Bitter Dish Anoxic Injury Of Vascular Endothelial Cells And The Protective Effects Of Pkc Delta/marcks Pathways Regulating

BackgroundProtein kinase C,referred to as PKC. Marcks (myristoylated alanine-rich C kinase substrate) is the specific substrate protein. Animal experiments and human clinical trials discovered PKC δ/MARCKS signal transduction pathway and cerebral isehemic injury are closely linked, the increased expression of PKC δ/Marcks pathway in the acute cerebral infarction onset. So whether the in vitro cell experiment hypoxia model have the same conclusion, traditional Chinese medicine of Kudiezi Injection in cells under hypoxia downregulates PKC8/MARCKS pathway activity so as to play a protective effect on hypoxic injury, its mechanism remains to be elucidated.ObjectiveStudy of PKCδ/MARCKS signal transduction pathway in in vitro experiments involved in the hypoxic injury; clarify the Kudiezi Injection to the neuroprotective effect by down-regulating PKC δ/MARCKS signal transduction pathway, provide exact basis for the further study of protective effect of traditional Chinese medicine in hypoxic isehemic injury.Methods1set up CoC12induction of human umbilical vein endothelial cells (HUVEC) anoxic damage model, and apply the PKC specific inhibitor Rottlerin, delta, bitter dish injection to intervene.2after the building of HUVEC and morphology observation, observe its drug intervention morphological changes over time.3application determined by MTT colorimetry to detect different point in time each activity of HUVEC cell model.4fixed protein imprinting method (Western Blot) caused by hypoxia model P-groups of cells after PKC delta, P-Marcks expressed in normal and drug intervention after the quantity.5cell immunohistochemical (IHC) method in the microscopic observation of PKC delta/MARCKS the distribution and expression of proteins in cells.6real-time fluorescent quantitative PCR method to detect PKC delta/MARCKS mRNA expression.7application SPSS17.0statistical software analysis to drug intervention groups if there is a difference compared with the normal and model group. Evaluation of bitter dish injection on cell hypoxia of protection.ResultAfter1cause HUVEC cell hypoxia model, each group had obvious morphological changes. Rottlerin injection group, bitter plate morphology and cell number on an improvement compared with model group, form, quantity is more.2determined by MTT colorimetric method shows the statistical data show that Rottlerin with bitter dish injection has obvious protective effect on cells.(P <0.01), no obvious difference between the two groups (P>0.05).3fixed protein imprinting method (Western Blot) according to P-PKC delta P MARCKS expressed in hypoxia injury model group increased, while Rottlerin, bitter dish injection can inhibit its expression.(P <0.01).4cell immunohistochemical (IHC) method in the microscopic observations show that P-PKC delta P-MARCKS, mainly distributed in the cytoplasm after hypoxia injury, staining obviously deepened, hints of oxygen makes its expression increases, Rottlerin with bitter dish injection can inhibit its activity and expression.Five real-time fluorescent quantitative PCR method showed that hypoxia injury model group P-PKC delta P-MARCKS mRNA expression increased significantly, while Rottlerin with bitter dish group can inhibit its mRNA expression (P <0.05).Conclusion1application of CoC12to establish in vitro cell hypoxia model, hypoxia can cause cell damage and morphological change, application of bitter dish injection and Rottlerin intervention can improve the morphological change is less.2application determined by MTT colorimetric method, fixed protein imprinting method (Western Blot) and cell immunohistochemical (IHC) method indicates CoC12induced hypoxia increases cellular damage, promote P-PKC delta P-Marcks activity and expression. And Rottlerin with bitter dish injection can reduce cell damage, inhibit P-PKC delta P Marcks activity and expression.3bitter dish oxygen injection intervention model cells can be lowered the activity of PKC delta/MARCKS signal transduction pathway, so as to have the protection of cell hypoxia effect, this effect might be brain protection treatment new targets.