| Tong Luo Jiu Nao injection (TLJN) consists of Geniposide and Panax notoginseng saponins (PNS).It is a kind of compound new drug for acute ischemic stroke,based on the theory of "toxic brain meridian damage". In stroke treatment, neither gardenoside nor PNS can compare with TLJN’s function, which plays the superiority of traditional Chinese compound prescriptions. Both in laboratory and in clinical trials, TLJN plays a key role of ischemic stroke treatment. Relative pharmacological study reveal the injection can inhibit the occurrence and development of cerebral ischemia through various channels.TLJN has obvious protective effect on brain microvascular endothelial cells(BMECs).Previous study to screen out the drug target gene by gene chip technology. We found that The expression of GluR3 mRNA in oxygen-glucose-deprived (OGD)-induced BMECs was down-regulated obviously by TLJN. In the traditional research,glutamate receptors involves the brain damage in neurons and glial cells.There were few reports about glutamate receptors’ existence and functions in BMECs. So according to the gene chip, exploring GluR3’s existence and functions is this research innovation points.Therefore this experiment on the basis of the previous studies,use Geniposide and PNS as control group, observe the influence of TLJN to the expression of GluR3 mRNA,DNA and downstream related protein in OGD-induced BMECs.TO reveal TLJN molecular targets of vascular protection,also interpret the advantages of compound prescriptions.Objective:To study the effects of TLJN and its active components on the expression and function of Glutamate receptor 3 (GluR3) in mimic ischemic BMECs. Also test the downstream protein expression related to GluR3, to reveal the molecular mechanism of TLJN’s protective function in strock.Mothods:1. SD rats were used to acquire,separate,culture,breed original cells by mature BMECs culture method. The third generation of BMECs were used and mimic cerebral ischemia in vitro was established by OGD method.2. TLJN consists of the main composition of gardenia and PNS. Therefore Geniposide and PNS as control groups in this study.The cells were divided into 5 groups.normal, model, Geniposide, PNS, TLJN. Each group was treat with OGD except normal. Three drug groups before the OGD and affter OGD with corresponding drugs, TLJN concentration is 4μl/ml, gardenoside final concentration is 33.2μg/ml; PNS concentration is 22μg/ml (gardenoside and PNS final concentration same with TLJN).After processing, use of PCR and Western blotting method to detect GluR3 mRNA and protein expression.3. Add GYKI52466 (GluR3-blocker) group on the basis of the above five groups.Using Western blotting methods to detect all groups’CD147 and MMP-9 expression of BMECs.To explore its terminal targets.4. Using Western blotting method to detect the expression of occludin in all groups.5. To verify the GluR3’s existence and positioning in BMECs, SD rats were selected to make middle cerebral artery occlusion model(MCAO).Using this brains to detect the position of GluR3 and occludin by immunohistochemical method.6. Using SPSS20.0 for statistical analysis,compares the single factor analysis of variance between groups, P<0.05 think the difference was statistically significant.Results:1. Based on the theory of the central dogma,the result of PCR and Western blotting reveal that GluR3 express in the BMECs. Immunohistochemical results verify its positioning.2. The effects of TLJN Injection and its components on the expression of GluR3 in mimic ischemic BECMs.The result of PCR and Western blotting show that the expression of GluR3 increase observably after OGD. Each treatment group compared with model group,could cut the expression of GluR3 in different degree.The effect of TLJN is better than others.3. The effects of TLJN Injection and its components on the expression of CD147 and MMP-9 in mimic ischemic BECMs.The result of Western blotting reveals that the expression of CD 147 and MMP-9 increase observably after OGD. Each treatment drugs could reduce those.TLJN’s result is better than others’. GluR3 blocker will cut the expression of CD 147 and MMP-9 in a certain extent.4. The effects of TLJN Injection and its components on the expression of occludin in mimic ischemic BECMs.Western blotting reveal that occludin expression have obvious increase after OGD. Each drug could cut down the two proteins in different degree,also the effect ofTLJN is better. The immunohistochemical results of animal experiments reveal the differences of occludin expression between each groups.Conclusion:1. This study found that GluR3 express in BECMs of SD rat. GluR3 expression level significantly increased after the OGD model.Some groups with GluR3 blockers GYKI52466 reflect that GluR3 raised CD147,with MMP-9 increased. Promote degradation of occluding. Explain GluR3 may participate in the ischemic brain injury.2. TLJN, Geniposide,PNS could cut down GluR3 expression effectively in OGD model. Also cut down CD 147 and MMP-9. Reduce the degradation of the BBB related proteins occluding, llustrates the drugs could reduce the expression of GluR3. GluR3 play the key role of protecting BBB after ischemic stroke.3. TLJN, Geniposide,PNS could regulate the expression of CD147n MMP-9、occludin obviously. TLJN had more optimized effect than its active components, which showed the therapeutic advantages of Chinese medicine compatibility. |
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