乳脂球-表皮生长因子8对脑缺血后小胶质细胞极化的调控作用机制研究

The mechanism by which epidermal growth factor 8 regulates microglia polarization in transient cerebral ischemia

目的探寻乳脂球-表皮生长因子8(MFG-E8)在脑缺血后小胶质细胞极化的调控作用和作用机制。方法选取C57BL/6小鼠40只,采用随机数字表法将其为假手术组、缺血组、重组小鼠乳脂球-表皮生长因子8(rmMFG-E8)组和rmMFG-E8+科利维林(Colivelin TFA)组,每组10只小鼠。缺血组、重组小鼠乳脂球-表皮生长因子8(rmMFG-E8)组和rmMFG-E8+科利维林(Colivelin TFA)组制作大脑中动脉梗阻再灌注模型(tMCAO),其中rmMFG-E8组和rmMFG-E8+Colivelin TFA组小鼠在造模成功后立即进行脑梗侧侧脑室立体定位注射,分别给予总量2μl,浓度0.4μg/μl的rmMFG-E8和总量2μl,浓度0.4μg/μl的rmMFG-E8+总量2μl,浓度5 pmol/μl的Colivelin TFA干预。造模成功1、3、5、7 d后采用行为学实验检测神经功能恢复情况,造模成功7 d后采用组织染色观察脑梗死灶体积比例,通过实时荧光定量聚合酶链式反应检测小胶质细胞M1极化标记物诱导型一氧化氮合酶(iNOS)、M2极化标记物精氨酸酶1(Arg1)和小鼠类几丁质酶3样分子(Ym1)、MFG-E8的基因表达水平,通过免疫印迹实验检测MFG-E8、磷酸化信号转导与转录因子3(p-STAT3)和细胞因子信号传导抑制因子-3(SOCS3)蛋白表达水平。结果行为学检测发现,缺血组、rmMFG-E8组和rmMFG-E8+Colivelin TFA组在造模成功1、3、5、7 d后的转棒时间和mNSS评分与假手术组同时间点比较,差异均有统计学意义(P<0.05)。造模成功7 d后,与假手术组比较,缺血组MFG-E8基因和蛋白表达均显著降低(P<0.05),M1极化标记物iNOS基因的表达显著增高(P<0.05),M2极化标记物Arg1和Ym1基因的表达显著下降(P<0.05),p-STAT3/STAT3蛋白的表达显著上调(P<0.05),SOCS3/GAPDH蛋白的表达显著下调(P<0.05)。造模成功7 d后,与缺血组比较,rmMFG-E8组的梗死灶体积显著缩小(P<0.05)。造模成功7 d后,rmMFG-E8+Colivelin TFA组iNOS基因的表达较rmMFG-E8组明显增加(P<0.05),Arg1和Ym1基因的表达明显降低(P<0.05),p-STAT3/STAT3的表达显著上调(P<0.05),SOCS3/GAPDH的表达显著下降(P<0.05)。结论MFG-E8可通过STAT3信号通路促进脑缺血后小胶质细胞向M2型极化,促进脑缺血小鼠神经功能的恢复。

Objective To explore the function of epidermal growth factor 8 in the polarization of microglia after ischemic brain injury and its mechanism.Methods Forty C57BL/6 mice were randomly divided into a sham operation group,an ischemia group,a recombinant mouse milk fat globule epidermal growth factor 8(rmMFG-E8)group and an rmMFG-E8+colivelin TFA group,each of 10.The middle cerebral artery occlusion and reperfusion model(tMCAO)was established in all except the sham operation group.Right after the modelling the mice in the rmMFG-E8 group were immediately injected with 2μL of 0.4μg/μL of rmMFG-E8 into the ventricle contralateral to the cerebral infarction.The rmMFG-E8+Colivelin TFA group was injected with the same dose of rmMFG-E8 plus 2μL of 5pmol/μL colivelin TFA.On the 1st,3rd,5th and 7th day after the modelling,neurological functioning was documented using behavioral tests.The volume proportion of the cerebral infarction was observed after tissue staining on the 7th day after the operation.The gene expression levels of M1 polarization marker-induced nitric oxide synthase(iNOS),M2 polarization marker arginase-1(Arg1)and mouse chitinase-like molecule 3(YM1)in the microglia were detected using real-time fluorescence quantitative polymerase chain reactions.The protein expression levels of MFG-E8,phosphorylated signal transduction and transcription factor 3(p-STAT3)and cytokine signal transduction inhibitor-3(SOCS3)were determined using western blotting.Results The behavior tests revealed significant differences between the sham operation group and the other groups on all four days.Compared with the sham operation group,the average expression of MFG-E8 gene and its protein,Arg1 and Ym1,and the SOCS3/GAPDH protein ratio had decreased significantly in the ischemic group,while the average expression of iNOS and the p-STAT3/STAT3 protein ratio had increased significantly.On the 7th day after the modelling,compared with the ischemic group,the infarct volume was significantly smaller in the rmMFG-E8 group.The average expression of iNOS and the average p-STAT3/STAT3 ratio in the rmMFG-E8+colivelin TFA group had increased significantly compared with the rmMFG-E8 group,while the average expression of Arg1 and Ym1,and the SOCS3/GAPDH ratio were significantly lower.Conclusion MFG-E8 promotes the polarization of M2-type microglia after cerebral ischemia through STAT3 signaling,promoting the recovery of neurological functioning.