原花青素对LPS/ATP诱导的NLRP3炎症小体激活及NF-κBp65磷酸化的抑制作用

Inhibitory effects of proanthocyanidin on LPS/ATP induced activation of NLRP3 inflammasome and phosphorylation of NF-κBp65

目的:研究原花青素对脂多糖(lipopolysaccharide,LPS)与腺嘌呤核苷三磷酸(adenosine triphosphate,ATP)联合诱导的小鼠小胶质细胞BV2 NOD样受体蛋白3(nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)炎症小体激活及核因子(nuclear factor,NF)-κBp65磷酸化的抑制作用。方法:以1.0μg/mL LPS、2.5μmol/L ATP诱导BV2细胞炎症损伤模型,用不同浓度原花青素(0.1、1.0、2.5、5.0μg/mL)干预细胞。采用噻唑蓝(thiazolyl blue tetrazolium bromide,MTT)法检测细胞存活率;比色法检测一氧化氮(nitric oxide,NO)释放水平;微量酶标法测乳酸脱氢酶(lactate dehydrogenase,LDH)活力;ELISA法检测白细胞介素(interleukin,IL)-1β、IL-18的分泌水平;Western blot法检测NLRP3、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC)、半胱氨酸天冬氨酸蛋白酶(caspase)-1、pro-caspase-1、p-NF-κBp65、NF-κBp65蛋白表达水平。结果:不同浓度的原花青素对BV2细胞存活率的影响与对照组相比差异无统计学意义(P>0.05)。与对照组相比,LPS/ATP诱导可增加BV2细胞NO、IL-1β、IL-18水平以及LDH活力(P<0.05),增加NLRP3、ASC、pro-caspase-1、caspase-1、p-NF-κBp65蛋白表达水平(P<0.05)。与LPS/ATP组相比,原花青素能降低BV2细胞NO、IL-1β、IL-18水平以及LDH活力(P<0.05);原花青素(1.0、2.5、5.0μg/mL)降低NLRP3、ASC、pro-caspase-1、caspase-1蛋白表达水平(P<0.05);NF-κB抑制剂BAY11-7082(5.0μmol/L)降低NF-κBp65磷酸化及NLRP3、ASC、pro-caspase-1、caspase-1蛋白表达水平(P<0.05)。结论:原花青素可以抑制LPS/ATP诱导的BV2细胞炎症因子分泌及NLRP3炎症小体激活,该作用与原花青素抑制LPS/ATP诱导条件下NF-κBp65磷酸化密切相关。

Objective:To observe the inhibitory effects of proanthocyanidin on activation of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome and phosphorylation of nuclear factor(NF)-κBp65 induced by lipopolysaccharide(LPS)and adenine nucleoside triphosphate(ATP)in mouse microglia(BV2).Methods:BV2 cells were stimulated with 1.0μg/mL LPS and 2.5μmol/L ATP,and treated with different concentrations of proanthocyanidin(0.1,1.0,2.5,5.0μg/mL).Cell viability was determined by thiazolyl blue tetrazolium bromide(MTT)assay.Nitric oxide(NO)release was detected by colorimetry.The activity of lactate dehydrogenase(LDH)was measured by microenzyme labeling method.The secretion of interleukin(IL)-1βand IL-18 were determined by ELISA.Expression of NLRP3,apoptosis-associated speck-like protein containing a CARD(ASC),caspase-1,pro-caspase-1,p-NF-κBp65,NF-κBp65 were detected by Western blot.Results:The effect of different concentrations of proanthocyanidin on the survival rate of BV2 cells was not statistically significant compared with the control group(P>0.05).Compared with the control group,LPS/ATP increased the secretions of NO,IL-1β,IL-18 and LDH activity(P<0.05),and the expressions of NLRP3,ASC,pro-caspase-1,caspase-1,p-NF-κBp65(P<0.05).Compared with the LPS/ATP group,proanthocyanidin reduced the secretions of NO,IL-1β,IL-18 and LDH activity of BV2 cells(P<0.05).In addition,proanthocyanidin(1.0,2.5,5.0μg/m L)decreased the expressions of NLRP3,ASC,pro-caspase-1 and caspase-1(P<0.05).Similarly,NF-κB inhibitor BAY11-7082(5.0μmol/L)reduced NF-κBp65 phosphorylation and the expressions of NLRP3,ASC,pro-caspase-1,and caspase-1(P<0.05).Conclusion:Proanthocyanidin can inhibit secretion of inflammatory factor and activation of NLRP3 inflammasome induced by LPS/ATP,which is closely related to the inhibition of phosphorylation of NF-κBp65 by proanthocyanidin in LPS/ATP induced status.