A Study On The Relationship Between Thymus Atrophy And Inflamm-aging And The Role Of MiRNA-146a-5p In It

Introduction:The thymus is an important immune organ.Lymphopoietic progenitor cells from bone marrow interact with thymus and thymic stromal cells,and then differentiate,develop and mature into T lymphocytes.Thymus atrophy will lead to immunosenescence and lower function of the immune cells,increasing the risk of cancer,autoimmune diseases and infections in the elderly.And these serious senile diseases may be accompanied by a chronic inflammatory state,called inflamm-aging.Inflamm-aging is considered to be one of the most significant consequences of immunosenescence,but the direct relationship between its occurrence and thymus atrophy is still unclear.Understanding the molecular mechanism of thymus atrophy and the relationship between thymus atrophy and inflamm-aging will provide theoretical basis for alleviating inflamm-aging and improving the health state of aged population.Methods:1.To establish an aging model to explore whether inflamm-aging exists in aged mice and the possible relationship between thymus atrophy and inflamm-aging.?1?Establishment of an aging model:2-mo old C57BL/6 female mice were subcutaneously injected with different doses of D-galactose daily for 8 weeks.The dose causing obvious thymus atrophy?compared with the natural aging mice aged 18-20 months?was screened for subsequent experiments;?2?Immunosenescence index measurement:CD4⁺CD8^-CD25⁺Foxp3⁺regulatory T cell?pTreg?levels were detected by flow cytometry;?3?Inflamm-aging index measurement:Blood CD3⁺CD8⁺CD28^-T cells and spleen CD3⁺CD4⁺TNF-?⁺T cells were detected by flow cytometry,serum IL-6 level was detected by ELISA,inflammatory cell infiltration in Lung was detected by HE staining;?4?Determination of thymus autoreactive T cell precursor level:The dynamic levels of CD4⁺CD44^hiKi67⁺and CD8⁺CD44^hiKi67⁺T lymphocytes were detected by flow cytometry;?5?Thymus negative selection function evaluation:CD4⁺CD8⁺,CD4⁺,CD8⁺lymphocytes and thymus Treg?tTreg?cells were detected by flow cytometry,western blot analysis was used to check thymus Aire protein expression.2.To explore the primary thymic stromal cells premature senescense induced by D-galactose and the possible role of miRNA-146a-5p in this process.?1?Primary cell culture:We extracted thymic stromal cells?TSC?from C57BL/6 mice less than 3 days old and cultured them;?2?TSC premature senescense caused by D-galactose:TSC were exposed to different concentration gradients?0-225mM?of D-galactose with different time?24h,48h,72h?,cell proliferation assay?MTS?was used to detect cell proliferation and activity,cell snescence was detected by the SA-galactosidase kit,the ROS level of cells was detected by the reactive oxygen species?ROS?assay kit,cell apoptosis and cycle arrest were detected by flow cytometry,western blot was used to detect the expression of senescence related proteins p16,p21,p53 and autophagy related proteins LC3,p62 and mTOR;?3?Level of miRNA-146a-5p was detected in premature senescence TSC:The level of miRNA-146a-5p was detected by Real Time PCR;?4?Effects of miRNA-146a-5p on the premature senescence of TSC:Transfection efficiency was tested by Real Time PCR after transfection of miRNA-146a-5p mimic or Negative Control?NC?into TSC using lipo RNA^imax,TSC was modified with miRNA-146a-5p mimic and/or D-galactose,and the effect of miRNA-146a-5p on TSC premature senescence was detected;?5?To dectect how did miRNA-146a-5p affect TSC premature senescence:Western blot was used to detect the level of autophagy and the miRNA-146a target gene TRAF6.Results:1.Aging mice had an inflamm-aging state,and thymus atrophy could directly aggravate this state.?1?Large doses of D-galactose could induce aging and lead to obvious thymus atrophy in mice,and the atrophy and the reduction of total thymus cells were equivalent to the natural aging mice?18-20 months old?;?2?Immunosenescence occurred in aging mice,and the proportion of peripheral Treg cells increased;?3?Inflamm-aging state was observed in aging mice,with increased proportion of peripheral senescent cells?CD3⁺CD8⁺CD28^-T cells?,increased proportion of peripheral CD3⁺CD4⁺TNF-?⁺inflammatory T lymphocytes,increased blood IL-6 level and inflammatory cell infiltration in the lungs;?4?Proportion of peripheral autoreactive T cells precursor(CD4⁺CD44^hiKi67⁺and CD8⁺CD44^hiKi67⁺)in aged mice were increased;?5?The proportion of CD4CD8 double positive lymphocytes decreased,while the proportion of CD4 and CD8 single positive lymphocytes increased,the tTreg cells of thymus was increased,and the thymus negative selection was damaged and self-saving occurred;?6?The expression of Aire protein in thymus of aged mice was reduced.2.miRNA-146a-5p alleviated stress induced premature senescence of primary TSC induced by D-galactose.?1?When the thymus primary TSC were exposed to D-galactose at the concentration of 225mM for 48h,their viability decreased significantly;?2?ROS of TSC was significantly increased with 225mM D-galactose exposure for 48h;?3?Stress induced premature senescence?SIPS?model of TSC was successfully made:When TSC were exposed to 0-225 mM D-galactose for 48h,cell apoptosis had no obvious change,cell cycle was arrested in G2 stage,SA-?-galactose glucoside enzyme levels increased gradually,cell senescence related protein p53,p16,p21 increased gradually,LC3II/LC3I protein which involved in autophagy were decreased,p62 protein and mTOR increased;?4?miRNA-146-5p decreased in TSC with SIPS;?5?miRNA-146a-5p mimic reduced SIPS of TSC:miRNA-146a-5p mimic reversed the decreased cell viability of TSC with SIPS,alleviated cell cycle arrest,reduced the level of SA-galactosidase,reduced the level of senescence related protein;?6?miRNA-146a-5p might alleviate the SIPS of TSC by targeting TRAF6 instead of increasing autophagy clearance:miRNA-146a-5p didn't reverse the decrease of autophagy of TSC,but reversed the increase of TRAF6.Conclusions:1.Inflamm-aging was observed in aged mice;2.When thymus atrophy happened,the negative selection was impaired,and peripheral self-reactive T cells precursor were increased,which could directly aggravate inflamm-aging;3.TSC was sensitive to ROS stimulation,and SIPS occurred when ROS accumulated;4.miRNA-146a-5p could reduce the SIPS of TSC;5.miRNA-146a-5p might play a role in delaying thymus atrophy,thereby alleviating inflamm-aging.