Study On The Protective Mechanism Of MBMSCs-EVs On Nicotine-induced Damage To Mouse Spermatogenic Cells

ObjectiveTo investigate the repair of nicotine-induced injury to germ cell 1(GC-1)by mouse bone marrow mesenchymal stem cell-derived extracellular vesicles(mBMSCs-EVs).To illustrate the potential clinical value of mBMSCs-EVs in male infertility,and to provide experimental evidence for the prevention and treatment of smoking-related diseases.Methods(1)The whole bone marrow method was used to isolate and culture mouse bone marrow mesenchymal stem cells(mBMSCs).And the third-generation of mBMSCs were used for cell morphology observation,surface antigen detection,and adipogenic and osteogenic differentiation experiments to identify different aspects of mBMSCs.(2)Differential ultracentrifugation was used to extract mBMSCs-EVs.The morphology of mBMSCs-EVs was observed by transmission electron microscope.The particle size distribution of mBMSCs-EVs was detected by nanoparticle tracking analysis technology.The expression of CD63 and CD9 on the surface of mBMSCs-EVs was detected by Western-blot.(3)The endocytosis of mBMSCs-EVs by GC-1 was observed with immunofluorescence analysis.(4)The effect of nicotine on the proliferation of GC-1 cells was detected by the MTT experiment.And the appropriate concentration of nicotine was screened for subsequent experiments.The MTT experiment was used to detect the effect of proliferation ability of mBMSCs-EVs on the GC-1 cells after nicotine-induced damage,the appropriate concentration of mBMSCs-EVs was also screened for the subsequent experiments.The plate colony formation experiment was used to detect the effect of mBMSCs-EVs on the colony forming ability of GC-1 cells after nicotine-induced injury.The scratch and transwell experiment were used to detect the effect of mBMSCs-EVs on the migration ability of GC-1 cells after nicotine-induced injury.Flow cytometry was used to detect the effect of mBMSCs-EVs on the apoptosis ability of GC-1 cells after nicotine-induced injury.RT-PCR was used to detect mBMSCs-EVs on the apoptosis-related mRNA expression of GC-1 cell after nicotine-induced injury.Western-blot was used to detect mBMSCs-EVs on the cell-related proteins expression of GC-1 after nicotine-induced injury.(5)Flow cytometry analyzer was used to detect the effect of deguelin on the antiapoptotic ability of mBMSCs-EVs.Western-blot was used to detect the cell-related proteins expression of mBMSCs-EVs on GC-1 in each group after the intervention of deguelin.Results(1)A typically long fusiform or polygonal cells were successfully isolated from mouse bone marrow.They were similar to fibroblasts in morphology.After multiple passaging and purification,flow cytometry was used to identify the surface markers of mBMSCs.The cultured cells were successfully differentiated into osteoblasts and adipocytes.(2)mBMSCs-EVs were microscopically observed as single cup-shaped or discshaped membranous nano-scale vesicles,with the diameter ranging from tens to hundreds of nanometers.The high expression of surface proteins of CD 63 and CD 9were detected in mBMSCs-EVs by Western-blot,in line with the general surface markers of EVs.(3)Immunofluorescence analysis was used to confirm that mBMSCs-EVs can be endocytosed by GC-1 cells.(4)The results of MTT,plate colony formation experiment,scratch experiment and transwell experiment showed that mBMSCs-EVs could promote the proliferation and migration of GC-1 cells after nicotine-induced injury.Flow cytometry analysis showed that mBMSCs-EVs could inhibit nicotine-induced GC-1 cell apoptosis.Western-blot showed that after treatment of nicotine-injured GC-1 cells by mBMSCsEVs,the expression of apoptotic proteins BAX and caspase-3 were decreased whilethe expression of anti-apoptotic protein Bcl-2 was increased.Furthermore,the expression of p-PI3 K and p-Akt were increased while the expression of PTEN was decreased.(5)Flow cytometry analysis showed that the anti-apoptotic ability of mBMSCsEVs was decreased after deguelin treatment.Western-blot showed that the expression of BAX and caspase-3 were increased after deguelin interfering with mBMSCs-EVs.Furthermore,the expression of anti-apoptotic protein Bcl-2 were decreased,and the expression of p-PI3 K and p-Akt were decreased while the expression of PTEN was increased.ConclusionUnder the condition of culture in vitro,mBMSCs-EVs promoted the proliferation and migration of GC-1 cells after nicotine-induced injury and inhibited nicotineinduced apoptosis of GC-1 cells through the PI3K/Akt pathway.