The Molecule Mechanism And Protective Effect Of CTRP1 On Neuron Damage Induced By Cerebral Ischemia And Reperfusion

Ischemic stroke is the most common type of stroke which is one of leading causes of death and disability in the worldwide.The critical treatment in clinic is timely restoration and improvement of cerebral blood supply.However,exacerbation of tissue injury companied with restoration of blood supply,which called cerebral ischemia reperfusion injury（CIRI）.Since the pathophysiology mechanism of CIRI is still unclear,the effective intervention strategies in clinic are still lacking.Therefore,it is important to explore the pathophysiology mechanism of CIRI deeply for finding new targets for prevention and treatment of CIRI.It has been reported that CTRP1 protected against cardiac injury induced by ischemia reperfusion injury via inhibition of cell apoptosis.In addition,a recent paper reported the circulating level of CTRP1 was increased in patients with acute ischemic stroke and CTRP1 attenuated microglia autophagy and inflammatory response by regulating the Akt/m TOR pathway in OGD/R treated BV2 cells.However,there is no report about the effect of CTRP1 on neurons.In this project,we established in vivo and in vitro experimental models to observe the effect of CTRP1 on neurons injury induced by CIRI and explore whether ERS-related apoptosis is involved in the mechanism of CTRP1.PartⅠEffect of CTRP1 on neuron injury induced by cerebral ischemic and reperfusion in ratsAims:To observe the CTRP1 expression in the brain and blood of rats subjected to MCAO/R;to explore the role and mechanism of CTRP1 in MCAO/R rats through intervention of CTRP1 overexpression.Methods:1.Establishment of MCAO/R model in ratsAdult male SD rats was anesthetized by intraperitoneal injection of4%chloral hydrate(400mg·kg^-1).Common carotid artery（CCA）,external carotid artery（ECA）and internal carotid artery（ICA）were dissected bluntly.The proximal end of CCA and ECA were ligated.A nylon thread coated with glue was inserted into ICA with a depth of about 2cm and pulled out for reperfusion after 90 mins ischemia.The reperfusion time was chosen in different experiments.2.Designment of groups（1）In order to detect expression changes of CTRP1 in brain and blood of rats:sham group,MCAO and reperfusion for 6h group,MCAO and reperfusion for 12h group,MCAO and reperfusion for 24h group,MCAO and reperfusion for 36h group,MCAO and reperfusion for 48h group,MCAO and reperfusion for 72h group.（2）In order to observe the effect of CTRP1 in MCAO/R rats:sham group,MCAO/R group,MCAO/R and empty lentivirus group（MCAO/R+LV-NC）,MCAO/R and CTRP1 overexpression lentivirus group（MCAO/R+LV-CTRP1）.（3）In order to explore the effect of CTRP1 on PERK signal pathway in MCAO/R rats:sham group,MCAO/R group,MCAO/R and empty lentivirus group（MCAO/R+LV-NC）,MCAO/R and CTRP1 overexpression lentivirus group（MCAO/R+LV-CTRP1）,MCAO/R and CCT020312 group（MCAO/R+CCT020312）,MCAO/R and CTRP1 overexpression lentivirus and CCT020312 group（MCAO/R+LV-CTRP1+CCT020312）.CCT020312is a selective activator of PERK.3.Observation indicators and methodsZea-longa scores was used to evaluate neurological deficits;TTC staining was used to determine infarction volume;HE staining was used to examine pathological changes in brain tissue;immunofluorescence was used to observe the location and expression of proteins;TUNEL was applied to analyze the apoptosis;q RT-PCR was used for detecting CTRP1m RNA expression;ELASA was used to detect CTRP1 content in blood;Co-immunoprecipitation was used to explore the interaction between CTRP1,CRP78 and PERK;Western blot was used to detect the protein expression.Results:1.Compared with sham group,the m RNA and protein expression of CTRP1 were significantly decreased in cortex of 12h and 24h-reprefusion group;while the protein level of CTRP1 in blood was elevated and the m RNA level was no significant changes in MCAO/R rats.The expression of CTRP1 located mainly at neuron,a small amount at microglia,and hardly at astrocyte.2.Compared with sham group,in the MCAO/R group,the infarction volume and neurological deficit scores were increased significantly;pathological injury was observably increased;the loss and apoptosis of neurons were increased.The expression of Bcl-2 was decreased and the expressions of BAX,CHOP,GRP78,ATF4,ATF6,PERK,IRE1awere increased significantly in MCAO/R rats.Compared with administration of LV-NC,the administration of LV-CTRP1 markedly decreased the infarct volume and neurological scores,alleviated brain injuries,reduced the loss of neurons,attenuated the apoptosis in brain observably;BAX and CHOP proteins expression were significantly decreased,Bcl-2 protein expression was increased;the expression of ERS maker proteins such as GRP78,ATF4,PERK were reduced significantly.There were no significant differences in the expression of ATF6,IRE1aand p-IRE1abetween MCAO/R+LV-NC group and MCAO/R+LV-CTRP1 group.3.COIP results revealed that there was interaction between CTRP1and GRP78 as well as interaction between CTRP1 and PERK.Compared with MCAO/R+LV-NC group,the binding of GRP78 and PERK was enhanced in MCAO/R+LV-CTRP1 group;the interaction between CTRP1and PERK was enhanced significantly,while the interaction between CTRP1 and GRP78 was not changed in MCAO/R+LV-CTRP1 group.4.Compared with MCAO/R+LV-NC group,the expression of GRP78,ATF4,BAX,CHOP and the expression ratio of p-e IF2α/e IF2α,p-PERK/PERK were down-regulated in MCAO/R+LV-CTRP1 group.Compared with MCAO/R+LV-CTRP1 group,in the MCAO/R+LV-CTRP1+CCT020312 group,the infraction volume and neurological deficit scores were increased significantly;the pathological damage worsen;the loss of neurons and apoptosis response were aggravated;the expression of BAX,CHOP,p-e IF2α/e IF2αwere increased significantly,while the changes of expression of GRP78,ATF4,p-PERK/PERK were no significant difference.Conclusion:1.CTRP1 was mostly expressed in neurons,small amount in microglia,and hardly in astroglia.The expression of CTRP1 were decreased significantly in cortex of MCAO/R rats;while the protein level of CTRP1 was increased significantly and the m RNA level was no significant changes in the blood of MCAO/R rats.2.CTRP1 overexpression protected against neuron injury in MCAO/R rats by inhibiting ERS related apoptosis.The protective effect was abolished by CCT020312.3.CTRP1 had significant effect on ERS by inhibiting PERK signal pathway,but no significant effect on ATF6 and IRE1asignal pathway.CTRP1 has binding to GRP78 and PERK and CTRP1 overexpression inhibited the dissociation of GRP78 and PERK.PartⅡEffect of CTRP1 on PC12 cell injury induced by OGD/RAims:To observe the CTRP1 expression changes in OGD/R treated PC12 cells;to explore the role and mechanism of CTRP1 in OGD/R treated PC12 cells through intervention of CTRP1.Methods:1.Establishment of OGD/R modelPC12 cells were cultured in sugar-free DMEM in an incubator containing 5%CO₂,94%N₂,1%O₂ at 37℃for oxygen glucose deprivation.After 1.5h,the sugar-free DMEM was replaced with complete medium.The PC12 cells were inoculated in an incubator with 5%CO₂at 37℃for different time to reoxygenation.2.Designment of groups（1）In order to detect the expression changes of CTRP1 in OGD/R treated PC12 cells:Normal group,OGD and reoxygenation 3h group,OGD and reoxygenation 6h group,OGD and reoxygenation 9h group,OGD and reoxygenation 12h group,OGD and reoxygenation 24h group,OGD and reoxygenation 48h group,OGD and reoxygenation 72h group.（2）In order to observe the effect of CTRP1 on PC12 cells injury induced by OGD/R:Normal group,OGD/R group,OGD/R+si-NC group,OGD/R+si-CTRP1 group,OGD/R+LV-NC group,OGD/R+LV-CTRP1group.（3）In order to explore the effect of CTRP1 on ERS in OGD/R treated PC12 cells:Normal group,OGD/R group,OGD/R+LV-NC group,OGD/R+LV-CTRP1 group,OGD/R+LV-NC+tunicamycin group,OGD/R+LV-CTRP1+tunicamycin group.Tunicamycin is an inducer of ERS.（4）In order to explore the effect of CTRP1 on PERK signal pathway in OGD/R treated PC12 cells:Normal group,OGD/R group,OGD/R+LV-NC group,OGD/R+LV-CTRP1 group,OGD/R+LV-CTRP1+CCT020312 group.CCT020312 is a selective activator of PERK.3.Observation indicators and methodsCell viability was analyzed by MTT;cell apoptosis was determined by TUNEL assay and flow cytometry;m RNA and protein expression were detected by q RT-PCR and Western blot respectively.Results:1.Compared with Normal group,the m RNA and protein expression of CTRP1 decreased significantly in OGD/R treated PC12 cells.2.Compared with Normal group,the cell viability of PC12 was decreased significantly and apoptosis was increased significantly in OGD/R group;compared with OGD/R intervention,the LV-NC and si-NC had no effect on cell viability and apoptosis.Compared with LV-NC,LV-CTRP1 markedly increased the cell viability and reduced apoptosis significantly;while,compared with si-NC,CTRP1 deficiency further decreased the cell viability and increased apoptosis significantly.3.Compared with LV-NC,LV-CTRP1 decreased the proteins expression of GRP78,CHOP,ATF4,BAX,significantly.Compared with LV-CTRP1,the combined intervention with CTRP1 and tunicamycin leaded a reduction of cell viability and a rise in apoptosis response;as well as elevation expression of GRP78,CHOP,ATF4,BAX in PC12 cells significantly.4.The treatment of OGD/R and LV-NC increased the expression of GRP78,CHOP,ATF4,BAX,p-PERK.Compared with LV-NC,LV-CTRP1decreased the protein expression of GRP78,CHOP,ATF4,BAX,p-PERK significantly.Compared with OGD/R+LV-CTRP1 group,in the OGD/R+LV-CTRP1+CCT020312 group,the cell viability was reduced,apoptosis was aggravated;the expression of BAX,CHOP,GRP78,ATF4,p-PERK were increased significantly.Conclusion:1.The m RNA and protein expression of CTRP1 were decreased significantly in OGD/R treated PC12 cells.2.CTRP1 protected PC12 cells against the injury induced by OGD/R.The effect of CTRP1 was blocked by tunicamycin and CCT020312.3.CTRP1 attenuated OGD/R treated PC12 cell injury by inhibiting ERS related apoptosis through PERK pathway.PartⅢEffect of CTRP1 on primary neuron injury induced by OGD/RAims:To observe the CTRP1 expression in primary cortical neurons subjected to OGD/R;to explore the role and mechanism of CTRP1 in primary cortical neurons subjected to OGD/R through intervention of CTRP1.Methods:1.Establishment of OGD/R modelThe primary cortical neurons were cultured in sugar-free DMEM in an incubator containing 5%CO₂,94%N₂,1%O₂ at 37℃for oxygen glucose deprivation.After 0.5h,the sugar-free DMEM was replaced with complete medium.The primary cortical neurons were inoculated in an incubator with5%CO₂ at 37℃for reoxygenation.2.Designment of groups（1）In order to detect the expression changes of CTRP1 in OGD/R treated primary cortical neurons:Control group,OGD/R group（OGD/R24h）.（2）In order to observe the effect of CTRP1 on primary neuron injury induced by OGD/R:Control group,OGD/R group,OGD/R+si-NC group,OGD/R+si-CTRP1 group,OGD/R+LV-NC group,OGD/R+LV-CTRP1group.（3）In order to explore the effect of CTRP1 on PERK signaling pathway in OGD/R treated primary cortical neurons:Control group,OGD/R group,OGD/R+LV-NC group,OGD/R+LV-CTRP1 group,OGD/R+LV-CTRP1+CCT020312 group.CCT020312 is a selective activator of PERK.3.Observation indicators and methodsCell viability was analyzed by MTT;cell apoptosis was determined by TUNEL assay;m RNA and protein expression were detected by q RT-PCR and Western blot respectively.Results:1.Compared with Control group,the CTRP1 protein expression was decreased significantly in OGD/R group.2.OGD/R treatment markedly decreased the cell viability,elevated the level of apoptosis,reduced the expression of Bcl-2 and significantly increased the expression of BAX,Cleaved-Caspase3,Caspase12,CHOP,GRP78,ATF4,PERK,p-PERK.Compared with OGD/R,LV-NC and si-NC had no effect on cell viability,apoptosis and proteins expression.Compared with intervention of LV-NC,LV-CTRP1 markedly increased the cell viability and reduced apoptosis,decreased the expression of BAX,Cleaved-Caspase3,Caspase12,CHOP,GRP78,ATF4,PERK,p-PERK,and elevated the protein expression of Bcl-2 significantly.Compared with intervention of si-NC,si-CTRP1 further significantly increased the expression of BAX,Cleaved-Caspase3,Caspase12,CHOP,GRP78,ATF4,PERK,p-PERK,and reduced the protein level of Bcl-2.3.Compared with OGD/R+LV-CTRP1 group,primary neurons in the OGD/R+CTRP1+CCT020312 group,the cell viability was decreased,apoptosis was aggravated;the Bcl-2 protein expression was decreased significantly,the expression of BAX,Cleaved-Caspase3,Caspase12,CHOP,GRP78,ATF4,PERK,p-PERK were up-regulated significantly.Conclusion:1.The protein expression of CTRP1 in OGD/R treated primary cortical neurons was down-regulated significantly.2.Overexpression of CTRP1 improved neuron injury via inhibiting apoptosis and PERK signal pathway;interference the expression of CTRP1aggravated apoptosis and PERK signal pathway.3.The protective effect of CTRP1 was reversed by CCT020312.4.CTRP1 played a protective role in OGD/R treated neuron injury via inhibiting apoptosis induced by PERK pathway.