基于PINK1-Parkin线粒体自噬通路探讨海昆肾喜含药血清对N2a/APP695细胞的神经保护作用  

The neuroprotective effects of Haikun Shenxi capsule medicated serum on N2a/APP695 cells based on PINK1-Parkin mitophagy pathway

作　　者：孙妍华 季敬 彭娇 何珊[1,2] 付先军 SUN Yan-hua;JI Jing;PENG Jiao;HE Shan;FU Xian-jun(College of Pharmacy,Shandong University of Traditional Chinese Medicine,Shandong University Key Laboratory of Marine Traditional Chinese Medicine,Shandong Traditional Chinese Medicine Omics Engineering Technology Research Center,Shandong University of Traditional Chinese Medicine,Jinan 250355,China;Marine Traditional Chinese Medicine Research Center,Shandong University Key Laboratory of Marine Traditional Chinese Medicine,Shandong Traditional Chinese Medicine Omics Engineering Technology Research Center,Shandong University of Traditional Chinese Medicine,Jinan 250355,China)

机构地区：[1]山东中医药大学药学院,山东济南250355 [2]海洋中药研究中心山东省高等学校海洋中药重点实验室山东中医药组学工程技术研究中心山东中医药大学,山东济南250355

出　　处：《中国药理学通报》2024年第3期461-468,共8页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金青年科学基金项目(No81903944);国家中医药管理局高水平中医药重点学科建设项目(No zyyzdxk-2023124);山东省中医药重点学科建设项目[(No 2022)4号]。

摘　　要：目的研究海昆肾喜(haikun shenxi,HKSX)含药血清对N2a/APP695细胞的神经保护作用及机制。方法制备HKSX含药血清,高效薄层色谱(high performance thin layer chromatography,HPTLC)对其成分进行分析。应用含药血清干预N2a/APP695细胞,MTT检测HKSX含药血清的细胞毒性;ELISA检测Aβ_(1-42)含量;JC-1法检测线粒体膜电位、DCFH-DA法检测活性氧(reactive oxygen species,ROS)、ATP检测试剂盒检测三磷酸腺苷(adenosine triphosphate,ATP)评价线粒体功能;免疫印迹法(Western blot)检测PTEN诱导的激酶1(PTEN induced putative kinase 1,PINK1)、Parkin、p62、微管相关蛋白1轻链3(microtubule-associated-proteinlight chain-3,LC3)及p-Tau S396蛋白的表达;设置线粒体自噬抑制剂Mdivi-1干预组,Western blot检测PINK1、Parkin、p-Tau S396蛋白表达,ELISA检测Aβ_(1-42)含量,验证HKSX的神经保护作用是否与线粒体自噬密切相关。结果与模型对照组(MC)相比,HKSX低、中、高剂量组Aβ_(1-42)含量明显降低,高剂量组尤为明显(P<0.01);线粒体膜电位及ROS含量明显降低(P<0.01)、ATP含量升高(P<0.01);PINK1、Parkin(P<0.01)及LC3Ⅱ(P<0.01)的表达增加,而p62的表达降低(P<0.01),p-Tau S396蛋白亦降低(P<0.01);而Mdivi-1的干预,在很大程度上抑制了HKSX对PINK1、Parkin蛋白的活化(P<0.01)及对p-Tau S396和Aβ_(1-42)的清除(P<0.01),表明HKSX的神经保护作用依赖于PINK1-Parkin信号通路。结论HKSX能够提高线粒体自噬相关蛋白PINK1、Parkin蛋白的表达,激活线粒体自噬,提高线粒体功能,抑制Aβ及p-Tau S396蛋白在神经细胞中的沉积,发挥神经保护作用,从而起到防治AD的效果。Aim To research the neuroprotective effect of Haikun Shenxi(HKSX)medicated serum on N2a/App695 cells and the underlying mechanism.Methods HKSX medicated serum was prepared and carbohydrate components in it was analyzed using high performance thin layer chromatography(HPTLC).N2a/App695 cells were intervened with HKSX medicated serum,the cytotoxicity of HKSX medicated serum was measured by MTT;Aβ_(1-42)content was detected by ELISA;mitochondrial function indicators including mitochondrial membrane potential(MMP),reactive oxygen species(ROS)and adenosine triphosphate(ATP)were detected using JC-1,DCFH-DA,and ATP detection kit respectively;the protein expression levels of PTEN-induced putative kinase 1(PINK1),Parkin,p62,microtubule-linked-proteinlight-chain-3(LC3)and p-Tau S396 were detected by Western blot;a mitophagy inhibitor Mdivi-1 intervention group was set up to verify whether the neuroprotective effect of HKSX was closely related to mitophagy through detecting the expression of PINK1,Parkin,p-Tau S396 by Western blot and the content of Aβ_(1-42)by ELISA.Results Compared with the MC group,the contents of Aβ_(1-42)in the low,medium and high dose groups of HKSX were significantly reduced,especially in the high dose group(P<0.01);the MMP and ROS levels were significantly reduced(P<0.01),while the ATP content significantly increased(P<0.01).Besides,the expression of PINK1,Parkin and LC3-Ⅱ(P<0.01)increased,while the expression of p62 significantly decreased(P<0.01),and p-Tau S396 protein also significantly decreased(P<0.01).However,the intervention of Mdivi-1 strongly reversed the activation of PINK1 and Parkin proteins(P<0.01)and clearance of Aβ_(1-42)(P<0.01)by HKSX,indicating that the neuroprotective effects of HKSX depended on PINK1 signaling pathway.Conclusions HKSX could increase the expression of mitophagy-related proteins PINK1 and Parkin,activate mitophagy,improve mitochondrial function,inhibit the deposition of Aβand p-Tau S396 proteins in nerve cells,exert a neuroprotective effect,thereby achieving

关 键 词：阿尔茨海默病 海昆肾喜含药血清 化浊排毒 线粒体自噬 PINK1-Parkin N2a/App695 

分 类 号：R-332[医药卫生] R284.1R329.24R394.2R745.7