基于PERK-eIF2α通路探讨苁蓉舒痉颗粒对帕金森病模型大鼠黑质纹状体内质网应激的调节作用

Regulation of Congrong Shujing Granules on Stress Response in Nigrostriatal Endoplasmic Reticulum of Parkinson's Disease Model Rats Based on PERK-eIF2αPathway

目的 研究苁蓉舒痉颗粒对鱼藤酮诱导的帕金森病(PD)模型大鼠黑质纹状体内质网应激蛋白激酶样内质网激酶(PERK)-真核翻译起始因子2α(eIF2α)通路的调节作用。方法 将100只健康SD大鼠随机分为正常组33只和造模组67只,造模组采用颈背部皮下注射鱼藤酮葵花油乳液建立PD模型,参照行为学评分标准鉴定造模成功后,随机分为模型组34只和治疗组33只。治疗组予苁蓉舒痉颗粒药液6.8 mL/(kg·d)灌胃,正常组、模型组予等量生理盐水灌胃,连续干预14 d。于灌胃第1、3、5、7、9、11、13、14天,采用平行反应监测(PRM)蛋白相对定量分析检测大鼠黑质纹状体eIF2α特异性肽段定量的变化;灌胃14 d后应用透射电镜观察3组大鼠黑质神经细胞超微结构;采用免疫组化法检测大鼠黑质PERK、p-PERK、eIF2α、p-eIF2α表达的平均光密度值。结果与正常组比较,在灌胃第13、14天模型组黑质纹状体eIF2α特异性肽段定量明显增高(P均<0.05),灌胃14 d后黑质神经细胞肿胀,内质网扩张,黑质PERK、p-PERK、eIF2α、p-eIF2α的平均光密度值增高(P<0.05,P<0.01);与模型组比较,治疗组在灌胃第5天黑质纹状体eIF2α特异性肽段定量明显增高(P<0.01),灌胃第13、14天时明显减低(P均<0.05),灌胃14 d后黑质神经细胞肿胀、内质网扩张等形态异常有所改善,黑质PERK、pPERK、eIF2α、p-eIF2α的平均光密度值明显降低(P<0.05,P<0.01)。结论 苁蓉舒痉颗粒可能通过动态调节内质网应激PERK-eIF2α通路,起到恢复内质网稳态的作用。

Objective:To study the regulating effect of Congrong Shujing(CRSJ)granules on the protein kinase-like ER kinase(PERK)-eukaryotic translation initiation factor 2 alpha(eIF2α)pathway in the nigrostriatum of Parkinson's disease(PD)model rats induced by rotenone.Methods:A total of 100 SD rats were randomly divided into normal group(n=33)and modeling group(n=67).The PD model was established in the modeling group by subcutaneous injection of rotenone sunflower oil emulsion into the neck and back.The rats with successful modeling according to the behavioral scoring criteria were randomly divided into model group(n=34)and treatment group(n=33).The treatment group was given 6.8 mL/(kg·d)CRSJ granules by gavage,while the normal group and model group were given equal volume of physiological saline,with continuous intervention for 14 days.On days 1,3,5,7,9,11,13,and 14 of gavage,parallel reaction monitoring(PRM)protein relative quantitative analysis was used to detect the quantification of eukaryotic translation initiation factor 2α(eIF2α)-specific peptides in the nigrostriatum of rats.After intragastric administration for 14 days,the ultrastructural changes of rat substantia nigra neurons were observed by transmission electron microscopy,and the mean optical density of PERK,p-PERK,eIF2α,and p-eIF2αin rat substantia nigra were detected by immunohistochemistry.Results:Compared with the normal group,the quantification of eIF2α-specific peptides in the nigrostriatum of the model group significantly increased on the 13th and 14th days of gavage(P<0.05).After intragastric administration for 14 days,the neurons in the substantia nigra of the model group were swollen,and the endoplasmic reticulum was expanded;the mean optical density of PERK,p-PERK,eIF2α,and p-eIF2αsignificantly increased than that of the normal group(P<0.05 or 0.01).Compared with the model group,the quantification of eIF2α-specific peptides in the nigrostriatum of the treatment group significantly increased on the 5th days of gavage(P<0.05),and significantly decreased on the 13th and 14th day of gavage(P<0.05).After intragastric administration for 14 days,the swelling of substantia nigra neurons and the expansion of endoplasmic reticulum were improved;the mean optical density of PERK,p-PERK,eIF2α,and p-eIF2αsignificantly decreased than that of the model group(P<0.05 or 0.01).Conclusion:CRSJ granules may dynamically regulate the PERK-eIF2αpathway of endoplasmic reticulum stress to restore endoplasmic reticulum homeostasis.