过硫谷胱甘肽可改善高脂食物导致的雄鼠低睾酮水平状态

Glutathione persulfide prevents high-fat diet induced down-regulation of testosterone biosynthesis

目的探究过硫谷胱甘肽(glutathione persulfate,GSSH)是否可以改善雄性肥胖小鼠的低睾酮水平,并探讨其作用机制。方法将45只小鼠平均分为3组,低脂饮食(low-fat diet,LFD)组:喂LFD 10周,随后45 d继续LFD同时每天腹腔注射生理盐水(normal saline,NS),记为LFD+NS组(n=15);高脂饮食(high-fat diet,HFD)组:喂HFD 10周,随后45 d继续HFD同时每天腹腔注射NS,记为HFD+NS组(n=15);HFD+GSSH组(n=15):HFD 10周,随后45 d HFD同时每天腹腔注射GSSH(200 mg/kg)。处理结束后所有小鼠眼眶取血,断颈处死小鼠并解剖取出睾丸,分别用ELISA、qPCR以及Western blotting的方法检测小鼠血清睾酮和丙二醛(malondialdehyde,MDA)水平、睾酮关键合成酶(StAR、3β-HSD、Cyp11a1、Cyp17a1)以及抗氧化蛋白(StAR、3β-HSD、NR5A1、EHD3)表达水平等。此外,用100μmol/L的GSSH处理小鼠睾丸碎片后检测睾酮合成酶表达水平。最后用50μmol/L和100μmol/L的GSSH分别处理小鼠睾丸间质TM3细胞株24 h后,加入100μmol/L的H_(2)O_(2),继续培养TM3细胞24 h,然后收集细胞检测NR5A1、SOD与Nrf2蛋白表达水平。结果①给药结束后,LFD+NS组、HFD+NS组与HFD+GSSH组小鼠的体质量分别是(30.67±1.22)g、(40.43±1.56)g、(33.30±0.95)g;HFD+NS组体质量增加了24.53%,给药前后差异有统计学意义(P=0.002),而LFD+NS组、HFD+GSSH组的体质量在给药前后差异均无统计学意义(均P>0.05)。②HFD+NS组小鼠睾酮浓度为(12.9±1.7)μg/L,显著低于LFD+NS组[(18.3±1.2)μg/L],差异有统计学意义(P=0.020);HFD+GSSH组小鼠睾酮浓度为(25.4±2.1)μg/L,显著高于HFD+NS组,差异有统计学意义(P=0.030)。RT-PCR检测结果显示,与LFD+NS组小鼠相比,HFD+NS组小鼠睾丸所有被检测的睾酮合成关键基因(StAR、3β-HSD、Cyp11a1及Cyp17a1)表达水平都显著下降(P=0.003、P=0.007、P<0.001、P<0.001)。这些基因的表达水平则在HFD+GSSH组小鼠睾丸中得到了恢复(P=0.002、P<0.001、P<0.001、P=0.006)。③与LFD+NS组小鼠[(9.00±1.59)nmol/mL]相比,HFD+NS组小鼠血清MDA水平[(10.61±1.73)nmol/mL]显著升高(P=0.016);与HFD+NS组小鼠相比,HFD+GSSH组小鼠血清MDA水平[(9.23±0.94)nmol/mL]下降,且具有统计学意义(P=0.048)。④HFD+NS组的肥胖小鼠睾丸中NR5A1、EHD3、StAR与3β-HSD蛋白水平与LFD+NS组小鼠相比明显下调(P=0.002、P=0.012、P=0.004、P=0.043),HFD+GSSH组小鼠睾丸中NR5A1、EHD3、StAR与3β-HSD蛋白水平与HFD+NS组的肥胖小鼠相比则显著上升(P<0.001、P=0.017、P=0.004、P<0.001)。⑤TM3细胞在H_(2)O_(2)的存在下,NR5A1、Nrf2与SOD的表达水平皆发生显著下调(P<0.001、P=0.002、P=0.004)。结论GSSH通过提高睾酮合成所需关键基因表达而改善HFD喂养的雄鼠睾酮水平。

ObjectiveTo investigate effects and underlying mechanisms of glutathione persulfate(GSSH)on the level of testosterone in male obese mice.MethodsTotally 45 mice were divided into 3 groups on average.Low-fat diet(LFD)+normal saline(NS)group:15 mice were fed with LFD for 10 weeks,followed by LFD together with daily intraperitoneal injection of saline for 45 d;high-fat diet(HFD)+NS group:15 mice were fed with high-fat diet for 10 weeks,followed by HFD and daily intraperitoneal injection of NS for 45 d;HFD+GSSH group:15 mice were fed with HFD for 10 weeks,followed by a HFD for 45 d and daily intraperitoneal injection of GSSH(200 mg/kg).After the treatment,all mice were killed with their necks-severed,testis and serum were taken out from the mice.Serum levels of testosterone and malondialdehyde(MDA),the mRNA levels of key enzymes for testosterone synthesis(StAR,3β-HSD,Cyp11a1 and Cyp17a1)were measured by RT-PCR.The testicular protein levels of StAR,3β-HSD,NR5A1 and EHD3 were measured by Western blotting assay.Protein levels of NR5A1,SOD and Nrf2 were measured in mouse Leydig TM-3 cells that were treated with 50μmol/L and 100μmol/L GSSH,respectively,following with treatment with 100μmol/L H_(2)O_(2).Results1)After treatment,the body weight of mice in HFD+GSSH group did not change significantly,while the body weight of mice in HFD+NS group raised by 24.53%(from 32.46 g to 40.43 g)during the 45-day-intraperitoneal injection(P=0.002).2)Serum level of testosterone in HFD+NS group[(12.9±1.7)μg/L]was significantly lower than that in LFD+NS group[(18.3±1.2)μg/L,P=0.020].However,serum level of testosterone in HFD+GSSH group was(25.42±2.1)μg/L,which was significantly higher than that in HFD+NS group(P=0.030).The RT-PCR test results showed that compared with LFD+NS group,the expression levels of all key genes involved in testosterone synthesis(StAR,3β-HSD,Cyp11a1,Cyp17a1)showed a significant decrease in HFD+NS group(P=0.003,P=0.007,P<0.001,P<0.001).The expression levels of these genes were restored in the mouse testes of HFD+GSSH group(P=0.002,P<0.001,P<0.001,P=0.006).3)Similarly,compared with LFD+NS group[(9.00±1.59)nmol/mL],the serum MDA level of HFD+NS group[(10.61±1.73)nmol/mL]raised significantly(P=0.016),while GSSH reversed the raised HFD+NS high level of serum MDA in HFD+GSSH group[(9.23±0.94)nmol/mL,P=0.048].4)Both levels of NR5A1,EHD3,StAR,and 3β-HSD were reduced in HFD+NS group(P=0.002,P=0.012,P=0.004,P=0.043),but their levels were significantly restored in HFD+GSSH group(P<0.001,P=0.017,P=0.004,P<0.001).5)The levels of NR5A1,Nrf2 and SOD were obviously down-regulated in TM3 cells treated with H 2O 2(P<0.001,P=0.002,P=0.004).ConclusionGSSH can raise serum level of testosterone in HFD-fed mice by up-regulating expression of genes which are important for testicular testosterone biosynthesis.