Dynamic Expression Of YAP In The Acute Stage Of Cerebral Ischemic Injury And Its Preliminary Mechanism In Microglia

The Hippo signaling pathway plays an important role in animal development,tissue regeneration and tumor inhibition.The pathway is involved in regulating the expression of related genes by regulating the activity of downstream transcriptional cofactor YAP(Yes-associated protein)/ TAZ(TAFAZZIN),which participates in a variety of cellular activities(cell metabolism,proliferation,differentiation or migration,etc.).At the same time,many upstream signals(cell polarity,mechanical signals,cell density,soluble factors and stress signals)participate in the regulation of Hippo pathway.At present,forensic research was looking for reliable identification molecules as objective evidence of injury or disease.Current studies have shown that YAP can promote the development of inflammation in macrophages in the gut,heart,liver,skin and other organs and show temporal changes.Therefore,YAP may have the potential to be an identification molecule.However,the localization and dynamic changes of YAP in microglia in ischemic brain injury have not been reported.The timing of the emergence of inflammatory cells and inflammatory factors and the level of inflammatory response play a delicate balancing role in the process of cerebral ischemia injury and repair.The purpose of this study was to observe the location and dynamic expression of YAP after cerebral ischemia injury in a rat model,and to preliminarily explore the role of YAP in inflammation.【Objective】1.To study the dynamic expression of YAP in the acute stage of cerebral ischemic injury.2.To observe the cell localization of YAP after cerebral ischemia injury.3.To investigate the effect of inhibiting YAP protein expression on the acute stage of cerebral ischemic injury and the role played in microglia.【Methods】In this study,a permanent middle cerebral artery occlusion(p MCAO)model was established in rats using the filament method.The activity of the key molecule YAP in the Hippo/YAP signaling pathway was downregulated by intracerebroventricular injection of verteporfin(VP).The study was divided into two parts: In the first part,the experimental animals were divided into three groups: Sham group,model 1-day group(M1d group),and model 3-day group(M3d group).In the second part,the experimental animals were divided into five groups: Sham group,model 1-day group(M1d group),model 3-day group(M3d group),inhibitor 1-day group(VP1d group),and inhibitor 3-day group(VP3d group).Neurological function was evaluated by Zea Longa score or mNSS score,the proportion of cerebral infarction and edema in rats was measured by TTC staining,cell types were determined by HE staining,protein expression was initially determined by immunohistochemistry,and relative content of target protein was detected by Western blot.Immunofluorescence was used to detect the localization of target proteins and bioinformatics was used to detect differential genes.【Result】1.In the acute stage of cerebral ischemic injury,YAP protein expression is up-regulated with the extension of time.The expression of YAP in the ischemic edge area showed no significant change compared with the control group 1 day after injury,and YAP protein was expressed in both astrocytes and microglia 3 days after injury,and microglia began to express YAP protein 3 days after injury.2.Compared with the 3-day model group,the 3-day blocker group significantly reduced the infarct size,suggesting that blocking YAP can reduce the infarct size when ischemic injury occurs.In the acute stage of ischemic injury,the expression of IL-1 decreased and CD206 increased after VP inhibited the expression of YAP,suggesting that YAP was involved in the polarization process of microglia and thus played a pro-inflammatory role.【Conclusion】1.The expression of YAP protein in the acute stage of cerebral ischemia injury is gradually increased,suggesting that YAP can be used as a reference index for infering the time of cerebral ischemia injury.2.In the acute stage of cerebral ischemic injury,YAP protein in microglia cells showed a gradual upward trend,suggesting that YAP played an important role in the pathological process of microglia-mediated inflammation.3.In the acute stage of ischemic injury,VP promotes M2-type polarization of microglia cells by inhibiting YAP,which contributes to injury repair.