人脐带间充质干细胞移植通过改善线粒体功能与减轻氧化应激修复雄性小鼠放射性生殖损伤OA

Objective To investigate the ameliorative effect of human umbilical cord mesenchymal stem cells(hUC-MSCs)on radiation-induced reproductive damage in male mice and its potential mechanisms.Methods After obtaining informed consent and signed consent forms from healthy full-term pregnant women in the Department of Obstetrics and Gynecology at Xijing Hospital of Air Force Medical University,umbilical cord tissue approximately from 6 to 8 cm in length from healthy full-term newborns was collected under sterile conditions in the operating room for isolating and culturing hUC-MSCs;45 healthy male C57BL/6 mice aged from 6 to 8 weeks with a body weight of 22±2 g were divided into Control+PBS(n=3),Control+MSCs(n=6),Rad+MSCs(n=6),Control(n=10),Rad(n=10),and Rad+hUC-MSCs(n=10)groups using a random number table method;the Rad,Rad+MSCs,and Rad+hUC-MSCs groups were subjected to whole-body single-dose irradiation using 60Co γ-ray at a dose rate of 1.28 Gy/min and a total dose of 6 Gy;immediately after irradiation,the Rad+MSCs and Rad+hUC-MSCs groups received a single tail vein injection of hUC-MSCs suspension(2×10⁶ cells/200 μL);the distribution of hUC-MSCs in testicular tissue was observed using a multimodal in vivo imaging system and confocal fluorescence microscopy;at 12 weeks post-irradiation,bilateral testes were harvested and weighed to calculate the testicular coefficient;sperm quality was assessed under a microscope;testicular morphology and structure were evaluated using Hematoxylin and Eosin(HE)staining;reactive oxygen species(ROS)and mitochondrial membrane potential(MMP)levels in testicular tissue were measured by flow cytometry;malondialdehyde(MDA),total superoxide dismutase(T-SOD),and catalase(CAT)contents were detected by colorimetry;testicular cell apoptosis was assessed using TUNEL staining;and Western blot was used to detect the expression levels of Parkin RBR E3 ubiquitin-protein ligase(Parkin),PTEN-induced putative kinase 1(PINK1),Beclin1,OPA1 mitochondrial dynamin-like GTPase(OPA1),dynamin-related protein 1(Drp1),Bcl2-associated X protein(Bax),and B cell lymphoma-2(Bcl-2).Results At 24 h and 48 h post-irradiation,multimodal in vivo imaging showed no fluorescent signals in the testicular tissue of the Control+PBS group,while fluorescent signals were observed in the Control+MSCs and Rad+MSCs groups;further confocal fluorescence microscopy revealed red fluorescence in the interstitial regions of testicular tissue in the Control+MSCs and Rad+MSCs groups,detecting fluorescence dye labeled MSCs signals;at 12 weeks post-irradiation,compared to the Control group,the Rad group showed significantly decreased sperm count(P＜0.05)and motility(P＜0.01),significantly increased malformation rate(P＜0.001),vacuolization and disordered arrangement of spermatogenic cells,reduced seminiferous tubule diameter and spermatogenic epithelium thickness(P＜0.001),elevated testicular tissue ROS levels(P＜0.05),significantly decreased MMP levels(P＜0.001),increased MDA content(P＜0.001),elevated T-SOD activity(P＜0.05),decreased CAT activity(P＜0.05),increased protein expression of Parkin(P＜0.01),PINK1(P＜0.05),Beclin1(P＜0.05),OPA1(P＜0.01),and Drp1(P＜0.05),increased TUNEL-positive cells(P＜0.001),and decreased Bcl-2/Bax ratio(P＜0.01);compared to the Rad group,the Rad+hUC-MSCs group exhibited significantly increased sperm count and motility(P＜0.05),significantly decreased malformation rate(P＜0.001),improved vacuolization and arrangement of spermatogenic cells,increased spermatogenic epithelium thickness(P＜0.01),reduced testicular tissue ROS levels(P＜0.05),significantly increased MMP levels(P＜0.01),decreased MDA content(P＜0.001),reduced T-SOD activity(P＜0.05),increased CAT activity(P＜0.05),decreased protein expression of Parkin(P＜0.05),PINK1(P＜0.05),Beclin1(P＜0.05),OPA1(P＜0.05),and Drp1(P＜0.05),reduced TUNEL-positive cells(P＜0.001),and increased Bcl-2/Bax ratio(P＜0.05).Conclusion hUC-MSCs may alleviate radiation-induced male reproductive damage by mitigating mitochondrial damage,reducing testicular tissue oxidative stress injury and cell apoptosis.