| Peripheral cells are one of the important components of neurovascular units.Under physiological conditions,pericytes have multiple functions such as promoting angiogenesis,maintaining brain capillary stability,and regulating cerebral microcirculation blood flow.After cerebral ischemia and reperfusion,the ischemic cascade reaction occurs in the brain tissue of the ischemic site,and the pericytes are activated to contract,compressing the capillaries in which they are located,causing microcirculation disorders,resulting in the "no reflow" phenomenon.Based on the changes in the role of peripheral blood cells in regulating capillary blood flow after ischemia-reperfusion,this study explores the therapeutic effect and mechanism of Qingkailing Injection on cerebral infarction microcirculation disorders using a mouse global cerebral ischemia-reperfusion model.the study.Objective(1)Demonstrate the protective effect of Qingkailing injection on the cerebral ischemia-reperfusion model mice.(2)To explore the effect of Qingkailing injection on the morphological and functional intervention of pericytes after cerebral ischemia-reperfusion and its mechanism of action.Method(1)Clean male C57BL/6 mice,weighing 20 to 25 g,were randomly divided into four groups.They were:sham operation group,model group,Qingkailing group,and edaravone group.The model group,Qingkailing group,and edaravone group mice were used to prepare a model of global cerebral ischemia-reperfusion.The sham operation group only isolated bilateral carotid arteries without vascular clamping.The four groups were administered at 0,4,12,and 24 hours after cerebral ischemia-reperfusion.Qingkailing group was intraperitoneally injected with Qingkailing injection(0.1ml/10g)diluted with double normal saline,and edaravone group was intraperitoneally injected with edaravone injection diluted with three times normal saline(0.1ml/10g),the sham operation group and the model group were injected with the same volume of normal saline at the same time.Twenty-four hours after modeling,the neurological scores of the four groups of mice were scored;the pathological changes in the brain tissue of the mice in each group were observed by HE staining and Nissl staining.To observe the protective effect of Qingkailing injection on the brain group of global cerebral ischemia model mice.(2)Clean male C57BL/6 mice,weighing 20-25 g,were randomly divided into four groups.They were:sham operation group,model group,Qingkailing group,and edaravone group.The model group.Qingkailing group,and edaravone group mice were used to prepare a model of global cerebral ischemia-reperfusion.The sham operation group only isolated bilateral carotid arteries without vascular clamping.The four groups were administered at 0,4,12,and 24 hours after cerebral ischemia-reperfusion.Qingkailing group was intraperitoneally injected with Qingkailing injection(0.1ml/10g)diluted with double normal saline,and edaravone group was intraperitoneally injected with edaravone injection diluted with three times normal saline(0.1ml/10g).the sham operation group and the model group were injected with the same volume of normal saline at the same time.Immunofluorescence staining was used to observe the changes of peripheral cell morphology in the ischemic region of brain tissue of mice in each group;Western-blot and ELISA methods were used to detect the expression of peripheral cell markers α-SMA and PDGFR-β in the brain tissue of mice in each group The change.To observe the effect of Qingkailing injection on morphology and function of pericytes after ischemia-reperfusion.(3)Clean male C57BL/6 mice,weighing 20-25 g,were randomly divided into four groups.They were:sham operation group,model group,Qingkailing group,and edaravone group.The model group,Qingkailing group,and edaravone group mice were used to prepare a model of global cerebral ischemia-reperfusion.The sham operation group only isolated bilateral carotid arteries without vascular clamping.The four groups were administered at 0,4,12,and 24 hours after cerebral ischemia-reperfusion.Qingkailing group was intraperitoneally injected with Qingkailing injection(0.1 ml/10g)diluted with double normal saline,and edaravone group was intraperitoneally injected with edaravone injection diluted with three times normal saline(0.1ml/10g),the sham operation group and the model group were injected with the same volume of normal saline at the same time.Western-blot method and ELISA method were used to detect the expression of pathway protein in RhoA/ROCK pathway in the brain tissue of mice in each group,and the effect of Qingkailing injection on RhoA/ROCK pathway after ischemia-reperfusion was observed.Result(1)Nerve function score:There was no neurological injury in the sham-operated mouse group.The spontaneous activities of the model group mice were reduced,their consciousness was significantly lower,their pain and sensation were weakened,their limb strength was weakened,their motor coordination and balance ability were deteriorated,and their scores were significantly lower than those of the sham operation group(P<0.001);Compared with the model group,the mice in the Qingkailing group had more spontaneous activities and better consciousness.The pain sensation,physical strength,motor coordination,and balance ability were better than the model group,and their scores were significantly higher than the model group(P<0.001).(2)HE staining:The brain tissue of the sham operation group was uniformly stained with HE,the tissue structure was complete,the cytoplasm was pale pink,the nucleus was dark blue,the nucleoli was clear,the hippocampus was neat and round,and the neurons were full.The brain tissue of the model group showed ischemic damage,a large number of vertebral body cells in the hippocampus hardened and atrophied,neurons atrophied and died,and capillary endothelial cells became enlarged.In the Qingkailing group,the brain tissue showed a trend of recovery.No hippocampal atrophy was observed in the hippocampus,and neuronal necrosis was mild.In the edaravone group,neuron necrosis and endothelial cell swelling were occasionally seen,and the necrotic recovery area was seen.(3)Nissl staining:The Nissl bodies were full and abundant in the sham operation group.Nissl bodies were absent in the model group,and the neuronal cell axis showed vacuole-like changes.The Nissl bodies in Qingkailing group are relatively full.The existence and extinction of the Nidella bodies in the edaravone group.(4)Double immunofluorescence staining:Pericytes and endothelial cells were co-localized outside the capillaries in the sham operation group,and the vascular structure was normal.Compared with the sham operation group,the peripheral cells of the model group contracted,the blood vessels were severely compressed and deformed,and the blood vessel lumen was locked.The edaravone group was similar to the Qingkailing group.Compared with the sham operation group,the pericytes contracted slightly,which caused a certain amount of compression on the blood vessels,but compared with the model group,the vascular lumen was not severely deformed.(5)Western-blot method to detect changes in expression of pericyte markers:Peripheral markers α-SMA and PDGFR-β were significantly increased in the model group(P<0.001).However,the expressions of peripheral cell markers α-SMA(P<0.001)and PDGFR-β(P<0.01)in the Qingkailing and Edaravone groups were significantly reduced.It was proved that Qingkailing injection can effectively inhibit pericyte activation.(6)ELISA method was used to detect the expression changes of pericyte marker protein:the expression of α-SMA and PDGFR-β was the lowest in the sham operation group.Compared with the sham operation group,the expression levels of α-SMA and PDGFR-β in the model group were significantly increased(P<0.001).Compared with the model group,the expression levels of α-SMA(P<0.001)and PDGFR-β(P<0.01)in the Qingkailing group were significantly reduced;α-SMA(P<0.001)and PDGFR-β(P<0.001)expression was significantly reduced.It was proved that Qingkailing injection can effectively inhibit pericyte activation.(7)Western-blot method was used to detect the changes in expression of RhoA/ROCK pathway proteins:the expressions of pathway proteins RhoA.ROCK,and MLC-2 in the sham group were the lowest.Compared with the sham operation group,the expression of RhoA,ROCK and MLC-2 in the model group increased significantly(P<0.001).Compared with the model group,the expression levels of RhoA(P<0.001),ROCK(P<0.001),and MLC-2(P<0.05)in the Qingkailing group were significantly reduced.Compared with the model group,the expressions of RhoA(P<0.001),ROCK(P<0.001),and MLC-2(P<0.01)in the edaravone group were significantly reduced.This indicates that Qingkailing injection can effectively inhibit the activation of this pathway.(8)ELISA method was used to detect the expression changes of RhoA/ROCK pathway proteins:the expression levels of pathway proteins RhoA,ROCK,and MLC-2 were the lowest in the sham operation group.Compared with the sham operation group,the expression of RhoA,ROCK and MLC-2 in the model group increased significantly(P<0.001).Compared with the model group,the expression levels of RhoA(P<0.01),ROCK(P<0.001),and MLC-2(P<0.001)in the Qingkailing group were significantly reduced.Compared with the model group,the expressions of RhoA(P<0.001),ROCK(P<0.001),and MLC-2(P<0.01)in the edaravone group were significantly reduced.This indicates that Qingkailing injection can effectively inhibit the activation of this pathwayConclusion(1)Qingkailing injection can significantly improve the neurological score and improve the pathological changes of ischemic brain tissue in mice with global cerebral ischemia-reperfusion model.With brain tissue and nerve function protection(2)Qingkailing injection can effectively inhibit the activation of RhoA/ROCK pathway and pericytes after cerebral ischemia and reperfusion,reduce the pressure of pericytes on cerebral capillaries,and improve the microcirculation disorder after cerebral ischemia and reperfusion. |
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