| Ojective:To study the effect of peroxynitrite (ONOO-) on nitrative modification of PPARy in vitro and in vivo, as well as the anti-nitrative property of Hydroxysafflor yellow A (HSYA).Methods:1. With the model of ONOO-induced brain tissue homogenate nitration in vitro,3groups were included:blank group, ONOO group and ONOO+HSYA (0.01,0.1and1mM) group. Protein nitration was characterized by Western blot using anti-3-nitrotyrosine (3-NT) antibody.2primary cortical neurons prepared from embryonic day17-20rats were cultured in vitro. Study was carried out with d-7neurons. Cells were divided into blank group, ONOO group, ONOO+glutamic acid (Glu) group, and HSYA intervention group. PPARy,3-NT, and Bcl-2protein expression was evaluated by Western blotting, and nitro-PPARy was observed by immunoprecipitation (IP) method. Immunofluorescence staining was used to determine the cellular localization of PPARy,3-NT and NF-Kb.3. adult male SD rats, underwent60-min middle cerebral artery occlusion followed by reperfusion of24h (MCAO/R), were randomly divided into sham group, vehicle group and the HSYA treatment group. In the HSYA treatment group,30min before MCAO,10mg/kg of HSYA was injected via tail vein. mNSS was used to evaluate the neurological function deficits. TTC staining was adopted to determine the volume of cerebral infarction. The expression of3-NT in ischemic brain was characterized by Western blot and Immunofluorescence staining to determine the degree of protein nitration; IP inspection was utilized for nitro-PPARy and immunofluorescence staining for the cellular localization of PPARγ,3-NT, and NF-kb.Results:1. in vitro experiments:(1) Compared with blank group, incubation of brain tissue homogenate with ONOO-for30min resulted in significant protein nitration, the expression of3-NT increased by94.90%, respectively. Compared with ONOO group, ONOO+HSYA groups (doses of0.01,0.1and1mM, respectively) showed dose-dependent inhibition of ONOO-induced protein nitration by15.68%,30.25%,79.22%respectively. The inhibition of HSYA at dose of1mM was most obvious, the differences were statistically significant (F=187.412, P<0.05; F=227.629, P<0.05).2. Primary cultured cortical neurons:ONOO induced marked3-NT expressionin in cells;Compared with that of ONOO-group, HSYA significantly reduced3-NT expression by31.20%; however, peroxynitrite have no effect on protein expression level of PPARy. IP inspection and immunofluorescence staining showed that neuronal PPARy can be nitrated and nitration inhibits PPARy nuclear translocation. HYSA does not affect the protein expression level of PPARy, but can decrease PPARy nitration and therefore enhance the content of nuclear PPARy.4. Experiments in vivo (the rat model of MCAO/R):(1) Compared with vehicle group, HSYA treatment caused41.22%(t=4.673, P<0.01) decrease in infact volume and improved mNSS performance(t=2.746, P<0.05).(2) The observation of protein nitration in brain ischemia which was characterized by Western blot and IHC revealed that nitrated protein was few in sham group while greatly induced in vehicle group. However, HSYA treatment significantly decreased the expression of3-NT, the differences were statistically significant (F=94.120, P<0.05; F=32.072, P<0.05).(3) With IP inspection and immunofluorescence staining, nitrated PPARy was found in the ischemic brain of MCAO/R rats and accumulated in the cytoplasm, indicating the decreased shift of PPARy from cytoplasm to nucleus. The retention of nitro-PPARy in the cytopalsma compartment was also accompanied by the down-regulation of Bcl-2expression.Conclusion:1. ONOO-can induce nitrotyrosine formation in brain tissue homogenate and primary cultured cortical neurons, HSYA has the inhibitory effect against ONOO-induced protein nitration.2. PPARy nitrative modification occurred in ONOO-treated cultured neurons and ischemic brain, resulting in the retention of PPARy in the cytosol compartment. HSYA significantly reduced the level of nitro-PPARy and increased the nuclear shift of PPARy.3. HSYA was neuroprotective against cerebal ischemia-reperfusion injury by showing the ability to decrease infarction volume and improve neurological function scores. It seems that the protective mechanism may be related to the inhibition of protein (PPARy) nitration. |
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