Senescence-associated Β-galactosidase Staining Research Articles

EZH2-H3K27me3-mediated silencing of mir-139-5p inhibits cellular senescence in hepatocellular carcinoma by activating TOP2A

BackgroundEpigenetic alterations play an important role in hepatocellular carcinoma (HCC) development. Enhancer of zeste homolog 2 (EZH2) is a well-known epigenetic modifier that functions as an oncogene in tumors by promoting the H3K27me3-mediated transcriptional repression of tumor suppressor genes. “Senescent cells” has been proposed as a possible core component of the hallmarks of cancer conceptualization. Induction of cell senescence and targeted elimination of these senescent tumor cells are new strategies for tumor therapy. However, the role of EZH2 in regulating cellular senescence remains poorly understood.MethodsBioinformatics analyses suggested that EZH2 and DNA topoisomerase II alpha (TOP2A) are coexpressed in tumors, including HCC. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analyses and gene set enrichment analyses (GSEA) suggests a correlation of EZH2 and TOP2A expression with cellular senescence in HCC. MicroRNA (miRNA) inhibitor and mimics, siRNA, PLKO-shRNA, and plenti6.3-miR-139 were used to upregulate or downregulate the expression of target genes. CCK8, EdU, clone formation, and senescence-associated β-galactosidase (SA-β-gal) staining assays were performed to assess cell proliferation and cellular senescence phenotypes. Dual-luciferase reporter and chromatin immunoprecipitation assays were performed to investigate the targeted binding and inhibition of TOP2A 3′ untranslated region (UTR) by miR-139-5p and the DNA enrichment of miR139-5p by EZH2 and H3K27me3. BALB/c nude mice were used to establish a xenograft tumor model and verify the phenotypes upon EZH2 and TOP2A silencing and miR-139 overexpression in vivo. In addition, tissue microarrays were used to analyze the expression patterns and correlations among EZH2, TOP2A, and miR-139-5p expression in HCC.ResultsBioinformatics analysis revealed that EZH2 and TOP2A are coexpressed in HCC. In vitro gain- and loss-of-function experiments showed that inhibition of EZH2 and TOP2A induces cellular senescence and inhibits proliferation of HCC cells. In vivo tumorigenesis assays indicated that EZH2 and TOP2A knockdown inhibits tumorigenesis by inducing cellular senescence. Mechanistically, EZH2 promotes TOP2A expression by regulating the H3K27me3-mediated epigenetic silencing of miR-139-5p. TOP2A is a direct target of miR-139-5p, and inhibition of miR-139-5p can reverse the promotion by EZH2 of TOP2A expression. The overexpression of miR-139-5p induces cellular senescence and inhibits proliferation of HCC cells both in vitro and in vivo. Clinically, expression of EZH2 and TOP2A are higher in HCC tissues than in normal tissues, and this high coexpression indicates a worse outcome of patients with HCC. Moreover, expression of EZH2 and TOP2A is significantly correlated with tumor differentiation grade, tumor invasion, and TNM stage in HCC. miR-139-5p expression is lower in HCC tumors than in normal tissues and is correlated with better prognosis of HCC patients.ConclusionsOur study revealed the role of the EZH2/miR-139-5p/TOP2A axis in regulating cellular senescence and cell proliferation in HCC, enriching the molecular mechanisms of EZH2-mediated epigenetic regulation in HCC. Therefore, our results provide insight into the therapeutic potential of targeting EZH2 to induce cellular senescence and then destroy senescent cells for HCC.Graphic

Phytic acid improves osteogenesis and inhibits the senescence of human bone marrow mesenchymal stem cells under high-glucose conditions via the ERK pathway

Hyperglycaemia causes impairment of osteogenic differentiation and accelerates stem cell senescence, resulting in weakened osteogenesis and disordered bone metabolism. Phytic acid (PA) is an antioxidant that is reportedly beneficial to bone homeostasis. The present study aims to clarify how PA affects the osteogenic capacity and cellular senescence of bone marrow mesenchymal stem cells (BMSCs) exposed to high-glucose environments, as well as the potential molecular mechanisms. Our results indicate that osteogenic differentiation in BMSCs cultivated in high-glucose conditions is enhanced by PA, as evidenced by increased alkaline phosphatase activity and staining, Alizarin Red S staining, osteogenic marker in in vitro studies, and increased osteogenesis in animal experiments. PA also prevented high-glucose-induced senescence of BMSCs, as evidenced by the repression of reactive oxygen species production, senescence-associated β-galactosidase staining, and P21 and P53 expression. Furthermore, it was found that PA rescued the high-glucose-inhibited expression of phosphorylated extracellular regulated protein kinases (p-ERK). The inhibition of ERK pathway by the specific inhibitor PD98059 blocked the PA-enhanced osteogenesis of BMSCs and promoted cell senescence. Our results revealed that PA enhances osteogenic differentiation and inhibits BMSC senescence in a high-glucose environment. In addition, the activation of the ERK pathway seems to mediate the beneficial effects of PA. The findings provide novel insights that could facilitate bone regeneration in patients with diabetes.

IL-37 counteracts inflammatory injury in the temporomandibular joint via the intracellular pathway.

Background: The temporomandibular joint is often afflicted by osteoarthritis (TMJOA), causing pain and dysfunction, which is particularly prevalent in the elderly population. IL-37 is effective in avoiding excessive inflammatory damage to the organism. This article investigates the role and mechanism of intracellular IL-37 in TMJOA. Methods: Enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, Western blotting, Senescence-associated β-galactosidase staining, immunofluorescence, and lentivirus were performed to elucidate the underlying mechanism. Results: The results confirmed that IL-37 in synovial cells decreased with aging. Inflammatory stimulus elevated intracellular IL-37 in synoviocytes, while lentiviral knockdown of IL-37 resulted in more inflammatory factor production. Dynamic changes of IL-37 were observed in the nucleus and supernatant. In addition, Caspease-1 inhibitor hindered intracellular IL-37 maturation, and Smad3 inhibitor caused the loss of nuclear translocation of mature IL-37. Transfection of synovial cells with IL-37-expressing lentivirus resulted in relief not only of synovitis but also of the cartilage damage and inflammation caused by synovitis. Conclusion: This study provides new insights into the intracellular anti-inflammatory mechanism of IL-37. It also confirms that IL-37 decreases with cellular senescence and that increasing intracellular IL-37 can effectively treat synovitis and synovitis-induced inflammatory damage to cartilage.

Senescent tumor cells in colorectal cancer are characterized by elevated enzymatic activity of complexes 1 and 2 in oxidative phosphorylation.

Cellular senescence is defined as an irreversible cell cycle arrest caused by various internal and external insults. While the metabolic dysfunction of senescent cells in normal tissue is relatively well-established, there is a lack of information regarding the metabolic features of senescent tumor cells. Publicly available single-cell RNA-sequencing data from the GSE166555 and GSE178341 datasets were utilized to investigate the metabolic features of senescent tumor cells. To validate the single-cell RNA-sequencing data, we performed senescence-associated β-galactosidase (SA-β-Gal) staining to identify senescent tumor cells in fresh frozen colorectal cancer tissue. We also evaluated nicotinamide adenine dinucleotide dehydrogenase-tetrazolium reductase (NADH-TR) and succinate dehydrogenase (SDH) activity using enzyme histochemical methods and compared the staining with SA-β-Gal staining. MTT assay was performed to reveal the complex 1 activity of the respiratory chain in in-vitro senescence model. Single-cell RNA-sequencing data revealed an upregulation in the activity of complexes 1 and 2 in oxidative phosphorylation, despite overall mitochondrial dysfunction in senescent tumor cells. Both SA-β-Gal and enzyme histochemical staining using fresh frozen colorectal cancer tissues indicated a high correlation between SA-β-Gal positivity and NADH-TR/SDH staining positivity. MTT assay showed that senescent colorectal cancer cells exhibit higher absorbance in 600 nm wavelength. Senescent tumor cells exhibit distinct metabolic features, characterized by upregulation of complexes 1 and 2 in the oxidative phosphorylation pathway. NADH-TR and SDH staining represent efficient methods for detecting senescent tumor cells in colorectal cancer.

Bleomycin-treated myoblasts undergo p21-associated cellular senescence and have severely impaired differentiation.

As we age, the ability to regenerate and repair skeletal muscle damage declines, partially due to increasing dysfunction of muscle resident stem cells-satellite cells (SC). Recent evidence implicates cellular senescence, which is the irreversible arrest of proliferation, as a potentiator of SC impairment during aging. However, little is known about the role of senescence in SC, and there is a large discrepancy in senescence classification within skeletal muscle. The purpose of this study was to develop a model of senescence in skeletal muscle myoblasts and identify how common senescence-associated biomarkers respond. Low-passage C2C12 myoblasts were treated with bleomycin or vehicle and then evaluated for cytological and molecular senescence markers, proliferation status, cell cycle kinetics, and differentiation potential. Bleomycin treatment caused double-stranded DNA breaks, which upregulated p21 mRNA and protein, potentially through NF-κB and senescence-associated super enhancer (SASE) signaling (p < 0.01). Consequently, cell proliferation was abruptly halted due to G2/M-phase arrest (p < 0.01). Bleomycin-treated myoblasts displayed greater senescence-associated β-galactosidase staining (p < 0.01), which increased over several days. These myoblasts remained senescent following 6days of differentiation and had significant impairments in myotube formation (p < 0.01). Furthermore, our results show that senescence can be maintained despite the lack of p16 gene expression in C2C12 myoblasts. In conclusion, bleomycin treatment provides a valid model of damage-induced senescence that was associated with elevated p21, reduced myoblast proliferation, and aberrant cell cycle kinetics, while confirming that a multi-marker approach is needed for the accurate classification of senescence within skeletal muscle.

Mesenchymal stem cells derived from the fibrotic tissue of atrophic nonunion or the bone marrow of iliac crest: A donor-matched comparison

PurposeAtrophic nonunion is one of the most difficult complications of fracture. The cellular factors that contribute to atrophic nonunion are poorly understood, and mesenchymal stem cells (MSCs) are recognized as the key contributor to bone formation. This study aimed to characterize the MSCs isolated from the fibrotic tissue of atrophic nonunion (AN-MSCs) from the perspective of proliferation, differentiation potential, senescence, and paracrine function. MethodsHuman atrophic fibrotic tissue was obtained from four donors aged 29–37 for isolating AN-MSCs, and donor-matched bone marrow acquired from the iliac crest for isolating MSCs (IC-MSCs) as control. The AN-MSCs or IC-MSCs in passage 3 were applied for the following evaluations. The surface markers expressed on the two cells were evaluated using flow cytometry. The proliferation of the two cells for up to 11 days was comparatively investigated. After osteogenic, chondrogenic, or adipogenic induction, multi-lineage differentiation of AN-MSCs or IC-MSCs was comparatively evaluated using lineage-specific stains and lineage-specific gene expression. Enzyme-linked immunosorbent assay (ELISA) assessment was applied to evaluate the paracrine function of AN-MSCs or IC-MSCs. Cellular senescence of AN-MSCs or IC-MSCs was evaluated using senescence-associated β-galactosidase (SA-β-gal) staining. ResultsAN-MSCs or IC-MSCs from the four donors showed morphologic and immunophenotypic characteristics of MSCs, with the expression of MSCs markers and negative expression of hematopoietic markers. In general, AN-MSCs showed similar proliferation and adipogenic capacity with IC-MSCs. In contrast, IC-MSCs showed significantly higher osteogenic and chondrogenic capacity compared to AN-MSCs. Moreover, the culture medium of IC-MSCs contains significantly higher levels of VEGF, TGF-β1, PDGF-BB, and IGF-1 than the culture medium of AN-MSCs. Lastly, the AN-MSCs are more prone to cellular senescence than the IC-MSCs. ConclusionsIn-vitro, AN-MSCs were similar to IC-MSCs in proliferation and adipogenic capacity, but inferior to IC-MSCs in osteogenic and chondrogenic capacity, paracrine function, and anti-senescence.

Multilineage differentiation potential in the infant adipose- and umbilical cord-derived mesenchymal stem cells.

This study aims to compare the biological properties of infant adipose-derived mesenchymal stem cells (infant ADSCs) from excised polydactyly fat tissue and umbilical cord-derived mesenchymal stem cells (UCSCs) in terms of proliferation and differentiation capabilities. The proliferation of infant ADSCs and UCSCs was analyzed by determining the fold changes of cell numbers and doubling time periods. The state of senescence and replicative stress was compared by analyzing the expression of age-related genes, senescence-associated β-galactosidase (SA-β-gal) staining, and phosphorylated histone variant H2AX (γH2AX) immunofluorescence staining. The expression levels of superoxide dismutase ( SODs ) and genes related to multilineage differentiation were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Differentiation levels were determined using histochemical staining, immunohistochemical staining, and immunofluorescence staining. Infant ADSCs exhibited higher proliferation rates and expression levels of SOD1 , SOD2 , and SOD3 at passages 3-5 compared with UCSCs. Senescence related genes ( p16 , p21 , and p53 ), SA-β-gal staining, and replicative stress analysis were reduced in infant ADSCs. The expression levels of chondrogenic genes ( COL2 and COL10 ), osteogenic genes ( RUNX2 and ALP ), adipogenic genes ( LPL ), and hepatogenic genes ( ALB and TAT ) in infant ADSC-differentiated cells were significantly higher than those in UCSCs. Histochemical and immunofluorescence staining confirmed these results. Only the expression levels of tenogenic genes ( MMP3 , DCN , and COL3 ) in infant ADSC-differentiated cells were lower than those in UCSCs. Infant ADSCs exhibit higher proliferation rates, reduced cellular senescence and replicative stress, better antioxidative activity, and higher differentiation potential toward chondrogenic, osteogenic, adipogenic and hepatogenic lineages than UCSCs.

P12.01.A TUMOR CELL SENESCENCE AND IMMUNE MICROENVIRONMENT IN GLIOBLASTOMAS

Abstract BACKGROUND Cellular senescence (CS) is a state of irreversible cell cycle arrest, and the expression of p16INK4a in cells is one of the reliable markers for CS. However, senescent cells are metabolically active with the senescence-associated secretory phenotype (SASP), which can influence the tissue microenvironment by paracrine signaling to the adjacent tumor, non-tumor, and immune cells. MATERIAL AND METHODS In the present study, we evaluated p16INK4a expression in glioblastoma and investigated its association with CS and SASPs. We analyzed the expression of p16INK4a in 73 glioblastomas by immunostaining. To examine the association of p16INK4a expression and CS, we performed senescence-associated β-galactosidase (SA-β-Gal) staining, a standard marker of CS, using glioblastoma cell lines and fresh frozen tumor tissues. For SASPs analysis, RNA sequencing with Gene Set Enrichment Analysis (GSEA) was performed. RESULTS Among 73 glioblastomas, twenty-eight cases (38.4%) revealed diffuse strong p16INK4a expression in tumor cells. The glioblastoma with diffuse p16INK4a expression (GMDP) patients were younger (52.4 vs. 59.2 years) and showed prolonged overall survival (median: 25.5 vs. 12.3 months) compared to those harboring negative expression. In vitro analysis, p16INK4a over-expressed glioblastoma cell line showed increased expression of SA-β-Gal which indicates CS. In addition, fresh frozen tissues from GMDP also revealed SA-β-Gal positivity. RNA sequencing analysis revealed that splicing or protein biosynthetic genes were enriched in GMDP, and GSEA showed significant enrichment of SASP genes (false discovery rate &amp;lt;0.05), especially chemokines associated with monocytes and macrophages. CONCLUSION Our data suggest that increased expression of p16INK4a in glioblastoma is associated with tumor cell senescence, and SASPs from senescent tumor cells could be one of the crucial modulators in the tumor immune microenvironment.

TRAF3IP2 drives mesenchymal stem cell senescence via regulation of NAMPT-mediated NAD biosynthesis

BackgroundThe cellular senescence of mesenchymal stem cells (MSCs) limits their application in regenerative medicine. This study aimed to clarify the role of TNF receptor-associated factor 3 interacting protein 2 (TRAF3IP2), a pro-inflammatory cytoplasmic adaptor protein, in regulating MSC senescence and to explore the potential mechanisms. Methods: MSC senescence was determined by senescence-associated β-galactosidase (SA-β-gal) staining. The expression of TRAF3IP2 and senescence-related proteins was detected by Western blotting. The nicotinamide adenine dinucleotide (NAD+) level and nicotinamide phosphoribosyl transferase (NAMPT) expression in MSCs was measured. Results: Compared with that in MSCs isolated from young donors (YMSCs), the expression of TRAF3IP2 was greatly increased in MSCs derived from aged donors (AMSCs). Overexpression of TRAF3IP2 accelerated YMSC senescence whereas downregulation significantly rescued cellular senescence. The protein level of NAMPT and the level of NAD+ were significantly decreased in AMSCs compared with YMSCs. Mechanistically, TRAF3IP2 induced MSC senescence via downregulation of NAMPT expression and NAD + level by inhibiting the AMPK signaling pathway. These effects were partially reversed by treatment with an AMPK or NAMPT activator. Conclusion: We revealed that TRAF3IP2 accelerated MSC senescence via downregulation of NAMPT-mediated NAD biosynthesis by mediation of the AMPK pathway, highlighting a novel means to rejuvenate senescent MSCs.

Quercetin ameliorates oxidative stress-induced senescence in rat nucleus pulposus-derived mesenchymal stem cells via the miR-34a-5p/SIRT1 axis.

Intervertebral disc degeneration (IDD) is a main contributor to low back pain. Oxidative stress, which is highly associated with the progression of IDD, increases senescence of nucleus pulposus-derived mesenchymal stem cells (NPMSCs) and weakens the differentiation ability of NPMSCs in degenerated intervertebral discs (IVDs). Quercetin (Que) has been demonstrated to reduce oxidative stress in diverse degenerative diseases. To investigate the role of Que in oxidative stress-induced NPMSC damage and to elucidate the underlying mechanism. In vitro, NPMSCs were isolated from rat tails. Senescence-associated β-galactosidase (SA-β-Gal) staining, cell cycle, reactive oxygen species (ROS), real-time quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, and western blot analyses were used to evaluated the protective effects of Que. Meanwhile the relationship between miR-34a-5p and Sirtuins 1 (SIRT1) was evaluated by dual-luciferase reporter assay. To explore whether Que modulates tert-butyl hydroperoxide (TBHP)-induced senescence of NPMSCs via the miR-34a-5p/SIRT1 pathway, we used adenovirus vectors to overexpress and downregulate the expression of miR-34a-5p and used SIRT1 siRNA to knockdown SIRT1 expression. In vivo, a puncture-induced rat IDD model was constructed, and X rays and histological analysis were used to assess whether Que could alleviate IDD in vivo. We found that TBHP can cause NPMSCs senescence changes, such as reduced cell proliferation ability, increased SA-β-Gal activity, cell cycle arrest, the accumulation of ROS, and increased expression of senescence-related proteins. While abovementioned senescence indicators were significantly alleviated by Que treatment. Que decreased the expression levels of senescence-related proteins (p16, p21, and p53) and senescence-associated secreted phenotype (SASP), including IL-1β, IL-6, and MMP-13, and it increased the expression of SIRT1. In addition, the protective effects of Que on cell senescence were partially reversed by miR-34a-5p overexpression and SIRT1 knockdown. In vivo, X-ray, and histological analyses indicated that Que alleviated IDD in a puncture-induced rat model. In summary, the present study provides evidence that Que reduces oxidative stress-induced senescence of NPMSCs via the miR-34a/SIRT1 signaling pathway, suggesting that Que may be a potential agent for the treatment of IDD.

Astrocyte senescence-like response related to peripheral nerve injury-induced neuropathic pain

BackgroundPeripheral nerve damage causes neuroinflammation, which plays a critical role in establishing and maintaining neuropathic pain (NeP). The mechanisms contributing to neuroinflammation remain poorly elucidated, and pharmacological strategies for NeP are limited. Thus, in this study, we planned to explore the possible link between astrocyte senescence and NeP disorders following chronic sciatic nerve injury.MethodsAn NeP animal model was established by inducing chronic constrictive injury (CCI) to the sciatic nerve in adult rats. A senolytic drug combination of dasatinib and quercetin was gavaged daily from the first postoperative day until the end of the study. Paw mechanical withdrawal threshold (PMWT) and paw thermal withdrawal latency (PTWL) were evaluated to assess behaviors in response to pain in the experimental rats. Senescence-associated β-galactosidase staining, western blot analysis, and immunofluorescence were applied to examine the levels of proinflammatory factors and severity of the senescence-like response in the spinal cord. Lipopolysaccharide (LPS) was administered to induce senescence of spinal astrocytes in primary cultures in vitro, to explore the potential impacts of senescence on the secretion of proinflammatory factors. Furthermore, single-cell RNA sequencing (scRNA-seq) was conducted to identify senescence-related molecular responses in spinal astrocytes under neuropathic pain.ResultsFollowing sciatic nerve CCI, rats exhibited reduced PMWT and PTWL, increased levels of spinal proinflammatory factors, and an enhanced degree of senescence in spinal astrocytes. Treatment with dasatinib and quercetin effectively attenuated spinal neuroinflammation and mitigated the hypersensitivities of the rats subjected to sciatic nerve CCI. Mechanistically, the dasatinib-quercetin combination reversed senescence in LPS-stimulated primary cultured astrocytes and decreased the levels of proinflammatory factors. The scRNA-seq data revealed four potential senescence-related genes in the spinal astrocyte population, and the expression of clusterin (CLU) protein was validated via in vitro experiments.ConclusionThe findings indicate the potential role of astrocyte senescence in neuroinflammation following peripheral nerve injury, and suggest that targeting CLU activation in astrocytes might provide a novel therapeutic strategy to treat NeP.Graphical abstract

Abstract P1039: Soluble Extracellular Matrix Protein Induces Senescence In Aortic Valve Interstitial Cells To Up-regulate Valvular Osteogenic Activity

Background: Calcific aortic valve disease (CAVD) is the most prevalent heart valve disease in the elderly, and elevated osteogenic activity of aortic valve interstitial cells (AVICs) play a critical role in CAVD progression to severe aortic valve stenosis. While soluble extracellular matrix (ECM) proteins, including biglycan and matrilin 2, have been found to up-regulate the osteogenic activity in human AVICs, the mechanism underlying the cellular and molecular mechanisms remain unclear. As greater densities of senescent AVICs were observed in human calcified aortic valves, we tested the hypothesis that soluble ECM protein induces senescence in AVICs to up-regulate valvular osteogenic activity. Methods and Results: Human AVICs were isolated from normal and CAVD aortic valves. Levels of P16 and P21 were assessed by immunoblotting, and senescent changes is identified using senescence-associated β-galactosidase (SA-β-gal) staining. AVICs from diseased valves displayed higher levels of P16 and P21, greater SA-β-gal staining and active calcium deposition. Soluble matrilin 2 (2.0 μg/ml) up-regulated the expression of P16 and P21, and enhanced SA-β-gal staining in normal AVICs, leading to greater calcium deposition. Further, knockdown of P16 markedly reduced calcium deposition in AVICs exposed to soluble matrilin 2. The impact of soluble matrlin 2 is associated with activation of the M6PR-ERK1/2 pathway. Blocking or knockdown of M6PR (M6P, 10 mM or siRNA, 1.0 nM), or inhibition of ERK1/2 (PD98059, 25 μM) attenuated the effect of soluble matrilin 2 on AVIC senescence and resultant up-regulation of osteogenic activity. Conclusion: AVICs from aortic valves of patients with CAVD have higher levels of senescence activity. Soluble matrilin 2, via the M6PR-ERK1/2 pathway, induces AVIC senescence to promote the osteogenic activity. Targeting the D-M6PR-ERK1/2 pathway may suppress AVIC senescence and attenuate aortic valve calcification associated with CAVD progression.

Preparation and evaluation of ovalbumin-fucoidan nanoparticles for nicotinamide mononucleotide encapsulation with enhanced stability and anti-aging activity.

Nicotinamide mononucleotide (NMN) has been recognized as a promising bio-active compound in relieving aging-related mitochondrial dysfunction. Self-assembled nanoparticles were prepared based on interaction between ovalbumin (OVA) and fucoidan to improve the stability and bio-accessibility of NMN. The OVA-fucoidan nanoparticles (OFNPs) displayed outstanding thermal stability and entrapment ability of NMN. The reactive oxygen species (ROS) analysis and senescence-associated β-galactosidase (SA-β-gal) staining characterization indicated that NMN encapsulated by OFNPs could effectively alleviate the cellular senescence of d-galactose-induced senescent cells. In vivo Caenorhabitis elegans experiment demonstrated that NMN-loaded OFNPs caused less accumulation of lipofuscin and protected NMN from thermal damage. Compared with free NMN, the NMN-loaded OFNPs prolonged lifespan from 28 to 31days, increased 26% reproductive ability, and improved 12% body length of Caenorhabitis elegans. The results indicated that the use of nanocarriers could be a good strategy to improve anti-oxidative stress and anti-aging ability of NMN.

Epigenetic modulation of Drp1-mediated mitochondrial fission by inhibition of S-adenosylhomocysteine hydrolase promotes vascular senescence and atherosclerosis

AimsVascular senescence, which is closely related to epigenetic regulation, is an early pathological condition in cardiovascular diseases including atherosclerosis. Inhibition of S-adenosylhomocysteine hydrolase (SAHH) and the consequent increase of S-adenosylhomocysteine (SAH), a potent inhibitor of DNA methyltransferase, has been associated with an elevated risk of cardiovascular diseases. This study aimed to investigate whether the inhibition of SAHH accelerates vascular senescence and the development of atherosclerosis. Methods and resultsThe case-control study related to vascular aging showed that increased levels of plasma SAH were positively associated with the risk of vascular aging, with an odds ratio (OR) of 3.90 (95% CI, 1.17–13.02). Elevated pulse wave velocity, impaired endothelium-dependent relaxation response, and increased senescence-associated β-galactosidase staining were observed in the artery of SAHH+/- mice at 32 weeks of age. Additionally, elevated expression of p16, p21, and p53, fission morphology of mitochondria, and over-upregulated expression of Drp1 were observed in vascular endothelial cells with SAHH inhibition in vitro and in vivo. Further downregulation of Drp1 using siRNA or its specific inhibitor, mdivi-1, restored the abnormal mitochondrial morphology and rescued the phenotypes of vascular senescence. Furthermore, inhibition of SAHH in APOE−/− mice promoted vascular senescence and atherosclerosis progression, which was attenuated by mdivi-1 treatment. Mechanistically, hypomethylation over the promoter region of DRP1 and downregulation of DNMT1 were demonstrated with SAHH inhibition in HUVECs. ConclusionsSAHH inhibition epigenetically upregulates Drp1 expression through repressing DNA methylation in endothelial cells, leading to vascular senescence and atherosclerosis. These results identify SAHH or SAH as a potential therapeutic target for vascular senescence and cardiovascular diseases.

Leukoreduced PRP enhanced proliferation and ECM production yet inhibited senescence, inflammation, and multi-differentiation potential of AFSCs by downregulating HMGB1

BackgroundThis study assessed the role and potential mechanism of platelet-rich plasma (PRP) in the progression of intervertebral disk degeneration (IVDD).MethodsAnnulus fibrosus (AF)-derived stem cells (AFSCs) from New Zealand white rabbits received the transfection with high mobility group box 1 (HMGB1) plasmids and the subsequent treatment with bleomycin, 10% leukoreduced PRP or leukoconcentrated PRP. Dying cells were indicated by immunocytochemistry analysis for senescence-associated β-galactosidase (SA-β-gal) staining. The proliferation of these cells was evaluated based on the population doubling time (PDT). The expressions of HMGB1, pro-aging and anti-aging molecules, extracellular matrix (ECM)-related catabolic/anabolic factors, and inflammatory genes at the molecular or transcriptional levels were quantified via Western blot or reverse transcription-quantitative PCR (RT-qPCR). Besides, the adipocytes, osteocytes, and chondrocytes were separately dyed by Oil Red O, Alizarin Red S, and Safranin O staining.ResultsBleomycin enhanced the senescent morphological changes and increased the PDT and the expressions of SA-β-gal, pro-aging molecules, ECM-related catabolic factors, inflammatory genes, and HMGB1 while suppressing the expressions of anti-aging and anabolic molecules. Leukoreduced PRP reversed the effects of bleomycin and inhibited the differentiation of AFSCs into adipocytes, osteocytes, and chondrocytes. Besides, HMGB1 overexpression offset the roles of leukoreduced PRP in AFSCs.ConclusionLeukoreduced PRP promotes cell proliferation and ECM production of AFSCs, while inhibiting their senescence, inflammation, and multi-differentiation potentials via downregulating HMGB1 expression.

Senescence-related gene c-Myc affects bladder cancer cell senescence by interacting with HSP90B1 to regulate cisplatin sensitivity.

Patients with advanced bladder cancer gradually become less sensitive to chemotherapeutic agents, leading to tumor recurrence. Initiating the senescence program in solid tumors may be an important means of improving short-term drug sensitivity. The important role of c-Myc in bladder cancer cell senescence was determined using bioinformatics methods. The response to cisplatin chemotherapy in bladder cancer sample was analyzed according to the Genomics of Drug Sensitivity in Cancer database. Cell Counting Kit-8 assay, clone formation assay, and senescence-associated β-galactosidase staining were used to assess bladder cancer cell growth, senescence, and sensitivity to cisplatin, respectively. Western blot and immunoprecipitation were performed to understand the regulation of p21 by c-Myc/HSP90B1. Bioinformatic analysis showed that c-Myc, a cellular senescence gene, was significantly associated with bladder cancer prognosis and sensitivity to cisplatin chemotherapy. c-Myc and HSP90B1 expression were highly correlated in bladder cancer. Reducing the level of c-Myc significantly inhibited bladder cancer cell proliferation, promoted cellular senescence, and enhanced cisplatin chemosensitivity. Immunoprecipitation assays confirmed that HSP90B1 interacted with c-Myc. Western blot analysis showed that reducing the level of HSP90B1 could redeem the p21 overexpression caused by c-Myc overexpression. Further studies showed that reducing HSP90B1 expression could alleviate the rapid growth and accelerate cellular senescence of bladder cancer cells caused by c-Myc overexpression, and that reducing HSP90B1 levels could also improve cisplatin sensitivity in bladder cancer cells. HSP90B1/c-Myc interaction regulates the p21 signaling pathway, which affects cisplatin chemosensitivity by modulating bladder cancer cell senescence.

Treatment of porcine ovarian follicles with tert-butyl hydroperoxide as an ovarian senescence model in vitro.

Ovarian aging is the main reason of female reproductive problems. Excessive oxidative stress can induce ovarian senescence and follicular atresia, thereby reducing the reproductive performance. Follicles were divided into five groups for in vitro culture based on the duration of stimulation with tert-butyl hydroperoxide (t-BHP)-control group and groups 1 h, 2 h, 6 h, and 12 h. The results revealed that the ratio of progesterone (P4) to estradiol (E2) was increased after 24 and 36 h of follicle culture, shifting follicles toward atresia (P < 0.05). Stimulated by 200 μM t-BHP, follicles showed progressive aging phenotype. Senescence-associated β-galactosidase staining (SA-β-Gal) showed a significant increase in the number of positive cells (P < 0.05). Reactive oxygen species were also significantly upregulated (P < 0.05). t-BHP treatment for 6 h induced significant increases in Caspase 3, P53, and Foxo1 mRNA and protein levels (P < 0.05) and significant decreases in SOD mRNA and protein levels (P < 0.05). Transcriptome sequencing analysis of the follicles showed that the aged and treatment groups were clustered together in hierarchical clustering. Correlation analysis indicated significant changes at the transcriptome level in the treatment groups versus the control group. The common differentially expressed genes in the treatment groups were enriched in three growth-factor signaling pathways associated with cell proliferation and apoptosis (P53, mTOR, and MAPK). In conclusion, induction of follicular senescence by treatment with 200 μM t-BHP for 6 h is an effective in vitro model to simulate ovarian senescence in sows.

Protective impact of nicotinamide mononucleotide (NMN) and platelet-rich fibrin (PRF) on replicative and radiation-induced senescence of human osteoblasts

The aim of this study was to investigate the cellular changes induced by spontaneous/replicative senescence and radiation in human osteoblasts (OBs), and the impact of cultivation with nicotinamide mononucleotide (NMN) and platelet-rich fibrin (PRF) on apoptosis, senescence-associated β-galactosidase staining (SA β-gal), and senescence-related gene expression using RT2 Profiler PCR array.The results showed that replicative OB aging follows a different pattern from that of radiation-induced cellular senescence. SA β-gal intensity score showed a significant elevation after spontaneous replicative aging of OB (agiT1) 7 days following the start of the experiment, compared with their initial control condition (T0) (T0 = 2.1 ± 0.47; agiT1 = 9.60 ± 1.56; p = 0.001). Concurrent treatment by NMN and PRF showed a protective effect on OBs undergoing replicative senescence, and reduced SA β-gal staining significantly (agiT1 = 9.60 ± 1.56; agiT1+PRF = 3.19 ± 0.52; agiT1+NMN = 3.38 ± 0.36; p < 0.001).These results provide evidence for the potential clinical implications of systematic NMN administration and local PRF application to prevent age-related bone disturbances in elderly patients.

METTL3-mediated m6A modification of SIRT1 mRNA inhibits progression of endometriosis by cellular senescence enhancing

BackgroundEndometriosis (EMs), the ectopic planting of functional endometrium outside of the uterus, is a leading cause of infertility and pelvic pain. As a fundamental mRNA modification, N6-methyladenosine (m6A) participates in various pathological processes. However, the role of m6A RNA modification in endometriosis remains unclear. The present study explores METTL3-mediated m6A modification and the mechanisms involved in endometriosis.MethodsThe dominant m6A regulators in EMs were analysed using RT‒PCR. Candidate targets and possible mechanisms of METTL3 were assessed by m6A-mRNA epitranscriptomic microarray and RNA sequencing. A primary ESCs model was employed to verify the effect of METTL3 on m6A modification of SIRT1 mRNA, and the mechanism was elucidated by RT‒PCR, Western blotting, MeRIP, and RIP assays. CCK-8 viability assays, Transwell invasion assays, EdU proliferation assays, wound healing migration assays, and senescence-associated β-galactosidase staining were performed to illuminate the potential biological mechanism of METTL3 and SIRT1 in ESCs in vitro. An in vivo PgrCre/ + METTL3 −/− female homozygous mouse model and a nude mouse xenograft model were employed to further investigate the physiologic consequences of METTL3-mediated m6A alteration on EMs.ResultsOur data show that decreased METTL3 expression significantly downregulates m6A RNA methylation levels in ESCs. Silencing m6A modifications mediated by METTL3 accelerates ESCs viability, proliferation, migration, and invasion in vitro. The m6A reader protein YTHDF2 binds to m6A modifications to induce the degradation of SIRT1 mRNA. SIRT1/FOXO3a signalling pathway activation is subsequently inhibited, promoting the cellular senescence of ESCs and inhibiting the ectopic implantation of ESCs in vitro and in vivo.ConclusionsOur findings demonstrate that METTL3-mediated m6A methylation epigenetically regulates the ectopic implantation of ESCs, resulting in the progression of endometriosis. Our study establishes METTL3-YTHDF2-SIRT1/FOXO3a as a critical axis and potential mechanism in endometriosis.

GuiLu-ErXian Glue extract promotes mesenchymal stem cells (MSC)-Induced chondrogenesis via exosomes release and delays aging in the MSC senescence process

Ethnopharmacological relevanceThe treatment of osteoarthritis (OA) patients is a challenging problem. Mesenchymal stem cells (MSCs) are multipotent cells and play key roles in regenerative medicine for cartilage degeneration. GuiLu-ErXian Glue (GLEXG) is an herbal remedy widely used in traditional Chinese medicine to treat joint pain and disability in elderly OA patients. However, the mechanisms of how GLEXG affects MSCs-induced chondrogensis remains to be elucidated. Aim of the studyThe aim of this study was to investigate the effects of GLEXG on MSC-derived chondrogenesis, both in vitro and in vivo and its potential mechanisms. MethodsUsing human MSC (hMSCs) as in vitro model, the effects of HPLC-profiled GLEXG water extract on chondrogenic differentiation were investigated by 3D spheroid cultures under chondrogenesis-inducing medium (CIM) condition. The chondrogenesis process was evaluated by measuring the sphere sizes, chondrogenesis-related genes expression by reverse transcription real-time PCR that targeted type II/X collagens, SOX9, aggrecan, and protein expression by immunostaining. Anti-TGF-β1 neutralization antibody was used for mechanistic study. Mono-iodoacetate (MIA) induced OA joint was used to evaluate the effects of GLEXG on in vivo model. MSCs-derived exosomes were purified for proteomics study and senescence process was evaluated by cumulative population doublings and senescence-associated β-Galactosidase staining. ResultsThe results showed that GLEXG enhanced hMSCs chondrogenesis and upregulated RNA expression of type II/X collagen, SOX9 and aggrecan at 0.1 μg/mL, 0.3 μg/mL in vitro. In vivo, GLEXG at the dose of 0.3 μg intraarticular (i.a.) injection rescued the MIA-induced cartilage defect. Proteomics and ingenuity pathway analysis obtained from MSCs-released exosomes suggested that senescence pathway was less activated in GLEXG group than in vehicle group. Besides, GLEXG was able to increase cumulative population doubling and delayed hMSCs senescence process after four passages in cultures. Conclusionwe conclude that GLEXG promotes in vitro MSC-induced chondrogenesis possibly via exosomes release and delays aging in the MSC senescence process and that treatment with GLEXG (0.3 μg, i.a.) rescued cartilage defects in rat OA knee model.