| Ojective:1. To study the effects of peroxynitrite (ONOO-) and heme/NaNO2/H2O2on brain tissue nitration and oxidation, and the influences of Hydroxysafflor yellow A (HSYA) on them in vitro, and to explore the protective mechanisms of HSYA on nitration and oxidation of damaged brain tissue.2. Using a rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) in vivo, to examine protein nitration, protein carbonylation, lipid peroxidation (LPO) and reduced glutathione (GSH) expression in rats exposed to cerebral ischemia-reperfusion injury (CIRI) and CIRI in combination with HSYA, as well as explore the protective mechanisms of HSYA on CIRI.Methods:1. To establish the models of brain tissue nitration and oxidation (ONOO-and heme/NaNO2/H2O2) in vitro, with brain tissue for substrate, divided into blank group, control group and HSYA intervention groups (doses of0.01,0.1and1mM, respectively). Protein nitration was characterized by Western blot using anti-3-nitrotyrosine (3-NT) antibody.2,4-dinitrophenylhydrazine (DNPH) was used for the quantification of protein carbonylation. The content of maleic dialdehyde (MDA), as the representative of LPO, was determined by the means of thibabituric acid (TBA).2. Healthy 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 brain ischemia HSYA was injected10mg/kg via tail vein. mNSS was used to evaluate the neurological function. TTC staining was adopted to determine the volume of cerebral infarction. The expression of3-NT in brain ischemia was characterized by Western blot and immunohistochemical staining (IHC) to determine the degree of protein nitration. DNPH was used for the quantification of protein carbonylation. The content of MDA, as the representative of LPO, was determined by the means of TBA. The content of GSH was evaluated by the means of5,5’-Dithio bis-(2-nitrobenzoic acid)(DTNB).Results:1. Experiments in vitro:(1) Compared with blank group, incubation of brain tissue with ONOO-or heme/NaNO2/H2O2for30min resulted in significant protein nitration which could be detectable by Western blot, the expression of3-NT increased by95.10%and62.98%, respectively. Compared with control group, HSYA intervention groups (doses of0.01,0.1and1mM, respectively) showed dose-dependent inhibition on ONOO--nduced protein nitration by14.58%,28.25%,75.22%respectively, and on heme/NaNO2/H2O2-triggered protein nitration by11.59%,34.48%,76.14%respectively. The inhibition of HSYA at dose of1mM was most obvious, the differences were statistically significant (F=166.412, P<0.05; F=227.629, P<0.05).(2) The treatment of brain tissue with ONOO-or heme/NaNO2/H2O2resulted in the significant formation of carbonyl groups. HSYA, at doses of0.1and1mM, dose-dependently decreased ONOO--induced protein carbonylation by26.13%and46.23%respectively, the difference was statistically significant (F=14.625, P<0.05). However, HSYA was relatively ineffective in protecting brain tissne from heme/NaNO2/H2O2-induced oxidative damage, the difference was not statistically significant (P>0.05).(3) The treatment of brain tissue with ONOO-or heme/NaNO2/H2O2resulted in the significant formation of LPO, while HSYA could dose-dependently inhibit the formation of LPO, especially at higher concentration (1mM), the differences were statistically significant (F=67.176, P=0.000; F=66.475, P<0.05).2. Experiments in vivo (a rat model of MCAO/R):(1) Compared with sham group,60min ischemia and24h reperfusion caused33.75%infact volume in average in ipsilateral hemisphere and mNSS increased. Compared with vehicle group, the infact volume of HSYA treatment group decreased by48.86%(t=5.673,P<0.01), mNSS also showed the same change (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 protein nitration had few expression in sham group while more in vehicle group, HSYA treatment significantly decreased the expression of3-NT, the differences were statistically significant (F=88.120, P<0.05; F=29.072, P<0.05).(3) The observation of protein carbonylation in brain ischemia which was characterized by the means of DNPH revealed that protein carbonylation had few expression in sham group while more in vehicle group. HSYA treatment significantly decreased the content of protein carbonyls by30.13%compared with vehicle group, the difference was statistically significant (F=11.166, P<0.05).(4) The observation of LPO in brain ischemia which was characterized by the means of TBA revealed that LPO had few expression in sham group while more in vehicle group. HSYA treatment significantly decreased the content of MDA by30.91%compared with vehicle group, the difference was statistically significant (F=49.662,P<0.05).(5) A certain content of GSH expressed in sham group. Compared with sham group, the content of GSH in brain homogenate was decreased by29.32%at24h of reperfusion following60min ischemia. Compared with vehicle group, HSYA treatment significantly creased the content of GSH by69.46%, the difference was statistically significant (F=8.384, P <0.05).Conclusion:1. ONOO-and heme/NaNO2/H2O2can induce brain tissue nitration and oxidation in different pathways, the influences of HSYA on them were also different. Protein nitration and lipid peroxidation were efficiently inhibited by HSYA regardless of the two pathways. HSYA dose-dependently inhibited ONOO--induced protein oxidation. However, HSYA ineffectively suppressed heme/NaNO2/H2O2-triggered protein oxidation.2. HSYA had protective effect on cerebal ischemia-reperfusion injury. It seems that the protective mechanism may be related to inhibit protein nitration, protein oxidation and lipid peroxidation, while enhance antioxidative defense capacities of brain tissue. |
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