Effects Of Salvianolate Lyophilized Injection On The Expression Of VEGF?IL-10 In MCAO Rats

Background and objectiveIschemic stroke is a major cause of mortality and long-term disability worldwide. What's more,it possesses the characteristics of high incidence, high morbidity and high mortality,which poses a serious threat to people's life and health.There are different mechanisms involved in the pathogenesis of stroke,including excitotoxicity, oxidative stress, inflammation, endothlial and microvascular dysfunction,cellular necrosis and apoptosis.In particular, inflammatory response,endothlial and microvascular dysfunction are major mechanisms in cerebral ischemia injury. An ischemic cascade comprised of a series of biochemical events is rapidly initiated within minutes after occlusion of a cerebral vessel.The inflammatry cytokines in acute ischemic stroke are released,such as tumor necrosis factor-?(TNF-?), the interleukin-10(IL-10), the interleukin-1(IL-1), the interleukin-20(IL-20), the interleukin-6(IL-6) and so on.While IL-10 is a important anti-inflammatory and neuroprotective cytokine,and it hleps to fight inflammation by suppressing the production of many proinflammatory molecules including TNF-?, IL-1?, and IL-8.Vascular endothelial growth factor(VEGF) is one of important neurotrophic factors.And it has been demonstrated to be up-regulated after ischemic stroke in animal models.Because of the complex pathophysiological mechanisms,the therapy about ischemic stroke become the focus of clinical work.In recent years,Chinese medicine has taken place great intterest among many researchers.Salvia(named “Danshen” in Chinese),a traditional Chinese medicine, has been widely used in the clinic to treat various microcirculatory disturbance-related diseases, such as cardiovascular disease, cerebrovascular disease, renal dysfunction, liver fibrosis, and diabetic vascular complications.And it has the characteristics of multi-targets, multi-channels in the treatment of cerebral infarction.Salvianolate lyophilized injection(SLI) is the major water-soluble compounds extracted from Danshen,and has many kinds of pharmacological activities.SLI is primarily composed of the Salvianolic acid B,the Salvianolic acid D,the Salvianolic acid E, the Rosmarinic acid and so on.Research shows that Salvianolic acid B can protect the blood-brain barrier against cerebral ischemia-reperfusion injury by reducing extravasation of immunoglobulin.Furthermore, Salvianolic acid B can improve blood flow, reduces oxidative injury, improves blood vessel endothelium function to achieve the aim of treatment acute ischemic stroke. Currently,the literatures about the research of SLI are rarely in the aspect of cerebral vascular protection at acute ischemic stroke.This study chooses the classical animal model: middle cerebral artery occlusion(MCAO).What's more,the successful MCAO rats are injected SLI(30mg/Kg) by intraperitoneal injection at 6h?12h?24h?48h four different time points.Then this study used the Zea-Longa method to access neurological deficits of the MCAO rats at different time points.The cerebral infarction size was assessed by 2,3,5-triphenyl-tetrazolium chloride(TTC) staining in MCAO rats at different time points. The morphological structure of cortical neurons were observed by HE staining. Also,VEGF and IL-10 expression in serum and brain tissues were analyzed with enzyme linked immunosorbent assay(ELISA),western blot and immunohistochemistry staining at different time points after ischemic stroke in MCAO rats. These results demonstrate that SLI has the neuroprotective function and anti-inflammatory in acute ischemic stroke. Materials and MethodsSelected healthy adult male Sprague-Dawley rats(120,weighing 250-280g) were provided by the Laboratory Animal Center of Zhengzhou University.These rats were randomly divided into three groups:the Sham group,the MCAO group,theMCAO+ SLI group.Then according to the ischemic stroke time,each group were divided into four subgroups at 6h?12h?24h?48h four time points.Then the left middle cerebral artery occlusion models were established according to reforming Longa method. The Sham group rats underwent the same procedure except for MCAO.After rats were awake,at the different time points,the MCAO+SLI group rats received the salvianolate lyophilized injection(30mg/Kg) by intraperitoneal injections(ip.).While the Sham group and the MCAO group rats were given isodose saline intraperitoneally. At 6h?12h?24h?48h four time points,the three group ischemic rats were accessed neurological deficits by Longa's 5-point scale.Rats with a score of 0 or 4 were excluded,a score of 1-3 points included in the experiment. The cerebral infarction size was assessed by TTC staining in three group rats at different time points. The serum level of VEGF?IL-10 were measured by ELISA. The brain tissues expression of VEGF?IL-10 were evaluated by Western blot method and immunohistochemistry staining at different time points.Then the morphological structure of cortical neurons were observed by HE staining at different time points after ischemic stroke. Result 1 Neurological deficits and TTC stainingAt different time points,the neurological deficits of sham group showed 0 score.There was no significantly statistical difference in both MCAOgroup and MCAO+SLI group at 6h?12h(P>0.05).While at 24h?48h,the neurological deficit scores of MCAO+SLI group were significantly lower than in the MCAO group,the difference was statistically significant(P<0.05).After 24 h of cerebral ischemia,theTTC staining results of MCAO group and the MCAO+SLI group rats:the middle cerebral artery territory shown white,while the white area of MCAO+SLI group rats was significantly small compared with the MCAO group,and the right side of the brain was red. The middle cerebral artery territory of the Sham group was red,and the right side of the brain was also red. 2 ELISA and Western blot analysis of the VEGF?IL-10 levels in serum and brain tissuesThe ELISA method result: at different ischemic time points,the serum concentration of VEGF?IL-10 of the Sham group was a little.The serum level of VEGF?IL-10 increased in the MCAO group and the MCAO+SLI group,compared with the sham group(P<0.05) at different time points.And furthermore,the level of VEGF?IL-10 in the MCAO+SLI group was higher than the MCAO group(P<0.05).The Western blot analysis method result:at different ischemic time points, the expressions of VEGF?IL-10 in ischemic brain tissues were a few in the Sham group.The expressions of VEGF?IL-10 in brain tissues raised in the MCAO group and the MCAO+SLI group,started to increase at 6h,reached the highest levels at 24 h and decreased lightly at 48 h.At different time points,the expression of VEGF?IL-10 in the MCAO+SLI group was higher more than the MCAO group(P<0.05). 3 HE staining and Immunohistochemistry StainingThe HE staining result:at different time points,the brain tissue structure of the Sham group was completed,and a majority of neuros had normal morphology,the neuros were arranged densely,clear nuclei and cytoplasm evenly.After cerebral ischemia,the periphery neuros of the infarct were degeneration necrosis,cytoplasm pyknosis and cytoplasm unevenly.However,the number of necrotic neurocytes was more in MCAO group,compared to the MCAO+SLI group at different time points.The immunohistochemistry stsining result:the microscopic observation of the cortex ischemia penumbra on the left side in rats showed:the expressions of VEGF?IL-10 in ischemic brain tissues of the Sham group were a few, and the positive cells were quite a few. The positive cells of VEGF and IL-10 in brain tissues increased in the MCAO group and the MCAO+SLI group,started to increase at 6h,reached the highest levels at 24 h and decreased lightly at 48 h.At different time points,the positive cells of VEGF?IL-10 in the MCAO+SLI group was higher more than the MCAO group(P<0.05). Conclusions1.Acute cerebral ischemia can effect the expression of VEGF and IL-10 in serum and brain tissue of rats,,6h?12h?24h?48h after cerebral ischemia, the expressions of VEGF and IL-10 displayed the trend of first rise then downward.2.The salvianolate lyophilized injection can up-regulate the VEGF ? IL-10 expression,reduce the number of degeneration necrosis,decrease the size of cerebral infarction and improve the neurological deficits.