| BackgroundAging is a combination of irreversible changes over time observed in all eukaryotic organisms at the level of molecules,cells,tissues,organs,and systems.Both male and female reproductive capacities decline with age.Now,with the development of modern society,couples prone to delay their reproductive years due to economic reasons,high standards of living,or career planning,making it of great significance to research the effects of aging on reproductive systems.In the process of aging,a great percentage of male gradually found decreased testicular endocrine function,with a decrease in the level of serum testosterone.Patients appear loss of libido,erectile dysfunction,reduced endurance,osteoporosis,increased body fat,cognitive function and memory decline,known as late-onset hypogonadism(LOH).It seriously affected the living standard and life quality of the patients.The decline of male testicular function is closely related with oxidative stress and the decrease of antioxidant capacity in the reproductive system.Vitamin D3 is closely related to male reproductive function.A lot of research confirms that vitamin D receptor(VDR)and vitamin D metabolic enzymes were expressed in the central and peripheral reproductive organs.Multiple clinical studies revealed that serum 25(OH)D levels were positively correlated with total testosterone and free testosterone in the body.The sperm count and vitality were significantly reduced in the VDR knockout mice.In addition,vitamin D have shown a effect of suppressing oxidative stress in other organs and tissues.Kelch-like epichlorohydrin-1(Keap1)/nuclear factor E2 correlation factor 2(Nrf2)/anti-oxidation reaction element(ARE)signaling pathway is the key pathway of endogenous antioxidant stress system.Nrf2 is one of the critical transcription factors involved in the regulation of the anti-oxidant defense system including GCLC,SOD2,NQO-1 and HO-1 through their recruitment to the antioxidant response element(ARE)in their gene promoters.Animal studies have shown that in the Nrf2 knockout mice,the reduced expression of numerous antioxidant molecules result in reduced antioxidant capacity and therefore a decreasingly testosterone level and poorly spermatogenesis.Animal studies have shown that active vitamin D can activate the Nrf2-ARE antioxidant pathway in nonobese type 2 diabetes rats and murine model of asthma.However,can vitamin D ameliorate the testicular function of D-galactose induced aging mice via activates Nrf2 pathway and its downstream transcription factor including GCLC,SOD2,NQO-1 and HO-1 expression? Our research aims to study vitamin D on the testicular function of aging mice and the possible mechanism by supplement calcitriol for 8 weeks.ObjectiveTo investigate the effect of calcitriol on the testicular function of D-galactose induced aging mice and the possible mechanism.Methods8 week old SD rats were randomly divided into two groups first.One group were subcutaneously injected with 15% D-galactose 500mg/(kg*d)for 8 weeks to establish aging rat model.The other group give the same dosage of physiological saline.After 8 weeks,aging rats were randomly divided into three groups: aging model group(DG group),aging model + low dose vitamin D group(LD group),aging model + high dose vitamin D group(HD group);The normal group divided into three group: normal control group(NC group),normal control group + low dose vitamin D(LC group),normal control group + high dose vitamin D(HD group).LD group,LC group were given 0.025ug/(kg*d)calcitriol soluble peanut oil in the stomach,HD group,HC group were given 0.1ug/(kg*d)calcitriol,while DG group and NG group were given the same dosage of peanut oil.8 weeks later,the rats were sacrificed,their body weight and testis weight were weighed up.Rat epididymis were taken for sperm count and motility,blood for serum calcium and testosterone.Tissue homogenate were for MDA and SOD tests.Testicular morphological changes were examined by light microscopy.The mRNA level of Nrf2 was detected by RT-PCR.The expressions and distributions of VDR,Nrf2 were detected by immunohistochemistry and immunofluorescence.The protein level of of VDR,Nrf2,GCLC,SOD2 were detected by western blot.Results1.Evalution of the aging rat modelCompared with NC group,DG group gradually appeared to be slow in action after injection of D-galactose,and the hair was yellow.After 8 weeks of D-galactose injection,the thymus index?hepar index were decreased compared with NC group.The testis index and serum testosterone were decreased in DG group compared with NC group.2.Effect of vitamin D on the general situation and serum testosterone levelThe testis index and serum testosterone were decreased in DG group compared with NC group(P<0.05).After supplement with vitamin D,LD group and HD group testis weight and testis index were significantly higher than those of DG group(P<0.05).But,there were no significant difference between LD group and HD group(P>0.05)testis weight and testis index.And the LC group and HC group have no difference compared with NC group(P>0.05)in testis weight and testis index.What is more,there were no differences in body weight among these groups(P>0.05).The serum testosterone were decreased in DG group compared with NC group(P<0.05).After supplement with vitamin D,LD group and HD group testis weight and testis index were significantly higher than those of DG group(P<0.05).Supplement with vitamin D,the serum calcium of LD group and HD group were higher than DG group(P<0.05),LC group and HC group higher than NC group(P<0.05).There were no differences in serum phosphorus among these groups(P>0.05).3.Effect of active vitamin D on the sperm parametersThe sperm count and motility were decreased in DG group compared with NC group(P<0.05).After supplement with vitamin D,LD group and HD group sperm count and motility were significantly higher than those of DG group(P<0.05).And the LC group and HC group have no difference compared with NG group(P>0.05).4.Effect of active vitamin D on testicular SOD and MDA levelCompared with the NC group,the DG group SOD level were lower and MDA level were higher(P<0.05),and after supplement with vitamin D,LD group and HD group showed SOD level were higher and MDA level were lower than DC group(P<0.05).5.Effect of active vitamin D on testicular morphologyHE staining: In the DG group,the spermatogenic cells were arranged loosely,the interstitial cell arranged loosely and the number were reduced.In NC group,the arrangement of spermatogenic cells were close and the stroma abundant.6.The expression of mRNA level of Nrf2 in testisRT-PCR showed,the mRNA level of Nrf2 were lower in the DG group compared with the NG group(P<0.05),LD group and the HD group were high than DG group in the mRNA expression of Nrf2(P<0.05).7.The expression of level of Nrf2?VDR?GCLC?SOD2 proteinCompared with the NC group,Nrf2?GCLC?SOD2 were lower in the DG group compared with the NC group(P<0.05),the LD group and the HD group were high than DG group in the protein expression of Nrf2?GCLC?SOD2 and VDR(P<0.05).8.The expression and localization of VDR?Nrf2 in testis.Immunohistochemistry and immuofluorescence results showed that VDR and Nrf2 were expressed in all levels of sperm cell,both in nucleus and cytoplasm.Compared with NC group,the expression of VDR and Nrf2 in DG group were significantly decreased,the expression of VDR and Nrf2 were higher in LD and HD group than in DG group.ConclusionVitamin D can ameliorate the testicular function of D-galactose induced aging rats,which may be associated with the antioxidant capacity of Nrf2/ARE signaling pathway. |
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