C5aR/C5L2在小胶质细胞炎症中的作用及机制

Effect and mechanism of C5aR/C5L2 in microglia inflammation

目的观察棕榈酸(PA)及C5aR拮抗剂(PMX53)干预后小胶质细胞的炎症改变,探讨C5a受体(C5aR)及C5L2在PA诱导的小胶质细胞炎症中的作用。方法取新生1日龄小鼠,分离脑组织,原代培养小胶质细胞并进行分离纯化及鉴定。纯化小胶质细胞随机分成3组:正常对照组、PA处理组、PA+PMX53处理组。分别应用Western blot检测PA及PA+PMX53干预后小胶质细胞C5aR、C5L2、p38MAPK蛋白表达;RT-PCR检测C5L2 mRNA表达及ELISA法检测IL-6表达变化。结果通过免疫组化鉴定,成功培养小胶质细胞。PA干预后,C5aR蛋白表达较对照组明显上升(P<0.001),而加入PMX53后,C5aR蛋白表达较PA组显著下降(P<0.001);PA干预后,C5L2蛋白及mRNA表达较对照组增加(P<0.001),而加入PMX53后,C5L2蛋白表达与PA组差异无统计学意义(P=0.978),而C5L2 mRNA表达较PA组明显上升(P<0.001);PA组较对照组p38MAPK蛋白的表达增加(P=0.002),PA+PMX53组p38MAPK蛋白表达较PA组降低(P=0.004);PA干预后,IL-6浓度较对照组显著升高(P<0.001),而PMX53干预后,PA+PMX53组较PA组IL-6浓度显著下降(P<0.001)。结论 PA可能通过C5a-C5aR激活MAPK信号途径中的p38MAPK通路,从而诱发小胶质细胞炎症反应,而C5L2在此过程中可能发挥抗炎作用。

Objective To observe the changes of inflammation in microglia after the intervention of palmitic acid(PA)and C5aR antagonist(PMX53),and to explore the effect of C5a receptor(C5aR)and C5L2 in cell inflammation of PA-induced microglia.MethodsNeonatal mice aged 1 day were selected,the brain tissue was isolated,and primary microglia cells were purified and identified.Purified microglial cells were randomly divided into 3 groups:normal control group,PA treatment group,PA+PMX53 treatment group.The protein expressions of C5aR,C5L2 and p38MAPK in microglia cells after the intervention of PA and PA+PMX53 were detected by Western blot.C5L2mRNA expression was detected by RT-PCR,and IL-6 expression was detected by ELISA.Results Microglia cells were successfully cultured by immunohistochemical identification.Compared with normal control group,C5aR protein expression was obviously increased after the intervention of PA(P<0.001);compared with PA group,C5aR protein expression was declined significantly after adding PMX53(P<0.001);compared with normal control group,C5L2 protein and mRNA expression was increased after the intervention of PA(P<0.001);compared with PA group,there was no statistically significant difference on C5L2 protein expression after adding PMX53(P=0.978),while the C5L2 mRNA expression increased obviously(P<0.001);p38MAPK protein expression in PA group was higher than normal control group(P=0.002),and increased than PA+PMX53 group(P=0.004);after the intervention of PA,IL-6 concentration significantly increased than the normal control group(P<0.001),while after the intervention of PMX53,the concentration of IL-6 in PA+PMX53 group was significant reduced than that in PA group(P<0.001).Conclusion PA can active p38MAPK pathway of MAPK signaling pathways by C5a-C5aR,thus induce microglia inflammation,and C5L2 may play an anti-inflammatory role in this process.