和厚朴酚抑制线粒体可溶蛋白诱导小胶质细胞活化的体外实验研究

Honokiol Inhibiting Microglia Activation Induced by MDP in vitro Experimental Study

目的:探讨线粒体可溶蛋白诱导小胶质细胞活化作用及和厚朴酚对小胶质细胞活化的影响。方法:培养BV2小胶质细胞,分空白对照组(control)、线粒体可溶蛋白组(MDP)、和厚朴酚干预组(HNK)。ELISA检测不同浓度线粒体可溶蛋白(MSP)刺激不同时间的细胞上清液中IL-6和TNF-α含量;RT-PCR半定量法测定各组转录因子Klf4的表达情况;倒置相差显微镜观察各组细胞形态变化。结果:1.ELISA:MSP(10μg/ml,100μg/ml)处理7h时,IL-6和TNF-α含量与control组比较显著升高(P<0.05);100μg/ml MSP处理1、3、5、7h时,IL-6和TNF-α表达与control组比较明显升高(P<0.05),且7h时HNK组含量明显低于MDP组(P<0.05)。2.RT-PCR结果显示MDP组KLP4的表达显著高于空白对照组和HNK组(P<0.05)。3.倒置相差显微镜观察示空白组胶质细胞形态呈静息状态;MDP组小胶质细胞的胞体变圆或者椭圆,突起消失,呈"阿米巴"状;HNK组活化状态的小胶质细胞明显减少。结论:线粒体可溶蛋白可以激活小胶质细胞,促进白细胞介素IL-6和TNF-α的释放,和厚朴酚能够有效地抑制线粒体可溶蛋白诱导的小胶质细胞活化,其机制可能是通过下调Klf4的表达发挥作用。

Objective： To investigate the activation of microglia induced by MSP and the activated process affected by Honokiol. Methods： Mice BV2 microglia in logarithmic growth phase were randomly divided into 3 groups.（1）control group： the cells were cultured with serum free medium;（2）MDP group：the cells were treated with MDP after cultured for 24 h.（3）HNK group： after pretreated with Honokiol for 30 min, MDP was added in the cells. ELISA： The concentration of IL-6 and TNF-α in culture supernatant of these group were determined; Semi-quantity RT-PCR method was used to analyze the dynamic expression of transcription factor Klf4 in different groups; Cellular morphological changes were observed under phase-contrast microscope. Results： ELISA： MSP（10 μg/ml, 100 μg/ml） treated for 7h. Compared with the control group, Il-6 and TNF-α content in cell supernatant have statistical significance（P〈 0.05）, suggesting microglia activation have certain concentration dependent manner; MSP（100 μg/ml） treated for 1,3,5,7 h. Compared with control group, Il-6 and TNF-α content in cell supernatant have statistical significance at each time point（P〈 0.05）, suggesting microglia activation have certain time dependent manner; MPS（100 μg/ml） treated for 7 h. Compared with the HNK group, Il-6 and TNF-α content in cell supernatant have statistical significance（P〈0.05）, suggesting Honokiol could decrease the expression of Il-6 and TNF-α.（2）RT-PCR：Klf4 expression level raised obviously after stimulating of microglia by MSP, and honokiol could downregulate Klf4 expression.（3）Morphological observation： Under phase-contrast microscope, microglia in the control group were in resting state; microglia in the MDP group, the cell body became round, neurites disappeared, presenting ＂Amoeba＂ shape; The activation of microglia in HNK group was reduced obviously. Conclusion： 1.MSP can induce microglia activation, and promote Il-6 and TNF-α release, and its activation effect is in the manner of concentration and time dependence. 2. Honokiol can effectively inhibit the microglia activation induced by MSP, the possible mechanism is to down-regulate Klf4 expression of the microglia.