| BackgroundSince Jenning reported the phenomenon of ischemia-reperfusion injury in 1960, this problem has become a severe challenge for doctors in the treatment area of myocardial infarction. In order to attenuate ischemia reperfusion and finally reduce infarct size of myocardium, making every effort to secure the cardiomyocytes, lots of interventions had been made in the clinical practice and experiments. In 1986, a significant breakthrough had been made in the field of IRI—ischemic preconditioning (IPC), which is considered to be the most effective intervention against IRI. But due to the unprediction of ischemic time and ethical constraint, ischemic preconditioning is confined in clinical application. The myocardium protection mechanism hasn’t been clarified. Therefore, researchers have never stopped investigating the internal mechanism of IPC, which is of great significance in prevention and treatment of ischemia/anoxia heart disease.As an endogenously adaptive mechanism of cells, moderate endoplasmic reticulum stress (ERS) activates the unfolded protein response (UPR), which triggers a series of signaling transduction pathways and increases the synthesis of chaperones, elevating cellular resistance to injury and promoting cells survival. When cells undergo irreversible ER stress and the homeostasis of the ER can not be restored, the UPR induces apoptosis of the damaged cells. IRE1-XBP1 is the most conserved ER stress adaptive signaling pathway across species. Its opening determins the fate of cells. XBP1 is an important transcription factor involved in myocardial protection of IRI. Rearchers found that inhibition of miRNA-30c-2-3p could increase the expression of XBP1 and attenuate cells death induced by ERS.Until now, there is no rearch whether miRNA-30c-2-3p and XBP1 paticipate in the underlying mechanism of IPC, providing cardioprotection. As a newly endogenous gene regulator, miRNA involved in signal regulatory pathways of the ERS, plays an important role in the cardioprotection of ischemia/anoxia preconditioning. We speculate that XBP1 induced by endogenous miRNA-30c-2-3p during preconditioning process contributes to protect cardiomyocytes against ischemic injury. And can we attenuate ischemia-reperfusion cell death in cardiomyocytes by down-regulating miRNA-30c-2-3p to increase the expression of transcription factor XBP1?In order to clarify this hypothesis, this in viro experiment was designed. The aim of this study was to make the roles of miRNA-30c-2-3p and XBP1 in the cardioprotection of anoxic preconditioning clearly, and evaluate that the inhibition of endogeneous miRNA-30c-2-3p modulating the expression level of XBP1 affected the cardiomyocytes against anoxia/reoxygenation injury. This experimental study provided an important evdience to the myocardial protection interventions and related drug discovery. It was divided into three parts:Part 1:Using primary cultures of neonatal rat cardiomyocytes to establish in vitro cardiomyocyte anoxia/reoxygenation injury model and anoxia preconditioning modelTo explore a mature, stable with high purity method for primary culture of neonatal rat cardiomyocytes in vitro and establish rat cardiomyocyte anoxia/reoxygenation injury model and anoxia preconditioning model with anaero-pack system combined with nitrogen.Neonatal SD rat cardiomyocytes were isolated from 1-to 3-day-old Sprague-Dawley rats using 0.125% trypsin and 0.1% type 1 collagenase digestions under 35℃ and purified by differential velocity adherent technique for 70 min with 5-BrdU treatment. The purity of cardiomyocytes was identificated by cardiac troponia T-SABC-Cy3 (POD) immunohistochemical with DAPI staining.The in viro neonatal rat cardiomyocytes anoxia/reoxygenation injury model was established by anaero pack combined with nitrogen.Cardiomyocytes cultured for 72 h were randomly divided into 7 groups:Sham group(control group), cultured as normal; 1h group, only anoxia for 1 h; 1h/6h group, anoxia for 1 h followed by 6 h reoxygenation; 3 h group, only anoxia for 3 h; 3h/6h group, anoxia for 3 h followed by 6 h reoxygenation; 6 h group, only anoxia for 6 h; 6 h/6 h group, anoxia for 6 h followed by 6 h reoxygenation. At the end of the experiment, lactate dehydrogenase (LDH) release rate was detected by the LDH Release Assay Kit and myocardial apoptosis was detected by the dead cell apoptosis Kit with Annexin V/propidine iodide (Annexin V/PI) for flow cytometry.The in viro neonatal rat cardiomyocytes anoxia preconditioning model was estabished by anaero pack combined with nitrogen. Cardiomyocytes cultured for 72 h were randomly divided into 6 groups:Sham group(control group), cultured as normal; AR group, anoxia for 3 h by 6 h reoxygenation; 15min*1 group, anoxia for 15 min followed by 15 min reoxygenation before AR (anoxia for 3 h by reoxygenation 6 h); 15min*2 group, two cycles of anoxia for 15 min followed by 15 min reoxygenation before AR; 30min*1 group, anoxia for 30 min followed by 30 min reoxygenation before AR; 30min*2 group, two cycles anoxia for 30 min followed by 30 min reoxygenation before AR; At the end of the experiment, lactate dehydrogenase (LDH) release rate was detected by the LDH Release Assay Kit and myocardial apoptosis was detected by the dead cell apoptosis Kit with Annexin V/propidine iodide (Annexin V/PI) for flow cytometry.Neonatal cardiomyocytes were active and contracted strongly. After cultured for 48-72h, cardiomyocytes contracted synchronously. The purity of cardiomyocytes was up to93.9±2.2%.The results from cardiomyocytes anoxia/reoxygenation injury models showed that compared to Sham group, the rate of LDH release and apoptosis were significantly higher, but the rate of normal cell was significantly lower in 1 h,1 h/6 h,3 h,3 h/6 h,6 h and 6 h/6 h groups. As compared to 1h group, the LDH release rate and apoptosis were higher, but the rate of normal cell was lower in 3 h and 6 h groups. There were not significantly differences in the LDH release rate, apoptosis, and the rate of normal cell between 1 h/6 h group and 1h group. As compared to 3 h group, the LDH release rate, apoptosis was significantly increased, but the rate of normal cell was significantly decreased in 6 h and 3h/6h groups. The LDH release rate, apoptosis, and the rate of normal cell did not differ between 6h/6h and 6h groups.The results from cardiomyocytes anoxia preconditioning models showed that compared to Sham group, the rate of LDH release and apoptosis were significantly higher, but the rate of normal cell was significantly lower in 15min*1,15min*2,30min*1, 30min*2 and AR groups. As compared to 30min*1 group, the LDH release rate and apoptosis were higher, but the rate of normal cell was lower in 15min*1,15min*2, 30min*2 and AR groups. There were not significantly differences in the LDH release rate, apoptosis, and the rate of normal cell between 15min*1 group and AR group. As compared to 15min*2 group, the LDH release rate and apoptosis were significantly increased, but the rate of normal cell was significantly decreased in AR groups; As compared to AR group, the LDH release rate and apoptosis were significantly increased, but the rate of normal cell was significantly decreased in 30min*2 group, and the LDH release rate, apoptosis was significantly decreased, but the rate of normal cell was significantly increased in 15min*2 group.Part 2:The roles of XBP1 and miRNA-30c-2-3p in mechanism of cardioprotection induced by anoxia preconditioning and targeted relationship between miRNA-30c-2-3p and XBP1The objective of this part study was to clarify the roles of XBP1 and miRNA-30c-2-3p in mechanism of cardioprotection induced by anoxia preconditioning and to verify whether XBP1 was one of the target genes of miRNA-30c-2-3p.The cultured cardiomyocytes were randomly divided into 2 groups:AR group, anoxia for 3 h followed by 6 h reoxygenation; APC group, anoxia for 30 min followed by 30 min reoxygenation before AR. At the time before AR, reoxygenation for 1 h,3 h and 6 h the expression of XBP1 mRNA, XBPls protein and miRNA-30c-2-3p were detected by real-time quantitative PCR and Western blotting.Cardiomyocytes were transfected with different concentrations of lentivirus carried on the miRNA-30c-2-3p antisense oligonucleotide or nonsense oligonucleotide for 48 h. Then, the best infection concentration of lentivirus was determined and the expression of XBP1 mRNA, XBPls protein and miRNA-30c-2-3p at the time of before AR, after reoxygenation 1 h,3 h and 6 h ware detected by using realtime-PCR and Western blotting.To measure a direct interaction between miRNA-30c-2-3p and its potential bi-nding site within XBP1 mRNA by using the dual luciferase report gene detection. The psiCHECKTM-2-reporter-XBP1-3’UTR-wt (XBP1-3’UTR-wt) vector or psiCHE-CKTM-2-reporter-XBP1-3’UTR mut (mut 3’UTR) vector or psiCHECKTM-2 was c o-transfected into H9c2 cells along with miRNA-30c-2-3p mimics or miRNA neg-ative control (miRNA-NC) and assayed for expression of a dual luciferase report-e r. It devided into 6 groups:①NC mimics+psiCHECKTM-2; ②NC mimics+psiC-HE CKTM-2-Xbp1-3’UTR-wt; ③NC mimics+psiCHECKTM-2-Xbpl-3’UTR-mut ④rno-miR-30c-2-3p mimics+psiCHECKTM-2﹔⑤no-miR-30c-2-3p mimics+psiCHE-CKTM-2-Xbp 1-3’UTR-wt; ⑥rno-miR-30c-2-3p mimics+psiCHECKTM-2-Xbp1-3’UTR-mu-t.The results showed that compared to before AR, the expression of XBP1 gene, XBP1s protein and miRNA-30c-2-3p were significantly increased after reoxygenation 1 h,3 h and 6 h in AR group, and peaked at the time of after reoxygenation 3 h. Compared to AR groups, the expression of XBP1 gene, XBP1s protein were significantly elevated and miRNA-30c-2-3p expression was significantly reduced after reoxygenation 1 h,3 h and 6 h in APC group, and peaked at the time of after reoxygenation 3 h.Using a fluorescence microscope to observe the cardiomyocytes infected with lentivirus. The proportion of GFP positive cells increased along with the increasing concentration of infected lentivirus. When infected with MOI 50, positive GFP cells took up more than 90%, there was not statistical difference between MOI 50 and MOI100. Thus, MOI 50 was determined as the most suitable concentration of lentivirus infection. After infected with lentivirus carried on miRNA-30c-2-3p inhibitor, the expression level of miRNA-30c-2-3p was significantly decreased and XBP1 was significantly increased before AR, after reoxygenation for 1 h,3 h and 6 h, the level of XBP1s protein was increased after reoxygenation for 1 h,3 h and 6 h.XBP1-3’UTR vector-transfected H9c2 cells showed a distinct decrease in luciferase activity when co-transfected with miR-30c-2-3p mimics, while no significant changes in luciferase activity was observed following the co-transfection of mut 3’UTR vector with miR-30c-2-3p mimics or miRNA-NC mimics.Part3:The study on the protection with the regulation of XBP1 by miRNA-30c-2-3p against in vitro rat cardimyocytes anoxia/reoxygenation injuryThe objective of this part study was to evaluate whether modulating XBP1 by inhibiting endogenous miRNA-30c-2-3p can protect cardiomyocytes against cardiomyocyte anoxia/reoxygenation injury and to futher clarify the relationship between miRNA-30c-2-3p and XBP1 in the cardioprotection mechanism of anoxia preconditioning.According to the different treatments, the cultured cardiomyocytes were randomly divided into 4 groups:Sham group, cultured as normal; AR group, anoxia for 3 h followed by 6 h reoxygenation; APC group, after anoxia for 30 min followed by 30 reoxygenation, then anoxia for 3 h followed by 6 h reoxygenation; INH group, infected with lentivirus miRNA-30c-2-3p inhibitor for 48 h, then anoxia for 3h followed by 6 h reoxygenation. Except INH group, the other three groups were infected with lentivirus miRNA-NC. At the end of the experiment, lactate dehydrogenase (LDH) release rate was detected by the LDH Release Assay Kit and cardiomyocyte apoptosis was detected by the dead cell apoptosis Kit with Annexin V-APC/7-AAD for flow cytometry. The expression of miRNA-30c-2-3p and XBP1 mRNA were detected by realtime-PCR, and XBP1s and BiP protein expression were detected by western blotting respectively.The results showed that compared to Sham group, both the rate of LDH release and apoptosis were significantly increased, but the rate of normal cell were significantly reduced in AR, APC and INH groups. Compared to AR group, the rate of LDH release was significantly decreased and apoptosis was significantly increased but the proportion of normal cells was significantly decreased in APC and INH groups. Compared with APC group, the rate of LDH release was significantly increased and apoptosis were significantly increased, but the rate of normal cell was significant lower in INH group. Compared with the Sham group, APC and AR groups’expression of miRNA-30-2-3p were significantly increased and INH group’s expression was significantly decreased. Compared with Sham group, other three groups’expression of XBP1 were significantly increased. Compared to the AR group, the expression of XBP1 was increased in APC and INH groups; Compared to the APC group, the expression of XBP1 were significantly increased in INH group.Compared with the Sham group, other three groups’expression of XBPls significantly increased;Compared to the AR group, the expression of XBPls was increased in APC and INH groups; Compared to the APC group, the expression of XBPls were significantly decreased in INH group.The expression tendency of BiP protein as same as XBP1s. The other three groups were significantly higher than the Sham group; Compared to the AR group, the expression of BiP was increased significantly in APC and INH groups; Compared to the APC group, the level of BiP was significant lower in INH group.Conclusions:Based on the results of all experiments, the following conclusions can be drawn:1. An in vitro rat cardiomyocytes anoxia/reoxygenation injury model can be successfully established by the means of anoxia for 3 h followed by 6 h reoxygenation and in vitro cardiomyocytes anoxia preconditioning model can be successfully established by the means of anoxia 30 min followed by reoxygenation 30 min before anoxia/reoxygenation injury with an anaero-pack combined with nitrogen. Anoxia preconditioning can provide cardioprotection and attenuate the cells apoptosis in vitro rat cardiomyocyte against anoxia/reoxygenation injury.2. XBP1 mRNA is a functional target gene of miRNA-30c-2-3p in rat cardiomyocytes. Expression of miRNA-30c-2-3p is down-regulated while XBP1 level is increased in rat cardiomyocytes by anoxia preconditioning compared with anoxia/reoxygenation injury,suggesting that anoxia preconditioning results in cardioprotection by inhibiting miRNA-30c-2-3p and increasing endoplamic reticulum stress related XBP1.3. Inhibition of endogenous miRNA-30c-2-3p can increase the expression of XBP1, leading to significant reduction of cardiomyocyte apoptosis induced by anoxia/reoxygenation injury.4. Expression of BiP can be affected by the modulation of miRNA-30c-2-3p to XBP1, indicated that BiP maybe one of the XBP1 downstream factors to protect cardiomyocytes from apoptosis. |
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