The Regulation Of PPARγ In The Injury Of Cerebral Ischemia/Reperfusion Or Hydrogen Peroxide Exposure

Objecitive: ① To examine the effects of 12/15-LOX-derived product 12s-hydroxyeicosatetraenoic acid(12s-HETE) on ischemia/reperfusion(I/R) injury in rats exposed to MCAO, and investigate the underlying mechanism of 12s-HETE through activation of PPARγ. ② To examine the regulatory effect of 12/15-LOX pathway on PPARγ in primary cultured cortical neurons exposed to oxygen-glucose deprivation(OGD). ③ To examine the negative regulation of hydrogen peroxide(H2O2) on PPARγ in primary cultured cortical neurons, especially the ERK1/2 pathway-mediated posttranslational modification of PPARγ.Method: ①We established the ischemia/reperfusion(I/R) model in Sprague Dawley(SD) rats using the method of middle cerebral artery occlusion(MCAO). For I/R intervention, rats were pretreated with the 12/15-LOX metabolite 12s-HETE 30 minutes before MCAO. The neuroprotective effects of 12s-HETE on I/R rats were scaled by modified neurological severity scores(m NSS) and TTC staining 24 hours after 60min-MCAO. PPARγ expression and activation(nuclear translocation), cleaved caspase3, and i NOS expression were evaluated by the methods of western blotting and immunohistochemistry staining. ②Primary cortical neurons from SD rats were used to establish an OGD model. 12s-HETE, the 12/15-lipoxygenase specific inhibitor(baicalein), or the 12/15-lipoxygenase antisense oligonucleotide(as ON-12/15-LOX) was added to OGD-treated neurons, respectively. PPARγ and 12/15-LOX expression were detected. PPARγ target genes expression and PPARγ-DNA specific binding activity were observed by the analysis of RT-PCR and peroxisome proliferator responsive element(PPRE) luciferase reporter vectors, respectively. ③Primary cortical neurons underwent 0~24-hour hydrogen peroxide(H2O2)(200μM) treatment, with or without ERK1/2 activation inhibitor(U0126 20μM or PD98059 10μM) 30min-pretreatmen. Total, cytosolic, and nuclear protein extracts were isolated respectively. Immunoflorescene staining or western blotting was explored to evaluate the expression of ERK1/2, phospho-ERK1/2, PPARγ, phospho-PPARγ and PPARγ, as well as the intracellular distribution of PPARγ.Results: ①Compared with I/R group, 12s-HETE treatment dramatically decreased the infarct size and improved the m NSS. 12s-HETE also increased PPARγ protein expression and nuclear translocation(activation), decreased i NOS and cleaved caspase3 protein expression in ischemic brain. ② 12/15-LOX and PPARγ total protein expression and PPARγ nuclear translocation increased significantly in OGD-treated neurons. 12s-HETE treatment increased PPARγ total protein and nuclear translocation, upregulated the m RNA levels of PPARγ target genes(lipoprotein lipase and acyl-Co A oxidase), and enhanced PPARγ-DNA binding activity, indicating the elevation of PPARγ transcriptional activity. On the contrary, inhibiton of 12/15-LOX by either the 12/15-LOX inhibitor(baicalein) or antisense oligonucleotides(as ON-12/15-LOX) treatment decreased both 12/15-LOX and PPARγ protein expression and PPARγ nuclear translocation. ③ A transient and robust activation of ERK1/2 was observed in H2O2-treated primary cortical neurons. The phosphorylation of PPARγ and a paralleled decrease of PPARγ nuclear translocation were also detected within 3h in H2O2-treated primary cortical neurons, which could be totally blocked by ERK1/2 inhibitors. From 6h to 24 h of H2O2 treatment, both PPARγ and phospho-PPARγ proteins decreased significantly.Conclusion: ①12/15-LOX metabolite 12s-HETE protects brain from cerebral ischemia-reperfusion injury in rats by upregulation of PPARγ and inhibition of proinflammatory and pro-apoptic factors. ②12/15-LOX pathway is possibly involved in activation of PPAR? by its metabolites in OGD treated neurons. ③ H2O2 treatment negatively regulated PPAR? in neuronal cells by decreasing PPAR? protein level and increasing ERK1/2-mediated PPAR? phosphorylation.