摘要
目的探讨1,25(OH)_(2)D_(3)对Aβ_(1-42)诱导阿尔茨海默病(Alzheimer′s disease,AD)细胞模型中细胞焦亡的抑制作用及其机制。方法将PC12细胞分为对照组、模型组、Caspase-1-siRNA组、NC-siRNA组、1,25(OH)_(2)D_(3)组、联合干预组。对照组仅用DMEM高糖培养基培养细胞;模型组用20μmol/L Aβ_(1-42)处理细胞以造模;Caspase-1-siRNA组先用50 nmol/L Caspase-1-siRNA处理细胞,再加入20μmol/L Aβ_(1-42);NC-siRNA组先用50 nmol/L NC-siRNA处理细胞,再加入20μmol/L Aβ_(1-42);1,25(OH)_(2)D_(3)组用100 nmol/L 1,25(OH)_(2)D_(3)干预后加入20μmol/L Aβ_(1-42);联合干预组先用50 nmol/L Caspase-1-siRNA处理细胞,然后用100 nmol/L 1,25(OH)_(2)D_(3)干预,最后加入20μmol/L Aβ_(1-42)。细胞免疫荧光检测凋亡相关斑点蛋白(ASC)蛋白的表达;Western blot检测细胞焦亡通路相关蛋白表达,包括:NOD样受体热蛋白结构域相关蛋白3(NLRP3)、天冬氨酸蛋白水解酶-1前体(pro-Caspase-1)、天冬氨酸蛋白水解酶-1(Caspase-1)、消皮素D-N端(GSDMD-N)、IL-1β、IL-1β前体(pro-IL-1β)、IL-18和IL-18前体(pro-IL-18)蛋白;吖啶橙/溴乙啶(AO/EB)染色检测细胞膜通透性。结果与对照组相比,模型组ASC蛋白荧光强度增强(P<0.01),细胞焦亡通路相关蛋白表达均增加(P<0.05),细胞膜通透性变大(P<0.01)。与模型组相比,1,25(OH)_(2)D_(3)组和Caspase-1-siRNA组ASC蛋白荧光强度减弱(P<0.01),pro-Caspase-1、Caspase-1、GSDMD-N、pro-IL-1β、IL-1β、pro-IL-18和IL-18蛋白表达均下调(P<0.01),细胞通透性减小(P<0.01)。与Caspase-1-siRNA组相比,联合干预组GSDMD-N和IL-18蛋白表达降低(P<0.01),细胞通透性减小(P<0.01)。结论Aβ_(1-42)能够诱导PC12细胞发生细胞焦亡现象。1,25(OH)_(2)D_(3)可以通过抑制Aβ_(1-42)诱导的PC12细胞发生焦亡来发挥其抗炎的神经保护作用,其机制与Caspase-1的抑制密切相关。
Objective To investigate the inhibitory effect of 1,25(OH)_(2)D_(3)on Aβ_(1-42)-induced pyroptosis in the cell model of Alzheimer′s disease(AD)and its mechanism.Methods The PC12 cells were divided into control group,model group,Caspase-1-siRNA group,NC-siRNA group,1,25(OH)_(2)D_(3)group and Caspase-1-siRNA+1,25(OH)_(2)D_(3)group.The PC12 cells in control group were cultured with DMEM high glucose medium.AD cell model was established with 20μmol/L Aβ_(1-42).The PC12 cells in Caspase-1-siRNA group were pretreated with 50 nmol/L Caspase-1-siRNA,and then given 20μmol/L Aβ_(1-42).The PC12 cells in NC-siRNA group were treated with 20μmol/L Aβ_(1-42)after intervention with 50 nmol/L NC-siRNA.The PC12 cells in 1,25(OH)_(2)D_(3)group were intervened with 100 nmol/L 1,25(OH)_(2)D_(3),followed by 20μmol/L Aβ_(1-42).The PC12 cells in Caspase-1-siRNA+1,25(OH)_(2)D_(3)group were firstly pretreated with 50 nmol/L Caspase-1-siRNA,and then added 100 nmol/L 1,25(OH)_(2)D_(3),followed by 20μmol/L Aβ_(1-42).Cellular immunofluorescence was used to observe the expression of apoptosis-associated speck-like protein containing a CARD(ASC).Western blot was used to detect the expressions of pyroptosis-related proteins,including NOD-like receptor thermal protein domain associated protein 3(NLRP3),pro-Caspase-1,cysteinyl aspartate specific proteinase 1(Caspase-1),N-terminal of gasdermin D(GSDMD-N),interleukin-1β(IL-1β),pro-IL-1β,interleukin-18(IL-18)and pro-IL-18.The cell membrane permeability was detected by acridine orange/ethidine bromide(AO/EB)staining.Results Compared with control group,the fluorescence intensity of ASC protein was enhanced in model group(P<0.01),the expressions of pyroptosis pathway-related proteins were increased(P<0.05),and the cell membrane permeability was enhanced(P<0.01).Compared with model group,the fluorescence intensity of ASC protein was decreased in 1,25(OH)_(2)D_(3)group and Caspase-1-siRNA group(P<0.01),the expressions of pro-Caspase-1,Caspase-1,GSDMD-N,pro-IL-1β,IL-1β,pro-IL-18 and IL-18
作者
李学敏
成乐
吕晨慧
王希
陈爽直
张骋
赵海峰
LI Xuemin;CHENG Le;L Chenhui;WANG Xi;CHEN Shuangzhi;ZHANG Cheng;ZHAO Haifeng(Department of Totoxicology,Shanxi Provincial Center for Disease Control and Prevention,Taiyuan 030012,China;Department of Nutrition and Food Hygiene,School of Public Health,Shanxi Medical University)
出处
《山西医科大学学报》
CAS
2024年第8期994-1000,共7页
Journal of Shanxi Medical University
基金
中央引导地方科技发展资金资助项目(YDZJSX20231A056)。
