邻苯二甲酸二乙基己酯及代谢产物MEHP对幼鼠睾丸组织TGF-β1表达和端粒酶活性的影响

Di-2-ethylhexyl phthalate and its metabolite single-ethylhexyl phthalate affect TGF-beta 1 expression and telomerase activity in the testis of young male rats

目的:观察邻苯二甲酸二乙基己酯(DEHP)及代谢产物邻苯二酸-单-2-乙基己酯(MEHP)对幼鼠睾丸组织转化生长因子-β1(TGF-β1)表达和端粒酶活性的影响,探讨DEHP和MEHP损害生精功能可能的机制。方法:生后2周龄的Wistar雄性幼鼠96只,随机分8组,每组12只。随机选择1组行生理盐水[0.9%NS0.2 ml/(kg.d),喂养3周]灌胃,作为正常对照组(NC组);再选1组行环磷酰胺[CTX 100 mg/(kg.d),喂养1周]灌胃,作为阳性对照组(PC组);余各组分别用DEHP、MEHP按低剂量[100 mg/(kg.d),喂养3周]、中剂量[200 mg/(kg.d),喂养2周]、高剂量[300 mg/(kg.d),喂养1周]灌胃制作动物模型。观察不同时期、不同剂量下睾丸组织精子形态变化,光镜下计数精子头部及畸形率;应用免疫组化SABC法及RT-PCR法检查睾丸组织TGF-β1的表达,并测定面密度;ELISA法检查睾丸组织中端粒酶活性。结果:①睾丸组织内精子形态变化:用药组精子数量减少,出现精子断头、无钩、双尾等畸形精子;在光镜下精子计数,与NC组比较,用药各组精子头部显著减少(P<0.05),畸形率增加(P<0.05),但高剂量短时间用药组与低剂量长时间用药组比较,差异无统计学意义(P>0.05)。②睾丸组织TGF-β1的表达变化:NC组低表达,DEHP及代谢产物MEHP染毒各组生精细胞内表达增多,PC组大量表达,阳性细胞呈黄褐色,主要分布于胞膜及胞质。NC组面密度为0.156 0±0.003 5、TGF-β1mRNA为1.51±0.20,PC组面密度为0.534 0±0.003 1、TGF-β1 mRNA为8.43±1.75;用药的DEHP组面密度均值为0.289 0±0.003 6、TGF-β1 mRNA为3.83±1.57,MEHP组面密度均值为0.284 0±0.003 1、TGF-β1 mRNA为3.51±1.41,用药各组与NC组、PC组比较差异有统计学意义(P<0.01),但高剂量短时间用药组与低剂量长时间用药组比较,差异无统计学意义(P>0.05);③睾丸组织中端粒酶活性:与NC组比较,用药各组睾丸组织中端粒酶活性下降(P<0.05),高剂量短时间用药组与低剂量长时间用药组比较,差异无统计学意义(P>0.05)。结论:DEHP及代谢产物MEHP对幼鼠生精功能有明显损害,其损害机制可能与DEHP及代谢产物MEHP诱导睾丸组织TGF-β1的表达水平升高、端粒酶活性降低有关。

Objective： To investigate the influences of di-2-ethylhexyl phthalate（DEHP） and its metabolite single-ethylhexyl phthalate（MEHP） on the expression of transforming growth factor-beta 1（TGF-β1） and telomerase activity in young male Wistar rats.Methods： Ninety-six 2-week-old male Wistar rats were equally randomized into a normal control（NC） group,a positive control（PC） group,and six experimental groups.Those of the NC group were intragastrically administered 0.9% normal saline at a dose of 0.2 ml per kg per d for 3 weeks,those in the PC group cyclophosphamide（CTX） at 100 mg per kg per d for 1 week,and those of the experimental groups DEHP and MEHP,respectively,at a low dose（100 mg per kg per d） for 3 weeks,a moderate dose（200 mg per kg per d） for 2 weeks,and a high dose（300 mg per kg per d） for 1 week.Then we observed the morphological changes of the testicular sperm and counted the sperm heads and their abnormity rate at different doses and times.We detected the expression of TGF-β1 in the testis tissue using immunohistochemical SABC and RT-PCR,measured the area density,and determined telomerase activity by ELISA.Results： Compared with the NC group,the experimental groups showed an obvious reduction in the total sperm count and number of sperm heads（P〈0.05） and a significant increase in the rate of teratosperm（P〈0.05）,such as decapitated,hookless,and double-tailed sperm.And there were no significant differences between the high-dose short-term and low-dose long-term medication groups（P〉0.05）.The expression of TGF-β1 was low in the NC group,high in the PC group,and obviously increased in the membrane and cytoplasm of spermatogenic cells of the experimental groups.The area density and TGF-β1 mRNA expression were 0.156 0±0.003 5 and 1.51±0.20 in the NC group,0.534 0±0.003 1 and 8.43±1.75 in the PC group,0.289 0±0.003 6 and 3.83±1.57 in the DEHP groups,and 0.284 0±0.003 1 and 3.51±1.41 in the MEHP groups.There were significant differences between the experimental and the other two groups（P〈0.01）,but not between the high-dose short-term and low-dose long-term medication groups（P〉0.05）.Telomerase activity was remarkably reduced in the experimental groups as compared with the NC group（P〈0.05）,but with no significant difference between the high-dose short-term and low-dose long-term medication groups（P〉0.05）.Conclusion： DEHP and its metabolite MEHP can evidently induce spermatogenic injury in young male rats,which may be associated with their induction of increased TGF-β1 expression and decreased telomerase activity in the rat testis.