1,25(OH)2D3对Aβ1-42诱导阿尔茨海默病细胞模型中细胞焦亡的抑制作用OACSTPCD
Inhibitory effect of 1,25(OH)2D3 on Aβ1-42-induced pyroptosis in the cell model of Alzheimer's disease
目的 探讨1,25(OH)2D3对Aβ1-42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)2D3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20 μmol/L Aβ1-42处理细胞以造模;Caspase-l-siRNA组先用50 nmol/L Caspase-l-siRNA 处理细胞,再加入 20 μmol/L Aβ1-42;NC-siRNA 组先用 50 nmol/L NC-siRNA 处理细胞,再加入 20 μmol/L Aβ1-42;1,25(OH)2D3 组用 100 nmol/L 1,25(OH)2D3干预后加入 20μmol/L Aβ1-42;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)2D3干预,最后加入20 μmol/L Aβ1-42.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-1 β、IL-1 β 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)2D3 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 Aβ1-42能够诱导PC12细胞发生细胞焦亡现象.1,25(OH)2D3可以通过抑制Aβ1-42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)2D3 on Aβ1-42-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)2D3 group and Caspase-1-siRNA+1,25(OH)2D3 group.The PC12 cells in control group were cultured with DMEM high glucose medium.AD cell model was established with 20 μmol/L Aβ1-42.The PC12 cells in Caspase-1-siRNA group were pretreated with 50 nmol/L Caspase-1-siRNA,and then given 20 μmol/L Aβ1-42.The PC12 cells in NC-siRNA group were treated with 20 μmol/L Aβ1-42 after intervention with 50 nmol/L NC-siRNA.The PC12 cells in 1,25(OH)2D3 group were intervened with 100 nmol/L 1,25(OH)2D3,followed by 20 μmol/L Aβ1-42.The PC12 cells in Caspase-1-siRNA+1,25(OH)2D3 group were firstly pretreated with 50 nmol/L Caspase-1-siRNA,and then added 100 nmol/L 1,25(OH)2D3,followed by 20 μmol/L Aβ1-42 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-1 β(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 D3 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 were downregulated(P＜0.01),and the cell membrane permeability was reduced(P＜0.01).Compared with Caspase-1-siRNA group,the expressions of GSDMD-N and IL-18 were decreased in Caspase-1-siRNA+1,25(OH)2D3 group(P＜0.01),and the cell membrane permeability was declined(P＜0.01).Conclusion Aβ1-42 could induce the cell pyroptosis in PC12 cells.1,25(OH)2D3 exerts its anti-inflammatory neuroprotective effect in PC12 cells by inhibiting Aβ1-42-induced pyroptosis,and the mechanism is closely related to the inhibition of Caspase-1.
李学敏;成乐;吕晨慧;王希;陈爽直;张骋;赵海峰
山西省疾病预防控制中心毒理科,太原 030012山西医科大学公共卫生学院营养与食品卫生学教研室
临床医学
1,25(OH)2D3Aβ1-42阿尔茨海默病细胞焦亡炎症Caspase-1
1,25(OH)2D3Aβ1-42Alzheimer's diseasepyroptosisinflammationCaspase-1
《山西医科大学学报》 2024 (008)
994-1000 / 7
中央引导地方科技发展资金资助项目(YDZJSX20231A056)
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