L-NAME对实验性隐睾生精细胞凋亡与p38MAPK表达的影响

The effect of N^w-nitro-L-arginine-methyl-ester on the spermatogenic cell apoptosis and activity of p38MAPK in rats experimental cryptorchidism

目的:通过研究一氧化氮合酶(NOS)抑制剂L-NAME对大鼠实验性隐睾生精细胞凋亡的调节及其对磷酸化p38MAPK表达的影响,揭示实验性隐睾生精细胞凋亡过程中NOS/NO与p38MAPK之间的关系。方法:雄性SD大鼠建立单侧隐睾模型,随机分为隐睾组、隐睾+生理盐水组、隐睾+L-NAME组、假手术组,术后第7天处死大鼠。硝酸还原酶法测定睾丸组织NO_x^-含量,化学比色法测定睾丸组织中NOS活性;免疫组化与Western blot检测磷酸化p38MAPK蛋白表达的变化;TUNEL原位末端标记法结合流式细胞术检测生精细胞凋亡。结果:各组大鼠左侧睾丸及假手术组左侧与右侧睾丸之间重量无明显差异,其余各组右侧隐睾均轻于左侧睾丸;隐睾+L-NAME组隐睾重量明显大于隐睾组及隐睾+生理盐水组,其睾丸组织中NO_x^-含量、NOS活性均低于假手术组和隐睾组;免疫组化显示磷酸化p38MAPK在假手术组睾丸生精上皮内表达阴性,其他三组表达阳性,且多见于凋亡的生精细胞,隐睾+L-NAME组的阳性表达弱于隐睾组和隐睾+生理盐水组。Western blot显示,四组均有磷酸化p38MAPK的条带,隐睾组和隐睾+生理盐水组表达最强,隐睾+L-NAME组次之,假手术组最弱。凋亡细胞百分比和凋亡指数在隐睾组及隐睾+生理盐水组最高,隐睾+L-NAME组次之,假手术组最低,差异均有显著性意义。结论:L-NAME可抑制生精细胞凋亡;L-NAME可抑制磷酸化p38MAPK的表达;一氧化氮可通过激活p38MAPK介导生精细胞凋亡。

Objective To investigate the inhibition effect of L-NAME on the expression of phosphorylating p38 MAPK during spermatogenic cell apoptosis in rats experimental cryptorchidism,reveal the relation of NOS/NO and p38 MAPK in the process of spermatogenic cells apoptosis.Methods The male rats(42day-old)were divided into four groups:sham pertion,cryptorchid,cryptorchid+saline,cryptorchid+L-NAME(Nw-nitro-L-arginine-methyl-ester).The rats were sacrificed on the 7th days after operation.NOx-(nitrite/nitrate)concentration in the right testis was measured by Greiss method.The activity of NOS in the right testis was measured by colorimetric method.The effect of L-NAME on apoptosis of spermatogenic cell in the unilateral cryptorchidism was evaluated by FCM and TUNEL.The protein expression level of phosphorylating p38 MAPK in testes were detected by Western blot and immunohistochemical assay accordingly.Results Administration of L-NAME could decrease NOx-concentration,the activity of NOS and spermatogenic cell apoptosis of the cryptorchid testes,and it could significantly inhibit weight lose of the cryptorchid.In the spermatogenic epithelium of the sham operation group there were no phosphorylating p38 MAPK expression which could be detected in the spermatogenic epithelium of the other three groups by immunohistochemical assay,but the expression of phosphorylating p38 MAPK in the testes of the cryptorchid group and the cryptorchid+saline group are higher than that of the cryptorchid testes treated with L-NAME.Western Blot analysis showed the protein expression of phosphorylating p38 MAPK in the testes of the cryptorchid group and the cryptorchid+saline group is higher than the other two group,the sham operation group is the lowest.Conclusion 1.Administration of L-NAME can inhibit spermatogenic cell apotosis.2.Administraton of L-NAME can inhibit the expression of phosphorylating p38MAPK.3.Nitric oxide may mediate spermatogenic cells apoptosis by activating p38MAPK pathway.