The Study Of The Protection Mechanism Of Danhong Injection And Its Main Effective Components Combination On Suckling Mouse Brain Microvascular Endothelial Cells Injured By Hypoxic

Objective This research adopts primary cultured brain microvascular endothelial cells(rBMEC)in vitro to mimic the blood-brain barrier model,with Danhong injection and its main effective components:Protocatechuic aldehyde,Danshensu,Salvianolic acid B and Hydroxysafflor yellow A compatibility as the research object,investigate the protection mechanism of that on brain microvascular endothelial cells injured by Hypoxic from the cellular level and molecular leve,further study on the compatibility rule,clarify the traditional Chinese medicine compound compatibility principle and scientific.Methods1.Establishing rBMEC cell model injured by Hypoxic and identification:taking suckling mouse cerebral cortex,adopting enzyme digestion twice,screening twice,gradient centrifugat-ion,rinsing,finally vaccinating to the culture bottle.After cells grow into compact monolayer,conducting immunological identify by Ⅷ factor antibody of rabbit anti rat and Ⅱ antibody.When cells grow into compact monolayer,reentering M199 culture medium without serum,then placeing cells in the anoxic tank,ventilating with 4%CO2,1%O2 and 95%N2 mixed gas,after15 min,continue to culture 4h to establish hypoxic model,then observing by microscope and transmission electron microscope.2.Toxicity experiment to determine each drug concentration:determining non toxic dose of nimodipine,Danhong injection and its main effective components on cells.Selecting appropriate concentration to groups:normal group,hypoxic model,high,middle and low dose group of Danhong injection,the effective components compatibility groups,nimodipine positive control group.3.Observing the oxidative damage effect on cells of Danhong injection and its main effective components:using colorimetry,TBA method,WST-1 method and Folin-phenol to detect the LDH content of supernatant,MDA level,SOD activity and total protein content in the cell,analyzing each group cell oxidative damage degree.4.Observing the inflammatory injury effect on cells of Danhong injection and its main effective components:Detecting cell MMP-9,ICAM-1 mRNA expression level by RT-PCR method to analyze each group cell inflammatory injury degree.5.Observing the apoptosis effect on cells of Danhong injection and its main effective components:adopting flow cytometry to measure the cells early apoptosis rate and cells cycle.Detecting cell P53 mRNA expression level by RT-PCR method to analyze each group cell apoptosis condition.Results1.Primary cells Observation,identification and the effect on cell shape of hypoxic:primary brain microvascular endothelial cells appearing shortshuttle-like or polygon,culturing after 7d,cells covered cultivate hole to form a typical paving stone appearance;Ⅷ factor andⅡ antibody determination,the nucleus of cells appears red fluorescence,and cytoplasmic appears strong green fluorescence;after 4h of Hypoxic condition,adherence cells shrinkage,turning round,falling off,cells volume significantly shrink,contour fuzzy,tight junctions significantly disappear,diastasising with the surrounding cells;appearing the typical phenomenon of cell early apoptosis by Observing with transmission electron microscope,the cell nucleus chromatin pyknosis and the cell nucleus chromatin significantly edge set.2.Toxicity experiment results:with the increase of volume,the impact of promoting cell proliferation enhance,100μL reach a plateau;confirming danhong injection dosage(100、50、25μL/mL),nimodipine dosage(200mg/L),each effective component concentration orthogonal compatibility 9 groups.3.Anti-oxidative damage results:nimodipine and 100,50μL/mL danhong injection significantly inhibit the content increase of LDH in the supernatant fluid,increase intracellular SOD activity,and decrease MDA Ievel;25μL/mL group has no obvious effect;Compatibility of 1,2,3,9 group significantly inhibit the content increase of LDH in the supernatant fluid;Compatibility of 1,7 group obviously enhance intracellular SOD activity;Compatibility of 1,2,9 group significantly reduce MDA leve.4.Anti-inflammatory damage results:nimodipine and 100,50μL/mL danhong injection significantly down-regulation the ICAM-1,MMP-9 mRNA expression of hypoxic cells;25μL/mL group can only down-regulation the MMP-9 mRNA expression;Compatibility of 1,3,6,9 group significantly down-regulation the MMP-9 mRNA expression;Compatibility 1,9 group significantly down-regulation the ICAM-1 mRNA expression.5.Anti-apoptosis damage results:nimodipine and 100,50μL/mL danhong injection significantly down-regulation the P53 mRNA expression to inhibit the rBMEC early apoptosis and G1/S phase block induced by hypoxia;25μL/mL group has no obvious effect;Compatibility of 1,6,9 group significantly inhibit the rBMEC early apoptosis;Compatibility of 1,9 group significantly inhibit theGl/S phase block;Compatibility 1,6,9 group significantly down-regulation theP53 mRNA expression.Conclusion This laboratory successfully cultures primary brain microvascular endothelial cells to use as a study in vitro model of the blood-brain barrier,successfully establishes hypoxia model,the research shows that Danhong injection and its main effective components compatibility have a obvious protection on brain microvascular endothelial cells injured by hypoxic,their mechanism of action maybe:can protect rBMEC cells complete structure and shape,ehance the ability of clearing oxygen free radicals,reduce the degree of lipid peroxidation,reduce oxidative stress damage;down-regulation the P53 mRNA expression to inhibit the rBMEC early apoptosis and G1/S phase block;down-regulation the ICAM-1 mRNA expression to induce adhesion,promote endothelial cell metastasis,reduce endothelial cell activity;down-regulation the MMP-9 mRNA expression to promote angiogenesis and so on.