Gallic acid attenuates thymic involution in the d-galactose induced accelerated aging mice

Highlights

• Gallic acid administration restored thymic involution in the d-galactose induced accelerated aging mice.

• Gallic acid promoted cells proliferation in the thymus of the d-galactose induced accelerated aging mice.

• Gallic acid led to the reduction of apoptotic cells in the thymus of the d-galactose induced accelerated aging mice.

• Gallic acid stimulated the expression of FoxN1 in the d-galactose induced accelerated aging mice.

Abstract

Senescence is an inevitable and complicated phenomenon. Age-associated thymic involution increases the risk of infectious diseases, which results in the immunosenescence and leads to a poor immune function. d-galactose (d-gal) can cause damages that resemble accelerated aging in mice. Gallic acid (GA), as one of the natural phenolic compounds, has been demonstrated to act in antioxidant and anti-tumor effects. In this study, we explored the effects of GA in preventing the age-related thymic involution and the alterations of the forkhead box protein N1 (FoxN1) in d-gal induced accelerated aging mice. The accelerated aging mice model was established by intraperitoneal injection d-gal for eight weeks and given GA with 200, 250, 500 mg/kg body weight per day, respectively, for six weeks. It showed that the d-gal-treated mice developed structural changes in the thymi compared to normal control mice. With supplement of GA, the mice restored the normal thymic anatomy, including the thickening cortex compartment and clearer cortico-medullary junction. The d-gal-treated mice showed a severe reduction in the number of thymocytes, GA mice also displayed the increased numbers of CD4 + T cells through flow cytometric analysis. GA treatment increased the proliferative cells by BrdU incorporation assay and reduced the numbers of apoptotic cells with FITC-12-dUTP labeling (TUNEL). The expression of FoxN1 was also found increased in GA treated mice by immunohistochemistry and quantitative reverse transcriptase PCR (qRT-PCR). Taken together, our results suggested that the administration of GA opposed the involution of thymus via stimulation of FoxN1 expression and proliferation of cells in a dose-dependent manner.

Introduction

Aging is associated with the changes in morphology and physiology of many organs and systems, including the immune system. Thymus is the primary central immune organ which provides a specialized environment for the development and maturation of T cells. The thymic epithelial cells (TECs) seem to be the most important for the thymic microenvironment (Anderson and Jenkinson, 2001). TECs can secrete many factors which are essential for the development of thymocytes. It has been reported that the thymus will degenerate with aging (Khalyavkin and Krut’ko, 2015) and the thymic microenvironment changes with age, including the structural disorder of the cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs) (Aw et al., 2008). The thymus degenerates with aging and T-cell generation declines, which called the age-associated thymic involution (Haynes et al., 2000). The involution of thymus leads to a poor immune function of the body, thus increasing the susceptibility to many infectious diseases and the risk of autoimmune disease and cancer (Aspinall and Andrew, 2000a). The immunosenescence has been closely associated with thymic involution and is marked by age-related alterations of thymic microenvironment (Aw et al., 2009).

Gallic acid (GA, 3,4,5-Trihydroxybenzoic acid), as one of the dietary polyphenol, mainly exists in many plants including gallnut, tea, grapes, berries and other fruits as well as in wine. GA has the function of antioxidant and can scavenge on free radicals (Lu et al., 2006; Goszcz et al., 2017). It has diverse effects through several pharmacological and biochemical pathways, which makes it a valuable additive as nutritional supplements to prevent the cancer risks (Verma et al., 2013). GA has anti-tumorigenesis on various tumor types by inhibiting the proliferation and migration or inducing cytotoxicity and apoptosis of cancerous cells (You and Park, 2010; Liao et al., 2018; Moghtaderi et al., 2018). It can also induce endothelium-dependent contraction and strongly inhibited the endothelium-dependent relaxation (Sanae et al., 2003). However, whether GA has an effect on age-associated thymic involution remains unknown.

The forkhead box protein N1 (FoxN1) is one of the members of forkhead/winged helix transcription factor family, which is specifically expressed in the membrane of TECs and the skin. FoxN1 regulates the development and functional maturation of TECs (Romano et al., 2013). It has been reported that the expression of FoxN1 increased during the embryonic period while decreased in the postnatal thymus (Ortman et al., 2002). Deficiency in FoxN1, resulted in a cystic thymic rudiment and a hairless phenotype (Nehls et al., 1994). Previous studies have demonstrated that the up-regulation of FoxN1 expression reversed age-related thymic involution (Zook et al., 2011; Bredenkamp et al., 2014; Rode et al., 2015).

Some promising interventions to prevent or even reverse age-related thymic involution and restore immune function with aging are currently under investigation. The approaches of restoring thymic morphology and improving immune function with age-related thymic involution involved in various mechanisms. It has been reported that exogenous melatonin can reduce the involution of postnatal thymus (Tian et al., 2003). Overexpression of FoxN1 restored thymic function (Zook et al., 2011; Ruan et al., 2014). Whether GA can prevent the age-associated thymic involution via FoxN1 needs to be further investigated.

d-galactose (d-gal) has been commonly used to induce accelerated senescence in rodent models (Wei et al., 2005; Chogtu et al., 2018, Majdi2018). In order to explore whether GA has an effect on age-associated thymic involution and elucidate its underlying mechanism, in the present study, we examined the effects of GA in preventing the age related thymic involution and the alterations of FoxN1 expression in d-gal induced accelerated aging mice. These results suggested that GA mediates protective properties and has the potential of suppressing the thymic involution.