| Objective:1.investigating how pretreatment cultured primary neonatal mice cardiomyocytes which is simulated myocardial ischemic with soluble epoxide hydrolase inhibitor, t-AUCB(trans-4-(4-(3-adamantan-1-yl-ureido)-cyclohexyloxy)-bensoic acid) influences inward rectifying potassium current(IKi).2. investigating the mechanism of t-AUCB regulate miR-1, to study whether PI3K/AKT pathway participates regulate microRNA-1.MethodsPart of the in vitro:We choose neonatal Kunmin mice, and culture primary cardiomyocytes and then deal with these cells on different conditions. The cultured cardiomyocates were divided into five groups:(l) control group (2)ago-miR-1group (3) ago-miR-1+t-AUCB group (4) ago-miR-1-NC group (5) ago-miR-1+DMSO group. At last we record IKi current in whole cell model.Part of the in vitro: 1. The Kunming male mice of8weeks old were divided into five groups:(1)drinking water+sham group (2) drinking water+myocardial infarction(drinking water+MI)(3) t-AUCB0.2mg/L+myocardial infarction(t-AUCB0.2mg/L+MI)(4)t-AUCB lmg/L+myocardial infarction(t-AUCB1mg/L+MI)(5) t-AUCB5mg/L+myocardial infarction(t-AUCB5mg/L+MI).After24hours,we get the ischemic region of mice’s heart. Then the expressions ofp-AKT,p-GSK3β of these tissue were tested by Western Blot.2. The Kunming male mice of8weeks old were administered with water contain t-AUCB of concentrations5mg/L for7days.7days later, they were operated myocardial infarction, and after the surgery,they were tale-injected with micrONTM mmu-miR-la-3p agomir of micrONTM mmu-miR-la-3p agomir-negative control. They were divided into6groups:(1) drinking water+sham group (2) drinking water+myocardial infarction(drinking water+MI)(3) drinking water+myocardial infarction+tale-injected with agomiR-1(drinking water+MI+agomiR-1)(4) drinking water+myocardial infarction+tale-injected with agomiR-1Neg Ctl (drinking water+MI+Neg Ctl)(5) t-AUCB5mg/L+myocardial infarction(t-AUCB5mg/L+MI)(6) t-AUCB5mg/L+myocardial infarction*tale-injected with agomiR-1(t-AUCB5mg/L+MI+agomiR-1). After24hours,we get the ischemic region of mice’s heart. Then the expressions of p-AKT,p?-GSK3β of these tissue were tested by Western Blot.3.The Kunming male mice of8weeks old were administered with water contain t-AUCB of concentrations5mg/L for7days.7days later, they were operated myocardial infarction, and after the surgery,they were tale-injected with PI3K inhibitor wortmannin. They were divided into five groups:(1) drinking water+sham group (2) drinking water+myocardial infarction(drinking water+MI)(3) t-AUCB5mg/L+myocardial infarction(t-AUCB5mg/L+MI)(4)t-AUCB5mg/L+myocardial infarction+tale-injected with0.3mg/kg wortmannin (t-AUCB5mg/L+MI+0.3W (tale-injected))(5) t-AUCB5mg/L+myocardial infarction+intraperitoneal-injected with0.3mg/kg wortmannin (t-AUCB5mg/L+MI+0.3W (intraperitoneal-injected)). After24hours, we get the ischemic region of mice’s heart. The expression of KCNJ2, GJA1mRNA of ischemic tissue were determined by Real-time PCR.ResultsPart of the in vitro:1. The characteristic of I-V curve of the current we tested is with the increase of voltage the current getting close to X axis which is in accordance with the features of I-V curve of inward rectifying potassium current IK1· 2. At-100mV, the Ik1current density of the control group was-4.62±0.24pA/pV.3. At-100mV, the IK1-current density of the ago-miR-1group was-0.05±0.06pA/pV,compared with control group, IKi current density was markedly decreased and has statistic significance (n=6, p<0.05).4. At-100mV, the IK1. current density of the ago-miR-1+t-AUCB group was-2.71±0.12pA/pV,compared with ago-miR-1group, IKi current density was markedly increased and has statistic significance (n=6, p<0.05).5. At-100mV, the IK1current density of the ago-miR-1-NC group was-3.94±0.27pA/pV, compared with control group without statistic significance (n=6, p>0.05).6. At-100mV, the IK1current density of the ago-miR-1+DMSO group was-0.16±0.04pA/pV, compared with ago-miR-1group without statistic significance (n=6, p>0.05).Part of the in vivo1.Compared with the drinking water+sham group, the level ofp-AKT, p-GSK3β was significantly lower in the ischemic region of mice hearts (n=6, all p<0.05).2.t-AUCB can dose-dependently elevate the level of p-AKT, p-GSK3β in the ischemic region of mice hearts (n=6, all p<0.05). 3. AgomiR-1could further decrease the level of p-AKT, p-GSK3p in the ischemic region of mice hearts.However, t-AUCB with the concentration of5mg/L can reverse the effects of agomiR-1(n=6, all p <0.05).4. compared with the control group, t-AUCB with the concentration of5mg/L could elevate the expression of the KCNJ2,GJAlmRNA,but the PI3K inhibitor wortmannin could abolish the effects(n=6, all p<0.05)Conclusion:Part of the in vitro1.Ik1current density is markedly decreased when the miR-1was overexpressioned;2.t-AUCB can reverse the effects of the IK1induced by the overexpression of miR-1.Above all:t-AUCB can regulate Ik1current by regulate the target gene KCNJ2mRNA of miR-1.Part of the in vivo1.The level of the p-AKT, p-GSK3β is decreased in the ischemic region of mice hearts.2.t-AUCB can dose-dependent reverse the decrease of the level of p-AKT,p-GSK3β in the ischemic region of mice hearts.3.micrONTM mmu-miR-la-3p agomir could make a further decrease of the level of p-AKT,p-GSK3β in the ischemic region of mice hearts.However, t-AUCB with the concentration of5mg/L can reverse the effects of micrONTM mmu-miR-1a-3p agomir.4.t-AUCB with the concentration of5mg/L can elevate the expression of the KCNJ2,GJA1mRNA,but the PI3K inhibitor wortmannin can reverse the elevated of the mRNA. Above all:t-AUCB regulate PI3K pathway via mir-1antiarrhythmias... |
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