【作者】 赵茜；

【导师】 张如意； 余建强；

【作者基本信息】 宁夏医科大学 ， 内科学， 2011， 硕士

【摘要】 目的通过建立雄性糖尿病大鼠生殖损伤的模型,观察氟伐他汀对糖尿病大鼠睾丸损伤的保护作用,并探讨氟伐他汀对糖尿病大鼠睾丸损伤保护作用的可能机制。方法选用SPF级雄性SD大鼠86只,随机取10只做为正常对照组,其余大鼠均腹腔注射STZ(65mg/kg)诱导糖尿病大鼠模型。将造模成功大鼠按血糖、体重均匀分为糖尿病模型对照组(DM)、氟伐他汀(6、18mg/kg)组和胰岛素组。氟伐他汀组每天灌胃1次,正常对照组及糖尿病模型组给予等体积生理盐水灌胃,胰岛素组给予精蛋白锌胰岛素皮下注射2U·kg-1·d-1,连续给药8周。8周末,处死动物,留取血清及睾丸组织,行血清睾酮(T)、睾丸组织MDA、SOD、GSH-Px检测,并在光镜下观察HE染色、TUNEL染色及免疫组织化学(Bcl-2,Bax)等指标,透射电镜下观察睾丸组织超微结构改变。结果1.氟伐他汀对糖尿病大鼠一般情况的影响:实验期间,糖尿病模型组大鼠除“三多一少”现象外,还出现多系统并发症的表现,死亡较多(41.6%);与糖尿病组比较,氟伐他汀各剂量组对外界反应相对灵敏,并发症程度较轻,死亡相对较少(36.4%和27.3%);胰岛素组大鼠的一般情况明显改善,死亡较少(27.3%)。2.氟伐他汀对糖尿病大鼠血糖、体重的影响:经8周药物干预后,糖尿病组大鼠空腹血糖显著高于正常组(P<0.01);氟伐他汀各组空腹血糖较糖尿病组有不同程度的下降(P<0.01);胰岛素组与正常组空腹血糖无显著性差异(P>0.05)。与正常组相比,糖尿病组大鼠体重显著减轻(P<0.01);氟伐他汀各组与糖尿病组大鼠体重无显著性差异(P>0.05);胰岛素组大鼠体重较糖尿病组显著增加(P<0.01),但不及正常组(P<0.01)。3.氟伐他汀对糖尿病大鼠睾丸质量、睾丸脏器系数的影响:糖尿病组大鼠睾丸质量、脏器系数较正常组显著降低(P<0.01);氟伐他汀各组睾丸质量及脏器系数高于糖尿病组,且18mg/kg组与糖尿病组存在显著性差异(P<0.01);胰岛素组大鼠睾丸质量低于正常组(P<0.01),但两组脏器系数无显著性差异(P>0.05)。4.氟伐他汀对糖尿病大鼠精子参数的影响:糖尿病组大鼠精子数量、精子活率显著少于正常组(P<0.01);氟伐他汀6mg/kg组精子数量、精子活率与糖尿病组无显著性差异(P>0.05),氟伐他汀18mg/kg组精子数量、精子活率均较糖尿病组显著增高(P<0.05或P<0.01),但不及胰岛素组(P<0.01)。胰岛素组精子数量、精子活率低于正常组(P<0.01)。5.氟伐他汀对糖尿病大鼠睾丸组织HE染色的影响:光镜下,正常组大鼠睾丸曲细精管饱满,各级生精细胞排列整齐,管腔内可见密集精子;糖尿病模型组大鼠曲细精管明显萎缩,生精细胞脱落、数量显著减少,大部分管腔内未见精子;氟伐他汀组曲细精管结构基本完整,6mg/kg组管腔内可见精子细胞核分裂像,18mg/kg组管腔内可见少量精子;胰岛素组曲细精管及各级生精细胞形态基本正常,管腔内可见较多精子。6.氟伐他汀对糖尿病大鼠睾丸生精细胞TUNEL染色的影响:凋亡细胞表达糖尿病组最多,并显著高于其余各组(P<0.01);氟伐他汀干预后,凋亡生精细胞数量较糖尿病组明显减少(P<0.01),且18mg/kg组优于6mg/kg组,但不及胰岛素组;胰岛素组凋亡细胞数量与正常组相近(P>0.05)。7.氟伐他汀对糖尿病大鼠睾丸组织电镜下超微结构的影响:糖尿病组大鼠睾丸间质细胞体积缩小,细胞质减少,线粒体减少、嵴溶解,内质网扩张,脂滴增加,间质细胞核固缩,染色质呈块状浓集;间质微血管内皮细胞增生、肿胀,基底膜显著增厚,可见大量纤维素样物质沉积。氟伐他汀组间质细胞细胞器增多,线粒体嵴结构较清晰,内质网增多,脂滴减少,细胞核染色质浓集、边集现象好转;间质微血管管腔通畅,血管内皮细胞形态明显好转,血管基底膜增厚现象明显减轻,纤维素样沉积明显减少,且18mg/ kg组优于6mg/kg组,接近于正常组。胰岛素组间质细胞、间质微血管超微结构与正常组接近。8.氟伐他汀对糖尿病大鼠血清睾酮的影响:糖尿病组大鼠睾酮水平较正常组显著降低(P<0.01)。氟伐他汀18mg/kg组睾酮水平较糖尿病组显著增高(P<0.01),但不及胰岛素组;胰岛素组睾酮水平低于正常组(P<0.05)。9.氟伐他汀对糖尿病大鼠睾丸组织SOD、GSH-PX活性和MDA含量的影响:与正常组大鼠相比,糖尿病模型组大鼠SOD水平显著降低(P<0.01)、GSH-PX、MDA水平显著增高(P<0.01);与糖尿病组大鼠相比,氟伐他汀各组SOD水平显著增高(P<0.01或P<0.05),GSH-PX水平、MDA水平显著下降(P<0.05或P<0.01);氟伐他汀各组SOD、GSH-PX、MDA水平与胰岛素组间无显著性差异(P>0.05)。10.氟伐他汀对糖尿病大鼠睾丸生精细胞Bax,Bcl-2免疫组化阳性细胞表达的影响:在正常组中,Bcl-2表达最明显,Bax表达最少,而糖尿病组较其余各组Bcl-2表达显著减少(P<0.01),Bax表达显著增加(P<0.01);氟伐他汀治疗组较糖尿病组Bcl-2表达显著增加,Bax表达显著减少(P<0.01),且18mg/kg组优于6mg/kg组。胰岛素组较氟伐他汀组Bcl-2表达增加、Bax表达减少(P<0.01),但与正常组比较还存在显著性差异(P<0.01)。结论1.氟伐他汀可减轻糖尿病大鼠的睾丸损伤,对睾丸组织具有保护作用。2.氟伐他汀对糖尿病大鼠睾丸损伤的保护作用机制可能与减轻糖尿病大鼠睾丸组织氧化应激水平以及调节生精细胞Bax,Bcl-2阳性细胞表达等有关。更多还原

【Abstract】 Objective To observe the protective effects of Fluvastatin on diabetic testis injury and explore the possible mechanism on the reproductive damage in diabetic male rats model.Methods 86 SPF Male SD rats were used in the experiment , 10 rats were taken as normal control group, the others were induced to diabetic model by injected STZ (65mg/kg) in intraperitoneal. The successful model rats were divided into diabetic group (DM), fluvastatin (6,18 mg / kg) group and insulin group according to their blood glucose and body weights. Fluvastatin groups were fed one time per day, the normal control group and diabetic model group were given equal volume of saline, and the insulin group was given protamine zinc insulin 2U·kg once a day in subcutaneously for 8 weeks. Animals were sacrificed after 8 weeks, the specimens of serum and testicular tissue from the rats were collected, the serum testosterone (T) and MDA, SOD, GSH-Px in testicular tissue were detected. The HE staining ,TUNEL staining and immunohistochemistry (Bcl-2 , Bax) indicators were observed under the light microscope, and the ultrastructural changes of testicular tissue were observed by transmission electron microscope.Results1.Effects of fluvastatin on the general situation of diabetic rats: during the experimental period, diabetic rats group showed typical symptoms of the complications of diabetes and higher mortality (41.6%). Compare to the diabetic group, the response of rats to the outside world was more sensitive, and with lesser complications and death rate (36.4% and 27.3%) in each dose of fluvastatin group. In insulin group, the general situation of rats significantly improved, and with lesser death ( 27.3%). 2. Effects of fluvastatin on blood glucose and body weight: after 8 weeks of drug intervention, fasting blood glucose level was significantly higher in the diabetic group than the normal control group (P <0.01); fasting blood glucose level in fluvastatin groups had different extent of decline compared with diabetic group (P <0.01); fasting blood glucose level had no significant difference in insulin group and normal control group (P> 0.05). Compared with normal control group, body weight reduced significantly in diabetic model rats (P <0.01); in each group of fluvastatin and diabetic rats there was no significant difference in body weight (P> 0.05); body weight of insulin group was significantly increased (P <0.01), but still less than the normal group (P <0.01).3. Effects of fluvastatin on diabetic rats testis mass and testicular coefficient: the quality and the coefficient of diabetic model rat testis were significantly lower than the normal control group (P <0.01); testis mass and coefficient in fluvastatin groups were higher than the diabetic model group and 18mg/kg group was significantly different with the diabetic mode group (P <0.01); testis mass was lower in insulin group than normal control group (P <0.01), but there were no significant difference in testis coefficient (P> 0.05).4. Effects of fluvastatin on sperm parameters in diabetic rats: the sperm count and sperm motility in diabetic model group were significantly less than the normal control group (P <0.01); fluvastatin 6mg/kg group had no significant difference with the diabetic mode group (P> 0.05) of sperm count and sperm motility rate, fluvastatin 18mg/kg group was significantly higher than the diabetic model group (P <0.05 or P <0.01)on these, but less than insulin group (P <0.01). Insulin group‘s sperm count and sperm motility had significant difference with the normal group (P <0.01).5. Effects of fluvastatin on HE staining of diabetic rat’s testicular tissue:under the light microscopy, the normal rat’s testis seminiferous tubules were full and neatly arranged in spermatogenic cell lumen and the sperm density; the diabetic model group rats’seminiferous tubules were atrophied, germ cells were significant reduced, no sperm in most of the lumens; structure of seminiferous tubules were integrity in fluvastatin groups: mitotic phase of sperm could be seen in 6mg/kg group and some sperms could be seen in 18mg/kg group. The morphologies of spermatogenic cells and seminiferous tubules in insulin group looked like as normal group, more sperm could be seen in the seminiferous tubules.6. Effects of fluvastatin on TUNEL staining of diabetic rats’spermatogenic cells: the amount of apoptotic cells was the most in the diabetic group, and significantly higher than other groups (P <0.01). After the intervention of fluvastatin, the number of apoptotic spermatogenic cells significantly decreased compared with diabetic model group (P <0.01), and it was better in the 18mg / kg group than 6mg/kg group, but still more than insulin group; The number of apoptotic cells in insulin group was similar to the normal group (P> 0.05).7. Effects of fluvastatin on testicular tissue ultrastructure of diabetic rats under the electron microscope: in diabetic rats, Leydig cells were shrunk, cytoplasmic reduced, mitochondrial reduced and cristae dissolved, endoplasmic reticulums were expanded, lipid droplets were increased , Leydig cell nuclear pyknosis , chromatin gathered; Interstitial microvascular endothelial cell proliferated and swelled, basement membrane thickened significantly, showing that a large number of cellulose-like substance deposited. In the fluvastatin group ,organelles increased in Leydig cells ,the structure of mitochondrial cristae became more clear, endoplasmic reticulum increased, lipid droplets, the Phenomenon of Leydig cell nuclear pyknosis and Chromatin gathered better than the diabetic group; The interstitial capillary lumen patency, significantly improved in vascular endothelial cells, vascular basement membrane thickening and fibrinoid deposition was significantly reduced. And the 18mg/kg group was better than 6mg/kg group, close to the normal group.The ultrastructure of interstitial cells and interstitial microvasculars in the Insulin group were close to the normal group.8. Effects of fluvastatin on serum testosterone levels in diabetic rats: testosterone levels in diabetic rats were significantly lower than the normal rats (P <0.01). Testosterone levels were higher in fluvastatin 18mg/kg group than the diabetic model group (P <0.01), but less than insulin group. The testosterone levels of insulin group were lower than the normal group (P <0.05).9. Effects of fluvastatin on testis tissue SOD, GSH-PX activity and MDA content in diabetic rats: compared with normal rats, SOD levels were significantly lower in diabetic rats than normal rats (P <0.01), GSH-PX and MDA levels were significantly higher than normal rats (P <0.01). SOD were significantly higher in the fluvastatin groups than the diabetic group (P <0.01 or P <0.05), GSH-PX level and MDA level were decreased significantly than the diabetic group (P < 0.05 or P <0.01); The fluvastatin groups have no significant differences with the Insulin group in SOD, GSH-PX and MDA level (P> 0.05).10. Effects of fluvastatin on Bax, Bcl-2 positive immunohistochemical expression of spermatogenic cells in diabetic rats: In the normal group, Bcl-2 expression was the most and Bax expression was the least, the Bcl-2 expression was significantly decreased and Bax expression was significantly increased in diabetic group than in the other groups (P <0.01). The Bcl-2 expression in fluvastatin group was significantly increased than in the diabetic group (P <0.01), and Bax expression was significantly decreased (P <0.01)than in the diabetic group, the 18mg / kg group was better than 6mg/kg group. Insulin group was better than the fluvastatin group (P <0.01), but there was significant difference with the normal group (P <0.01).Conclusion1.Fluvastatin can reduces testicular tissue Injury of diabetic rats, it has a protective effect on testicular tissue.2.The Protective mechanism of fluvastatin on the testicular tissue in diabetic rats may related to the reduced oxidative stress and regulation of germ cell Bax, Bcl-2 positive cells更多还原