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目的:研究GDNF和雌二醇(E2)联合使用对6-羟基多巴胺(6-OHDA)所致的多巴胺能神经细胞损伤的影响并探讨其作用的机制。方法:新生2~3 d的雄性SD大鼠行常规中脑脑片培养,6-OHDA诱导损伤,脑片分为8组:正常对照组、溶剂对照组、E2组、Wortmannin(PI3-K特异性阻断剂)+E2组、GDNF组、Wortmannin+GDNF组、GDNF+E2组、Wortmannin+GDNF+E2组。免疫荧光染色观察黑质致密部(SNc)酪氨酸羟化酶(TH)的表达情况;Western Blot检测各组脑片中Bcl-2及Bad的表达。结果:免疫荧光染色结果显示,在GDNF+E2组,TH的表达比单用GDNF或E2时显著升高,差异有统计学意义(P<0.05);阻断PI3-K/Akt信号通路后,TH的表达与未阻断组相比降低,差异有统计学意义(P<0.05)。Western Blot检测结果显示,在GDNF+E2组,Bcl-2的表达比单用GDNF或E2时升高,而Bad的表达比单用GDNF或E2时降低,差异均有统计学意义(P<0.05);阻断PI3-K/Akt信号通路后,Bcl-2的表达与未阻断组相比降低,而Bad的表达是升高的,差异有统计学意义(P<0.05)。结论:GDNF和E2联合使用对6-OHDA所致的黑质多巴胺能神经细胞损伤有保护作用,其机制可能是通过调节凋亡相关蛋白Bcl-2和Bad的表达变化实现的。
Objective: To investigate the effect of GDNF combined with estradiol (E2) on dopaminergic neuronal injury induced by 6-hydroxydopamine (6-OHDA) and to explore its mechanism. Methods: Male Sprague-Dawley rats aged 2 ~ 3 days were cultured in normal brain slices and induced by 6-OHDA. Brain slices were divided into 8 groups: normal control group, solvent control group, E2 group, Wortmannin + E2 group, GDNF group, Wortmannin + GDNF group, GDNF + E2 group and Wortmannin + GDNF + E2 group. The expression of tyrosine hydroxylase (TH) in substantia nigra pars compacta (SNc) was observed by immunofluorescence staining. The expression of Bcl-2 and Bad in each group was detected by Western Blot. Results: The results of immunofluorescence staining showed that the expression of TH in GDNF + E2 group was significantly higher than that in GDNF alone or E2 group (P <0.05). After PI3-K / Akt signaling pathway was blocked, The expression of TH decreased compared with the non-blocked group (P <0.05). The results of Western Blot showed that in GDNF + E2 group, the expression of Bcl-2 was higher than that of GDNF alone or E2, while the expression of Bad was lower than that of GDNF or E2 alone (P <0.05 ). After blocking the PI3-K / Akt signaling pathway, the expression of Bcl-2 decreased compared with that of the non-blocked group, while the expression of Bad increased. The difference was statistically significant (P <0.05). Conclusion: The combination of GDNF and E2 can protect the substantia nigra dopaminergic neurons induced by 6-OHDA. The mechanism may be through the regulation of the expression of Bcl-2 and Bad.