Regulation Of MiR-497 And PPARδ/β On Neural Injury After Brain Ischemia

Cerebral vascular diseases are the third leading cause of death in the world, among which over 70% are ischemic stroke. Because of limited on treatment, the prevalence of ischemic stroke bears a burden individually and universally by severe outcome. Lots of factors including exitotoxicity, increasing of free radical spices, and the inflammation are believed to contribute to the pathology of stroke. Therefore, new therapeutic methods are valuable for clinical service.There are two parts in the thesis, which shows the study about the regulation of miR-497 and the effects of peroxisome proliferator-activated receptorδ/β(PPARδ/β) on neural injury after brain ischemia.MicroRNA (miRNA) is a group of short and non-coded RNA that involves in physiology, pathology and diseases of central nervous system (CNS) especially. In Part I, the regulation of miR-497 on neural injury after brain ischemia was studied. By real-time PCR, we identified the miR-497 was increased dramatically after middle cerebral artery occlusion (MCAO) model in mice. In order to study the effect of miR-497 in cells apoptosis, we employed oxygen glycogen deprivation (OGD) model to induce cells death. We determined that miR-497 contributed to neruo2a (N2A) cellular line apoptosis in OGD model by down regulating Bcl-2 and Bcl-w. With luciferase assay, we proofed that miR-497 bond to the 3'UTR of Bcl-2 and Bcl-w. To conclusion, in the in vitro data, we confirmed that miR-497 accelerated apoptosis after OGD model by degrading Bcl-2 and Bcl-w, which are key anti-apoptosis genes in neural protection. In order to study the role of miR-497 in neural injury after brain ischemia, we silenced the miR-497 in brain by using miR-497 antagomir. Through the lateral ventricle injection, miR-497 antagomir effectively inhibited the expression level of miR-497 in brain, but the proteins of Bcl-2 and Bcl-w increased. MCAO models were performed 24h after injection of miR-497 antagomir, then the brain was incised to 8 slices at the 23rd hour following the model operation. All of the section samples were stained with triphenyltetrazolium chloride (TTC). By calculating total infarct volume, cortex infarct volume and subcortex volume, we found that the treatment of miR-497 antagomir decreased all of these parameters. At the same time, the treatment improved the neurobiological deficit on mice after MCAO. In this part, we investigated the role of miR-497 in neural apoptosis with in vitro and in vivo models and discovered its pro-apoptosis function. The regulation of miR-497 on Bcl-2 and Bcl-w, which are the key genes regulating the mitochondrial apoptosis pathway, was meaningful in treatment of brain ischemia.Peroxisome proliferator-activated receptorδ/β(PPARδ/β) is a nuclear receptor by acting as a transcriptional factor. PPAR8/P plays an important role in energy regulation as well as it is shown that activating of PPARδ/βrescues endothelial cells on several apoptosis models. Considering the function of cerebral vascular endothelial cells after stroke, which maintain blood-brain barrier, we investigated the role of PPARδ/βin ischemia-induced endothelial injury in Part II. After lateral ventricle injection of GW501516 (a kind of PPARδ/βagonist), the infarct volume of brain in mice was significantly decreased comparing with sham group. By intravenous injection of Evans blue, the experiments showed that the permeability of microvascular vessels in modeled brain was protected following the GW501516 treatment, which meant that the brain-blood barrier (BBB) was maintained by activating of PPARδ/β. The mechanism was suggested that the endothelial cells in microvascular vessels were less apoptosis comparing with sham group since the functional protein of gene Bcl-2 was increased whereas the activity of Capase-3 and DNA fragmentation were down-regulated. Moreover, the expression of miR-15a, of which target gene is Bcl-2, was decreased after GW501516 treatment. Therefore, it was implied that the activating of PPARδ/βprotect brain tissue after MCAO neural-injury by inhibiting the apoptosis of endothelial cells.PartⅠThe regulation of miR-497 on neural-injury after brain ischemiaObjectiveCurrently, the mechanism of neural apoptosis following the brain ischemia is unclear yet. Therefore, the effective treatment of brain ischemia is limited. It was reported that the miRNAs were involved in multi physiology and pathology processes including apoptosis, especially the miRNAs changed dramatically after cerebral ischemia. However, the detail mechanism of these changes and what kinds of specific miRNA involved in stroke are not well known until now. In this part, we investigated the regulation mechanism of miRNA on brain ischemia.Methods1. At the 23rd hour after MCAO, the brain tissue on the penumbra was detected by Western Blot with multi apoptosis-related miRNAs, which were compared with sham group.2. Oxygen and glucose deprivation (OGD) model was performed when the Neuro2A (N2A) cells were in 70-80% confluence. At Oh,4h and 24h after OGD, the cells apoptosis were detected by LDH (lactate dehydrogenase) and MTT (3-[4,5-dimethythiazol-2-y1]-2,5-diphenyltetrazolium bromide), and also the miR-497 or Bcl-2/Bcl-w were respectively detected by real-time PCR or Western Blot.3. Transferred the miR-497 mimic or miR-497 inhibitor into the N2A, the protein level of Bcl-2 and Bcl-w in the cells was then examined. Or the cells were in exposure to OGD and then the apoptosis situation were assayed by LDH and MTT at Oh,4h and 24h following the OGD.4. Plasmids included 3'UTR and mutant of 3'UTR on Bcl-2 or Bcl-w were constructed into a Luciferase vector. After Co-transfer the vector with miR-497 mimic, fluorescence were examined 48h later.5. At the 23rd hour after MCAO, the brain tissue on penumbra were accessed for detecting protein of Bcl-2 and Bcl-2.6. According previous reports, the antisense oligonucleotides for silencing miR-497 was generated and named as miR-497 antagomir.7. Continuous intracerebroventricular injection of miR-497 antagomir lateral ventricle was performed on mice at the 24th hour before MCAO with micro-pump. There were three groups in the experiment including CSF (cerebrospinal fluid, as Sham), eGFP (enhanced green fluorescin protein, as control), and antagomir (as treatment).8. At the 24th hour after injection, brain tissue was isolated for detecting expression of miR-497 and protein level of Bcl-2 and Bcl-w.9. At the 23rd hour after MCAO, the brains of mice were sliced into 8 sections and then they were stained with TTC. Calculating the infarct volume of total area, cortex, and subcortex.10. At the 23rd hour after MCAO, neurobiological deficit was evaluated by method of "5 score 6 grades".11. A two-tail t-test was employed for the statistics evaluation when make a comparison between two group. One way ANOVA following Bonferroni's post-hoc test was employed when comparison among different groups. Kruskal Wallis test was employed when analysis the neurobiological deficit among groups.Result1. At the 23rd hour after MCAO, the expression of miR-497 increased in the brain penumbra. At the 24th hour after OGD, N2A generated more miR-497 than non-OGD;2. Over-expression of miR-497 contributed to apoptosis of N2A with OGD. In the opposition, repression of miR-497 inhibited apoptosis of N2A.3. The fluorescence value decreased after co-transferring the miR-497 mimic with 3'UTR of Bcl-2 and Bcl-w, however, the mutant of 3'UTR on Bcl-2 and Bcl-w didn't show the decrease.4. The protein level of Bcl-2 and Bcl-w was down-regulated after MCAO;5. With the miR-497 antagomir treatment, the miR-497 expression was decreased in brain whereas the protein of Bcl-2 and Bcl-w were increased;6. With the miR-497 antagomir treatment, at the 23rd hour after MCAO model, the infarct volumes of total area, cortex, and subcortex in brain were decreased comparing with Sham and control.7. With the miR-497 antagomir treatment, at the 23rd hour after MCAO, the neurobiological deficit was improved in the model mice.Conclusion1. Both the OGD in vitro and MCAO in vivo increased the expression level of miR-497 in N2A neural cells or brain;2. The miR-497 contributed the apoptosis of N2A neural cells in OGD. The mechanism was suggested as the degradation of target genes including Bcl-2 and Bcl-w.3. Repression of miR-497 improved the ethology of mice after MCAO.PartⅡActivation of PPARδ/βprotects ischemia-induced endothelial injuryObjectivePPARδ/βbelongs to the first subfamily of nuclear receptors, and the member 2 in group C (NR1C2). There are three members such as a, y, andδ/βin PPAR family, which show the different function and distribution respectively. PPAR8/P can be activated by fatty acid and its metabolites. The main functions of PPARδ/βwere discovered as the accelerated fatty acid metabolism, the increased energy expenditure and the improved insulin resistance. GW501516, the agonist of PPARδ/β, has been used as the clinical trials on treatment of hyperlipidemia, but its function in circulation system and CNS is unknown now.After occur of ischemia, the apoptosis of endothelial cells in the cerebral vascular vessel significantly contributed the damage of BBB(brain-blood barrier). Then the permeability increased dramatically, which coursed the unbalance of physiology situation of neurons. There was the report that activation of PPARδ/βcould protect endothelial cells in several apoptosis models. Here, we investigated the effects of GW501516 as activator of PPARδ/βon endothelial injury after brain ischemia.Methods:1. With stereotaxic apparatus, we injected the GW501516 into the lateral ventricle with micro-pump in a concentration of 10μg/μl at speed of 1μl/h. The injection was performed at the 24th hour before MCAO. There were three groups in this experiment, including Non-MCAO surgery as Sham, injection of CSF as control and injection of GW501516 as treatment.2. At the 23rd hour after MCAO, the brain was incised into 8 slices and then stained with TTC. The infarct volume was calculated.3. At the 23rd hour after MCAO,0.1ml of 4% Evans blue was injected intravenously through the mice tail vein. And then, the amount of Evans blue in the mice brain was detected at the 1st hour following injection.4. At the 23rd hour after MCAO, the brain tissue were isolated and protein level of Bcl-2, activity of Caspase-3 and expression of miR-15a were respectively detected.5. A two-tail t-test was employed for the statistics evaluation when make a comparison between two group. One way ANOVA following Bonferroni's post-hoc test was employed when comparison among different groups.Result1. Comparing with Sham group, the treatment of GW501516 decreased the infarct volume of brain in model mice.2. After the treatment of GW501516, there were less amount of Evans blue in the treated brain than in the control.3. At the 23rd hour after MCAO, the data showed that the activity of Caspase-3 in endothelial cells were decreased accompanying with GW501516 treatment and DNA fragmentation inhibit, comparing with the sham. Also it was found that the expression level of Bcl-2 protein was increased. However, the expression of miR-15a, of which target gene is as Bcl-2, was repressed.Conclusion1. GW501516 protected the brain tissue and the BBB after cerebral ischemia, comparing with the sham group.2. Activation of PPARδ/βrescued the endothelial cells after brain ischemia by increasing the express level of Bcl-2 protein, which maybe was the cause of miR-15a repression.