MFGE8在缺血性脑损伤中表达及对巨噬细胞极化的调控作用

The expression of MFGE8 in ischemic brain injury and its regulation of macrophage polarization

目的探究蛋白质酪氨酸激酶2(MFGE8)在缺血性脑损伤(IBI)中的表达及对巨噬细胞极化的调控作用。方法ELISA检测缺血性脑卒中患者和大脑中动脉闭塞(MCAO)大鼠模型外周血中MFGE8蛋白表达;IF检测MCAO模型MFGE8在脑组织中的定位表达;流式细胞术检测脑组织巨噬细胞极化比例;采用稳转Mfge8的N2a神经元细胞(Mfge8CA)培养上清处理BV-2小胶质细胞,流式细胞术检测巨噬细胞极化比例;免疫印迹检测MFGE8、αv/β3-整合素、FAK、NF-κB、ERK1/2、Jnk1/2、p38、PI3K、AKT、m TOR蛋白表达。结果IBI患者和MCAO模型大鼠外周血中MFGE8含量明显低于对照组(P=0.0446,P=0.0259);MFGE8与神经元细胞标志(Neu N)高度共定位表达;MCAO模型大鼠脑组织中M1型(CD45+F4/80+i NOS+Arginase1-)巨噬细胞比例明显高于对照组(P=0.0004);M2型(CD45+F4/80+i NOS-Arginase1+)巨噬细胞比例明显低于对照组(P<0.0001);Mfge8CA组N2a神经细胞系培养上清处理的BV-2小胶质细胞M1型巨噬细胞比例明显低于野生组(WT,P=0.0230);M2型巨噬细胞比例明显高于WT(P<0.0001);Mfge8CA组BV-2小胶质细胞αv/β3-整合素、FAK、p-P85、P85、p-AKT(Ser473)、p-mTOR(Ser2481)和p-mTOR(Ser2488)蛋白表达明显高于WT组。Mfge8CA组BV-2小胶质细胞p-P65蛋白表达明显低于WT组。结论MFGE8高表达于IBI患者外周血,神经元细胞来源的MFGE8可能通过激活PI3K/AKT/m TOR信号促进BV-2小胶质细胞M2型巨噬细胞极化,并抑制M1型巨噬细胞极化。

Objective To investigate the expression of protein tyrosine kinase 2( MFGE8) in patients with ischemic brain injury( IBI) and its regulation on macrophage polarization. Methods ELISA was used to detect the expression of MFGE8 protein in peripheral blood of patients with ischemic stroke and middle cerebral artery occlusion( MCAO) rat models;IF was used to detect the localization and expression of MFGE8 in brain;BV-2 microglia was treated with the culture supernatant of N2 a neuronal cells( Mfge8 CA) stably transformed with Mfge8. The polarization ratio of macrophages was detected by flow cytometry. Western blot detection Mfge8,αv/β 3-integrin,FAK,NF-κB. ERK1/2,JNK1/2,P38,PI3 K,AKT,m TOR protein expression. Results The relative content of MFGE8 in peripheral blood of IBI patients and MCAO model rats was significantly lower than that of the control group( Ctrl,P = 0. 0446,P = 0. 0259). MFGE8 was highly co-localized with neuron cell marker( Neu N);the proportion of M1 type( CD45 + F4/80 + i NOS + Arginase1-) macrophages in the brain tissue of MCAO model was significantly higher than that in the Ctrl( P = 0. 0004). The proportion of M2 type( CD45+ F4/80 + i NOS-Arginase1 +) macrophages was significantly lower than that of the Ctrl( P < 0. 0001). The proportion of M1 macrophages of BV-2 microglia after supernatant of N2 a( mfge8 CA) treatment was significantly lower than that of Wild type( WT,P = 0. 0230). The proportion of M2 macrophages was significantly higher( P < 0. 0001). The protein expressions of α v/β3-integrin,FAK,p-P85,P85,p-AKT( Ser473),p-mTOR( Ser2481) and p-mTOR( Ser2488) in BV-2 microglia after supernatant of N2 a( mfge8 CA) treatment were significantly higher than those in WT group. The expression of p-P65 protein was significantly lower than that in WT group. Conclusion MFGE8 is highly expressed in peripheral blood of patients with IBI. MFGE8 derived from neuronal cells may promote BV-2 microglia M2 macrophages polarization by activating PI3 K/AKT/m TOR signals,and inhibit the polarization of M1 macrophages.