| Objectives: In the present study,we investigated whether oral administration of LBP in streptozotocin(STZ)-included type-1 diabetes male mice would reverse the testicular dysfunction and explored potential relationship with PI3K/Akt/mTOR mediated excessive autophagy and apoptosis signal pathway.Methods: 1.To observe the protective effects of LBP on diabetic testicular dysfunction in male mice.(1)ICR mice were administered daily via intra-peritoneal(i.p.)injection of streptozotocin(STZ)in a sodium citrate buffer(0.1 mol/L of citrate,p H 4.5)at a dose of 45 mg/kg body weight(b.w.)for 5 consecutive days.(2)A drop of blood was collected from the tail vein and glucose levels were monitored with a glucometer.(3)The sperm parameters measured by white blood cell(WBC)chamber by measuring the indexes of sperm count,sperm motility,sperm viability and the sperm-head count.(4)The levels of serum testosterone(T),follicular stimulating hormone(FSH),and luteinizing hormone(LH)were assayed by ELISA.(5)The testes,epididymis and seminal vesicle were weighed by precise analytical balance,and the organ coefficients were calculated.(6)Antioxidant enzyme activity and content of malondialdehyde(MDA)and reactive oxygen species(ROS)level were assessed by the method of spectrophotometry in testis tissues.(7)Histopathological impairments were analyzed by and hematoxylin-eosin(HE)staining in diabetic testicular tissues.(8)Johnsen’s score was used to categorize the spermatogenesis.(9)Ultrastructural changes in diabetic testicular tissues were evaluated by Electron Microscopy.2.To explore the possible protective mechanism of LBP ameliorated diabetic testicular dysfunction via inhibition of PI3K/Akt/mTOR pathway mediated excessive autophagy and apoptosis in mice.(1)Autophagosomes and autophagic vesicles in testicular cells was evaluated by transmission electron microscope(TEM).(2)Immunofluorescence detected the effects of LBP on the activation of Beclin-1 and LC3 I in diabetic testicular tissue in mice.(3)Western blot analysis were used to examine the expression of p-PI3K、p-Akt、Beclin-1、LC3I/LC3II、Bcl-2、Caspase-3、Bax in testicular tissues.(4)Reverse transcriptase–polymerase chain reaction(RT-PCR)was used to quantify the mRNA level of Beclin-1 and LC3 I in testicular tissues.Results: 1.Protective effects of LBP on testicular dysfunction in streptozotocin-induced diabetic male mice.Compare to the diabetic group,(1)The blood glucose levels of the LBP(20 and 40 mg/kg)had a significant decrease at the end of experiment(P<0.01).(2)LBP(20 and 40 mg/kg)and sildenafil citrate(SC)treatment significantly increased sperm count(P<0.01),sperm motility(P<0.05,P<0.01),sperm viability(P<0.01)and the sperm-head counts(P<0.01,P<0.05),and significantly decreased the abnormal sperm count(P<0.01)compared with the vehicle group.(3)LBP(40 mg/kg)and sildenafil citrate(SC)significantly marked up-regulation serumT(P<0.01 for all),FSH(P<0.01,P<0.05)and LH(P < 0.05,P<0.01).(4)LBP(20,40mg/kg)treatment of the diabetic mice considerably increased the weight of reproductive organs,ameliorated its histological and ultrastructural appearance to different degrees.(5)Johnsen’s scores were statistically significantly higher after the administration of LBP(20 and 40 mg/kg)compared with the diabetic group(P < 0.01 for all).(6)LBP(40 mg/kg)and SC group increased the activity of SOD,CAT and GSH-Px and reduced the content of MDA and ROS(P<0.01).2.LBP ameliorated diabetic testicular dysfunction via inhibition of PI3K/Akt/mTOR pathway mediated excessive autophagy and apoptosis in mice.Compared to diabetic group(1)LBP(40 mg/kg)significantly decreased the number of autophagy and autophagic vesicles in germ cells.(2)LBP(40 mg/kg)significantly increased immunoreactivity of Beclin-1 and LC3 I in diabetic testicular tissues.(3)The protein expression levels of p-PI3K、p-Akt、Beclin-1、LC3I/LC3II、Bcl-2 markedly increased and Caspase-3 and Bax significantly decreased in LBP 40 mg/kg group(P<0.01).(4)The mRNA expression of Beclin-1 and LC3 I were significantly reduced(P < 0.01)in LBP 40 mg/kg group.Conclusions: 1.LBP exerts protective effects on streptozotocin-induceddiabetic testiculardysfunctionin male mice.2.The protective effects of LBP on diabetic male testicular dysfunction,which is likely via inhibition of PI3K/Akt/mTOR pathway mediated excessive autophagyand germ cell apoptosis. |
