Sun Jing-Jun, Liu Ying, Ye Zhu-Rong
Department of Pathology, Shanghai Medical College, Fudan University, Shanghai, China.
Neurosci Bull. 2008 Aug;24(4):231-43. doi: 10.1007/s12264-008-0430-x.
The present study aimed to explore the role of P2Y(1) receptor in glial fibrillary acidic protein (GFAP) production and glial cell line-derived neurotrophic factor (GDNF) secretion of astrocytes under ischemic insult and the related signaling pathways.
Using transient right middle cerebral artery occlusion (tMCAO) and oxygen-glucose-serum deprivation for 2 h as the model of ischemic injury in vivo and in vitro, immunofluorescence, quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, enzyme linked immunosorbent assay (ELISA) were used to investigate location of P2Y(1) receptor and GDNF, the expression of GFAP and GDNF, and the changes of signaling molecules.
Blockage of P2Y(1) receptor with the selective antagonist N(6)-methyl-2'-deoxyadenosine 3',5'-bisphosphate diammonium (MRS2179) reduced GFAP production and increased GDNF production in the antagonist group as compared with simple ischemic group both in vivo and in vitro. Oxygen-glucose-serum deprivation and blockage of P2Y(1) receptor caused elevation of phosphorylated Akt and cAMP response element binding protein (CREB), and reduction of phosphorylated Janus kinase2 (JAK2) and signal transducer and activator of transcription3 (STAT3, Ser727). After blockage of P2Y(1) receptor and deprivation of oxygen-glucose-serum, AG490 (inhibitor of JAK2) reduced phosphorylation of STAT3 (Ser727) as well as expression of GFAP; LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), decreased phosphorylation of Akt and CREB; the inhibitor of mitogen-activated protein kinase kinase1/2 (MEK1/2) U0126, an important molecule of Ras/extracellular signal-regulated kinase (ERK) signaling pathway, decreased the phosphorylation of JAK2, STAT3 (Ser727), Akt and CREB.
These results suggest that P2Y(1) receptor plays a role in the production of GFAP and GDNF in astrocytes under transient ischemic condition and the related signaling pathways may be JAK2/STAT3 and PI3-K/Akt/CREB, respectively, and that crosstalk probably exists between them.
本研究旨在探讨P2Y(1)受体在缺血损伤条件下对星形胶质细胞中胶质纤维酸性蛋白（GFAP）生成及胶质细胞源性神经营养因子（GDNF）分泌的作用及其相关信号通路。
采用短暂性大脑中动脉闭塞（tMCAO）和氧-葡萄糖-血清剥夺2小时作为体内外缺血损伤模型，运用免疫荧光、定量实时逆转录-聚合酶链反应（RT-PCR）、蛋白质印迹法、酶联免疫吸附测定（ELISA）来研究P2Y(1)受体和GDNF的定位、GFAP和GDNF的表达以及信号分子的变化。
与单纯缺血组相比，在体内和体外实验中，使用选择性拮抗剂N(6)-甲基-2'-脱氧腺苷3',5'-双磷酸二铵（MRS2179）阻断P2Y(1)受体后，拮抗剂组中GFAP生成减少，GDNF生成增加。氧-葡萄糖-血清剥夺及P2Y(1)受体阻断导致磷酸化Akt和环磷酸腺苷反应元件结合蛋白（CREB）升高，磷酸化Janus激酶2（JAK2）和信号转导及转录激活因子3（STAT3，Ser727）降低。阻断P2Y(1)受体并进行氧-葡萄糖-血清剥夺后，AG490（JAK2抑制剂）降低了STAT3（Ser727）的磷酸化水平以及GFAP的表达；磷脂酰肌醇3-激酶（PI3-K）抑制剂LY294002降低了Akt和CREB的磷酸化水平；丝裂原活化蛋白激酶激酶1/2（MEK1/2）抑制剂U0126（Ras/细胞外信号调节激酶（ERK）信号通路的重要分子）降低了JAK2、STAT3（Ser727）、Akt和CREB的磷酸化水平。
这些结果表明，P2Y(1)受体在短暂缺血条件下对星形胶质细胞中GFAP和GDNF的生成起作用，相关信号通路可能分别为JAK2/STAT3和PI3-K/Akt/CREB，且它们之间可能存在相互作用。
