饮食诱导糖调节受损大鼠睾丸细胞抗氧化水平、细胞周期进程和凋亡的变化

目的:通过检测长程高脂饮食诱导的糖调节受损(IGR)大鼠模型睾丸细胞抗氧化水平、细胞周期进程、坏死、凋亡以及钙离子浓度(Ca2+]i)和线粒体膜电位(Δψm)的变化,探讨IGR对雄性生殖的损伤和机制。方法:雄性Wistar大鼠随机分为正常对照组(10只)和IGR模型组(30只),通过长程高脂饮食连续喂养20周建立IGR模型;苏木精-伊红(HE)染色检测大鼠睾丸生精细胞病理学改变;生物化学方法检测丙二醛(MDA)含量、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)活性;碘化丙啶(PI)单染流式细胞术检测细胞周期进程的变化;AnnexinⅤ-FITC凋亡试剂盒检测细胞凋亡;Fluo-3和Rhodamine探针标记流式细胞术检测Ca2+]i和Δψm的变化。结果:Wistar大鼠高脂饮食连续喂养20周后,其中12只大鼠空腹血糖控制在6.1～7.0 mmol/L之间和/或糖负荷2 h血糖在7.8～11.1 mmol/L之间,成模率达到40%。显微镜下可见正常对照组睾丸组织切片中较多正处于分裂的精母细胞和精子细胞,而IGR模型组则未见或较少见正处于分裂的细胞。与正常对照组比较,IGR模型组睾丸组织中MDA含量显著增高,SOD、CAT和GSH-Px活性则显著降低(P<0.05或P<0.01)。IGR模型组睾丸G0/G1期细胞百分率显著降低,而G2/M期细胞百分率显著升高(P<0.05或P<0.01),S期细胞百分率未见明显变化;IGR模型组正常睾丸细胞百率比未见明显改变,坏死睾丸细胞百分率显著降低,而凋亡睾丸细胞百分率显著升高(P<0.05和P<0.01)。同时,IGR模型组睾丸细胞Ca2+]i显著升高,而Δψm显著降低(P<0.01)。结论:IGR能够引起大鼠睾丸生精细胞分裂障碍,可能机制涉及氧化损伤增加、抗氧化酶活性降低、细胞G2/M期阻滞、Ca2+]i升高、Δψm降低以及睾丸细胞凋亡。

Changes in the antioxidant level,cell cycle progression and apoptosis of testicular cells in rats with diet-induced impaired glucose regulation

Objective: To detect the changes of the antioxidant level,cell cycle progression,necrosis and apoptosis,calcium ion concentration(i) and mitochondrial membrane potential(Δψm) in the model rats of impaired glucose regulation(IGR) induced by long-range high-fat diet,and to explore IGR-induced male reproductive injury and its mechanisms.Methods: Forty male Wistar rats were randomly divided into a normal control(n = 10) and an IGR model group(n = 30),and the IGR model was established by 20 weeks of long-range high-fat diet.Pathological changes in the rat spermatogenic cells were detected by HE staining;the content of malondialdehyde(MDA) and activities of superoxide dismutase(SOD),catalase(CAT) and glutathione peroxidase(GSH-Px) were measured with biochemical methods;changes in the cell cycle progression,necrosis and apoptosis were determined using flow cytometry with propidium iodide(PI) dyeing and the Annexin Ⅴ-FITC kit,respectively,and i and Δψm were detected by flow cytometry with Fluo-3 and Rhodamine probe labeling,respectively.Results: After 20 weeks of continuous high-fat diet,fasting blood glucose was kept at 6.1-7.0 mmol/L and blood glucose at 7.8-11.1 mmol/L after 2 h glucose load in 12 rats,with a 40% success rate of modeling.Lots of dividing spermatocytes and spermatids were seen in the tissue sections of the normal control rats under the microscope,but few or none in the IGR models.Compared with the normal controls,the IGR model rats showed remarkably increased MDA content and decreased SOD,CAT and GSH-Px activities in the testis tissue(P < 0.05 or P < 0.01),reduced G0/G1 cells and increased G2/M cells(P < 0.05 or P < 0.01),decreased necrotic cells and increased apoptotic cells(P < 0.05 or P < 0.01),increased i and decreased Δψm(P < 0.01),but no significant changes in the percentages of S cells and normal cells.Conclusion: IGR can cause spermatogenic cell division disorder in rats,which may be attributed to increased oxidative damage,decreased antioxidant enzyme activities,G2/M phase arrest,i elevation,Δψm reduction,and apoptosis of testicular cells.