| Objective To investigated the effects of betaine(BET)on streptozotocin(STZ)-induced diabetic spermatogenic dysfunction and its underlying mechanisms in male mice.Methods 1.To observe the protective effects of BET on STZ-induced diabetic spermatogenic dysfunction in male mice(1)STZ was used to construct a mouse model of type 1 diabetes,and it was divided into Control group,DM group,DM + Clomiphene citrate(CC)(5mg/kg)group,and DM + BET(200,400,800mg/kg)group,after 8 weeks of treatment.The weight,testis weight,epididymis weight and seminal vesicle gland weight of diabetic mice were measured by weighing method,and testicular,epididymal and seminal vesicle gland organ coefficients were calculated;Determination of sperm parameters(sperm count,sperm motility,sperm viability and the sperm abnormality)in diabetic mice by white blood cell chamber;Detection of DNA damage in sperm of diabetic mice by single cell gel electrophoresis;Observation of pathological changes in testis tissue of diabetic mice by HE staining;Transmission electron microscopy was used to observe the ultrastructure of sertoli cells in diabetic mice.(2)A high glucose(HG,80mM)-induced TM4 sertoli cells injury model was established and divided into control group,HG group(80mmol/L),BET group(0.05mmol/L),PDTCsubthreshold dose group(0.01?M),PDTC subthreshold dose(0.01?M)+ BET subthreshold dose(0.02mmol/L)group,treatment for 48 h.CCK-8 method was used to detect the effect of BET on the survival rate of HG-induced TM4 sertoli cells;Single-cell gel electrophoresis was used to detect the degree of DNA damage to HG-induced TM4 sertoli cells;ELISA was used to detect the effect of BET on the content of INH-B in TM4 sertoli cells induced by HG.2.To explore the protective mechanism of BET on spermatogenic dysfunction in diabetic mice(1)Effect of BET on TNF-? and iNOS contents in testicular tissues of diabetic mice by ELISA;Western blot methods used to estimate the protein expression of p38 MAPK,p-p38 MAPK,NF-?Bp65,and p-NF-?Bp65 in the teaticular tissue.(2)ELISA was used to detect the effect of BET on TNF-? and iNOS levels in HG-induced TM4 Sertoli cells;Western blot methods used to estimate the protein expression of p38 MAPK,p-p38 MAPK,IKK-?,p-IKK-?,NF-?Bp65,and p-NF-?Bp65 in TM4 Sertoli cells;Immunofluorescence was used to detect the effect of BET on nuclear translocation of NF-?B p65 in TM4 Sertoli cells induced by HG;Real-time fluorescence quantitative method was used to detect the mRNA expression of p38 MAPK and NF-?B p65 in TM4 Sertoli cells.Results 1.Observe the protective effect of BET on spermatogenic dysfunction in diabetic mice1 Compared with diabetes model group,the BET(800mg/kg)group can significantly increase the weight of diabetic mice,testes,epididymis,and seminal vesicles(P <0.01),and increase testicular,epididymis,and seminal vesicle gland organ coefficients(P <0.01).In addition,compared with diabetes model group,the BET(800mg/kg)group can significantly increase the sperm count,sperm motility,and sperm viability of diabetic mice(P <0.01),significantly reduce the sperm abnormality of diabetic mice(P <0.01),and improve testicular cells DNA damage in diabetic mice;Compared with the diabetic group model,the BET(800mg/kg)group can significantly reduce the pathological damage of testicular tissue indiabetic mice,increase the number of testicular sertoli cells and reduce the pathological damage of testicular sertoli cells in diabetic mice.2 Compared with the high glucose model group,the BET(0.05mmol/L)group can significantly increase the survival rate of TM4 sertoli cells(P <0.01),and can improve the DNA damage of TM4 sertoli cells induced by HG;Compared with the high glucose model group,BET(0.05mmol/L or BET 0.02mmol/L subthreshold dose + PDTC 0.01?M subthreshold dose)can increase the content of INH-B in TM4 sertoli cells(P <0.05).2.To explore the protective mechanism of BET on spermatogenic dysfunction in diabetic mice(1)The results of ELISA showed that BET(800mg/kg)group reduced the level of iNOS and TNF-? in the testis tissue with compared with the diabetes model group(P < 0.01);The results of western blot showed that BET(800mg/kg)group reduced the protein expression level of p38 MAPK,p-p38 MAPK,NF-?Bp65,and p-NF-?Bp65 protein in testis tissue with compared with the diabetes model group(P <0.01).(2)The results of ELISA experiments showed that the BET(0.05mmol/L or BET 0.02mmol/L subthreshold dose + PDTC 0.01?M subthreshold dose)can reduce the expression of TNF-?and iNOS in TM4 sertoli cells with compared with the HG model group(P <0.01,P <0.05);Western Blot experiments showed that the BET(800mg/kg)group can significantly reduce the protein expression of p38 MAPK,p-p38 MAPK,IKK-?,p-IKK-?,NF-?Bp65,and p-NF-?Bp65 in TM4 sertoli cells with compared with the HG model group(P <0.01);The immunofluorescence results showed that the BET(0.05mmol/L or BET 0.02mmol/L subthreshold dose + PDTC 0.01?M subthreshold dose)group could inhibit the migration of NF-?B p65 into the nucleus;The results of qRT-PCR experiments showed that the BET(0.05mmol/L or BET 0.02mmol/L subthreshold dose + PDTC 0.01?M subthreshold dose)group can reduce the mRNA expression of p38 MAPK and NF-?Bp65 in TM4 sertoli cells with compared with the HG model group(P <0.01).Conclusions 1.The Protective effects of BET on STZ-induced spermatogenic dysfunction in diabetic male mice.2.The protective effect of BET on spermatogenic dysfunction in DM mice is related to the inhibition of p38MAPK/NF-?B signaling pathway. |
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