Effect And The Mechanism Of Hydrogen-rich Water On The Angiogenesis In Penumbra Brain Tissue With Traumatic Brain Injury Rats

Part 1 The effect of hydrogen-rich water on the CD34 in penumbra brain tissue of traumatic brain injury-challenged ratsObjective To determine the effect of hydrogen-rich water on the CD34 and angiogenesis in the penumbra tissue of brain injury of rats.Methods a total of 54 adult male Sprague-Dawley(SD)rats were randomly divided into three groups: sham-operated,TBI(Traumatic brain injury),and TBI+HW(Traumatic brain injury+ hydrogen-rich water).Each group is divided into 24 h,3d and 7d three subgroups.Sham group Don’t impact.After inducing TBI,TBI+HW group intraperitoneal inject hydrogen-rich water(5 ml/kg),and one times a day until to die.Sham group and TBI group at the same time point intraperitoneal inject same amount of saline solution.Observe neurological severity scores(NSS)for neurologic deficits,hematoxylin-eosin staining for pathological changes.immunohistochemical analysis to observe the expression of CD34+cells,then counting the newborn blood capillary sprouts around the traumatic brain tissue by CD34+ cells markers.western blot analysis for the expression of CD34.Results 3d and 7d after injury,NSS of TBI+HW rats significantly decreased;at 24 h and 3d after injury,pathological changes in lesion boundary brain tissue of TBI rats were characterized by obvious hemorrhagic necrosis,severe brain edema,loose neural substrates,and the pathological changes were more obvious at 3d.In comparison with the TBI group,edema volume was lower in TBI+HW rats.At 7d after injury,newborn blood capillary hyperplasia in TBI+HW was significantly higher than that in TBI.In comparison with the TBI group,the expression of CD34 protein in TBI+HW significantly increased than that in TBI at 7d after injury.In comparison with the sham group,the expression of CD34 protein in TBIand TBI+HW group significantly increased at different time-points.Conclusion Hydrogen-rich water promotes CD34+cells home to the site of injured tissue and enhancing angiogenesis,then to improve clinical outcomes during brain functional recovery.Part 2 The effect of hydrogen-rich water on the expression of HIF-1α and VEGF in penumbra brain tissue of traumatic brain injury-challenged ratsObjective To determine the effect of hydrogen-rich water on the HIF-1αand VEGF in the penumbra tissue of brain injury of rats.Methods a total of 54 adult male Sprague-Dawley(SD)rats were randomly divided into three groups: sham-operated,TBI(Traumatic brain injury),and TBI+HW(Traumatic brain injury+ hydrogen-rich water).Each group is divided into 24 h,3d and 7d three subgroups.Sham group Don’t impact.After inducing TBI,TBI+HW group intraperitoneal inject hydrogen-rich water(5 ml/kg),and one times a day until to die.Sham group and TBI group at the same time point intraperitoneal inject same amount of saline solution.Observe the brain water content.western blot analysis and RT-PCR for the expression of HIF-1αand VEGF.Results Compare with sham group,at 24 h and 3d after injury,the brain water content of TBI group and TBI+HW group is increased significantly,In comparison with the TBI group,the brain water content of TBI+HW group is decreased significantly.In comparison with the TBI group,the expression of HIF-1αand VEGF protein and m RNA in TBI+HW significantly increased at each time after injury.In comparison with the sham group,the expression of HIF-1α and VEGF protein and m RNA in TBI and TBI+HW group significantly increased at different time-points.Conclusion Hydrogen-rich water promotes the expression of HIF-1α and VEGF in penumbra brain tissue with TBI rats.Part3 Hydrogen-rich water promote the Angiogenesis in penumbra brain tissue of traumatic brain injury rats by PI3K/AKT patnwayObjective To explore the molecule mechanism of hydrogen-rich water promoting the angiogenesis in traumatic brain injury rats penumbra brain tissues.Methods a total of 30 adult male Sprague-Dawley(SD)rats were randomly divided into five groups:sham-operated,TBI(Traumatic brain injury),TBI+HW(Traumatic brain injury+hydrogen-rich water),TBI+LY(Traumatic brain injury+ LY294002),TBI+LY+HW(Traumatic brain injury+ LY294002+hydrogen-rich water).Sham group Don’t impact.After inducing TBI,TBI+HW group and TBI+LY+HW group intraperitoneal inject hydrogen-rich water(5 ml/kg),and one times a day until to die.Sham group,TBI group and TBI+LY group at the same time point intraperitoneal inject same amount of saline solution.TBI+LY group and TBI+LY+HW group Intraventricular injection LY29400220 min before trauma,and tail vein inject LY294002 one times a day until to die.Immunohistochemical analysis to observe the expression of CD34 + cells,then counting the newborn blood capillary sprouts around the traumatic brain tissue by CD34+cells markers.western blot and PCR analysis for the expression of PI3K、AKT、HIF-1α、VEGF.Results Comparison with Sham group,the expression of PI3K、AKT、HIF-1α、VEGF and the number of newborn blood capillary in TBI group,TBI+HW group,TBI+LY group and TBI+LY+HW group significantly increased.In comparison with the TBI group,the expression of PI3K、AKT、HIF-1α、VEGF in TBI+HW rats is significantly higher,and the number of newborn blood capillary increased.In comparison with the TBI+LY group and TBI+LY+HW group,TBI group and TBI+HW group PI3K、p-AKT、HIF-1α、VEGF significantly increased.The number of newborn blood capillary sprouts around the traumatic brain tissue in TBI+LY group and TBI+LY+HW group is significantly decreased than TBI group and TBI+HW group.Conclusion hydrogen-rich water can up-regulate the PI3K/AKT signal pathway in penumbra traumatic brain tissue,increased the expression of HIF-1α,up-regulate the VEGF,then promote the angiogenesis in traumatic brain injury rats brain tissues.the possible mechanisms for improving angiogenesis in traumatic brain injury rats by PI3K/AKT signal pathway.