Protect Effect And Mechanism Of Salvianolic Acids On The Blood-brain Barrier Permeability On Mice With Acute Focal Cerebral Ischemia Injury.

Objective: Stroke is one of the leading causes of death and disability worldwide, with the most frequent prevalence of ischemic stroke. Brain edema is a leading cause of death after stroke. Vasogenic edema is caused by uncontrolled fluid leakage from the blood to the brain parenchyma through a weakened blood–brain barrier(BBB) and contributes to an actual net volume increase of the brain,which often leads to death. Oxidative stress produces BBB damage and leads to vasogenic edema,what is well-proven pathogenic mechanisms.The aggression of reactive oxygen species(ROS) after brain ischemia contributes to BBB disruption by attacking directly or triggerring molecular pathways related to the dysfunction of ion transporters in the cell membrane and those related to increased vascular permeability. The development of effective therapeutic strategies,what aim at reducing brain edema,may reduce death and disability from stroke. Thus, drugs protecting the BBB may be a promising management strategy for treatment of ischemic stroke.Our previous studies found that antioxidant drugs, such as polydatin,bicyclol and paeonol could ameliorate the permeability of BBB after cerebral infarction.Salvianolic acids(SAs) is water-soluble polyphenolic antioxidants extracted from traditional Chinese herb Danshen, which is widely used for the prevention and treatment of myocardial ischemia diseases. SAs is mainly composed of salvianolic acid B(SAB),salvianolic acid D,salvianolic acid E, rosmarinic acid and lithospermic acid.Most of them are characteristic of antioxidant effect in many pharmacological actions. SAB has been confirmed of protective effect on cerebral ischemia in animal experiments. This study aims to detect the neuroprotective effect of SAs in mouse permanent middle cerebral artery occlusion model(p MCAO)with SAB as positive control. Neurological deficit, so as infarction volume, brain edema and BBB leakage,is to be evaluated after SAs and SAB intervention. We target to explore the protective effect of SAs and to investigate its possible mechanism.Methods: Male and healthy CD-1 mice were subjected to p MCAO, as described by Longa previously. Experiment 1 was conducted to to detect neuroprotection of SAs in the acute phase of cerebral ischemia.Seventy-two mice were randomly assigned to four groups(18 mice in each group): Sham operated group(Sham+0.9% saline), p MCAO group(p MCAO + 0.9% saline), SAs group(p MCAO + SAs 15mg/kg), SAB group(p MCAO + SAB 10mg/kg). SAs or SAB was administrated through intraperitoneal injection immediate after brain ischemia happened. In the cases of p MCAO and Sham group, equal volume of 0.9% saline was administered in the same manner. Six mice of each group was injected 2% evans blue(EB) solution(4ml/kg) through tail vein. EB leakage was detect by scenery photometer at 24 h after p MCAO.The other 12 mice were conducted to evaluate neurological deficit, to measure brain water content,to analyze infarct size with 2, 3, 5-triphenyltetrazolium chloride(TTC) at 24 h after occlusion.Experiment 2 was to investigate possible mechanisms of SAs on ischemia brain. Six mice of each group were used to examine the oxidative response by testing the activities of superoxide dismutase(SOD) and malondialdehyde(MDA) content in ischemic cerebral hemisphere.Claudin-5,occludin,MMP-9,and AQP-4 were detected by western blot(3 mice of each group).Results:1 SAs reduced neurological deficits.All mice were detected the scores of neurologic deficit on a 6-point scale at 24 h after p MCAO,except those in evans blue testing.Mann–Whitney U test analysis was used to do statistical analysis. Mice in p MCAO group, SAs group and SAB group performed a leftpalsy. Compared with p MCAO group, the scores in SAs group and SAB group were lowered significantly(P<0.05). And there were no significant differences in neurological deficit scores between SAs group and SAB group.2 SAs ameliorated the brain edema.In Sham operated group, brain water content of ipsilateral hemisphere was 79.67%±1.89%. In p MCAO group, the water content increased to 84.42% ±2.87% at 24 h after occlusion. In SAs group and SAB group, the brain water content was reduced significantly compared with p MCAO group(p MCAO vs. SAs,SAB :84.42%±2.87% vs. 82.49%±0.89%, 81.81%±1.68%, P<0.05).However,no statistical difference was observed between these two groups.(n = 6 in each group).3 SAs reduced the infarct volume.There was extensive lesion in both striatum and lateral cortex in p MCAO group. In SAs group and SAB group, the infarct volume were significantly reduced after p MCAO( p MCAO vs. SAs,SAB:54.67%±3.14% vs. 42.88%±2.75%,37.88%±1.29%,P<0.05). No significant difference was found in infarct volume between SAs group and SAB group.4 SAs ameliorated BBB permeability.As expected, an extensive EB leakage was found in mice from the p MCAO groups. The protective effects of SAs was observed by examination of EB content at 24 h.Compared with the p MCAO,significantly lower EB extravasation was observed in SAs group and SAB group(P<0.05). No significant difference was found in EB leakage between SAs group and SAB group.5 SAs improved the ability of resistance to oxidative damage.In this experiment, compared with sham group, total SOD and CU-Zn SOD activity of p MCAO group in ipsilateral hemisphere homogenate were significantly decreased at 24 h after ischemia. Both in SAs group and SAB group,activities of two kinds of SOD were obviously improved,meanwhile MDA content decreased.The difference was statistically significant(P<0.05). And there was no significant difference between SAs group and SAB group.In short,SAs could improve the ability of resistance to oxidative damage in the nervous system by scavenging free radical.The protective effect is equivalent of SAB.6 SAs affected the protein expression of claudin-5,occludin,MMP-9and AQP-4It showed us that the protein expression of claudin-5 and cccludin decreased after 24 hours in p MCAO group,while MMP-9 and AQP-4 protein level increased. In SAs group and SAB group, the protein expression of claudin-5 and cccludin were up-regulated,while expression of MMP-9 and AQP-4 protein were decreased.They all had significant differences compared with p MCAO group(P<0.05). While in SAs group and SAB group, no significant difference was found.Conclusions: Above all, our findings showed that administration of SAs in cerebral ischemia mice could protect brain by ameliorate neurological deficits,brain edema,infarct volumes. Further, SAs could alleviate BBB leakage,protected the brain by down-regulation of MMP-9 and AQP-4 and up-regulation of claudin-5 and cccludin.At the same time,activity of SOD was improved and MDA content was reduced. The protective effect is equivalent of SAB.We deduce that SAs protect the focal ischemia brain by resistance to oxidative damage and then ameliorate the permeability of BBB, alleviate cerebral edema. Thus, SAs may represent a potentially beneficial therapeutic medicine for focal cerebral ischemic stroke.