降钙素基因相关肽对LPS并ATP诱导小胶质细胞NLRP3炎症小体激活影响

EFFECT OF CALCITONIN GENE-RELATED PEPTIDE ON THE ACTIVATION OF NLRP3 INFLAMMASOME INDUCED BY LPS AND ATP IN MICROGLIA

目的 探讨降钙素基因相关肽(CGRP)对脂多糖(LPS)联合三磷酸腺苷(ATP)诱导的BV2小胶质细胞NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体活化的抑制作用。方法 通过检测不同方法处理的各组细胞NLRP3蛋白含量,确定LPS联合ATP激活小胶质细胞最佳模型并筛选CGRP最佳作用浓度。实验分为对照组、模型组以及CGRP组,采用蛋白免疫印迹法检测细胞NLRP3、凋亡相关的斑点样蛋白(ASC)、半胱氨酸天冬氨酸蛋白酶-1(caspase-1)的表达,酶联免疫吸附试验法检测细胞内炎性因子白细胞介素1β(IL-1β)的表达。结果BV2小胶质细胞NLRP3炎症小体激活最佳模型的制备方法为先加入100μg/L的LPS作用12 h,再加入5 mmol/L的ATP继续作用45 min;CGRP抑制NLRP3炎症小体激活的最佳浓度为10μg/L。模型组细胞NLRP3、ASC、caspase-1、IL-1β的蛋白表达较对照组明显升高,CGRP组细胞上述蛋白表达较模型组明显降低,差异均有显著性(F=7.261~151.232,P<0.05)。结论 CGRP可能通过抑制BV2小胶质细胞NLRP3炎症小体激活,发挥抗炎神经保护作用。

Objective To investigate the inhibitory effect of calcitonin gene-related peptide(CGRP) on the activation of nod like receptor thermoprotein domain associated protein 3(NLRP3) inflammatory bodies in BV2 microglia induced by lipopolysaccharide(LPS) combined with adenosine triphosphate(ATP). Methods By detecting the content of NLRP3 protein in cells with different treatment methods, the best model of LPS combined with ATP activated microglia was determined, and the best concentration of CGRP was screened. The experiment was divided into control group, model group and CGRP group. The protein expressions of NLRP3, apoptpsis-associated speck-like protein containing(ASC) and cysteinyl aspartate specific protease-1(caspase-1) were detected by Western blot;Quantitative detection of intracellular inflammatory factor interleukin-1β protein expression by enzyme-linked immunosorbent assay. Results The best model of NLRP3 inflammasome activation in BV2 microglia was prepared with 100 μg/L LPS used for 12 h, and then 5 mmol/L ATP was added for 45 min;the optimal concentration of CGRP to inhibit NLRP3 inflammasome activation was 10 μg/L. Compared with the control group, the expression of NLRP3, ASC, caspase-1 and IL-1β in model group was significantly higher;compared with the model group, the expression of NLRP3, ASC, caspase-1 and IL-1β decreased significantly(F=7.261-151.232, P<0.05). Conclusion CGRP can play an anti-inflammatory and neuroprotective role by inhibiting the activation of NLRP3 inflammatory bodies in BV2 microglia.