绿原酸抑制NF-κB/NLRP3炎性体通路减轻LPS诱导小胶质细胞神经炎症损伤研究

Chlorogenic Acid Reducing LPS-induced Neuroinflammatory Injury in Microglia by Inhibiting NF-κB/NLRP3 Inflammasome Pathway

目的:探究绿原酸(CGA)对脂多糖(LPS)诱导的小胶质细胞神经炎症损伤及核因子κB(NF⁃κB)/Nod样受体家族含pyrin结构域蛋白3(NLRP3)炎性体通路的影响。方法:培养小胶质细胞BV2,3⁃(4,5⁃二甲基噻唑⁃2)⁃2,5⁃二苯基四氮唑溴盐(MTT)法检测不同浓度(0,1,2,4,8 mmol·L^(-1))CGA对BV2细胞活力的影响;再次培养BV2细胞,依次分为对照(Control)组、LPS组、脂多糖和药物溶剂对照[LPS+二甲亚砜(DMSO)]、脂多糖和阳性药物对照(LPS+Positive)组以及脂多糖和绿原酸处理(LPS+CGA)组。免疫荧光法检测肿瘤坏死因子α(TNF⁃α)和白细胞介素(IL)⁃1β表达情况;ELISA法检测TNF⁃α、IL⁃1β、IL⁃6、IL⁃10、IL⁃12和诱导型一氧化氮合成酶(iNOS)含量;免疫印迹分析NF⁃κB/NLRP3炎性体通路和凋亡相关蛋白水平;流式细胞仪检测细胞凋亡率。结果:与CGA 0 mmol·L^(-1)比较,CGA 1,2,4 mmol·L^(-1)对BV2细胞增殖活力的影响无统计学意义(P>0.05),CGA 8 mmol·L^(-1)显著降低BV2细胞增殖活力(P<0.05)。LPS诱导后细胞凋亡率升高,细胞上清液中TNF⁃α、IL⁃1β、IL⁃6、IL⁃12和iNOS分泌量增多,IL⁃10分泌量减少,细胞中NF⁃κB p65、磷酸化NF⁃κB抑制蛋白(p⁃IκBα)、NLRP3、含半胱氨酸的天冬氨酸蛋白水解酶1(Caspase⁃1)和B细胞淋巴瘤/白血病⁃2(Bcl⁃2)相关X蛋白(Bax)蛋白水平上调,细胞中Bcl⁃2蛋白水平下调(P<0.05)。添加CGA可明显改善LPS对BV2细胞的影响(P<0.05)。结论:CGA通过抑制NF⁃κB/NLRP3炎性体通路减轻LPS诱导的BV2细胞神经炎症损伤。

Objective:To explore the effects of chlorogenic acid(CGA)on lipopolysaccharide(LPS)-induced neuroinflammatory injury in microglia and nuclear factor kappa-B(NF-κB)/Nod-like receptor family pyrin domain-containing protein 3(NLRP3)inflammasome pathway.Methods:Microglial cells BV2 were cultured,and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)method was used to detect the effects of CGA at different concentrations(0,1,2,4 and 8 mmol·L^(-1))on the viability of BV2 cells;BV2 cells were cultured again and divided into control(Control)group,LPS group,lipopolysaccharide and drug solvent control[(LPS+dimethyl sulfoxide(DMSO)]group,lipopolysaccharide and positive drug control(LPS+Positive)group,and lipopolysaccharide+chlorogenic acid treatment(LPS+CGA)group.Immunofluorescence method was used to detect the expression of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL^(-1)β);ELISA method was used to detect the contents of TNF-α,IL^(-1)β,interleukin-6(IL-6),interleukin-10(IL^(-1)0),interleukin-12(IL^(-1)2)and inducible nitric oxide synthase(iNOS);Western blot was used to analyze the levels of NF-κB/NLRP3 inflammasome pathway and apoptosis-related proteins;flow cytometry was used to detect cell apoptosis rate.Results:Compared with 0 mmol·L^(-1) CGA,1,2 and 4 mmol·L^(-1) CGA had no statistically significant effect on the proliferation of BV2 cells(P>0.05),and 8 mmol·L^(-1) CGA significantly reduced the proliferation of BV2 cells(P<0.05).After LPS induction,the rate of apoptosis increased,the secretion of TNF-α,IL^(-1)β,IL-6,IL^(-1)2 and iNOS in the cell supernatant increased,the secretion of IL^(-1)0 decreased,the protein levels of NF-κB p65,p-IκBα,NLRP3,Caspase-1 and Bax in the cells were up-regulated,and the protein level of Bcl-2 in the cells were down-regulated(P<0.05).Adding CGA could significantly relieved the effects of LPS on BV2 cells(P<0.05).Conclusion:CGA can reduce LPS-induced neuroinflammatory injury of BV2 cells by inhibiting NF-κB/NLRP3 inflammasome pathway.