title: Probenecid protects against oxygen-glucose deprivation injury in primary astrocytes by regulating inflammasome activity.
abstract: Inflammation is extremely important in the development of cerebral ischemia/reperfusion injury. Pannexin 1 (Panx1) channel has been reported to activate inflammasome in astrocytes and be involved in ischemic injury, but this damage effect is reversed by a Panx1 inhibitor-probenecid. However, the mechanism of probenecid protects against cerebral ischemia/reperfusion injury remains unclear. In present study, we hypothesized that probenecid protected astrocytes from ischemia/reperfusion injury in vitro by modulating the inflammasome. Primary cultured neocortical astrocytes were exposed to oxygen-glucose deprivation/reoxygenation (OGD/RX) and probenecid was added in this model. Viability and nuclear morphology of astrocytes, production of reactive oxygen species (ROS), protein expressions of NLRP3 (NOD-like receptor protein 3), caspase-1, and AQP4 (Aquaporins 4), as well as release of cellular HMGB1 and IL-1β were observed to evaluate the effect and mechanisms of probenecid on OGD/reoxygenated astrocytes. Probenecid did not affect cell viability at concentrations of 1, 5, 10, and 100μM but induced significant astrocytes death at 500μM. Probenecid inhibited cell death and ROS generation in astrocytes subjected to 6h of OGD and 24h of reoxygenation. The expression levels of NLRP3, caspase-1, and AQP4 increased after 6h of OGD, but probenecid treatment attenuated this increase. Moreover, the extracellular release of IL-1β and HMGB1 from OGD/reoxygenated astrocytes increased significantly. However, treatment by probenecid resulted in substantial reduction of these proteins levels in extracellular space. In conclusion, The Panx1 inhibitor, probenecid, which was administered before OGD, provided protective effects on the OGD/reoxygenation model of cultured astrocytes by modulating inflammasome activity and downregulating AQP4 expression.
authors.affiliation: Department of Neurosurgery, Renmin Hospital of Wuhan University, Hubei, China.
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Jian
authors.affiliation: Department of Anesthesia, the First Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang, China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Ding
authors.affiliation: Cardiovascular Surgery, Renmin Hospital of Wuhan University, Hubei, China.
authors.forename: Hongping
authors.initials: H
authors.lastname: Deng
authors.affiliation: Department of Neurosurgery, Renmin Hospital of Wuhan University, Hubei, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Neurosurgery, Renmin Hospital of Wuhan University, Hubei, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Yi
authors.affiliation: Department of Neurosurgery, Renmin Hospital of Wuhan University, Hubei, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Anesthesia, the First Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang, China.
authors.forename: Shengmei
authors.initials: S
authors.lastname: Zhu
authors.affiliation: Central laboratory, Renmin Hospital of Wuhan University, Hubei, China. Electronic address: ljgu0908@yahoo.com.
authors.forename: Lijuan
authors.initials: L
authors.lastname: Gu
authors.affiliation: Department of Neurosurgery, Renmin Hospital of Wuhan University, Hubei, China; Central laboratory, Renmin Hospital of Wuhan University, Hubei, China. Electronic address: xiaoxingxiong@whu.edu.cn.
authors.forename: Xiaoxing
authors.initials: X
authors.lastname: Xiong
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism*; D002545:Brain Ischemia / Q000150:complications / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D017630:Connexins / Q000037:antagonists & inhibitors; D004660:Encephalitis / Q000209:etiology / Q000517:prevention & control; D005947:Glucose / Q000378:metabolism; D058847:Inflammasomes / Q000187:drug effects*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors; D018696:Neuroprotective Agents / Q000008:administration & dosage*; D010100:Oxygen / Q000378:metabolism; D061251:Primary Cell Culture; D011339:Probenecid / Q000008:administration & dosage*; D017382:Reactive Oxygen Species / Q000378:metabolism; D015427:Reperfusion Injury / Q000150:complications / Q000378:metabolism
chemical_list: D017630:Connexins; D058847:Inflammasomes; D009419:Nerve Tissue Proteins; D018696:Neuroprotective Agents; C503631:Panx1 protein, mouse; D017382:Reactive Oxygen Species; D005947:Glucose; D011339:Probenecid; D010100:Oxygen
keywords: Cerebral ischemia/reperfusion injury; HMGB1; Inflammasome; NLRP3; Pannexin 1