补肾止颤方调控miR-296-5p/GSDMD通路抑制帕金森病大鼠神经元焦亡机制研究

Research on Mechanism of Bushen Zhichan Decoction(补肾止颤方)Regulating miR-296-5p/GSDMD Pathway to Inhibit Neuronal Charring in Rats with Parkinson's Disease

目的从焦亡角度探讨补肾止颤方保护帕金森病(Parkinson's disease,PD)大鼠中脑黑质区神经元的作用以及可能机制。方法将60只雄性SD大鼠随机分为正常组,模型组,补肾止颤方低(15 g·kg-1)、中(30 g·kg-1)、高(60 g·kg-1)剂量组,美多芭组(0.2 g·kg-1)。除正常组外,余各组予背部皮下注射蛋白酶体抑制因子I(proteasome inhibitors I,PSI)溶液构建慢性PD模型。模型组与正常组予等体积生理盐水灌胃,余各组予对应药物剂量灌胃,连续干预3周。采用行为学观察爬杆实验评估各组大鼠行为学改变,予透射电镜观察大鼠中脑黑质区神经元细胞结构变化;实时荧光定量聚合酶链式反应(real time quantitative PCR,RT-PCR)检测大鼠中脑黑质区miR-296-5P及Gasdermin D(GSDMD)蛋白、天冬氨酸特异性半胱氨酸蛋白酶-1(Caspase-1)、白细胞介素-1β(interleukin-1β,IL-1β)的mRNA表达;蛋白免疫印迹法(Western blot)检测α-突触核蛋白(α-synuclein,α-syn)、酪氨酸羟化酶(tyrosine hydroxylase,TH)蛋白表达;双荧光素酶报告基因技术测定miR-296-5p与GSDMD上的靶向调控作用。结果与正常组比较,余各组大鼠出现自主活动减少、全身不自主震颤、行走迟缓等症状,悬挂实验评分下降(P<0.05);电镜下可见神经元细胞呈不同程度的水肿,部分细胞膜结构模糊,细胞核呈不规则形,细胞中α-syn、Caspase-1、IL-1β、GSDMD mRNA表达明显升高(P<0.05),TH及miR-296-5p表达水平明显下降(P<0.05)。与模型组比较,补肾止颤方低、中、高剂量组大鼠全身不自主震颤、行走迟缓等症状改善,悬挂实验评分升高(P<0.05),细胞水肿减轻,细胞中α-syn、Caspase-1、IL-1β、GSDMD mRNA表达呈不同程度下降(P<0.05),TH及miR-296-5p表达水平升高(P<0.05),以补肾止颤方高剂量组改变显著;荧光素酶报告显示miR-296-5p可靶向调控GSDMD的表达。结论补肾止颤方能改善PD大鼠的运动协调能力及受损的神经元细胞,其机制可能与调控miR-296-5p/GSDMD通路,下调Caspase-1蛋白及炎性因子水平,减轻神经炎症反应,从而抑制PD大鼠神经元细胞焦亡有关。

Objective To explore the protective effect of Bushen Zhichan Decoction(补肾止颤方)on neurons in substantia nigra of midbrain in Parkinson's disease(PD)rats and its possible mechanism.Methods Sixty male SD rats were randomly divided into normal group,model group,Bushen Zhichan Decoction low(15 g·kg-1),medium(30 g·kg-1),high(60 g·kg-1)dose groups and Madopar group(0.2 g·kg-1).Except the normal group,each group was injected subcutaneously with proteasome inhibitor I(PSI)solution on the back to construct the chronic PD model.The model group and the normal group were given the same volume of normal saline,and the corresponding drugs were given to each group for 3 weeks.The behavioral changes of rats in each group were evaluated by behavioral observation,roller test and climbing rod test,and the structural changes of neurons in the substantia nigra of rats were observed by transmission electron microscopy.RT-qPCR was used to detect the mRNA expression of miR-296-5P,GSDMD,Caspase-1 and IL-1βin the substantia nigra of rats.Western blot was used to detect the expressions ofα-syn and TH proteins.The dual luciferase reporter gene technology was used to determine the targeted regulation of miR-296-5p and GSDMD.Results Compared with those of the normal group,the rats in each group showed symptoms such as reduced autonomic activity,systemic involuntary tremor and walking retardation.The scores of rolling axis test and suspension test were decreased(P<0.05).Under the electron microscope,the neuron cells showed varying degrees of edema,some cell membrane structures were fuzzy and the nuclei were irregular.The mRNA expressions ofα-syn,Caspase-1,IL-1βand GSDMD in the cells were significantly increased(P<0.05),and the expressions of TH and miR-296-5p were significantly decreased(P<0.05).Compared with those of the model group,the rats in the low,medium and high dose groups of Bushen Zhichan Decoction showed improved symptoms such as systemic involuntary tremor and walking retardation,increased scores of rolling test and suspension test(P<0.05),reduced cell edema,decreased mRNA expression ofα-syn,Caspase-1,IL-1βand GSDMD in cells to varying degrees(P<0.05),and increased expression levels of TH and miR-296-5p(P<0.05).The changes in the high dose group of Bushen Zhichan Decoction were obvious.The luciferase report showed that miR-296-5p could regulate the expression of GSDMD.Conclusion Bushen Zhichan Decoction can improve the motor coordination ability of PD rats and protect the damaged neuron cells.The mechanism may be related to the regulation of miR-296-5p/GSDMD pathway,down-regulation of Caspase-1 protein and inflammatory factor levels,and reduction of nerve inflammatory response,thereby inhibiting pyroptosis of neuron cells in PD rats.