The Study On The Effect And Mechanism Of Phenolic Compounds Extracted From Gastrodia Elata Blume.on The BEnd.3 OGD/Rep Injury Model

Objective:Cerebral ischemia-reperfusion injury is a complex pathophysiological process,among the many theories,the brain microvascular cell injury has occupied the central position.Our previous researches showed that phenolic compounds extracted from ethyl acetate of Gastrodia elata Blume.had significant brain protection on cerebral ischemia injury in rats,whose mechanisms were related to anti-inflammation and endothelium-dependent vasodilatation,which indicated that Gastrodia effective ingredients may protect brain from destruction by activate PI3K/AKt signal pathway.Thus this project intended to duplicate cell Oxygen-glucose deprivation/reperfusion(OGD/Rep)injury model to detect cell viability by MTT method,to detect LDH,NO and inflammatory mediators by kits,to detect signature proteins of TJ by immunofluorescence,and to detect MMPs by fluorescence quantitative PCR so as to clearly recognize the protective mechanism of phenolic compounds and to detect related factors of PI3K/AKt signaling pathway(AKt,p-AKt,e NOS,p-e NOS)to further discuss the mechanism of Gastrodia effective ingredients protect brain from destruction.Methods:1.Protective effect of phenolic compounds extracted from Gastrodia elata Blume.on the b End.3 OGD/Rep injury model.1.1 Duplicated OGD/Rep injury model by three gas incubator,and detectedb End.3 cell viability.1.2 Duplicated OGD/Rep injury model and detected whether phenolic compounds extracted from Gastrodia elata Blume.had protective effect by MTT method.Detected occludin and claudin-5 protein by immunofluorescence.Detected NO and LDH by kits.Detected MMP-2/9 by PCR.2.Effect of phenolic compounds on inflammatory mediators in the b End.3OGD/Rep injury model.Detected TNF-?,IL-6,and IL-1? by kits to explore the effect of phenolic compounds extracted from Gastrodia elata Blume.on inflammatory mediators.3.Effect of phenolic compounds on PI3K/AKt signaling pathway in the b End.3 OGD/Rep injury model.Duplicated OGD/Rep injury model,detected AKt,p-AK,e NOS and p-e NOS to explore the effect of phenolic compounds extracted from Gastrodia elata Blume.on PI3K/AKt signal pathway by western blot detection.Results:1.Phenolic compounds extracted from Gastrodia elata Blume.have protective effect on the b End.3 OGD/Rep injury model.1.1 Compared with normal group,OGD 6h/Rep 4h were carried out on the b End.3 OGD/Rep injury significantly(P < 0.001),and cell viability was(56.95 ±1.99)%,so the follow-up experiments selected this conditional modeling.1.2 Phenolic compound B,C,and D could elevate cell survival rate(P<0.05,P<0.01).Phenolic compound B,C and D could elevate occludin protein and claudin-5protein expression(P < 0.05 or P < 0.01);phenolic compound H could elevate occludin protein expression(P < 0.001).Phenolic compound B could elevate NO expression(P < 0.05).Phenolic compound B,C,D and H could decrease LDH expression(P<0.05 or P<0.01).Phenolic compound B,C,D and H could decrease LDH expression(P<0.01).2.Effect of phenolic compounds on inflammatory mediators in the b End.3OGD/Rep injury model.Phenolic compound B,C,D could decrease IL-6 and IL-1? expression(P<0.05 or P<0.01).3.Effect of phenolic compounds on PI3K/AKt signaling pathway by western blot detection.Phenolic compound C: treatment with LY294002,AKt and e NOS protein expression had no influence,but p-AKt/AKt,and p-e NOS/e NOS rate were significantly lower(P<0.05 or P<0.01).Conclusion:1.Phenolic compounds B,C,D,and H extracted from Gastrodia elata Blume.could elevate TJ protein(occludin and claudin-5)expression and cell survival rate,promote cells release NO,and decrease LDH expression to resist OGD/Rep injury.2.Phenolic compound C extracted from Gastrodia elata Blume.resist OGD/Rep injury by activation of PI3K/AKt signal pathway.