STAT3调控NLRP3炎症小体在麦芽酚铝致BV2细胞炎症反应中的作用
Role of STAT3 activated NLRP3 inflammasomes in BV2 cell inflammatory response induced by maltol aluminum
-
摘要:
背景 铝激活信号转导和转录激活因子3（STAT3）致小胶质细胞活化核苷酸结合和寡聚化结构域样受体蛋白3（NLRP3）炎症小体产生炎症反应并造成神经毒性。
目的 探讨STAT3调控NLRP3炎症小体在麦芽酚铝（Al(mal)3）致小鼠小胶质细胞株（BV2）细胞炎症反应中的作用。
方法 选取BV2细胞株，利用Al(mal)3染毒和STAT3拮抗剂C188-9干预，实验分为5组：对照组，Al(mal)3低、中、高剂量组（40、80和160 μmol·L−1 Al(mal)3），C188-9干预组（10 μmol·L−1 C188-9+160 μmol·L−1 Al(mal)3）。采用CCK8检测细胞活力；采用Western blotting检测BV2细胞M1/M2型标志物CD68/CD206的表达，以及STAT3、p-STAT3、NLRP3、cleaved-casepase-1、衔接蛋白凋亡相关斑点样蛋白（ASC）表达量；采用ELISA检测促炎因子白细胞介素（IL）-1β、IL-18和抗炎因子IL-10的含量。
结果 细胞活力测定显示：随着染铝浓度的增加，各剂量组细胞活力逐渐降低。与对照组相比，Al(mal)3高剂量组细胞活力下降18%（
P ＜0.05）；与Al(mal)3高剂量组相比，C188-9干预组细胞活力升高14%（P ＜0.05）。与对照组相比，Al(mal)3低、中、高剂量组CD68的表达分别升高19%、20%、21%（P ＜0.05），Al(mal)3高剂量组CD206的表达降低25%（P ＜0.05）。与Al(mal)3高剂量组相比，C188-9干预组CD68的表达水平降低9%（P ＜0.05），而CD206的表达水平升高22%（P ＜0.05）。与对照组相比，Al(mal)3高剂量组p-STAT3蛋白表达量和p-STAT3/STAT3值分别增加129%和127%（P <0.05）。与Al(mal)3高剂量组相比，C188-9干预组p-STAT3蛋白表达量和p-STAT3/STAT3值分别降低55%和54%（P <0.05）。NLRP3炎症小体测试结果显示，与对照组相比，Al(mal)3高剂量组NLRP3蛋白表达量增加75%（P <0.05），Al(mal)3中、高剂量组cleaved-casepase-1蛋白表达量分别增加28%、35%（P <0.05），Al(mal)3低、中、高剂量组ASC表达量分别增加22%、25%、53%（P <0.05）。与Al(mal)3高剂量组相比，C188-9干预组NLRP3、cleaved-casepase-1、ASC蛋白表达量分别降低30%、19%、32%（P <0.05）。与对照组相比，IL-1β在Al(mal)3中、高剂量组含量分别增加18%、21%（P <0.05），IL-18在Al(mal)3高剂量组的含量增加10%（P <0.05）。与Al(mal)3高剂量组相比，C188-9干预组IL-18的含量降低23%（P <0.05）。抗炎因子IL-10的含量在各组差异无统计学意义（P >0.05）。结论 铝能引起BV2小胶质细胞的炎症反应并且以促炎反应为主，其机制可能与STAT3调控NLRP3炎症小体分泌炎症因子有关。
Abstract:Background Aluminum activates signal transducer and activator of transcription 3 (STAT3), causing microglial nucleotide-binding and oligomerization domain-like receptors protein 3 (NLRP3) inflammasome activation and inflammatory responses and producing neurotoxicity.
Objective To explore the role of STAT3 regulated NLRP3 inflammasomes in the inflammatory response of mouse microglia cell line (BV2) cells induced by maltol aluminum Al(mal)3.
Methods BV2 cells were assigned to five groups: one control group, three Al(mal)3 exposure groups (low, medium, and high doses at 40, 80, and 160 μmol·L−1 Al(mal)3 respectively), and one C188-9 (STAT3 antagonist) intervention group 10 μmol·L−1 C188-9 +160 μmol·L−1 Al(mal)3. Cell viability was detected by CCK8. The expression of M1/M2 type markers, i.e. CD68/CD206, STAT3, p-STAT3, NLRP3, cleaved-casepase-1, and apoptosis-associated speck-like protein (ASC) in BV2 cells were detected by Western blotting, and proinflammatory cytokines interleukin (IL)-1β and IL-18, and anti-inflammatory cytokine IL-10 were determined by ELISA.
Results The results of cell viability assay showed that cell viability gradually decreased with the increase of Al(mal)3 dose. Compared with the control group, the cell viability of the Al(mal)3 high-dose group was decreased by 18% (
P <0.05); compared with the Al(mal)3 high-dose group, the cell viability of the C188-9 intervention group was significantly elevated by 14% (P <0.05). Compared with the control group, the expression levels of CD68 in the Al(mal)3 low-, medium-, and high-dose groups were elevated by 19%, 20%, and 21%, respectively (P <0.05); the expression level of CD206 in the Al(mal)3 high-dose group was decreased by 25% (P <0.05). Compared with the Al(mal)3 high-dose group, the expression level of CD68 in the C188-9 intervention group was reduced by 9% (P <0.05), whereas the expression level of CD206 was elevated by 22% (P <0.05). Compared with the control group, the p-STAT3 protein expression and the p-STAT3/STAT3 ratio in the Al(mal)3 high-dose group increased by 129% and 127%, respectively (P <0.05). Compared with the Al(mal)3 high-dose group, the p-STAT3 protein expression and the p-STAT3/STAT3 ratio in the C188-9 intervention group were decreased by 55% and 54%, respectively (P >0.05). Compared with the control group, the expression level of NLRP3 protein increased by 75% in the Al(mal)3 high-dose group (P <0.05), the expression levels of cleaved-casepase-1 protein increased by 28% and 35% in the Al(mal)3 medium- and high-dose groups (P <0.05), and the expression levels of ASC increased by 22%, 25%, and 53% in the Al(mal)3 low-, medium- and high-dose groups (P <0.05), respectively. Compared with the Al(mal)3 high-dose group, the expression levels of NLRP3, cleaved-casepase-1, and ASC proteins in the C188-9 intervention group decreased by 30%, 19%, and 32%, respectively (P <0.05). Compared with the control group, the levels of IL-1β in the Al(mal)3 medium- and high-dose groups increased by 18% and 21%, respectively (P <0.05), and the level of IL-18 in the Al(mal)3 high-dose group increased by 10% (P <0.05). Compared with the Al(mal)3 high-dose group, the IL-18 levels were reduced by 23% in the C188-9 intervention group (P <0.05). The content of anti-inflammatory factor IL-10 did not differ significantly between groups (P >0.05).Conclusion Aluminum can induce inflammatory responses in BV2 microglia and is predominantly pro-inflammatory, and the mechanism may involve STAT3 regulation of NLRP3 inflammasome secretion of inflammatory factors.
下载:
