Qin Xia, Sun Zhen-Quan, Dai Xiao-Jing, Mao Shan-Shan, Zhang Jiao-Li, Jia Meng-Xing, Zhang Yong-Mei
Xuzhou Medical College, China.
Neurol Res. 2012 May;34(4):379-89. doi: 10.1179/1743132812Y.0000000028.
The neuroprotective effects of pituitary adenylate cyclise-activating polypeptide (PACAP) have been well documented in vivo and in vitro. However, the mechanisms by which PACAP protected microglia from ischemic/hypoxic injury via inhibition of microglia activation remain unclear. Toll-like receptor 4 (TLR4) plays a considerable role in the induction of innate immune and inflammatory responses. The purpose of this study is to investigate the effect of PACAP on the oxygen and glucose deprivation (OGD)/reoxygenation BV2 microglia and to explore the role of TLR4/myeloid differentiation protein 88 (MyD88)/nuclear factor-kappa B (NF-kappaB) pathway in the neuroprotective effects of PACAP.
We conducted OGD/reoxygenation by placing BV2 microglia into an airtight chamber and in glucose-free medium. BV2 microglia cell viability was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] assay. Western blot was utilized to detect TLR4, MyD88 expression, inhibitory protein of NF-kappaB (IkappaB) phosphorylation/degradation, NF-kappaB activation. Level of tumor necrosis factor-alpha (TNF-alpha) in culture medium was measured with enzyme-linked immunosorbent assay (ELISA). Apoptosis was determined by flow cytometry.
We found that pretreatment with PACAP to BV2 cells immediately before OGD/reoxygenation significantly alleviated microglia hypoxic injury. PACAP inhibited upregulation of TLR4, MyD88 and NF-kappaB in BV2 microglial cells exposed to OGD/reoxygenation. PACAP administration also significantly reduced the production of proinflammatory cytokines and apoptosis in BV2 microglia exposed to OGD/reoxygenation.
Pretreatment with PACAP inhibited activation of the TLR4/MyD88/NF-kappaB signaling pathway and decreased inflammatory cytokine levels, as well as apoptosis in microglia, thereby attenuating microglia hypoxic injury. Our results suggested that TLR4-mediated MyD88-dependent signaling pathway contributed to neuroprotection of PACAP to microglia against OGD/reoxygenation.
垂体腺苷酸环化酶激活多肽（PACAP）的神经保护作用在体内和体外均已得到充分证明。然而，PACAP通过抑制小胶质细胞激活来保护其免受缺血/缺氧损伤的机制仍不清楚。Toll样受体4（TLR4）在诱导先天性免疫和炎症反应中起重要作用。本研究的目的是探讨PACAP对氧糖剥夺（OGD）/复氧BV2小胶质细胞的影响，并探讨TLR4/髓样分化蛋白88（MyD88）/核因子-κB（NF-κB）信号通路在PACAP神经保护作用中的作用。
我们通过将BV2小胶质细胞置于密闭培养箱中并在无糖培养基中培养来进行OGD/复氧。通过MTT [3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐] 法测定BV2小胶质细胞的活力。利用蛋白质免疫印迹法检测TLR4、MyD88的表达、NF-κB抑制蛋白（IkappaB）的磷酸化/降解以及NF-κB的激活。用酶联免疫吸附测定法（ELISA）测量培养基中肿瘤坏死因子-α（TNF-α）的水平。通过流式细胞术检测细胞凋亡。
我们发现，在OGD/复氧前立即用PACAP预处理BV2细胞可显著减轻小胶质细胞的缺氧损伤。PACAP抑制了暴露于OGD/复氧的BV2小胶质细胞中TLR4、MyD88和NF-κB的上调。给予PACAP还显著降低了暴露于OGD/复氧的BV2小胶质细胞中促炎细胞因子的产生和细胞凋亡。
用PACAP预处理可抑制TLR4/MyD88/NF-κB信号通路的激活，降低炎症细胞因子水平以及小胶质细胞的凋亡，从而减轻小胶质细胞的缺氧损伤。我们的结果表明，TLR4介导的MyD88依赖性信号通路有助于PACAP对小胶质细胞抵抗OGD/复氧的神经保护作用。
