摘要
目的探讨抗衰老蛋白Klotho对睡眠剥夺小鼠神经元凋亡的调控作用和潜在机制。方法体外培养小鼠神经元细胞,用200μg·mL-1的β-淀粉样蛋白(aβ)孵育细胞(aβ组),用等量PBS孵育细胞(对照组),用200μg·mL-1的aβ联合100μg·mL-1的Klotho联合孵育细胞(Klotho+aβ组)。CCK-8法检测各组细胞增殖活力。流式细胞术检测细胞凋亡。Western blot检测细胞中克罗托蛋白(Klotho)、沉默信息调节因子1(Sirt1)、核因子E2相关因子2(Nrf2)、磷酸化核因子E2相关因子2(p-Nrf2)的表达。将45只成年雌性小鼠随机分为5组,包括正常组(Con组)、睡眠剥夺组(SD组)、Klotho+SD组(预先30 min海马区注10 mg·kg^(-1)Klotho)、EX527+Klotho+SD组(预先30 min海马区注射10 mg·kg^(-1)Klotho和1.5 mg·kg^(-1)的Sirt1抑制剂EX527)、ML385+Klotho+SD组(预先30 min海马区注射10 mg·kg^(-1)Klotho和4.0 mg·kg^(-1)的Nrf2抑制剂ML385)。除正常组外其余小鼠被放置于自动睡眠剥夺系统中以5 r/min旋转的方式诱导20 h建立睡眠剥夺小鼠模型。试剂盒法检测小鼠海马组织中活性氧(ROS)、丙二醛(MDA)、超氧化物歧化酶(SOD)的水平,Western blot检测海马组织中Sirt1、Nrf2、p-Nrf2、Bax、cleaved-caspase3、Bcl-2的表达。结果与对照组比较,aβ组增殖活力降低,细胞凋亡率增加,Klotho、Sirt1、p-Nrf2的表达降低(^(均)P<0.05)。与aβ组比较,Klotho+aβ组增殖活力增加,细胞凋亡率降低,Klotho、Sirt1、p-Nrf2的表达增加(^(均)P<0.05)。与Con组比较,SD组海马组织中ROS、MDA、Bax、cleaved-caspase3的水平上调,Sirt1、p-Nrf2、SOD、Bcl-2的水平均下调(^(均)P<0.05)。与SD组比较,Klotho+SD组海马组织中ROS、MDA、Bax、cleaved-caspase3的水平均下调,Sirt1、p-Nrf2、SOD、Bcl-2的水平均上调(^(均)P<0.05)。与Klotho+SD组比,EX527+Klotho+SD组海马组织中ROS、MDA、Bax、cleaved-caspase3的水平均上调,Sirt1、p-Nrf2、SOD、Bcl-2的水平均下调(^(均)P<0.05)。ML385+Klotho+SD组海马组织中ROS、MDA、Bax、cleaved-caspase3的水平均上调,p-Nrf2、SOD、Bcl-2的水平均下调(^(均)P<0.05)。结论Klotho通过激活Sirt1/Nrf2信号通路抑制睡眠剥夺小鼠的神经元凋亡。
Objective To investigate the regulatory effect and potential mechanism of the anti-aging protein Klotho on neuronal apoptosis in sleep-deprived mice.Methods Mouse neuronal cells were cultured in vitro,incubated with 200μg·mL^(-1)of Aβ(Aβgroup),incubated with an equal amount of PBS(control group),and co-incubated with 200μg·mL^(-1)of Aβand 100μg·mL^(-1)of Klotho(Klotho+Aβgroup).Cell proliferation activity was detected using the CCK-8 method.Flow cytometry was used to detect cell apoptosis.Western blot was performed to detect the expression of Klotho,silent information regulator 1(Sirt1),nuclear factor erythroid 2-related factor 2(Nrf2),and phosphorylated-Nrf2(p-Nrf2)in cells.Forty-five adult female mice were randomly divided into 5 groups,including the normal group(Con group),sleep deprivation group(SD group),Klotho+SD group(previously injected with 10 mg·kg^(-1)Klotho in the hippocampal region 30 minutes before),EX527+Klotho+SD group(previously injected with 10 mg·kg^(-1)Klotho and 1.5 mg·kg^(-1)Sirt1 inhibitor EX527 in the hippocampal region 30 minutes before),and ML385+Klotho+SD group(previously injected with 10 mg·kg^(-1)Klotho and 4.0 mg·kg^(-1)Nrf2 inhibitor ML385 in the hippocampal region 30 minutes before).Except for the normal group,the rest of the mice were placed in an automatic sleep deprivation system to induce a sleep-deprived mouse model by rotating at 5 rpm for 20 hours.The levels of ROS,MDA,and SOD in the mouse hippocampal tissue were measured using assay kits,and the expression of Sirt1,Nrf2,p-Nrf2,Bax,cleaved-caspase3,and Bel-2 in the hippocampal tissue was detected by Western blot.Results Compared with the control group,the Aβgroup showed decreased cell proliferation activity,increased cell apoptosis rate,and decreased expression of Klotho,Sirt1,and p-Nrf2(^(all)P<0.05).Compared with the Aβgroup,the Klotho+Aβgroup showed increased cell proliferation activity,decreased cell apoptosis rate,and increased expression of Klotho,Sirt1,and p-Nrf2(^(all)P<0.05).Compared with the Con group,the SD group showed upregulated levels of ROS,MDA,Bax,cleaved-caspase3,and downregulated levels of Sirt1,p-Nrf2,SOD,and Bel-2(^(all)P<0.05).Compared with the SD group,the Klotho+SD group showed downregulated levels of ROS,MDA,Bax,cleaved-caspase3,and upregulated levels of Sirt1,p-Nrf2,SOD,Bcl-2(^(all)P<0.05).Compared with the Klotho+SD group,the EX527+Klotho+SD group showed upregulated levels of ROS,MDA,Bax,cleaved-caspase3,and downregulated levels of Sirt1,p-Nrf2,SOD,Bcl-2(^(all)P<0.05).The ML385+Klotho+SD group showed upregulated levels of ROS,MDA,Bax,cleaved-caspase3,and downregulated levels of p-Nrf2,SOD,Bcl-2(^(all)P<0.05)compared with the Klotho+SD group.Conclusion Klotho inhibits neuronal apoptosis in sleep-deprived mice by activating the Sirt1/Nrf2 signaling pathway.
作者
徐佳骏
韩炜
刘丽丹
彭颜晖
Xu Jiajun;Han Wei;Liu Lidan;Peng Yanhui(Department of Neurology,the Sixth Affiliated Hospital of Xinjiang Medical University,Urumqi 830002,China)
出处
《脑与神经疾病杂志》
2025年第7期402-408,共7页
Journal of Brain and Nervous Diseases
基金
新疆维吾尔自治区自然科学基金资助项目(2023D01C225)。
