血清淀粉样蛋白A对小胶质细胞迁移的影响及机制研究
Effect of serum amyloid A on microglial migration and its mechanism
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摘要: 探究血清淀粉样蛋白A（SAA）对小胶质细胞迁移的影响及机制。采用Transwell小室法检测SAA诱导原代小胶质细胞和鼠源N9小胶质细胞的迁移能力。用Transwell检测甲酰肽受体2（FPR2）拮抗剂和TLR2中和抗体对SAA诱导N9小胶质细胞迁移的影响。实时荧光定量PCR检测SAA作用N9小胶质细胞后，FPR2和Toll样受体2（TLR2）的表达变化。Western blot检测SAA对N9小胶质细胞下游信号通路激酶表达的影响。用Transwell检测信号通路抑制剂对SAA诱导的N9小胶质细胞迁移的影响。结果显示：SAA以浓度依赖性方式促进原代小胶质细胞和N9小胶质细胞迁移。FPR2拮抗剂和TLR2中和抗体抑制了SAA诱导的N9小胶质细胞迁移。SAA促进了N9小胶质细胞内FPR2和TLR2受体的mRNA转录水平增加。SAA刺激N9小胶质细胞丝裂原活化蛋白激酶（MAPKs）和核因子κB（NF-κB）信号通路的激活，表现为细胞外调节蛋白激酶（ERK）、p38和c-Jun氨基末端激酶（JNK）磷酸化水平增加，I?Bα表达水平降低。p38、JNK和NF-κB信号通路抑制剂抑制了SAA诱导的N9小胶质细胞迁移。组间差异有统计学意义（P<0.05）。研究结果表明，SAA通过作用FPR2和TLR2受体，激活下游p38、JNK和NF-κB信号通路，进而诱导小胶质细胞迁移。Abstract: To investigate the effect of serum amyloid A (SAA) on microglial migration and its mechanism,the migration ability of SAA-induced primary microglia and murine N9 microglia,and the effect of formyl peptide receptor 2 (FPR2) antagonist and TLR2 neutralizing antibody on SAA-induced migration of N9 microglia were all examined by Transwell assay. The expression changes of FPR2 and Toll-like receptor 2 (TLR2) in N9 microglia after SAA stimulation were detected by real-time PCR. The effect of SAA on the expression of downstream signaling pathway kinases in N9 microglia was detected by Western blot. The effect of signaling pathway inhibitors on SAA-induced N9 microglial migration was examined by Transwell assay. The results showed that SAA promoted the migration of primary microglia and N9 microglia in a concentration-dependent manner. FPR2 antagonist and TLR2 neutralizing antibody inhibited SAA-induced N9 cell migration. SAA promoted increased mRNA transcript levels of FPR2 and TLR2 in N9 microglia,and stimulated the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB) signaling pathways in N9 microglia,as shown by increased extracellular regulated protein kinase (ERK),p38 and c-Jun N-terminal kinase (JNK) phosphorylation levels and decreased IκBα expression levels. Inhibitors of p38,JNK,and NF-κB signaling pathways inhibited SAA-induced migration of N9 microglia. The difference between the groups was statistically significant (P<0.05). These results indicate that SAA activates downstream p38,JNK,and NF-κB signaling pathways by acting on FPR2 and TLR2,thereby inducing microglia migration.
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Keywords:
- serum amyloid A /
- microglia /
- migration /
- receptor /
- signaling pathway
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