枸杞多糖对2,4-二氯苯氧乙酸所致雌性大鼠生殖系统损伤的保护作用

Protective effect of Lycium barbarum polysaccharide on reproductive system injury of female rats induced by 2,4-dichlorophenoxy acetic acid

[背景]2,4-二氯苯氧乙酸(2,4-D)作为阔叶杂草除草剂和植物生长调节剂而被广泛应用,相关研究显示2,4-D具有神经毒性、内分泌干扰性、遗传毒性与致癌性以及生殖毒性等。[目的]了解2,4-D暴露对雌性大鼠生殖系统的影响,初步探讨枸杞多糖(LBP)的保护作用及可能机制。[方法]SPF级雌性SD大鼠24只,每组6只,随机分为空白对照组(去离子水1 mL·d^(–1))、染毒组(75 mg·kg^(-1)2,4-D)、枸杞多糖对照组(50 mg·kg^(-1) LBP)、枸杞多糖干预组(75 mg·kg^(-1)2,4-D+50 mg·kg^(-1) LBP)。每天经口灌胃1次,连续染毒28d。每隔2d测量体重1次,染毒结束后取卵巢及子宫组织称重并计算脏器系数;采用苏木精-伊红染色法(HE)检测卵巢及子宫组织病理改变;采用ELISA法检测血清中雌二醇(E2)水平;使用相关试剂盒测定血清中氧化损伤[超氧化物歧化酶(SOD)、谷胱甘肽过氧化酶(GSH-Px)及丙二醛(MDA)]水平;TUNEL荧光染色法检测卵巢及子宫组织细胞凋亡情况;Western blotting法检测卵巢组织中死亡受体通路相关蛋白Fas、FasL、FADD、Pro-Caspase-8、Cleaved-Caspase-8、Pro-Caspase-3、Cleaved-Caspase-3表达量。[结果]与空白对照组相比,染毒组大鼠卵巢组织结构异常,处于不同发育阶段的卵泡数量减少,形态出现改变,且闭锁卵泡数量增多;子宫内膜不完整,可见内膜缺失,出现不同程度的胞核假复层现象,固有层腺体数减少。与染毒组相比,枸杞多糖干预组大鼠卵巢组织结构相对较完整,处于不同发育阶段的卵泡数量增加,结构相对完整,排列紧密;子宫组织结构相对较完整,内膜缺失现象及胞核假复层现象减少。各组间SOD、GSH-Px、E2水平,MDA浓度差异均有统计学意义(F=86.1、26.2、43.3、22.3,均P<0.01)。与空白对照组相比,染毒组血清SOD、GSH-Px、E2水平降低(P>0.01),MDA􀀁浓度增加(P>0.01);与染毒组相比,枸杞多糖干预组血清SOD、GSH-Px、E2水平升高(P>0.01),MDA􀀁浓度降低(P>0.01)。各组间卵巢及子宫组织细胞凋亡率差异均有统计学意义(F=64.8、55.5,均P<0.01)。与空白对照组相比,染毒组大鼠卵巢及子宫组织细胞凋亡率升高(P>0.01);与染毒组相比,枸杞多糖干预组大鼠卵巢及子宫组织细胞凋亡率降低(P>0.01)。各组间卵巢组织内死亡受体通路相关蛋白表达差异均有统计学意义(均P<0.05)。与空白对照组相比,染毒组大鼠卵巢组织内Fas、FasL、FADD、Pro-Caspase-8、Cleaved-Caspase-8、Pro-Caspase-3、Cleaved-Caspase-3表达升高(P<0.05或0.01);与染毒组相比,枸杞多糖干预组大鼠卵巢组织内Fas、FasL、FADD、Pro-Caspase-8、Cleaved-Caspase-8、Pro-Caspase-3、Cleaved-Caspase-3表达降低(P<0.05或0.01)。[结论]2,4-D能够引起雌性大鼠发生氧化应激进一步介导Fas-FasL通路引发细胞凋亡而造成雌性大鼠生殖系统损伤。枸杞多糖能降低雌性大鼠氧化应激水平,下调Fas-FasL通路相关蛋白表达,减少生殖细胞凋亡,以达到生殖保护的目的。

[Background]2,4-Dichlorophenoxyacetic acid(2,4-D)is widely used as a broad-leaved herbicide and plant growth regulator.Related studies have shown that 2,4-D has neurotoxicity,ability to disrupt endocrine function,genotoxicity,carcinogenicity,and reproductive toxicity.[Objective]This experiment is conducted to investigate the effect of 2,4-D exposure on reproductive system of female rats,and to preliminarily explore the potential ameliorative effect of Lycium barbarum polysaccharide(LBP)and its possible mechanism.[Methods]Twenty-four SPF female SD rats with six rats in each group were randomly divided into a blank control group(deionized water 1 mL·d^(−1)),an exposure group(75 mg·kg^(-1)2,4-D),an LBP control group(50 mg·kg^(-1) LBP),and an LBP intervention group(75 mg·kg^(-1)2,4-D+50 mg·kg^(-1) LBP).The rats were given intragastric administration once a day for 28 consecutive days.Body weight was measured every two days.After exposure,ovary and uterus were weighed and organ coefficients were calculated;the pathological changes of ovary and uterus were detected by hematoxylin-eosin staining(HE);the level of estradiol(E2)in serum was detected by ELISA;the levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),and malondialdehyde(MDA)in serum were measured by corresponding kits;the apoptosis of ovarian and uterine cells was detected by TUNEL fluorescence staining;and the protein expression levels of Fas,FasL,FADD,Pro-Caspase-8,Cleaved-Caspase-8,Pro-Caspase-3,and Cleaved-Caspase-3 in ovarian tissues were detected by Western blotting.[Results]Compared with the blank control group,the ovarian structure of the exposure group was abnormal,the number of follicles at different developmental stages decreased,morphological changes were observed,and the number of atretic follicles increased;the endometrium was incomplete,with different degrees of nuclear pseudostratification and decreased number of glands in lamina propria.Compared with the exposure group,the ovarian structure of the LBP intervention group was complete,and the follicles at different developmental stages increased in amount,remained intact,and were arranged closely;the uterine structure was relatively intact,showing decreased endometrial loss and nuclear pseudostratification.There were significant differences in the levels of SOD,GSH-Px,E2,and MDA among the four groups(F=86.1,26.2,43.3,and 22.3,all P<0.01).Compared with the blank control group,the levels of serum SOD,GSH-Px,and E2 decreased in the exposure group(P<0.01),while the concentration of MDA increased(P<0.01).Compared with the exposure group,the levels of serum SOD,GSH-Px,and E2 in the LBP intervention group increased(P<0.01),and the concentration of MDA decreased(P<0.01).There were significant differences in the apoptosis rates of ovarian and uterine cells among the four groups(F=64.8,55.5,both P<0.01).Compared with the blank control group,the apoptosis rates of ovarian and uterine cells increased in the exposure group(P<0.01).Compared with the exposure group,the apoptosis rates of ovarian and uterine cells decreased in the LBP intervention group(P<0.01).There were significant differences in the expression levels of death receptor pathway-related proteins in ovarian tissues among the four groups(all P<0.05).Compared with the blank control group,the expression levels of Fas,FasL,FADD,Pro-Caspase-8,Cleaved-Caspase-8,Pro-caspase-3,and Cleaved-Caspase-3 were increased in the exposure group(P<0.05 or 0.01).Compared with the exposure group,the expression levels of above proteins were decreased in the LBP intervention group(P<0.05 or 0.01).[Conclusion]The study findings reveal that 2,4-D can induce oxidative stress and further mediate Fas-FasL pathway to induce apoptosis,resulting in reproductive system damage in female rats.LBP can reduce the oxidative stress level,down-regulate the expression of Fas-FasL pathway-related proteins,and reduce the apoptosis of germ cells,therefore protecting reproductive system of female rats.