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目的观察油酸(oleic acid,OA)及C5a受体(C5aR)拮抗剂(PMX53)干预后小胶质细胞的炎性改变,探讨C5a-C5aR在油酸诱导的小胶质细胞炎症中的作用。方法取新生1 d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5组,分别为正常对照组、OA 100μmol/L处理组、OA 100μmol/L+PMX53 100 nmol/L处理组、OA 200μmol/L处理组、OA 200μmol/L+PMX53 100 nmol/L处理组。应用Western印迹、ELISA法检测不同浓度OA及OA+PMX53干预后小胶质细胞Iba-1、C5aR蛋白及TNF-α表达变化。结果成功培养小胶质细胞,且纯度≥99%。不同浓度OA干预后,Iba-1、C5aR蛋白表达及TNF-α浓度较对照组明显升高(P<0.01);OA+PMX53干预组较OA组Iba-1及TNF-α浓度有所下降(P<0.01);OA+PMx53干预组较OA组C5aR蛋白表达有所下降(OA100 vs.OA100+PMX53,P<0.05,OA200 vs.OA200+PMX53,P<0.01)。200μmol/L OA干预组较100μmol/L OA干预组Iba-1、C5aR蛋白表达及TNF-α浓度高(P<0.01)。结论应用C5aR拮抗剂可以抑制油酸诱导的小胶质细胞的炎症反应,提示C5a-C5aR参与了油酸诱发的炎症反应,推测C5aR拮抗剂在神经系统炎性损伤中具有神经保护作用。
Objective To investigate the inflammatory changes of microglia after the intervention of oleic acid (OA) and C5a receptor antagonist (PMX53), and to explore the role of C5a-C5aR in oleic acid-induced microglial inflammation . Methods Newborn 1-day-old mice were isolated, brain tissue was isolated and primary cultured microglia were isolated, purified and identified. The purified microglia were randomly divided into 5 groups: OA 100μmol / L, OA 100μmol / L + PMX53 100 nmol / L, OA 200μmol / L, OA 200μmol / L + PMX53 100 nmol / L treatment group. The changes of Iba-1, C5aR protein and TNF-α in microglia were detected by Western blotting and ELISA assay after treated with different concentrations of OA and OA + PMX53. Results The microglia were cultured successfully with purity ≥99%. After intervention with different concentrations of OA, the expression of Iba-1 and C5aR protein and the concentration of TNF-α were significantly higher than those of the control group (P <0.01); the concentrations of Iba-1 and TNF-α in the OA + PMX53 intervention group were decreased P <0.01). The expression of C5aR protein in OA + PMx53 group was lower than that in OA group (OA100 vs. OA100 + PMX53, P <0.05, OA200 vs. OA200 + PMX53, P <0.01). The expression of Iba-1 and C5aR protein and the concentration of TNF-α in 200μmol / L OA intervention group were higher than those in 100μmol / L OA intervention group (P <0.01). Conclusion C5aR antagonist can inhibit oleic acid-induced microglial inflammatory response, suggesting that C5a-C5aR is involved in the inflammatory response induced by oleic acid. It is speculated that C5aR antagonist has neuroprotective effect on nervous system inflammatory injury.