Inhibitory effects of proanthocyanidin on LPS/ATP induced activation of NLRP3 inflammasome and phosphorylation of NF⁃κBp65
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摘要:
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1.0 μg/mL LPS、2.5 μmol/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、5.0 μg/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、
Abstract:
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 1.0 μg/mL LPS and 2.5 μmol/L ATP，and treated with different concentrations of proanthocyanidin（0.1，1.0，2.5，5.0 μg/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）-1β and 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（1.0，2.5，5.0 μg/mL） decreased the expressions of NLRP3，ASC，pro-caspase-1 and caspase-1（P < 0.05）. Similarly，NF-κB inhibitor BAY11-7082（5.0 μmol/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.
