雷公藤内酯醇对脑缺血再灌注大鼠小胶质细胞M1/M2型极化的影响

Effect of Triptolide on the M1/M2 Polarization of Microglia in Rats with Cerebral Ischemia-reperfusion

目的:探讨雷公藤内酯醇(TPL)对脑缺血再灌注(CIR)大鼠小胶质细胞M1/M2型极化的影响。方法:选取192只SD大鼠设假手术组、模型组、丁苯酞(6 mg/kg)组和TPL低、中、高(0.2、0.4、0.8 mg/kg)剂量组,每组32只。除假手术组外,其余5组采用线栓阻断大脑中动脉2 h的方法复制CIR大鼠模型。各组均于造模前3 d开始给药,1次/d,再灌注24 h后,检测大鼠神经功能、脑梗死率、脑含水量,观察缺血侧皮层神经元病理学变化;酶联免疫吸附试验法(ELISA)检测缺血侧皮层组织炎症介质含量,免疫荧光标记法检测小胶质细胞表型M1型标志物CD86/离子钙结合衔接分子-1(Ionized Calcium-binding Adapter Molecule-1,Iba-1)、M2型标志物CD206/Iba-1,蛋白质免疫印迹法检测Toll样受体4(TLR4)、核因子κB、p-核因子κB蛋白表达。结果:与模型组比较,TPL中、高剂量组和丁苯酞组神经功能缺失评分、脑梗死率、脑含水量显著降低(P<0.05);缺血侧皮层神经元病理学变化明显改善,肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、细胞间黏附分子-1(ICAM-1)含量显著降低而IL-10含量显著升高,CD86/Iba-1阳性细胞率显著降低,CD206/Iba-1阳性细胞率显著升高(P<0.05);缺血侧皮层组织Toll样受体4(TLR4)、p-核因子κB表达显著下调,p-核因子κB/核因子κB比值显著降低(P<0.05)。除神经功能缺失评分、脑含水量、IL-1β含量外,TPL高剂量组对其他指标的影响均优于丁苯酞组(P<0.05)。结论:TPL可抑制大鼠CIR损伤,可能与促进小胶质细胞M1型向M2型极化,抑制TLR4/核因子κB通路活化及炎症反应有关。

Objective:To investigate the effect of triptolide(TPL)on the M1/M2 polarization of microglia in rats with cerebral ischemia-reperfusion(CIR).Methods:A total of 192 SD rats were divided into sham operation group,model group,butylphthalide(6 mg/kg)group and TPL low,medium,and high dose(0.2,0.4,0.8 mg/kg)groups,with 32 rats in each group.Except for the rats in sham operation group,the rats in other groups underwent CIR modeling by occluding the middle cerebral artery with a suture for 2 hours.Drug administration was initiated in all groups 3 days before modeling,once a day.After 24 hours of reperfusion,the neurological function,cerebral infarction rate,brain water content,pathological changes in the ischemic penumbra cortex were assessed.The levels of inflammatory factors in ischemic cerebral cortex were detected by enzyme-linked immunosorbent assay(ELISA).Immunofluorescence labeling was employed to detect the expression of M1-type marker CD86/(Ionized Calcium-binding Adapter Molecule,Iba-1),M2-type marker CD206/Iba-1 in microglia,and Western blot was conducted to assess the protein expression of toll-like receptor 4(TLR4),nuclear factor-κB(NF-κB),and p-NF-κB.Results:Compared with the model group,the TPL medium and high-dose groups,as well as the butylphthalide group,showed significantly reduced neurological deficit scores,cerebral infarction rates,and brain water contents(P<0.05);the pathological changes of cortical neurons in the ischemic penumbra were significantly improved.The levels of TNF-α,IL-1β,ICAM-1 were significantly decreased and the level of IL-10 was significantly increased.The positive rate of CD86/Iba-1 cells significantly decreased,and the positive rate of CD206/Iba-1 cells significantly increased(P<0.05).The expressions of TLR4 and p-NF-κB in the ischemic cortex tissue were significantly down-regulated,and the p-NF-κB/NF-κB ratio significantly decreased(P<0.05).Except for neurological deficit score,brain water content and IL-1βlevel,the effects of high dose TPL on other indicators were better than those of Butylphthalide group(P<0.05).Conclusion:TPL can inhibit CIR injury in rats,which may be related to promoting the polarization of microglia from M1 type to M2 type,inhibiting the activation of TLR4/NF-κB pathway and suppressing inflammatory responses.