The Codonopsis Composition Of Rat Brain Microvascular Endothelial Cells During Hypoxia / Hypoglycemic Injury Protective Effect And Mechanism

1. objectiveThe brain is the most impressionable organ for oxygen lack. Tissue injury from ischemia causes significant damage to brain tissue and produces serious influence on its function .Brain microvascular endothelial cell(BMEC) is a primary target of ischemic insults in the brain. Brain microvascular endothelial cell injury following ischemia may exacerbate brain damage and contribute to the breakdown of blood-brain-barrier (BBB) leading to increased vascular permeability and vasogenic brain edema. For the past few years,"nerve-blood-vessel unit"was proposed by many researchers, Which they think should be considered as a whole factor."Nerve-blood-vessel unit"consists of neurocytes,endothelial cells,astrocyte and much extracellular matrix aroud brain tissue ,which sustain the integrality of brain . Since past years,we payed more attention to the neurocytes in the field of ischemic cerebrovascular disease ,other than endothelial cells. But many studies show that if we do some effective measures to protect endothelial cells instead of activating neurotization only,maybe,we could get better clinical effect . Oxygen-glucose deprivation (OGD) presents an in vitro model of ischemic insult and BMEC apoptosis has been shown in entailing OGD. However, the characteristics of cerebral endothelial cell death after ischemia are to be elucidated.Apoplexy is the chief one of the fulminating diseases in traditional Chinese medicine (TCM) and one of the three kinds of main diseases in modern medicine, but couldn't reach perfect effect by modern therapeutics as well.Treatment of ischemic apoplexy at the acute stage has important and direct influence on prognosis and the recovery of deficit of nevere function ,therefore ,it has caught the attention of all the medical field .Doctors of TCM always pay attention to removing phlegm ,dispersing blood stasis ,removing phlegm and biood stasis together ,purging ,clean away heat and detoxifying because of the different opinion on the pathogenic factors playing the key role in the disease .All the treatment laid stress on eliminateing pathogenic factors and neglected strengthening the body resistance ,so professors Sun Su Lun studied classical document ,contemporary progress of TCM and Western medicine ,and then put forward a brand-new standpoint of pathogenesis and a treatment of ischemic apoplexy at the acute stage according to clinical experiences under the guidance of the concept of the organism as a whole together with diagnosis and treatment based on an overall analysis of signs and symptoms. This research project advanced the therapeutic principle of"supporting health energy, strengthening body resistance and protecting brain"for the treatment in acute stage of apoplexic stroke in connection with that of "eliminating pathogenic factors" widely accepted in TCM to complete into integrated therapeutic system. In acute stage of apoplexic stroke, the therapeutic principle of"supporting health energy, strengthening body resistance and protecting brain"accords with Zhong Jing's therapeutic point"prevent well before deficiency and once a disease appears ,it should be diagnosed and treated as early as possible so as to arrest its progress.",which has important meaning in the second /third levels of prevention on ischemic apoplexy.The therapeutic principle of"supporting health energy, strengthening body resistance and protecting brain"doesn't mean cure slowly ,Conversely its key therapeutic point is to reinforce urgently and may have immediate effect.Clinical and empirical studies confirm the therapy of Codonopsis pilosula(Franch.)Nannf. in ischemia damage,for example,our laboratory ever do some researchs to confirm the strikingtherapy of three components of Codonopsis pilosula(Franch.)Nannf(total saponin of Radix Codonopsis,saponin L-1 of Radix Codonopsis,saponin SFE of Radix Codonopsis)on rat cranial nerves and astrocytes in ischemic damage . So we replicate oxygen-glucose deprivation model with BMEC of rat to simulate hypoxic/ischemic insults in order to study various enzyme,chemical transmitter,gave off by suffered BMEC,death,apptosis and Caspase-3. The study provide a new idea and a new way for studying the mechanism of Codonopsis pilosula(Franch.)Nannf. treating brain injury caused by ischemic insults2. method2.1 Primary extracting and culturing cerebral microvascular endothelial cell of rats, then applying morphology and the presence of factorⅧrelative antigen to identify cells .2.2 Deciding the asepsis final concentration, protection final concentration and gradient protection final concentration of the three components of Codonopsis pilosula(Franch.)Nannf. by MTT.2.3 To investigate protective effects of the three components of Codonopsis pilosula(Franch.)Nannf. on BMEC cultured in oxygen-glucose deprivation circumstance,damaged BMEC were determined by measuring the leaking of LDH(normal biochemistry method) , the contents of NO(nitrate reductase method) and MDA(xanthine oxidase method),and the activity of SOD(thio-barbituric acid method) in the supernatant.2.4 The double stain of Hoechst 33342 and propidium iodide(PI) with fluorescence microscope to detect the necrosis and apoptosis of the BMEC after different concentrations of different drugs on the oxygen-glucose deprivation injury.2.5 Using the immunocytochemical method and techniques of image half- quantitative analysis to detect the mean optical density of Caspase-3 expression in BMEC after different concentrations of different drugs on the oxygen-glucose deprivation injury.2.6 Adopting SPSS10.0 to carry out statistics and variance analysis.3. Result3.1 Some capillary segments can be found by sieving twice , cells from capillary growing monoptychially and mixing together in the course of cells extraction. the correlated antigen ofⅧfactor is positive. All these facts prove that the cells extracted are brain microvascular endothelial cell of rat.3.2 cell survival rate(%):Compared with the model group, the cell survival rate increased significantly in the protective groups of Nimodipine ,the middle and low)concentraion group of total saponin of Radix Codonopsis, the low concentraion group of saponin L-1 of Radix Codonopsis and the low concentraion group of saponin SFE of Radix Codonopsis(P<0.01).3.3 cell dead rate(%)and apoptosis rate (%)by double stain in situ3.3.1 cell dead rate(%): Compared with the model group,the cell dead rate decreased significantly in the protective groups of Nimodipine,the middle and low concentraion group of total saponin of Radix Codonopsis, the low concentraion group of saponin L-1 of Radix Codonopsis and the low concentraion group of saponin SFE of Radix Codonopsis(P<0.01).3.3.2 cell apoptosis rate (%):①Compared with the model group,the cell apoptosis rate decreased significantly in the protective groups of Nimodipine,the middleconcentraion group of total saponin of Radix Codonopsis, the low concentraion group of saponin L-1 of Radix Codonopsis(P<0.01);②Compared with the model group,the cell apoptosis rate decreased in the groups of the middle concentraion group of saponin L-1 of Radix Codonopsis and the low concentraion group of saponin SFE of Radix Codonopsis(P<0.05).3.4 LDH release rate(%):①Compared with the model group, LDH release rate can be inhibited significantly in the protective groups of Nimodipine,the middle and low concentraion group of total saponin of Radix Codonopsis, the low concentraion group of saponin L-1 of Radix Codonopsis and the low concentraion group of saponin SFE of Radix Codonopsis(P<0.01);②Compared with the model group(23.36±2.60), LDH release rate can be inhibited in the groups of the middle concentraion group of saponin L-1 of Radix Codonopsis and the middle concentraion group of saponin SFE of Radix Codonopsis(P<0.05).3.5 NO content(μmol/L)in the supernatant: Compared with the model group,NO content in the supernatant decreased significantly in the protective groups of Nimodipine,the middle and low concentraion group of total saponin of Radix Codonopsis, the low concentraion group of saponin L-1of Radix Codonopsis and the low concentraion group of saponin SFE of Radix Codonopsis(P<0.01).3.6 the activity of SOD(U/mL) and MDA content(nmol/mL)in the supernatant:3.6.1 the activity of SOD(U/mL):①Compared with the model group, the activity of SOD in the supernatant increased significantly in the protective groups of Nimodipine,the middleconcentraion group of total saponin of Radix Codonopsis, the low concentraion group of saponin L-1 of Radix Codonopsis and the low concentraion group of saponin SFE of Radix Codonopsis(P<0.01);②Compared with the model group, the activity of SOD in the supernatant increased in the groups of the low concentraion group of total saponin of Radix Codonopsis, the middle concentraion group of saponin L-1 of Radix Codonopsis and the middle concentraion group of saponin SFE of Radix Codonopsis(P<0.05).3.6.2 MDA content(nmol/mL):①Compared with the model group, MDA content in the supernatant decreased significantly in the protective groups of Nimodipine,the middleconcentraion group of total saponin of Radix Codonopsis, the low concentraion group of saponin L-1 of Radix Codonopsis and the low concentraion group of saponin SFE of Radix Codonopsis(P<0.01);②Compared with the model group, MDA content in the supernatant decreased in the groups of the low)concentraion group of total saponin of Radix Codonopsis, the middle concentraion group of saponin L-1 of Radix Codonopsis and the middle concentraion group of saponin SFE of Radix Codonopsis(P<0.05).3.7 Caspase-3 expression:①Compared with the model group, the mean optical density of Caspase-3 expression decreased significantly in the protective groups of Nimodipine,the middle and low concentraion group of total saponin of Radix Codonopsis, the middle and low concentraion group of saponin L-1 of Radix Codonopsis and the low concentraion group of saponin SFE of Radix Codonopsis(P<0.01);②Compared with the model group, the mean optical density of Caspase-3 expression decreased in the group of the middle concentraion group of saponin SFE of Radix Codonopsis(P<0.05).4. conclusionThe oxygen-glucose deprivation model can imitate ischemia injury fairly good,so it can increase leaking of LDH,NO and MDA content and decrease SOD activity obviously.Meanwhile , the necrosis ,apoptosis and the activity of Caspase-3 of the BMEC on the oxygen-glucose deprivation injury are significantly increased. From my studies ,we can see that the three components of Codonopsis pilosula(Franch.)Nannf. could effectively block the damage mechanisms bove-mentioned, reverse these changes ,thus protecting the endothelial cells and relievingischemia damage . Analyze the experimental result in detail :① the middle and low concentraion group of total saponin of Radix Codonopsis, the low concentraion group of saponin L-1 of Radix Codonopsis and the low concentraion group of saponin SFE of Radix Codonopsis may have a better protection on BMEC on the oxygen-glucose deprivation injury, (vs model group,p<0.01) ;②the middle concentraion group of saponin L-1 of Radix Codonopsis and the middle concentraion group of saponin SFE of Radix Codonopsis may have a protection,but no better than upper groups (vs model group, 0.010.05) ;④we also find that the high concentraion group of saponin SFE of Radix Codonopsis may make a serious damage on the BMEC on the oxygen-glucose deprivation injury(vs model group,p<0.01).