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目的:为探讨Sonic Hedgehog(Shh)信号通路激活剂,2,6,9-三取代嘌呤化合物(purmorphamine,PM)对帕金森病(Parkinson’s disease,PD)模型动物中小鼠身体的协调能力、多巴胺能神经元及小胶质细胞细胞活性的影响及可能机制。方法:将47只C57BL/6J雄性8周小鼠随机分为control组、PM组、MPTP组、PM+MPTP组,使用腹腔注射1-methyl-4-phenyl 1,2,3,6-tet-rahydropyridine(MPTP)制作小鼠的帕金森病模型。应用悬杆实验、免疫组织化学和实时荧光定量PCR(real-time quantitative PCR,Q-PCR)技术分别检测小鼠的运动协调能力,酪氨酸羟化酶(tyrosine hydroxylase,TH)阳性的多巴胺(DA)能神经元的形态、数目,Ionized calcium binding adapter molecule 1(Iba1)阳性的小胶质细胞的形态以及炎症因子TNF-α的表达情况。结果:(1)悬杆实验显示:在预处理PM 24 h后MPTP诱导的PD模型小鼠中,与MPTP组相比,PM+MPTP组小鼠后爪抓到横杆的时间明显缩短(P<0.01);而与control组相比,MPTP组中小鼠后爪抓到横杆的时间显著延长(P<0.01)。(2)TH免疫组织化学结果显示:control组TH+DA能神经元染色很深,胞体密集,突起明显;与control组相比,MPTP组TH+DA能神经元的数量明显减少,细胞质着色变浅,突起变短或者消失(P<0.01);与MPTP组相比,PM+MPTP组TH+DA能神经元不仅使损失细胞的数目明显减少,而且细胞质染色变深,突起有所增多(P<0.01)。(3)Iba1免疫组织化学染色显示:与control相比,MPTP组小胶质细胞变大、形态不规则、突起变粗;与MPTP组相比,PM+MPTP组小胶质细的胞体形态较小,突起呈现细长较多。(4)Q-PCR检测结果显示:与control组相比,MPTP组TNF-α和Iba1mRNA表达明显增加(P<0.01);与MPTP组对比,PM+MPTP组表达的TNF-α和Iba1 mRNA水平明显降低(P<0.01)。结论:PM可改善PD模型小鼠的运动协调能力,保护多巴胺能神经元,其机制可能与降低小胶质细胞的炎症水平有关。
OBJECTIVE: To investigate the synergistic effect of Sonic Hedgehog (Shh) signaling pathway activator, 2,6,9-tri-substituted purine (PM) on the body of mice in Parkinson’s disease (PD) Effect of neuronal and microglial cell activity and its possible mechanism. Methods: Forty-seven C57BL / 6J male 8-week-old mice were randomly divided into control group, PM group, MPTP group and PM + MPTP group. Rat model of Parkinson’s disease was made with rahydropyridine (MPTP). The ability of motor coordination, tyrosine hydroxylase (TH) -positive dopamine (TH) in mice were detected by using the hanging rod test, immunohistochemistry and real-time quantitative PCR (Q-PCR) DA neurons, morphology, number of Irog1-positive microglia and the expression of inflammatory cytokines TNF-α. Results: (1) The experimental results of the hanging rod showed that in MPTP-induced PD model mice, the time of grasping the crossbar of PM + MPTP group was significantly shorter than that of MPTP group (P <0.01). Compared with the control group, the time for grasping the crossbar in the hind paw in the MPTP group was significantly longer (P <0.01). (2) The TH immunohistochemistry results showed that the TH + DA neurons in the control group were stained darker, with dense cell bodies and prominent protrusions. Compared with the control group, the number of TH + DA neurons in the MPTP group was significantly decreased, (P <0.01). Compared with MPTP group, TH + DA neurons in PM + MPTP group not only significantly decreased the number of lost cells, but also stained cytoplasm became deeper and the number of protrusions increased <0.01). (3) Immunohistochemical staining of Iba1 showed that compared with control group, microglia in MPTP group became larger, irregular in shape and thicker in protuberance. Compared with MPTP group, microglial cell morphology in PM + MPTP group was more Small, protruding slender show more. (4) The results of Q-PCR showed that compared with control group, the expression of TNF-α and Iba1 mRNA in MPTP group was significantly increased (P <0.01). Compared with MPTP group, the levels of TNF-αand Iba1 mRNA Significantly lower (P <0.01). Conclusion: PM can improve the motor coordination of PD mice and protect the dopaminergic neurons. The mechanism may be related to the reduction of inflammation in microglia.