Cellular Senescence and Ageing.

Cellular senescence has become a subject of great interest within the ageing research field over the last 60 years, from the first observation in vitro by Leonard Hayflick and Paul Moorhead in 1961, to novel findings of phenotypic sub-types and senescence-like phenotype in post-mitotic cells. It has essential roles in wound healing, tumour suppression and the very first stages of human development, while causing widespread damage and dysfunction with age leading to a raft of age-related diseases. This chapter discusses these roles and their interlinking pathways, and how the observed accumulation of senescent cells with age has initiated a whole new field of ageing research, covering pathologies in the heart, liver, kidneys, muscles, brain and bone. This chapter will also examine how senescent cell accumulation presents in these different tissues, along with their roles in disease development. Finally, there is much focus on developing treatments for senescent cell accumulation in advanced age as a method of alleviating age-related disease. We will discuss here the various senolytic and senostatic treatment approaches and their successes and limitations, and the innovative new strategies being developed to address the differing effects of cellular senescence in ageing and disease.

The combination of mitogenic stimulation and DNA damage induces chondrocyte senescence.

OBJECTIVE: Cellular senescence is a phenotypic state characterized by stable cell-cycle arrest, enhanced lysosomal activity, and the secretion of inflammatory molecules and matrix degrading enzymes. Senescence has been implicated in osteoarthritis (OA) pathophysiology; however, the mechanisms that drive senescence induction in cartilage and other joint tissues are unknown. While numerous physiological signals are capable of initiating senescence, one emerging theme is that damaged cells convert to senescence in response to sustained mitogenic stimulation. The goal of this study was to develop an in vitro articular cartilage explant model to investigate the mechanisms of senescence induction. DESIGN: This study utilized healthy cartilage derived from cadaveric equine stifles and human ankles. Explants were irradiated to initiate DNA damage, and mitogenic stimulation was provided through serum-containing medium and treatment with transforming growth factor ß1 and basic fibroblastic growth factor. Readouts of senescence were a quantitative flow cytometry assay to detect senescence-associated ß galactosidase activity (SA-ß-gal), immunofluorescence for p16 and γH2AX, and qPCR for the expression of inflammatory genes. RESULTS: Human cartilage explants required both irradiation and mitogenic stimulation to induce senescence as compared to baseline control conditions (7.16% vs 2.34% SA-ß-gal high, p = 0.0007). These conditions also resulted in chondrocyte clusters within explants, a persistent DNA damage response, increased p16, and gene expression changes. CONCLUSIONS: Treatment of cartilage explants with mitogenic stimuli in the context of cellular damage reliably induces high levels of SA-ß-gal activity and other senescence markers, which provides a physiologically relevant model system to investigate the mechanisms of senescence induction.

Effects and Related Mechanisms of the Senolytic Agent ABT-263 on the Survival of Irradiated A549 and Ca9-22 Cancer Cells.

Senolytic agents eliminate senescent cells and are expected to reduce senescent cell-mediated adverse effects in cancer therapy. However, the effects of senolytic agents on the survival of irradiated cancer cells remain unknown. Here, the effects of the senolytic agent ABT-263 on the survival of irradiated A549 and Ca9-22 cancer cells were investigated. ABT-263 was added to the culture medium after irradiation. SA-ß-gal activity and cell size, which are hallmarks of cell senescence, were evaluated using a flow cytometer. The colony-forming assay and annexin V staining were performed to test cell survival. We first confirmed that radiation increased the proportion of cells with high SA-ß-gal activity and that ABT-263 decreased it. Of note, ABT-263 decreased the survival of irradiated cancer cells and increased the proportion of radiation-induced annexin V+ cells. Furthermore, the caspase inhibitor suppressed the ABT-263-induced decrease in the survival of irradiated cells. Intriguingly, ABT-263 decreased the proportion of SA-ß-gal low-activity/large cells in the irradiated A549 cells, which was recovered by the caspase inhibitor. Together, these findings suggest that populations maintaining the ability to proliferate existed among the irradiated cancer cells showing senescence-related features and that ABT-263 eliminated the population, which led to decreased survival of irradiated cancer cells.

Preadipocyte secretory factors differentially modulate murine macrophage functions during aging which are reversed by the application of phytochemical EGCG.

The present study aimed at evaluating the role of senescent cell microenvironment as an extrinsic causal factor for altered age-associated macrophage functions, and that whether such changes could be ameliorated by the application of tea catechin epigallocatechin gallate (EGCG). To ascertain this, we analyzed the impact of secretory metabolites of proliferating (P) and senescent (S) preadipocyte cells on the induction of phenotypic and functional characteristics associated with aging in macrophages isolated from young (YM) and old (OM) C57BL/6J mice. The role of EGCG as alleviator of preadipocyte media-induced senescence and inflamm-aging was evaluated in OM. Results revealed strong age-related dysregulation in macrophage functions as evident by decreased CD11b expression, enhanced expression of cytokines (IL-6/TNF-α/IL-1ß/IL-10) and cell cycle inhibitors p53/p21WAF1/p16Ink4a, as well as augmentation of M2 phenotype (Arg1/Msr1/Mrc1) and SA-ß-gal activity. Ex vivo exposure of macrophages (YM and OM) to secretory factors of preadipocytes induced differential effects, and treatment with S culture media largely showed an augmentation of senescent phenotype, particularly in the YM. Pretreatment with EGCG (10 µM) to OM caused a dramatic reversal of both age-associated and preadipocyte media-induced changes as evident from upregulation of CD11b and ROS levels, inhibition of inflammatory makers, attenuation of p53/p21WAF1/p16Ink4a expression and SA-ß-gal activity. Our results indicate vital role of adipose tissue-mediated extrinsic factors in shaping macrophage phenotype and functions during aging. It is also apparent that EGCG is a promising candidate in developing preventive therapies aimed at alleviating macrophage inflamm-aging and senescence that may help curb incidences of inflammatory disorders in elderly.

Comparison of several commonly used detection indicators of cell senescence.

Cell senescence is the state of irreversible growth arrest that can be triggered by a variety of different cellular stresses. Currently, the commonly used detection indicators involved in the study of cell senescence include senescence-associated ß-galactosidase, Clusterin, Telomeres/Telomerase, senescence-associated heterochromatin foci, senescence-associated secretory phenotype, senescence marker protein-30, tumor suppressor genes p53 and p16, and other indicators such as Ki67 and decoy receptor 2. These indicators are widely used in the study of cell senescence, each with its own characteristics, advantages, and disadvantages. This review summarizes several commonly used cell senescence indicators and compares their accuracy, credibility, specificity, and the scope of their potential application.

MicroRNA-145 induces the senescence of activated hepatic stellate cells through the activation of p53 pathway by ZEB2.

Activation of quiescent hepatic stellate cells (HSCs) is the major event in liver fibrosis, along with enhancement of cell proliferation and overproduction of extracellular matrix. Recent findings suggest that senescence of activated HSCs might limit the development of liver fibrosis. The p53, a guardian of the genome is associated with liver fibrosis, has been shown to regulate HSCs senescence. In this study, we report that microRNA-145 (miR-145) and p53 were downregulated in vivo and in vitro, concomitant with the enhanced expression of zinc finger E-box binding homeobox 2 (ZEB2). In addition, overexpression of miR-145 and p53 led to upregulation of the number of senescence-associated ß-galactosidase-positive HSCs and the expression of senescence markers p16 and p21, along with the reduced abundance of HSC activation markers α-smooth muscle actin and type I collagen in activated HSCs. Furthermore, silencing of ZEB2 promoted senescence of activated HSCs. Moreover, we also demonstrated that miR-145 specifically targeted the 3'-untranslated regions of ZEB2. In vitro promoter regulation studies show that ZEB2 could bind to the E-box of the p53 promoter as well as inhibit its promoter activity and thus suppress the expression of p53, which in turn repressed activated HSCs senescence. Taken together, our results describe a novel miR-145-ZEB2-p53 regulatory line might participate in the senescence of activated HSCs and might carry potential therapeutic targets for restraining liver fibrosis.

Dental pulp stem cells senescence and regenerative potential relationship.

Uncomplicated treatments for pulpitis and periodontitis continues to be challenging and regenerative approaches could meet this contingency. Dental pulp stem cells (DPSCs) represent a good candidate for oral recovering therapies. Here, we investigated changes in morphology, proliferation, and in vitro differentiation toward mesenchymal and neuronal phenotypes of human DPSCs harvested from differently aged donors. Aging is a physiologic phenomenon occurring with time that hamper body's capability to maintain homeostasis also affecting the functional reserve. Cytofluorimetric, immunohistochemical, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and western blot analyses were performed to gain insight for successful regenerative strategies in elderly. We observed a decline in DPSCs proliferation and differentiation potential with age. Interestingly, these cells behaved differently under osteogenic or odontogenic stimuli, showing different age-related mineralization capabilities. Similarly, neurogenic differentiation decreased with age. In conclusion, our observations represent a valid tool for the development of tailored regenerative strategies in an aging society.

The proteomic study of serially passaged human skin fibroblast cells uncovers down-regulation of the chromosome condensin complex proteins involved in replicative senescence.

Dermal fibroblast is one of the major constitutive cells of skin and plays a central role in skin senescence. The replicative senescence of fibroblasts may cause skin aging, bad wound healing, skin diseases and even cancer. In this study, a label-free quantitative proteomic approach was employed to analyzing the serial passaged human skin fibroblast (CCD-1079Sk) cells, resulting in 3371 proteins identified. Of which, 280 proteins were significantly changed in early passage (6 passages, P6), middle passage (12 passages, P12) and late passage (21 passages, P21), with a time-dependent decrease or increase tendency. Bioinformatic analysis demonstrated that the chromosome condensin complex, including structural maintenance of chromosomes protein 2 (SMC2) and structural maintenance of chromosomes protein 4 (SMC4), were down-regulated in the serially passaged fibroblast cells. The qRT-PCR and Western Blot experiments confirmed that the expression of these two proteins were significantly down-regulated in a time-dependent manner in the subculture of human skin fibroblasts (HSFb cells). In summary, we used serially passaged human skin fibroblast cells coupled with quantitative proteomic approach to profile the protein expression pattern in the temporal progress of replicative senescence in HSFb cells and revealed that the down-regulation of the chromosome condensin complex subunits, such as SMC2 and SMC4, may play an important role in the fibroblast senescence.

Increased chemo-sensitivity by knockdown coilin expression involved acceleration of premature cellular senescence in HeLa cells.

Coilin is a marker protein of the Cajal body (CB). Cajal bodies, functional nuclear structure, play important roles for the maturation of telomerase mRNAs. However, whether CB participates in the process of cell senescence is unknown. Cisplatin is a frequently used drug for the chemotherapy for various cancers, which was recently reported to be able to induce premature senescence of tumor cells. In this study, we found that when HeLa cells were treated with 2 µg/ml cisplatin for 4 days, stagnant cell growth, especially in cells stained positive of SA-ß-gal, was accompanied with significant changes in CB morphologies. The removal of cisplatin allowed the recovery of normal CB appearance, but was not able to restore cells from senescent states. Knocking down coilin expression by siRNA attenuated the growth and reduced the viability of treated cells, and the decreased rate of CB formation correlated with increased staining of SA-ß-gal. Interestingly, when coilin knocked-down cells exposed to cisplatin, the drug sensitivity as shown by the reduction of cell viability was significantly increased compared to the control siRNA transfection groups. Overexpression of coilin phosphomutants increased SA-ß-gal fluorescence following treatments with cisplatin as compared to the wild type coilin transfection. Our results indicated that coilin was an important functional player that involved in cisplatin-induced premature cell senescence. It suggested that the modulation of coilin expression could be considered as a potential anti-tumor strategy to increase the sensitivity of chemotherapy through which drug-induced cell senescence was accelerated.

Identification of miR-494 direct targets involved in senescence of human diploid fibroblasts.

Cellular senescence is a permanent cell cycle arrest triggered by different stimuli. We recently identified up-regulation of microRNA (miR)-494 as a component of the genetic program leading to senescence of human diploid IMR90 fibroblasts. Here, we used 2-dimensional differential gel electrophoresis (2D-DIGE) coupled to mass spectrometry to profile protein expression changes induced by adoptive overexpression of miR-494 in IMR90 cells. miR-494 induced robust perturbation of the IMR90 proteome by significantly (P≤0.05) down-regulating a number of proteins. Combination of mass spectrometry-based identification of down-regulated proteins and bioinformatic prediction of the miR-494 binding sites on the relevant mRNAs identified 26 potential targets of miR-494. Among them, computational analysis identified 7 potential evolution-conserved miR-494 targets. Functional miR-494 binding sites were confirmed in 3'-untranslated regions (UTRs) of 4 of them [heterogeneous nuclear ribonucleoprotein A3 (hnRNPA3), protein disulfide isomerase A3 (PDIA3), UV excision repair protein RAD23 homolog B (RAD23B), and synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP)/heterogeneous nuclear ribonucleoprotein Q (hnRNPQ)]. Their reduced expression correlated with miR-494 up-regulation in senescent cells. RNA interference-mediated knockdown of hnRNPA3 and, to a lesser extent, RAD23B mirrored the senescent phenotype induced by miR-494 overexpression, blunting cell proliferation and causing up-regulation of SA-ß-galactosidase and DNA damage. Ectopic expression of hnRNPA3 or RAD23B slowed the appearance of the senescent phenotype induced by miR-494. Overall, these findings identify novel miR-494 direct targets that are involved in cellular senescence.

Senescence-associated ß-galactosidase activity marks the visceral endoderm of mouse embryos but is not indicative of senescence.

Senescence-associated ß-galactosidase (SA-ß-gal) activity is widely used as a marker of cellular senescence and as an indicator of organismal aging. Here, we report that SA-ß-gal activity is present in the visceral endoderm layer of early postimplantation mouse embryos in predictable patterns that vary as the embryo progresses in development. However, determination of the mitotic index and analysis of the expression of Cdkn1a (p21), a marker of senescent cells, do not indicate cellular senescence. Instead, analysis of embryos in culture revealed the presence of SA-ß-gal activity in apical vacuoles of visceral endoderm cells likely a reflection of acidic ß-galactosidase function in these organelles. SA-ß-gal serves as a practical marker of the dynamics of the visceral endoderm that can be applied to developmental as well as functional studies of early mammalian embryos.

Nesfatin-1 attenuated lipopolysaccharide-induced inflammatory response and senescence in human dental pulp cells.

Lipopolysaccharide (LPS)-triggered damage in human dental pulp cells (hDPCs) is associated with the progression of gingivitis, which is inflammation of the gingival tissue. Nesfatin-1 is a peptide secreted by neurons and peripheral tissues. Here, we report a novel property of Nesfatin-1 in ameliorating LPS-induced inflammatory response and senescence in hDPCs. First, we demonstrate that Nesfatin-1 repressed LPS-triggered expression of inflammatory factors. Secondly, Nesfatin-1 restored telomerase activity and the expression of human telomerase reverse transcriptase (hTERT) and telomeric repeat binding factor 2 (TERF2) against LPS. Senescence-associated ß-galactosidase (SA-ß-gal) staining assay revealed that Nesfatin-1 attenuated LPS-induced cellular senescence in hDPCs. We also found that Nesfatin-1 increased telomerase activity in LPS-challenged hDPCs. It is also shown that Nesfatin-1 reduced the expression of plasminogen activator inhibitor-1 (PAI-1) and p16. Additionally, LPS stimulation reduced the expression of SIRT1, which was rescued by Nesfatin-1. However, the silencing of sirtuin1 (SIRT1) abrogated the protective property of Nesfatin-1 in preventing cellular senescence, implying that the function of Nesfatin-1 is regulated by SIRT1. Taken together, our findings suggest that Nesfatin-1 might possess a protective effect against gingivitis.

Senolytic therapeutics: An emerging treatment modality for osteoarthritis.

Osteoarthritis (OA), a chronic joint disease affecting millions of people aged over 65 years, is the main musculoskeletal cause of diminished joint mobility in the elderly. It is characterized by lingering pain and increasing deterioration of articular cartilage. Aging and accumulation of senescent cells (SCs) in the joints are frequently associated with OA. Apoptosis resistance; irreversible cell cycle arrest; increased p16INK4a expression, secretion of senescence-associated secretory phenotype factors, senescence-associated ß-galactosidase levels, secretion of extracellular vesicles, and levels of reactive oxygen and reactive nitrogen species; and mitochondrial dysregulation are some common changes in cellular senescence in joint tissues. Development of OA correlates with an increase in the density of SCs in joint tissues. Senescence-associated secretory phenotype has been linked to OA and cartilage breakdown. Senolytics and therapeutic pharmaceuticals are being focused upon for OA management. SCs can be selectively eliminated or killed by senolytics to halt the pathogenesis and progression of OA. Comprehensive understanding of how aging affects joint dysfunction will benefit OA patients. Here, we discuss age-related mechanisms associated with OA pathogenesis and senolytics as an emerging modality in the management of age-related SCs and pathogenesis of OA in preclinical and clinical studies.

Senescence marker protein 30 inhibits angiotensin II-induced cardiac hypertrophy and diastolic dysfunction.

BACKGROUND AND OBJECTIVE: Senescence marker protein 30 (SMP30) is assumed to behave as an anti-aging factor. Recently, we have demonstrated that deficiency of SMP30 exacerbates angiotensin II-induced cardiac hypertrophy, dysfunction and remodeling, suggesting that SMP30 may have a protective role in the heart. Thus, this study aimed to test the hypothesis that up-regulation of SMP30 inhibits cardiac adverse remodeling in response to angiotensin II. METHODS: We generated transgenic mice with cardiac-specific overexpression of SMP30 gene using α-myosin heavy chain promoter. Transgenic mice and wild-type littermate mice were subjected to continuous angiotensin II infusion (800 ng/kg/min). RESULTS: After 14 days, heart weight and left ventricular weight were lower in transgenic mice than in wild-type mice, although blood pressure was similarly elevated during angiotensin II infusion. Cardiac hypertrophy and diastolic dysfunction in response to angiotensin II were prevented in transgenic mice compared with wild-type mice. The degree of cardiac fibrosis by angiotensin II was lower in transgenic mice than in wild-type mice. Angiotensin II-induced generation of superoxide and subsequent cellular senescence were attenuated in transgenic mouse hearts compared with wild-type mice. CONCLUSIONS: Cardiac-specific overexpression of SMP30 inhibited angiotensin II-induced cardiac adverse remodeling. SMP30 has a cardio-protective role with anti-oxidative and anti-aging effects and could be a novel therapeutic target to prevent cardiac hypertrophy and remodeling due to hypertension.

Comparative analysis of markers for H2O2-induced senescence in renal tubular cells.

To address what marker(s) is/are most suitable for determining renal cell senescence, cell area, granularity, cycle shift/arrest, SA-ß-Gal, SIRT1 and p16 were evaluated after inducing senescence in HK-2 cells with 0.2-0.8 mM H2O2. Only cell area and granularity concentration-dependently increased at all time-points, whereas SA-ß-Gal, SIRT1 and p16 showed significant coefficient of determination (R2) at two time-points. Cell granularity had significant correlation coefficient (R) with other six, whereas SA-ß-Gal had significant R with five, and cell area, SIRT1 and p16 had significant R with four others. Comparing to SA-ß-Gal, other markers had significantly lower fold-changes only at 72-h with 0.8 mM H2O2, whereas p16 provided greater fold-changes at 48-h with 0.4 and 0.8 mM H2O2. Therefore, cell area, granularity, SA-ß-Gal and p16 may serve as the most suitable markers for determining H2O2-induced senescence in HK-2 renal cells, whereas other markers can be also used but with inferior quantitative precision.

Flow Cytometry-based Method for Efficient Sorting of Senescent Cells.

Cellular senescence is a reprogrammed cell state triggered as an adaptative response to a variety of stresses, most often those affecting the genome integrity. Senescent cells accumulate in most tissues with age and contribute to the development of several pathologies. Studying molecular pathways involved in senescence induction and maintenance, or in senescence escape, can be hindered by the heterogeneity of senescent cell populations. Here, we describe a flow cytometry strategy for sorting senescent cells according to three senescence canonical markers whose thresholds can be independently adapted to be more or less stringent: (i) the senescence-associated-ß-galactosidase (SA-ß-Gal) activity, detected using 5-dodecanoylaminofluorescein Di-ß-D-galactopyranoside (C12FDG), a fluorigenic substrate of ß-galactosidase; (ii) cell size, proportional to the forward scatter value, since increased size is one of the major changes observed in senescent cells; and (iii) cell granularity, proportional to the side scatter value, which reflects the accumulation of aggregates, lysosomes, and altered mitochondria in senescent cells. We applied this protocol to the sorting of normal human fibroblasts at the replicative senescence plateau. We highlighted the challenge of sorting these senescent cells because of their large sizes, and established that it requires using sorters equipped with a nozzle of an unusually large diameter: at least 200 µm. We present evidence of the sorting efficiency and sorted cell viability, as well as of the senescent nature of the sorted cells, confirmed by the detection of other senescence markers, including the expression of the CKI p21 and the presence of 53BP1 DNA damage foci. Our protocol makes it possible, for the first time, to sort senescent cells from contaminating proliferating cells and, at the same time, to sort subpopulations of senescent cells featuring senescent markers to different extents. Graphical abstract.

Chemical constituents with senolytic activity from the stems of Limacia scandens.

While screening senotherapeutics from natural products, seven undescribed chemicals, two syringylglycerol derivatives, two cyclopeptides, one tigliane analogue, and two chromone derivatives, as well as six known compounds were isolated from the stems of Limacia scandens. The structures of compounds were elucidated through spectroscopic data analysis, including 1D and 2D NMR, HRESIMS, and CD data. All compounds were tested in replicative senescent human dermal fibroblasts (HDFs) to determine their potential as senotherapeutic agents to specifically target senescent cells. One tigliane and two chromones derivatives showed senolytic activity, indicating that senescent cells were selectively removed. Especially, 2-{2-[(3'-O-ß-d-glucopyranosyl)phenyl]ethyl}chromone is expected to be a potential senotherapeutics by inducing HDF death, inhibiting the activity of senescence-associated ß-galactosidase (SA-ß-gal) and expressing senescence-associated secretory phenotype (SASP) factors.

11-Cl-BBQ, a select modulator of AhR-regulated transcription, suppresses lung cancer cell growth via activation of p53 and p27Kip1.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and functions as a tumour suppressor in different cancer models. In the present study, we report detailed characterization of 11-chloro-7H-benzimidazo[2,1-a]benzo[de]iso-quinolin-7-one (11-Cl-BBQ) as a select modulator of AhR-regulated transcription (SMAhRT) with anti-cancer actions. Treatment of lung cancer cells with 11-Cl-BBQ induced potent and sustained AhR-dependent anti-proliferative effects by promoting G1 phase cell cycle arrest. Investigation of 11-Cl-BBQ-induced transcription in H460 cells with or without the AhR expression by RNA-sequencing revealed activation of p53 signalling. In addition, 11-Cl-BBQ suppressed multiple pathways involved in DNA replication and increased expression of cyclin-dependent kinase inhibitors, including p27Kip1 , in an AhR-dependent manner. CRISPR/Cas9 knockout of individual genes revealed the requirement for both p53 and p27Kip1 for the AhR-mediated anti-proliferative effects. Our results identify 11-Cl-BBQ as a potential lung cancer therapeutic, highlight the feasibility of targeting AhR and provide important mechanistic insights into AhR-mediated-anticancer actions.

Whole-mount Senescence-Associated Beta-Galactosidase (SA-ß-GAL) Activity Detection Protocol for Adult Zebrafish.

Senescence-associated beta-galactosidase (SA-ß-GAL) is an enzyme that accumulates in the lysosomes of senescent cells, where it hydrolyses ß-galactosides. With p16, it represents a well-recognized biomarker used to assess senescence both in vivo and in cell culture. The use of a chromogenic substrate, such as 5-bromo-4-chloro-3-indoyl-ß-d-galactopyranoside (X-Gal), allows the detection of SA-ß-GAL activity at pH 6.0 by the release of a visible blue product. Senescence occurs during aging and is part of the aging process itself. We have shown that prematurely aged zebrafish accumulate senescent cells detectable by SA-ß-GAL staining in different tissues, including testis and gut. Here, we report a detailed protocol to perform an SA-ß-GAL assay to detect senescent cell accumulation across the entire adult zebrafish organism ( Danio rerio ). We also identify previously unreported organs that show increased cell senescence in telomerase mutants, including the liver and the spinal cord.

Structural Characterization of Gracilariopsis lemaneiformis Polysaccharide and Its Property in Delaying Cellular Senescence.

The sulfated polysaccharide was isolated from the purified G. lemaneiformis polysaccharide (PGP), and its property in delaying H2O2-induced 2BS cellular senescence was studied. The results showed that PGP was a linear polysaccharide containing alternating α-(1 → 3)- and ß-(1 → 4)-galactopyranose units. Most of the sulfate groups are at C6 of the -(1 → 4)-α-D-Galp, and a small part of them are at C3 and C6. PGP pretreatment could decrease SA-ß-gal-positive cells and prevent the formation of senescence-associated heterochromatic foci (SAHF) induced by H2O2 in a dose-dependent manner. It is speculated that PGP may delay aging by downregulating the expression of p21 and p53 genes. The finding provides new insights into the beneficial role of G. lemaneiformis polysaccharide (GP) on retarding senescence process.