| A significant decline in testosterone(T)and free testosterone(FT)in middle and aged men.Lots of senescence phenomenon and clinical illness are closely associated with the hormone change,e.g,increase of abdominal visceral-fat mass,metabolic syndrome,atherosclerosis,and osteoporosis.Evidence suggests that sex hormone levels of human are direct regulated by the hypothalamus-pituitary-testis axis(HPT),which is the primary central regulation of reproductive Function.Generally,the hypothalamic gonadotropin-releasing hormone(GnRH)neurons are the key output cells of central nervous network(CNN)to control the reproductive capacity of mammals,rodents and other animals,which are responsible for integrating multiple internal steady-state and external environmental signals,maintaining an individual's fertility.The majority of the GnRH neurons send projections to the median eminence,which are interconnected to form a functional unit termed the GnRH pulse generator and are responsible for intermittent release of GnRH.When the concentration of GnRH in the pituitary portal vein increased,the secretion of luteinizing hormone(LH)and follicle-stimulating hormone(FSH)in the anterior pituitary lobe,that is the main central pathway of serum T regulation.Recent evidence suggests that acupuncture therapies modulates serum hormone levels and has anti-senility effects,however,the mechanisms remain unclear.We have observed that electro-acupuncture(EA)and mild moxibustion(MM)at the BL23(Shenshu)and CV4(Guanyuan)acupoints could significantly increased serum T and FT levels.In this study,we hypothesize that coordinated activity of HPT appears to regulate GnRH secretion thereby the serum sex hormone levels be affected in anti-senescence process of central mechanisms of acupuncture and moxibustion intervention.Objectives1.To observe the effects of warm-needling acupuncture,mild moxibustion and electro-acupuncture therapies on testicular cell morphology and serum sex hormone levels of senescent male rats.2.To observe the effects of warm-needling acupuncture,mild moxibustion and electro-acupuncture therapies on genes and proteins related to HPT reproductive endocrine system in senescent male rats,to provide a deeper molecular understanding of acupuncture and moxibustion therapies on the central regulation of reproductive senescence in male rats.Methods1.We defined 95% confidence intervals for adult SD rat serum T and FT as the biochemical standard values.Blood T and FT levels were significantly lower than the standard value for aged SD rats included in our experiments.Forty 20-month-old rats were selected and randomly assigned into five groups: aged rat group(AR),warm-needling acupuncture-treated group(WNA),mild moxibustion-treated group(MM),electroacupuncture-treated group(EA),and drug-treated group(DR).Eight adult rats were used as the adult control group(AC),8 rats in each group.The acupuncture and moxibustion treatments were performed at the bilateral “shenshu”(BL23)and “guanyuan”(CV4)for 15 min,continuous treatment five days a week with two days off.Rats in the DR group were subcutaneously injected in the abdomen with testosterone propionate(7 mg/kg every third day)for eight weeks.Rats in the AR and AC groups were injected with 0.9% saline solutions,with the same volume as the DR group.2.After treatments,testis was processed for paraffin embedding and sectioned to 5-?m thickness for subsequent periodic acid Schiff(PAS)staining for light microscopy.3.Serum T,FT,GnRH,LH and FSH levels in each group were assessed by enzyme-linked immunosorbent assay(ELISA).4.The activity of GnRH-R in testicular tissues was assessed by immunohistochemistry;The expression of HPT-related proteins in the hypothalamus,pituitary,testicle(Kp,GnRH-R,GPR54 and GPR147)were determined by Western blot assay(WB).The expression of KISS-1/GPR54 and RFRP-3/GPR147 mRNA in hypothalamus,GnRH-R mRNA in pituitary and testicle tissues were detected by real-time quantitative PCR detecting system(Q-PCR).Results1.As shown by the PAS staining results,the testicular cells of adult male rats were nearly elliptical,seminiferous tubules were regular,the boundary membrane was intact,and the spermatogenic cells of all levels were arranged in an orderly manner.The most prominent changes in the testicular cells of senescent rats were morphological changes,including thickening of basement membrane,atrophy of seminiferous tubules,spermatogenic cell abscission,degeneration and atrophy of leydig cells.The degree of morphological changes was lower in the WNA,MM,EA and DR groups than in the AR group,the number of testicular leydig cells in the DR group was decreased.2.As shown by ELISA results,compared with the AC group,the levels of T,FT and GnRH were decreased(all P?0.05);Compared with the AR group,the levels of serum T,FT and GnRH in the WNA,MM,EA and DR groups were significantly higher than those in the AR group(all P?0.05),the level of GnRH in WNA group was significantly higher than that in DR group(P?0.05),mean FSH and LH did not differ among groups(all P?0.05).3.As shown by immunohistochemistry results,GnRH-R immunoreactivity was clearly present in leydig cells(LC),GnRH-R immunoreactivity(IR)was not observed in spermatogenic cells,positive cells showed diffuse cytoplasmic staining with reinforcement at the membrane level.A significant reduction in the number of GnRH-R IR was seen in the AR group,compared with the AC group(P?0.05).Mean IOD analysis showed that the immunoreactivity of GnRH-R appeared at a low level in the AR group,while staining increased significantly in the WNA,MM,EA,and DR groups.The immunoreactivity of Gn RH-R in the WNA,MM and EA-treated groups was significantly higher than that of the AR and DR groups(P?0.05).Compared with the AC group,the protein and mRNA expression of Kp,GPR54,GPR147 and RFRP-3 mRNA in the hypothalamus of the AR group was significantly decreased(all P?0.05).In contrast,there were no significant differences in Kp,GPR54,GPR147 protein and mRNA expression and RFRP-3 mRNA expression between the DR and AR groups(all P?0.05),though there was significant enhancement in the WNA,MM,and EA groups(all P?0.05).No significant difference in the protein and mRNA expression of GnRH-R in the pituitary was detected among groups(P ? 0.05).Compared with the AC group,the protein and mRNA expression of GnRH-R in testis of the AR group was significantly decreased(all P?0.05),the protein expression of GPR147 in the testis of the AR group was significantly increased(P ? 0.05);Compared with the AR group,the protein and m RNA expression of GnRH-R in testis of the WNA,MM and EA groups were significantly increased(all P?0.05),GPR54 in the testis of the WNA,MM and EA groups was significantly increased(P?0.05),the protein expression of GPR147 was significantly decreased(P?0.05).However,there were no significant differences in GnRH-R protein and mRNA expression in the testis between the DR and AR groups(all P?0.05).Conclusion1.WNA,MM and EA played a role in delaying the thickening of testis cells,the abscission of spermatogenic cells and the degeneration and atrophy of interstitial cells.Testosterone replacement therapy(TRT)may lead to the decrease of LC and the degeneration of function.2.The decrease of serum T and FT in senescent male rats may be associated with the decrease of GnRH-R in LC.WNA,MM and EA improved serum GnRH levels via regulation of the HPT in senescent male rats.To promote the secretion of GnRH and the expression of Gn RH-R protein and mRNA in testicular tissue;GnRH may act directly on the LC of testis,promoted the secretion of TC,and played a role in delaying senescence.3.The effect of WNA,MM,EA and TRT on adding serum T and FT was similar,but drugs may not directly activate GnRH synthesis and secretion,raising testosterone levels only.WNA,MM,EA are suitable for long-term use in clinic,and the curative effect of WNA and MM is better than that of EA. |
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