| Background:Acute myocardial infarction(AMI)is a common cardiovascular event.The morbidity and mortality of myocardial ischemia reperfusion injury(MIRI)induced by AMI increased year by year.In addition,with the continuous innovation and popularization of interventional cardiology and surgery,the post-treatment MIRI cannot be ignored.MIRI disease has become a common and frequently occurring disease in clinical work,which can cause ultrastructural changes,metabolic dysfunction and apoptosis of myocardial cells,affect myocardial function,metabolism and cardiac electrophysiology,and seriously induce myocardial infarction.So the prevention and reduction of MIRI plays an important role in reducing mortality and improving the prognosis of patients.The mechanism of MIRI is complex,and most studies have suggested that the mechanism may be related to oxidative stress,intracellular calcium overload,inflammatory response,energy metabolism disorder and apoptosis.MicroRNA(microRNA,miRNA)is a small non-coding RNA with a length of 19-25 nucleotides,which is a rich class of gene regulatory molecules in multicellular organisms and affects the expression of many protein-coding genes.MiRNA negatively regulates gene expression by specific binding to target mRNA,inhibiting translation or degradation of target mRNA.There is increasing evidence that miRNAs are involved in regulating many basic cellular behaviors such as cell differentiation,proliferation,growth,migration,and apoptosis.Studies have found that miRNAs are involved in almost all pathophysiological processes of the heart,and play an irreplaceable role in various pathophysiological processes such as myocardial fibrosis and vascular regeneration,and may be a important key regulatory factor.MiR-93 was the first miR-17 microRNA cluster to be discovered and closely related to the early evolution of vertebrates.Studies have shown that decreased miR-93 expression can significantly improve cerebral ischemia-reperfusion injury,but there are few studies on the role of miR-93 in MIRI.Transcription factor NF-E2 related factor 2(Nrf2)is a key transcriptional molecule that regulates intracellular oxidative stress response,which activates the expression of downstream antioxidant target genes by binding with antioxident response elements(ARE).Studies have shown that Nrf2 signaling pathway is the main pathway to regulate the expression of antioxidant and phase ? metabolic enzymes.And it has a protective effect on reducing cerebral ischemia reperfusion injury.Therefore,it is speculated that Nrf2 may play an important role in the fight against oxidative stress in MIRI.At present,Nrf2 is a potential research hotspot in the prevention and treatment of cardiovascular diseases.Therefore,in this study,a rat cardiomyocytes model of OGD/R was constructed to study and explore the mechanism of miR-93 regulated the apoptosis of OGD/R cardiomyocytes by Nrf2 signaling pathway.The expression of miR-93 and its possible mechanism were investigated and verified in rat MIRI models.The project consists of the following two parts:First part:The expression of miR-93 in the OGD/R model of cardiomyocytes and the mechanism of regulating apoptosis in the OGD/R cardiomyocytes;Second part:To verify the expression and possible mechanism of miR-93 in the myocardial tissue of MIRI rats.Part ? The role of miR-93 targeting Nrf2 signaling pathway on OGD myocardial cellObjective:To establish a model of oxygen and glucose deprivation in cardiomyocytes and analyze the role and mechanism of miR-93 in cardiomyocytes.Methods:(1)Primary cell culture:cardiomyocytes of neonatal SD rat were cultured until the 5th day.(2)The morphology of cardiomyocytes was observed under an optical microscope,and the purity of cardiomyocytes was identified by immunofluorescence.(3)Modeling and grouping:The cells were cultured in the sugar-free medium,and incubated into a three-gas incubator(0.5%O2,5%CO2,94.5%N2)for oxygen-glucose deprivation to simulate myocardial ischemic injury in vivo.The experimental groups were divided into the normal control group(NC group);OGD/R 1h group;OGD/R 2h group;OGD/R 3h group according to the time of oxygen and glucose deprivation.(4)The morphology of OGD/R cardiomyocytes in each group was observed by transmission electron microscopy.(5)Survival rate of OGD/R cardiomyocytes in each group was determined by trypan blue staining.(6)Lactate dehydrogenase(LDH),creatine kinase(CK-MB)and cardiac troponin(cTnI)activity were detected by lactate release method.(7)The expression of miR-93 in OGD/R cardiomyocytes in each group was determined by real time flurescent quantitative PCR(RT-qPCR).(8)TargetScan and miRDB target gene prediction software were used to predict that Nrf2 might be a target gene of miR-93 through comprehensive bioinformatics analysis,and the relationship between miR-93 and Nrf2 genes was verified using double luciferase reporter gene assay.(9)The model of OGD/R 2h group were used for subsequent experiments.The NC group and OGD/R group were transfected with miR-93 antagomir and nc-antagomir,respectively.The expression of miR-93 and Nrf2 genes in each transfection group was detected by RT-qPCR.(10)Flow cytometry was used to detect the apoptosis rate of cardiomyocytes in each transfection group.(11)The expression of protein Nrf2,caspase-3 and cleaved caspase-3 in each transfection group was determined by western blot.Results:(1)Immunofluorescence observation showed that the purity of primary cardiomyocytes was more than 90%,which could be used to establish the OGD/R model.(2)Transmission electron microscopy observation showed that the degree of mitochondrial swelling in the OGD/R 1h group was mild,and the clear cristae structure could be observed.The mitochondria in the OGD/R2h group were obviously swollen,some vacuoles were denaturing,the cristae were broken but to a lesser extent,the transmittance zone was visible in the matrix,the sarcoplasmic reticulum was expanded,and the muscle filaments were arranged in disorder.The myocardium in OGD/R3h group showed obvious rupture of cell membrane,adherent detachment growth,severe swelling of mitochondria,rupture of partial cell membrane,and dissolution of myofilaments.(3)Trypan-blue staining results showed that the survival rate of cardiomyocytes in each OGD/R group was significantly lower than that in NC group,and the survival rate of cardiomyocytes decreased with the prolongation of oxy-glucose deprivation time.(4)LDH,CK-MB and cTnI activities of cardiomyocytes in OGD/R groups were significantly higher than that in NC group(P<0.05),and the longer duration of oxygen and glucose deprivation,the more significant the increase of LDH activity.(5)RT-qPCR detected the expression level of miR-93 in the myocardial cells of each group.The results showed that compared with the NC group,the expression level of miR-93 of OGD/R groups were significantly increased(P<0.05),and the expression level of miR-93 was also gradually increased with the increase of the time of oxygen and glucose deprivation(P<0.05).(6)TargetScan and miRDB target gene prediction software analyzed that Nrf2 might be one of the target gene of miR-93,and the double luciferase reporter gene test confirmed that Nrf2 was a target gene of miR-93.(7)In OGD/R cardiomyocytes,the expression level of miR-93 in miR-93 antagomir group was significantly lower than that in NC-antagomir group,while the expression level of Nrf2 mRNA and protein were significantly higher than that in NC-antagomir group(P<0.05).It was suggested that miR-93 could negatively regulate the mRNA and protein expression of Nrf2 gene in OGD/R cells.(8)In OGD/R cardiomyocytes,the apoptosis rate of miR-93 antagomir group was significantly lower than that of NC-antagomir group.Meanwhile,western blot analysis of also found that expression level of cleaved caspase-3 was significantly lower than that in NC-antagomir group,suggesting that inhibition of mir-93 could significantly inhibit apoptosis of OGD/R cardiomyocytes.Conclusions:(1)The model of OGD/R cardiomyocytes was successfully established by in vitro culture,and the cardiomyocytes cultured of oxygen-glucose deprivation for 2h and reoxygenated for 6h was selected as a appropriate model of OGD/R cardiomyocytes,laying a foundation for the subsequent experiments.(2)The expression level of miR-93 in OGD/R cardiomyocytes was significantly increased.(3)Bioinformatics software predicted and double luciferase reporter gene experiment verified that Nrf2 was a target gene of miR-93,and that miR-93 could negatively regulate the expression of Nrf2.(4)miR-93 was involved in the regulation of apoptosis of cardiomyocytes,and inhibition of miR-93 expression could inhibit the apoptosis of OGD/R cardiomyocytes.(5)In OGD/R cardiomyocytes,miR-93 regulated the apoptosis of OGD/R cardiomyocytes by targeting the Nrf2 signaling pathway.Part 2 Expression and role of miR-93 in MIRI rat modelObjective:To analyze the expression of miR-93 in MIRI rat model and its role.Methods:(1)Animal grouping:40 SD rats were randomly divided into 4 groups,10 in each group:Sham group,ischemia reperfusion model(MIRI)group,miR-93 antagomir group,NC-antagomir group.Before the establishing the MIRI model,rats in the sham group and MIRI group were injected with 0.5ml normal saline through the tail vein,and rats in miR-93 antagomir group and NC-antagomir group were injected with miR-93 antagomir and antagomir negative control through the tail vein,respectively,with the final concentration of 80mg/kg,once a day for a total of 7d.MIRI rats model were established 2h after the last injection.(2)LDH activity was detected by lactate release method,CK-NB activity and cTnI in arterial blood of rats were detected by double-antibody sandwich method.(3)After reperfusion 24h,the myocardial tissue(ischemic area)was stained with HE,and the pathologic change was observed by optical microscopy.(4)After reperfusion 24h,the size of the myocardial infarct was determined by TTC staining.(5)Expression level of miR-93 and its target gene Nrf2 mRNA in rat myocardial tissue in each group was detected by RT-qPCR,and expressions of Nrf2 protein and apoptosis related proteins caspase-3,cleaved caspase-3,Bcl-2 and Bax were detected by western blot.(6)The levels of oxidative stress indicators:activity of superoxide dismutase(SOD),malondialdehyde(MDA)and glutathione peroxidase(GSH-Px)in the myocardial tissues of the rat in each group were measured.(7)The apoptosis in each group were detected by TUNEL.Results:(1)miR-93 was highly expressed in MIRI group,while the expression level in miR-93 antagomir group was significantly lower than that in MIRI group and NC-antagomir group.The expression level of Nrf2 mRNA and protein in MIRI group was significantly lower than that in sham group(P<0.05).The expression of Nrf2 mRNA and protein in miR-93 antagomir group were significantly higher than those in MIRI group and NC-antagomir group(P<0.05).The results indicated that the expression of miR-93 in MIRI myocardial tissue increased,while the expression of Nrf2 decreased,and expression of miR-93 was negatively correlated with Nrf2.(2)HE staining showed that there were significant damage in some myocardial tissue of rats in the MIRI group,including necrosis,inflammatory cell infiltration,cell edema and disordered arrangement.The damage of myocardial tissue in miR-93 antagomir group was less severe than that in MIRI group,showing reduction of inflammatory cell infiltration,mild edema,and fewer necrotic cardiomyocytes.(3)TTC staining showed that there was no myocardial infarction in the rats in sham group,while there were significant myocardial infarction in the MIRI group and NC-antagomir group.The myocardial infarction area in miR-93 antagomir group was significantly lower than that of NC-antagomir group(P<0.05).(4)The serum levels of CK-MB,LDH and cTnI in MIRI group were significantly higher than that in sham group(P<0.05),but no significant difference was found between MIRI group and NC-antagomir group.The serum levels of CK-MB,LDH and cTnI in the miR-93 antagomir group were significantly lower than those in MIRI group and NC-antagomir group(P<0.05).It suggested that inhibiting miR-93 could reduce the degree of myocardial injury.(5)The oxidative stress index results showed that compared with sham group,MDA level in MIIRI group increased,while SOD and GSH-Px activity decreased.Compared with MIRI group and NC-antagomir group,the level of MDA miR-93 antagomir group was decreased,while the activity of SOD and GSH-Px were significantly increased(P<0.05).It suggested that inhibiting miR-93 could inhibit oxidative stress in rat myocardial tissue.(6)TUNEL assay showed that there was fewer apoptotic cardiomyocytes in sham group,while apoptosis index in MIRI group was significantly increased(P<0.05).The apoptosis index of cardiomyocytes in miR-93 antagomir group was significantly lower than that in NC-antagomir group(P<0.05).(7)Western blot results showed that the expression of caspase-3,cleaved caspase-3 and Bax proteins were significantly increased,while the expression of Bcl-2 protein was significantly decreased(P<0.05).The expression of caspase-3,cleaved caspase-3 and Bax proteins in the myocardial tissue in miR-93 antagomir group were significantly lower,while the expression of Bcl-2 protein was significantly higher than those in NC-antagomir group and MIRI group(P<0.05).The results suggested that inhibiting miR-93 could inhibit the apoptosis of cardiomyocytes in rats.Conclusions:(1)In the MIRI rats,the expression of miR-93 was high,while the expression of Nrf2 was low,showing a negative correlation.(2)Inhibiting miR-93 could reduce ischemia-reperfusion injury and the oxidative stress response in myocardial tissue in MIRI rats.(3)In the MIRI rats,inhibiting miR-93 could inhibit the apoptosis of myocardial cells and reduce myocardial ischemia-reperfusion injury.The mechanism may be related to the negative regulation of Nrf2 signaling pathway by miR-93. |
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