摘要
背景:法舒地尔对阿尔茨海默病小鼠脑内的线粒体动力学有调节作用,并且可以抑制神经炎症,但能否调节线粒体自噬和NLRP3炎症小体进而减轻β-淀粉样蛋白毒性尚不清楚。目的:探究法舒地尔对β-淀粉样蛋白1-42诱导的人源神经母细胞瘤细胞株SH-SY5Y凋亡和线粒体自噬以及NLRP3炎症小体的调节作用。方法:将SH-SY5Y细胞接种于孔板内,细胞贴壁后分3组干预:对照组不加入任何药物,模型组加入20μmol/L β-淀粉样蛋白1-42,法舒地尔组同时加入20μmol/L β-淀粉样蛋白1-42与15 mg/L法舒地尔,干预24 h后,采用MTT法检测细胞活性,TUNEL染色检测细胞凋亡,qRT-PCR和Western blot检测凋亡相关蛋白表达,免疫荧光染色和Western blot检测线粒体自噬相关蛋白表达,免疫荧光染色和Western blot检测NLRP3炎症小体相关蛋白表达。结果与结论:(1)与对照组比较,模型组细胞活性降低、凋亡率升高(P<0.05);与模型组比较,法舒地尔组细胞活性升高、凋亡率降低(P<0.05);(2)qRT-PCR和Western blot检测结果显示,与对照组比较,模型组细胞Bax mRNA与蛋白表达升高(P<0.05),Bcl-2 mRNA与蛋白表达降低(P<0.05);与模型组比较,法舒地尔组细胞Bax mRNA与蛋白表达降低(P<0.05),Bcl-2 mRNA与蛋白表达升高(P<0.05);(3)免疫荧光染色和Western blot检测结果显示,与对照组相比,模型组细胞PINK1、帕金森病蛋白和LC3蛋白表达降低(P<0.05),p62蛋白表达升高(P<0.05);与模型组相比,法舒地尔组细胞PINK1、帕金森病蛋白和LC3蛋白表达升高(P<0.05),p62蛋白表达降低(P<0.05);(4)免疫荧光染色和Western blot检测结果显示,与对照组相比,模型组细胞NLRP3、ASC、Caspase-1和白细胞介素1β蛋白表达升高(P<0.05);与模型组相比,法舒地尔组细胞NLRP3、ASC、Caspase-1和白细胞介素1β蛋白表达降低(P<0.05);(5)结果表明,法舒地尔可以减轻β-淀粉样蛋白1-42诱导的SH-SY5Y细胞凋亡,其机制可能与激活线粒体自噬且抑制NLRP3炎症小体激活有关。
BACKGROUND:Fasudil has a regulatory effect on mitochondrial dynamics in the brain of Alzheimer’s disease mice and can inhibit neuroinflammation,but whether it can reduce the toxicity ofβ-amyloid protein by regulating mitophagy-NLRP3 inflammasome pathway remains unclear.OBJECTIVE:To investigate the regulatory effect of fasudil onβ-amyloid 1-42-induced apoptosis and mitophagy and NLRP3 inflammasome in human derived neuroblastoma cell line SH-SY5Y cells.METHODS:SH-SY5Y cells were inoculated into the pore plate.After adhesion,cells were divided into three groups for intervention:No drug was added to the control group;20μmol/Lβ-amyloid 1-42 was added to the model group,and 20μmol/Lβ-amyloid 1-42 and 15 mg/L fasudil were added to the fasudil group at the same time.After 24 hours of intervention,the cell activity was detected by MTT assay and apoptosis was detected by TUNEL staining.The expression of apoptosis-related proteins was detected by qRT-PCR and western blot assay.The expression of mitochondrial autophagy related proteins was detected by immunofluorescence staining and western blot assay.The expression of NLRP3 inflammasome related proteins was detected by immunofluorescence staining and western blot assay.RESULTS AND CONCLUSION:(1)Compared with control group,the cell activity of the model group was decreased and the apoptosis rate was increased(P<0.05).Compared with model group,cell activity in the fasudil group was increased and apoptosis rate was decreased(P<0.05).(2)The results of qRT-PCR and western blot assay showed that compared with the control group,the expression of Bax mRNA and protein was increased in the model group(P<0.05),while the expression of Bcl-2 mRNA and protein was decreased(P<0.05).Compared with the model group,the expression of Bax mRNA and protein was decreased(P<0.05),and the expression of Bcl-2 mRNA and protein was increased(P<0.05)in the fasudil group.(3)The results of immunofluorescence staining and western blot assay showed that compared with the control group,the expressions of PINK1,Parkinson’s disease protein and LC3 protein were decreased(P<0.05),while the expression of p62 protein was increased(P<0.05)in the model group.Compared with model group,the expression levels of PINK1,Parkinson’s disease protein,and LC3 protein were increased(P<0.05),while the expression of p62 protein was decreased(P<0.05)in the fasudil group.(4)The results of immunofluorescence staining and western blot assay showed that compared with the control group,the expression levels of NLRP3,ASC,Caspase-1,and interleukin1βprotein were increased in the model group(P<0.05).Compared with the model group,the expression levels of NLRP3,ASC,Caspase-1,and interleukin1βwere decreased in the fasudil group(P<0.05).(5)The results show that fasudil can reduce the apoptosis of SH-SY5Y cells induced byβ-amyloid 1-42,and its mechanism may be related to the activation of mitophagy and the inhibition of NLRP3 inflammasome activation.
作者
郭敏芳
张慧宇
章培军
苏琴
贾思玮
尉杰忠
Guo Minfang;Zhang Huiyu;Zhang Peijun;Su Qin;Jia Siwei;Yu Jiezhong(Institute of Brain Science/Key Laboratory of Molecular Cellular Immunology in Datong,Shanxi Datong University,Datong 037009,Shanxi Province,China;Research Center of Neurobiology,Key Research Laboratory of Benefiting Qi for Acting Blood Circulation Method to Treat Multiple Sclerosis of State Administration of Traditional Chinese Medicine,Shanxi University of Chinese Medicine,Jinzhong 030619,Shanxi Province,China;First Affiliated Hospital of Shanxi Datong University/Datong Fifth People’s Hospital,Datong 037009,Shanxi Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2025年第23期4939-4946,共8页
Chinese Journal of Tissue Engineering Research
基金
山西省基础研究计划项目(20210302123476),项目负责人:郭敏芳
山西省基础研究计划项目(20210302123478),项目负责人:张慧宇
山西省2022年度“四个一批”科技兴医创新计划项目(2022XM33),项目负责人:尉杰忠
山西省中医药科研课题计划项目(2023ZYYB2042),项目负责人:尉杰忠
山西大同大学大学生创新创业训练计划项目(XDC2022174),项目负责人:郭敏芳。
