Cellular Senescence Signaling Pathways Research Articles

Mechanism of ergosterol peroxide on MCF-7 breast cancer cells based on network pharmacology and in vitro experiments

This study investigated the effects of ergosterol peroxide(EP) on the proliferation and apoptosis of MCF-7 breast cancer cells, explored its possible mechanisms of action, and verified the effects and mechanisms by in vitro experiments. Network pharmaco-logy was used to screen the target proteins of EP and construct target networks and protein-protein interaction(PPI) networks to predict the potential target proteins and related pathways involved in EP anti-breast cancer effects. The MTT assay was performed to measure the inhibitory effect of EP on MCF-7 cell proliferation, and the colony formation assay was used to assess the cell cloning ability. Flow cytometry and laser confocal microscopy were employed to evaluate cell apoptosis, mitochondrial membrane potential and reactive oxygen species(ROS) levels. Western blot analysis was conducted to examine the expression levels of B-cell lymphoma 2(Bcl-2), Bcl-2-associated X protein(Bax), cytochrome C(Cyt C), caspase-7, cleaved caspase-7, phosphatidylinositol 3-kinase(PI3K), and se-rine/threonine kinase B(AKT) in MCF-7 cells treated with EP. The results of network pharmacology prediction yielded 173 common targets between EP and breast cancer; the results of Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis showed that EP treatment for breast cancer mainly affected the signaling pathways such as cancer pathway, PI3K-AKT signaling pathway, cellular senescence signaling pathway, and viral carcinogenesis pathway; and the MTT assay results showed that the viability of MCF-7 cells in the EP group was significantly lower than that in the control group, exhibiting a time-and concentration-dependent trend, and EP can inhibit colony formation of MCF-7 breast cancer cells. Treatment with 10, 20, and 40 μmol·L~(-1) EP for 24 h resulted in a significant increase in the total apoptosis rate of MCF-7 cells, a significant decrease in mitochondrial membrane potential, and a significant increase in ROS levels. In addition, treatment with EP led to an upregulation of Cyt C, Bax, and cleaved caspase-7 protein expression, and a downregulation of p-PI3K, p-AKT, and Bcl-2 protein expression in MCF-7 cells. Studies have shown that EP inhibits MCF-7 breast cancer cell proliferation and reduces colony formation by a mechanism that may be related to the PI3K-AKT pathway mediating the mitochondrial apoptotic pathway.

Oleuropein Supplementation Ameliorates Long-Course Diabetic Nephropathy and Diabetic Cardiomyopathy Induced by Advanced Stage of Type 2 Diabetes in db/db Mice.

Previous studies have reported the therapeutic effects of oleuropein (OP) consumption on the early stage of diabetic nephropathy and diabetic cardiomyopathy. However, the efficacy of OP on the long-course of these diabetes complications has not been investigated. Therefore, in this study, to investigate the relieving effects of OP intake on these diseases, and to explore the underlying mechanisms, db/db mice (17-week-old) were orally administrated with OP (200 mg/kg bodyweight) for 15 weeks. We found that OP reduced expansion of the glomerular mesangial matrix, renal inflammation, renal fibrosis, and renal apoptosis. Meanwhile, OP treatment exerted cardiac anti-fibrotic, anti-inflammatory, and anti-apoptosis effects. Notably, transcriptomic and bioinformatic analyses indicated 290 and 267 differentially expressed genes in the kidney and heart replying to OP treatment, respectively. For long-course diabetic nephropathy, OP supplementation significantly upregulated the cyclic guanosine monophosphate-dependent protein kinase (cGMP-PKG) signaling pathway. For long-course diabetic cardiomyopathy, p53 and cellular senescence signaling pathways were significantly downregulated in response to OP supplementation. Furthermore, OP treatment could significantly upregulate the transcriptional expression of the ATPase Na+/K+ transporting subunit alpha 3, which was enriched in the cGMP-PKG signaling pathway. In contrast, OP treatment could significantly downregulate the transcriptional expressions of cyclin-dependent kinase 1, G two S phase expressed protein 1, and cyclin B2, which were enriched in p53 and cellular senescence signal pathways; these genes were confirmed by qPCR validation. Overall, our findings demonstrate that OP ameliorated long-course diabetic nephropathy and cardiomyopathy in db/db mice and highlight the potential benefits of OP as a functional dietary supplement in diabetes complications treatment.

RNA-Seq and miRNA-Seq data from Epstein-Barr virus-infected tree shrews reveal a ceRNA network contributing to immune microenvironment regulation

ABSTRACT Epstein-Barr virus (EBV) infection in humans is ubiquitous and associated with various diseases. Remodeling of the immune microenvironment is the primary cause of EBV infection and pathogenesis; however, the underlying mechanism has not been fully elucidated. In this study, we used whole-transcriptome RNA-Seq to detect mRNAs, long non-coding RNAs (lncRNA), and microRNA (miRNA) profiles in the control group, 3 days, and 28 days after EBV infection, based on the tree shrew model that we reported previously. First, we estimated the proportion of 22 cell types in each sample using CIBERSORT software and identified 18 high-confidence DElncRNAs related to immune microenvironment regulation after EBV infection. Functional enrichment analysis of these differentially expressed lncRNAs primarily focused on the autophagy, endocytosis, and ferroptosis signalling pathways. Moreover, EBV infection affects miRNA expression patterns, and many miRNAs are silenced. Finally, three competing endogenous RNA regulatory networks were built using lncRNAs that significantly correlated with immune cell types, miRNAs that responded to EBV infection, and potentially targeted the mRNA of the miRNAs. Among them, MRPL42-AS-5 might act as an hsa-miR-296-5p “sponge” and compete with target mRNAs, thus increasing mRNA expression level, which could induce immune cell infiltration through the cellular senescence signalling pathway against EBV infection. Overall, we conducted a complete transcriptomic analysis of EBV infection in vivo for the first time and provided a novel perspective for further investigation of EBV-host interactions.

Bioinformatics-based discovery of intervertebral disc degeneration biomarkers and immune-inflammatory infiltrates.

Intervertebral disc degeneration (IVDD) is a common chronic disease in orthopedics, and its molecular mechanisms are still not well explained. This study's objective was to bioinformatics-based discovery of IVDD biomarkers and immune-inflammatory infiltrates. The IVDD illness gene collection was gathered from GeneCards, DisGeNet, and gene expression profiles were chosen from the extensive Gene Expression Omnibus database (GSE124272, GSE150408, and GSE153761). The STRING database was used to create a network of protein-protein interactions, while the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases were used for functional enrichment analysis. Using hub genes, the immune cell infiltration between IVDD patient samples and control tissues was examined. Finally, quantitative polymerase chain reaction and Western blot experiments were used to verify the expression of hub genes. A total of 27 differentially expressed hub genes were identified by bioinformatics. According to GO and KEGG analyses, hub genes were prominent in immunological responses, chemokine-mediated signaling pathways, and inflammatory responses, with the key signaling pathways engaged in cellular senescence, apoptosis, Th1 and Th2 cell differentiation, and Th17 cell differentiation. Immune cell infiltration research revealed that T cells, lymphocytes, B cells, and NK cells were decreased in IVDD patients while monocytes, neutrophils, and CD8 T cells were increased. The expression levels of the senescence hub genes SP1, VEGFA, IL-6, and the apoptosis key gene CASP3 were considerably greater in the IVDD model group than in the control group, according to in vitro validation. In conclusion, the cellular senescence signaling pathway, the apoptosis signaling pathway, and associated hub genes play significant roles in the development and progression of IVDD, this finding may help direct future research on the senescence signaling route in IVDD.

Biochemical Targets and Molecular Mechanism of Matrine against Aging.

The aim of this study is to explore the potential targets and molecular mechanism of matrine (MAT) against aging. Bioinformatic-based network pharmacology was used to investigate the aging-related targets and MAT-treated targets. A total of 193 potential genes of MAT against aging were obtained and then the top 10 key genes (cyclin D1, cyclin-dependent kinase 1, Cyclin A2, androgen receptor, Poly [ADP-ribose] polymerase-1 (PARP1), histone-lysine N-methyltransferase, albumin, mammalian target of rapamycin, histone deacetylase 2, and matrix metalloproteinase 9) were filtered by the molecular complex detection, maximal clique centrality (MMC) algorithm, and degree. The Metascape tool was used for analyzing biological processes and pathways of the top 10 key genes. The main biological processes were response to an inorganic substance and cellular response to chemical stress (including cellular response to oxidative stress). The major pathways were involved in cellular senescence and the cell cycle. After an analysis of major biological processes and pathways, it appears that PARP1/nicotinamide adenine dinucleotide (NAD+)-mediated cellular senescence may play an important role in MAT against aging. Molecular docking, molecular dynamics simulation, and in vivo study were used for further investigation. MAT could interact with the cavity of the PARP1 protein with the binding energy at -8.5 kcal/mol. Results from molecular dynamics simulations showed that the PARP1-MAT complex was more stable than PARP1 alone and that the binding-free energy of the PARP1-MAT complex was -15.962 kcal/mol. The in vivo study showed that MAT could significantly increase the NAD+ level of the liver of d-gal-induced aging mice. Therefore, MAT could interfere with aging through the PARP1/NAD+-mediated cellular senescence signaling pathway.

Multi-Omics Analysis Reveals the Potential Effects of Maternal Dietary Restriction on Fetal Muscle Growth and Development.

In terms of fetal muscle growth, development, and health, maternal nutrition is a crucial influence, although the exact biochemical mechanism by which this occurs is still not fully understood. To examine the potential impacts of maternal dietary restriction on fetal muscle development, the sheep maternal dietary restriction model was developed for this study. In our study, 12 pregnant ewes were evenly split into two experimental groups and fed either 75% or 100% of a maternal nutrient. In addition, a multi-omics analysis was used to study the embryonic longissimus dorsis on gestational days (GD) 85 and 135. The fetal weight at GD 135 was significantly below normal due to the maternal restricted diet (p < 0.01). When fetuses were exposed to the dietary deficit, 416 mRNAs and 40 proteins were significantly changed. At GD 85, the multi-omics analysis revealed that maternal dietary restriction led to a significant up-regulation of the cell cycle regulator CDK2 gene in the cellular senescence signaling pathway, and the results of the qRT-PCR were similar to the multi-omics analysis, which showed that SIX1, PAX7, the cell cycle factors CDK4 and CDK6, and the BCL-2 apoptosis factor were up-regulated and several skeletal muscle marker genes, such as MYF5 and MyoD were down-regulated. At GD 135, maternal dietary restriction blocks the muscle fiber differentiation and maturation. The multi-omics analysis revealed that the TEAD1 gene was in the Hippo signaling pathway, the muscle marker genes MYF5 and MyoG were significantly down-regulated, and the TEAD1 binding of the down-regulated VGLL3 gene might be potential mechanisms affecting myofiber differentiation and maturation. Knocking down the CDK2 gene could inhibit the proliferation of primary embryonic myoblasts, and the expression levels of cell cycle regulatory factors CDK4 and CDK6 were significantly changed. Under low nutrient culture conditions, the number of myoblasts decreased and the expression of CDK2, CDK6, MYF5, PAX7 and BCL-2 changed, which was in perfect agreement with the multi-omics analysis. All of the findings from our study helped to clarify the potential effects of maternal dietary restriction on fetal muscle growth and development. They also provided a molecular foundation for understanding the molecular regulatory mechanisms of maternal nutrition on fetal muscle growth and development, as well as for the development of new medications and the management of related metabolic diseases.

Yiqi Huayu decoction alleviates bleomycin-induced pulmonary fibrosis in rats by inhibiting senescence.

Overview: In treating pulmonary fibrosis (PF), traditional Chinese medicine (TCM) has received much attention, but its mechanism is unclear. The pharmacological mechanisms of TCM can be explored through network pharmacology. However, due to its virtual screening properties, it still needs to be verified by in vitro or in vivo experiments. Therefore, we investigated the anti-PF mechanism of Yiqi Huayu Decoction (YHD) by combining network pharmacology with in vivo experiments. Methods: Firstly, we used classical bleomycin (BLM)-induced rat model of PF and administrated fibrotic rats with YHD (low-, medium-, and high-dose). We comprehensively assessed the treatment effect of YHD according to body weight, lung coefficient, lung function, and histopathologic examination. Second, we predict the potential targets by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) combined with network pharmacology. In brief, we obtained the chemical ingredients of YHD based on the UHPLC-MS/MS and TCMSP database. We collected drug targets from TCMSP, HERB, and Swiss target prediction databases based on active ingredients. Disease targets were acquired from drug libraries, Genecards, HERB, and TTD databases. The intersecting targets of drugs and disease were screened out. The STRING database can obtain protein-protein interaction (PPI) networks and hub target proteins. Molecular Complex Detection (MCODE) clustering analysis combined with enrichment analysis can explore the possible biological mechanisms of YHD. Enrichment analyses were conducted through the R package and the David database, including the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Reactome. Then, we further validated the target genes and target proteins predicted by network pharmacology. Protein and gene expression detection by immunohistochemistry, Western blot (WB), and real-time quantitative PCR (rt-qPCR). Results: The results showed that high-dose YHD effectively attenuated BLM-induced lung injury and fibrosis in rats, as evidenced by improved lung function, relief of inflammatory response, and reduced collagen deposition. We screened nine core targets and cellular senescence pathways by UHPLC-MS/MS analysis and network pharmacology. We subsequently validated key targets of cellular senescence signaling pathways. WB and rt-qPCR indicated that high-dose YHD decreased protein and gene expression of senescence-related markers, including p53 (TP53), p21 (CDKN1A), and p16 (CDKN2A). Increased reactive oxygen species (ROS) are upstream triggers of the senescence program. The senescence-associated secretory phenotypes (SASPs), containing interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and transforming growth factor-β1 (TGF-β1), can further exacerbate the progression of senescence. High-dose YHD inhibited ROS production in lung tissue and consistently reduced the SASPs expression in serum. Conclusion: Our study suggests that YHD improves lung pathological injury and lung function in PF rats. This protective effect may be related to the ability of YHD to inhibit cellular senescence.

Whole-Transcriptome Analysis of Non-Coding RNA Alteration in Porcine Alveolar Macrophage Exposed to Aflatoxin B1.

Aflatoxin B1 (AFB1) is a type of mycotoxin produced by the fungi Aspergillus flavus and Aspergillus parasiticus and is commonly found in cereals, oils and foodstuffs. In order to understand the toxic effects of AFB1 exposure on Porcine alveolar macrophages (3D4/2 cell), the 3D4/2 cells were exposed to 40 μg/mL AFB1 for 24 h in vitro, and several methods were used for analysis. Edu and TUNEL analysis showed that the proliferation of 3D4/2 cells was significantly inhibited and the apoptosis of 3D4/2 cells was significantly induced after AFB1 exposure compared with that of the control group. Whole-transcriptome analysis was performed to reveal the non-coding RNA alteration in 3D4/2 cells after AFB1 exposure. It was found that the expression of cell-cycle-related and apoptosis-related genes was altered after AFB1 exposure, and lncRNAs and miRNAs were also significantly different among the experimental groups. In particular, AFB1 exposure affected the expression of lncRNAs associated with cellular senescence signaling pathways, such as MSTRG.24315 and MSTRG.80767, as well as related genes, Cxcl8 and Gadd45g. In addition, AFB1 exposure affected the expression of miRNAs associated with immune-related genes, such as miR-181a, miR-331-3p and miR-342, as well as immune-related genes Nfkb1 and Rras2. Moreover, the regulation networks between mRNA-miRNAs and mRNA-lncRNAs were confirmed by the results of RT-qPCR and immunofluorescence. In conclusion, our results here demonstrate that AFB1 exposure impaired proliferation of 3D4/2 cells via the non-coding RNA-mediated pathway.

Cingulin b Is Required for Zebrafish Lateral Line Development Through Regulation of Mitogen-Activated Protein Kinase and Cellular Senescence Signaling Pathways.

Cingulin, a cytoplasmic element of tight junctions (TJs), is involved in maintenance of the integrity of epithelial and endothelial cells. However, the role of cingulin in the development of auditory organs remains unclear. Zebrafish is popular as a model organism for hearing research. Using the whole mount in situ hybridization (WISH) experiment, we detected the expression of cingulin b in the posterior lateral line system (PLLs) of zebrafish. We traced the early development progress of zebrafish PLLs from 36 hpf to 72 hpf, and found that inhibition of cingulin b by target morpholinos resulted in severe developmental obstruction, including decreased number of neuromasts, reduced proliferative cells in the primordium, and repressed hair cell differentiation in the neuromasts. To examine the potential mechanism of cingulin b in the development of zebrafish PLL neuromasts, we performed RNA-seq analysis to compare the differently expressed genes (DEGs) between cingulin b knockdown samples and the controls. The KEGG enrichment analysis revealed that MAPK signaling pathway and cellular senescence were the key pathways with most DEGs in cingulin b-MO morphants compared to the Control-MO embryos. Furthermore, quantitative RT-PCR analysis confirmed the findings by RNA-seq that the transcript levels of cell cycle negative regulators such as tp53 and cdkn1a, were remarkably upregulated after inhibition of cingulin b. Our results therefore indicated an important role of cingulin b in the development of auditory organs, and MAPK signaling pathway was inhibited while cellular senescence pathway was activated after downregulation of cingulin b. We bring forward new insights of cingulin by exploring its function in auditory system.

Multi-Omics Analysis of Microglial Extracellular Vesicles From Human Alzheimer's Disease Brain Tissue Reveals Disease-Associated Signatures.

Alzheimer’s disease (AD) is the most common cause of dementia, yet there is no cure or diagnostics available prior to the onset of clinical symptoms. Extracellular vesicles (EVs) are lipid bilayer-delimited particles that are released from almost all types of cell. Genome-wide association studies have linked multiple AD genetic risk factors to microglia-specific pathways. It is plausible that microglia-derived EVs may play a role in the progression of AD by contributing to the dissemination of insoluble pathogenic proteins, such as tau and Aβ. Despite the potential utility of EVs as a diagnostic tool, our knowledge of human brain EV subpopulations is limited. Here we present a method for isolating microglial CD11b-positive small EVs from cryopreserved human brain tissue, as well as an integrated multiomics analysis of microglial EVs enriched from the parietal cortex of four late-stage AD (Braak V-VI) and three age-matched normal/low pathology (NL) cases. This integrated analysis revealed 1,000 proteins, 594 lipids, and 105 miRNAs using shotgun proteomics, targeted lipidomics, and NanoString nCounter technology, respectively. The results showed a significant reduction in the abundance of homeostatic microglia markers P2RY12 and TMEM119, and increased levels of disease-associated microglia markers FTH1 and TREM2, in CD11b-positive EVs from AD brain compared to NL cases. Tau abundance was significantly higher in AD brain-derived microglial EVs. These changes were accompanied by the upregulation of synaptic and neuron-specific proteins in the AD group. Levels of free cholesterol were elevated in microglial EVs from the AD brain. Lipidomic analysis also revealed a proinflammatory lipid profile, endolysosomal dysfunction, and a significant AD-associated decrease in levels of docosahexaenoic acid (DHA)-containing polyunsaturated lipids, suggesting a potential defect in acyl-chain remodeling. Additionally, four miRNAs associated with immune and cellular senescence signaling pathways were significantly upregulated in the AD group. Our data suggest that loss of the homeostatic microglia signature in late AD stages may be accompanied by endolysosomal impairment and the release of undigested neuronal and myelin debris, including tau, through extracellular vesicles. We suggest that the analysis of microglia-derived EVs has merit for identifying novel EV-associated biomarkers and providing a framework for future larger-scale multiomics studies on patient-derived cell-type-specific EVs.

Proteomic Analysis of Hypoxia-Induced Senescence of Human Bone Marrow Mesenchymal Stem Cells.

Methods Hypoxia in hBMSCs was induced for 0, 4, and 12 hours, and cellular senescence was evaluated by senescence-associated β-galactosidase (SA-β-gal) staining. Tandem mass tag (TMT) labeling was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for differential proteomic analysis of hypoxia in hBMSCs. Parallel reaction monitoring (PRM) analysis was used to validate the candidate proteins. Verifications of signaling pathways were evaluated by western blotting. Cell apoptosis was evaluated using Annexin V/7-AAD staining by flow cytometry. The production of reactive oxygen species (ROS) was detected by the fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). Results Cell senescence detected by SA-β-gal activity was higher in the 12-hour hypoxia-induced group. TMT analysis of 12-hour hypoxia-induced cells identified over 6000 proteins, including 686 differentially expressed proteins. Based on biological pathway analysis, we found that the senescence-associated proteins were predominantly enriched in the cancer pathways, PI3K-Akt pathway, and cellular senescence signaling pathways. CDK1, CDK2, and CCND1 were important nodes in PPI analyses. Moreover, the CCND1, UQCRH, and COX7C expressions were verified by PRM. Hypoxia induction for 12 hours in hBMSCs reduced CCND1 expression but promoted ROS production and cell apoptosis. Such effects were markedly reduced by the PI3K agonist, 740 Y-P, and attenuated by LY294002. Conclusions Hypoxia of hBMSCs inhibited CCND1 expression but promoted ROS production and cell apoptosis through activating the PI3K-dependent signaling pathway. These findings provided a detailed characterization of the proteomic profiles related to hypoxia-induced senescence of hBMSCs and facilitated our understanding of the molecular mechanisms leading to stem cell senescence.

Bioinformatics Analysis of Dysregulated MicroRNA-Messenger RNA Networks in Small Cell Lung Cancer

The present study aimed to identify a key module of differentially expressed miRNAs (DE-miRNAs) together with the corresponding differentially expressed mRNAs (DE-mRNAs) within small cell lung cancer (SCLC). Linear models were applied to ascertain the DE-miRNAs and DE-mRNAs in SCLC versus matched non-carcinoma samples obtained from the RNA expression datasets of GSE19945, GSE74190 and GSE6044. The common DE-miRNAs were identified using the Venn plot. Then, 3 databases were used to retrieve the DE-miRNAs target genes, and the intersection was taken for validating the shared target genes. Besides, Cytoscape was utilized for constructing the miRNAmRNA network for SCLC. Finally, a key module of five DE-miRNAs and four hub genes was determined based on the degree. In addition, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted for exploring those hub genes in terms of their functions along with the involved signal transduction pathways. Altogether 106 shared DE-miRNAs were identified, which were used to predict 63 common target genes. In addition, a key module of five DE-miRNAs (hsa-miR-17-5p, hsa-miR-20a-5p, hsa-miR-20b-5p, hsa-miR-93-5p and hsa-miR- 106b-5p) and four hub genes (SOX4, DPYSL2, TGFBR2 and F3) were extracted from the miRNAmRNA network according to their degree. Finally, the hub genes were subjected to GO as well as KEGG analysis, which revealed that cell cycle G1/S phase transition, the extracellular matrix, and cellular senescence signaling pathways exerted vial parts during SCLC progression. A key module of five DE-miRNAs and four hub genes may be potentially used as clinical biomarkers to predict SCLC.

Bioinformatic framework for analysis of transcription factor changes as the molecular link between replicative cellular senescence signaling pathways and carcinogenesis.

Cellular senescence is a natural condition of irreversible cell cycle arrest and apoptotic resistance that occurs in cells exposed to various stress factors, such as replicative stress or overexpression of oncogenes. Unraveling the complex regulation of senescence in cells is essential to strengthen senescence-related therapeutic approaches in cancer, as cellular senescence plays a dual role in tumorigenesis, having both anti- and pro-tumorigenic effects. In our study we created a model of replicative cellular senescence, based on transcriptomic data, including an extra intermediate time-point prior to cells entering senescence, to elucidate the interplay of networks governing cellular senescence with networks involved in tumorigenesis. We reveal specific changes that occur in transcription factor activity at different timepoints before and after cells entering senescence and model the signaling networks that govern these changes.

Derivation of Cell-Engineered Nanovesicles from Human Induced Pluripotent Stem Cells and Their Protective Effect on the Senescence of Dermal Fibroblasts.

Stem cells secrete numerous paracrine factors, such as cytokines, growth factors, and extracellular vesicles. As a kind of extracellular vesicle (EV), exosomes produced in the endosomal compartment of eukaryotic cells have recently emerged as a biomedical material for regenerative medicine, because they contain many valuable contents that are derived from the host cells, and can stably deliver those contents to other recipient cells. Although we have previously demonstrated the beneficial effects of human induced potent stem cell-derived exosomes (iPSC-Exo) on the aging of skin fibroblasts, low production yield has remained an obstacle for clinical applications. In this study, we generated cell-engineered nanovesicles (CENVs) by serial extrusion of human iPSCs through membrane filters with diminishing pore sizes, and explored whether the iPSC-CENV ameliorates physiological alterations of human dermal fibroblasts (HDFs) that occur by natural senescence. The iPSC-CENV exhibited similar characteristics to the iPSC-Exo, while the production yield was drastically increased compared to that of iPSC-derived EVs, including exosomes. The proliferation and migration of both young and senescent HDFs were stimulated by the treatment with iPSC-CENVs. In addition, it was revealed that the iPSC-CNEV restored senescence-related alterations of gene expression. Treatment with iPSC-CENVs significantly reduced the activity of senescence-associated-β-galactosidase (SA-β-Gal) in senescent HDFs, as well as suppressing the elevated expression of p53 and p21, key factors involved in cell cycle arrest, apoptosis, and cellular senescence signaling pathways. Taken together, these results suggest that iPSC-CENV could provide an excellent alternative to iPSC-exo, and be exploited as a resource for the treatment of signs of skin aging.

Genetic Ablation of p16INK4a Does Not Protect against Cellular Senescence in Mouse Models of Chronic Obstructive Pulmonary Disease/Emphysema.

Cigarette smoke (CS) affects DNA damage and cellular senescence signaling pathways in the pathogenesis of chronic obstructive pulmonary disease (COPD). p16INK4a (p16: a cyclin-dependent kinase inhibitor) is a key marker of cellular senescence, which is induced by CS in lung cells. It is thought that removal of p16 attenuates premature aging by removing senesced cells. However, the role of p16 in CS-induced stress-induced premature senescence (SIPS) and senescence-associated secretory phenotype (SASP) during the development of COPD/emphysema is not known. We hypothesize that p16 regulates cellular senescence and DNA damage/repair molecular signaling targets during chronic CS-induced inflammation and airspace enlargement in mouse models of COPD. We used p16 global knockout (KO) and p16 lung epithelial cell-specific KO (p16CreCC10) mice to determine whether p16 removal in lung epithelium augments or protects against cellular senescence (SIPS and SASP) in chronic CS- and elastase-induced development of COPD/emphysema in mice. p16 KO mice exposed to chronic CS and p16 lung epithelial cell-specific KO mice exposed to elastase did not show attenuation of lung inflammation, altered lung function, or airspace enlargement. p16 KO and p16CreCC10 exposed to CS and elastase showed increases in lung senescence-associated β-galactosidase activity. Thus, removal of p16-positive cells did not protect against airspace enlargement and decline in lung function induced in COPD mouse models. Our findings suggest that p16 is not the only key player associated with CS-induced cellular senescence phenotypes (SIPS and SASP), decline in lung function, and airspace enlargement in COPD/emphysema.

Ginsenoside Rg1 enhances the resistance of hematopoietic stem/progenitor cells to radiation-induced aging in mice

To investigate the effects of ginsenoside Rg1 on the radiation-induced aging of hematopoietic stem/progenitor cells (HSC/HPCs) in mice and the underlying mechanisms. Male C57BL/6 mice were treated with ginsenoside Rg1 (20 mg·kg(-1)·d(-1), ip) or normal saline (NS) for 7 d, followed by exposure to 6.5 Gy X-ray total body irradiation. A sham-irradiated group was treated with NS but without irradiation. Sca-1(+) HSC/HPCs were isolated and purified from their bone marrow using MACS. DNA damage was detected on d 1. The changes of anti-oxidative activities, senescence-related markers senescence-associated β-galactosidase (SA-β-gal) and mixed colony-forming unit (CFU-mix), P16(INK4a) and P21(Cip1/Waf1) expression on d 7, and cell cycle were examined on d 1, d 3, and d 7. The irradiation caused dramatic reduction in the number of Sca-1(+) HSC/HPCs on d 1 and the number barely recovered until d 7 compared to the sham-irradiated group. The irradiation significantly decreased SOD activity, increased MDA contents and caused DNA damage in Sca-1(+) HSC/HPCs. Moreover, the irradiation significantly increased SA-β-gal staining, reduced CFU-mix forming, increased the expression of P16(INK4a) and P21(Cip1/Waf1) in the core positions of the cellular senescence signaling pathways and caused G1 phase arrest of Sca-1(+) HSC/HPCs. Administration of ginsenoside Rg1 caused small, but significant recovery in the number of Sca-1(+) HSC/HPCs on d 3 and d 7. Furthermore, ginsenoside Rg1 significantly attenuated all the irradiation-induced changes in Sca-1(+) HSC/HPCs, including oxidative stress reaction, DNA damage, senescence-related markers and cellular senescence signaling pathways and cell cycle, etc. Administration of ginsenoside Rg1 enhances the resistance of HSC/HPCs to ionizing radiation-induced senescence in mice by inhibiting the oxidative stress reaction, reducing DNA damage, and regulating the cell cycle.