Salvia Miltiorrhiza Depside Effects On The Expression Of TNF-α And IL-10 For Focal Cerebral Ischemia Of Rat

Background and PurposeWith the progress and development of society and echnology,People face with work pressure, pollution, genetic factors and many other issues,the occurrence of cerebrovascular disease is also increased.All the time,Cerebrovascular disease have a high incidence, high morbidity and high recurrence rate and a series of characteristics.Ischemic stroke accounted for fifty percent to sixty-five percent of cerebral apoplexy,which affecte people’s health and life seriously, and the physical dysfunction caused by stroke severely restricts the ability of the post-stroke patients resume the Ability of daily life for themselves.The incidence of ischemic stroke involves many aspects. In recent years, the studies on inflammatory response in ischemic cerebrovascular disease is increasing.When the cerebral ischemia attack,the cerebral vessels blood flow reduction or interruption of blood flow, and tissue damage occurs,hemodynamic changes in a short time,appears inflammatory edema and leukocyte exudation, adhesion, and massive release of the inflammatory mediators to follow, and then trigger further inflammatory response, and cause further damage to brain tissue.In the inflammatory reaction,the activated partial blood vessel endothelial cells, macrophages, smooth muscle cells can produce a variety of cytokines,some cytokines can activate the macrophages like tumour necrosis factor-α(TNF-α),some cytokines can regulate lymphocyte activation, differentiation, proliferation like interleukin-10( IL-10) 、 interleukin-2( IL-2).The activated macrophages produce large amounts of TNF-α, interleukin-1(IL-1), interleukin-8(IL-80) and other cytokines, and these cytokines can promote the Increasement of the number of adhesion moleculesand and adhesion ability,and thereby promoting the leukocyte adhesion to vascular endothelium further,block the microcirculation,and produce the secondary injury.TNF-α is a kind of proinflammatory cytokine。Barone FC in rats of ischemic injury odel, injecte a certain amount of TNF- α into the lateral ventricle of ats, the results showed that the volume of cerebral infarction increased significantly,and the extent of brain damage increased significantly.IL-10 is a multi-functional negative regulator in immune response,which mainly produced by activated B cells, macrophages, monocytes,but in the brain it is produced by the microglial cells with immunomodulatory effects. IL-10 is a kind of anti inflammatory cytokine,Which has antagonistic inflammatory mediators and can alleviate the inflammatory reaction.Drugs currently used in the clinic,such as statins and aspirin,which not only can lower blood pressure, anti-platelet aggregation, but also has anti-inflammatory effects.Research on Chinese traditional medicine of traditional Chinese medicine on ischemic cerebrovascular disease are also many. In which salvia miltiorrhiza, panax pseudo-ginseng, ginkgo biloba extract, safflower, have the Characteristics of multi-target, multi-channel, in the treatment of stroke. Salria miltiorrhiza depside is a kind of water-soluble active ingredient in salvia miltiorrhiza,with a variety of phar MCAOlogical activities,Such as anti- platelet aggregation and adhesion, anti- atherosclerotic, scavenging free radicals, protect nerve cells and improve circulation.In the research of salria miltiorrhiza depside on patients with acute cerebral infarction,Wang Zhizhong found salria miltiorrhiza depside can reduce the levels of MCP-1 and IL-10 in patients with acute cerebral infarction. Hao Yanchao observed that salria miltiorrhiza depside can reduce the levels of fibrinogen and C-reactive protein, thereby reducing cerebral blood viscosity and inflammation. In the research of platelet function in patients with cerebral infarction,Gao Hui[5] found salvianolate can reduce platelet aggregation and adhesion.In the research of salria miltiorrhiza depside on patients with cerebral infarction,Shen Yan[6] found salria miltiorrhiza depside ave the function of protecting the vascular endothelial function. Currently, salria miltiorrhiza depside in the study of cerebral vascular disease, mainly focused on the aspect of brain protection, anti-platelet mechanism, improve circulation,the literature about the research in the aspect of inflammatory reaction is rarely.This topic uses the left middle cerebral artery ischemia in rats(Middle cerebral artery occlusion, MCAO) model, using enzyme-linked immunosorbent assay(ELISA) and Western blotting techniques detection methods(Western Blot),Observe,brain edema, neurological function score and the changes of the content of TNF-α and IL-10 in brain injury tissue and serum of the rat acute with salria miltiorrhiza depside,research the inflammation mechanism of salvianolate in ischemic cerebrovascular diseases,in order to confirm the neuroprotective effect in ischemic cerebrovascular disease. Materials and MethodsHealthy adult male SD rats,which provided by the Medicine Laboratory Animal Supply Center of Zhengzhou University,Weighing about 200 ~ 300 g,Were randomly divided into 3 groups, each group of 36 animals,establishment of the left middle cerebral artery occlusion(MCAO) model of cerebral ischemia with reforming Longa method in rat,Set the administration time was 2h, 24 h, 48 h after surgery, intervention group(MCAO+Sal):received alria miltiorrhiza depside 30 mg / kg by intraperitoneal injections when the rats were awake after successful modeling;Model group(MCAO):received he same amount of physiological saline by intraperitoneal injections when the rats were awake after successful modeling;Sham group(Sham): after the rats which surgery without pluging wire in were awake, received the same amount of physiological saline by intraperitoneal injection.Set 2h, 12 h, 24 h, 48 h four time as the time points,three groups of rats were scored immediately after waking behavior in rats using a modified scoring method Longa,a score of 2-4 points included in the experiment, the other removed,decapitate the animals in the group after the score,measure the water content of the rat brain tissue by using wet and dry weight method,measure the Content of TNF-α and IL-10 in serum by enzyme-linked immunosorbent assay(ELISA) and detect the changes of the levels of the TNF-α and IL-10 in cerebral infarction area and surrounding by using Western Blot method. Results 1. Neurobehavioral scoresAll the scores of Sham group were 0. There was no statistically significance between MCAO+Sal group and MCAO group in 2h and 12 h after surgery(P>0.05).Compared with MCAO group, the score of MCAO+Sal group was in decline, and the difference was statistically significant in 24 h and 48 h after surgery(P<0.05). 2. Brain water contentThere was no significant difference on the right side of the brain in three groups at each time point.The brain water content of both MCAO+Sal group and MCAO group was significantly increased(P<0.05).There was no statistically significance between MCAO+Sal group and MCAO group on the left side of the brain in 2h and 12 h after surgery(P>0.05).Compared with MCAO group, the brain water content of MCAO+Sal group was in decline, and the difference was statistically significant in 24 h and 48 h after surgery(P<0.05). 3. Detect the changes of the content of TNF-alpha and IL-10 by Enzyme-linked immunosorbent assay and Western Blot Assay.Comparison of the content of IL-10 :Compared MCAO+ Sal group with MCAO group, at the time of 2h time point, showed no significant difference, at 12 h, 24 h, 48 h time point the results of comparison were statistically significant(P <0.05);compared MCAO+ Sal group with MCAO group,no significant difference in 2h and 12 h time point,there are significant differences in the expression of the content between 24 h and 48 h time point(P<0.05).Comparison of the content of TNF-α:Compared with Sham group, the TNF-α levels of MCAO + Sal group and MCAO group were increased;Compared with MCAO group,there is no significant difference at 2h time point,the content was significantly reduced with statistical significance at 12 h, 24 h, 48 h time point(P<0.05).4. The content of IL-10 had little expression in 2h, 12 h, increased Significantly in 24 h,continue to increase in 48 h, the rate of increase was gentle; the content of TNF-α in 12 h was higher than the other three time points, and decreased after 12 h. ConclusionsSalria miltiorrhiza depside can reduce Neurobehavioral scores, cerebral edema,suggesting Salria miltiorrhiza depside has neuroprotective effect on ischemic cerebrovascular disease,Its protective mechanism may relate to exert antiinflammatory effects by increasing the content of IL-10 and reducing the content TNF-α in ischemic brain tissue.