枸杞多糖对正己烷染毒大鼠卵巢Nrf2信号通路的影响

Effects of Lycium Barbarum Polysaccharide on Nrf2 Signaling Pathway on Ovary of Rat Treated with n-Hexane

目的探讨Nrf2信号通路在正己烷致卵巢毒性中的作用和枸杞多糖(LBP)对其的拮抗作用。方法 48只SD雌性大鼠,按体重随机分为8组,每组6只,分别为:对照(生理盐水)组,不同剂量的正己烷(675、1350、2700 mg/kg)单独染毒组,LBP(50 mg/kg)对照组,LBP(50 mg/kg)+不同剂量的正己烷(675、1350、2700 mg/kg)联合处理组。实验第1天对正己烷单染组和联合处理组进行腹腔注射正己烷单次染毒,对照组及LBP对照组腹腔注射生理盐水,第2~4天给对照组和正己烷单染组灌胃生理盐水,LBP对照组和联合处理组大鼠灌胃LBP 50 mg/kg,均1次/d,持续3 d,第5天处死动物,解剖取卵巢检测超氧化物岐化酶(SOD)活性、谷胱甘肽过氧化物酶(GSH-Px)活性、丙二醛(MDA)含量,Real-time PCR检测核因子E2相关因子2(Nrf2)、谷氨酸半胱氨酸连接酶催化亚基(GCLC)、血红素加氧酶-1(HO-1)、醌氧化还原酶1(NQO1)的mRNA水平,Western blot检测Nrf2蛋白表达水平。结果与对照组相比,各剂量正己烷染毒组大鼠卵巢GSH-Px活性均显著下降(均P<0.05),1350和2700 mg/kg正己烷染毒组大鼠卵巢SOD活性显著下降(均P<0.05),2700 mg/kg正己烷染毒组大鼠卵巢MDA含量显著升高(P<0.05);1350、2700 mg/kg正己烷染毒组大鼠卵巢Nrf2 mRNA和蛋白表达水平均显著升高(均P<0.05),且HO-1、NQO1、GCLC mRNA的表达水平也均显著升高(均P<0.05)。与未加LBP同剂量组相比,LBP+2700 mg/kg正己烷联合处理组大鼠卵巢SOD活性显著升高,LBP+1350、2700 mg/kg正己烷联合处理组大鼠卵巢Nrf2 mRNA和蛋白表达水平均显著下降(均P<0.05),且HO-1、NQO1、GCLC mRNA的表达水平也显著下降(均P<0.05)。结论在该实验条件下,正己烷可引起大鼠卵巢组织发生氧化应激,激活Nrf2信号通路,进而上调其下游Ⅱ相解毒酶的表达;LBP能缓解正己烷介导的氧化应激,拮抗Nrf2及下游Ⅱ相解毒酶的活化。

Objective To investigate the role of Nrf2 signaling pathway in n-hexane-induced ovarian toxicity and the antagonism of LBP in this toxicity.Methods Sprague Dawley(SD)female rats were randomly divided into 8 groups according to weight:control group,and 675,1350,2700 mg/kg n-hexane groups,50 mg/kg LBP group,and LBP+675,1350,2700 mg/kg n-hexane groups.On the first day,three n-hexane groups and three LBP+n-hexane groups were intraperitoneally injected with 675,1350,2700 mg/kg n-hexane(10 mg/kg)solution once,control group and LBP group were intraperitoneally injected with physical saline(10 mg/kg).From day 2 to day 4,control group and three n-hexane groups were given physical saline[10 mg/(kg·day)]by gavage,LBP group and LBP+n-hexane groups were given LBP(50 mg/kg)solution[10 mg/(kg·day)]by gavage.On day 5,the animals were sacrificed to obtain the ovaries.The activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)and the contents of malondialdehyde(MDA)were measured,the mRNA expression of Nrf2,catalyticsubunit of glutamate-cysteineligase(GCLC),heme oxygenase-1(HO-1),quinone oxidoreductase 1(NQO1)was detected by real-time PCR and the protein expression of Nrf2 by Western Blotting.Results The activity of GSH-Px was significantly decreased in the ovaries of rats treated with n-hexane at 670 mg/kg(P<0.05),and the activities of SOD and GSH-Px were significantly decreased in the ovaries of rats treated with n-hexane at 1350,2700 mg/kg(P<0.05),and the content of MDA was significantly increased in the ovaries of rats treated with n-hexane at 2700 mg/kg(P<0.05).Compared with the control group,the mRNA expression of Nrf2,HO-1,NQO1,GCLC and the protein expression of Nrf2 were significantly increased in the ovaries of rats in the 1350,2700 mg/kg n-hexane groups(P<0.05).Compared with the same dose groups without adding LBP,the activity of SOD was significantly increased in the ovaries of rats treated with 2700 mg/kg n-hexane+LBP(P<0.05).The mRNA expression levels of Nrf2,HO-1,NQO1,GCLC and the protein expression of Nrf2 were significantly decreased in the ovaries treated with 1350,2700 mg/kg n-hexane+LBP group(P<0.05).Conclusion A certain exposure dose of n-hexane can induce the oxidative stress in the ovaries of rats,activate Nrf2 signaling pathway,and up-regulate the expression of downstream phase Ⅱ detoxification enzymes.LBP can alleviate the n-hexane-mediated oxidative stress and antagonize the activation of Nrf2 and downstream phase Ⅱ detoxification enzymes.