Toll-like receptor 4 signaling is involved in PACAP-induced neuroprotection in BV2 microglial cells under OGD/reoxygenation.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Xuzhou Medical College, China.
Xia
X
Qin
Zhen-Quan
ZQ
Sun
Xiao-Jing
XJ
Dai
Shan-Shan
SS
Mao
Jiao-Li
JL
Zhang
Meng-Xing
MX
Jia
Yong-Mei
YM
Zhang
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D020925:Hypoxia-Ischemia, Brain / Q000378:metabolism*; D051379:Mice; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D051197:Toll-Like Receptor 4 / Q000378:metabolism*
D018696:Neuroprotective Agents; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D051197:Toll-Like Receptor 4