Protective Effects Of Resveratrol On Thymus In D-galactose-induced Senescence Accelerated Mice

Background and ObjectiveThe thymus provides a specialized microenvironment for development and maturation of naive T cells.The microenvironment includes thymus stromal cells,extracellular matrix,etc.The thymic epithelial cells(TECs),as an important component of the thymic microenvironment,are essential for the differentiation and selective development of T cells.The age-dependent thymic atrophy occurs after puberty,which would reduce thymus volume,output of T cell,TECs,and increase the numbers of fat cells,etc.D-galactose(D-gal)can lead to neurodegeneration,involution of immune function and reproductive function.Resveratrol(RSV)has been found to have immunomodulatory effects.However,it remains indefinite whether RSV affects the thymic microenvironment and prevents thymic involution.The transcription factors Forkhead box protein N1(Fox N1)regulates the differentiation and proliferation of TECs;and the Autoimmune regulator(Aire)regulates the development and maturation of medullary thymic epithelial cells.However,the effects of RSV on Fox N1 and Aire functions still remain unclear.Therefore,in this study,we mainly explored whether RSV has protective effects on involutional thymi in the D-galactose-induced senescence accelerated mice,and whether RSV plays a role in regulating Aire and Fox N1 in order to provide theoretical basis for mechanisms of thymic involution.Methods 1.Fifty ICR mice(2 months old)were divided randomly into five groups(10 per group): The control group,D-gal group,and RSV-treated groups with three doses (25,50,100 mg/kg.d).D-gal induced senescence accelerated mouse model was established by intraperitoneal injection of D-gal(120 mg/kg.d)dissolved in 0.9%saline solution for 8 weeks.Different doses of RSV were administered by intragastric gavage after two weeks.Bromodeoxyuridine(Brd U)(120 mg/kg)was inject intraperitoneally,3h later,the mice were sacrificed and fresh thymi were obtained.2.Frozen and paraffin sections of the thymus tissues were performed after fixation in 4% paraformaldehyde,as well as DNA and RNA of the thymus tissues were extracted.3.Thymocytes were isolated for flow cytometry to observe changes of thymocyte subsets(n=4).4.Frozen sections were stained with senescence-associated β-galactosidase(SA-β-Gal)to observe the senescence cells in the thymi(n=3).5.Apoptosis in those thymi were observed by deoxynucleotidyl transferase-mediated d UTP nick end labeling(Tunel)staining(n=4).6.The thymic morphological changes were observed via HE staining,and theexpression of Brd U,Aire and Fox N1 were observed by immunohistochemistry(n=4).7.The m RNA expression of Aire and Fox N1 was detected by q RT-PCR.8.DNA was converted with bisulfite,the methylation status of the promoter region of Aire and Fox N1 genes were detected by methylation-specific PCR;And the methylation sites of Cp G in the promoter region of Aire gene were detected by bisulfite sequencing PCR.Results 1.Compared with the control group,D-gal can induce thymic atrophy,which showed that the thymus volume became smaller and the division between cortex and medulla became obscured.2.Compared with the control group,the thymi of the D-gal-treated mice showed proliferating cells were reduced,senescence and apoptosis cells were increased, and the expression of Aire and Fox N1 was decreased.3.Compared with the control group,the promoter region methylation degree of Aire and Fox N1 was increased,the unmethylation degree was decreased.Methylation sites of Cp G in the promoter region of Aire gene were increased.4.After RSV administration,it showed an increase in thymus volume and a distinct division between cortex and medulla.5.After RSV administration,proliferating cells were increased,senescence and apoptosis cells were decreased,and the expression of Aire and Fox N1 in the thymus was increased.Meanwhile,the promoter region methylation degree of Aire and Fox N1 was decreased,the unmethylation degree was increased.Conclusions 1.D-galactose can cause thymic structural changes and reduce the expression of Aire and Fox N1.2.Resveratrol has protective effects on thymic damage induced by D-galactose;and it may maintain the thymus functions via stimulating the expression of Aire and Fox N1.