JAK2-STAT3途径介导凝血酶诱导的小胶质细胞iNOS的激活致多巴胺能神经元变性

JAK2-STAT3 Signaling Pathway Mediates Thrombin-induced Microglial iNOS Action and Degeneration of Dopaminergic Neurons in vitro

目的探讨JAK2-STAT3途径与凝血酶诱导多巴胺能神经元变性作用的关系。方法20U/ml凝血酶或AG490(10μmol/L)预处理+凝血酶(20U/ml)处理原代培养的小胶质细胞或中脑腹侧混合培养体系,用Westernblot检测小胶质细胞P-JAK1、P-JAK2、JAK2、STAT3、P-STAT3的表达;免疫荧光观察多巴胺能神经元的变性;Westernblot、RT-PCR及激光共聚焦观察iNOS的表达。结果凝血酶处理小胶质细胞2h后诱导JAK2、STAT3的磷酸化,2h和4h分别增加小胶质细胞iNOS的转录和表达,并且24h诱导中脑培养体系多巴胺能神经元变性。而AG490预处理显著地减少对应凝血酶处理组小胶质细胞JAK2和STAT3的磷酸化,抑制iNOS的表达,并且缓解多巴胺能神经元的变性。结论JAK2-STAT3途径介导凝血酶诱导的小胶质细胞iNOS的激活,导致多巴胺能神经元变性。

Objective To explore the relation between JAK2-STAT3 signaling pathway and thrombin-induced degeneration of dopaminergic neurons in vitro. Methods After 20 U/ml thrombin or AG490 preconditioning （10 μmol/L） ＋ thrombin-treated primary rat microglial ceils or co-cultures at indicated time points, Western blot analysis was performed to examine production of P-JAK1, P-JAK2, JAK2, P-STAT3, STAT3. TH immunofluorescence staining examined effect of thrombin on dopaminergie neurons of ventral mesencephalons cultures. Western blot and RT-PCR evaluated the levels of iNOS of microglia. Confoeal double immunofluoreseence staining evaluated the cellular location of iNOS expression. Results After thrombin-treated primary rat microglial cells, thrombin rapidly activated JAK2 and induced phosphorylation of STAT3 within 2-12 h. In addition, thrombin increased transcription and expression of iNOS at 2 h and 4 h respectively, and iNOS localized within microgila 12 h after thrombin treatment, at 24 h induced neurodegeneration of dopaminergic neurons derived from microglia in mesencephalic cultures. However, AG490, a JAK inhibitor, markedly reduced activation of JAK2 and STAT3 in thrombin-treated microglia, also inhibited thrombin-induced transcription and expression of iNOS, moreover at 24 h rescued dopaminergic neurons. Conclusion These results suggest that JAK2-STAT3 signaling pathway plays a critical role in mediating thrombin-induced iNOS action of mieroglia and degeneration of dopaminergic neurons in vitro.