| Objectives:1. To examine PPAR isotypes (PPARa, PPARβ/δ, and PPARγ) expression, in rats exposed to cerebral I/R and I/R in combination with pan-PPAR co-agonist, as well as explore the significance of altered PPARs expression in the context of brain injury.2. To evaluate the alteration in protein expression and nuclear translocation of endogenous PPARγafter focal cerebral I/R in rats, and the influence of PPARγagonist and antagonist to nuclear translocation, and to investigate the meaning of PPARγnuclear translocation in brain ischemia reperfusion injury.3. To examine 12/15-LOX and its metabolite 15-HETE expression in rats exposed to cerebral I/R, and the influence of 12/15-LOX antagonist and metabolite to expression, and to investigate the meaning of 12/15-LOX pathway in PPARγexpression.Methods:1. Adult male SD rats underwent 60min middle cerebral artery occlusion followed by a 24-hour reperfusion (MCAO/R), receiving either vehicle (I/R-group) or bezafibrate (6mg/kg) treatment (Beza-group) 30min before operation. mNSS.was used to evaluate the neurological function. TTC staining was adopted to determine the volume of cerebral infarction. The expressions of PPAR isotypes were characterized by immunohistochemical staining (IHC) and Western blot, respectively. Furthermore, the spatial localizations of PPAR isotypes were detected with respect to ipsilateral ischemic (core and penumbra) and contralateral non-ischemic hemisphere. 2. Adult male SD rats underwent 60min middle cerebral artery occlusion followed by 2,4,8 or 24 hour reperfusion (MCAO/R), receiving either vehicle (I/R-group) or rosiglitazone (6mg/kg) treatment (Ros-group) or GW9662 (4mg/kg) treatment (GW9662-group) 30min before operation. mNSS was used to evaluate the neurological severity. TTC staining was adopted to determine the volume of cerebral infarction. The expressions of PPARγwas characterized by immunohistochemical staining (IHC) and Western blot, respectively. The nuclear translocation of PPARγwas characterized by immunoflorescene staining.3. Adult male SD rats underwent 60min middle cerebral artery occlusion followed by a 24-hour reperfusion (MCAO/R), receiving either vehicle (I/R-group) or Baicalein treatment (Baicalein-group) or 15-hydroxyeicosatetraenoic (15-HETE) treatment (15-HETE-group) 30min before operation. Immunoflorescene staining and Western blot were used to evaluate expression of 12/15-LOX and PPARy. The content of 15-HETE was evaluated by enzyme immunoassay.Results:1. Compared with sham-group,60min ischemia and 24 h reperfusion caused (1) 44.30% infarct volume in average in ipsilateral hemisphere and mNSS increased, P<0.01; (2) marked up-regulations of all 3 PPAR isotypes expression evaluated by IHC (t=8.63,9.29,13.62, and P<0.01) and Western blot (PPARa by 1.47-fold, PPARδ/βby 3.52-fold, and PPARy by 2.25-fold;t=8.16,9.24,6.43; and P=0.000); meanwhile, dramatical increase in PPARs staining in the ischemia-affected region of the ipsilateral MCA territory, and in particular to the ischemic penumbra. No detectable increase in number and intensity was observed in the contralateral (non-ischemic) hemisphere. Compared with I/R-group, the pan-PPAR co-agonists bezafibrate (Beza) (1) dramatically decreased infarct volume by 54.36% in average (t=7.69, P=0.000), mNSS decreased too, P<0.01; (2) Bezafibrate treatment further increased the alterations of all PPARs expression, which were induced by the exposure of I/R, as determined by IHC (t=7.36,5.64,10.50, and P=0.000) and Western blot (PPARa by 95.45%, PPARβ/δby 183.47%, and PPARγby224.61%; r=13.02,17.52,13.64, and P=0.000); (3) the PPARs immunoreactive expressions were observed in not only the ipsilateral but the contralateral hemisphere of Beza-group.2. (1) Compared with Sham-group, I/R-group①PPARy whole protein expression:As shown with Western blot, the PPARy whole protein expression of 2h-reperfusion hadn't detectable change,t=-0.701, P>0.05; however, the PPARy whole protein expression of 4,8 and 24h-reperfusion all up-regulated in a reperfusion time-dependent way (t=-15.749,-16.715,-22.839, P<0.05); we can observe the same change with immunohistochemical staining;②PPARy component protein expression:as shown with Western blot, in Sham-group, PPARy expressed in both cytosol and nuclear fractions. A increase of PPARy in the nucleus (t=-11.343,-27.191,-24.861,-25.75; P<0.05) could be noted with a simultaneous reduction in the cytosol (t=7.197,9.031,10.777,13.78; P<0.05) following I/R, a time-dependent enhancement in PPARy translocation by the analysis of cytosol and nuclear level of PPARγat 2,4,8 and 24h of reperfusion; we can observe the same change with immunoflorescene staining; (2)Compared with I/R(60min/24h)-group,①the infarct volume of Ros-group decreased by 48.27% and the infarct volume of GW9662-group increased by 39.52%, mNSS also showed the same change, P<0.05;②PPARy whole and component protein expression:as shown with Western blot, rosiglitazone dramtically increased I/R-induced PPARy total protein(t=-23.465, P<0.05) and nuclear protein level (t=-33.788, P<0.05), and decreased I/R-induced PPARy cytosol protein level(t=6.428, P<0.05); GW9662 dramtically decreased I/R-induced PPARy total protein(t=29.403, P<0.05) and nuclear protein level (t=22.414, P<0.05), and decreased I/R-induced PPARy cytosol protein level(t=-6.826, P<0.05); we can observe the same change with immunoflorescence staining; 3. (1) Compared with Sham group, as shown with Western blot, the expression of 12/15-lipoxygenase (12/15-LOX) whole protein was enhanced in I/R(60min/24h) group (t=-8.195, P<0.05); the localization of 12/15-LOX was major in ischemic penumbra as shown with immunoflorescence staining, and consistent with PPARy; and as shown with enzyme immunoassay, the content of 15-HETE in I/R group was dramatically enhanced compared with Sham group (t=-12.787, P<0.05). (2) As shown with Western blot, the majority of PPARy localized in the nucleus under the I/R condition, however, different dose of Baicalein treatment significantly suppressed I/R-induced PPARy nuclear accumulation (150mg/kg,t=15.361, P<0.05; 200mg/kg,t=26.922, P<0.05) and maintained PPARy cytoplasmic retention (150mg/kg,t=-17.579, P<0.05; 200mg/kg,t=-25.719, P<0.05), furthermore, Baicalein also inhibited the up-regulation of PPARy total protein expression (150mg/kg,t=7.253, P<0.05; 200mg/kg,t=8.61, P<0.05); Baicalein 200mg/kg treatment suppressed I/R-induced increase in PPARy total protein expression(t=2.773, P<0.05; t=11.650, P<0.05) and nuclear translocation(t=4.313, P<0.05; t=37.468, P<0.05) with vehicle-treated I/R rats at both 4h and 24h after ischemia, and maintained PPARy cytoplasmic retention (t=4.313, P<0.05; t=-8.246, P<0.05); (3) Compared with I/R(60min/24h) group, 12/15-LOX metabolite 15-HETE treatment dramatically enhanced I/R-induce increase in PPARy total protein expression (t=-23.884, P<0.05) and nuclear translocation (t=-15.36, P<0.05), and suppressed PPARγcytoplasmic retention (t=5.86, P<0.05). Conclusion:1. Cerebral I/R injury increases the expression all 3 PPAR isotypes and activation of PPARs by pan-PPAR agonist not only reduces ischemic size but further enhances the alteration of PPARs. The up-regulation of PPARs expression may represent the compensatory self-protection against brain I/R injury. 2. (1) Cerebral I/R could up-regulated PPARy expression-and promoted its nuclear' translocation, the combined and final effect of these changes is to bring about an elevation of nuclear PPARγ, which probably associates with the enchancement of its nuclear activity; (2) The onset of PPARy nuclear translocation was earlier than that of the up-regulation of protein expression, indicating that nuclear translocation of PPARγwas a more direct and quick response to I/R; (3) The supplementation of rosiglitazone, the exogenous PPARγligand, promoted I/R-induced PPARγalterations, whereas the treatment with PPARγantagonist GW9662 inhibited the changes of PPARγ, indicate that I/R-induced changes of PPARγresulting from the activation by endogenous ligands; 3. When ischemia reperfusion, the expression and activity of 12/15-LOX increased; 12/15-LOX inhibitor reduced and metabolite 15-HETE enhanced I/R-induced PPARγchanges, it seems that the mechanism underlying I/R-induced changes of PPARγmay be related to, at least partlγ,12/15-LOX pathway.-...
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