Objective To investigate the effect of advanced glycation end products (AGEs) on testosterone pro-duction of rat Leydig cells and its underlying mechanism. Methods First, the primary Leydig cells were isolated, purified and cultured in vitro. The stromal cells were identified by 3β-HSD immunocytochemistry. Second, the effects of various concentrations of AGEs (25μg/mL, 50μg/mL, 100μg/mL and 200μg/mL) and BSA (200μg/mL) on Leydig cell viability were evaluated by MTT. Finally, Leydig cells were pre-incubated with various concentrations of AGEs and 200 μg/mL BSA for 12 h. Then, the culture medium was replaced with fresh medium containing human chorionic gonadotropin (hCG, 4 U/mL) with or without the same concentrations AGEs and BSA. The control group did not contain AGEs and BSA. The secretion of testosterone induced by human chorionic gonadotropin (hCG) was determined by ELISA. The pro-tein expression levels of ERK1/2 phosphorylationwere were measured by western blot. Results MTT data indicated that the viability of the Leydig cells treated with AGEs (concentrations≤200μg/mL) was not significantly altered. ELISA and western blot showed that AGEs could remarkably suppress testosterone production and decrease the phosphorylation levels of p-ERK1/2 induced by hCG in a concentration-dependent manner in rat Leydig cells compared with the control group (P<0.01). Conclusion AGEs decrease testosterone secretion in rat Leydig cells by inhibiting ERK1/2 phosphorylation.%目的：探讨非酶糖基化终末产物(AGEs)对大鼠睾丸间质细胞睾酮分泌的影响及其潜在机制。方法首先，原代培养大鼠睾丸间质细胞，采用3β-HSD免疫细胞化学方法对间质细胞进行鉴定；其次，MTT法测定不同浓度的AGEs (25μg/mL、50μg/mL、100μg/mL、200μg/mL)及200μg/mL BSA作用Leydig细胞后的细胞活力；最后，用不同浓度的AGEs及200μg/mL BSA预处理Leydig细胞12 h，之后更换含相应浓度AGEs或BSA的培养基并加入终浓度为4 U/mL的hCG，其中对照组不含AGEs和BSA，ELISA法和Western blot分别检测睾酮分泌量和p-ERK1/2的表达量。结果 MTT法表明AGEs (≤200μg/mL)对Leydig细胞的活力在本实验所研究的时间内没有影响；ELISA法和Western blot结果显示，不同浓度AGEs处理后，hCG诱导的Leydig细胞睾酮合成量和ERK1/2蛋白的磷酸化都呈现浓度依赖性下降，与对照组相比，差异有统计学意义(P<0.01)。结论 AGEs通过抑制ERK1/2的磷酸化降低大鼠Leydig cells睾酮的分泌。
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