Age-dependent Alterations Of Sox2 And Nanog In The Thymus And Effects Of Gallic Acid On Them

Background and ObjectiveAs an important central immune organ,thymic involution,begins after puberty,was caused by the gradual loss of thymic epithelial cells(TECs).With the increase of age,the thymus showed aging atrophy,which was mainly manifested by the shrinkage of thymus volume,decline of T cell output,decrease of thymic epithelial cells,and increase of adipocytes.Age-related thymus degeneration represents one of the most recognizable characteristics of the aging immune system and is thought to play a significant role in immunosenescence.Immunosenescence is closely related to the atrophy of the thymus,which is associated with an increase in the number of apoptotic cells and senescent cells in the aging thymus,which leads to a decline in T cell output and thymic degradation.Studies have shown that thymic epithelial progenitor cells(TEPC)are found in the thymus of adult mice.The presence of TEPC and/or stem cells is the subject of a long debate in this field.Nanog(Homeobox transcription factor Nanog)is a transcription factor which contains a homeodomain.Nanog’s pluripotency-related properties have been applied to cell reprogramming to generate induced pluripotent stem cells or cause metabolic changes.Recent studies have shown that Nanog plays an important role in regulating stem cell pluripotency.At the same time,the overexpression of Nanog resulted in a significant increase in lifespan.Sox2 and Nanog are recognized as stem cell transcription factors,regulating the self-renewal and differentiation of stem cells.Sox2(Sex determining region Y-box2)is a stem cell transcription factor of the SOX family.The SOX gene is expressed in a restricted space-time manner and is involved in stem cell biology,organogenesis and development,which plays a role in conferring adult stem cell characteristics.Sox2 regulates and maintains self-renewal and pluripotency in embryonic stem cells,and Sox2 is expressed in late-stage embryonic mouse progenitor cells.Sox2 is not only expressed in stem cells and progenitor cells,but also expressed in several tissues of mice and humans.As a biological marker of aging,its expression is reduced with aging in several tissues of mice and humans.Gallic acid(GA)is a natural product of polyphenols,mainly exists in different kinds of plants,such as fruits,vegetables,spices and tea.GA is currently known to have a variety of biological effects,such as anti-inflammatory,anti-microbial,and so on.GA has anti-cancer activity to a variety of cancer cells,and has anti-diabetic and antioxidant properties.However,there are not many reports about the effects of GA on thymus atrophy.Whether GA can delay the aging of the thymus and promote the regeneration of thymus needs to be further explored.This study mainly explored whether GA has a protective effect on thymic aging,and whether it can delay the thymic aging and promote thymic regeneration by affecting the expression of stem factors Sox2 and Nanog.The stem factors Sox2 and Nanog are essential in maintaining the pluripotency of progenitor cells.Studies have reported that Sox2 and Nanog are expressed in mTEC,suggesting the presence of epithelial progenitor cells in the adult thymus.How is the expression of Sox2 and Nanog in adult thymus,and whether GA can affect the expression of stem factors Sox2 and Nanog,by mobilizing epithelial progenitor cells,and promoting thymic regeneration,thus protecting the thymus from aging remains unknown.Therefore,we first detected the expression positions and changes of Sox2 and Nanog in the thymus of normal developing mice.Secondly,we administered GA to 6-month-old mice for 6 weeks,and detected the expression of Sox2 and Nanog,and explored whether GA can protect thymus from atrophy by regulating Sox2 and Nanog expression.D-galactose(D-gal)acute attenuating mouse model was established to detect the expression of Sox2 and Nanog and cells proliferation in the thymus after GA intervention.We try to explore whether GA play roles in delaying thymic aging and promoting its regeneration through Sox2 and Nanog.Since tissue repair is usually driven by stem cells or progenitor cells,this study is expected to provide important theoretical basis for the mechanism of thymic regeneration.Methods1.Experimental grouping①Kunming mice(1 M,6 M,9 M,12 M),five mice in each group.②Kunming mice(6 M,n=20)were randomly divided into two groups,6M+CMC(carboxymethyl cellulose sodium)group which was used as the control group and 6M+GA group in which GA was administered for 6 weeks.③Kunming mice(2 M,n=30)were randomly divided into three groups,10 in each group:Control group,D-gal group,and GA group.D-gal group and GA group were intraperitoneally injected with D-gal(120 mg/kg.d)for 8 weeks to establish a model of acute aging in mice.Since the third week,GA(250 mg/kg/day)was administered to the stomach for 6 weeks.The above animals were intraperitoneally injected with BrdU(Bromodeoxyuracil riboside,120 mg/kg)one month before sacrifice.Mice were anesthetized and thymus tissue was taken.2.Thymic histological analysis:Paraffin sections were prepared from thymic tissues,and the morphological and structural changes of thymus were observed by HE staining.The expressions of K5(mTEC marker),K8(cTEC marker),BrdU(Lable stem cells in Brdu retaining assay),Sox2 and Nanog was observed by immunohistochemistry(n=5).Immunofluorescence was used to observe the expression of K5 and K8 in thymus of D-gal treated aging mice.3.Detection of thymic senescent cells:Frozen sections were prepared from thymic tissue,and the senescent cells in the thymus were observed by senescence-associatedβ-galactosidas(SA-β-Gal)staining(n=5).4.Thymus cell subset ratio analysis:Thymus tissue was ground,thymus cells were separated by 400 mesh screen,and the percentage of thymocyte subtypes(CD4+,CD8+)was observed by flow cytometry(n=5).5.Thymus gene expression analysis:RNA was extracted from thymus tissues of different groups,and the mRNA expression levels of Sox2 and Nanog were detected by qRT-PCR(n=5).Experimental result1.Expression of Sox2 and Nanog in the thymus of natural aging mice:① As the age increases,the thymus volume and the thymus index decreased,the fatty and senescent cells increased,the structural integrity was destroyed and the distinction between the cortex and medulla was obscured.②BrdU-expressing positive cells continued to decrease with age,and were mainly distributed at the cortex-medulla junction and medulla,indicating that thymic adult stem cells existed at the cortex-medulla junction.③ It showed that the expression of Sox2 and Nanog decreased with age,and the expression positions were mainly distributed at the junction of cortex and medulla by immunohistochemistry.The expression levels of Sox2 and Nanog mRNA were also decreased with age by qRT-PCR.2.Expressions of Sox2 and Nanog in 6-month-old GA-administered thymus:① The thymus volume and thymus index were significantly higher than those in the control group.After administration,the cortex thickened,the dermatodermal boundary was obvious,and the senescent cells decreased significantly compared with the control group.②After GA administration,BrdU positive cells were significantly higher than those of control mice.The expressions of Sox2 and Nanog were significantly higher than those of control mice by immunohistochemistry and the mRNA expression of Sox2 and Nanog was significantly higher than that of control mice by qRT-PCR.3.Effect of GA on the expression of Sox2 and Nanog in thymus of D-gal treated mice:① The thymus volume and thymus index of control group and GA group were higher than those of D-gal group.In the D-gal group,the thymus tissue atrophy and the demarcation of the cortex and medulla were unclear,while in the control group and the GA group,the junction of the cortex and pulp was obvious,the cortex was thicker,and the thymus structure was more complete.In the D-gal group,there were more senescent cells in the thymus,and the senescent cells decreased after administration of GA.②The disorder of T cell subtype was presented in D-gal group,and the state of T cell subtype disorder was improved after GA administration by flow cytometry.③BrdU Label experiments showed that BrdU-positive cells had higher expression levels in the Control group and GA,while the D-gal group had less retention and was basically undetectable.④The expression of Sox2 and Nanog was significantly reduced after D-gal treatment,and the expression was significantly increased after GA administration by immunohistochemistry.The mRNA expression of Sox2 and Nanog in the D-gal group was also found significantly reduced,and the expression was significantly increased after GA administration by qRT-PCR.Conclusion1.Sox2,Nanog showed age-dependent changes in the thymus.Gallic acid can upregulate the expression of Sox2 and Nanog in the thymus.2.Sox2,Nanog and BrdU-positive cells were found to be more in the area of cortex-medulla junction in the thymi of mice,which indicated that thymic adult stem cells/progenitor cells may exist at the cortex-medulla junction of the thymi.3.Gallic acid may protect the thymus from age-related atrophy and D-galactose-induced thymus damage,and promote thymus regeneration by increasing the expression of Sox2 and Nanog.