目的 研究安菲博肽(anfibatide,ANF)对脑缺血再灌注损伤后小胶质细胞M1/M2极化及神经炎症的影响。方法 取SD大鼠64只,随机分为正常组,模型组,Toll样受体4(TLR4)阻断剂TAK-242(2 mg·kg-1),ANF(4 μg·kg-1)组,每组16只。线栓法建立大鼠局灶性脑缺血再灌注损伤模型,缺血2 h后恢复再灌,连续尾静脉注射给药5 d。氯化三苯基四氮唑(TTC)染色测定脑梗死体积,神经功能缺损评分(mNSS)方法进行神经功能缺损评分;免疫荧光及Western blot测定脑组织中CD86/CD206蛋白表达。取BV-2细胞,建立糖氧剥夺/复糖复氧(OGD/R)模型, 分对照组、OGD/R组、TAK-242 1 μmol·L-1组和ANF 0.1、0.2、0.4 μg·mL-1组,缺糖缺氧6 h后,复糖复氧24 h并用相应药物进行干预。免疫荧光及实时定量PCR(RT-PCR)测定各组BV-2细胞CD86/CD206蛋白及肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、IL-10、转化生长因子-β(TGF-β)基因表达。结果 与模型组比较,ANF可明显减少脑梗死体积,改善大鼠神经功能缺损,明显降低脑组织中CD86蛋白表达,明显升高CD206蛋白表达。与对照组比较,ANF 0.1、0.2、0.4 μg·mL-1可明显降低BV-2中CD86免疫荧光表达,明显降低CD86、IL-1β基因表达,明显升高CD206基因表达,0.2、0.4 μg·mL-1可明显升高CD206免疫荧光表达,明显降低TNF-α表达;升高IL-10、TGF-β基因表达水平。结论 ANF可通过抑制小胶质细胞M1极化、促进M2极化,从而减轻脑缺血再灌注损伤后神经炎症反应。
Abstract
OBJECTIVE To study the effect of anfibatide(ANF) on microglia M1/M2 polarization and neuroinflammation after cerebral ischemia/reperfusion injury. METHODS Sixty-four SD rats were randomly divided into four groups: sham, model, TAK-242(2 mg·kg-1, antagonist of toll-like receptor 4, TLR4), and ANF 4 μg·kg-1 group. The occlusion model of middle cerebral artery(MCAO) was established by thread embolism method. Reperfusion was resumed after 2 h of ischemia, and the drug was administered continuously by tail vein for 5 d. The neurological deficit was evaluated by mNSS and the infarct volume was measured by TTC staining. The expressions of CD86, CD206 proteins in brain tissue were determined by immunofluorescence and Western-blot method.BV-2 cells were used to establish OGD/R model and divided into control, OGD/R, TAK-242 1 μmol·L-1, ANF 0.1, 0.2, 0.4 μg·mL-1 group. After 6 h of hypoxia, reoxygenation was performed for 24 h and corresponding drugs were used for intervention. The expressions of CD86, CD206 proteins were determined by immunofluorescence, and CD86, CD206, TNF-α, IL-1β, IL-10 and TGF-β genes in BV-2 cells were detected by real-time quantitative PCR(RT-PCR). RESULTS Compared with the model group, ANF could significantly reduce the cerebral infarction volume, decrease the neurological deficit and the expression of CD86 protein in brain tissue, and significantly increase the expression of CD206 protein. In vitro, compared with the control group, ANF 0.1, 0.2, 0.4 μg·mL-1 significantly decreased the immunofluorescence expression of CD86 in BV-2, significantly reduced the expression of CD86, IL-1β mRNA, obviously increased the expression of CD206 mRNA, and ANF 0.2, 0.4 μg·mL-1 obviously increased the immunofluorescence expression of CD206.Furthermore, remarkably inhibited the expression of TNF-α mRNA, significantly promoted the expressions of IL-10, TGF-β mRNA. CONCLUSION ANF can inhibit inflammation after cerebral ischemia-reperfusion injury, which may be related to promoting the polarization of the microglia from M1 to M2 phenotype.
关键词
安菲博肽 /
脑缺血再灌注 /
炎症 /
小胶质细胞 /
极化
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Key words
anfibatide /
cerebral ischemia reperfusion /
inflammation /
microglia /
polarization
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中图分类号：
R965
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参考文献
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脚注
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基金
安徽省自然科学基金资助(2108085MH322)
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