摘要
背景:目前研究证实,灵孢多糖可促进神经退行性相关疾病的神经再生。神经退行性疾病的发生与线粒体功能失调密切相关,但灵孢多糖对神经退行性疾病的细胞凋亡及线粒体功能的调控作用尚不明确。目的:探索灵孢多糖对过氧化氢诱导的SH-SY5Y细胞凋亡及线粒体功能障碍的调控作用及机制。方法:SH-SY5Y细胞分为3组:对照组,过氧化氢组,灵孢多糖组。对照组细胞正常培养,过氧化氢组细胞用300μmol/L过氧化氢处理24 h,灵孢多糖组先用300μg/μL灵孢多糖干预一两个小时,然后加入300μmol/L过氧化氢干预24 h,干预结束后用JC-1试剂盒检测线粒体膜电位,TUNEL染色试剂盒检测细胞凋亡情况,丙二醛试剂盒和超氧化物歧化酶试剂盒检测丙二醛和超氧化物歧化酶活性,免疫荧光染色法和Western blot法检测凋亡、线粒体动力学相关蛋白的表达。结果与结论:①与对照组相比,过氧化氢组线粒体膜电位和超氧化歧化酶活性显著降低,细胞凋亡率、丙二醇水平显著增高,差异均有显著性意义(P<0.05);与过氧化氢组相比,灵孢多糖组线粒体膜电位和超氧化歧化酶活性显著增高,细胞凋亡率、丙二醛水平显著下降,差异均有显著性意义(P<0.05);②与对照组相比,过氧化氢组促凋亡蛋白Bax和Caspase-3的表达显著增加,抗凋亡蛋白Bcl-2的表达显著下降,差异均有显著性意义(P<0.05);与过氧化氢组相比,灵孢多糖组促凋亡蛋白Bax、Caspase-3的表达显著降低,抗凋亡蛋白Bcl-2的表达显著增加,差异均有显著性意义(P<0.05);③与对照组相比,过氧化氢组线粒体分裂蛋白Fis1与线粒体动力学蛋白p-Drp1的表达均显著增高,线粒体融合蛋白OPA1、Mfn1、Mfn2的表达均显著降低,差异均有显著性意义(P<0.05);与过氧化氢组相比,灵孢多糖组线粒体分裂蛋白Fis1与线粒体动力学蛋白p-Drp1的表达显著降低,线粒体融合蛋白OPA1、Mfn1、Mfn2的表达显著增高,差异均有显著性意义(P<0.05);④以上结果证实,灵孢多糖可通过改善线粒体功能障碍以减轻过氧化氢诱导的SH-SY5Y细胞氧化应激损伤和细胞凋亡。
BACKGROUND:Current studies have confirmed that Ganoderma lucidum polysaccharides can promote nerve regeneration in neurodegeneration-related diseases.The occurrence of neurodegenerative diseases is closely related to mitochondrial dysfunction,but the role of Ganoderma lucidum polysaccharides on the regulation of apoptosis and mitochondrial function in neurodegenerative diseases is not yet clarified.OBJECTIVE:To explore the regulatory effects and mechanisms of Ganoderma lucidum polysaccharides on apoptosis and mitochondrial dysfunction in H_(2)O_(2)-induced SH-SY5Y cells.METHODS:SH-SY5Y cells were divided into three groups:control group,H_(2)O_(2)group,and Ganoderma lucidum polysaccharides group.Cells in the control group were normally cultured.Cells in the H_(2)O_(2)group were treated with 300μmol/L H_(2)O_(2)for 24 hours.In the Ganoderma lucidum polysaccharides group,the intervention with 300μg/L Ganoderma lucidum polysaccharides was conducted first for 1-2 hours,followed by the addition of 300μmol/L H_(2)O_(2)for 24 hours.The mitochondrial membrane potential was detected by JC-1 kit.Apoptosis was detected by TUNEL staining kit.The activities of malondialdehyde and superoxide dismutase were detected by malondialdehyde test kit and superoxide dismutase test kit,respectively.The apoptosis and expression of mitochondrial dynamics-related proteins were detected by immunofluorescence staining and western blot assay.RESULTS AND CONCLUSION:(1)Compared with the control group,the mitochondrial membrane potential and superoxide dismutase activity were significantly reduced,as well as apoptotic rate and malondialdehyde levels were significantly increased in the H_(2)O_(2)group(P<0.05).After treatment with Ganoderma lucidum polysaccharides,the membrane potential and superoxide dismutase activities were significantly increased,and apoptotic rate and malondialdehyde levels were significantly reduced compared with the H_(2)O_(2)group(P<0.05).(2)The expression levels of pro-apoptotic proteins Bax and Caspase-3 were significantly increased,but the expression of anti-apoptotic protein Bcl-2 was significantly decreased in the H_(2)O_(2)group compared with the control group(P<0.05).Compared with the H_(2)O_(2)group,the levels of Bax and Caspase-3 were significantly decreased,but the expression of anti-apoptotic protein Bcl-2 was significantly increased in the Ganoderma lucidum polysaccharides group(P<0.05).(3)Compared with the control group,the expression of mitochondrial splitting proteins Fis1 and p-Drp1 was significantly increased,but the expression of mitochondrial fusion proteins OPA1,Mfn1,and Mfn2 was decreased in the H_(2)O_(2)group(P<0.05).Compared with the H_(2)O_(2)group,Fis1 and p-Drp1 expression was significantly reduced,but the expression levels of OPA1,Mfn1,and Mfn2 were significantly increased in the Ganoderma lucidum polysaccharides group(P<0.05).(4)The above results confirm that Ganoderma lucidum polysaccharides can attenuate H_(2)O_(2)-induced oxidative stress damage and apoptosis in SH-SY5Y cells by ameliorating mitochondrial dysfunction.
作者
李雁冰
王记委
刘晓琴
郭敏芳
牛晓洁
孟涛
苏琴
王瀚斌
杨立志
马存根
尉杰忠
Li Yanbing;Wang Jiwei;Liu Xiaoqin;Guo Minfang;Niu Xiaojie;Meng Tao;Su Qin;Wang Hanbin;Yang Lizhi;Ma Cungen;Yu Jiezhong(Research Center of Neurobiology/The 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;Institute of Brain Science,Shanxi Datong University,Datong 037009,Shanxi Province,China;Shanxi Guorun Pharmaceutical Co.,Ltd.,Datong 038100,Shanxi Province,China;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
2024年第25期4041-4047,共7页
Chinese Journal of Tissue Engineering Research
基金
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
山西省基础研究计划青年科学研究项目(20210302124276),项目负责人:刘晓琴
山西大同大学横向项目(2022001),项目负责人:刘晓琴
山西大同大学校级科研项目(2022Q21),项目负责人:刘晓琴
山西省卫生健康委科研项目(2021168),项目负责人:尉杰忠
大同市平台计划项目(2022082),项目负责人:尉杰忠。