| 1.Study objectiveTo investigate the effect of Buyanghuanwu Decoction(BHD)preconditioning on platelet activation and platelet proteins Akt Ser473,Akt Thr308 and PDKl in acute cerebral ischemia reperfusion rats,which is to explore the relationship between the mechanism of BHD anti-platelet activation and PI3K/Akt signaling pathway.2.Study methods84 male SD rats were randomly divided into 7 groups,BHD-A group,BHD-B group,BHD-C group(29.69,14.84,7.42g/kg),clopidogrel(7.81×10-3 g/kg)group(CG),acute cerebral ischemia reperfusion model group(MG),sham operation control group(SG),blank control group(BG).Every rat in medication groups was given by gavage with corresponding drug,once a day for 14 days.Rats in other groups were given by gavage with administrated equivalent distilled water.The rat model of occlusion of the middle cerebral artery(MCAO)reperfusion was established with suture-occluded method at day 14.SG only ligatured unilateral carotid artery.After 2 hours of middle cerebral artery occlusion and 6 hours reperfusion,the nervous function deficit scores was evaluated.Then all the rats were executed and extracted samples for later detections.3.Study contents3.1 After all the rats awaked,the nervous function deficit scores was evaluated according to the method of four score distributed by five rating levels.3.2 All the rats were executed and extracted brain tissue.Then the brain tissue would be cut into slices of 2mm and stained with 2%triphenyltetrazolium chloride(TTC).After the brain tissue fixed by 4%paraformaldehyde for 24 hours,each slices would be taken photos.The area of cerebral infarction would be alculated with Image-Pro plus analysis software.The cerebral infarction area percentage of rats = the sum of each section of infarction area/the sum of cross section area*100%.3.3 ELISA was used in the detection of plasma sCD40L and the expression of platelet activation marker CD62P in whole blood studied by flow cytometry(FCM),which is to investigate the effect of BHD inhibit platelet activation.3.4 The key proteins expression level of Akt Ser473?308Akt Thr308?PDK1 in platelet-rich plasma of PI3K/Akt signaling pathway were studied by western bloting,a semi-quantitative detection method.4.Study results4.1 Compare with SG and BG,the nervous function deficit scores and the area of cerebral infarction in MG were significantly increased(P<0.01).Compare with MG,All BHD groups and clopidogrel group decrease the nervous function deficit scores and the area of cerebral infarction(P<0.05).4.2 Plasma sCD40L and platelet CD62P in BG is the lowest(P<0.05 or P<0.01).Compare with SG,sCD40L and platelet CD62P was significantly increased in MG(P<0.05 or P<0.01).Compare with MG,BHD groups and clopidogrel group significantly decreased plasma sCD40L and the expression of platelet CD62P(P<0.05 or P<0.01).4.3 Compare with SG,The phosphorylation level of Akt Ser473,Akt Thr308,PDK1 proteins in MG was significantly improved(P<0.01).However,BHD groups and clopidogrel group were obviously lower than MG(P<0.05 or P<0.01).4.4 The effect of inhibitting platelet activation and regulating key proteins in PI3K/Akt signaling pathway of different concentration of BHD-A,BHD-B,BHD-C groups(29.69,14.84,7.42g/kg)in rats with acute cerebral ischemia reperfusion do not present a obvious dose-effect relationship.5.Conclusion5.1 Buyanghuanwu Decoction preconditioning is not only able to alleviate the nervous function deficit behavior but also reduce the area of cerebral infarction in acute cerebral ischemia reperfusion rats.It may play a role in brain protection.5.2 Buyanghuanwu Decoction is able to decrease the expression of plasma sCD40L and platelet CD62P in acute cerebral ischemia reperfusion rats,which reveals that BHD can inhibit platelet activation.5.3 Buyanghuanwu Decoction is able to decrease the phosphorylation level of proteins Akt Ser473?Akt Thr308?PDK1 in acute cerebral ischemia reperfusion rats,which prompt that BHD can inhibit PI3K/Akt signaling pathway to anti-platelet activation. |
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