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目的观察小胶质细胞(MG)在缺氧、缺糖(OGD)模型或组织型纤溶酶原激活剂(tPA)干预下被激活的主要信号通路,探讨神经源性丝氨酸蛋白酶抑制剂(NSP)抑制MG进而产生神经保护作用的可能机制。方法体外原代培养大鼠皮层MG并鉴定,建立OGD模型。将MG随机分为正常组、tPA组(经终浓度为0.05μg/μL的tPA干预24h)、OGD 3h组(经OGD模型干预3h)、NSP+tPA组(经终浓度为0.025μg/μL的NSP预处理0.5h后,再以终浓度为0.05μg/μL的tPA干预24h)和NSP+OGD 3h组(经终浓度为0.025μg/μL的NSP预处理0.5h后,再经OGD模型干预3h)。采用免疫荧光双标染色法检测各干预组丝裂原活化蛋白激酶(MAPK)通路磷酸化蛋白的表达情况和核转录因子(NF)-κB的核移位情况。采用免疫印迹法检测各组MAPK通路磷酸化蛋白表达量的变化。结果 MG纯度鉴定结果显示纯度>95%,OGD模型建立成功。免疫组织化学法检测结果显示,MAPK途径的磷酸化细胞外信号调节激酶(pERK)1/2、磷酸化p38(pp38)在正常组MG均有少量表达,且在tPA组和OGD 3h组的表达均显著升高;MAPK途径的磷酸化c-Jun氨基末端激酶(pJNK)在正常组MG有表达,但在tPA组和OGD 3h组的表达无明显变化。Western免疫印迹法检测结果显示,干预组(tPA组和OGD 3h组)pERK1/2、pp38和pJNK的表达量均较正常组显著升高(P值均<0.05)。NSP预处理组(NSP+tPA组和NSP+OGD 3h组)pERK1/2、pp38和pJNK的表达量均较干预组显著降低(P值均<0.05)。免疫荧光双标染色法检测结果显示,正常组MG中可见NF-κB(P65)蛋白主要表达于细胞质,细胞胞突显示清晰。tPA组和OGD 3h组MG中NF-κB发生明显的核移位现象,主要表达于细胞核,且细胞缺少胞突。NSP+tPA组和NSP+OGD 3h组MG中NF-κB的核移位现象明显减轻。结论大鼠肉瘤(Ras)/细胞外信号调节激酶(ERK)、c-Jun-氨基末端激酶(JNK)/应激活化蛋白激酶(SAPK)和p38通路对于MG在tPA和OGD干预下的激活均起作用,其中ERK1/2和p38发挥更重要的作用。tPA和OGD干预MG后,NF-κB发生明显的核移位,此通路被激活,经NSP预处理后,各条信号通路均被不同程度抑制。
Objective To observe the activation of microglial cells (MG) under the condition of hypoxia, hypoglycemia (OGD) or tissue plasminogen activator (tPA), and to explore the role of neuropeptide serine protease inhibitors (NSP ) Inhibits the MG and then the possible mechanism of neuroprotection. Methods Primary cultured rat cortical MG was identified and established OGD model. MG were randomly divided into normal group, tPA group (tPA intervention of 0.05μg / μL final concentration for 24h), OGD 3h group (intervention of OGD model for 3h), NSP + tPA group (final concentration of 0.025μg / μL NSP pretreatment 0.5h, and then with a final concentration of 0.05μg / μL tPA intervention 24h) and NSP + OGD 3h group (final concentration of 0.025μg / μL NSP pretreatment 0.5h, then OGD intervention 3h ). The expression of phosphorylated MAPK pathway and the nuclear translocation of NF-κB were detected by double-stained immunofluorescence staining in each intervention group. Western blotting was used to detect the phosphorylation of MAPK pathway in each group. Results The purity of MG was> 95%. The OGD model was established successfully. The results of immunohistochemistry showed that phosphorylated extracellular signal-regulated kinase (pERK) 1/2 and phosphorylated p38 (pp38) of MAPK pathway were slightly expressed in normal MG group, and were significantly higher in tPA group and OGD 3h group (PJNK) in the MAPK pathway were up-regulated in normal MG group, but there was no significant change in tPA group and OGD 3h group. Western blotting results showed that the expressions of pERK1 / 2, pp38 and pJNK in the intervention group (tPA group and OGD 3h group) were significantly higher than those in the normal group (all P <0.05). The expression of pERK1 / 2, pp38 and pJNK in NSP pretreatment group (NSP + tPA group and NSP + OGD 3h group) were significantly lower than those in intervention group (all P <0.05). Immunofluorescence double-stained staining results showed that in the normal group, the expression of NF-κB (P65) protein was mainly expressed in the cytoplasm and the cell cytoplasm was clearly displayed. The nuclear translocation of NF-κB was observed in the MG of the tPA group and the OGD 3h group, which was mainly expressed in the nucleus, and the cells lacked the cytoplasm. NSP + tPA group and NSP + OGD 3h MG nuclear NF-κB nuclear translocation was significantly reduced. Conclusion The activation of rat sarcoma (Ras) / extracellular signal-regulated kinase (ERK), c-Jun-amino-terminal kinase (JNK) / stress activated protein kinase (SAPK) Play a role, of which ERK1 / 2 and p38 play a more important role. After tPA and OGD were interfered with MG, nuclear translocation of NF-κB was obvious, and this pathway was activated. After pretreatment with NSP, all signaling pathways were inhibited to some extent.