摘要
目的探讨α7烟碱乙酰胆碱受体(α7nAChR)对PD模型大鼠的神经保护作用及其调控机制。方法将48只SPF级8周龄SD大鼠按随机数字表法分为正常对照组、PD模型组、α7nAChR空载组和α7nAChR过表达组,每组12只。后3组大鼠采用6-羟多巴胺(6-OHDA)诱导制备PD模型。造模后4周,后2组大鼠分别脑立体定位注射2μLα7nAChR空载体慢病毒和过表达慢病毒。正常对照组大鼠不做任何处理。注射后2周采用旋转实验检测大鼠的行为学变化;采用HE和尼氏染色检测大鼠右侧黑质致密部(SNc)中神经元的形态学变化;采用TUNEL染色检测大鼠右侧SNc中细胞凋亡情况;采用免疫荧光双标染色检测大鼠右侧SNc中酪氨酸羟化酶(TH)和α-突触核蛋白(α-Syn)的表达;采用ELISA实验检测大鼠右侧SNc中丙二醛(MDA)、4-羟基壬烯醛(4-HNE)的含量;采用蛋白质印迹法检测大鼠右侧SNc中铁死亡相关蛋白[铁蛋白重链1(FTH1)、Sigma受体1(S1R)、谷胱甘肽过氧化物酶4(GPX4)、长链脂酰辅酶A合成酶4(ACSL4)、溶质载体家族7成员11(SLC7A11)],钙调素依赖性蛋白激酶2(CAMKⅡ)/细胞外信号调节激酶(ERK)通路相关蛋白[磷酸化CAMKⅡ(p-CAMKⅡ)、磷酸化ERK(p-ERK)、磷酸化Kirsten大鼠肉瘤病毒癌基因同源物(p-KRAS)]的表达。结果(1)与正常对照组比较,PD模型组、α7nAChR空载组、α7nAChR过表达组大鼠的旋转圈数较多,差异均有统计学意义(P<0.05);与PD模型组比较,α7nAChR过表达组大鼠的旋转圈数较少,差异均有统计学意义(P<0.05)。(2)HE染色和尼氏染色结果显示,PD模型组大鼠右侧SNc多巴胺能神经元数量减少,尼氏体减少,伴有神经元分布紊乱、核凝聚或肿胀;α7nAChR过表达组的多巴胺能神经元外观明显改善,形态正常,细胞变性较少。(3)TUNEL染色结果显示,与正常对照组比较,PD模型组、α7nAChR空载组、α7nAChR过表达组大鼠的细胞凋亡率较高,差异均有统计学意义(P<0.05);与PD模型组比较,α7nAChR过表达组大鼠的细胞凋亡率较低,差异均有统计学意义(P<0.05)。(4)免疫荧光双标染色结果显示,与正常对照组(α-Syn:303.61±48.40、TH:13985.80±1956.06)比较,PD模型组、α7nAChR空载组、α7nAChR过表达组大鼠右侧SNc中α-Syn的表达增加(分别为4310.40±518.43、3846.60±524.47、1033.55±59.98),TH的表达降低(分别为760.97±57.26、842.55±113.41、8101.82±1171.85),差异均有统计学意义(P<0.05);与PD模型组比较,α7nAChR过表达组大鼠右侧SNc中α-Syn的表达降低,TH的表达增加,差异均有统计学意义(P<0.05)。(5)ELISA实验检测结果显示,与正常对照组比较,PD模型组、α7nAChR空载组、α7nAChR过表达组大鼠右侧SNc中4-HNE和MDA的含量升高,差异均有统计学意义(P<0.05);与PD模型组比较,α7nAChR过表达组大鼠右侧SNc中4-HNE和MDA的含量降低,差异均有统计学意义(P<0.05)。(6)蛋白质印迹法检测结果显示,与正常对照组比较,PD模型组、α7nAChR空载组、α7nAChR过表达组大鼠右侧SNc中FTH1、S1R、GPX4、SLC7A11蛋白的表达降低,ACSL4蛋白的表达增加,差异均有统计学意义(P<0.05);与PD模型组比较,α7nAChR过表达组大鼠右侧SNc中FTH1、S1R、GPX4、SLC7A11蛋白的表达增加,ACSL4蛋白的表达降低,差异均有统计学意义(P<0.05)。与正常对照组比较,PD模型组、α7nAChR空载组、α7nAChR过表达组大鼠右侧SNc中p-KRAS、p-CAMKⅡ、p-ERK蛋白的表达增加,差异均有统计学意义(P<0.05);与PD模型组比较,α7nAChR过表达组大鼠右侧SNc中p-KRAS、p-CAMKⅡ、p-ERK蛋白的表达降低,差异均有统计学意义(P<0.05)。结论α7nAChR可能通过调控CAMKⅡ/ERK通路与铁死亡相关蛋白对PD模型大鼠产生神经保护作用。
Objective To explore the neuroprotective effect ofα7 nicotinic acetylcholine receptor(α7nAChR)on rat models of Parkinson's disease(PD)and its underlying mechanism.Methods Forty-eight 8-week-old SPF-grade SD rats were randomly divided into a normal control group,a PD model group,anα7nAChR empty vector group and anα7nAChR overexpression group,with 12 rats in each group.PD models in the latter 3 groups of rats were established by 6-hydroxydopamine(6-OHDA).Four weeks after modeling,rats in the latter 2 groups were injected with 2μLα7nAChR overexpression lentivirus or empty vector lentivirus through stereotactic intracerebral injection,while rats in the normal control group did not receive any treatment.Two weeks after injection,the behavioral changes of these rats were detected by apomorphine-induced rotation test;the right substantia nigra pars compacta(SNc)was prepared and performed the following experiments:hematoxylin-eosin(HE)staining and Nissl staining were used to detect the neuron morphological changes,TUNEL was used to detect the neuron apoptosis,fluorescent double labeling was used to detect the expressions of tyrosine hydroxylase(TH)andα-synuclein(α-Syn),ELISA was used to detect the expressions of 4-hydroxynonenal(4-HNE)and malondialdehyde(MDA),and Western blotting was used to detect the expressions of ferroptosis-related proteins(ferritin heavy chain 1[FTH1],Sigma receptor 1[S1R],glutathione peroxidase 4[GPX4],long chain acyl-coa synthetase 4[ACSL4],solute carrier family 7 member11[SLC7A11]),and the expressions of proteins related to CAMKII/ERK pathway(phosphorylated calmodulin kinaseⅡ[p-CAMKⅡ],phosphorylated extracellular signal regulated kinase[p-ERK],and phosphorylated Kirsten rat sarcoma viral oncogene homolog[p-KRAS]).Results(1)Compared with the normal control group,the PD model group,α7nAChR empty vector group andα7nAChR overexpression group had significantly larger number of rotations(P<0.05);compared with the PD model group,theα7nAChR overexpression group had significantly smaller number of rotations(P<0.05).(2)HE staining and Nissl staining showed that the PD model group had decreased number of dopaminergic neurons and Nissl bodies,accompanied by neuronal distribution disorder,nuclear condensation or swelling;theα7nAChR-overexpression group had obviously improved appearance of dopaminergic neurons,with normal morphology and less cell degeneration.(3)TUNEL results showed that compared with the normal control group,the PD model group,α7nAChR empty vector group,andα7nAChR overexpression group had significantly higher apoptosis rate(P<0.05);compared with the PD model group,theα7nAChR overexpression group had statistically lower apoptosis rate(P<0.05).(4)Double immunofluorescent staining results showed that compared with the normal control group(303.61±48.40,13985.80±1956.06),the PD model group,α7nAChR empty vector group andα7nAChR overexpression group had significantly increasedα-Syn expression(4310.40±518.43,3846.60±524.47 and 1033.55±59.98)and statistically decreased TH expression(760.97±57.26,842.55±113.41 and 8101.82±1171.85)in the right SNc(P<0.05);compared with the PD model group,theα7nAChR overexpression group had significantly decreasedα-Syn expression and increased TH expression in the right SNc(P<0.05).(5)ELISA results showed that the 4-HNE and MDA expressions in the right SNc of the PD model group,α7nAChR empty vector group andα7nAChR overexpression group were significantly higher than those in the normal control group(P<0.05);the 4-HNE and MDA expressions in theα7nAChR overexpression group were significantly lower than those in the PD model group(P<0.05).(6)Western blotting results showed that compared with the normal control group,the PD model group,α7nAChR empty vector group andα7nAChR overexpression group had significantly decreased FTH1,S1R,GPX4,and SLC7A11 protein expressions,and statistically increased ACSL4 protein expression in the right SNc(P<0.05);compared with the PD model group,theα7nAChR overexpression group had significantly increased FTH1,S1R,GPX4,and SLC7A11 protein expressions and decreased ACSL4 protein expression in the right SNc(P<0.05).Compared with the normal control group,the PD model group,α7nAChR empty vector group andα7nAChR overexpression group had significantly increased p-KRAS,p-CAMKII,and p-ERK protein expressions in the right SNc(P<0.05);compared with the PD model group,theα7nAChR overexpression group had significantly decreased p-KRAS,p-CAMKII,and p-ERK protein expressions in the right SNc(P<0.05).Conclusion Theα7nAChR may exert neuroprotective effect on PD rat models by regulating the CAMKII/ERK pathway and ferroptosis-related proteins.
作者
潘燕
胡歆
裴静
童书杰
Pan Yan;Hu Xin;Pei Jing;Tong Shujie(Department of Neurology,Fifth Affiliated Hospital of Xinjiang Medical University,Urumuqi 830011,China)
出处
《中华神经医学杂志》
北大核心
2025年第6期561-571,共11页
Chinese Journal of Neuromedicine
基金
新疆维吾尔自治区自然科学基金(2022D01C570)
新疆神经系统疾病研究重点实验室项目(XJDX1711-2422)。
