Berberine ameliorates cellular senescence and extends the lifespan of mice via regulating p16 and cyclin protein expression

Increased mortality in COVID-19 often associates with microvascular complications. We have recently shown that SARS-CoV-2 spike protein promotes an inflammatory cytokine IL-6/IL-6R induced trans-signaling response and alarmin secretion. Virus infected or spike transfected human epithelial cells exhibited an increase in senescence state with the release of senescence associated secretory proteins (SASP) related inflammatory molecules. Introduction of BRD4 inhibitor AZD5153 to senescent epithelial cells reversed this effect and reduced SASP related inflammatory molecule release in TMNK-1 or EA hy926 as representative human endothelial cell line, when exposed to cell culture medium (CM) derived from A549 cells expressing SARS-CoV-2 spike protein, also exhibited a senescence phenotype with enhanced p16, p21, SA-β-galactosidase expression, and triggered SASP pathways. Inhibition of IL-6 trans-signaling by Tocilizumab and inhibition of inflammatory receptor signaling by the BTK inhibitor Zanubrutinib, prior to exposure of CM to endothelial cells, inhibited p21 and p16 induction. We also observed an increase in reactive oxygen species (ROS) in A549 spike transfected and endothelial cells exposed to spike transfected CM. ROS generation in endothelial cell lines was reduced after treatment with Tocilizumab and Zanubrutinib. Cellular senescence was associated with an increased level of the endothelial adhesion molecules, VCAM-1 and ICAM-1 with in vitro leukocyte attachment potential. Inhibition of senescence or SASP function prevented VCAM-1/ICAM-1 expression and leukocyte attachment. Taken together, we identified that the exposure of human endothelial cells to cell culture supernatant derived from SARS-CoV-2 spike protein expression displayed cellular senescence markers, leading to enhanced leukocyte adhesion. Importance: The present study was aimed at examining the underlying mechanism of extrapulmonary manifestations of SARS-CoV-2 spike protein associated pathogenesis, with the notion that infection of the pulmonary epithelium can lead to mediators that drive endothelial dysfunction. We utilized SARS-CoV-2 spike protein expression in cultured cells of human hepatocytes (Huh7.5) and pneumocytes (A549) to generate conditioned culture media (CM). Endothelial cell lines (TMNK-1 or EA hy926 ) treated with CM exhibited increase in cellular senescence markers by a paracrine mode, and lead to leukocyte adhesion. Overall, the link between these responses in endothelial cell senescence, and a potential contribution to microvascular complication in productively SARS-CoV-2 infected humans is implicated. Furthermore, the use of inhibitors (BTK, IL-6 and BRD4) showed reverse effect in the senescent cells. These results may support the selection of potential adjunct therapeutic modalities to impede SARS-CoV-2 associated pathogenesis.

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Chapter 27 Analysis of DNA Content and Cyclin Protein Expression in Studies of DNA Ploidy, Growth Fraction, Lymphocyte Stimulation, and the Cell Cycle

Methods in Cell Biology - Flow Cytometry Second Edition, Part A ◽

10.1016/s0091-679x(08)61732-x ◽

1994 ◽

pp. 421-435 ◽

Cited By ~ 19

Author(s):

Zbigniew Darzynkiewicz ◽

Jianping Gong ◽

Frank Traganos

Keyword(s):

Cell Cycle ◽

Protein Expression ◽

Dna Content ◽

Dna Ploidy ◽

Lymphocyte Stimulation ◽

Growth Fraction ◽

Cyclin Protein

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Glucose Oxidase Induces Cellular Senescence in Immortal Renal Cells through ILK by DownregulatingKlothoGene Expression

Oxidative Medicine and Cellular Longevity ◽

10.1155/2015/416738 ◽

2015 ◽

Vol 2015 ◽

pp. 1-13 ◽

Cited By ~ 7

Author(s):

Nuria Troyano-Suárez ◽

María del Nogal-Avila ◽

Inés Mora ◽

Patricia Sosa ◽

Susana López-Ongil ◽

...

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Protein Expression ◽

Glucose Oxidase ◽

Cellular Senescence ◽

Renal Cells ◽

Long Time ◽

P16 Expression ◽

Expression And Activity ◽

In Cells

Cellular senescence can be prematurely induced by oxidative stress involved in aging. In this work, we were searching for novel intermediaries in oxidative stress-induced senescence, focusing our interest on integrin-linked kinase (ILK), a scaffold protein at cell-extracellular matrix (ECM) adhesion sites, and on theKlothogene. Cultured renal cells were treated with glucose oxidase (GOx) for long time periods. GOx induced senescence, increasing senescence associatedβ-galactosidase activity and the expression of p16. In parallel, GOx increased ILK protein expression and activity. Ectopic overexpression of ILK in cells increased p16 expression, even in the absence of GOx, whereas downregulation of ILK inhibited the increase in p16 due to oxidative stress. Additionally, GOx reducedKlothogene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition. We demonstrated a direct link between ILK andKlothosince silencing ILK expression in cells and mice increasesKlothoexpression and reduces p53 and p16 expression in renal cortex. In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reducesKlothoexpression. We hereby present ILK as a novel downregulator ofKlothogene expression.

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Ku86 alleviates human umbilical vein endothelial cellular apoptosis and senescence induced by a low dose of ionizing radiation

Journal of International Medical Research ◽

10.1177/0300060518805302 ◽

2019 ◽

Vol 47 (2) ◽

pp. 893-904 ◽

Cited By ~ 1

Author(s):

Kai Wu ◽

Zejin Chen ◽

Qing Peng ◽

Guojian Chen ◽

Weihong Yan ◽

...

Keyword(s):

Ionizing Radiation ◽

Protein Expression ◽

Cellular Senescence ◽

Umbilical Vein ◽

Dependent Manner ◽

Low Doses ◽

Human Umbilical Vein ◽

Expression Levels ◽

Associated Proteins ◽

Umbilical Vein Endothelial Cells

Objective The aim of this study was to observe the effect of Ku86 on cellular senescence and apoptosis induced by various doses of ionizing radiation in human umbilical vein endothelial cells (HUVECs). Methods Senescence-associated β-galactosidase activity was detected to evaluate cell senescence. Apoptosis was determined by flow cytometry and a caspase enzyme determination kit. p16Ink4a, Sirt1, superoxide dismutase 2 (SOD2), xanthine oxidase (XOD), and Bcl-2 protein expression levels were measured by western blotting. Results Low doses of ionizing radiation induced cellular senescence and apoptosis in a dose-dependent manner. The Ku86 protein was negatively correlated with ionization intensity. After transfection of Ku86 with a vector (pcDNA 3.1), or interference with siRNA (si-Ku86), apoptosis/senescence and related protein expression were observed. Western blot results revealed that this induction of senescence was associated with activated Sirt1 and SOD2, and downregulation of p16Ink4a and XOD in 0.2 Gy ionizing radiation. The expression levels of apoptosis-associated proteins, such as Bcl-2, cleaved caspase-3, caspase-8, and caspase-9, were significantly altered in both the presence and absence of Ku86 with ionizing radiation (0.2 Gy). Conclusions Our study revealed that Ku86 overexpression inhibits HUVEC apoptosis and senescence induced by low doses of ionizing radiation.

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DPY30 regulates pathways in cellular senescence through ID protein expression

The EMBO Journal ◽

10.1038/emboj.2013.159 ◽

2013 ◽

Vol 32 (16) ◽

pp. 2217-2230 ◽

Cited By ~ 17

Author(s):

Elisabeth Simboeck ◽

Arantxa Gutierrez ◽

Luca Cozzuto ◽

Malte Beringer ◽

Livia Caizzi ◽

...

Keyword(s):

Protein Expression ◽

Cellular Senescence ◽

Id Protein

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Spermidine induces cytoprotective autophagy of female germline stem cells in vitro and ameliorates aging caused by oxidative stress through upregulated sequestosome-1/p62 expression

Cell & Bioscience ◽

10.1186/s13578-021-00614-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Xiaoyan Yuan ◽

Geng. G. Tian ◽

Xiuying Pei ◽

Xiaopeng Hu ◽

Ji Wu

Keyword(s):

Oxidative Stress ◽

Stem Cells ◽

Protein Expression ◽

Cellular Senescence ◽

Critical Role ◽

Autophagic Flux ◽

Germline Stem Cells ◽

Sequestosome 1 ◽

Female Germline

Abstract Background Autophagy is required for oogenesis and plays a critical role in response to aging caused by oxidative stress. However, there have been no reports on regulation of cytoprotective autophagy in female germline stem cells (FGSCs) in response to aging caused by oxidative stress. Results We found that Spermidine (SPD) significantly increased protein expression of autophagy markers microtubule-associated protein 1 light chain 3 beta-II (MAP1LC3B-II/LC3B-II) and sequestosome-1/p62 (SQSTM1/p62), and evoked autophagic flux in FGSCs. Moreover, SPD increased the number and viability of FGSCs in vitro. Further, we found that SPD significantly reduced basal or hydrogen peroxide (H2O2)-induced up-regulated protein expression of the aging markers, cyclin dependent kinase inhibitor 2A (p16/CDKN2A) and tumor protein 53 (p53). After knockdown of p62 in FGSCs, p16 protein levels were significant higher compared with controls. However, protein p16 levels were not significantly changed in p62 knockdown FGSCs with SPD treatment compared with without SPD. Moreover, SPD significantly changed the expression of autophagy-related genes and pathways in FGSCs, as shown by bioinformatics analysis of RNA sequencing data. Additionally, SPD significantly inhibited AKT/mTOR phosphorylation. Conclusions SPD induces cytoprotective autophagy in FGSCs in vitro and ameliorates cellular senescence of FGSCs induced by H2O2. Furthermore, SPD can ameliorate cellular senescence of FGSCs through p62. SPD might induce autophagy in FGSCs via the PI3K/Akt pathway. Our findings could be helpful for delaying aging of female germ cells due to oxidative stress and preserving female fertility.

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Lamin B receptor: role on chromatin structure, cellular senescence and possibly aging

Biochemical Journal ◽

10.1042/bcj20200165 ◽

2020 ◽

Vol 477 (14) ◽

pp. 2715-2720

Author(s):

Susana Castro-Obregón

Keyword(s):

Cellular Senescence ◽

Nuclear Membrane ◽

Chromatin Structure ◽

Cholesterol Synthesis ◽

Reductase Activity ◽

Chimeric Protein ◽

Nuclear Lamina ◽

Lamin B ◽

Inner Nuclear Membrane ◽

Lamin B Receptor

The nuclear envelope is composed by an outer nuclear membrane and an inner nuclear membrane, which is underlain by the nuclear lamina that provides the nucleus with mechanical strength for maintaining structure and regulates chromatin organization for modulating gene expression and silencing. A layer of heterochromatin is beneath the nuclear lamina, attached by inner nuclear membrane integral proteins such as Lamin B receptor (LBR). LBR is a chimeric protein, having also a sterol reductase activity with which it contributes to cholesterol synthesis. Lukasova et al. showed that when DNA is damaged by ɣ-radiation in cancer cells, LBR is lost causing chromatin structure changes and promoting cellular senescence. Cellular senescence is characterized by terminal cell cycle arrest and the expression and secretion of various growth factors, cytokines, metalloproteinases, etc., collectively known as senescence-associated secretory phenotype (SASP) that cause chronic inflammation and tumor progression when they persist in the tissue. Therefore, it is fundamental to understand the molecular basis for senescence establishment, maintenance and the regulation of SASP. The work of Lukasova et al. contributed to our understanding of cellular senescence establishment and provided the basis that lead to the further discovery that chromatin changes caused by LBR reduction induce an up-regulated expression of SASP factors. LBR dysfunction has relevance in several diseases and possibly in physiological aging. The potential bifunctional role of LBR on cellular senescence establishment, namely its role in chromatin structure together with its enzymatic activity contributing to cholesterol synthesis, provide a new target to develop potential anti-aging therapies.

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