小檗碱通过抑制CDK5/P25表达逆转MMP+诱导帕金森病细胞模型损伤

Berberine Reverses the Injury of MMP+-induced Parkinson’s Disease Cell Model by Inhibiting the Expression of CDK5/P25

目的探究小檗碱对1-甲基-4-苯基吡啶离子(MMP+)诱导的帕金森病(PD)细胞模型损伤的保护机制。方法用不同浓度MMP+(10、25、50、100、200、400μmol·L^(-1))或小檗碱(2.5、5.0、7.5、10.0、12.5、15.0μmol·L^(-1))处理小鼠中脑多巴胺能神经元(MN9D)48 h,最终选择MPP^(+)200μmol·L^(-1)诱导剂量和小檗碱10.0μmol·L^(-1)干预剂量进行后续实验。随机分为对照组(PD细胞不做任何处理)、MPP^(+)组(MPP^(+)200μmol·L^(-1)处理)和MPP^(+)+小檗碱组(小檗碱10μmol·L^(-1)预处理12 h,再以MPP^(+)200μmol·L^(-1)孵育48 h)。应用MTT实验和流式细胞术检测各组的细胞存活率和凋亡率;蛋白免疫印记法检测各组间calpain、caspase-3、CDK5、P35和P25蛋白的表达情况。结果MPP^(+)组呈剂量依赖性抑制MN9D细胞增殖。MPP^(+)+小檗碱组细胞存活率高于MPP^(+)组(t=6.837,P=0.002),细胞凋亡率低于MPP^(+)组(t=14.223,P<0.001)。MPP^(+)+小檗碱组calpain、caspase-3相对表达水平均低于MPP^(+)组(均P<0.001)。P35蛋白相对表达水平高于MPP^(+)组(P<0.001)。结论小檗碱逆转MPP^(+)刺激MN9D细胞的P35向P25转化,抑制calpain和caspase-3蛋白的表达,对MPP^(+)诱导的MN9D细胞损伤发挥保护作用。

Aim To study the protective mechanism of berberine on MMP+-induced injury of Parkinson’s disease cell model.Methods Mouse midbrain dopaminergic neurons(MN9D)were treated with different concentrationof MMP+(10,25,50,100,200,400μmol·L^(-1))or berberine(2.5,5.0,7.5,10.0,12.5,15.0μmol·L^(-1))for 48 h,and the concentration of 200μmol·L^(-1) MPP^(+)and 10.0μmol·L^(-1) berberine were used for the follow-up experiments.The neurons were randomly divided into a control group,a 200μmol·L^(-1) MPP^(+)group and a MPP^(+)+berberine group(first pretreated with 10μmol·L^(-1) of berberine for 12 h,then incubated with 200μmol·L^(-1) MPP^(+)for 48 h).MTT experiment and flow cytometry were used to detect the survival rate and apoptosis rate of cells between the different groups.Protein immunoblotting experiment was used to detect the expression of calpain,caspase-3,CDK5,p35 and p25 proteins between the different groups.Results MPP^(+)inhibited the proliferation of MN9D cells in a dose-dependent manner.The survival rate of cells in the MPP^(+)+berberine group was higher than that in the MPP^(+)group(t=6.837,P=0.002),and the apoptosis rate was lower than that in the MPP^(+)group(t=14.223,P<0.001).The relative expression levels of calpain and caspase-3 in the MPP^(+)+berberine group were lower than that in the MPP^(+)group(P<0.001).The relative expression level of p35 protein was higher than that of the MPP^(+)group(P<0.001).Conclusion Berberine reverses the MPP^(+)stimulation of the p35 to p25 conversion of MN9D cells,can inhibit the expression of CDK5/p25,weaken the expression of calpain and caspase-3 proteins,and plays a protective effect on MPP^(+)-induced MN9D cell damage.