Effect And Mechanism Of Methamphetamine Abuse On Spermatogenesis And Erectile Function In Men

Background:Methamphetamine(METH)is a new synthetic drug,which was got by younger with the fastest growth and largest number of abusers in China.Studies showed that long-term abuse can cause sexual problem such as erectile dysfunction and premature ejaculation.Animal experiments have confrmed that METH exposure can inhibit testicular spermatogenesis.However,the molecular mechanism leading to impaired spermatogenesis and erectile dysfunction is unclear.Substance and energy metabolism are the characteristics of maintaining life and physiological activities.The abuse of METH can cause the changes of endocrine,immunity and metabolism,and affect the substance and energy metabolism due to oxidative stress,immune response and cell autophagy,which finally lead to the abnormality of mental and physiological activities.Therefore,oxidative stress,substance and energy metabolism as the entry points may be the key to elucidate the molecular mechanism of spermatogenesis and erectile function damaged by METH abuse.The study aimed to explore the effects and mechanisms of METH exposure on spermatogenic and erectile function from the perspective of substance and energy metabolism.Part Ⅰ Investigation on spermatogenic and erectile function of male abusers with METH at early stage of abstinenceObjective:To evaluate the spermatogenic and erectile function of male abusers with METH at the early stage of abstinence and to explore those influencing factors via the cross-sectional survey.Methods:(1)At the early stage of abstinence,the baseline and blood drawing data of 45 male METH abusers and 50 healthy men were collected,and the score of Self-Estimation Index of Erectile Function-No Sexual Intercourse(SIEF-NS)were investigated by questionnaire.(2)The above data and SIEF-NS score between the two groups were compared.(3)Correlation test was used to perform univariate analysis of inhibin B and SIEF-NS scores in the two groups.(4)Multiple stepwise regression analysis was used to analyze the influencing factors of inhibin B and SIEF-NS scores in the two groups.Results:(1)Compared with the control group,the levels of the inhibinB,SIEF-NS score,fasting blood glucose(FBG),cortisol,leptin and adenosine deaminase(ADA)decreased(P<0.05)and the levels of ACTH and 8-OHdG increased in the abuse group(P<0.05).(2)In METH abuse group,the factors related to inhibin B were FBG,testosterone(TT),prolactin,follicle stimulating hormone(FSH),ADA,8-OHdG and SIEF-NS,and the factors related to SIEF-NS score were prolactin,FBG,TT,FSH,ADA,8-OHdG and inhibin B.(3)In METH abuse group,the influencing factors of inhibinB were FBG(β=0.780,P<0.001),lnADA(β=0.244,P<0.001)and InLeptin(β=0.129,P=0.006<0.05),and the influencing factors of SIEF-NS score were lnADA(β=0.768,P<0.001)and ln8OHdG(β=-0.202,P<0.001).Conclusions:(1)Long-term METH abuse can lead to the inhibition of spermatogenic function,which is mainly related to the decrease of blood glucose(fasting).(2)Long-term METH abuse can lead to the inhibition of erection,which is mainly related to low energy metabolism.Part Ⅱ Study on the effect of METH exposure on spermatogenic function in ratsObjective:To explore the effect and mechanism of METH exposure on spermatogenesis from the view of substance(mainly carbohydrate)metabolism via establishing the rat model of METH exposure.Methods:(1)40 male SD rats were randomly divided into short-term control group,short-term exposure group,chronic control group and chronic exposure group.The rats were given intraperitoneal injection of METH solution in different periods with gradually increasing dose,while the corresponding control group was injected with the same volume of normal saline,and the availability of the model was evaluated at the end of the model.(2)Assessment of testicular spermatogenesis and local oxidative stress:The serum levels of inhibin B were detected by ELISA.Testicular and epididymal tissues were taken for determining testicular index,sperm count and motility,malondialdehyde(MDA)and superoxide dismutase(SOD),and testicular spermatogenic cell apoptosis.(3)Detection of blood glucose and its regulatory factors:The blood glucose concentration was determined by colorimetric method.The serum levels of corticotropin releasing hormone(CRH),corticotropin(ACTH),corticosterone(CORT)and 8 hydroxydeoxyguanosine(8-OHdG)were detected by ELISA.Mineralocorticoid receptor(MR)and glucocorticoid receptor(GR)protein expression in adrenal tissue were detected by Western-blot.(4)Non-targeted metabolomics for testis:The testicular tissues in the short-term exposure and the short-term control groups were detected by non-targeted metabonomics,the differential metabolites and metabolic pathways were analyzed,and the target molecules with significant differences between the two groups were screened and summarized.(5)Test for testicular glycolysis:The mRNA levels of hypoxia inducible factor 1α(HIF-1α),glucose transporter 1(GLUT1),hexokinase 1(HK1)and lactate dehydrogenase C(LDHC)in testes were detected by qRT-PCR,and the protein expression and localization of HIF-1α,GLUT1,HK1 and LDHC in testis were detected by Western-blot and immunohistochemistry.Results:(1)The models of short-term and chronic METH exposure were successfully established.(2)Compared with the short-term control group,there were no significant changes in blood glucose(P>0.05),but the levels of 8-OHdG,CRH,ACTH and CORT increased,the protein expression of MR and GR in the adrenal glands were up-regulated in the short-term exposure group(P<0.05).Compared with the chronic control group,the levels of blood glucose and CORT decreased,the levels of 8-OHdG,CRH and ACTH increased,the protein expression of MR and GR in the adrenal glands were down-regulated in the short-term exposure group(P<0.05).(3)Compared with the short-term control group,there were no significant changes in the sperm count and motility,inhibin B,MDA and SOD activity,and the apoptotic bodies of spermatogenic cells in the short-term exposure group(P>0.05).Compared with the chronic control group,the levels of inhibin B,sperm count and motility and SOD activity decreased(P<0.05),the apoptotic bodies of spermatogenic cells and the level of MDA increased(P<0.05).(4)Metabonomic analysis showed that the significant differences were affected by carbohydrate metabolism importantly,including galactose metabolism,fructose and mannose metabolism and glycolysis.We concluded that the target molecules leading to metabolic differences were HIF-lα,GLUT1,HK1 and LDHC.(5)Compared with the short-term control group,the mRNA and protein expression of HIF-lα,GLUT1,HK1 and LDHC in testis were all up-regulated(P<0.05)in short-term exposure group.Compared with the chronic control group,the mRNA and protein expression of GLUT1,HK1 and LDHC in testis decreased(P<0.05)in chronic exposure group,but there was no significant difference for mRNA and protein expression of HIF-lα(P>0.05).(6)Compared with the short-term exposure group,the levels of serum 8-OHdG and MDA in testis were still up-regulated in the chronic exposure group,and the activity of SOD in testis decreased,but the expression of HIF-1α in testis was down-regulated(P<0.05).Conclusions:(1)Hypoglycemia caused by abnormal blood glucose regulation was an important cause of spermatogenic function damage after chronic METH exposure in rats,which might be related to adrenocortical dysfunction.(2)The important mechanism of spermatogenic dysfunction after chronic METH exposure is that the decompensation of oxidative stress mediated by HIF-1α in the testis reduces the expression of GLUT 1,which leads to the inhibition of glycolysis during spermatogenesis.Part:Ⅲ Study on the effect of METH exposure on erectile function in ratsObjective:To explore the effect and mechanism of METH exposure on erectile function from the view of energy metabolism via establishing the rat model of METH exposure and withdrawal.Methods:(1)40 SD rats possessing erection were randomly divided into acute control group,acute exposure group,chronic control group,chronic exposure group and abstinence group.The rats were given intraperitoneal injection of METH solution in different periods,while the corresponding control rats were injected with the same volume of normal saline;the duration of withdrawal was 8 days after addiction in the withdrawal group and the observation time was the same as that in the chronic control group.The exposure and withdrawal model were evaluated.(2)The state of penile erection was dynamically observed during establishing models,and the erectile function was measured by electrical stimulation of cavernous nerve(by bR=based intracavernous pressure/basal mean arterial pressure).(3)The level of serum 8-OHdG was determined by ELISA,and the levels of succinate dehydrogenase(SDH)and adenosine triphosphate(ATP)in corpus cavernosum tissue were measured.(4)The protein expression of adenosine 2b receptor(A2bR),PI3K,pAktl and endothelial nitric oxide synthase(eNOS)in corpus cavrnosum were detected by Western-blot.The mRNA levels of HIF-1α,A2bR and eNOS in corpus cavernosum were determined by qRT-PCR.The localization and expression of A2bR and eNOS in corpus cavernosum were detected by immunohistochemistry.Results:(1)The models of acute and chronic METH exposure and withdrawal were successfully established.(2)Spontaneous erection or ejaculation occurred in the acute exposure group on the 4th-5th day after exposure,in the chronic exposure group and the abstinence group on the 8-9th day after exposure.The phenomenon of spontaneous erection or ejaculation disappeared on the 17th or 18th day after exposure in the chronic exposure group and the abstinence groups.In the abstinence group,penile erection reappeared at the end of withdrawal by subcutaneous administration of apomorphine hydrochloride solution.(3)Compared with the acute control group,there was no significant change for bR(P>0.05),the serum 8-OHdG and the SDH and ATP levels in corpora cavernosa increased(P<0.05)in the acute exposure group.Compared with the chronic control group,the levels of bR,8-OHdG,and SDH and ATP in corpora cavernosa significantly decreased(P<0.05)in the chronic exposure group;there were no significant changes(P>0.05)in bR,8-OHdG levels,SDH and ATP levels in corpora cavernosa in the abstinence group.(4)Compared with the acute control group,the protein expression of HIF-1α,A2bR,PI3K,pAkt1 and eNOS,the mRNA expression of HIF-1α,A2bR and eNOS in corpora cavernosa increased in the acute exposure group(P<0.05).Compared with the chronic control group,the protein expression of A2bR,PI3K,pAkt1 and eNOS,the mRNA expression of HIF-1α,A2bR and eNOS in corpora cavernosa decreased in the chronic exposure group(P<0.05),but there was no significant difference in the mRNA and protein expression of HIF-1α(P>0.05).There were no significant differences for the protein expression of HIF-1α,A2bR,PI3K,pAkt1 and eNOS,the mRNA expression of HIF-1α,A2bR and eNOS in corpora cavernosa in the abstinence group(P>0.05).Conclusions:(1)Local oxidative stress in the penis increased and the expression of HIF-1α was up-regulated after acute METH exposure,which might further activate the A2bR-PI3K/AKT-eNOS signal pathway.(2)The penile erectile function was inhibited after chronic METH exposure and could return to the normal state following withdrawal,which were related to the changes of energy metabolism and the expression of A2bR-PI3K/AKT-eNOS signal pathway in penile tissue.