| ObjectiveIschemic stroke is a common disease of the central nervous system.Cerebral ischemiareperfusion injury(CIRI)is due to treatment or the body’s own compensatory mechanisms to achieve reperfusion of the ischemic region,leading to further damage to tissues and organs.T lymphocyte-mediated inflammatory response is an important cause of CIRI,and the IL-33/ST2 axis is closely related to the T lymphocyte immune response.Current clinical treatment strategies for ischemic stroke are limited,and acupuncture has achieved good clinical efficacy in recent years,while exosomes have been shown to exerting a neuroprotective effect to alleviate CIRI by suppressing the inflammatory response.This thesis focuses on the effects of electroacupuncture combined with human induced pluripotent stem cells-derived small extracellular vesicles(iPSC-EVs)on neuromotor function and IL-33/ST2 axis-mediated T-lymphocyte immune inflammatory response in mice with cerebral ischemia-reperfusion injury,providing new sights and experimental basis for the clinical treatment of ischemic stroke.MethodsHealthy male C57BL/6 mice(6-8 weeks old)were randomly divided into 6 groups:sham-operated(Sham)group,model(MCAO)group,sham-acupuncture(Sham Acu)group,electroacupuncture(EA)group,iPSC exosome(iPSC-EVs)group and electroacupuncture combined with iPSC exosome(EA+iPSC-EVs)group.There were 10 mice in each group.The middle cerebral artery occlusion(MCAO)was used to simulate the cerebral ischemia-reperfusion process according to the random number table method.The experiment was conducted in several times,and 64 mice were used in the experiment and 4 died.In the Sham group,only the right common,internal and external carotid arteries were isolated without inserting a wire plug;in the MCAO group,the right middle cerebral artery was occluded with a wire plug for 1 h and reperfused for 72 h.The MCAO model was prepared without any treatment;in the Sham Acu group,after successful modeling,acupuncture was performed at a non-transverse non-acupuncture point(10 mm above the anterior superior iliac spine)without receiving electroacupuncture;in the EA group,acupuncture needles were stuck into the mice at "Baihui" acupoint(GV20,on the midline of the head and the line connecting the apices of both auricles)and left "Zusanli"acupoint(ST36,5 mm distal to the head of the fibula beneath the stifle and 2 mm lateral to the tibial tuberosity)at a depth of 2-3 mm.EA stimulation was carried out for 30 min at 2 Hz(intensity,1 mA)frequency with continuous waves using an electroacupuncture device;iPSC-EVs group mice were injected iPSC exosomes in the tail vein immediately after successful modeling,20ug/each,once a day;EA+iPSC-EVs group performed electroacupuncture intervention and tail vein injection of iPSC exosomes once a day after successful modeling,respectively.Mice in the sham-operated and model groups were only grasped and stimulated once daily without any intervention.The particle size,particle concentration and surface markers of iPSC exosomes were detected by Flow NanoAnalyzer,and exosome morphology was observed by transmission electron microscopy.TTC(2,3,5-triphenyltetrazolium chloride)staining and TUNEL staining were used to evaluate brain infarct volume and neuronal apoptosis in the ischemic area of the brain in mice.The neurological function and behavior of mice were assessed by Clark neurological function score,cylinder test and open field test.The number of peripheral T lymphocyte subpopulations(Th1,Th2,Th17,Treg)in mice were detected by flow cytometry.The expression of IL-33/ST2 axis proteins and changes in astrocytes and microglia in mouse brain were detected by immunofluorescence and Western Blot.To verify the scientific hypothesis that electroacupuncture combined with iPSC exosomes can lighten inflammatory injury in cerebral ischemia/reperfusion by interfering with IL-33/ST2 axis and regulate the immune response of T-lymphocytes,and provide a new research idea and experimental basis for the prevention and treatment of cerebral ischemia/reperfusion injury by electroacupuncture combined with iPSC exosomes.Results1.Effects of electroacupuncture combined with iPSC-EVs on neuromotor function in MCAO miceThe Clark neurological function score assesses neurological impairment in mice in seven areas:body symmetry,gait,climbing,spin test,forelimb symmetry,forced spin,and whisker responsiveness.The higher the score,the greater the impairment.The results showed that the neurological function score was significantly higher in the model group compared with the sham-operated group(P<0.001),while electroacupuncture alone,iPSCEVs alone or the combination of both treatments significantly reduced the Clark score(P<0.001).There was no significant difference among the three intervention groups(P>0.05),and there was a significant difference between the electroacupuncture group and the sham acupuncture group(P<0.001).The results of the cylinder test showed that the electroacupuncture group,the iPSC-EVs group and the combined intervention of both had different degrees of improvement on neurological function in mice with cerebral ischemiareperfusion for 72 h(P<0.05 for the electroacupuncture group,P<0.001 for the iPSC-EVs group and P<0.001 for the EA+iPSC-EVs group),and there was a significant difference between the electroacupuncture group and the sham acupuncture group(P<0.05).The results of the open field test showed that both the electroacupuncture group and the iPSCEVs group could improve the motor dysfunction of MCAO mice(P<0.05 and P<0.01),but the improvement effect of the combined intervention of both was more significant(P<0.001).There was no statistical difference between the sham acupuncture group and the model group(P>0.05).2.Effects of electroacupuncture combined with iPSC-EVs on brain infarct volume and neuronal apoptosis in MCAO miceThe TTC staining results showed that the infarct volume of MCAO mice increased significantly after 72 h of cerebral ischemia-reperfusion(P<0.001),while the infarct volume of MCAO mice in the cortex,striatum or the whole hemisphere was significantly reduced after electroacupuncture of GV20,ST36 and tail vein injection of iPSC-EVs(P<0.001).The effect of the combined intervention group was significantly better than that of the electroacupuncture and iPSC-EVs groups(P<0.001).Both electroacupuncture alone and iPSC-EVs injection in the tail vein alone could effectively improve neuronal apoptosis and alleviate brain injury(P<0.001).Among them,electroacupuncture combined with iPSC-EVs had a more obvious therapeutic effect(P<0.001).There was no statistical difference between the sham acupuncture group and the model group(P>0.05),and there was a significant difference between the electroacupuncture group and the sham acupuncture group(P<0.001).3.Effects of electroacupuncture combined with iPSC-EVs on the immune response of the T-lymphocytes in MCAO miceFlow cytometry results showed that the number of Th1(IFN-γ+Th)cells and Th17(IL17+Th)was significantly elevated in MCAO mice after 72 h of cerebral ischemiareperfusion(P<0.001),and that treatment with electroacupuncture of GV20,ST36 and tail vein injection of iPSC-EVs significantly suppressed this abnormal response(P<0.01),especially after the combination of both treatments(P<0.001).No such treatment effect was observed in the sham acupuncture group(P>0.05).In contrast,electroacupuncture combined with iPSC-EVs was able to significantly increase the number of Treg(Foxp3+Treg)cells in MCAO mice and suppress the inflammatory response(P<0.01).While the number of Th2(IL-4+Th)cells did not differ significantly between the groups(P>0.05).4.Effects of electroacupuncture combined with iPSC-EVs on the expression of IL33/ST2 axis in MCAO miceImmunofluorescence and Western Blot results showed that the number of IL-33positive cells and protein expression levels in the ischemic region of the brains of MCAO mice were significantly increased after 72 h of cerebral ischemia-reperfusion,and this trend was effectively reduced after electroacupuncture and iPSC-EVs.Compared with the electroacupuncture group alone and iPSC-EVs group alone,the electroacupuncture combined with iPSC-EVs group was able to suppress IL-33 expression more significantly(P<0.001).Meanwhile,the elevation of IL-33 caused by cerebral ischemia-reperfusion was accompanied by the activation of astrocytes in the brain,and the immunofluorescence results suggested that the number of GFAP-positive cells in the infarct area of MCAO mice was significantly increased,while both electroacupuncture and iPSC-EVs interventions significantly reduced the number of GFAP-positive cells(P<0.05),and this effect was more obvious in the combined treatment group(P<0.001).In addition,IL-33 was found to be mainly expressed in astrocytes by immunofluorescence co-localization.Similarly,the expression trend of ST2 in the ischemic region of MCAO mice was similar to that of IL33,and the up-regulated ST2 levels were significantly reversed by electroacupuncture and iPSC-EVs,especially in the combined treatment group(P<0.001).There was no statistical difference between the sham acupuncture group and the model group(P>0.05),and there was a significant difference between the electroacupuncture group and the sham acupuncture group(P<0.001).5.Effects of electroacupuncture combined with iPSC-EVs on the activation of microglia in MCAO miceDuring cerebral ischemia-reperfusion,microglia in the infarct area were rapidly activated and proliferated,and the activated microglia gradually became larger and rounder in morphology,and the branches were gradually reduced.Immunofluorescence results showed that after 72 h of cerebral ischemia-reperfusion,the overall percentage of microglia in the infarcted area and the percentage of activated microglia in the resting state of MCAO mice were significantly increased(P<0.001),while the percentage of resting microglia was significantly decreased(P<0.001).All three treatment groups significantly reversed these indices,but the improvement was more significant in the electroacupuncture combined with iPSC-EVs group compared with the electroacupuncture alone and iPSC-EVs alone groups(P<0.001).There was no statistical difference between the sham acupuncture group and the model group(P>0.05),and there was a significant difference between the electroacupuncture group and the sham acupuncture group(P<0.001).Conclusion1.Electroacupuncture of GV20 and ST36,iPSC-EVs and both combined interventions could improve neurological function scores,attenuated neurological deficits,reduced cerebral infarct volume,improved neuronal apoptosis and attenuated brain injury in mice after 72 h of cerebral ischemia-reperfusion injury.The protective effects of electroacupuncture in mice with cerebral ischemia-reperfusion injury were comparable to those of iPSC-EVs,and the effects of combined interventions was superior to those of electroacupuncture alone and iPSC-EVs alone.Sham acupuncture had no cerebral protective effect.2.Electroacupuncture of GV20 and ST36,iPSC-EVs and both combined interventions had no significant effect on Th2 cells,but could down-regulate the number of Th1 and Th17 cells,the three intervention groups had comparable effects,and there was a significant difference between electro-acupuncture and sham acupuncture.The combined intervention of both could up-regulate the number of Treg cells and suppress the inflammatory response after 72 h of cerebral ischemia-reperfusion injury.The effect of both combined interventions was better than that of electroacupuncture alone and iPSC-EVs alone.3.Electroacupuncture of GV20 and ST36,iPSC-EVs and both combined interventions could regulate the expression of IL-33/ST2 axis,inhibit the activation of astrocytes and microglia,and reduce the inflammatory response in mice with cerebral ischemia/reperfusion injury.The effects of electroacupuncture and iPSC-EVs were comparable,and the combined effect of both interventions was significantly better than electroacupuncture alone and iPSC-EVs alone.Innovation1.Innovation of intervention:Based on the intervention of electroacupuncture of GV20 and ST36 for cerebral ischemia-reperfusion injury,this thesis used tail vein injection of iPSC-EVs combined with electroacupuncture as a new intervention,and compared the effects of electroacupuncture,iPSC-EVs and the combined intervention of both.A sham acupuncture group was also established.This provides an experimental basis for future exploration of the possibility of combining acupuncture with exosomes in stroke treatment.2.Theoretical innovation:This thesis explored the effects of electroacupuncture of GV20 and ST36 and tail vein injection of iPSC-EVs on the expression of IL-33/ST2 axis and immunomodulation in brain tissue of mice with cerebral ischemia-reperfusion injury.No reports related to this study were found after a literature search for new information. |
PDF Full Text Request