目的研究羟基红花黄色素A(HSYA)对体外亚铁血红素/亚硝酸钠/过氧化氢(heme/NaNO2/H2O2)途径引起脑组织蛋白质硝基化的抑制作用。方法模拟体内heme/NaNO2/H2O2硝基化途径,分别以牛血清白蛋白和脑组织蛋白为硝基化底物,分为对照组及药物干预组,HSYA的终浓度依次为0.01mmol/L、0.1mmol/L、1mmol/L。以Westernblotting法检测蛋白质酪氨酸硝基化水平。结果Heme/NaNO2/H2O2途径可引起牛血清白蛋白和脑组织蛋白显著的硝基化修饰,加入不同浓度HSYA后,蛋白质硝基化水平均呈剂量依赖性地降低,其中以1mmol/LHSYA的抑制作用最明显,与对照组相比,差异具有统计学意义(P均〈0.05)。结论HSYA可剂量依赖性地抑制heme/NaNO2/H2O2途径在体外对脑组织蛋白的硝基化修饰;提示HSYA抑制蛋白质硝基化反应可能是其对抗脑血管疾病与神经系统变性疾病的分子机制之一。
Objective To study the inhibitory effect of Hydroxysafflor yellow A (HSYA) on brain protein nitration induced by heme/NaNO2/H2O2 system in vitro. Methods To simulate in vivo heme/NaNO2/H2O2 system-induced nitra- tive pathway, with bovine serum albumin (BSA)and brain protein for nitrification substrates respectively, preincubated with or without HSYA ( at final concentrations of 0.01,0.1, lmmoL/L) at 37℃ for 5min, and then Heroin, NaNO2/ H2O2 were added, the mixture was further incubated at 37℃ for 30 min,which could be detectable by Western blotting using anti-nitro- tyrosine antibody. Results Heme/NaNO2/H2O2 system could induce significant protein tyrosine nitrative modification to BSA and brain protein in vitro. HSYA pretreatment showed a very strong capacity to dose-dependently inhibitionion in 3-ni- trotyrosine formation, especially at highest concentration (1 retool/L) , the differences between with and without HSYA were statistically significant ( P 〈 0.05, for all). Conclusion HSYA could dose-dependently inhibit brain protein nitrative mod- ification induced by heme/NaNO2/H2O2 system in vitro,which may be one of the molecular mechanisms to against cerebro- vascular and neurodegenerative diseases.