重组球形脂联素通过调节自噬及内质网应激对糖尿病小鼠睾丸的保护作用

Protective effect of recombinant spherical adiponectin on testis of diabetic mice by regulating autophagy and endoplasmic reticulum stress

目的观察外源性重组球形脂联素对糖尿病小鼠睾丸组织中自噬及内质网应激水平的影响,探讨其保护糖尿病小鼠睾丸的作用机制。方法48只雄性C57BL/6小鼠随机分为对照组(NC组,n=8)及糖尿病造模组(n=40)。采用腹腔注射链脲佐菌素制备1型糖尿病小鼠模型,共24只小鼠造模成功。将造模成功的糖尿病小鼠随机分为糖尿病组(DM组)、糖尿病脂联素低剂量组(DLA组)及高剂量组(DHA组),每组8只。DLA组、DHA组分别腹腔注射重组球形脂联素5、15μg/(kg·d),NC组、DM组腹腔注射等量生理盐水。于实验第8周末,采用葡萄糖氧化酶法检测空腹血糖水平,酶联免疫吸附法(ELISA)测定血清睾酮、胰岛素、卵泡刺激素(FSH)及黄体生成素(LH)水平;测量睾丸质量并计算睾丸指数,检测精子活动率及数量;HE染色观察睾丸组织形态变化;RT-PCR法测定自噬指标微管相关蛋白1轻链3(LC3)、自噬效应蛋白Beclin1、p62及内质网应激指标葡萄糖调节蛋白-78(GRP78)、CCAAT增强子结合蛋白同源蛋白(CHOP)、半胱氨酸天冬氨酸特异性蛋白酶-12(Caspase-12)mRNA的表达。结果与NC组比较,DM组小鼠睾丸组织出现生精小管变形,生精细胞及支持细胞数目减少,管腔内精子数目减少,部分管腔可见生精细胞脱落等病理改变,体重、睾丸质量、精子活动率、精子数量、血清胰岛素、睾酮、FSH及LH水平降低(P<0.05),LC3及Beclin1 mRNA表达水平降低(P<0.05),p62、GRP78、CHOP及Caspase-12 mRNA表达水平明显升高(P<0.05)。经重组球形脂联素治疗后,DLA组及DHA组上述指标均出现不同程度的改善,且DHA组改善情况优于DLA组。结论糖尿病小鼠睾丸自噬水平降低,内质网应激水平升高;重组球形脂联素可能通过调节自噬及内质网应激对糖尿病小鼠睾丸产生保护作用,且这种保护作用可能呈剂量依赖性。

Objective To analyze the effect of exogenous recombinant spherical adiponectin on the autophagy and endoplasmic reticulum stress in testis of diabetic mice induced by streptozotocin,and explore the mechanism of such protective effect to the testis of diabetic mice.Methods Forty-eight male C57 BL/6 mice were randomly divided into normal control group(NC,n=8)and diabetes group(n=40).Diabetic mouse model was established by intraperitoneal injection of streptozotocin,and 24 mice were successfully established.Diabetic mice were randomly divided into diabetic group(DM,n=8),diabetic adiponectin low dose group(DLA,n=8),and diabetic adiponectin high dose group(DHA,n=8).Mice in DLA group and DHA group were given intraperitoneal injection of recombinant spherical adiponectin at dose of 5μg/(kg·d)and 15μg/(kg·d)respectively.Mice in NC group and DM group were given intraperitoneal injection of the same amount of normal saline.After 8 weeks of adiponectin treatment,blood samples were taken to measure fasting blood glucose with glucose oxidase method.Enzyme-linked immunosorbent assay(ELISA)was performed to determine serum testosterone,insulin,follicular stimulating hormone(FSH)and luteinizing hormone(LH).Testicular weight was measured,and the testicular index(proportion by weight of testis/body)was calculated.Sperm count and mobility were measured.Morphological changes of testicular tissues were observed by HE staining.The mRNA expressions of endoplasmic reticulum stress(GRP78,CHOP,Caspase-12)and autophagy(LC3,Beclin1 and p62)were determined by real-time PCR.Results Compared with NC group,it was found in DM group that seminiferous tubules were deformed,the number of spermatogenic cells and Sertoli’s cells reduced,the number of sperm in the tubular lumen reduced,and spermatogenic cells sloughed in some tubular lumens.The body weight,testicular weight,sperm motility,sperm count,and the levels of serum insulin,testosterone,LH and FSH significantly decreased in DM group than those in NC group(P<0.05).The mRNA expression of Beclin1 and LC3 were significantly decreased(P<0.05),and of p62,GRP78,CHOP and Caspase-12 were significantly increased in the testis of DM group compared with NC group(P<0.05).Recombinant spherical adiponectin significantly reversed the changes mentioned above in the testis of diabetic mice,and the effect was better in DHA group than in DLA group.Conclusions The level of autophagy is decreased and the level of endoplasmic reticulum stress is increased in the testis of diabetic mice.Recombinant spherical adiponectin may have the protective effect on the testis of diabetic mice by regulating autophagy and endoplasmic reticulum stress,and the protective effect of adiponectin may be dose-dependent.