Epigallocatechin-3-gallate ameliorates lipopolysaccharide-induced acute thymus involution in mice via AMPK/Sirt1 pathway.

The thymus, a site to culture the naïve T lymphocytes, is susceptible to atrophy or involution due to aging, inflammation, and oxidation. Epigallocatechin-3-gallate (EGCG) has been proven to possess anti-inflammatory, antioxidant, and antitumor activity. Here, we investigate the effects of EGCG on thymic involution induced by lipopolysaccharide (LPS), an endotoxin derived from Gram-negative bacteria. The methodology included an in vivo experiment on female Kunming mice exposed to LPS and EGCG. Morphological assessment of thymic involution, immunohistochemical detection, and thymocyte subsets analysis by flow cytometry were further carried out to evaluate the potential role of EGCG on the thymus. As a result, we found that EGCG alleviated LPS-induced thymic atrophy, increased mitochondrial membrane potential and superoxide dismutase levels, and decreased malondialdehyde and reactive oxygen species levels. In addition, EGCG pre-supplement restored the ratio of thymocyte subsets, the expression of autoimmune regulator, sex-determining region Y-box 2, and Nanog homebox, and reduced the number of senescent cells and collagen fiber deposition. Western blotting results indicated that EGCG treatment elevated LPS-induced decrease in pAMPK, Sirt1 protein expression. Collectively, EGCG relieved thymus architecture and function damaged by LPS via regulation of AMPK/Sirt1 signaling pathway. Our findings may provide a new strategy on protection of thymus from involution caused by LPS by using EGCG. And EGCG might be considered as a potential agent for the prevention and treatment of thymic involution.

Thymic tuft cells: potential "regulators" of non-mucosal tissue development and immune response.

As the leading central immune organ, the thymus is where T cells differentiate and mature, and plays an essential regulatory role in the adaptive immune response. Tuft cells, as chemosensory cells, were first found in rat tracheal epithelial, later gradually confirmed to exist in various mucosal and non-mucosal tissues. Although tuft cells are epithelial-derived, because of their wide heterogeneity, they show functions similar to cholinergic and immune cells in addition to chemosensory ability. As newly discovered non-mucosal tuft cells, thymic tuft cells have been demonstrated to be involved in and play vital roles in immune responses such as antigen presentation, immune tolerance, and type 2 immunity. In addition to their unique functions in the thymus, thymic tuft cells have the characteristics of peripheral tuft cells, so they may also participate in the process of tumorigenesis and virus infection. Here, we review tuft cells' characteristics, distribution, and potential functions. More importantly, the potential role of thymic tuft cells in immune response, tumorigenesis, and severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) infection was summarized and discussed.

Apilimod enhances specific productivity in recombinant CHO cells through cell cycle arrest and mediation of autophagy.

Chinese hamster ovary (CHO) cells are expected to acquire the ability to produce higher recombinant therapeutic protein levels using various strategies. Genetic engineering targeting the cell cycle and autophagy pathways in the regulation of cell death in CHO cell cultures has received attention for enhancing the production of therapeutic proteins. In this study, we examined the small-molecule compound apilimod, which was found to have a positive influence on recombinant protein expression in CHO cells. This was confirmed by selective blocking of the cell cycle at the G0/G1 phase. Apilimod treatment resulted in decreased expression of cyclin-dependent kinase 3 (CDK3) and Cyclin C and increased expression of cyclin-dependent kinase suppressor p27Kip1, which are critical regulators of G1 cell cycle progression and important targets controlling cell proliferation. Furthermore, total transcription factor EB (TFEB) was lower in apilimod-treated CHO cells than in control cells, resulting in decreased lysosome biogenesis and autophagy with apilimod treatment. These multiple effects demonstrate the potential of apilimod for development as a novel enhancer for the production of recombinant proteins in CHO cell engineering.

Regeneration and proliferation of cardiomyocytes and its microRNA regulatory mechanisms.

Myocardial regeneration is identified as a concept at histological level. The core content is to increase the number of cardiomyocytes (CMs), so as to maintain the steady state of CMs under pathological or physiological conditions and ensure the normal cardiac function. In this review, we discussed the relevant factors involved in the regeneration of CMs, generalized in mice, large mammals and human. During different development stages of mammalian hearts, CMs showed several controlling and growth modes on the physiological or pathological state: mitosis, hypertrophy, nuclear polyploidy and multinucleation, amitosis and etc. We also discussed the mechanisms of specific microRNAs implicated in the cardiac development, as well as disease-induced apoptosis in CMs and the process of re-entering cell cycle after injury. It is hoped that this review will contribute to a deeper understanding of therapeutic approaches for myocardial regeneration after injury.

Metformin ameliorates thymus degeneration of mice by regulating mitochondrial function.

As the main lymphoid organ, the thymus degenerates with age. The loss of thymic epithelial cells is mainly related to thymus degeneration and reduced T cells development. As an insulin sensitizer, metformin is a first-line drug for the treatment of diabetes and has been shown to prolong the lifespan of mice, but the mechanism is still unclear. In this study, we explored the therapeutic effect of metformin on thymus degeneration in the accelerated aging mice, which was established by intraperitoneal injection D-galactose (120 mg/kg/day) for eight weeks. Metformin was intragastrically given with 100 or 300 mg/kg body weight per day, respectively, for six weeks. Histological examination showed that metformin administration could alleviate thymus atrophy caused by D-galactose. In addition, metformin therapy increased mitochondrial membrane potential, with a reduction in mitochondrial reactive oxygen species, MDA and SOD levels, and restored mitochondrial balance through enhanced expression of dynamin-related protein 1 (Drp1). Furthermore, metformin altered T lymphocyte subsets and cellular senescent cells; the expression of FoxN1, Aire and Sox2 of thymic epithelial cells also increased. Thus, metformin presented a positive effect on thymic degeneration through improving mitochondrial function. Taken together, these findings revealed an unexpected complexity in the anti-aging of this widely used drug.

Dosage effects of resveratrol on thymus involution in D-galactose-treated mice.

The thymus regulates a specific microenvironment for the growth and maturation of naive T cells. Involution of immune function was an important factor during body aging. Preventing the senescence of immune organs has become a major medical issue. Resveratrol (RSV) has been proved to delay the aging of many organs including the thymus. However, the underlying mechanism remains indefinite and the dosages of RSV on thymus involution need to be further clarified. In the current study, the senescence-accelerated mice were produced using d-galactose for two months. RSV at different dosages (25, 50, 100 mg kg-1  day-1 ) was then administered. The alteration of the thymic morphological structure was observed. It showed that three dosages of RSV significantly decreased cellular senescence of the thymus and no dosage difference was detected. For cellular proliferation and apoptosis of the thymus, 50 and 25 mg/kg per day of RSV displayed the best effects on cellular proliferation and apoptosis in the thymus, respectively. Furthermore, 50 mg/kg per day of RSV increased the expression of FoxN1 both at transcription and translation levels. These findings indicated that RSV could delay thymus atrophy in a dosage-dependent pattern and FoxN1 might involve in the beneficial mechanism of RSV, which was of great significance for the enhancement of thymic health and organic immunity. PRACTICAL APPLICATIONS: Resveratrol has been proved to delay aging of many organs including of thymus. In the present study, we explored the dosage of resveratrol on thymus involution and the expression of transcription factors forkhead box protein N1 (FoxN1) in the senescenceaccelerated mice induced by D-galactose. The results indicated that resveratrol could delay thymus atrophy in a dosage-dependent pattern within a certain dose range, and higher RSV concentration may have drug toxicity, which suggests that the dosage of RSV requires attention, And FoxN1 might involve in the beneficial mechanism of resveratrol supplement, which was of great significance to explore the mechanism for the enhancement of thymus immunity.

Curcumin protects thymus against D-galactose-induced senescence in mice.

Senescence-related decline of thymus affects immune function in the elderly population and contributes to the prevalence of many relevant diseases like cancer, autoimmune diseases, and other chronic diseases. In this study, we investigated the therapeutic effects of curcumin, an agent that could counter aging, and explored its optimal intake and the alteration of autoimmune regulator (Aire) after curcumin treatment in the D-galactose (D-gal)-induced accelerated aging mice. ICR mice were intraperitoneally injected with D-gal for 8 weeks to establish the accelerated aging model and given curcumin with 50, 100, and 200 mg/kg body weight per day by gavage, respectively, for 6 weeks. It indicated that the D-gal-treated mice developed structural changes in the thymi compared with the control group without D-gal and curcumin treatment. As the supplements of curcumin, it resulted in a restoration of the normal thymic anatomy with an increase of proliferating cells and a reduction of apoptotic cells in the thymi of the D-gal-induced aging model mice. Curcumin administration could also expand the expression level of Aire from mRNA level and protein level. The current study demonstrated that curcumin could ameliorate senescence-related thymus involution via upregulating Aire expression, suggesting that curcumin can rejuvenate senescence-associated alterations of thymus induced by D-gal accumulation.

Resveratrol ameliorates thymus senescence changes in D-galactose induced mice.

The thymic microenvironment plays an important role in the development of T cells. A decrease of thymic epithelial cells is the main cause of age-related thymic atrophy or degeneration. Resveratrol (RSV), a phytoalexin produced from plants, has been shown to inhibit the adverse effects of dietary obesity on the structure and function of the thymus. D-Galactose (D-gal) can induce accelerated aging in mice. In the present study, young mice (2 months old) were injected with D-gal (120 mg/kg/day) for 8 consecutive weeks to construct an accelerated aging model. Compared with normal control mice, the thymus epithelium of the D-gal treated mice had structural changes, the number of senescent cells increased, the number of CD4+ T cells decreased, and CD8+ T cells increased. After RSV administration by gavage for 6 weeks, it was found that RSV improved the surface phenotypes of D-gal treated mice, and recovered thymus function by maintaining the ratio of CD4+ to CD8+ cells. It also indicated that RSV enhanced the cell proliferation and inhibited cell senescence. Increased autoimmune regulator (Aire) expression was present in the RSV treated mice. The lymphotoxin-beta receptor (LTßR) expression also increased. These findings suggested that RSV intake could restore the alterations caused by D-gal treatment in the thymus via stimulation of Aire expression.

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

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.

Enhancing expression level and stability of transgene mediated by episomal vector via buffering DNA methyltransferase in transfected CHO cells.

Nonviral episomal vectors present attractive alternative vehicles for gene therapy applications. Previously, we have established a new type of nonviral episomal vector-mediated by the characteristic motifs of matrix attachment regions (MARs), which is driven by the cytomegalovirus (CMV) promoter. However, the CMV promoter is intrinsically susceptible to silencing, resulting in declined productivity during long-term culture. In this study, Chinese hamster ovary (CHO) cells and DNA methyltransferase-deficient (Dnmt3a-deficient) CHO cells were transfected with plasmid-mediated by MAR, or CHO cells were treated with the DNA methylation inhibitor 5-Aza-2'-deoxycytidine. Flow cytometry, plasmid rescue experiments, fluorescence in-situ hybridization (FISH), and bisulfite sequencing were performed to observe transgene expression, its state of existence, and the CpG methylation level of the CMV promoter. The results indicated that all DNA methylation inhibitor and methyltransferase deficient cells could increase transgene expression levels and stability in the presence or absence of selection pressure after a 60-generation culture. Plasmid rescue assay and FISH analysis showed that the vector still existed episomally after long-time culture. Moreover, a relatively lower CMV promoter methylation level was observed in Dnmt3a-deficient cell lines and CHO cells treated with 5-Aza-2'-deoxycytidine. In addition, Dnmt3a-deficient cells were superior to the DNA methylation inhibitor treatment regarding the transgene expression and long-term stability. Our study provides the first evidence that lower DNA methyltransferase can enhance expression level and stability of transgenes mediated by episomal vectors in transfected CHO cells.