Effects Of Sancai Lianmei Particle On Autophagy And Apoptosis In Testicular Spermatogenic Cells Of Diabetic Mice From The Perspective Of Oxidative Stress

Diabetic spermatogenic dysfunction is a common complication of male diabetes mellitus,which is also the main cause of male infertility.Studies have shown that the morbidity of infertility in male diabetic patients is about 35.1%.With the increased mobility of diabetes and younger incidence,the reproductive complications induced by diabetes become an important cause of male infertility,and oxidative stress injury is closely related to it,but the specific mechanism is not clear and effective approaches to prevent diabetic spermatogenic dysfunction have not been available."Xiaoke" is the name of diabetes in Traditional Chinese Medicine(TCM),and Yin deficiency and dryness heat are the basis of TCM Dialectics.Diabetes patients suffer from chronic illness,which consumes Qi and injures Yin,and Qi deficiency causes deficit essence and weak transportation.It is characterized by deficiency of both Qi and Yin,Yin damaging the yang,so called "Yang transformation into Qi and Yin transformation into shape".Yin deficiency causes excessive internal heat,in which Yin fluid is not enough to raise the Jing,leading to impotence and fatigue,and infertility.Sancai Lianmei(SCLM)Particle consists of Renshen(Radix Ginseng),Tiandong(Radix Asparagi Cochinchinensis),Dihuang(Radix Rehmanniae),Wumei(Fructus Mume),Rougui(Cortex Cinnamomi Cassiae),and Huanglian(Rhizoma Coptidis).SCLM Particle prescription follows the principle of nourishing Yin and clearing heat,harmonizing Yin and Yang,and it could also Tonify Qi and spleen,cultivating soil and producing essence.In our previous experiments,SCLM Particle has been testified that it could reduce blood glucose and antioxidant function.Also,we found that SCLM Particle can effectively improve the sexual function,semenquality and TCM symptoms of diabetic patients in clinical observation.In this study,SCLM Particle was used to treate spermatogenic dysfunction of diabetic mice in order to observe its effects on spermatogenic function,and it is also studied that the effects of SCLM Particle on mitochondrial autophagy and apoptosis in spermatogenic cells of diabetic mice via Nrf2/HO-1 oxidative stress signaling pathway.Type 2 diabetes mellitus(T2DM)mice model was constructed by high-fat diet and intraperitoneal injection of streptozotocin(STZ),and the mice were divided into four groups: normal control group(Ctrl),diabetic model group(DM),SCLM group(DM/SCLM)and metformin group(DM/DMBG).Among them,6 g/kg of SCLM Particle is administrated to mice everyday in DM/SCLM group,and 200 mg/kg of metformin was given to DM/DMBG group,and the mice were treated with same amount of saline in other groups for 4 weeks.After 4 weeks’ treatment,blood glucose,glycated serum protein(GSP),Serum insulin(INS)and other metabolic indicators were detected,and the serum FSH,LH,and T were measured to evaluate the changes of sex hormones.The epididymis was taken to detect sperm parameters,and HE staining was used to observe the pathological changes of testicular tissue,however,the ultrastructural changes of spermatogenic cells were observed by transmission electron microscopy(TEM).ELISA was used to detect SOD,MDA,8-OHdG in testicular tissue to evaluate the changes of oxidative stress indicators,and the Nrf2/HO-1signaling pathway was detected by Western blot and qPCR.Autophagy-related proteins Beclin-1,LC-3,P62 were detected by Western blot and qPCR and autophagosome was observed by TEM,in order to evaluate the changes of mitochondrial autophagy in germ cells.The spermatogenic cell apoptosis in testeis was also detected by apoptosis related genes of Bcl-2,Bax and TUNEL staining.The results showed that SCLM Particle could effectively reduce fasting blood glucose(FBG),GSP and improve insulin resistance in T2 DM mice.SCLM Particle could increase the expression of antioxidant enzyme SOD in testis tissue of T2 DM mice,and reduce the expression of oxidative stress products of MDA and 8-OHdG.It also enhanced the level of serum T,and increased testicular index of T2 DM mice.The sperm density,sperm motility and sperm survival rate were also increased,butspermatogenic cell apoptosis in testicular tissue was significantly decreased,leading to the improvement of spermatogenesis function in T2 DM mice.Moreover,SCLM Particle activated Nrf2/HO-1 signaling pathway,in which oxidative stress injury was alleviated in testis of T2 DM mice.The expression of autophagy marker proteins of Beclin-1,LC3 and P62 were decreased and autophagosomes were reduced in spermatogenic cells,in which excessive autophagy was inhibited.The expression of anti-apoptosis gene Bcl-2 was increased and pro-apoptotic gene Bax was decreased,leading to the reduction of cell apoptosis in the testis of T2 DM mice,and the spermatogenic function was improved by SCLM Particle.In summary,SCLM Particle could effectively alleviate testicular oxidative damage and germ cell apoptosis in T2 DM mice,which may be associated with the upregulation of Nrf2/HO-1 oxidative stress signal pathway and inhibition of excessive autophagy of spermatogenic cells.Therefore,results from the present study support the possibility that SCLM Particle may be used as a potential agent for treatment of diabetic infertility,which has good social and economic value,and provides new ideas for the treatment of diabetic spermatogenic dysfunction by TCM.