针刺调节Shh/Ptch1信号通路对帕金森认知障碍模型大鼠的神经保护作用研究

Study on Neuroprotective Effect of Acupuncture Modulating Shh/Ptch1 Signaling Pathway on Cognitive Impairment in Rat Models of Parkinson's Disease

目的:探讨针刺治疗是否可通过调节Shh/Ptch1信号通路进而改善帕金森(PD)大鼠认知功能。方法:将SD大鼠随机分为对照组、模型组、针刺组、针刺+环巴胺(Shh抑制剂)组、西药组(盐酸多奈哌齐)及针刺+西药组。采用Morris水迷宫检测空间学习记忆能力;ELISA法检测大鼠脑组织肿瘤坏死因子-α(TNF-α)、白细胞介素(IL-6、IL-10及IL-1β)水平;商品化试剂盒检测脑组织丙二醛(MDA)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)水平;HE染色观察大鼠海马组织形态变化;蛋白质印迹法检测大鼠海马组织活化半胱氨酸蛋白酶(Cleaved-caspase-3)、B淋巴细胞瘤(Bcl-2)、Bcl-2相关X蛋白(Bax)、音猬因子(Shh)及补缀同源物1(Ptch1)蛋白表达。结果:与对照组相比,模型组大鼠神经元有明显损伤,Morris水迷宫潜伏期时间、海马组织中TNF-α、IL-6、IL-1β、MDA水平、Cleaved-caspase-3及Bax蛋白表达水平显著升高,滞留时间和穿越平台次数、IL-10水平、SOD、CAT活性、Bcl-2、Shh及Ptch1蛋白表达水平显著降低(P<0.05);与模型组相比,针刺组、西药组和针刺+西药组大鼠神经元损伤显著减轻,Morris水迷宫潜伏期时间、海马组织中TNF-α、IL-6、IL-1β、MDA水平、Cleaved-caspase-3及Bax蛋白表达水平显著降低,滞留时间和穿越平台次数、IL-10水平、SOD、CAT活性、Bcl-2、Shh及Ptch1蛋白表达水平显著升高(P<0.05);与针刺组相比,针刺+环巴胺组大鼠神经元损伤显著加重,Morris水迷宫潜伏期时间、海马组织中TNF-α、IL-6、IL-1β、MDA水平、Cleaved-caspase-3及Bax蛋白表达水平显著升高,滞留时间和穿越平台次数、IL-10水平、SOD、CAT活性、Bcl-2、Shh及Ptch1蛋白表达水平显著降低(P<0.05);针刺组和西药组各检测指标差异均无统计学意义(P>0.05);与针刺组和西药组相比,针刺西药组大鼠神经元损伤显著减轻,Morris水迷宫潜伏期时间、海马组织中TNF-α、IL-6、IL-1β、MDA水平、Cleaved-caspase-3及Bax蛋白表达水平显著降低,滞留时间和穿越平台次数、IL-10水平、SOD、CAT活性、Bcl-2、Shh及Ptch1蛋白表达水平显著升高(P<0.05)。结论:针刺治疗可降低PD认知障碍大鼠炎症反应和氧化应激,减轻神经元损伤,进而改善其认知功能,可能是通过激活Shh/Ptch1信号通路实现的。

Objective:To explore whether acupuncture treatment can improve cognitive function in Parkinson's disease(PD)rats by regulating the Shh/Pth1 signaling pathway.Methods:SD rats were randomly divided into the control group,the model group,the acupuncture group,the acupuncture plus Cyclopentamine(Shh inhibitor)group,the Western medication group(Donepezil Hydrochloride),and the acupuncture plus Western medication group.Spatial learning and memory abilities were detected by Morris water maze;the levels of TNF-α,IL-6,IL-10 and IL-1βin brain tissues were detected by ELISA;the levels of MDA,SOD and CAT in brain tissues were detected by the commercial reagent kit;the morphological changes of hippocampal tissues were observed by HE staining;the protein expressions of Cleaved-caspase-3,Bcl-2,Bax,Shh and Ptch1 in hippocampal tissues were detected by Western Blotting.Results:Compared with those in the control group,the neurons showed significant damage;the latency time of the Morris water maze was increased;the levels of TNF-α,IL-6,IL-1βand MDA in hippocampal tissues and protein expressions of Cleaved-caspase-3 and Bax were obviously increased;the residence time and the times of platform crossings were decreased;the level of IL-10,the activities of SOD and CAT,and the protein expressions of Bcl-2,Shh and Ptc1 were significantly reduced in the model group(P<0.05).Compared with those in the model group,the neuronal damage of the rats was significantly reduced;the latency time of Morris water maze was shortened;the levels of TNF-α,IL-6,IL-1βand MDA in hippocampal tissues were decreased;the protein expressions of Cleaved-caspase-3 and Bax were obviously reduced;the residence time and the times of platform crossings were increased;the level of IL-10,the activities of SOD and CAT,the protein expressions of Bcl-2,Shh and Ptc1 were obviously increased in the acupuncture group,the Western medication group,and the acupuncture plus Western medication group(P<0.05).Compared with those in the acupuncture group,the neuronal damage of the rats was significantly increased;the latency time of Morris water maze was prolonged;the levels of TNF-α,IL-6,IL-1βand MDA were increased;the protein expressions of Cleaved-caspase-3 and Bax in the hippocampus were significantly increased;the residence time and the times of platform crossings were reduced;the level of IL-10 level,the activities of SOD and CAT,the protein expressions of Bcl-2,Shh and Ptch1 were significantly decreased in the acupuncture plus Cyclopamine group(P<0.05).There were no statistical differences in all detection indicators between the acupuncture group and the Western medication group(P>0.05).Compared with those in the acupuncture group and the Western medication group,the neuronal damage was significantly reduced;the latency time of Morris water maze was shortened;the levels of TNF-α,IL-6,IL-1βand MDA were decreased;the protein expressions of Cleaved-caspase-3 and Bax were significantly reduced;the residence time and the times of platform crossing were increased;the level of IL-10,the activities of SOD and CAT,the protein expressions of Bcl-2,Shh and Ptch1 were significantly increased in the acupuncture plus Western medication group(P<0.05).Conclusion:Acupuncture can reduce inflammation and oxidative stress,as well as alleviate neuronal damage in rats with PD cognitive impairment,thereby improve their cognitive function,which possibly through activating the Shh/Ptch1 signaling pathway.