OBJECTIVE:To investigate whether microRNA-489 could promote cardiomyocyte apoptosis through targeting inhibition of SPIN1, thus participating in the development of myocardial ischemia-reperfusion injury.MATERIALS AND METHODS:MicroRNA-489 expression in H9c2 cells induced with hypoxia/reoxygenation (H/R) was determined by quantitative real-time polymerase chain reaction (qRT-PCR). With microRNA-489 overexpression in H/R H9c2 cells, activities of lactate dehydrogenase (LDH), methane dicarboxylic aldehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were detected using relative commercial kits, respectively. The regulatory effect of microRNA-489 on the proliferation and apoptosis of H/R H9c2 cells was assessed through cell counting kit-8 (CCK-8) assay and flow cytometry (FCM), respectively. Through conducting a dual-luciferase reporter gene assay, we evaluated the binding condition between microRNA-489 and SPIN1. Protein expressions of apoptotic genes in H/R H9c2 cells with microRNA-489 overexpression were determined by Western blot. Finally, the regulatory role of microRNA-489 in PI3K/AKT pathway was detected through Western blot.RESULTS:QRT-PCR data showed that microRNA-489 was highly expressed in H/R H9c2 cells than those in normoxic control. Overexpression of microRNA-489 in H/R H9c2 cells increased the activities of LDH, MDA and GSH-PX, while decreased the activities of SOD. MicroRNA-489 overexpression markedly inhibited the proliferative rate but accelerated apoptosis of H/R H9c2 cells. Western blot results indicated that protein expressions of pro-apoptotic genes Bax and cytochrome C upregulated, whereas anti-apoptotic gene Bcl-2 downregulated after overexpression of microRNA-489 in H/R H9c2 cells. We confirmed that microRNA-489 could target to SPIN1 and inhibit its expression. After overexpression of microRNA-489 in H/R H9c2 cells, PI3K/AKT pathway was inhibited, which was further reversed by PI3K/AKT pathway agonist SC79. Besides, SC79 treatment also reversed the regulatory effect of overexpressed microRNA-489 on cellular behaviors of H/R H9c2 cells.CONCLUSIONS:MicroRNA-489 inhibits the proliferation and accelerates apoptosis of cardiomyocytes after MIRI by targeting inhibition of SPIN1 via inactivating PI3K/AKT pathway. MicroRNA-489 may be a potential therapeutic target for MIRI.

Recent studies indicate that over-activation of Cdk5 is a crucial pro-death signal and Cdk5 activity inhibition provides neuroprotection in animal stroke models. However, Cdk5 inhibitors are reported to affect physiological functions of Cdk5 and lead to serious side effects. Therefore, targeting Cdk5 or its activators without affecting physiological functions of Cdk5 is a therapeutic strategy for ischemic brain injury. In this study, we examined Cdk5 activity in a rat hypoxia/ischemia (HI) injury model. Cdk5 expression was not changed after HI injury, but Cdk5 activity significantly increased, which was demonstrated by the increased phorsphorylation-phosphorylation of Tau and glucocorticoid receptor (GR), two downstream signals of Cdk5. We further showed that the levels of Cdk5 activators p35 and p39 decreased after HI injury, while p25, which is converted from p35 and has a higher activator activity on Cdk5, increased markedly after HI injury. P5, a 24-residue mimetic peptide of p35, was reported to specifically inhibit the p25/Cdk5 signal pathway in an Alzheimer's disease model. P5-TAT, which can cross the blood-brain barrier and cell membrane facilitated by TAT protein, was used in our study. We found that p5-TAT treatment did not change the levels of p35, p39, and p25, but reduced the phorsphorylation of Tau and GR, suggesting the inhibition of the p25/Cdk5 by the peptide p5-TAT. This was supported by the fact that p5 interacted with Cdk5, but not with Cdk5 activators. In addition, p5-TAT reduced cleaved caspase-3 level, a marker of neuronal apoptosis. We further demonstrated that p5-TAT pre-treatment reduced cerebral infarct volume; even when p5-TAT was delayed to be administered at 24h after HI injury, p5-TAT still promoted long-term functional recovery. Therefore, Cdk5 inhibition by the small peptide p5-TAT or its derivatives is a promising therapeutic strategy for the treatment of ischemic brain injury including hypoxic-ischemic encephalopathy and stroke.

Objective. This study was designed to investigate the effect of the overexpression of heme oxygenase-1 (HO-1) on the immunoregulation in the model of abdominal cardiac xenotransplantation from the guinea pig to the rat.Materials and methods. To increase the expression of HO-1, both donors and recipients were injected with heme through the abdomen before the operation. The donors (guniea pigs) and the recipients (Sprague-Dawley [SD] rats) were divided randomly into three groups: group A, the heart from a guinea pig transplanted into the abdomen of an SD rat; group B, the recipients were injected with Chinese cobra venom factor (CVF) into the abdomen (40 mu g/kg and 60 mu g/kg 24 hours later) prior to transplantation; group C, CVF + HO-1 high-expression group: donors and recipients were abdominally injected with heme (75 mu mol/kg for 2 days before transplantation). The mean survival time (MST), pathological changes, the positive area of HO-1 in the grafted hearts, as well as the expressions of C-C chemokine receptor 5 (CCR5), intercellular adhesion molecule-1 (ICAM-1), and natural killer (NK) cell activity in recipients.Results. 1. The MST was longest in group C treated with heme. 2. The pathologic changes of hyperacute rejection were showed on the donor heart in group A, while delayed xenograft rejection changes took place on donor heart in other groups. 3. Compared with group B, The HO-1 positive area in the donor hearts of group C was significantly higher. (P < .05). 4. The lever of ICAM-1 and CCR5 in the peripheral blood of recipients (pg/mL) was attenuated in group C injected with heme. 5. Compared with group B, the activity of NK cell in the peripheral blood of recipients was much lower in group C (P < .05).Conclusion. The MST was prolonged by increasing expressions of HO-1, but acute vascular rejection was not completely overcome. Activation of vascular endothelial cells could be decreased by strengthening the expression of HO-1. NK cell activity was weakened by reinforced expression of HO-1.

The nuclear factor-kappaB (NF-κB) in cardiac vascular endothelial cells (type II VEC) is a key factor that activates delayed xenograft rejection (DXR), and therefore inhibition of NF-κB gene expression may alleviate post-transplant rejection. siRNA technology was used to inhibit NF-κB p65 gene expression in ICR mice. After jugular vein injection of siRNA/in vivo-jetPEI complex, fluorescence levels of FAM-labeled siRNA in hearts and lungs were much higher after jugular vein injection than tail vein injection, suggesting more efficient siRNA delivery to the heart through the jugular vein. The amount of FAM fluorescence of hearts increased to the highest level between 48 and 72 hours after injection, and decreased gradually 1 week after injection. A minimum dose of 6 nmol NF-κB p65 siRNA and a siRNA/in vivo-jetPEI ratio of 6 (N/P = 6) were required for in vivo siRNA-mediated gene silencing in the heart. Under these conditions, application of siRNA/in vivo-jetPEI complexes from the jugular vein successfully suppressed NF-κB p65 expression in the heart. The same strategy can be applied to heart transplant animal models to protect against NF-κB gene-related type II VEC activation and xenograft rejection.