p16INK4a Levels Research Articles

A new model and precious tool to study molecular mechanisms of macrophage aging.

The accumulation of senescent cells, characterized by a senescence-associated secretory phenotype (SASP), contributes to chronic inflammation and age-related diseases (ARD). During aging, macrophages can adopt a senescent-like phenotype and an altered function, which promotes senescent cell accumulation. In the context of aging and ARD, controlling the resolution of the inflammatory response and preventing chronic inflammation, especially by targeting macrophages, must be a priority. Aging being a dynamic process, we developed a model of in vitro murine peritoneal macrophage aging. Our results show that macrophages cultured for 7 or 14 days exhibit a senescence-like phenotype: proliferation decrease, the levels of cyclin-dependent kinase inhibitors p16INK4A and p21CIP1 and of pro-inflammatory SASP components (MCP-1, IL-6, IL-1β, TNF-α, and MMP-9) increase, phagocytosis capacity decline and glycolytic activity is induced. In our model, chronic treatment with CB3, a thioredoxin-1 mimetic anti-inflammatory peptide, completely prevents p21CIP1 increase and enables day 14 macrophages to maintain proliferative activity.We describe a new model of macrophage aging with a senescence-like phenotype associated with inflammatory, metabolic and functional perturbations. This model is a valuable tool for characterizing macrophage aging mechanisms and developing innovative strategies with promising therapeutical purpose in limiting inflammaging and ARD.

Transcriptome-wide profiling for melanocytes derived from newborn and adult human epidermis with enhanced proliferation.

The senescence-associated protein p16INK4A acts as a limiter element in cell-cycle progression. The loss of p16INK4A function is causally related to cellular immortalization. The increase in p16INK4A levels with advancing age was demonstrated in melanocytes. However, the characteristic difference between young and senescent melanocytes affecting immortalization of melanocytes remains unclear. In this study, we generated 10 different cell lines in total from newborn (NB) and adult (AD) primary normal human epidermal melanocytes (NHEM) using four different methods, transduction of CDK4R24C and cyclin D1 (K4D), K4D with TERT (K4DT), SV40 T-antigen (SV40T), and HPV16 E6 and E7 (E6/E7) and performed whole transcriptome sequencing analysis (RNA-Seq) to elucidate the differences of genome-wide expression profiles among cell lines. The analysis data revealed distinct differences in expression pattern between cell lines from NB and AD although no distinct biological differences were detected in analyses such as comparison of cell morphology, evaluation of cell proliferation, and cell cycle profiles. This study may provide useful in vitro models to benefit the understanding of skin-related diseases.

Anti-aging effects of Lacticaseibacillus paracasei PS117 on cognitive and intestinal health in naturally-aged mice: A focus on senescence-related proteins and microbiota composition

The rising global aging population underscores the urgency of maintaining the health and well-being of the elderly while reducing the healthcare burden. Anti-aging probiotics have emerged as a promising strategy. This study identified a novel anti-senescence probiotic, Lacticaseibacillus paracasei PS117 (PS117). The effects of PS117 and heat-treated PS117 (HT-PS117) supplementation on cognitive function of naturally-aged male mice were investigated. It was found that PS117 supplementation improved the cognitive performance of aged mice in the Y-maze test. Furthermore, the level of senescence-related protein p16INK4a (p16) were reduced, while anti-senescence protein sirtuin 1 (Sirt1) were increased in the hippocampus. In addition, there was an overall improvement in the intestinal function. Distinct changes in the gut microbiota were also identified, suggesting a potential contribution to the beneficial effects of PS117 supplementation. In conclusion, these results suggest that PS117 supplements could improve cognitive and intestinal functions in naturally-aged mice, while HT-117 improves only intestinal function, possibly by improving the gut microbiota composition.

Prevention of UVB-Induced Photoaging by an Ethyl Acetate Fraction from Allomyrina dichotoma Larvae and Its Potential Mechanisms in Human Dermal Fibroblasts.

Allomyrina dichotoma larvae (ADL) is an insect type that is used ethnopharmacologically to treat various diseases; however, its use as an antiaging treatment has not been widely studied. Previously, we found that an ethyl acetate (EA) fraction derived from an ADL extract (ADLE) has a high polyphenol content and antioxidant properties. In this study, we identified the underlying molecular mechanism for the protective effect of the EA fraction against UVB-induced photodamage in vitro and ex vivo. UVB treatment increased intracellular reactive oxygen species levels and DNA damage; the latter of which was significantly decreased following cotreatment with the EA fraction. Biological markers of aging, such as p16INK4a, p21WAF1, and senescence-associated β-gal levels, were induced by UVB treatment but significantly suppressed following EA-fraction treatment. UVB-induced upregulation of matrix metalloproteinase (MMP)-1 and downregulation of COL1A1 were also reversed by EA-fraction treatment in both cells and a 3D skin model, which resulted in increased keratin and collagen deposition. Moreover, EA-fraction treatment inhibited the phosphorylation of MAPKs (p38, ERK, and JNK) and nuclear factor (NF-)-kB and decreased the levels of inflammatory cytokines in UVB-treated cells. The results indicate that an EA fraction from ADLE ameliorates UVB-induced degradation of COL1A1 by inhibiting MMP expression and inactivating the MAPK/NF-κB p65/AP-1 signaling pathway involved in this process.

Sodium valproate affects the expression of p16INK4a and p21WAFI/Cip1 cyclin‑dependent kinase inhibitors in HeLa cells.

p16INK4a and p21WAF1/Cip1 are cyclin-dependent kinase inhibitors involved in cell cycle control, which can function as oncogenes or tumor suppressors, depending on the context of various extracellular and intracellular signals, and cell type. In human papillomavirus-induced cervical cancer, p16 INK4a shows oncogenic activity and functions as a diagnostic marker of cervical neoplasia, whereas p21 WAF1/Cip1 acts as a tumor suppressor and its downregulation is associated with the progression of malignant transformation. Several histone deacetylase (HDAC) inhibitors promote the positive and negative regulation of a number of genes, including p16 INK4a and p21 WAF1/Cip1; however, the effects of sodium valproate (VPA) on these genes and on the proteins they encode remain uncertain in HeLa cervical cancer cells. In the present study, these effects were investigated in HeLa cells treated with 0.5 or 2 mM VPA for 24 h, using reverse transcription-quantitative PCR, confocal microscopy and western blotting. The results revealed a decrease in the mRNA expression levels of p16 INK4a and a tendency for p16INK4a protein abundance to decrease in the presence of 2 mM VPA. By contrast, an increase in the protein expression levels of p21WAF1/Cip1 was detected in the presence of 0.5 and 2 mM VPA. Furthermore, VPA was confirmed to inhibit HDAC activity and induce global hyperacetylation of histone H3. Notably, VPA was shown to suppress p16 INK4a, a biomarker gene of cervical carcinoma, and to increase the abundance of the tumor suppressor protein p21WAF1/Cip1, thus contributing to the basic knowledge regarding the antitumorigenic potential of VPA. Exploration of epigenetic changes associated with the promoters of p16 INK4a and p21 WAF1/Cip1, such as histone H3 methylation, may provide further information and improve the understanding of these findings.

Topical application of a BCL-2 inhibitor ameliorates imiquimod-induced psoriasiform dermatitis by eliminating senescent cells

BackgroundPsoriasis is an inflammatory skin disease with unclear pathogenesis and unmet therapeutic needs. ObjectiveTo investigate the role of senescent CD4+ T cells in psoriatic lesion formation and explore the application of senolytics in treating psoriasis. MethodsWe explored the expression levels of p16INK4a and p21, classical markers of cellular senescence, in CD4+ T cells from human psoriatic lesions and imiquimod (IMQ)-induced psoriatic lesions. We prepared a senolytic gel using B-cell lymphoma 2 (BCL-2) inhibitor ABT-737 and evaluated its therapeutic efficacy in treating psoriasis. ResultsUsing multispectrum immunohistochemistry (mIHC) staining, we detected increased expression levels of p16INK4a and p21 in CD4+ T cells from psoriatic lesions. After topical application of ABT-737 gel, significant alleviation of IMQ-induced psoriatic lesions was observed, with milder pathological alterations. Mechanistically, ABT-737 gel significantly decreased the percentage of senescent cells, expression of T cell receptor (TCR) α and β chains, and expression of Tet methylcytosine dioxygenase 2 (Tet2) in IMQ-induced psoriatic lesions, as determined by mIHC, high-throughput sequencing of the TCR repertoire, and RT-qPCR, respectively. Furthermore, the severity of psoriatic lesions in CD4creTet2f/f mice was milder than that in Tet2f/f mice in the IMQ-induced psoriasis model. ConclusionWe revealed the roles of senescent CD4+ T cells in developing psoriasis and highlighted the therapeutic potential of topical ABT-737 gel in treating psoriasis through the elimination of senescent cells, modulation of the TCR αβ repertoire, and regulation of the TET2-Th17 cell pathway.

Inhibition of mTOR prevents glucotoxicity-mediated increase of SA-beta-gal, p16INK4a, and insulin hypersecretion, without restoring electrical features of mouse pancreatic islets

An over-activation of the mechanistic target of rapamycin (mTOR) pathway promotes senescence and age-related diseases like type 2 diabetes. Besides, the regenerative potential of pancreatic islets deteriorates with aging. Nevertheless, the role of mTOR on senescence promoted by metabolic stress in islet cells as well as its relevance for electrophysiological aspects is not yet known. Here, we investigated whether parameters suggested to be indicative for senescence are induced in vitro in mouse islet cells by glucotoxicity and if mTOR inhibition plays a protective role against this. Islet cells exhibit a significant increase (~ 76%) in senescence-associated beta-galactosidase (SA-beta-gal) activity after exposure to glucotoxicity for 72 h. Glucotoxicity does not markedly influence p16INK4a protein within 72 h, but p16INK4a levels increase significantly after a 7-days incubation period. mTOR inhibition with a low rapamycin concentration (1 nM) entirely prevents the glucotoxicity-mediated increase of SA-beta-gal and p16INK4a. At the functional level, reactive oxygen species, calcium homeostasis, and electrical activity are disturbed by glucotoxicity, and rapamycin fails to prevent this. In contrast, rapamycin significantly attenuates the insulin hypersecretion promoted by glucotoxicity by modifying the mRNA levels of Vamp2 and Snap25 genes, related to insulin exocytosis. Our data indicate an influence of glucotoxicity on pancreatic islet-cell senescence and a reduction of the senescence markers by mTOR inhibition, which is relevant to preserve the regenerative potential of the islets. Decreasing the influence of mTOR on islet cells exposed to glucotoxicity attenuates insulin hypersecretion, but is not sufficient to prevent electrophysiological disturbances, indicating the involvement of mTOR-independent mechanisms.

Senolytic effects on dental pulp stem cell's proliferation and differentiation during long-term expansion

Objective: To investigate the impact of intermittent senescent cell clearance on the proliferation and differentiation of dental pulp stem cells (DPSC) in long-term, large-scale expansion, and to explore strategies for maintaining the youthful state of DPSC in vitro. Methods: Human-derived dental pulp stem cells were isolated from healthy permanent teeth extracted for orthodontic or impeding eruption reasons, provided by the Department of Oral and Maxillofacial Surgery at West China Hospital of Stomatology, Sichuan University. Long-term, large-scale in vitro expansion of DPSC was conducted. The study compared young DPSC (passage 5) with aged DPSC (passage 25) using cellular senescence-associated β-galactosidase staining, colony formation assay, and Alizarin Red S staining for osteogenic differentiation induction. To assess the differences between the two cell populations in terms of senescence and amplification and differentiation ability. Medicine screening for the most effective senolytic was compared among 5 common senolytics [Navitoclax (ABT-263), curcumin, dasatinib, fisetin, and quercetin]. The clearance efficacy was compared using cellular senescence-associated β-galactosidase staining to reflect the changes in senescent cell ratio. The senolytic with the highest efficacy was chosen for further experiments. The passage at which the proportion of senescent cells significantly increased was identified, and the selected senolytic was administered three times at three-generation intervals from that passage to remove senescent cells. Both the control and senolytic-treated groups were estimated by fluorescence cellular senescence-associated β-galactosidase staining, real-time fluorescence quantitative PCR (RT-qPCR), colony formation assay, wound healing assay, and Alizarin Red S staining for osteogenic differentiation induction. Subcutaneous heterotopic osteogenesis was performed in nude mice and the grafts were analyzed by HE staining and alkaline phosphatase (ALP) immunohistochemical staining. Results: The proportion of senescent cells increased as the expansion extended, leading to decreased proliferation and osteogenic differentiation ability of senescent DPSC compared to young DPSC (P<0.05). Senescent DPSC exhibited altered mRNA expression levels of senescence-related genes, including p21, p16INK4a, IL-6, and Ki67 (P<0.001). Among the five senolytics, ABT-263 had the biggest decreases in the proportion of senescent cells. After intermittent ABT-263 treatment during expansion, the proportion of senescent cells in the senolytic-treated group [(6.72±2.34)%] was significantly lower than that in the control group [(31.82±0.57)%] (P<0.001). RT-qPCR confirmed that compared with the control group, mRNA expressions of p21, p16INK4a, and IL-6 in the senolytic-treated group were significantly decreased (P<0.05), while mRNA expressions of Ki67 were significantly increased (P<0.01). Furthermore, the cell healing ability and osteogenic differentiation ability of the senolytic-treated group were higher than those of the control group (P<0.05). In vivo experimental results indicated that the relative new bone area [(2.36±0.48)%] after DPSC transplantation in the senolytic-treated group was greater than that in the control group [(1.00±0.46)%] (P<0.05), and the expression of ALP was higher than that in the control group (P<0.01). Conclusions: ABT-263 can effectively eliminate senescent cells in long-term large-scale DPSC expansion. Continuous treatment with ABT-263 during cultivation can maintain the proliferation and differentiation ability of DPSC both in vivo and in vitro.

Determining the relationship of p16INK4a and additional molecular markers of aging with clinical frailty in hematologic malignancy

PurposeOlder adults with hematologic malignancies (HM) have unique challenges due to age and fitness. The primary aim of this pilot study was to benchmark the ability of multiple biomarkers of aging (p16, epigenetic clocks, T cell gene expression profiles, and T cell receptor excision circles (TREC) to identify frailty as measured by a clinical impairment index (I2) in patients with HM.Methods70 patients newly diagnosed with HM had peripheral blood T lymphocytes (PBTL) analyzed for p16INK4a expression using the OSU_Senescence Nanostring CodeSet. PBTL epigenetic age was measured using 7 epigenetic clocks, and TREC were quantified by qRT-PCR. A composite clinical impairment index (I2) was generated by combining values from 11 geriatric metrics (Independent Activities of Daily Living (iADL), physical health score, Short Physical Performance Battery (SPPB), Body Mass Index (BMI), Eastern Cooperative Oncology Group (ECOG) performance status, self-reported KPS, Blessed Orientation Memory Concentration (BOMC), polypharmacy, Mental Health Inventory (MHI)-17, Medical Outcomes Study (MOS) subscales). Clinical frailty was defined as a score of 7 or greater on the I2.ResultsAge-adjusted p16INK4a was similar in newly diagnosed patients and healthy controls (p > 0.1). PBTL p16INK4a levels correlated positively with the Hannum [r = 0.35, 95% CI (0.09–0.75); p adj. = 0.04] and PhenoAge [r = 0.37, 95% CI (0.11–0.59); p adj. = 0.04] epigenetic clocks. The discrimination ability of the I2 model was calculated using the area under the receiver operating characteristic curve (AUC). After adjusting for chronologic age and disease group, baseline p16INK4a [AUC = 0.76, 95% CI (0.56–0.98); p = 0.01], Hannum [AUC = 0.70, 95% CI (0.54–0.85); p = 0.01], PhenoAge [AUC = 0.71, 95% CI (0.55–0.86); p = 0.01], and DunedinPACE [AUC = 0.73, 95% CI (0.57–0.88); p = < 0.01] measures showed the greatest potential to identify clinical frailty using the I2.ConclusionsOur pilot data suggest that multiple blood-based aging biomarkers have potential to identify frailty in older adults with HM.Implications for Cancer SurvivorsWe developed the I2 index to quantify impairments across geriatric domains and discovered that PBTL p16, Hannum, PhenoAge, and DunedinPACE are promising indicators of frailty in HM.

New biflavonoids isolated from Xylia kerrii leaves extract with selective cytotoxicity against MH7A human rheumatoid arthritis synovial fibroblast cells.

Three new biflavonoids (1-3) and two known flavonoids (4, 5) were isolated from Xylia kerrii collected in Thailand. Compounds 1-5 showed selective cytotoxicity against the rheumatoid fibroblast-like synovial MH7A cell line, and these compounds showed weak cytotoxicity against the human lung synovial fibroblast WI-38 VA13 sub 2 RA cell line. Notably, compound 1 was highly selective toward MH7A cells with an IC50 value of 6.9μM, whereas the IC50 value for WI-38 VA13 sub 2 RA cells was > 100μM. The western blotting analysis of MH7A cells treated with compound 1 showed increased CDKN2A /p16INK4A and caspase-8 levels.

Endogenous sulfur dioxide deficiency as a driver of cardiomyocyte senescence through abolishing sulphenylation of STAT3 at cysteine 259

ObjectiveCardiomyocyte senescence is an important contributor to cardiovascular diseases and can be induced by stressors including DNA damage, oxidative stress, mitochondrial dysfunction, epigenetic regulation, etc. However, the underlying mechanisms for the development of cardiomyocyte senescence remain largely unknown. Sulfur dioxide (SO2) is produced endogenously by aspartate aminotransferase 2 (AAT2) catalysis and plays an important regulatory role in the development of cardiovascular diseases. The present study aimed to explore the effect of endogenous SO2 on cardiomyocyte senescence and the underlying molecular mechanisms. Approach and resultsWe interestingly found a substantial reduction in the expression of AAT2 in the heart of aged mice in comparison to young mice. AAT2-knockdowned cardiomyocytes exhibited reduced SO2 content, elevated expression levels of Tp53, p21Cip/Waf, and p16INk4a, enhanced SA-β-Gal activity, and elevated level of γ-H2AX foci. Notably, supplementation with a SO2 donor ameliorated the spontaneous senescence phenotype and DNA damage caused by AAT2 deficiency in cardiomyocytes. Mechanistically, AAT2 deficiency suppressed the sulphenylation of signal transducer and activator of transcription 3 (STAT3) facilitated its nuclear translocation and DNA-binding capacity. Conversely, a mutation in the cysteine (Cys) 259 residue of STAT3 blocked SO2-induced STAT3 sulphenylation and subsequently prevented the inhibitory effect of SO2 on STAT3-DNA-binding capacity, DNA damage, and cardiomyocyte senescence. Additionally, cardiomyocyte (cm)-specific AAT2 knockout (AAT2cmKO) mice exhibited a deterioration in cardiac function, cardiomegaly, and cardiac aging, whereas supplementation with SO2 donors mitigated the cardiac aging and remodeling phenotypes in AAT2cmKO mice. ConclusionDownregulation of the endogenous SO2/AAT2 pathway is a crucial pathogenic mechanism underlying cardiomyocyte senescence. Endogenous SO2 modifies STAT3 by sulphenylating Cys259, leading to the inhibition of DNA damage and the protection against cardiomyocyte senescence.

CDKN2A promoter methylation enhances self-renewal of glioblastoma stem cells and confers resistance to carmustine

BackgroundGlioblastoma, a highly aggressive form of brain cancer, poses significant challenges due to its resistance to therapy and high recurrence rates. This study aimed to investigate the expression and functional implications of CDKN2A, a key tumor suppressor gene, in glioblastoma cells, building upon the existing background of knowledge in this field.MethodQuantitative reverse transcription PCR (qRT-PCR) analysis was performed to evaluate CDKN2A expression in U87 glioblastoma cells compared to normal human astrocytes (NHA). CDKN2A expression levels were manipulated using small interfering RNA (siRNA) and CDKN2A overexpression vector. Cell viability assays and carmustine sensitivity tests were conducted to assess the impact of CDKN2A modulation on glioblastoma cell viability and drug response. Sphere formation assays and western blot analysis were performed to investigate the role of CDKN2A in glioblastoma stem cell (GSC) self-renewal and pluripotency marker expression. Additionally, methylation-specific PCR (MSP) assays and demethylation treatment were employed to elucidate the mechanism of CDKN2A downregulation in U87 cells.ResultCDKN2A expression was significantly reduced in glioblastoma cells compared to NHA. CDKN2A overexpression resulted in decreased cell viability and enhanced sensitivity to carmustine treatment. CDKN2A inhibition promoted self-renewal capacity and increased pluripotency marker expression in U87 cells. CDKN2A upregulation led to elevated protein levels of p16INK4a, p14ARF, P53, and P21, which are involved in cell cycle regulation. CDKN2A downregulation in U87 cells was associated with high promoter methylation, which was reversed by treatment with a demethylating agent.ConclusionOur findings demonstrate that CDKN2A downregulation in glioblastoma cells is associated with decreased cell viability, enhanced drug resistance, increased self-renewal capacity, and altered expression of pluripotency markers. The observed CDKN2A expression changes are mediated by promoter methylation. These results highlight the potential role of CDKN2A as a therapeutic target and prognostic marker in glioblastoma.

Senescence marker p16INK4a expression in patients with multiple sclerosis

ObjectivesTelomere attrition is associated with disability accumulation and brain atrophy in multiple sclerosis (MS). Downstream of telomere attrition is cellular senescence. We sought to determine differences in the cellular senescence marker p16INK4a expression between MS and healthy control participants and the association of p16INK4a expression with MS disability and treatment exposure. MethodsPatients meeting diagnostic criteria for MS and healthy controls were recruited for a cross-sectional pilot study. RNA was extracted from peripheral blood mononuclear cells (PBMCs) and p16INK4a expression levels were measured using qRT PCR. Spearman correlation coefficients and regression models were applied to compare expression levels to chronological age, assess case control differences, and determine associations with clinical outcome measures. ResultsFifty-two participants with MS (67 % female, ages 25–70) and 38 healthy controls (66 % female, ages 23–65) were included. p16INK4a levels were not linearly correlated with chronological age in MS (rhos = −0.01, p = 0.94) or control participants (rhos = 0.02, p = 0.92). Higher median p16INK4a levels were observed in the >50-year age group for MS (0.25, IQR 0.14–0.35) vs. controls (0.12, IQR 0.05–0.15) and in this age group B cell depletion therapy was associated with lower expression levels. p16INK4a expression was not associated with any of the measured MS disability outcomes. DiscussionCaution is needed with using p16INK4a expression level from PBMCs as an aging biomarker in MS participants, given lack of correlation with chronological age or large associations with clinical outcomes.

TARGETING P21CIP1-HIGHLY-EXPRESSING SENESCENT CELLS TO IMPROVE HEALTHSPAN IN PROGEROID MICE

Abstract Aging is the leading risk factor for a variety of chronic diseases. Emerging evidence suggests that modulating fundamental aging processes such as cellular senescence may delay the onset of most chronic conditions as a group and increase healthy lifespan. Cellular senescence refers to the essentially irreversible growth arrest that occurs when cells experience stress. One common feature of senescent cells is the high expression level of p16Ink4a and/or p21Cip1. We and others have demonstrated the causal roles of p16Ink4a-highly-expressing (p16high) senescent cells in various age-related conditions. However, the role of p21high senescent cells in aging remains largely unknown. To address these questions, we have generated and validated a novel p21-Cre mouse model containing p21 promoter driving inducible Cre. By crossing with floxed mice, we managed to monitor, sort, image, eliminate, or modulate p21high cells in vivo. We investigated the maximal walking speed and grip strength of klotho (kl)-/- progeroid mice weekly starting from 5 weeks old until death. We showed that clearance of p21high cells can improve physical function in kl-/- mice for a long time period. Additionally, inactivation of NF-κB specifically in p21high cells also can improve tissue function in kl-/- mice. This study will provide invaluable research tools and pinpoint the role of cellular senescence in age-related tissue dysfunction.

Senescence ofbone marrow mesenchymal stem cells in Wistar malerats receiving normal chow/high-calorie diets with/without vitamin D.

Bone marrow mesenchymal stem cells (BM-MSCs) have a momentous function in the composition of the bone marrow microenvironment because of their many valuable properties and abilities, such as immunomodulation and hematopoiesis. The features and actions of MSCs are influenced by senescence, which may be affected by various factors such as nutritional/micronutrients status, e.g., vitamin D. This study aimed to examine the effects of a high-calorie diet (HCD) with/without vitamin D on BM-MSCs senescence. In the first phase, 48 middle-aged rats were fed a normal chow diet (NCD, n = 24) and an HCD (n = 24) for 26 weeks. Afterward, the rats in each group were randomly divided into three equal subgroups. Immediately, eight-rat from each diet group were sacrificed to assess the HCD effects on the first phase measurements. In the second phase, the remaining 4 groups of rats were fed either NCD or HCD with (6 IU/g) or without vitamin D (standard intake: 1 IU/g); in other words, in this phase, the animals were fed (a) NCD, (b) NCD plus vitamin D, (c) HCD, and (d) HCD plus vitamin D for 4 months. BM-MSCs were isolated and evaluated for P16INK4a, P38 MAPK, and Bmi-1 gene expression, reactive oxygen species (ROS) levels, SA-β-gal activity, and cell cycle profile at the end of both phases. After 26 weeks (first phase), the ROS level, SA-β-gal-positive cells, and cells in the G1 phase were significantly higher in HCD-fed rats than in NCD-fed ones (P < 0.05). HCD prescription did not significantly affect cells in the S and G2 phases (p > 0.05). Compared with the NCD-fed animals, P16INK4a and P38 MAPK gene expression were up-regulated in the HCD-fed animals; also, Bmi-1 gene expression was down-regulated (P < 0.05).BM-MSCs from vitamin D-treated rats (second phase) exhibited reduced mRNA levels of P16INK4a and P38 MAPK genes and increased Bmi-1 mRNA levels (all P<0.05). Vitamin D prescription also declined the percentage of SA-β-gal-positive cells, ROS levels, and the cells in the G1 phase and increased the cells in the S phase in both NCD and HCD-fed animals (P <0.05). The reduction of the cells in the G2 phase in rats fed with an NCD plus vitamin D was statistically non-significant (P=0.128) and significant in HCD plus vitamin D rats (P=0.002).HCD accelerates BM-MSCs senescence, and vitamin D reduces BM-MSCs senescence biomarkers.

SIRT1 regulates osteoblast senescence through SOD2 acetylation and mitochondrial dysfunction in the progression of Osteoporosis caused by Cadmium exposure

Environmental Cadmium (Cd) is a toxicant with widespread exposure, documented adverse effects on bone homeostasis, and makes the onset of osteoporosis (OP), one of the age-related chronic diseases an enormous burden to modern societies worldwide. Aging is the largest risk factor for a multitude of age-related diseases and osteoblasts senescence reduces bone formation and is a key factor for osteoporosis. Despite anti-aging molecules the nuclear silent information regulator of transcription 1 (SIRT1) actions in chondrocytes and bone cells are critical for normal skeletal development and homeostasis, much less is known about the role of SIRT1 in osteoporosis. Here, we aim to demonstrate that SIRT1 mediates osteoblasts' senescence response to OP caused by Cd. The senescent osteoblasts accumulation and their viability were analyzed after Cd exposure. To explore the effects and mechanism of SIRT1 in Cd-induced osteoblastic senescence, we generated SIRT1-overexpressed osteoblast and SIRT1 conditional overexpression in the rat femur. Meanwhile, the OP rat model was established by removing bilateral ovaries. We found decreased SIRT1 expression and senescent osteoblasts accumulation after Cd exposure. Meanwhile, Cd exposure increased P53, P16INK4a, and P21CIPI proteins level, triggered DNA damage response (DDR) through the phosphorylation of ATM and H2AX, and caused mitochondrial dysfunction by the increased acetylation of SOD2 and excessive mitophagy. SIRT1 overexpression attenuated DDR and mitochondrial dysfunction and downregulated the increase of hall makers senescence caused by Cd in osteoblasts. We found overexpression of osteoblastic SIRT1 protects against Cd-induced senescence, which is likely driven by ATM-mediated DDR and SOD2ace-mediated mitochondrial dysfunction. Our study demonstrates the mechanism of SIRT1 in mediating bone homeostasis via senescence. Further mechanistic studies using specific SIRT1 mutations elucidating how SIRT1 modulates bone cell senescence, will provide new therapeutic strategies for human osteoporosis.

P16INK4a and pRb expression in laryngeal squamous cell carcinoma with and without infection by EBV or different genotypes of HPV: a retrospective study

BackgroundLaryngeal squamous cell carcinoma (LSCC) represents one of the principal tumors of the head and neck. Human papillomavirus (HPV) and Epstein–Barr virus (EBV) are considered risk factors for the development and the clinical prognosis of LSCC. High levels of p16INK4a are suggested as a surrogate marker of HPV or EBV infection in some head and neck tumors but in LSCC is still controversial. Furthermore, pRb expression may be considered an additional biomarker but it has not been clearly defined. This work aimed to compare the expression of pRb and p16INK4a as possible biomarkers in tumor tissues with and without infection by EBV or different genotypes of HPV from patients with LSCC.MethodsTumor samples from 103 patients with LSCC were previously investigated for the presence and genotypes of HPV using the INNO-LiPA line probe assay and for the infection of EBV by qPCR. p16 INK4a and pRb expression was assessed by immunohistochemistry.ResultsOf the 103 tumor samples, expression of p16INK4a was positive in 55 (53.4%) and of this, 32 (56.1%) were positive for HPV whereas 11 (39.3%) were EBV positive but both without a significantly difference (p > 0.05). pRb expression was positive in 78 (75.7%) and a higher frequency of this expression was observed in HPV negative samples (87.0%) (p = 0.021) and in high-risk HPV negative samples (85.2%) (p = 0.010). No difference was observed when comparing pRb expression and EBV infection status (p > 0.05).ConclusionOur results support the suggestion that p16INK4a is not a reliable surrogate marker for identifying HPV or EBV infection in LSCC. On the other hand, most of our samples had pRb expression, which was more frequent in tumors without HPV, suggesting that pRb could indicate HPV negativity. However, more studies with a larger number of cases are required, including controls without LSCC and evaluating other molecular markers to determine the real role of p16INK4a and pRb in LSCC.

Nuclear translocation of YAP drives BMI-associated hepatocarcinogenesis in hepatitis B virus infection.

Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) development and progression. The aim of this study was to mechanistically investigate the involvement of Hippo signalling in HBV surface antigen (HBsAg)-dependent neoplastic transformation. Liver tissue and hepatocytes from HBsAg-transgenic mice were examined for the Hippo cascade and proliferative events. Functional experiments in mouse hepatoma cells included knockdown, overexpression, luciferase reporter assays and chromatin immunoprecipitation. Results were validated in HBV-related HCC biopsies. Hepatic expression signatures in HBsAg-transgenic mice correlated with YAP responses, cell cycle control, DNA damage and spindle events. Polyploidy and aneuploidy occurred in HBsAg-transgenic hepatocytes. Suppression and inactivation of MST1/2 led to the loss of YAP phosphorylation and the induction of BMI1 expression in vivo and in vitro. Increased BMI1 directly mediated cell proliferation associated with decreased level of p16INK4a , p19ARF , p53 and Caspase 3 as well as increased Cyclin D1 and γ-H2AX expression. Chromatin immunoprecipitation and the analysis of mutated binding sites in dual-luciferase reporter assays confirmed that the YAP/TEAD4 transcription factor complex bound and activated the Bmi1 promoter. In chronic hepatitis B patients, paired liver biopsies of non-tumour and tumour tissue indicated a correlation between YAP expression and the abundance of BMI1. In a proof-of-concept, treatment of HBsAg-transgenic mice with YAP inhibitor verteporfin directly suppressed the BMI1-related cell cycle. HBV-associated proliferative HCC might be related to the HBsAg-YAP-BMI1 axis and offer a potential target for the development of new therapeutic approaches.

Physical exercise attenuates age-related muscle atrophy and exhibits anti-ageing effects via the adiponectin receptor 1 signalling.

Although the adiponectin signalling exerts exercise-mimicking effects, whether this pathway contributes to the anti-ageing benefits of physical exercise has not been established yet. Swim exercise training and wheel running were used to measure lifespan in the nematode Caenorhabditis elegans and skeletal muscle quality in mice, respectively. Muscle weight, muscle fibre cross-sectional area (CSA) and myonuclei number were used to evaluate muscle mass. RNA sequencing (RNA-Seq) analysis of skeletal muscle in exercised mice was used to study the underlying mechanisms. Western blot and immunofluorescence were performed to explore autophagy- and senescence-related markers. The C.elegans adiponectin receptor PAQR-1/AdipoR1, but not PAQR-2/AdipoR2, was activated (3.55-fold and 3.48-fold increases in p-AMPK on Days 1 and 6, respectively, P<0.001), which was involved in lifespan extension in exercised worms. Exercise training increased skeletal muscle mass index (1.29-fold, P<0.01), muscle weight (1.75-fold, P<0.001), myonuclei number (1.33-fold, P<0.05), muscle fibre CSA (1.39-fold, P<0.05) and capillary abundance (2.19-fold, P<0.001 for capillary density; 1.58-fold, P<0.01 for capillary number) in aged mice. Physical exercise reduced protein (2.94-fold, P<0.001) and mRNA levels (1.70-fold, P<0.001) of p16INK4a , a marker for cellular senescence, in skeletal muscle of aged mice. These beneficial effects of exercise on skeletal muscle of mice were dependent on AdipoR1. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for differentially expressed genes in skeletal muscle between exercised mice with and without AdipoR1 knockdown by RNA-Seq analysis revealed that several KEGG pathways, such as 'AMPK signalling pathway' (P<0.001), 'FOXO signalling pathway' (P<0.001) and 'autophagy' (P<0.001) were overrepresented. Knockdown of FoxO3a inhibited exercise-mediated beneficial effects on skeletal muscle quality of mice by inhibiting autophagy/mitophagy (3.81-fold reduction in LC3-II protein, P<0.001; 1.53-fold reduction in BNIP3 protein, P<0.05). Knockdown of daf-16, the FoxO homologue in C.elegans, reduced autophagy (2.77-fold and 2.06-fold reduction in GFP::LGG-1 puncta in seam cells and the intestine, respectively, P<0.05) and blocked lifespan extension by exercise in worms. Our findings provide insights into how the AdipoR1 pathway has an impact on the anti-ageing benefits of exercise and implicate that activation of the AdipoR1 signalling may represent a potential therapeutic strategy for reducing age-related loss of skeletal muscle.

Examination of age-related changes in the submandibular glands of male mice.

Salivary gland hypofunction adversely affects the oral environment and daily life by causing dry mouth (xerostomia). Senescence-related atrophy of salivary gland tissues is one cause of xerostomia, and it is particularly common among the elderly. However, the underlying mechanism is poorly understood, and no treatment has been established. Therefore, we examined age-related changes in senescence-associated secretory phenotype (SASP) factors, which regulate stemness and cellular senescence, in mouse submandibular glands. We analyzed the submandibular glands of 6-week-old (young group, n = 6) and 82-week-old mice (aged group, n = 6). We performed salivary flow rate measurements, histological analysis including immunohistochemistry, and quantitative real-time PCR. The salivary flow rate was significantly lower in the aged group than in the young group. In addition, immunostaining and quantitative real-time PCR illustrated that aquaporin-5 and α-amylase expressions were significantly decreased in aged mice, indicating salivary gland hypofunction. c-Kit and cytokeratin 5 expressions were also significantly decreased in this group, suggesting that the regenerative abilities of the submandibular glands were reduced because of decreased stem and progenitor cell counts. Furthermore, the levels of p16INK4a and p21 (the senescence markers) and TGF-β1 and IL-6 (SASP factors) were significantly increased in mice, suggesting that senescence had been promoted. The decreased numbers of stem and progenitor cells and increased levels of SASP factors might be associated with age-related changes in mouse submandibular glands. These results might facilitate the development of treatments for senescence-related submandibular gland hypofunction.