| The thymus gradually degenerates in early life and rapidly atrophies with age,leading to the destruction of the thymic microenvironment,a decrease in naive T-cell production,a decline in immune function,and a weakening of tumor immune surveillance.However,the pathological mechanism of thymic atrophy is difficult to define.It has been extensively reported that thymus atrophy is caused by age-related degeneration of thymus epithelial cells.Recent studies have shown that mitochondrial dysfunction occurs during aging,and the absence of the mitochondrial inner membrane fusion protein Opal affects metabolic homeostasis and epithelial aging.However,the role of Opal in thymus involution remains unclear.In vivo studies revealed that Opal expression shows an age-related decline during thymic degeneration.And the Opal defect leads to the blurring of the thymic cortex and medulla,reduction of thymic epithelial cells,and accelerated thymic atrophy.Furthermore,in vitro studies demonstrate that oxidative stress-induced Opal cleavage in thymic epithelial cells is consistent with that in the aging mouse model.We identify that oxidative damage-induced Opal cleavage,leading to mitochondrial morphological changes,mitochondrial DNA damage,and increased ROS production,is an important factor in promoting thymic atrophy.Although irreversible thymic atrophy is considered to be a traditional aging process,exploring the role and molecular mechanism of Opal in age-related thymic atrophy and thymic epithelial cell senescence provides a theoretically effective target for delaying thymic atrophy and maintaining immune function.Objective:The aim of this study is to investigate the effect of Opal-mediated mitochondrial damage on thymic epithelial cell senescence and thymic age-related degeneration.Methods:(1)To explore the age-related degeneration of the thymus,C57BL/6 mice at one week,eight weeks,and 12 months of age are utilized to represent young,adolescent,and old.The HE stain is used to observe the structural changes of thymus tissue in mice at different ages.Using immunofluorescence co-localization staining to examine the expression of mitochondria in thymic epithelial cells.Using Western blot to detect the age-related expression of the Opal protein.(2)A mouse model of Opal specifically knocked out in thymic epithelial cells was constructed.The thymus volume and weight of WT and Opal-cKO mice at 8 weeks were detected.The structural changes of thymus tissues were observed in WT and Opal-cKO mice by a HE stain.Using Flow cytometry to compare the proportion of CD4+,CD8+,DP,and DN cells associated with T cells and the proportion of thymic epithelial cells between WT and Opal-cKO mice.(3)The thymic epithelial cell line with Opal gene expression silenced was constructed.After induction of senescence by Adriamycin(ADM)treatment,β-galactosidase stain,and oil red stain are performed to observe the senescence and adipogenesis of the interference cell line and the control cell line.Investigate the role of Opal in senescent cells.(4)The cleavage of Opal protein in mouse thymic epithelial cells at 1 week,8 weeks,and 12 months of age by Western blot was detected.The cleavage of Opal is detected in ADM-induced senescent cells in vitro.Using mitochondrial red fluorescent probe to monitor mitochondrial morphology by fluorescence microscopy.(5)To explore the influence of Opal on oxidative aging.Opal protein cleavage changes in thymic epithelial cells are observed after induction of oxidative stress with different concentrations of H2O2.Mitochondrial morphological changes are observed by the mitochondrial red fluorescent probe.The degree of mitochondrial DNA damage during oxidative stress was detected through qPCR,and the content of ROS by immunofluorescence was detected.Results:(1)The thymus volume of mice varies in different age groups.The thymus volume reaches its maximum in adolescence and then degeneration with age.(2)In aged mice,the thymus tissue structure was disordered,the number of mitochondria in thymic epithelial cells is reduced,and the expression of Opal is decreased,while the expression of Opal in thymocytes changed little.(3)The thymic epithelial cells specific Opal-deficient mice accelerated thymic atrophy.In adolescence,the thymic volume is extremely reduced,the thymic cell number is reduced,the structure of the cortex and medulla is disordered,and the thymic adipocytes are increased.(4)The thymic epithelial cell line with Opal interference is more prone to senescence and increased lipid droplets than the control cells,consistent with the animal experiment results.(5)In the early stage of thymic degeneration,Opal was cleaved in thymic epithelial cells,resulting in the increase of S-Opal,but Opal cleavage was not changed in thymocytes.(6)In vitro experiments verified that S-Opal increased in ADM-induced thymic epithelial cell senescence model,also accompanied by mitochondrial morphology change,and mitochondrial fragmentation.(7)Oxidative stress is determined to promote the cleavage of Opal,leading to mitochondrial fragmentation,mitochondrial DNA damage,and increased ROS production,leading to the aging of thymic epithelial cells and thymic atrophy.Conclusion:Decreased expression of mitochondrial dynamics-related protein Opal specifically promotes thymic atrophy and degeneration in thymic epithelial cells.The specific mechanism is that in the process of age-related thymic degeneration,thymic epithelial cells are susceptible to oxidative stress,which leads to the increase of mitochondrial stress Opal shear,resulting in mitochondrial fragmentation and mitochondrial DNA damage,and the production of more ROS,resulting in a vicious circle,which leads to the aging of thymic epithelial cells,the disorder of cortical and medullary structure,the increase of fatty accumulation,and the atrophy of the thymus.This study provides a research basis and points the way forward in the exploration of alleviating age-related thymic atrophy and promoting the rejuvenation" of the aged thymus. |
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