白藜芦醇联合大豆异黄酮改善衰老大鼠海马组织氧化应激致细胞凋亡作用

Effects of resveratrol combined with soy isoflavones on apoptosis induced by oxidative stress in hippocampus of aging model rats

目的探讨白藜芦醇(resveratrol,Res)与大豆异黄酮(soy isoflavone,SIF)单独或联合对衰老大鼠海马组织氧化应激引起的细胞凋亡的作用。方法60只SD雌性大鼠按体重随机分为6组:假手术组、衰老模型组、Res干预组(80 mg/kg)、SIF干预组(160 mg/kg)、联合干预组(80 mg/kg Res+160 mg/kg SIF)和戊酸雌二醇阳性对照组。卵巢摘除术合并D-半乳糖腹腔注射建立衰老大鼠模型,各干预组灌胃相应受试物,每天1次,连续12周。分别用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法[terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling,TUNEL法]和透射电镜观察大鼠海马组织细胞凋亡和线粒体超微结构的改变;化学比色法检测大鼠海马组织抗氧化物超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)的活力及脂质过氧化产物丙二醛(malonaldehyde,MDA)的含量;蛋白质免疫印迹法(Western blot)检测线粒体呼吸链细胞色素C氧化酶Ⅰ(cytochrome C oxidaseⅠ,COXⅠ)和凋亡相关蛋白B细胞淋巴瘤/白细胞基因伴随蛋白X(Bcl-2 associated X protein,Bax)、B细胞淋巴瘤/白血病-2蛋白(B-cell leukemia/lymphoma 2,Bcl-2)及细胞色素C的表达。结果与假手术组相比,衰老模型组大鼠TUNEL阳性细胞数增多,凋亡指数(apoptotic index,AI)升高;线粒体空泡样变严重,线粒体完整率降低;海马组织SOD、CAT及GSH-Px活力降低,MDA水平升高;COXⅠ蛋白表达降低,Bax和细胞色素C表达升高,Bcl-2/Bax的比值降低,差异均有统计学意义(P<0.05)。与衰老模型组相比,Res组、SIF组和Res+SIF组及戊酸雌二醇组大鼠TUNEL阳性细胞数减少,AI显著降低;线粒体结构均有所改善,线粒体完整率升高;海马组织SOD及GSH-Px活力显著升高,MDA水平显著降低,COXⅠ蛋白表达上调,Bax和细胞色素C表达下调(P<0.05或P<0.01)。与Res和SIF单独干预组比较,Res+SIF联合干预组AI降低,线粒体完整率升高,SOD及GSH-Px活力显著升高,MDA水平显著降低,COXⅠ蛋白表达上调,Bax和细胞色素C蛋白表达下调,Bcl-2/Bax比值升高(P<0.05)。各组大鼠Bcl-2蛋白表达差异无统计学意义(P>0.05)。结论Res和SIF单独及联合干预均可改善衰老模型大鼠海马组织氧化应激引发的细胞凋亡,联合作用更为显著。改善作用可能与提高大鼠抗氧化能力,上调大鼠海马组织COXⅠ蛋白的表达和Bcl-2/Bax蛋白比值,抑制细胞色素C的释放有关。

OBJECTIVE To investigate the effect of resveratrol(Res)combined with soy isoflavones(SIF)on apoptosisinduced by oxidative stress in hippocampus in aging model rats.METHODS Sixty female SD rats were randomly divided into the Sham control group,aging model group,Res treatment group,SIF treatment group,Res combined with SIF treatment group and estrogen replacement therapy group(ERT group).Rats with aging were induced by bilateral ovariectomy combined with intraperitoneal injection of D-galactose.TUNEL and transmission electron microscopy were used to observe apoptosis and ultrastructural changes of mitochondrion in hippocampus,respectively.The activities of superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GSH-Px)and content of Malonaldehyde(MDA)were detected in the hippocampal homogenate.The protein expressions of cytochrome C oxidase(COX)Ⅰ,Bcl-2 associated X protein(Bax)、B-cell leukemia/lymphoma 2(Bcl-2)and cytochrome C were detected by Western blot.RESULTS Compared with the Sham control group,the number of TUNEL-positive cells and the apoptotic index(AI)in the model group increased.Marked increase of mitochondrial swelling and vacuolation,decrease of mitochondrial integrity were also observed in the model group.Additionally,the levels of SOD,CAT and GSH-Px were decreased and the level of MDA was increased in the model group in the hippocampus,Bax and cytochrome C protein expression increased,and the COXⅠprotein expression and the ratio of Bcl-2/Bax decreased(P<0.05).In compared with the model group,the number of TUNEL-positive cells and AI significantly decreased,mitochondrial integrity improved in all of the treatment groups,the COXⅠprotein expression significantly up-regulated,Bax and cytochrome C protein expression downregulated,and the protein expression ratio of Bcl-2/Bax increased(P<0.05 or P<0.01).Compared with the Res alone and SIF alone treatment group,Res combined SIF treatment decreased the AI and Bax,cytochrome C protein expression,moreover,increased the mitochondrial integrity rate,COXⅠand Bcl-2/Bax protein expression ratio(P<0.05).There was no statistically significant difference in Bcl-2 protein expression among all the groups(P>0.05).CONCLUSION Res and SIF alone and in combination decreased apoptosisinduced by oxidative stress in hippocampus of aging model rats,and beneficial effect in the Res combined SIF treatment group is more significant than that in the alone administration.Improving the antioxidant capacity,increasing the protein expressions of COXⅠandthe ratio of Bcl-2 and Bax,and inhibiting the release of cytochrome C may be the mechanisms by which Res and SIF improve apoptosis in aging rats.