锌对亚慢性砷中毒雄性SD大鼠睾丸抗氧化力的影响

Effect of zinc to anti-oxidation capacity of testis in male SD rats with sub-chronic arsenic poisoning

目的探讨锌对砷致大鼠睾丸损伤中相关保护作用的机制,为预防砷的生殖毒性及辅助性治疗提供理论依据。方法清洁级SD雄性大鼠30只,体重200 g左右,随机分为3组,分别为对照组(蒸馏水)、砷组和砷+锌组。对照组、砷组分别给亚砷酸钠0 mg/L、60 mg/L,砷加锌组每日分别予浓度为60 mg/L As(As为亚砷酸钠)+227 mg/L Zn(Zn为硫酸锌)的水溶液,采用自由饮水方式进行染毒,连续染毒12周,每天称体重。染毒结束后处死大鼠,摘取睾丸组织、称重;分光光度法检测睾丸组织匀浆中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)及丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)、一氧化氮合酶(NOS)的活力及一氧化氮(NO)的含量。光镜下观察睾丸形态变化,实时荧光定量聚合酶链反应(Real-Time PCR)测定各组大鼠睾丸的金属硫蛋白-1(MT-1)、金属硫蛋白-2(MT-2)基因表达情况。结果 HE染色结果显示,对照组大鼠睾丸结构可见精子发育良好、生精上皮细胞排列有序,睾丸间质中血管完整。砷组大鼠睾丸,可见其生精小管中生精上皮坏死及空泡化,睾丸间质水肿、坏死出血。锌+砷组大鼠睾丸一些生精小管中逐渐出现正常组织结构。与对照组比较,砷组睾丸MDA含量显著增加(P<0.01);砷+锌组睾丸MDA浓度显著低于砷组(P<0.01)。与对照组比较,砷组睾丸SOD活力显著降低(P<0.01);砷+锌组中睾丸SOD活力显著高于砷组(P<0.01)和对照组(P<0.05)。与对照组比较,砷组睾丸GSH-Px的活力显著下降(P<0.01);与砷组比较,砷+锌组睾丸GSH-Px活力升高(P<0.01)。与对照组比较,砷组睾丸CAT的活力显著下降(P<0.01);与砷组比较,砷+锌组睾丸CAT活力升高(P<0.01),与对照组比较,砷组NOS活性显著性降低(P<0.01)。与对照组比较,砷组睾丸MT-1、MT-2基因表达水平明显上调了3.60倍和1.90倍;而砷+锌组与对照组比较,大鼠睾丸MT-1表达水平也上调2.04倍,MT-2没有明显的变化;但砷+锌组与砷组比较,砷+锌组MT-1、MT-2基因表达明显下调(P<0.01)。结论 1.亚慢性砷中毒可增强睾丸组织氧化应激效应,导致大鼠睾丸MT-1、MT-2基因表达上调;而砷暴露的同时补锌,其MT-1、MT-2基因表达下调。2.补锌后机体可能通过调节MT-1、MT-2基因表达,改善睾丸的抗氧化状态等机制,阻止砷的积累和锌的耗损,从而缓解大鼠砷暴露所致的睾丸损伤。

[Objective]To investigate the protective mechanism of zinc on arsenic-induced testicular trauma in rats,provide theoretical basis for the prevention of reproductive toxicity of arsenic and adjuvant therapy.[Methods]Thirty male SD rats（200 grams of body weight）were randomly divided into control group（distilled water）,arsenic（As）group and arsenic＋zinc（As ＋Zn）group. The rats in control group and As group were given 0 mg/L and 60 mg/L sodium arsenite respectively,and rats in As＋Zn group were given60 mg/L sodium arsenite and 227 mg/L zinc sulfate. The animals were exposed by the drinking water for 12 weeks,and measured the body weight every day. Rats were killed after the end of the exposure,to collect and weigh the testis. The levels of SOD,CAT,MDA,GSH-Px and NOS,and NO content in testis tissue were determined by spectrophotometric method. The morphological changes of testis were observed under light microscope. Real-Time PCR was used to test the expression of MT-1 and MT-2 in testis.[Results]HE stained results revealed that the control group had normal testicular histology with evidence of spermatogenesis,well-organized distribution of cells in the seminiferous epithelium and intact blood vessels in the interstitium. As-exposed rats exhibited hemorrhage and edema in the interstitial space,necrosis and disintegration of spermatocytes from basement membrane. While As＋Zn group rats showed a histological picture of testicular tissue with active spermatogenesis in most of seminiferous tubules. MDA level in testis of As group was significantly higher than that of control group（P0.01）,while MDA level in testis of As＋Zn group was significantly lower than that of As group（P0.01）. SOD activity in testis of As group was significantly lower than that of control group（P0.01）,while SOD activity in testis of As ＋Zn group was significantly higher than that of As group（P0.01）and control group（P0.05）. GSH-Px activity in testis of As group was significantly lower than that of control group（P0.01）,while GSH-Px activity in testis of As＋Zn group was significantly higher than that of As group（P0.01）. CAT activity in testis of As group was significantly lower than that of control group（P0.01）,while CAT activity in testis of As＋Zn group was significantly higher than that of As group（P0.01）. NOS activity of As group was significantly lower than that of control group（P0.01）. The expression level of MT-1 and MT-2 in testis of As group respectively increased by 3.60 times and 1.90 times as compared with those in control group. The expression level of MT-1 in testis of As ＋Zn group increased by2.04 times as compared with that in control group,but there was no significant difference in expression level of MT- 2.The expression levels of MT-1 and MT-2 in testis of As ＋Zn group were significantly lower than those of As group（P 0.01）.[Conclusion]1. Subacute arsenic poisoning can enhance the oxidative stress effect of testis tissue,and induce the increase expression of MT-1 and MT-2 in testis of rats. However,zinc supplement while arsenic exposure may decrease the expression of MT-1 and MT-2.2. Zinc supplement can relieve the testicular injury induced by arsenic exposure in rats through regulating expression of MT-1 and MT-2,ameliorating the testicular antioxidant status,and preventing arsenic accumulation and zinc deprivation.