| Background:At present,the problem of aging society is becoming more and more serious,and the problem of androgen deficiency in elderly men has gradually become a focus of attention.Male late-onset hypogonadism(LOH)refers to middle-aged and older men increasing with age A group of clinical syndromes arising from the progressive decrease in serum testosterone levels.Reduced testosterone secretion is one of the most important features of LOH,and it is also a necessary condition for the diagnosis of LOH.Recent studies have shown that there is an endocrine pathway parallel to the hypothalamic-pituitary-gonad axis,which can act on the endocrine function of the testis,which involves the osteogenic hormone osteocalcin.Osteocalcin(OC)is a marker protein specifically expressed after osteoblast differentiation and maturation,and is regarded as a marker of bone renewal and bone transformation.Osteocalcin has two forms of expression in the human body: fully carboxylated osteocalcin(c OC)and uncarboxylated osteocalcin(uc OC).Fully carboxylated osteocalcin can help it combine with hydroxyapatite,maintain the normal mineralization rate of bone,and play an important role in bone metabolism.Another uncarboxylated osteocalcin is released in the blood and has biological activity.Studies have shown that uncarboxylated osteocalcin is closely related to male reproduction.Bone can regulate the function of testicular interstitial cells through uncarboxylated osteocalcin,and affect the spermatogenic function of testis through changes in testosterone levels.However,its specific The relevant mechanism is not yet clear.In this study,D-galactose was injected subcutaneously to build a rat aging model to study the effect of uncarboxylated osteocalcin on testosterone synthesis in elderly male rats,hoping to use osteocalcin to clinically treat male delayed hypogonadism Provide research foundation.Purpose:An aging rat model was established to investigate the effect of uncarboxylated osteocalcin on testosterone synthesis in testicular stromal cells of elderly male rats.Methods:1.Three-month-old male SD rats were randomly divided into two groups,one of which was subcutaneously injected with 10% D-galactose solution 300 mg · kg-1 · d-1 subcutaneously for 8 weeks to produce subacute rats In the aging model,another group was injected with the same dose of normal saline for 8 weeks as a control group.After 8 weeks,the rats were killed by de-neck method,and the body weight and testis weight were weighed separately to calculate the testicular organ index;the orbital vein blood was taken for testosterone hormone test and the testis tissue was fixed with formaldehyde solution for HE section staining observation;the rats were taken Testicular tissues were tested for SOD and MDA content,and whether the rat aging model was successfully established based on the test results..2.The rat testis tissues were taken,shredded and digested with type Ⅳ collagenase and then separated and purified with primary density testicular stromal cells using percoll solution gradient density centrifugation,and the testicular stromal cells were identified by 3β-HSD staining method.3.Stimulate rat testicular stromal cells with different mass concentrations of 0(equivalent PBS solution),1,3 ng / m L non-carboxylated osteocalcin,and collect the culture medium 24 hours later to testosterone concentration using a testosterone ELISA test kit;Fluorescence quantitative PCR method was used to detect the expression of key testosterone synthesis enzymes such as St AR,Cyp11 a,Cyp17 in testicular interstitial cells.Result1.Identification of subacute rat aging model: After 4 weeks of injection of D-galactose solution,rats gradually showed a decrease in drowsiness,curly hair loss of gloss and even partial shedding.After 8weeks,it was found that the rats in the model group had significantly reduced diet,atrophy,slow action,and slow response.Compared with the normal control group,the serum testosterone level of the model group decreased(P <0.05),and the testicular index decreased(P <0.05).The measurement of serum oxidative stress indicators showed that compared with the normal control group,the SOD enzyme activity of the model group decreased(P <0.05),and the MDA content increased(P <0.05).HE staining showed that the structure of the testis tissue of the control group was complete,the spermatogenic tubules were full,the number of spermatogenic cells was relatively large and neatly arranged,the sperm content in the tube was relatively high,and the number of interstitial cells was relatively large.In the model group,the structure of the testis tissue was disordered,and the spermatogenic tubule epithelium was damaged and atrophied.The number of spermatogenic cells was relatively small and arranged irregularly.The number of spermatozoa in the tube was reduced,and the interstitial cells were rare.2.Cultivation and identification of testicular stromal cells: The extracted primary testicular stromal cells are cultured in the culture medium for 12 hours,and most of the cells begin to adhere to the growth.At this time,the testicular stromal cells are clustered,and most of them appear as Round or spindle-shaped,the testicular stromal cells will produce antennae from within 48 hours.The testicular stromal cells cultured for 48 hours were stained with 3β-HSD,and there was a large amount of blue substance in the cytoplasm of the positive cells.3.The effect of uncarboxylated osteocalcin on the level of testosterone secretion in rat testicular stromal cells: compared with the control group of rat testicular stromal cells added with the same amount of PBS solution The level of testosterone secreted by the cells decreased(P <0.05);the testes stromal cells of the model group were treated with 1,3 ng / m L unfusogenic osteocalcin,respectively,compared with the testes treated with the same amount of PBS The level of testosterone in the supernatant of cell culture fluid of interstitial cells and osteocalcin-treated group was increased(P <0.05),but it was still lower than the level of testosterone in testicular interstitial cells of control group(P <0.05).The above results indicate that the testosterone synthesis ability of the testicular stromal cells in the aging model group decreased significantly compared with the control group,and after uc OC treatment,its testosterone synthesis ability could be significantly improved.4.The experimental results show that compared with the model testis interstitial cells added with the same amount of PBS solution,the St AR,Cyp11 a,Cyp17 of the model testis interstitial cells of the model group treated with 1 ng / ml non-carboxylated osteocalcin The expression of genes increased significantly;the expression of St AR,Cyp11 a,and Cyp17 genes in the testicular stromal cells of the model group after treatment with 3 ng / ml non-carboxylated osteocalcin significantly increased;Carboxylated osteocalcin can promote the expression of testosterone synthesis key enzymes in the testicular stromal cells of the aging rats in the model group,and may be related to the dose concentration to a certain extent.ConclusionThis study successfully constructed a D-galactose-induced aging rat model,demonstrating that uncarboxylated osteocalcin can promote D-galactose-induced synthesis of testosterone in testicularmesenchymal cells of elderly rats,and this effect may be Non-carboxylated osteocalcin is caused by the expression of key enzymes that promote the synthesis of testosterone. |
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