阿片相关雄激素缺乏症的外周机制及胰岛素样生长因子1的治疗作用

Peripheral mechanism of opioid-induced androgen deficiency and therapeutic effect of insulin-like growth factor 1

目的探讨阿片相关雄激素缺乏症(OPIAD)的外周机制及胰岛素样生长因子1(IGF1)对OPIAD的治疗作用。方法雄性成年C57BL/6小鼠sc给予吗啡5 mg·kg^(-1)或等体积生理盐水,4 h后取外周血和睾丸组织。分离雄性成年小鼠睾丸曲细精管组织培养24 h后进行以下分组:(1)正常对照组和IGF1组(IGF1100μg·L^(-1),孵育12,24和48 h);(2)正常对照组与阿片μ受体激动剂DAMGO组(DAMGO 10μmol·L^(-1),孵育48 h);(3)正常对照组、IGF1组(IGF1100μg·L^(-1),孵育48 h)、DAMGO组(DAMGO 10μmol·L^(-1),孵育48 h)和IGF1+DAMGO组(同时加入DAMGO 10μmol·L^(-1)和IGF1100μg·L^(-1),孵育48 h);正常对照组、溶剂组(0.01%DMSO,孵育24 h)、IGF1组(IGF1100μg·L^(-1),孵育24 h)、鬼臼苦素(PPP)组(PPP 1μmol·L^(-1),孵育24 h)和PPP+IGF1组(PPP 1μmol·L^(-1)孵育2 h后,加入IGF1100μg·L^(-1)继续培养24 h)。用超高效液相色谱-串联质谱法检测小鼠血清和睾丸组织及曲细精管组织培养基中的睾酮含量,用ELISA检测小鼠血清和睾丸组织及曲细精管组织培养基中IGF1含量,用荧光定量PCR检测曲细精管组织中睾酮合成相关酶mRNA水平。结果小鼠实验中,吗啡组小鼠血清和睾丸组织中睾酮和IGF1含量较正常对照组显著降低(P<0.05)。离体实验中:(1)IGF1孵育24和48 h组曲细精管培养基中睾酮含量均较相应时间正常对照组明显升高(P<0.01)。(2)DAMGO组曲细精管培养基中IGF1含量较正常对照组显著降低(P<0.01)。(3)与正常对照组相比,IGF1组曲细精管培养基中睾酮含量明显升高(P<0.01),DAMGO组降低(P<0.05);IGF1组和IGF1+DAMGO组曲细精管组织中胆固醇合成急性调节蛋白(Star)和17β-羟基类固醇脱氢酶3(17βHsd3)mRNA水平显著升高(P<0.05),DAMGO组3β-羟化类固醇脱氢酶1(3βHsd1)mRNA水平显著降低(P<0.01)。与DAMGO组相比,IGF1+DAMGO组培养基中睾酮含量显著升高(P<0.01);IGF1+DAMGO组曲细精管组织中Star和17βHsd3 mRNA水平显著升高(P<0.05)。PPP+IGF1组曲细精管培养基中睾酮含量与IGF1组相比明显降低(P<0.01),而与PPP组相比无明显变化。结论OPIAD存在阿片μ受体介导的外周机制。IGF1通过其受体促进睾酮的合成分泌,可纠正DAMGO引起的睾酮合成分泌抑制。

OBJECTIVE To investigate the peripheral mechanism of opioid-induced androgen deficiency(OPIAD)and the therapeutic effect of insulin-like growth factor 1(IGF1)against OPIAD.METHODS C57 BL/6 male adult mice were sc injected with morphine(5 mg·kg^(-1))and saline before the serum and testicular tissue were collected after 4 h.The seminiferous tubules of adult male mice were isolated and cultured for 24 h,and then divided into different groups for the following experiments:(1)Normal control and IGF1 group(incubated with IGF1100μg·L^(-1) for 12,24 or 48 h).(2)Normal control and DAMGO group(incubated with DAMGO 10μmol·L^(-1) for 48 h).(3)Normal control,IGF1 group(incubated with IGF1100μg·L^(-1) for 48 h),DAMGO group(incubated with DAMGO 10μmol·L^(-1) for 48 h)and IGF1+DAMGO group(co-incubated with DAMGO 10μmol·L^(-1) and IGF1100μg·L^(-1) for 48 h).(4)Normal control,vehicle(incubated with 0.01%DMSO for 24 h),IGF1(incubated with IGF1100μg·L^(-1) for 24 h),picropodophyllin(PPP)(incubated with PPP 1μmol·L^(-1) for 24 h)and PPP+IGF1(incubated with PPP 1μmol·L^(-1) for 2 h,then co-incubated with IGF1100μg·L^(-1) for 24 h)group.Testosterone contents in the serum and testicular homogenate of mice and the culture supernatant of seminiferous tubules were measured by ultra high performance liquid chromatography-tandem mass spectrometry(UPLC-MS).IGF1 contents in the serum and testicular homogenate of mice and the culture supernatant of seminiferous tubules were measured by ELISA assay.The mRNA levels of testosterone synthesis-related enzymes in seminiferous tubules were measured by qRT-PCR.RESULTS In vivo:Compared to normal control group,the contents of testosterone and IGF1 in the serum and testicular homogenate of mice in morphine groups were significantly decreased(P<0.05).In vitro:Compared to normal control group,the testosterone contents in the culture supernatant of seminiferous tubules were increased in IGF124 h-and 48 hincubation groups(P<0.01).Compared to normal control group,the IGF1 contents in the culture supernatant of seminiferous tubules were decreased in DAMGO group(P<0.01).Compared to normal control group,the testosterone content in the culture supernatant of seminiferous tubules was increased significantly in IGF1 group(P<0.01)but decreased in DAMGO group(P<0.05).The mRNA levels of Star and17βHsd3 in tissue of seminiferous tubules were enhanced in IGF1 group and IGF1+DAMGO group(P<0.05),but 3βHsd1 mRNA levels were inhibitted in DAMGO group(P<0.01).Compared with DAMGO group,the testosterone content in the culture supernatant of seminiferous tubules was increased significantly in IGF1+DAMGO group(P<0.01),and the mRNA levels of Star and 17βHsd3 in tissue of seminiferous tubules were enhanced in IGF1+DAMGO group(P<0.05).Compared with IGF1 group,the testosterone content was decreased in IGF1+PPP group(P<0.01).There was no significant difference in concentrations of testosterone between PPP group and IGF1+PPP group.CONCLUSION The peripheral mechanism mediated byμreceptor is involved in OPIAD.IGF1 can promote the synthesis and secretion of testosterone through IGF1 receptor,and alleviate the inhibition of the synthesis and secretion of testosterone caused by DAMGO.