| Objective To investigate the regulation of P2Y12 receptor signal transduction pathway and SCF/c-kit signal pathway infocal cerebral ischemia reperfusion ratmodel by Naoluoxintong,a representative of Yiqi Huoxue Tongluo method,and to explore its effects on coagulation,fibrinolysis and angiogenesis in focal cerebral ischemia.Methods The study was divided into two sections.SD rats were randomly divided into sham group,model group,Tongxinluo group and Naoluo Xintong group,control group for arterial separation,only the rest of the modified line plug method is used to establish the middle cerebral artery occlusion reperfusion(MCAO/R)on the right side of brain ischemia rat model(N=24,divided into 3,7,14 d three subgroups,the drug lavage).In section 1,HE staining and dyeing nissl bodies respectively were applied to observe inflammation and cell necrosis,immunofluorescence detection of ischemia lateral frontal and parietal cortex Platelet ADP receptor subunit(P2Y12)protein levels,immunohistochemical staining method to detect ischemia lateral frontal and parietal cortex half dark with Phospholipase C(PLC),Phosphoinositide 3kinase(PI3K),the expression of Tumor Necrosis Factor(TNF-?),Western blot method to detect ischemia lateral frontal and parietal cortex Protein kinase B(AKT)protein levels in the frontal and parietal cortex.In section 2,rat Regional cerebral blood flow(r BCF)was detected by laser doppler,immunohistochemical staining method to detect ischemia lateral frontal and parietal cortex half dark with Vascular Endothelial Growth Factor(VEGF),Angiopoietins-1(Ang-1),the expression of endothelial cell marker(CD34),Western blot method to detect ischemia lateral frontal and parietal cortex Tyrosine kinase growth factor receptor(C-kit),the protein level of Stem cell factor(SCF),SCF gene chip detection kit signaling pathways/C-43 gene.Results In section 1?HE and nissl body staining showed that the method of invigorating qi and activating blood circulation alleviated the inflammation and cell necrosis in the frontal and parietal cortex of rats with cerebral ischemia reperfusion,and the necrotic nissl bodies in the penumbra zone of the ischemic cortex were reduced and the state was improved.?P2Y12 immunofluorescence displayed:Compared with group J,the expression of P2Y12 in group M was significantly increased on day 3,day 7 and day 14(P<0.01).P2Y12 immunofluorescence displayed:Compared with group M,the expression of P2Y12 in group T was significantly decreased on day 3(P<0.05),and on day 7 and 14(P<0.01).The expression of P2Y12 in group q was significantly decreased on day 3,7 and 14(P<0.01).Compared with group T,the expression of P2Y12 in YQ group was not statistically significant(p>0.05).Compared with group J,the PLC positive area in group M was significantly increased on day 3,day 7 and day 14(P<0.01).?Immunohistochemical results showed that:(1)PLC: Compared with the M group,the PLC cortical positive area of the other groups was significantly reduced on day 3 and day 7(P<0.01),while that of the YQ group was significantly reduced on day 14(P<0.01).Compared with group T,YQ group was significantly reduced on day 3 and day 7(P<0.01),while YQ group was significantly reduced on day 14(P<0.05).And contrast on group J,the average optical density of PLC in group M was decreased on day 3(P<0.05),and the expression on day7 and day 14 was not statistically significant(P>0.05).Compared with group M,the average optical density of PLC in other groups showed no statistical significance on day 3,day 7 and day 14(P>0.05).(2)PI3K:Compared with group J,the cortical positive area of PI3 K in group M was significantly increased on day 3,day 7 and day 14(P<0.01).Compared with group M,the cortical positive area of PI3 K in group T was significantly reduced on day 3(P<0.01),and no statistical significance was found on day 7 or 14(P>0.05).The cortical positive area of PI3 K in group YQ was significantly reduced on day 3 or 7(P<0.01),and no statistical significance was found on day 14(P>0.05).Compared with group T,YQ group presented no statistical significance on day 3,day 7 and day 14(P>0.05).Compared with group J,the mean optical density of PI3 K in group M decreased significantly on day 3,day 7 and day 14(P<0.01).Compared with group M,the mean optical density of PI3 K increased on day 3 in group T(P<0.05),but did not change significantly on day 7 and day 14.The mean optical density of PI3 K increased significantly on day 3 and day 7 in group YQ(P<0.01),but did not change significantly on day 14.Compared with group T,YQ increased on day 3 and 7(P<0.05),while no statistical significance was found on day 14(P>0.05).(3)TNF-?:Compared with group J,TNF-? cortical positive area was significantly increased on day 3 and 7(P<0.01),and increased on day 14(P<0.05).TNF-? cortical positive area was significantly decreased at day 3,day 7 and day 14 in T group and YQ group compared with M group(P<0.01).Compared with group T,YQ group presented no statistical significance on day 3,day 7 and day 14(P>0.05).Compared with group J,the mean optical density of TNF-? in group M was significantly decreased on day 3(P<0.01),and no statistical significance was found on day 7 or 14(P>0.05).Compared with group M,the mean optical density of TNF-? in group T was significantly increased on day 3 and day 7(P<0.01),but not statistically significant on day 14(P>0.05),and significantly increased on day 3(P<0.01),but not statistically significant on day 7 and day 14(P >0.05).Compared with group T,YQ group was significantly reduced on day 3and 7(P<0.01),while no statistical significance was found on day 14(P>0.05).?Western blot results revealed: Compared with group J,AKT protein expression in group M was significantly increased on day 3,7 and 14(P<0.01).Compared with M group,AKT and YQ skin group were significantly reduced on day 3,day 7 and day 14(P<0.01).Compared with group T,the day 14 of YQ group was decreased(P<0.05).Experiment 2 ?r CBF Compared with group J,r CBF of group M was significantly reduced at 5min on day 3,day 7 and day 14 after insertion(P<0.01).There was no significant difference among the other groups(p>0.05).r CBF test results show:Compared with group J,r CBF in group M was significantly reduced at on day 3,day 7and day 14 before sampling(P<0.01).Compared with group M,r CBF was significantly increased in group T and YQ on on day 3,day 7 and day 14 before sampling(P<0.01).Compared with group T,YQ group presented no statistical significance(P>0.05).?Immunohistochemical results:(1)VEGF: Compared with group J,the cortical positive area of VEGF in group M was significantly decreased on day 3 and 7(P<0.01),while no statistical significance was found on day 14(P>0.05).Compared with group M,the cortical positive area of VEGF in group T and YQ was significantly increased on day 3and 7(P<0.01),while that in group T was significantly increased on day 14(P<0.05)and that in group YQ was significantly increased on day 14(P<0.01).Compared with group T,YQ group increased in day 3 and day 14(P<0.05),and significantly increased in day 7(P<0.01).Compared with group J,mean optical density of VEGF cortex in group M was significantly increased on day 3 and 7(P<0.01),while no statistical significance was found on day 14(P>0.05).Compared with the M group,the mean optical density of the cortex of the T group and the YQ group was significantly decreased on day 3 and day 7(P<0.01),while no statistical significance was found on day 14(P>0.05).Compared with group T,there was no statistical significance in YQ skin group on day 3,day 7 and day 14(P>0.05).(2)Ang-1 Compared with group J,Ang-1 cortical positive area in group M was significantly decreased on day 3,day 7and day 14(P<0.01).Compared with group M,the Ang-1 cortical positive area in group T and YQ was significantly increased on day 3,day 7 and day 14(P<0.01).Compared with group T,YQ group was significantly increased on day 3 and 7(P<0.01),while no statistical significance was found on day 14(P>0.05).Compared with group J,the Ang-1 cortical mean optical density in group M was significantly increased on day3 and day 7(P<0.01),but not on day 14(P>0.05).Compared with group M,the Ang-1cortical mean optical density in group T was significantly decreased on day 3(P<0.01),and on day 7(P<0.05),but was not statistically significant on day 14(P>0.05);in group YQ,the Ang-1 cortical mean optical density was significantly decreased on day 3 and 7(P<0.01),but was not statistically significant on day 14(P>0.05).Compared with group T,YQ group presented no statistical significance on day 3,day 7 and day 14(P>0.05).(3)CD34: Compared with group J,CD34 cortical positive area in group M was significantly reduced on day 3 and 7(P<0.01),while no statistical significance was found on day 14(P>0.05).Compared with group M,CD34 cortical positive area was significantly increased in group T and YQ on day 3,day 7 and day 14(P<0.01).Compared with group T,YQ was significantly increased on day 3 and day 14(P<0.05),and significantly increased on day 7(P<0.01).Compared with group J,the average optical density of CD34 cortical in group M was significantly increased on day 3(P<0.01),while no statistical significance was found on day 7 or 14(P>0.05).Compared with group M,the average optical density of CD34 cortical in group T and YQ was significantly decreased on day 3(P<0.01),and no statistical significance was found on day 7 or 14(P>0.05).Compared with group T,YQ group presented no statistical significance on 3d,7d and 14d(P>0.05).?Western blot results:(1)c-kit:Compared with group J,c-kit protein expression in group M was significantly decreased on day 3,day 7 and day 14(P<0.01).Compared with group M,c-kit protein expression was significantly increased on day 3,7 and 14 in group T and YQ(P<0.01).Compared with group T,YQ group was significantly increased on day 3,day 7 and day14(P<0.01).(2)SCF: Compared with group J,SCF protein expression in group M was significantly decreased on day 3,7 and 14(P<0.01).Compared with group M,SCF protein expression in group T increased on day 3(P<0.05),and increased significantly on day 7 and 14(P<0.01).SCF protein expression in YQ group was significantly increased on 3d,7d and 14d(P<0.01).Compared with group T,YQ group was significantly increased on day 3,day 7 and day 14(P<0.01).?SCF/c-kit gene chip detection results showed that Mvs J: Cma1 gene expression was significantly different(Fold up-or down-regulation >2).Tvs M: Grb7 gene expression was significantly different(Fold up-or down-regulation >2).Yvs M: gene expressions of Cma1,Grb7,Vav1 and Fes were significantly different(Fold up-or down-regulation >2).Conclusion Naoluo Xintongcould reduce the expression of P2Y12,PLC,PI3 K,TNF-and AKT-related proteins in the P2Y12 signaling pathway,regulate coagulation and fibrinolysis,reduce inflammation and improve cerebral ischemia reperfusion injury.In the acute stage of cerebral ischemia reperfusion,the anti-inflammatory effect of Naoluo Xintongwas better than that of Tongxinluo.In addition,by increasing r CBF in the SCF/ c-kit signaling pathway,Naoluo Xintong enhances the expression of VEGF,ang-1,CD34,c-kit and scf-related proteins,promotes the angiogenesis of ischemic brain tissue,improves the blood perfusion flow in local brain tissue,and promotes the structural and functional recovery of damaged brain tissue. |
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