Inhibition Of Soluble Epoxide Hydrolase With RNA Interference Influence Acute Myocardial Ischemic Necrosis

Background and ObjectivesAcute myocardial ischemic necrosis has negative impact on systolic and diastolic function of heart, and leads to heart failure, arrhythmias and sudden death. In recent years, inhibition of soluble epoxide hydrolase (sEH) has been found a promising therapeutic strategy for cardiovascular disease. Several researches have suggested that inhibition of sEH facilitates the recovery of myocardial contractile function due to myocardial ischemia, and reduces myocardial infarct size (IS), ameliorates ventricular remodeling, and protects from developing pressure overload-induced heart failure. Previous studies have suggested that inhibition of sEH can result in beneficial effects via reduction in cellular apoptosis following myocardial infarction. However, there are seldom reports about application of RNA interference (RNAi) technology to inhibiting sEH, and then plays protective role in cardiovascular disease. RNAi is a highly specific and effective mechanism of post-transcriptional gene silencing mediated by double-stranded small interfering RNA (siRNA). SiRNA molecules is capable of specifically degrading the target mRNA to effectively inhibit the subsequent protein expression, only a much lower concentration of siRNA is needed. The effects of siRNA on mRNA degradation are efficient and specific.In this study, plasmids containing specific sEH siRNA sequences have been constructed and transferred into the primary cultured mice cardiomyocytes, to select the siRNA plasmids having the best silence effect to sEH. And to investigate the effects of inhibiting sEH on cardiomyocytes apoptosis induced by isoproterenol (ISO). A myocardial ischemic necrosis model of mice was established by enterocoelia injection of high dose isoproterenol, before that the mice were treated with the siRNA plasmids. We measure the expression of sEH, and the expression of apoptosis-related genes, such as Bcl-2, Bax in myocardium of mice. Myocardial ischemia and ventricular remodeling after inhibiting sEH have been investigated in present study. Possible mechanisms also have been explored.Part one:Isolation and identification of the primary cultured BALB/c mice neonatal cardiacmyocytesObjective:To establish a method for isolating, purifying and culture primary cardiomyocytes from neonatal BALB/c mice.Method:Myocardial tissues from neonatal BALB/c mice 1 to 3 day old were digested with 0.125% trypsin and 0.1% collagenase I. The cardiomyocytes were collected in DMEM medium, which contains 15% fetal bovine serum. Purification of cardiomyocytes was exerted by differential adhesion and the use of 5-bromodeoxyuridine. Immunohistochemistry staining of a-actin was used to identify cardiomyocytes.Results:Some cardiomyocytes began to beat spontaneously after 16-24 hours culture. After 2-3 days culture, all cells got together connected gradually, beat rhythmically and synchronously. By immunohistochemistry staining of a-actin, almost all cells were confirmed as cardiomyocytes.Conclusion:High quantity and survival primary cardiomyocytes of BALB/c mice could be effectively and reliably cultured by this method.Part two:Knockdown of soluble epoxide hydrolase by RNA interference in mice cardiomyocytesObjective:To construct target at soluble epoxide hydrolase (sEH) gene-specific recombinant plasmids pSUPER.retro.neo and selectively knockdown the expression of sEH in primary mice cardiomyocytes by RNA interference (RNAi), in order to select the plasmids having the best silence effect to sEH.Method:Two pairs of siRNA sequences that target at sEH gene were designed, in order to construct the siRNA plasmids specific to sEH gene (EH-1, EH-2). There were plasmids carrying a nonspecific siRNA coding sequence (PCN plasmids) used as the negative control group (PCN group) and the blank control group (c group). Then the plasmids were transfected into primary cultured mice cardiomyocytes by FuGENE HD. The mRNA and protein expressions of sEH were analyzed by RT-PCR and Western blotting respectively.Result: The expressions of mRNA and protein of sEH of EH-2 group were significantly decreased than that of control group, PCN and EH-1 group (P<0.01). There were no difference of expressions of mRNA and protein of sEH in control group, PCN and EH-1 group. EH-2 had the best silence effect to sEH. Therefore it was renamed as EH-R.Conclusion:Recombinant siRNA plasmids had been successfully constructed. RNA interference could selectively knockdown sEH expression in primary cultured cardiomyocytes.Part three:The effect of inhibition of soluble epoxide hydrolase with RNA interference on isoproterenol induced cardiomyocytes apoptosisObjective: To knockdown the expression of sEH in mice cardiomyocytes with RNA interference (RNAi) by EH-R. To investigate the effects of inhibition of sEH on apoptosis-related genes and isoproterenol (ISO) induced cardiomyocytes apoptosis.Method:The plasmid EH-R, which was specific to sEH gene and showed the best silence effect to sEH, was selected to knockdown the expression of sEH in mice cardiomyocytes. Plasmid carrying a nonspecific siRNA coding sequence (PCN) has been used as the negative control. Then the plasmids were transfected into primary cultured cardiomyocytes by FuGENE HD. ISO at concentration of 10μmol/L has been choosed to induce cardiomyocytes apoptosis. Cardiomyocytes were divided into four groups:control group, ISO group, PCN+ISO group and EH-R+ISO group.Rates of cardiomyocytes apoptosis were determined by flow cytometery. The protein expression levels of Bcl-2 and Bax were detected by Western blotting.Results:Compared to that of control, ISO treatement led to obviously increase cardiomyocytes apoptosis, decreased Bcl-2 expression level, and increased Bax expression level (P<0.01). However, compared to ISO group and PCN+ISO group, obviously decreased cardiomyocytes apoptosis, increased Bcl-2 expression level, and decreased Bax expression level have been found in EH-R+ISO group (P<0.01).Conclusion:EH-R could selectively knockdown sEH expression in cultured cardiomyocytes.It also signifcantly increased the expression of Bcl-2, and relieved cardiomyocytes apoptosis induced by ISO.Part four:The effect of inhibition of soluble epoxide hydrolase with RNA interference on acute myocardial ischemic necrosis induced by isoproterenolObjective: To investigate the influence of silencing soluble epoxide hydrolase (sEH) gene of BALB/c mice with RNA interference (RNAi) on myocardial ischemic necrosis and impared cardiac function induced by Isoproterenol (ISO). And observe the impact of inhibiting sEH on expression levels of apoptosis-related genes.Methods:A myocardial ischemic necrosis model of BALB/c mice was established by enterocoelia injection of isoproterenol (20mg/kg/d) for three days. After that BALB/c mice were divided into four groups:control group (c group, no ISO treat), myocardial ischemic necrosis group (MI group) and myocardial ischemia treated with negative plasmid group (PCN group), myocardial ischemia treated with EH-R plasmid group (EH-R group). Ejection fraction (EF%),fractional shortening (FS%),left ventricular end diastolic diameters (LVEDd) of mice heart were measured by echocardiography. Pathological changes of myocardial ischemic necrosis were observed through myocardial tissue stained with HE. The protein expression levels of Bcl-2,Bax and sEH were detected by Western blotting.Results:Compared with C group, the myocardial ischemic necrosis groups have increased Bax expression levels and decreased Bcl-2 expression levels (P<0.01), and myocardial ischemic necrosis was also more obvious, and showed impared heart function including EF%,FS% decreased, LVEDd increased (P<0.01). However compared with MI group and PCN group, EH-R group has decreased Bax expression levels and increased Bcl-2 expression levels (P<0.01), and myocardial ischemic necrosis size was significantly reduced, and EF%, FS% increased, LVEDd decreased (P<0.01).Conclusion:RNAi could inhibit the expression of sEH in vivo, and increased the expression of antiapoptotic gene, such as Bcl-2. In the end, it ameliorated myocardial ischemic injury and heart dysfunction induced by ISO.