| BackgroundIschemic stroke refers to the necrosis or softening of local brain tissue caused by ischemia and hypoxia due to stenosis or occlusion of cerebral blood supply artery,which is the most common type of stroke,accounting for about 80%of stroke.High morbidity,disability,mortality and recurrence rate are the main clinical features.The current clinical treatment of IS is mainly intravenous thrombolysis in the acute phase.However,the narrow time window of thrombolytic drugs and the contraindication of drug use limit the wide application of thrombolytic therapy,and many patients may miss the best treatment opportunity,ultimately leading to death or brain dysfunction such as body movement,sensation and cognition in the later period of survivors,severely affecting their quality of life.It is therefore an urgent problem to find more effective targets to promote the recovery of neural function.Animal and clinical studies have shown that changing the excitability of motor cortex neurons can promote the recovery of neurological function after stroke.In the field of neuroscience,designer receptors exclusively activated by designer drugs(DREADDs)are the most commonly used chemical genetics techniques to excite or inhibit target neurons.Chemical genetics refers to the method of using genetic principles to modify biological macromolecules so that they interact with previously unrecognized small molecule chemical agonists.Selective activation or inhibition of neuronal excitability using DREADDs tools in animal models of cerebral ischemia can affect the prognosis of cerebral ischemia,but the mechanism is unknown.Extracellular vesicles(EVs)are vesicular bodies with a bilayer lipid membrane structure that are secreted by cells and play an important role in the process of information transfer between cells.After ischemic stroke,the activity of glutamatergic synapses increases,which induces the release of EVs.EVs preferentially combine with neighboring neurons,and promote information transfer between neurons.The effect of EVs released by neuronal activation after ischemic stroke on the inflammatory response and neurological dysfunction has not been reported.In this study,chemical genetics technology was used to inhibit or excite primary motor cortex(M1)neurons,to explore the role of regulating the excitability of M1 neurons on the injured side in the reconstruction of neurological function after ischemic stroke,and to clarify the role and mechanism of miRNA in EVs released by activation of M1 neurons in the neural dysfunction and inflammatory response after ischemic stroke in mice.Methods1.The transient middle cerebral artery occlusion(MCAO)model of C57BL/6 mice was established by suture method to simulate human ischemic stroke.The Sham operation group(Sham group)did not insert a suture,and the remaining steps were consistent with the model group.The experiment was divided into two groups:Sham group and MCAO group.Neurological deficit score(mNSS score),Negative geotaxis experiment,Kondziella’s inverted screen experiment and Grid-walking experiment were used to evaluate neurological impairment.TTC staining was used to detect the area of cerebral infarction.TUNEL staining was used to detect the level of apoptosis.Immunofluorescence staining was used to detect microglia/macrophages activation.Western blot was used to detect the expression of neuronal activation markers((Immediate early genes:c-Fos),inflammatory factors(IL-1β,TNF-α,Arginase-1(Arg-1))and p-NF-κB..The expression of c-Fos was detected by immunohistochemistry.The number of CaMKIIα+c-Fos+ cells and GAD67+c-Fos+cells was detected b by immunofluorescence and flow cytometry.2.Two weeks before the establishment of MCAO mouse model,adeno-associated viruses:AAV9-hSyn-HA-hM3D(Gq)-IRES-mCitrine(hM3Dq)and AAV9-hSyn-HA-hM4D(Gi)-IRES-mCitrine(hM4Di)were injected into the M1 by brain stereotactic injection technique.The experiment was divided into four groups:hM3Dq+MCAO+Vehicle group,hM3Dq+MCAO+CNO group,hM4Di+MCAO+Vehicle group and hM4Di+MCAO+CNO group.Clozapine N-oxide(CNO)(1 mg/kg)or vehicle(once every 24 h,3 times in total)was injected intraperitoneally 3 d before MCAO,CNO or vehicle was injected for the fourth time 30 min before MCAO,and CNO or vehicle was injected for the fifth time 23.5 h after MCAO.Behavior,cerebral infarction area,apoptosis,microglia/macrophages activation,inflammatory factors,c-Fos and p-NF-κB were detected.3.EVs were extracted from M1 of Sham group and MCAO group by ultracentrifugation.The experiment was divided into two groups:Sham-Ml-EVs group and MCAO-M1-EVs group.The differential miRNA in EVs of Sham-Ml-EVs group and MCAO-M1-EVs group were analyzed by miRNA expression profile.4.PC12 cells were used to construct an in vitro neuron "ischemia-reperfusion" injury model,namely oxygen-glucose deprivation/eoxygenation(OGD/R)model.The experiment was divided into two groups:Normal group and OGD/R group.CCK8 was used to detect cell viability,Fura-2 was used to detect intracellular Ca2+concentration([Ca2+]i),and Western blot was used to detect c-Fos expression.5.The EVs in the supernatant of PC 12 cells were extracted by ultracentrifugation.The experiment was divided into two groups:normal cultured PC 12 cells-derived EVs(Nor-NEVs group)and OGD/R PC12 cells-derived EVs(OGD-N-EVs group).The expression of miR-100-5p in EVs was detected by qRT-PCR.6.PC 12 cells were treated with Nor-N-EVs and OGD-N-EVs.The experiment was divided into three groups:Normal group,Nor-N-EVs group and OGD-N-EVs group.Cell viability was detected.7.BV2 cells and primary microglia were treated with Nor-N-EVs and OGD-N-EVs.The experiment was divided into three groups:Control group,Nor-N-EVs group and OGD-N-EVs group.The expression of IL-1β,Arg-1 and p-NF-κB was detected.8.PC12 cells transfection plasmids:pCMV-HA-hM3D(Gq)-EGFP-FRT-Hyg(hM3Dq)plasmid and pCMV-CMV-mCherry-hM4D(Gi)-3×HA-Puro(hM4Di)plasmid.The experiment was divided into four groups:hM3Dq-plasmid+Vehicle group,hM3Dq-plasmid+CNO group,hM4Di-plasmid+Vehicle group and hM4Di-plasmid+CNO group.The intracellular[Ca2+]i,miR-100-5p and c-Fos were detected.9.PC12 cells were transfected with negative control(NC)and miR-100-5p mimics(mimics).The experiment was divided into two groups:NC group and mimics group.Cell viability,intracellular[Ca2+]i and c-Fos were detected.10.BV2 cells were transfected with NC、mimics、miR-100-5p mut G6-A(mut G6-A)、miR-100-5p mut G20-A(mut G20-A)、miR-100-5p mut U7-A(mut U7-A)、miR-100-5p mut U18-A(mut U18-A)、miR-100-5p mut U19-A(mut U19-A)、miR-100-5p mut U21-A(mut U21-A)、miR-100-5p mut G6G20U7U18U19U21-AAAAAA(mut G6G20U7U18U19U21AAAAAA).The experiment was divided into nine groups:NC group、mimics group、mut G6-A group、mut G20-A group、mut U7-A group、mut U18-A group、mut Ui9-A group、mut U21-A group 和 mut G6G20U7U18U19U21—AAAAAA group.The expression of IL-1β and pNF-κB was detected.11.HEK293T cells were transfected with pCMV-EGFP-TLR7(human)-Neo(TLR7)plasmid and mimics/mut G6G20U7U18U19U21-AAAAAA.The interaction between miR-100-5p and TLR7 and the expression of miR-100-5p and IL-1β were detected.12.BV2 cells were transfected with NC/mimics and TLR7 siRNA.The experiment was divided into two groups:NC+TLR7 siRNA group and mimics+TLR7 siRNA group.The expression of IL-1β and p-NF-κB was detected.13.PC 12 cells were transfected with NC/mimics and TLR7 siRNA.The experiment was divided into two groups:NC+TLR7 siRNA group and mimics+TLR7 siRNA group.Cell viability was detected.14.Two days before the establishment of MCAO mouse model,control antagomiR and miR-100-5p antagomiR(100 pmol)were injected into the lateral ventricle by stereotactic injection technique.The experiment was divided into two groups:control antagomiR group and miR-100-5p antagomiR group.Behavior,cerebral infarction area,apoptosis,microglia/macrophages activation were detected.Results1.Inhibiting M1 neuronal activity attenuated ischemic stroke-induced brain injuryThe results of immunofluorescence and flow cytometry showed that CAMKIIα+c-Fos+cells and GAD67+c-Fos+cells in M1 of MCAO mice were significantly increased,suggesting that acute ischemia induced excitatory neurons and inhibitory neurons in M1 to be activated.Inhibiting M1 neuronal activity by hM4Di+CNO can reduce cerebral infarction area and neuronal apoptosis,inhibit microglia/macrophages activation,improve neurologieal deficit signs,enhance forelimb muscle strength and improve sensorimotor function and coordination in MCAO mice.However,the use of hM3Dq+CNO to promote M1 neuronal activity did not affect cerebral infarct area,neuronal apoptosis,microglia/macrophages activation,and neurological dysfunction in MCAO mice.2.OGD-N-EVs induced microglial inflammatory response and decreased neuronal activityOGD/R treatment can up-regulate the levels of intracellular[Ca2+]i and c-Fos in PC 12 cells,suggesting that OGD/R can activate neurons.EVs from PC 12 cells were collected to incubate neurons and microglia in vitro.The results showed that EVs could be ingested by neurons and microglia.Compared with Nor-N-EVs group,OGD-N-EVs treatment induced the increase of IL-1β expression and the decrease of Arg-1 expression in microglia.Compared with Nor-NEVs group,OGD-N-EVs treatment induced the decrease of neuron activity.3.The expression of miR-100-5p was increased in MCAO-M1-EVs and OGD-N-EVsThe results of miRNA expression profile showed that 7 miRNA were significantly upregulated in MCAO-M1-EVs group compared with Sham-Ml-EVs group.The up-regulated miR-100-5p is abundant in Ml-EVs,and the expression level of miR-100-5p is positively correlated with the infarction area.Consistent with the results of animal experiments,the expression of miR-100-5p in OGD-N-EVs was also significantly up-regulated.Activation of PC 12 cells with hM3Dq-plasmid+CNO could up-regulate the expression of miR-100-5p,while hM4Di-plasmid+CNO did not affect the expression of miR-100-5p in PC 12 cells.4.MiR-100-5p U18U19G20 motif induced microglial inflammatory response and decreased neuronal viabilityMiR-100-5p mimics(mimics)were used to overexpress miR-100-5p in BV2 cells.The showed that miR-100-5p、mut G6-A、mut U7-A and mut U21-A promoted IL-1β and p-NF-κB expression,inhibited Arg-1 expression,but not mut G20-A,nut U18-A,mut U19-A and mut C6G20U7U18U19U21-AAAAAA.MiR-100-5p mimics(mimics)were used to overexpress miR-100-5 p in PC 12 cells.The results showed that miR-100-5p induced PC 12 cell viability decline,but mut C6G20U7U18U19U21-AAAAAA had no such effect.5.Inhibiting M1 neuronal activity inhibited ischemic stroke-induced activation of NF-κB signaling pathwayIschemic stroke induced an increase in the expression of p-NF-κB in the M1,inhibiting M1 neuronal activity by hM4Di+CNO reduced ischemic stroke-induced p-NF-κB expression.However,the use of hM3Dq+CNO to promote M1 neuronal activity did not affect p-NF-κB expression.6.MiR-100-5p binds to and activates TLR7In HEK293T cells,miR-100-5p mimics(mimics)was used to overexpress miR-100-5p,miR-100-5p mut C6G20U7U18U19U21-AAAAAA was used to overexpress miR-100-5p mut,TLR7 plasmid was used to overexpress TLR7.RNA binding protein immunoprecipitation results showed that miR-100-5p binds to TLR7.Meanwhile,compared with the mimics group,the expression of IL-1β in the TLR7 plasmid+mimics group was significantly increased.Compared with TLR7 plasmid group,the expression of IL-1β in TLR7 plasmid+mimics group was significantly increased.7.MiR-100-5p induces microglial inflammatory response and neuronal viability reduction via TLR7 pathwayMiR-100-5p mimics(mimics)were used to overexpress miR-100-5p in BV2 cells,and TLR7 siRNA was used to inhibit TLR7 expression.The results showed that there was no significant difference in the expression of IL-1β and p-NF-κB between NC+TLR7 siRNA group and mimics+TLR7 siRNA group.MiR-100-5p mimics were used to overexpress miR-100-5p in PC 12 cells,and TLR7 siRNA was used to inhibit TLR7 expression.The results showed that there was no significant difference in cell viability between NC+TLR7 siRNA group and mimics+TLR7 siRNA group.8.Inhibition of miR-100-5p expression reduced ischemic stroke-induced brain injuryInhibition of miR-100-5p expression in MCAO mice significantly reduced cerebral infarction area and neuronal apoptosis,inhibited microglia/macrophages activation,improved neurological deficit signs,enhanced forelimb muscle strength,and improved sensorimotor function and coordination.Conclusion1.Cerebral ischemia can induce the activation of neurons in M1 on ipsilateral hemisphere.Inhibiting M1 neuronal activity of ipsilateral hemisphere with the hM4Di efficiently alleviated infarct area and neuro-inflammation,and improved motor recovery in ischemic stroke mice.2.MiR-100-5p in EVs released by OGD/R induced neuronal activation recognizes and binds TLR7 of microglia through U18U19G20 motif,and then activates TLR7 and downstream NF-κB signal pathway,inducing an inflammatory response;at the same time,miR-100-5p induced apoptosis by activating TLR7 of neurons.3.Inhibition of miR-100-5p expression reduced cerebral infarction area and neuroinflammation and promoted neurological function recovery in mice with ischemic stroke.Innovative topics1.The effects of neuronal activation in the M1 of the ipsilateral on neurological function and inflammatory response in mice with IS were clarified for the first time.2.It is clarified that miR-100-5p in EVs released by neuronal activation is one of the key molecules that induce microglia/macrophages to pro-inflammatory polarization and neuronal damage after IS.3.It is systematically clarified that miR-100-5p binds to and activates TLR7 via the U18U19G20 motif,and then induces microglia/macrophages activation and neuronal damage. |
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