The Protective Effect Of BMSCs Derived Exosomes On Testis Ischemia-reperfusion Injury

Objective:To investigate the protective effect of the rat bone marrow mesenchymal stem cells(BMSCs)derived exosomes on testis ischemia-reperfusion injury.Methods:The rat BMSC was isolated,cultured and identified in the original culture.By observing the growth of the adherent cells form,using flow cytometry instrument to detect cell surface specific protein markers,and to the osteoblast and into fat cell differentiation induced to confirm successful isolation and culture of bone marrow mesenchymal stem cells.mass culture BMSCs by serum without exosomes and collecting cell culture supernatant,methods of collecting cell supernatant by ultracentrifugation to extract exosomes derived from BMSCs,and the material with nano particle tracking analyzer(NTA)to analyze the size,transmission electron microscope(TEM)and the morphology of Western blot to detect the typical biomarkers to identify extraction is exosomes.To construct the testicular ischemia reperfusion injury(IRI)model in male SD rats,24 healthy adolescent male SD rats were randomly divided into three groups.A:Sham group(Sham group),ischemia reperfusion group(I/R group)was divided into two groups:group B:physiological saline treatment group(I/R+NS),group C:BMSCs source exosomes(100ug/ml)treatment group(I/R+ BMSCs-exo).The exosomes marked with PKH67 was injected into the rat body with tail vein injection,and were cultured with the spermatogenic cells(gc-1).Finally,then the rats were twisted testes(left)testis,Confocal observation was used to observe the distribution of exocrine in the testis.The histopathological structure of each group was examined by optical microscopy and the scoring of HE tissue structure and the uptake of spermatogenic cells were observed as well.Using biochemical method to detect the testis tissue superoxide dismutase(SOD),malondialdehyde(MDA),nitric oxide short(NOS:tNOS and iNOS),total antioxidant capacity(T-AOC)and Catalase(CAT)content.At the same time,the expression level of Inflammatory related protein high mobility group B1(HMGB1),and apoptosis related protein cysteine aspartate 3(Caspases-3),BAX,B lymphocytic-2 gene(BCL-2)in testicular tissues of each group were detected by Western blotting.Results:BMSCs was successfully isolated and cultured from the bone marrow of the rat,and the exocrine secreted by BMSCs was successfully extracted.In the animal model of ischemia reperfusion injury,the testicular morphology of group A(Sham)is normal,the structure is distinct,and the germ cells are arranged orderly,with normal spermatophore and spermatogenic cells.Compared with the sham operation group,group B(I/R+NS)had the most significant testicular tissue damage,showing a large amount of exudation and cavitation in the lumen.Spermatocyte arrangement disorder,cell level is not clear,spermatozoon epithelium or a certain degree of loss or detachment.while in the treatment group C(I/R+BMSCs-exo),although the disorder testicular structure but there is a certain level of germ cells arranged closely,and out of order form spermatogenic cell cavity epithelial cells to a certain extent decreased with statistical significance in the organizational structure of HE score(p<0.05).Microscopically,spermatogonial cells can absorb exosomes,and exosomes can reach the testicles through the blood-testes barrier.Testicular tissue biochemical indicator detection,in group B(I/R + NS)organization of the content of MDA and NOS(tNOS and iNOS)higher than group A(Sham)with statistical significance(p<0.01)and SOD,T AOC and CAT levels compared with group A(Sham)is statistically lower(p<0.01).But after exosome treatment C group(I/R+BMSCs-exo),when compared to normal saline group B(I/R+NS),The content of MDA and NOS(tNOS and iNOS)in the tissues decreased significantly(p<0.05),and the levels of SOD,t-aoc and CAT were significantly increased(p<0.05).Protein expression in the testis tissue was detected by Western blot,compared with group A(Sham),Caspases-3,Bax and HMGB1 in group B(I/R+NS)expression level was significantly increased(p<0.001)and the expression level of BCL-2 was decreased(p<0.05).In group C(I/R+BMSCs-exo),the expression level of Caspases-3,Bax and HMGB1 was decreased(p<0.01)and the expression level of BCL-2 increased to some extent(p<0.05).Conclusion:The exosomes derived from bone marrow mesenchymal stem cells(BMSCs)has the protective effect of antioxidation,reduces inflammation and apoptosis on testicular ischemia-reperfusion injury.To provide a new direction and research basis for the subsequent treatment of testicular torsion reduction and its related complications.