| Background: Ejaculation is a highly integrated and coordinated process.The regulation of ejaculation involves multiple neurophysiological pathways and multiple neurotransmitters in the peripheral and central nervous system.In recent years,andrologists have been devoted to the study of the mechanism of ejaculatory regulation.Premature ejaculation is the most common ejaculatory dysfunction in men,and it is also one of the most widely studied diseases in Andrology.Serotonin(5-HT)is the most important inhibitory neurotransmitter in central ejaculatory regulation.Increasing the concentration of central 5-HT could delay ejaculation.However,clinical experience shows that selective serotonin reuptake inhibitors(SSRIs)can not effectively delay ejaculation in about 20%-30% patients with premature ejaculation,indicating that the regulation of central 5-HT concentration by blocking 5-HT transporter(5-HTT)can not fully explain the mechanism of ejaculatory regulation,and the level of central 5-HT may be affected by other factors.Because the peripheral 5-HT cannot pass through the blood-brain barrier,the central 5-HT mainly comes from the 5-HT neurons in the brain,meanwhile the tryptophan hydroxylase 2(TPH2)is the only rate-limiting enzyme in the synthesis process of central 5-HT.Studies have shown that central 5-HT neurons are located in the raphe nucleus,which is near the midline of the brainstem.Related researches in rodents and human brains have confirmed that TPH2 is specifically expressed in the raphe nucleus.This is an important finding for central 5-HT related diseases,which makes it possible to avoid the confounding effects caused by peripheral 5-HT effectively.Brain-derived neurotrophic factor(BDNF)is the most important member of the neurotrophic factor family and is widely distributed in the central nervous system of mammals.It mainly binds to its high-affinity receptor tyrosine kinase receptor B(Trk B),by activating downstream signal transduction pathways,promoting the growth,differentiation,survival and synaptic plasticity of neurons,and participating in learning,memory,cognition,emotion and other functional activities.The classic "neuronutrition theory" holds that damage to neuroplasticity and cell regeneration may be the pathophysiological basis of many diseases.As one of the most common neurotrophic factors,BDNF plays an important role in the development and maintenance of the central nervous system.It could supply nutrition for a variety of neurons such as serotonergic,dopaminergic and cholinergic neurons and multiple brain areas such as raphe nucleus,prefrontal cortex and hippocampus.Previous studies about BDNF mainly focused on mental and psychological disorders,some of which suggested that there is a close relationship between BDNF and 5-HT.So far,there is only one preliminary clinical study involving the field of ejaculatory regulation,and the results suggest that compared with healthy controls,the level of serum BDNF in premature ejaculation patients is significantly lower,while it does not involve the expression of BDNF in the central nervous system and the role and possible mechanism of BDNF in ejaculatory regulation.Due to methodological and ethical limitations,it is difficult to obtain the BDNF level of human brain,the rat model of ejaculatory dysfunction becomes the best choice for basic research of premature ejaculation.According to the distribution of intravaginal ejaculation latency(IELT)in the population,Waldinger et al.proposed the ejaculatory distribution theory,that is,IELT is continuous and similar to normal distribution.Furthermore,variability in ejaculation frequency(EF)in male rodents also exhibits this biological continuity and fits with the ejaculatory distribution theory.Thus,rapid and sluggish ejaculators in male rodents corresponds to premature ejaculation and delayed ejaculation in humans.Based on the above,we hypothesize that endogenous BDNF may regulate the concentration of central 5-HT by affecting the expression of TPH2 in raphe nucleus,and then participate in the regulation of ejaculation.The successful conduct of this study may reveal the role and mechanism of BDNF in the regulation of ejaculation,and provide a new theoretical basis for exploring the pathogenesis and treatment of premature ejaculation.PART 1 Expression of BDNF in rats with different ejaculatory functionsObjective: This part aims to study the expression levels of BDNF in the central nervous system(4 ejaculation-related brain regions including hippocampus,prefrontal cortex,raphe nucleus and striatum),spinal cord and serum of rats with different ejaculatory functions.Methods: Based on the ejaculatory distribution theory,rats with different ejaculation functions were screened out through copulatory experiments.According to the distribution of EF values,male rats were classified as sluggish,normal and rapid groups,6 rats in each group.After the rats in 3 groups were anesthetized,3ml of venous blood was collected from the inferior vena cava and centrifuged to obtain serum.Then,the rats were perfused transcardially with 200 ml 0.9% Na Cl solution.Brain and spinal cord samples were removed,and brain structure dissection of the hippocampus,frontal cortex,raphe nucleus and corpus striatum was performed on ice.The tissue samples were snap frozen in liquid nitrogen and stored at-80°C until the analyses.We performed enzyme-linked immunosorbent assay(ELISA)to quantitatively detect the levels of BDNF in each brain region,spinal cord and serum of rats in 3 groups.Results: Consistent with the results in patients with premature ejaculation,the concentration of serum BDNF decreased significantly from the sluggish rats to normal and rapid rats.Besides,in both brain regions and spinal cord,the sluggish group had the highest BDNF levels,while the rapid group had the lowest BDNF levels.Regression analyses of the expression of BDNF presented positive linear correlations between serum and brain(r = 0.958,P < 0.001),and between serum and spinal cord(r = 0.967,P < 0.001),respectively.Conclusions: Our findings suggested insufficient BDNF in the central nervous system may lead to rapid ejaculation.The current study adds to the evidence that BDNF is involved in the regulation of ejaculation.PART 2 Mechanisms of endogenous BDNF involved in central ejaculatory regulationObjective: Given that 5-HT neurons in the brain are located in raphe nucleus,our follow-up study was localized to raphe nucleus.The aim of this section was to investigate the relationship between BDNF,TPH2 and 5-HT expression in raphe nucleus and the possible mechanisms involved in the cnetral regulation of ejaculation.Methods: In this part,5-HT levels were determined by enzyme-linked immunosorbent assay(ELISA).Real-Time Quantitative PCR and Western blot analyses were used to measure the level of TPH2 m RNA and the expression of TPH2 protein(the rate-limiting enzyme in central 5-HT synthesis)in raphe nucleus,respectively.Meanwhile,the expression of TPH2 in raphe nucleus of each group was verified by immunohistochemistry.Results: The primary finding of our study was that both BDNF and 5-HT were significantly decreased in raphe nucleus of rapid ejaculators.There was a strong positive linear correlation between the levels of BDNF and 5-HT(r = 0.944,P <0.001).Further results showed that decreased TPH2 gene expression accompanied by TPH2 protein was shown in rapid ejaculators with lower BDNF level.Conclusions: Our results suggest the deficiency of endogenous BDNF induces the downregulation of TPH2 expression and the decrease of 5-HT synthesis in raphe nucleus of rapid ejaculator rats.BDNF may serve as a promising biomarker in patients with premature ejaculation.PART 3 Effect of aerobic exercise on the expression of endogenous BDNF and the regulation of ejaculation: a plot study in ratsObjective: Exercise greatly benefits brain health and behaviors improvement.Some studies suggest that regular aerobic exercise could improve endogenous BDNF expression in the brain and increase neuroplasticity.The purpose of this section is not only to explore the effect of exercise on ejaculation control,but also to make a preliminary assessment of the use of aerobic exercise as a complementary strategy to dapoxetine treatment in rapid ejaculators.Methods: We selected another 12 rapid ejaculators with the same method as the first part and randomly divided them into 4 groups: Ctrol(control)group,Ex(aerobic exercise)group,Dapo(acute dapoxetine 60mg/kg b.w.)group,and Ex+Dapo(aerobic exercise plus acute dapoxetine 60mg/kg b.w.)group.Six rounds of mating test were conducted in 4 groups of rats after the interventions.At the end of the test,the rats were killed.3 ml blood was taken and the raphe nucleus was dissected.The changes in ejaculatory parameters of the rats before and after the intervention were compared,and the serum cortisol levels and the levels of BDNF and 5-HT in raphe nucleus were measured by ELISA.Results: The primary finding of our study was that both aerobic exercise and acute dapoxetine could enhance ejaculation control and prolong ejaculation latency in rapid ejaculator rats.The ejaculation delay effect of aerobic exercise was nearly equivalent to that of acute dapoxetine.Meanwhile,both aerobic exercise and dapoxetine treatment could lead to increased expression of the BDNF and 5-HT in raphe nucleus of rapid ejaculators.Moreover,the two interventions,when applied together,may further up-regulate the expression of the BDNF-5-HT duo in a complementary manner.Conclusions: The present study verified aerobic exercise improves ejaculation control by upregulating the BDNF-5-HT duo and can be used as a complementary treatment to dapoxetine in rapid ejaculator rats.Clinicians are encouraged to try exercise prescriptions to rapid ejaculators.PART 4 Aerobic exercise regulates ejaculation by up-regulating the expression of TPH2 in the raphe nucleus through the BDNF/Trk B pathwayObjective: To explore the effect of aerobic exercise on the expression of BDNF,Trk B,TPH2 and 5-HT in the raphe nucleus of normal ejaculators,and to provide theoretical basis for the mechanism of aerobic exercise improving ejaculatory control in rats.Methods: Sixteen rats with stable ejaculation were selected from Part 3 and randomly divided into control group(Ctrol group),Trk B blocking group(K252a group),aerobic exercise group(Ex group),aerobic exercise plus Trk B blocking group(Ex+K252a group).The training intensity of Ex group was 15m/min,and the duration of exercise was 1h/day,6 days/week,a total of 6 weeks.Compare the changes of ejaculatoty parameters of rats in different group before and after intervention.The expression of BDNF,Trk B and TPH2 m RNA in the raphe nucleus were measured by RT-q PCR;The levels of BDNF,Trk B and TPH2 proteins in the raphe nucleus were detected by WB.The levels of 5-HT in raphe nucleus were measured by ELISA.Results: First,we found that after Trk B was blocked by K252 a,the expression of TPH2 was down-regulated significantly.Meanwhile,the rats showed shortened EL and increased EF,which indicated that Trk B was involved in ejaculation regulation.Secondly,we found that aerobic exercise can further enhance the ejaculation control ability of normal ejaculation rats,promote the expression of BDNF/Trk B,and further up-regulate the expression of TPH2.Finally,our results also showed that when aerobic exercise and K252 a were combined,the ejaculatory control of rats in the Ex+K252a group was significantly lower than that in the Ex group.Although the expression level of BDNF/Trk B in the raphe nucleus was not significantly different from that in the Ex group,the expression of TPH2 was significantly decreased in the Ex+K252a group.Conclusions: BDNF/Trk B pathway is involved in the central regulation of ejaculation.Aerobic exercise can promote the expression of BDNF/Trk B pathway in the raphe nucleus,and increase the synthesis of central 5-HT by inducing the up-regulation of TPH2 expression,which plays an important role in enhancing the control of ejaculation and prolonging the ejaculation latency. |
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