Efficacy Evaluation And Mechanism Of QSYQ Prescription Via Regulating Platelet-leukocyte Aggregates Against Thrombotic Stroke Injury

Objective:Ischemic stroke accounts for 87%of the incidence of stroke.Intravenous thrombolysis and thrombectomy are the main clinical treatment strategies for restoring blood supply to brain tissue.The research group has previously confirmed the protective effect of QSYQ Formula on ischemic cerebral ischemia/reperfusion injury,but whether QSYQ formula and its components of YQ and HX can partially restore blood supply to brain tissue,thus reducing brain tissue injury remains unclear.QSYQ is composed of astragalus membranaceus,salvia miltiorrhiza,Panax notoginseng and Healthy sesame oil.Among them,YQ component consist Astragalus astragalus,HX component is composed of salvia miltiorrhiza and Panax notoginseng.Based on the previous study on stroke in the laboratory,this study adopted the acute stage of thrombotic stroke model,and took the interaction between immune system and hemostatic system as the entry point to explore the pharmacodynamic effects and key mechanisms of QSYQ formula and its components of YQ and HX in this animal model.In addition,in vitro experiment,we used two methods to verify the in vivo experiment results and discusses the synergy/difference effect of YQ and HX:b End.3（mouse cerebral microvascular endothelial cells）oxygen deprivation of sugar/reoxygenation（OGD/R）model,thrombin induced platelet-leukocyte aggregates（PLAs）model.In order to provide a theoretical basis for the treatment of embolic stroke with QSYQ formula.Methods:1.The acute stage model of cerebral stroke was established by thrombus method,in which the thrombus formed in the capillary was injected into the middle cerebral artery through the internal carotid artery to cause thromboembolic stroke.SD rats were randomly divided into sham operation group,model group,QSYQ group,YQ group and HX group.Drugs or normal saline were given by continuous intragastric administration for 7 days before modeling.After 24 h modeling by thrombus method,the pharmacodynamic effects of QSYQ formula and its components of YQ and HX were evaluated by cerebral infarction volume,nerve function defect score and peripheral circulating blood smear.2.The key target of formatting PLAs was obtained through literature retrieval,based on the method of thrombosis cerebral apoplexy.Blood samples were used to detect the level of key targets by RT-PCR.We used immunofluorescence（IF）and western blot（WB）to investigate the key target protein levels of platelet-leukocyte aggregate in thrombosis stroke model.By the carotid artery thrombosis model of Fe Cl₃,the effects of QSYQ formula,YQ and HX components on inhibiting thrombosis and recanalizing thrombosis were investigated.Circulating leukocyte were ablated by injection of anti-Ly6G to investigate whether the antithrombotic effect of QSYQ Formula was partially mediated by platelet-leukocyte interaction.ICR mice were randomly divided into sham operation group,model group,anti-Ly6G group,QSYQ group,YQ group and HX group.Drugs or normal saline were given by continuous intragastric administration for 7 days before modeling.The anti-Ly6G group was intraperitoneally injected with anti-Ly6G antibody（10mg/kg）one day before modeling,while the other groups were intraperitoneally injected with Ig G2a homologous as control（10mg/kg）.Whether QSYQ formula and its components of YQ and HX can inhibit thrombosis and recanalize blood vessels was investigated by dynamic carotid blood flow,vascular occlusion time,final blood flow,number of circulating leukocyte and proportion of circulating activated platelets.3.In vitro,the 50%inhibitory concentration（IC50）of QSYQ on b End.3 cells was determined by CCK8.BEnd.3 cells were deprived of oxygen and sugar for 6 hours and reoxygenated for 24 hours.Drugs were added at the time of reoxygenation.Fresh extracted and stained platelets or leukocyte were added after reoxygenation,and the cell adhesion rate was detected by high content screening system（Operetta）after incubation.In another vitro experiment,platelets and leukocyte were co-incubated for 15 min in the presence of drugs,then co-incubated 20 min in the presence of agonist thrombin and antibody dye.Flow cytometry was used to detect the formation of PLAs and the expression of key proteins.Results:1.Cerebral infarction volume,neurological deficit score and peripheral blood PLAs were significantly increased 24 h after thrombosis modeling.QSYQ,YQ and HX groups can significantly reduce the cerebral infarction volume and neurological deficit score of thrombus rats,and the two components have synergistic effect.QSYQ could significantly reduce the number of PLAs,while both YQ and HX groups tended to decrease the number of PLAs.2.RT-PCR results showed that QSYQ,YQ and HX groups could significantly reduce the m RNA expression levels of key target genes for PLAs formation,such as MAC-1,PSGL-1,TLR2 and CD40.IF results of brain tissue showed that QSYQ group could significantly reduce the expression of CD62P and PSGL-1,and WB results further proved the regulation of PSGL-1.The regulation of PSGL-1 in YQ group was more prominent than that of HX component,while the regulation of CD62P in HX group was better than that of YQ component,indicating that YQ component and HX component played different roles in brain protection through PLAs.In the Fe Cl₃ carotid artery thrombosis model,QSYQ,YQ and HX groups can significantly prolong the time of vascular occlusion,improve the final blood flow,and significantly reduce the number of immune cells and platelet activation in peripheral blood,without affecting the hemostatic function.Anti-Ly6G group effectively ablated circulating leukocyte in vivo,significantly prolonged vascular occlusion time,improved final blood flow,and significantly activated platelet state.3.CCK8 results showed that 0.1mg/m L and 0.25mg/m L QSYQ did not inhibit the activity of cerebral endothelial microvascular cells.When the dose of QSYQ was increased to0.5 mg/m L or above,the cell viability was significantly inhibited,and the half inhibitory concentration was 0.6783 mg/m L.The results of OGD/R model cell adhesion experiment showed that QSYQ formula could significantly reduce the adhesion of platelet and leukocyte to endothelial cells.The effect of YQ and HX components was weaker than that of the whole formula,and the two components had a synergistic effect.Flow cytometry results showed that QSYQ formula and its components of YQ and HX could significantly reduce the formation of PLAs.QSYQ group could significantly reduce the protein levels of CD62P and PSGL-1 in PLAs.YQ group had a more prominent contribution to PSGL-1 regulation,which further verified the results of in vivo experiments.Conclusion:1.In this experiment,QSYQ play a protective role in cerebral infarction rats with thrombotic stroke,effectively reducing the volume of cerebral infarction and neurological function score.CD62P/PSGL-1 mediated PLAs formation was revealed and verified as the key mechanism of QSYQ in reducing brain injury in acute stage of thrombotic stroke rats.2.The potential synergistic/differential effect between YQ group and HX group was revealed.The YQ component of Astragalus membranaceus focuses on the regulation of leukocyte related receptors in the immune system,while the HX component of Salvia miltiorrhiza notoginseng focuses on the regulation of platelet function in the hemostatic system.