Regulatory Effect Of WIP1 In Hypoxic Brain Injury

Brain is an oxygen-sensitive organ. The serious anoxia of brain leads to irreversible injuries including impairments on cognitive,language,action capabilities and so on, which even become life-threatening. The brain anoxia is often caused by natural environment(e.g. hypoxia in plateau environment) and pathological status(e.g. ischemic hypoxia). In the above mentioned two common types of brain anoxia, the inflammation occurred in hypoxic condition plays critical roles, which results in mass cytotoxic substance release and accelerates neural cells death and brain injury. However, the regulatory genes of hypoxic inflammation induced brain injury are understood limitedly. According to the previous research, WIP1 phosphotase can directly relieve the inflammation by dephorylating the key sites of p38 and NF-κB, in normoxia, but whether WIP1 is able to regulate hypoxic inflammation induced brain injury and ischemic stroke is unknown. In this study, we explore the functions of WIP1 in hypoxic inflammation induced brain injury and ischemic stroke, and the following findings were made: 1. Establishing a murine model of plateau hypoxic inflammation induced brain injury.We chose the widely used laboratory animal--C57BL/6 mice and treated mice with the hypobaric chamber mimicked the hypoxic environment in 6000-meter paleatu and plus intraperitoneal injection of low dose LPS to induce the hypoxic inflammation and associated brain injury. Our evaluation results on this model showed:(a) Inflammation-related cytokines increased significantly in the peripheral circulation: the ELISA tests showed that the levels of pro-inflammatory cytokines IL-6, TNF-α, and anti-inflammatory cytokine IL-10 in mice significantly increased to various degrees.(b) Pathologic changes appeared in the brain tissue: cortex and hippocampus injuries appeared obviously after treatment, including cell swelling, pericellular spaces widening, angiogenesis, shrunken neurons with darkly stained pyknotic nuclei and so on. Strikingly, we found that compared to cortex and hippocampus, the nuclei ventrales posteriors thalami was more sensitive to this hypoxic lesion, demonstrating disordered cells arrangement, and this phenomenon was not seen in cortex and hippocampus.(c) Compensatory angiogenesis appeared in the brain tissue: Using immunofluorescence staining, we found the expression level of CD31 in cortex, hippocampus and the nuclei ventrales hypothalamic nuclei regions was up-regulated to different extent, indicating vessel hyperplasia appeared to counter hypoxic inflammation stress.(d) The cognitive capabilities of mice was damaged more seriously: using the locomotion video track and new object recognition detection system, we found the mobility and cognitive of mice declined more significantly. 2. Identifying WIP1 as a neural-protective regulator to hypoxic inflammation induced brain injury.Using above established mice model, we proved that, in hypoxic inflammation induced brain injury:(a) WIP1 knockout mice inflammation-related factors increased more significantly in peripheral circulation: through ELISA tests, the pro-inflammatory cytokines IL-6, IL-1β, TNF-α and anti-inflammatory factor IL-10 were found to increase higher levels.(b) microglia activation in WIP1 knockout mice brain was more obvious: utilizing Iba-1 immunohistochemistry assays, the activation of microglia in cortex, hippocampus, and the nuclei ventrales posteriors thalami regions were determined to have higher levels than wildtype counterparters’.(c) inflammations responese in the brain of WIP1 knockout mice were more severe, and the expression level of inflammatory cytokines increased more significently: ELISA tests showed that, the expression levels of pro-inflammatory cytokines IL-6, IL-1β, TNF-α in WIP1 knockout mice brain tissue lysate increased more obviously.(d) brain tissue structure lesions in WIP1 knockout mice were more severe: Brain tissue H&E staining results showed that more serious injuries of organizational structure appeared in the WIP1 knockout mice, including more serious cellar swelling、pericellular spaces widening、neuronal shrinkage with karyopycnosis stained in cortex and hippocampus, and nuclei ventrales posteriors thalami region.(e) The cognitive capabilities of the WIP1 knockout mice was damaged more seriously: using the locomotion video track and new object recognition detection system, we found the mobility and cognitive of WIP1 knockout mice declined more significantly. 3. Preliminarily unclosing the neural-protective role of WIP1 gene in ischemic brain injury.This study explored the role of WIP1 gene in ischemic brain injury, and obtained some preliminary results:(a) the expression level of WIP1 increased in the ischemic brain tissue: Utilizing the MACO permanent middle cerebral artery occlusionmodel and the Western Blot tests, the WIP1 expression in the ischemic side was found to be significantly higher than its in the control side.(b) clarifying the expression profile of WIP1 in neural cells: Ischemic stimulus was simulated by oxygen glucose deprivation(OGD) in the primary cultured neurons, astrocytes and microglias, and the changes of WIP1 expression in various cells were observed. Western Blot results showed that, WIP1 mainly expressed in the microglial cells, and its expression significantly increased after OGD treatment, also the same verification results were got in microglial cell line BV2.(c) constructing the stable cell strain BV2 overexpressing WIP1: The stable cell strains BV2 that overexpressed WIP1 were successfully constructed with lenti-viral packaging technology, and the inflammation functions of stable cell strains BV2 were detected by LPS and OGD stimulus respectively. The ELISA results showed that WIP1 overexpression can significantly inhibit pro-inflammatory cytokines TNF-α secreting.(d) investigating the role of WIP1 in permanent cerebral ischemia brain injury: The preliminary results show that, WIP1 has a protective role in the ischemic brain injury, and WIP1 deletion will aggravate this injury.In summary, this study first determines the function of WIP1 that regulate brain injury induced by hypoxic inflammation. WIP1 deletion significantly elevates inflammatory response in the plateau hypoxic, and exacerbates brain injuries. These findings proves that WIP1 is a neural protective regulatory to pathological process of brain injury caused by hypoxic inflammation.