低剂量棉酚与甾体激素联合应用对生精细胞凋亡和iNOS蛋白表达的影响

Influence of combination regimen of low-dose gossypol acetic acid with steroid hormones on apoptosis of germs and iNOS expression

目的研究低剂量棉酚与甾体激素联合应用（棉甾联合用药）对大鼠睾丸生精细胞凋亡和诱导型一氧化氮合酶（iNOS）蛋白表达的影响。方法本实验采用大鼠喂服棉酚12.5mg/（kg.d）＋激素[去氧孕烯125μg/（kg.d）＋炔雌醇25μg/（kg.d）＋十一酸睾丸酮100mg/（kg.d）]联合用药的方式,与单独喂服相同剂量的棉酚或激素的大鼠及喂服甲基纤维素溶剂的大鼠相对照,通过原位缺口末端标记（TUNEL）染色,观察生精细胞凋亡的情况;通过免疫组织化学染色和免疫印迹法,观察诱导型一氧化氮合酶（iNOS）蛋白表达的变化。结果TUNEL染色显示,在正常对照组,阳性着色主要分布在Ⅶ、Ⅷ、Ⅸ期生精小管的残余体中,偶见于粗线期精母细胞中。棉甾联合用药组和激素组,阳性着色主要分布在粗线期精母细胞中,随用药时间的延长,阳性着色的细胞数量增多（P〈0.01）。iNOS免疫组织化学显示,在正常对照组,在残余体、精原细胞和粗线期精母细胞中的表达具有期依赖性。在Ⅶ、Ⅷ、Ⅸ期生精小管,iNOS主要在残余体中表达;在Ⅸ-Ⅻ期生精小管,精原细胞和粗线期精母细胞的核中可见少量表达。在棉甾联合用药组,iNOS在残余体中的表达,随用药时间的延长表达量降低（P〈0.05）;iNOS在粗线期精母细胞、精原细胞胞核中的表达,随用药时间的延长表达量增高（P〈0.05）。iNOS在正常对照组的间质细胞中持续表达,棉甾联合用药组4周降至最低,3时间点表达量无统计学差别。结论iNOS蛋白在低剂量棉酚加甾体激素联合应用大鼠睾丸中各部位的表达变化趋势不一致,分别产生不同的功能;生精细胞凋亡数增加,可能与iNOS的表达增多有关。

Objective To investigate the influence on apoptosis of germs and expression of inducible nitric oxide synthase （iNOS） expression in male rats testis by administered with combination regimen of low-dose gossypol acetic acid（ GA）with steroid hormones（desogestre/ethinylestradiol/testosterone undecanote（DSG/E/TU）. Methods Adult male rats were randomly divided into four groups. Group GH ： rats were fed orally with GA [ 12.5 mg （ kg. day） ] and DSG [ 125μg/（ kg. day ） ] / E [ 25 μg/（ kg. day ） ]/TU [100mg/（kg. day）]; Group G： rats were administered with a single dose of GA [ 12.5mg/（kg. day）]; Group H： rats were administered with DSG[ 125μg/（kg. day）], E[25μg/（kg. day）]and TU[ 100mg/（kg. day）] ; Group C： rats only received vehicle （1% methyl cellulose）. The testes were removed at 4,6,8 weeks respectively. TUNEL staining was observed. The expressions of iNOS were detected by immunohistochemical staining and Western blotting. Results The TUNEL positive stainings were found in the residual bodies in the methylcelluose-treated group（C） and in the pachytene spcrmatocytes nuclei in gossypol combined with steroid hormones group（GH）. The amount of apoptosis cells were much larger in the GH group than that in the control group with treatment time increasing（ P 〈 0.01 ）. iNOS was expressed constitutively in Leydig cells and in a stage-dependent manner in residual bodies, spermatogonium and pachytene spermatocyte in the normal testis. Compared with group C, expression was decreased in residual bodies（ P 〈 0.05） and increased in spermatogonium and pachytene spermatocyte（ P 〈 0.05） in GH group. Conclusion The effect of low-dose gossypol acetic acid with steroid hormones to iNOS＇ s expression was different. The studies suggest a role for iNOS in spermatogenesis and germs apoptosis.