Study On The Mechanism Of Adscs Repairing The Damage Of Di-2-ethylhexyl Phthalate(DEHP) To Mouse Spermatogonia Cells

Objective:To investigate the possible mechanism of spermatogonal GC-1 spg injured by di-2-ethylhexyl phthalate（DEHP）and the repair effects of adipose-derived stem cells（ADSCs）and adipose stem cells-derived extracellular vesicles（ADSCs-EVs）on GC-1 spg after injury,whitch may provide a certain experimental basis for the clinical treatment of male spermatogenic system drug/toxic oxidative damage.Methods:（1）ADSCs were extracted and subcultured to 3～5 generations for experiment.The ADSCs were identified by experiments.（2）ADSCs-EVs were extracted by ultrafast centrifugal method,and the particle size distribution of ADSCs-EVs was detected by nanoparticle particle size tracking analysis（NTA）.The morphology of ADSCs-EVs was observed by transmission electron microscope（TEM）.The expressions of surface marker proteins CD 9 and CD63 of ADSCs-EVs were detected by Western-blot assay.（3）GC-1spg cells were treated with 5 different concentrations of DEHP through CCK8 assay;Transwell assay;Clonal formation assay;Flow cytometry;Western-blot assay;The contents of MDA,Fe³⁺and GSH in GC-1spg cells were determined by chemical spectrophotometry.The effects of DEHP on the proliferation,migration,cloning and apoptosis of GC-1spg as well as the mechanisms related to iron death were investigated in a series of experiments including mitochondrial membrane potential detection and reactive oxygen species detection.（4）Establish ADSCs co-culture system,and explore the repair effects of ADSCs on proliferation,migration,cloning,apoptosis and ferroptosis related mechanisms of GC-1spg after injury through the above series of experiments.（5）Cell immunofluorescence assay was conducted to observe the internalization of ADSCs-EVs by GC-1spg cells,and to explore the repair effect of ADSCs-EVs on the proliferation,migration,cloning and apoptosis of GC-1spg after injury,as well as the mechanism related to ferroptosis.（6）Through the above series of experiments,the effects of ferroptosis inhibitor（Ferrostatin-1）,on the proliferation,migration,cloning and apoptotic ability of GC-1spg after injury and the mechanisms related to iron death were detected.Results:（1）A class of fusiform or polygonal cells similar to fiber cells were successfully isolated from adipose tissue,and their morphology was more typical after multiple subpassage.After induction by osteoblast inducer,they could be directed to differentiate into osteoblasts and adipocytes.（2）TEM showed the shape of a single"cup-shaped"nanofilm vesicle,and NTA showed that the diameter of the vesicle was between tens and hundreds of nanometres.Western-blot assay detected the expression of ADSCs-EVs unique surface markers CD9 and CD 63,which met the identification criteria of ADSCs-EVs.（3）CCK8,transwell and clonogenesis assay showed that DEHP inhibited the proliferation,migration and clonogenesis of GC-1spg after treatment with 5 different concentrations of DEHP.Flow cytometry showed that DEHP could promote apoptosis of GC-1spg.Chemical spectrophotometry showed that DEHP increased the accumulation of Fe³⁺and decreased the contents of MDA and GSH in GC-1spg.The mitochondrial membrane potential test showed that DEHP decreased the mitochondrial membrane potential in GC-1spg.Reactive oxygen species（ROS）in GC-1spg increased with DEHP.Western-blot assay showed that DEHP decreased the expression of ferroptosis related proteins GPX4 and x CT in GC-1spg.（4）Cell immunofluorescence assay showed that ADSCs-EVs could be internalized and ingested by GC-1spg.（5）According to the above experiments,both ADSCs and ADSCs-EVs promoted the proliferation,migration and cloning ability of injured GC-1spg,inhibited cell apoptosis,reduced the contents of Fe³⁺and reactive oxygen species in injured GC-1spg,and restored the expressions of mitochondrial membrane potential,MDA,GSH,GPX4 and x CT to pre-injury.（6）After treatment with ferroptosis inhibitor,the biological characteristics of injured GC-1spg and the above cellular indexes were restored to the pre-injury.Conclusions:DEHP can inhibit the proliferation and migration of GC-1spg through ferroptosis pathway,and ADSCs and ADSCs-EVs can block the ferroptosis pathway to repair the injured GC-1spg cells.