PC12细胞常规培养并分为空白组、模型组、天麻钩藤饮含药血清低、中、高剂量组,空白组和模型组加空白血清,其他3组加10%的天麻钩藤饮含药血清,孵育30 min后,模型组和含药血清组再加500μmol·L^(-1)MPP^+共孵育48 h后收集细胞,采用Cell Titer 96Aoueous MTS Reagent powder(MTS法)检测细胞存活率。后续实验分为5组,即:空白组,MPP^+组,MPP^++含药血清高剂量组,MPP^++SP600125组,MPP^++含药血清高剂量+SP600125组,采用Annexin VFITC/PI双染色法检测细胞凋亡,分光光度法检测细胞半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)活性,蛋白免疫印迹法(Western blot)检测JNK,p-JNK,c-Jun,p-c-Jun蛋白表达水平。结果:与空白组比较,模型组细胞活力明显降低(P<0.05);与模型组比较,10%的天麻钩藤饮低、中、高剂量含药血清组细胞活力显著增加(P<0.05)。后续试验中,与空白组比较,MPP^+组细胞凋亡率,Caspase-3活性,p-JNK和p-c-Jun蛋白表达明显升高(P<0.05)。与MPP^+组比较,MPP^++含药血清高剂量组,MPP^++SP600125组均能够使细胞凋亡率,Caspase-3活性,p-JNK和p-c-Jun蛋白表达明显降低(P<0.05),而MPP^++含药血清高剂量+SP600125组的作用明显高于MPP^++含药血清高剂量组(P<0.05),但不高于MPP^++SP600125组。JNK和c-Jun蛋白表达无明显变化。结论:天麻钩藤饮含药血清对MPP^+诱导的PC12细胞的凋亡具有保护作用,其机制可能与JNK信号通路密切相关。
Objective： To examine the effect of Tianma Gouteng Yin-containing serum on 1-methyl-4- phenylpyridinium （MPP＋） -induced apoptosis of PC12 cells, and explore the neuroprotective effect of c-Jun N- terminal kinase （JNK） signaling pathway. Method： A total of 32 male SD rats were randomly divided into control group and high, middle and low-dose Tianma Gouteng Yin-containing serum groups （22.8, 11.4, 5.7 g·kg-1）. The rats were intragastrically given Tianma Gouteng Yin to prepare Tianma Gouteng Yin-containing serum. The control group was given the same volume of normal saline. The proliferation of PC12 cells was cultured and divided into the blank group, the model group, and high, middle and low-dose Tianma Gouteng Yin-containing serum groups. The blank group and the model group were given blank serum, while the other three groups were given 10% Tianma Gouteng Yin-containing serum incubated for 30 rain. The model group and Tianma Gouteng Yin- containing serum groups were additionally given 500 μmol·L-1 MPP＋ and co-cultured for 48 h. The cell viability was measured by Cell Titer 96 ~ Aoueous MTS Reagent powder （MTS）. The follow-up experiment was divided into five groups, namely blank group, MPP＊ group, MPP ＋ ＋ high-dose Tianma Gouteng Yin-containing serum group, MPP＊ ＋ SP600125 group, and MPP ＋ SP600125 ＋ high-dose Tianma Gouteng Yin-containing serum group. Apoptosis was analyzed speetmphotometry. The by flow cytometry. Cysteine aspartate protease-3 （Caspase-3） activity was detected by expressions of JNK, p-JNK, c-Jun, p-c-Jun protein were detected by Western blot. Result： Compared with the blank control, cell viability was significantly decreased in the model group （P 〈 0. 05）. Compared with the model group, cell viability was significantly increased in high, middle and low-dose Tianma Gouteng Yin-containing serum groups （ P 〈 0. 05 ）. In the follow-up experiment, compared with blank control, apoptosis, Caspase-3 activity, and expressions of p-JNK and p-c-Jun protein were significantly increased in the MPP＊ group （ P 〈 0. 05 ）. Compared with the MPP ＋ group, poptosis, Caspase-3 activity, and expressions of p-JNK and p-c-Jun protein were significantly decreased in the MPP ＋ ＋ high-dose Tianma Gouteng Yin-containing serum group and MPP＊ ＋ SP600125 group （P 〈 0. 05）. Co-treatment with SP600125 and high-dose Tianma Gouteng Yin-cnntaining serum resulted in a slight increase in neuroprotection, which was significantly greater than high-dose Tianma Gouteng Yin-containing serum treatment alone （ P 〈 0. 05 ）, but not significantly greater than SP600125 treatment alone. The expressions of JNK and c-Jun protein showed no significant change. Conclusion： Tianma Gouteng Yin-containing serum has the neuroprotective effect against MPP ＋ -induced apoptosis, which may be closely related to the JNK signaling pathway.
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2017年第21期141-146,共6页
Chinese Journal of Experimental Traditional Medical Formulae
基金
黑龙江省教育厅2016年度基本科研业务费项目(2016-KYYWF-0854)
