Exosomes Derived From BMSCs Ameliorate Cyclophosphamide-induced Testosterone Deficiency By Regulating The Autophagy Of Leydig Cells

Background:At present,the cancer incidence rates around the globe are on the rise,and human health has been severely challenged.As the treatment methods continue to enhance,the therapeutic effect of some malignant tumors frequently occurring in children and adolescents has also been significantly improved,the significant reductions in mortality have been achieved.In the case of these young cancer patients,chemotherapy is considered one of the most effective treatments.Cyclophosphamide(CP)is a chemotherapeutic drug and immunosuppressive agent widely used in clinical practice,and its side effects cannot be easily ignored.Many studies have shown that long-term use of CP will cause dysfunction or failure of Leydig cells(LCs),thus causing testosterone deficiency(TD).For these patients with TD,AUA and EAU guidelines recommend testosterone replacement therapy,but this treatment method still has many side effects.Therefore,this study aims to explore a more effective and safe treatment method,so we first explored the role of bone marrow mesenchymal stem cells derived exosomes(BMSCs-exo)in cyclophosphamide induced testosterone deficiency(CPTD).Methods:Firstly,we isolated BMSCs from C57BL/6 mice,and identified their morphology,differentiation potential and surface markers.Secondly,we extracted BMSCs-exos by differential centrifugation,and identified their morphology,particle size and surface marker proteins.Further successfully constructing the model of CPTD in vivo and in vitro,we examined the effects and probed the molecular mechanisms of BMSCs-exos on CPTD by detecting the expression levels of genes and proteins related to autophagy and testosterone synthesis.Meanwhile,the testosterone concentration in serum and cell-conditioned medium and the photophosphorylation protein levels of adenosine monophosphate-activated protein kinase(AMPK)and mammalian target of rapamycin(mTOR)were measured.Results:Our results suggest that we successfully extracted and identified BMSCs and BMSCs exos.BMSCs-exos could be absorbed by LCs through the blood-testosterone barrier in mice,promoting autophagy in LCs and improving the CP-induced low serum testosterone levels.BMSCs-exos inhibited cell death in CP-exposed LCs,regulated the AMPK-mTOR signaling pathway to promote autophagy in LCs,and then improved the low testosterone synthesis ability of CP-induced LCs.Moreover,the autophagy inhibitor,3-Methyladenine(3-MA),significantly reversed the therapeutic effects of BMSCs-exos.Discussion:BMSCs-exos could promote LCs autophagy by regulating the AMPK-mTOR signaling pathway,thereby ameliorating CPTD.This study provides novel evidence for the clinical improvement of CPTD using BMSCs-exos.