MiR-211-5p Alleviates Focal Cerebral Ischemia-reperfusion Injury In Rats By Down-regulating The Expression Of COX2

Part ? Effects of mi R-211-5p on brain damage in MCAO/R-treated ratsObjective:In order to screen out mi RNAs with significant expression changes i in the brain of rats subjected to focal cerebral ischemia-reperfusion?MCAO/R?;To observe the changes of mi R-211-5p and COX2 m RNA expressions in MCAO/R-treared rats;To observe the effects of mi R-211-5p on brain damage,COX2 expression and its downstream products,and the related inflammatory factors in MCAO/R-treated rats.Method: 1.Establishment of middle cerebral artery occlusion/reperfusion?MCAO/R?rat model Ischemic stroke was produced using the MCAO/R method as previously reported.Rats were deeply anesthetized with intraperitoneal injection of sodium pentobarbital?40 mg/kg?.Blunt dissection was performed to expose the right common carotid artery?CCA?,external carotid artery?ECA?,and internal carotid artery?ICA?.A nylon suture?Jialing,Guangzhou,China?was inserted into the CCA until it blocked the origin of the middle cerebral artery.90 mins after MCAO,the suture was withdrawn to reperfusion.The sham group received the same surgical without occlusion.Laser Doppler flowmetry?Periflux System 5000,Sweden?was used to monitor the cerebral blood flow?CBF?in ischemia?regional CBF ? 80 of baseline?and reperfusion?regional CBF ? 70 of baseline?.Animals dying before the chosen endpoint or no infarction except for sham were eliminated.2.Experimental group The experimental animals were divided into three batches.In order to screen out mi RNAs with significant expression differences,the first batch was divided into three groups: normal group,sham group,24 h group?after MCAO operation,reperfusion for 24h?,n=3 per group.For the time course study,SD rats were randomly separated into 6 groups:?1?sham group,?2?6h group?after MCAO operation,reperfusion for 6h?,?3?12h group?after MCAO operation,reperfusion for 12h?,?4?24h group?after MCAO operation,reperfusion for 24h?,?5?48h group?after MCAO operation,reperfusion for 48h?,?6?72h group?after MCAO operation,reperfusion for 72h?,n=3 per group.The brains of rats were removed at 6h,12 h,24h,48 h and 72 h after MCAO/R.For the mechanism study,SD rats were randomly separated into 6 groups:?1?sham group,?2?MCAO/R+vehicle group,?3?MCAO/R+mi R-211-5p agomir group,?4?MCAO/R+agomir negative control group,?5?MCAO/R+mi R-211-5p antagomir group,?6?MCAO/R+antagomir negative control group,n=10 per group.The brains of rats were removed at 24 h after MCAO/R.3.observation indicators and methods The changes of blood flow were detected by color Doppler flowmetry during modeling;the changes were evaluated by neurological deficit score 0-5;infarct size was calculated by TTC staining;The pathological changes of hippocampus and cortex were observed by HE staining.The changes of COX2 in neurons were observed by immunofluorescence.The contents of PGD₂,PGE2,TNF-? and IL-1? in hippocampus and cortex of rats were detected by ELISA.SOD activity and MDA content in hippocampus and cortex of rats were detected by chemical enzymology;mi R-211-5p and COX2 m RNA were detected by q PCR;COX2 protein expression was detected by Western blot.Result: 1.Compared with the normal group,there was no significant difference in the sham group.Compared with the sham group,mi R-211-5p was significantly down-regulated in the cortex of 24-hour reperfusion group,and up-regulated in the hippocampus.2.With the length of reperfusion,mi R-211-5p showed a trend of decreasing first and then increasing in the cortex,nadiring at 12 hour;mi R-211-5p showed a trend of increasing first and then decreasing in hippocampus,peaking at 12 h;mi R-211-5p also showed a trend of increasing first and then decreasing in PC12 cells,reaching a peak at 48 hours.COX2 had a tendency to rise first and then decrease both in vitro and in vivo.3.Compared with the vehicle group,the neurological deficit score and infarct volume of the mi R-211-5p agomir group were significantly reduced,the cortical and hippocampal vacuolization,nuclear deep staining and pyknosis were significantly reduced;the exspressions of COX2 m RNA and protein in cortex and hippocampus were markly decreased;The contents of PGD₂,IL-1? and TNF-? was significantly decreased in the cortex,and the contents of PGE2,IL-1? and TNF-? in the hippocampus were significantly decreased.Compared with the vehicle group,the neurological deficit score and infarct volume of the mi R-211-5p antagomir group were significantly increased,the cortical and hippocampal vacuolization,nuclear deep staining and pyknosis were significantly increased;the exspressions of COX2 m RNA and protein in cortex and hippocampus were markly increased;The contents of PGD₂,IL-1? and TNF-? was significantly increased in the cortex,and the contents of PGE2,IL-1? and TNF-? in the hippocampus were significantly increased.There was no significant difference between the agomir NC group and the antagomir NC group compared with the vehicle group.At the same time,there was no significant difference in SOD activity and MDA content between the groups.Conclusion: 1.The expression of mir-211-5p in the cortex and plasma of MCAO/ r-treated rats was significantly decreased,while the expression in the hippocampus was significantly increased.2.mi R-211-5p expression was negatively correlated with the expression of COX2 m RNA,mi R-211-5p has a neuroprotective effect on CIRI,and its mechanism may be related to inhibiting the expression of COX2 and thereby reducing the levels of downstream products and inflammatory factors..Part ? Effects of mi R-211-5p on the damage of OGD/R-treated PC12 cellsObjective: To observe the effects of mi R-211-5p on PC12 cell injury subjected to OGD/R,and to explore its possible mechanism.Method: 1.PC12 cell culture and establishment of PC12 cell damage model induced by OGD/R The rat pheochromocytoma-12?PC12?cell line is regarded as an established neuron-like system that can be used as a cell model for neuron.PC12 cells were cultured in high-glucose Dulbecco's modified Eagle's medium?DMEM,Gibco?supplemented with 10 % heat-inactivated fetal bovine serum?FBS,Gibco?,100 U/m L penicillin,and 100 mg/m L streptomycin in a humidified atmosphere of 5 % CO2 at 37?.The culture media of PC12 cells was replaced with glucose-free DMEM before oxygen-glucose deprivation?OGD?.Then the cells were put into the incubator filled with 1.0% oxygen,5% CO2 and 94% N2 for 2h.After OGD,the cells were cultured in normal culture medium with oxygen.2.Experimental group To investigate the effects of mi R-211-5p on OGD/R-treated PC12 cell injury and analyze its possinble mechanism,PC12 cells were divided into six groups: normal group,OGD/R group,OGD/R+mi R-211-5p mimic group,OGD/R+mimic negative control?NC?group,OGD/R+mi R-211-5p inhibitor group,OGD/R+inhibitor NC group.The mi R-211-5p-related reagent was administered to the lateral ventricle for the day before OGD/R.In order to investigate whether the effects of mi R-211-5p was reversed by COX2 si RNA,PC12 cells was divided into four groups: normal group,OGD/R group,OGD/R+mi R-211-5p inhibitor group,OGD/R+ mi R-211-5p inhibitor + COX2 si RNA group.3.Main observation indicators and methods: Cell proliferation and survival were detected by MTT assay and LDH assay;cell apoptosis was detected by flow cytometry;mi R-211-5p and COX2 m RNA were detected by q-PCR;COX2 protein expression was detected by Western blot;The contents of PGE2,PGD₂ was detected by ELISA;dual luciferase reporter assay was used to detect the binding site of mi R-211-5p on COX2 m RNA.Results: 1.Compared with the normal group,the cell apoptosis rate and LDH release rate of OGD/R group increased significantly,the proliferation rate decreased significantly,The exspressions of COX2 m RNA and protein increased significantly,and the contents of PGD₂ and PGE2 increased significantly.Compared with the OGD/R group,the cell apoptosis rate and LDH release rate of mi R-211-5p mimic group decreased significantly,the proliferation rate increased significantly,the exspressions of COX2 m RNA and protein decreased significantly,the contents of PGD₂ and PGE2 decreased significantly.Compared with the OGD/R group,the cell apoptosis rate and LDH release rate of mi R-211-5p inhibitor group increased significantly,the proliferation rate decreased significantly,the exspressions of COX2 m RNA and protein increased significantly,the contents of PGD₂ and PGE2 increased significantly.Compared with the OGD/R group,there was no significant difference between the mimic NC group and the inhibitor NC group.2.Compared with the mi R-211-5p inhibitor group,the release rate of LDH in the mi R-211-5p inhibitor+COX2 si RNA group was significantly decreased,the proliferation rate was significantly increased,and the expressions of COX2 m RNA and protein was significantly decreased.3.Dual luciferase reporter experiment confirmed that there are binding sites for mi R-211-5p in the non-coding region of COX2 m RNA.Conclusions:1.The expressions of COX2 and its downstream products significantly increased in PC12 cell was damaged by OGD/R treatment.2.Mi R-211-5p mimic had protective effect on OGD/R-treated cell damage and reduced the expressions of COX2 and its downstream products;mi R-211-5p inhibitor aggravates PC12 cell damage caused by OGD/R,and increase the expressions of COX2 and its downstream products.These results indicates that mi R-211-5p may be involved in OGD/R injury by regulating COX2.3.Bioinformatics analysis and dual luciferase assay confirmed that there are binding sites for mi R-211-5p in the non-coding region of COX2 m RNA.he effect of mi R-211-5p inhibitor on OGD/R can be reversed by COX2 si RNA.These results suggest that mi R-211-5p protects against CIRI through targeting the endogenous expression of COX2...