脑心通调控NLRP3/Caspase-1通路抑制脂多糖诱导的BV2细胞焦亡

Naoxintong Inhibits LPS Induced BV2 Cell Pyroptosis by NLRP3/Caspase-1 Signaling Pathway

目的:探索脑心通醇提物对脂多糖(LPS)诱导的小胶质细胞BV2焦亡的影响,并通过NOD样受体热蛋白结构域3(NLRP3)/半胱氨酸蛋白酶-1(Caspase-1)通路阐释其可能的作用机制。方法:LPS(1 mg·L^-1)体外诱导BV2细胞的同时,加入不同质量浓度脑心通醇提物(2,10,50 mg·L^-1)干预后,分别采用MTS法检测细胞活性,实时荧光定量PCR(Real-time PCR)检测白细胞介素-1β(IL-1β),肿瘤坏死因子-α(TNF-α)和NLRP3的mRNA水平;免疫荧光法检测NLRP3蛋白表达的情况;蛋白免疫印迹法(Western blot)检测NLRP3/Caspase-1通路关键蛋白表达。结果:与空白组比较,LPS(1 mg·L^-1)能明显降低BV2细胞活性,显著升高IL-1β,TNF-α及NLRP3 mRNA表达水平(P<0.01),且可提升NLRP3蛋白表达水平以及Caspase-1剪切体和前体蛋白的比值(Caspase-1 p20/Caspase-1)。与LPS组比较,脑心通醇提物(2,10,50 mg·L^-1)干预后,逆转了LPS导致的细胞活性降低(P<0.01),50 mg·L^-1脑心通醇提物可显著降低IL-1β,TNF-α及NLRP3 mRNA表达水平(P<0.05,P<0.01),显著降低LPS诱导的NLRP3的高表达以及Caspase-1 p20/Caspase-1(P<0.01)。结论:脑心通能明显抑制LPS诱导的小胶质细胞焦亡,其作用机制与NLRP3/Caspase-1通路密切相关。

Objective:To explore the effect of Naoxintong ethanol extract(NXT)on pyroptosis of BV2 microglia cells induced by lipopolysaccharide(LPS),and to explain the mechanism of pyroptosis based on NOD like receptor thermoprotein domain 3(NLRP3)/cysteine-proteinase-1(Caspase-1)pathway.Method:BV2 cells was treated with different concentrations of NXT(2,10,50 mg·L^-1)after induced by LPS(1 mg·L^-1)in vitro.Real-time PCR was used to detect mRNA expression of pro-inflammatory cytokine such as interleukin 1 beta(IL-1β),tumor necrosis factor(TNF)-α,and NLRP3.Western bolt and immunofluorescence were used to observe the protein expression of NLRP3/Caspase-1 signaling pathway.Result:Compared with control group,after LPS(1 mg·L^-1)stimulation,BV2 cells viability was decreased.The mRNA expression levels of IL-1β,TNF-αand NLRP3 were significantly elevated(P<0.01),the protein levels of NLRP3 and Caspase-1 p20/Caspase-1 were also increased.After given NXT(2,10,50 mg·L^-1),BV2 cells viability reversed which induced by LPS.Compared with LPS group,the mRNA expression of IL-1β,TNF-αand NLRP3 reduced obviously with given50 mg·L^-1 NXT(P<0.05,P<0.01),significantly inhibited NLRP3 high protein expression and Caspase-1 p20/Caspase-1 expression(P<0.01).Conclusion:NXT can inhibit LPS induced pyroptosis of BV2 cells and the mechanism may closely related to NLRP3/Caspase-1 signaling pathway.