Effects Of Kudiezi Injection On The Expression Of NF-κB In Cerebral Microvascular Endothelial Cells In Ischemia-reperfusion Injury

It has been shown in the clinical practice,that in the acute phase of stroke,patients often exhibit obvious heat and stasis,in addition to the main symptoms of stroke,such as fever,flushing,abdominal distension,short red urine,dry knot stool,crimson or dark purple tongue,dry yellow fur and so on.Even serious ones can develop into high fever,irritability or coma.The heat-and-blood-stasis syndrome characterization is obvious.The heat is mainly caused by liver and kidney deficiency,with accumulated internal injury and abnormal Qi activity,which bring the endogenous pathogenic heat.Qi and blood stagnate in the brain and the pathological products accumulate in the body,which also leads to the birth of heat.The heat and stasis cement with each other,then harm the cerebral collaterals and functions.The key of the acute stroke treatment is clearing away heat,relieveing the toxin and activating blood circulation.Kudiezi Injection,one kind of modern Chinese medicine,whose main components are adenosine and flavonoids,has the effect of clearing away heat and relieveing the toxin,cooling and activating blood,discharging pus and relieving the pain.It has been widely used in the clinical treatment of acute stroke,significantly reducing the neural function defect of patients and improving the ability of daily living.Our team has found,in the preliminary animal experiments,that Kudiezi Injection can inhibit inflammatory reaction after cerebral infarction in rats.But at the cellular level,the anti-inflammatory mechanism in brain microvascular endothelial cells still needs to be further studied.Nuclear factor-kappa B(NF-κB)is an important inflammatory transcription factor in cerebral ischemia reperfusion.Once activated,it can combine with DNA sites,and mediates the expression of genes related to inflammation,resulting the waterfall-like inflammatory cascade.It is considered to be the"switc" of a series of inflammatory reaction after cerebral injury.This experiment used Kudiezi Injection as the research object and used rat brain microvascular endothelial cells as the carrier,and established a cell model of oxygen-glucose deprivation/reoxygenation(OGD/R)to mimic ischemia reperfusion injury.At the cellular level,we used the key transcription factor——NF-kappaB as a breakthrough point,studying the regulation mechanism of Kudiezi Injection on ischemia reperfusion injury of brain micro-vascular endothelial cells.[Objective]We aim to establish the oxygen-glucose deprivation/reoxygenation(OGD/R)cell injury model and explore the dynamic change of cell viability of cerebral microvascular endothelial cell under the ischemia and reperfusion injury,and study the effect of Kudiezi Injection on the expression of NF-kappa B in brain micro-vascular endothelial cells induced by oxygen-glucose deprivation/reoxygenation,and try to explain the possible inflammatory mechanism of "heat-clearing-and-blood-activating method" in treating stroke stasis-heat syndrome.[Methods]Rat brain micro-vascular endothelial cells were cultured in vitro.The cell injury model was established by oxygen-glucose deprivation/reoxygenation.First,the cells were cultured for 6 hours in oxygen-and-glucose-deprivated environment by injecting 95%N2 and 5%CO2 gas mixture and replacing culture medium with Krebs-Ringer Bicarbonate buffer which contained no glucose.Then,the cells were moved to normal culture condition with 95%air and 5%CO2 and normal culture medium,which was the procedure of reoxygenation.There were three groups:control group,OGD/R model group and Kudiezi Injection group.For Kudiezi Injection group,different concentrations of Kudiezi Injection were applied to cells from the oxygen-glucose deprivation stage.First,MTS method was used to measure the cell viability,screening the best time point and medicine concentration.After 6 hours’ oxygenglucose deprivation,cells were reoxygenated for 0 h,3 h,6 h,12 h,24 h,48 h.MTS was used to measure the cell viability,and thus the best time point was selected.From the oxygenglucose deprivation stage,2%,4%,8%Kudiezi Injection were applied to model cells and cell viability was measured by MTS method to screen the best medicine concentration.The expression of NF-kappa B were observed by immunofluorescence staining and tested by Western blot in total protein,cytoplasmic protein and nuclear protein seperately.[Results]1.Compared with control group,the cell viability of groups of each time point of OGD 6 h/R decreased significantly(P<0.01).With reoxygenation time prolonging,cell viability first decreased and then increased,and reached the lowest when OGD 6 h/R 3 h.At this time point,the most serious cell damage acurred.So the model was determined to be OGD 6 h/R 3 h.2.Compared with OGD 6 h/R 3 h model group,cell viability of Kudiezi Injection groups(KDZ 2%,4%,8%)were significantly increased(P<0.01,P<0.05).Among them,cell viability of KDZ 4%was the highest.3.Immunofluorescence staining to observe the expression of NF-kappa B:Compared with control group,the expression of NF-kappa B of OGD 6 h/R 3 h group significantly increased(P<0.01).Compared with OGD 6 h/R 3 h group,the expression of NF-kappa B of KDZ 4%group decreased significantly(P<0.01).4.Western blot to test NF-kappa B protein expression:(1)In total protein:Compared with control group,NF-kappa B protein expression of OGD 6 h/R 3 h group increased(P<0.05).Compared with OGD 6 h/R 3 h group,the expression of NF-kappa B of KDZ 4%group decreased(P<0.05).(2)In cytoplasmic protein:There was no significant difference of NF-kappa B protein expression among control group,OGD 6 h/R 3 h group and KDZ 4%group(P>0.05).(3)In nuclear protein:Compared with control group,NF-kappa B protein expression of OGD 6 h/R 3 h group significantly increased(P<0.01).Compared with OGD 6 h/R 3 h group,the expression of NF-kappa B of KDZ 4%group significantly decreased(P<0.01).[Conclusion]1.The viability of rat brain micro-vascular endothelial cell under ischemia and reperfusion injury changed dynamically.With the reperfusion time prolonging,cell viability decreased first and then increased,and the bottom was at the OGD 6 h/R3 h time point when abnomal high expression of NF-kappa B was observed.2.Kudiezi Injection can down-regulate the abnormal high expression of NF-kappa B in rat brain micro-vascular endothelial cells under ischemic reperfusion injury,and to a certain extent,can inhibit the activation of NF-kappa B,reducing its transfer into nuclei.3.The mechanism of Kudiezi Injection treating stroke of heat and stasis syndrome by clearing away heat and activating blood circulation may be related to its regulation of inflammation in cerebral ischemia and reperfusion injury.