The Effects And Mechanism Of ?-caryophyllene On Attenuating Acute Ischemic Stroke And Post-stroke Cognitive Impairment By Regulating CI-AMPARs Membrane Recruitment

Acute ischemic stroke has the characteristics of high morbidity,high disability rate,high mortality rate and high recurrence rate,and is the main cause of disability in the elderly.But up to now,its pathophysiological mechanism is still not completely clear,and no effective method for its treatment has been found.Therefore,it is of great economic significance and social value to further elucidate the pathogenesis of acute ischemic stroke,search for new therapeutic targets and develop safe and effective drugs.Many studies have shown that excitatory toxicity caused by glutamate released excessively into intercellular space after cerebral ischemia plays an important role in neuronal cell death after cerebral ischemia injury.The excessive glutamate mainly activates two postsynaptic membrane receptors,NMDARS and AMPARs.Glu A2,a subunit of AMPARs,inhibits Ca²⁺permeability,which determines the permeability of AMPARs to Ca²⁺.According to whether AMPARs contains Glu A2,AMPARs can be divided into CI-AMPARs and CP-AMPARs.After cerebral ischemia injury,a large number of Ca²⁺enter the pyramidal neurons in the relatively fragile hippocampal CA1 region through AMPARs with altered permeability,causing intracellular calcium overload and triggering a lethal response of the neurons.In addition,AMPARs mediate most of the fast excitatory synaptic transmission in the central nervous system(CNS),and play an important role in learning and memory.It was found that the increase of calcium influx in neurons after cerebral ischemia also aggravates the neurodegenerative process after cerebral ischemia.Synaptic plasticity regulated by long term potentiation(LTP)and long term depression(LTD)is considered to be the physiological basis of learning and memory.Recent results showed that CI-AMPARs are transported and recruited from extra-synapses and/or intra-cells to the surface of the hippocampal synaptic membrane,thereby altering the calcium ion flow and the current on the synaptic membrane,disturbing the LTP and LTD processes,and thus injuring the cognitive related learning and memory processes.However,it is unclear whether CI-AMPARs play a role in cognitive impairment after acute ischemic stroke.The endogenous cannabinoid system was first found to play an important role in regulating behavior,such as addiction,anxiety,and ingestion,etc.Later studies found that the cannabinoid system plays an important role in the pathophysiological mechanisms of cerebral ischemia reperfusion and some neurodegenerative diseases.Our group found that?-caryophyllene(BCP),an Cannabinoid receptor?type agonist,that could combine with the CB2 receptor completely and protect the nerve injury and alleviate the learning and memory ability of animals after cerebral ischemia,but its mechanism is still unclear.Considering that CB2 receptor agonists modulate Ca²⁺flow in pyramidal neurons in rodent medial prefrontal cortex,it is demonstrated that CB2 receptor may modulate ion homeostasis and neuronal excitability.Therefore,we speculate that the protective effect ofCB2 receptor(BCP)on acute ischemic stroke and post-stroke cognitive dysfunction may be related to its regulation of membrane transport of CI-AMPARs.According to this scientific hypothesis,we took OGD/R and acute ischemic stroke model(MCAO),by MTT test,flow cytometry apoptosis test,cell morphology observation,neurobehavioral score,rod rotation test,TTC staining,H&E staining,MWM water maze,object recognition task,Immunofluorescence double standard,flow detection of[Ca2+]i,patch clamp electrophysiological techniques,and analysis clinical acute ischemic stroke patients samples and clinical data.From the level of cells,animals and clinical samples,on the basis of demonstrating the role of?-caryophyllene on the protection of acute ischemic stroke and post-stroke,we study the mechanisms of BCP facilitating CI-AMPAR translocation to the synaptic membrane and recruitment of these receptors and the relationship between its role on the protection of acute ischemic stroke and post-stroke.Methods and results:1.?-caryophylene alleviate acute ischemic stroke and post-stroke cognitive dysfunction(1)Oxygen-glucose deprivation/reoxygenation(OGD/R)of the mouse neuron HT-22 cells was divided into Control group,Control+10?M BCP group(Normal control),OGD/R+5?M BCP group,OGD/R+10?M BCP group,OGD/R+20?M BCP group;After 24h,48h,72h,a cellular level protective effect on OGD/R in neurons of BCP was observed by MTT proliferation detection,flow cytometry detection of cell apoptosis,observation of cell morphology under the microscope.(2)(1)The 6-8week old male C57BL/6 mouse were randomly divided into sham surgery group,sham+BCP 72mg/kg group(Normal control),ischemia model+solvent group,ischemia model+BCP 36mg/kg group,ischemia model+144mg/kg group.After administration of drug for 6consecutive days,the 6 experimental mouses in every group were used to establish the acute ischemic stroke model by the middle cerebral artery occlusion(MCAO)on the 6th day.(2)After 1 hour of acute cerebral ischemia and 7 days of reperfusion,to observe the effect of BCP on nerve function of mice after MCAO,the animals were accessed by the modified Longa 0-5 scale,the modified neurological severity Score(m NSS)and the rod rotation test.TTC staining was used to detect the cerebral infarct volume of mice in each group,and H&E staining was used to observe the pathological structure changes of nerve cells in the hippocampus of mice in each group,to verify and analyze the protective effect of BCP on acute ischemic stroke mice in vivo,the results demonstrated that BCP has a dose-dependent protective effect on brain injury in acute ischemic stroke mice.(3)After 1 hour of acute cerebral ischemia and 7 days of reperfusion,Morris water maze experiment includes space positioning navigation experiment,space exploration experiment to detect the escape latency,the number of times that crossing the platform and the time of target quadrant,and object recognition experiment were performed to evaluate learning and memory function in each group and verify the protection of BCP on learning and memory impairment after acute ischemic stroke.The results showed that BCP could significantly improve the escape latency,the number of target platform,the activity time of target quadrant and the object recognition index of MCAO mice.In other words,BCP could significantly improve the cognitive dysfunction of MCAO mice.2.BCP facilitates CI-AMPARs translocation to the synaptic membrane and recruitment of these receptors in acute ischemic stroke(1)Western blots were used to detect the surface levels of the AMPAR subunit Glu A2 and total Glu A2 expression in each group.We found that BCP treatment is essential for translocation of CI-AMPARs to the synaptic membrane and the recruitment of these receptors during in OGD/R cell model.(2)We detected the[Ca²⁺]i using calcium fluorescent probe Fluro-3AMi and tested by laser confocal and flow cytometry.We observed that an increase of cellular[Ca²⁺]I in OGD/R group.However,the BCP can inhibit the[Ca²⁺]i of OGD/R model.(3)After membrane protein extraction,western blotting and immunofluorescence co-localization assay used to detect the expression changes of Glu A2 in membrane proteins and total protein in each group.The results showed that the accumulation of CI-AMPARs on the surface of synaptic membranes was reduced in MCAO model,while BCP administration could increase the expression of CI-AMPARs on the surface of synaptic membranes after MCAO.These results indicate that BCP treatment is essential for translocation of CI-AMPARs to the synaptic membrane and the recruitment of these receptors during acute ischemic stroke.(4)Changes in synaptic excitatory currents after MCAO and BCP treatment were analyzed using the patch clamp technique.Compared with the sham group,the amplitude and frequency of synaptic excitatory currents were higher in the MCAO group.Compared with the MCAO group,the amplitude and frequency of synaptic excitatory currents were lower in the MCAO+BCP group.The results indicated that BCP can inhibit the AMPAR-mediated m EPSCs in the hippocampus neuron induced by acute ischemic stroke.3.PKA-dependent membrane surface recruitment of CI-AMPARs is crucial for BCP-mediated protection against acute ischemic stroke and post-stroke cognitive impairment(1)To elucidate the effect of BCP on the c AMP/PKA signaling pathway in the OGD/R and MCAO model,we assessed the expression of c AMP with an enzyme-linked immunosorbent assay(ELISA)and the expression of phosphorylated PKA(p-PKA),PKA,phosphorylated CREB(p-CREB),CREB,and BDNF by western blotting.There was a sharp downregulation of c AMP,p-PKA,p-CREB,and BDNF level was observed in the OGD/R and MCAO group compared with those in the Control group.However,the BCP-treated groups showed less degradation of c AMP,p-PKA,p-CREB,and BDNF expression.These results indicated that BCP activated the c AMP/PKA pathway in OGD/R and MCAO model.(2)Using PKA inhibitors-H-89,the cells were divided into Control group,OGD/R group,OGD/R+10?M BCP group,and OGD/R+10?M BCP+H-89 group.Using Western Blot detected the expression of Glu A2 in the cell membrane surface and total protein,flow cytometry detector and laser confocal detected[Ca²⁺]i by using Fluro-3 AM.We can found that PKA inhibitors H-89 can reverse the role of BCP promoting CI-AMPARs membrane translocation to the synaptic membrane and recruitment in OGD/R cell model.So,the c AMP/PKA pathway is involved in BCP-induced facilitation of CI-AMPAR translocation to the synaptic membrane and recruitment of these receptors during OGD/R.(3)C57BL/6 male mice were divided into Sham group,MCAO group,MCAO+72 mg/kg BCP group,MCAO+72 mg/kg BCP+H-89 group.To determine whether the c AMP/PKA pathway is involved in the BCP-induced facilitation of CI-AMPARs translocation to the synaptic membrane and recruitment of these receptors in MCAO model,a western blot assay and confocal immunofluorescence analysis were performed.The western blot results showed that compared with the MCAO+BCP group,the MCAO+BCP+H-89 group exhibited significantly reduced the expression of the Glu A2 AMPAR subunit on the surface of synaptic membranes.Moreover,laser confocal immunofluorescence analysis also showed that compared with the MCAO+BCP group,the MCAO+BCP+H-89 group exhibited reduced colocalization of Glu A2 and synaptophysin,with a level similar to that observed in the MCAO group.We also found that the PKA inhibitor H-89 produced greater disruption the BCP-induced inhibition of AMPAR-mediated m EPSCs in the hippocampus neuron than in animals treated with BCP alone by patch clamp electrophysiological detection.These results showed that H-89 can abolish BCP-mediated CI-AMPAR translocation to the synaptic membrane.Taken together,BCP promote the transport of CI-AMPARs membrane surface in MCAO animal model is the style of PKA-dependent.(4)The expression changes of key molecules in the c AMP/PKA signaling pathway between all groups cells and mice were detected by ELISA and western blotting technology.(5)OGD/R mouse neuron HT-22 cells was divided into Control group,OGD/R group,OGD/R+10?M BCP group,OGD/R+10?M BCP group+H-89.Using MTT proliferation detection,flow cytometry detection of cell apoptosis,observation of cell morphology under the microscope methods,we found that H-89 can reverse the protective effect of BCP for OGD/R neurons cells.So,facilitation of CI-AMPAR translocation to the synaptic membrane surface,which depends on the c AMP/PKA pathway,is required for BCP-mediated protection against OGD/R.(6)Mice were divided into Sham group,MCAO group,MCAO+72mg/kg BCP group,MCAO+72 mg/kg BCP+H-89 group.Using modified Longa,m NSS,rota-rod test,TTC staining,H&E staining methods,we found that H-89 significantly disrupted BCP-induced neuroprotection against acute ischemic stroke.Compared with the MCAO+BCP group,the neurological scores increased and the time spent on rota-rod test decreased.And cerebral infarction volume were increased and a large number of neuronal cells died in the hippocampal CA1 region in the MCAO+BCP+H-89 group.These results indicated that the PKA-dependent membrane surface recruitment of CI-AMPARs is involved in BCP-induced neuroprotection in acute ischemic stroke.(7)The mechanism of BCP on the spatial learning and memory abilities of post-stroke mice were examined using the MWM test and object recognition task.In the MCAO+BCP+H-89 group,we found that H-89could counteract the shorter escape time induced by BCP treatment.And H-89 could reverse the effects induced by BCP induced-the platform crossings and time spent in the target quadrant.These results demonstrated that translocation of CI-AMPARs to the synaptic membrane surface depends on the c AMP/PKA pathway,and this translocation is required for BCP-mediated protection against post-stroke cognitive impairment.(8)Serum samples were collected from 30 patients with acute ischemic stroke before and after thrombolysis.The c AMP level were detected by ELISA.We found that the level of c AMP was up-regulated after thrombolysis in acute ischemic stroke patients together analysis of the clinical case data of the patients.In conclusion,the main results of this study are as follows:(1)The role of BCP-mediated the neuroprotection for acute ischemic stroke and against post-stroke cognitive impairment was clarified;(2)It was found for the first time that BCP facilitates CI-AMPARs translocation to the synaptic membrane and recruitment of these receptors in acute ischemic stroke;(3)It was found that the PKA-dependent membrane surface recruitment of CI-AMPARs is involved in BCP-induced neuroprotection in acute ischemic stroke and regulating LTP and LTD to improve the learning and memory abilities in post-stroke cognitive impairment.The results of this study lay a foundation for further elucidating the role and molecular mechanism of BCP in the protection of acute ischemic stroke and post-stroke cognitive dysfunction,and provide a new idea for finding new targets for the treatment of acute ischemic stroke and developing safe and effective drugs.