scholarly journals Mitomycin C Induces Apoptosis in Rheumatoid Arthritis Fibroblast-Like Synoviocytes via a Mitochondrial-Mediated Pathway

2015 ◽  
Vol 35 (3) ◽  
pp. 1125-1136 ◽  
Author(s):  
Chuqi Yan ◽  
Dechao Kong ◽  
Dong Ge ◽  
Yanming Zhang ◽  
Xishan Zhang ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Viability ◽  
Mitomycin C ◽  
Apoptotic Cell ◽  
Chronic Inflammatory Disease ◽  
Cell Counting ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Control Treatment ◽  
Induced Apoptosis ◽  
Fibroblast Like Synoviocytes

Background/Aims: Rheumatoid arthritis (RA) is a systemic chronic inflammatory disease characterised by prominent synoviocyte hyperplasia and a potential imbalance between the growth and death of fibroblast-like synoviocytes (FLS). Mitomycin C (MMC) has previously been demonstrated to inhibit fibroblast proliferation and to induce fibroblast apoptosis. However, the effects of MMC on the proliferation and apoptosis of human RA FLS and the potential mechanisms underlying its effects remain unknown. Methods: Cell viability was determined using the Cell Counting Kit-8 assay. Apoptotic cell death was analysed via Annexin V-FITC/PI double staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling. The production of intracellular reactive oxygen species (ROS) was assessed via flow cytometry, and the changes in mitochondrial membrane potential (ΔΨm) were visualized based on JC-1 staining via fluorescence microscopy. The expression of apoptosis-related proteins was determined via Western blot. Results: Treatment with MMC significantly reduced cell viability and induced apoptosis in RA FLS. Furthermore, MMC exposure was found to stimulate the production of ROS and to disrupt the ΔΨm compared to the control treatment. Moreover, MMC increased the release of mitochondrial cytochrome c, the ratio of Bax/Bcl-2, the activation of caspase-9 and caspase-3, and the subsequent cleavage of poly(ADP-ribose) polymerase. Conclusion: Our findings suggest that MMC inhibits cell proliferation and induces apoptosis in RA FLS, and the mechanism underlying this MMC-induced apoptosis may involve a mitochondrial signalling pathway.

scholarly journals MiRNA-506 inhibits rheumatoid arthritis fibroblast-like synoviocytes proliferation and induces apoptosis by targetting TLR4

2019 ◽  
Vol 39 (5) ◽  
Author(s):  
Dan Li ◽  
Qingchen Zhou ◽  
Gaojian Hu ◽  
Gang Wang
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Counting ◽  
Luciferase Assay ◽  
Qrt Pcr ◽  
Cycle Arrest ◽  
Tnf Α ◽  
Proliferation And Apoptosis ◽  
Synovial Tissues ◽  
Induced Apoptosis ◽  
Fibroblast Like Synoviocytes

Abstract Fibroblast-like synoviocytes (FLSs) play a crucial role in rheumatoid arthritis (RA) pathogenesis. While miRNA (miR)-506 has been implicated in the progression of multiple diseases, its role in RA remains to be explored. The present study evaluated the function of miR-506 in the regulation of RA-FLSs. FLSs were prepared from RA and healthy synovial tissues. The expression of miR-506 was measured by quantitative real time PCR (qRT-PCR). The effects of miR-506 on RA-FLSs proliferation and apoptosis were detected by cell counting Kit-8 and flow cytometry assays, respectively. The determination of TNF-α, IL-6, and IL-1β concentrations in RA-FLSs supernatant were done by ELISA. The levels of miR-506 were detected to be significantly lower in the synovial tissues and FLSs of RA than in the synovial tissues and FLSs of healthy controls. The miR-506 up-regulation in RA-FLSs significantly inhibited the proliferation and promoted cell cycle arrest at the G0/G1 phase. The overexpression of miR-506 induced apoptosis, along with an increase in activities of caspase-3 and -8. A target gene Toll-like receptor 4 (TLR4) under the direct regulation of miR-506 was identified through the luciferase assay, qRT-PCR and western blot analysis. Forced overexpression of TLR4 in the rescue experiments showed that TLR4 effectively reversed the effect on proliferation and apoptosis in miR-506-overexpressing RA-FLSs. Thus, miR-506 may be a potential target for RA prevention and therapy of RA.

Inhibition of Histone Deacetylase 6 Protects Hippocampal Cells Against Mitochondria-mediated Apoptosis in a Model of Severe Oxygen-glucose Deprivation

2019 ◽  
Vol 19 (9) ◽  
pp. 673-682 ◽  
Author(s):  
Panpan Chang ◽  
Yuzi Tian ◽  
Aaron M. Williams ◽  
Umar F. Bhatti ◽  
Baoling Liu ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Cytochrome C ◽  
Cell Viability ◽  
Histone Deacetylase ◽  
Caspase 3 ◽  
Cell Counting ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Histone Deacetylase 6 ◽  
Phosphorylated Akt ◽  
Ldh Release

Background: Histone deacetylase (HDAC) 6 inhibitors have demonstrated significant protective effects in traumatic injuries. However, their roles in neuroprotection and underlying mechanisms are poorly understood. This study sought to investigate the neuroprotective effects of Tubastatin A (Tub-A), an HDAC6 inhibitor, during oxygenglucose deprivation (OGD) in HT22 hippocampal cells. Methods: HT22 hippocampal cells were exposed to OGD. Cell viability and cytotoxicity were assessed by cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) release assay. Cellular apoptosis was assessed by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Mitochondria membrane potential was detected using JC-1 dye. Expressions of acetylated α-tubulin, α-tubulin, cytochrome c, VDAC, Bax, Bcl- 2, cleaved caspase 3, phosphorylated Akt, Akt, phosphorylated GSK3β and GSK3β were analyzed by Western blot analysis. Results: Tub-A induced acetylation of α-tubulin, demonstrating appropriate efficacy. Tub-A significantly increased cell viability and attenuated LDH release after exposure to OGD. Furthermore, Tub-A treatment blunted the increase in TUNEL-positive cells following OGD and preserved the mitochondrial membrane potential. Tub-A also attenuated the release of cytochrome c from the mitochondria into the cytoplasm and suppressed the ratio of Bax/Bcl-2 and cleaved caspase 3. This was mediated, in part, by the increased phosphorylation of Akt and GSK3β signaling pathways. Conclusion: HDAC 6 inhibition, using Tub-A, protects against OGD-induced injury in HT22 cells by modulating Akt/GSK3β signaling and inhibiting mitochondria-mediated apoptosis.

scholarly journals MiR-140-3p inhibits the cell viability and promotes apoptosis of synovial fibroblasts in rheumatoid arthritis through targeting sirtuin 3

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Beibei Zu ◽  
Lin Liu ◽  
Jingya Wang ◽  
Meirong Li ◽  
Junxia Yang
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Viability ◽  
Cell Apoptosis ◽  
Caspase 3 ◽  
Pearson Correlation ◽  
Fibrous Tissue ◽  
Synovial Fibroblasts ◽  
Cell Counting ◽  
Sirtuin 3 ◽  
Fibrous Tissues

Abstract Background Synovial fibroblasts (SFs) with the abnormal expressions of miRNAs are the key regulator in rheumatoid arthritis (RA). Low-expressed miR-140-3p was found in RA tissues. Therefore, we attempted to investigate the effect of miR-140-3p on SFs of RA. Methods RA and normal synovial fibrous tissue were gathered. The targets of miR-140-3p were found by bioinformatics and luciferase analysis. Correlation between the expressions of miR-140-3p with sirtuin 3 (SIRT3) was analyzed by Pearson correlation analysis. After transfection, cell viability and apoptosis were detected by cell counting kit-8 and flow cytometry. The expressions of miR-140-3p, SIRT3, Ki67, Bcl-2, Bax, and cleaved Caspase-3 were detected by RT-qPCR or western blot. Results Low expression of miR-140-3p and high expression of SIRT3 were found in RA synovial fibrous tissues. SIRT3 was a target of miR-140-3p. SIRT3 expression was negatively correlated to the expression of miR-140-3p. MiR-140-3p mimic inhibited the MH7A cell viability and the expressions of SIRT3, Ki67, and Bcl-2 and promoted the cell apoptosis and the expressions of Bax and cleaved Caspase-3; miR-140-3p inhibitor showed an opposite effect to miR-140-3p mimic on MH7A cells. SIRT3 overexpression not only promoted the cell viability and inhibited cell apoptosis of MH7A cells but also reversed the effect of miR-140-3p mimic had on MH7A cells. Conclusions The results in this study revealed that miR-140-3p could inhibit cell viability and promote apoptosis of SFs in RA through targeting SIRT3.

scholarly journals Neuroprotective Effects of Alpha-Mangostin on MPP+-Induced Apoptotic Cell Death in Neuroblastoma SH-SY5Y Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Prachya Janhom ◽  
Permphan Dharmasaroja
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Caspase 3 ◽  
Nuclear Dna ◽  
Apoptotic Cell ◽  
Apoptotic Cell Death ◽  
Concomitant Treatment ◽  
Ros Production ◽  
Cellular Models ◽  
Induced Apoptosis

In vitrostudies have shown that extracts from mangosteen (Garcinia mangostanaLinn.) act as antioxidants and cytoprotective agents against oxidative damage. The protective effect of alpha-mangostin, the major xanthone found in the pericarp of the mangosteen, in cellular models of Parkinson’s disease (PD), has not been investigated. This study aims to investigate whether alpha-mangostin could protect SH-SY5Y neuroblastoma cells from MPP+-induced apoptosis. The effects of alpha-mangostin on MPP+-induced cell death were evaluated with a cell viability assay, staining for nuclear DNA morphology, flow cytometry for apoptotic cells and reactive oxygen species (ROS) production, quantitative real-time PCR for the expression of p53, Bax, and Bcl-2, and western blot analysis for cleaved caspase-3. Concomitant treatment with alpha-mangostin attenuated the effect of MPP+on cell viability and apoptotic cell death. Alpha-mangostin reduced ROS formation induced by MPP+. Bax/Bcl-2 expression ratio and expression of p53 were significantly lower in cells cocultured with alpha-mangostin and MPP+. The cotreated cells showed a significant decrease in activated caspase-3 compared with MPP+treatment alone. Our data suggest that cytoprotection of alpha-mangostin against MPP+-induced apoptosis may be associated with the reduction of ROS production, modulating the balance of pro- and antiapoptotic genes, and suppression of caspase-3 activation.

scholarly journals Potentiation of neurite outgrowth and reduction of apoptosis by immunosuppressive agents: implications for neuronal injury and transplantation

2006 ◽  
Vol 20 (5) ◽  
pp. 1-7 ◽  
Author(s):  
Jason Sheehan ◽  
Anne Eischeid ◽  
Randi Saunders ◽  
Nader Pouratian
Keyword(s):  
Cyclosporin A ◽  
Neurite Outgrowth ◽  
Apoptotic Cell ◽  
Immunosuppressive Agents ◽  
Neuronal Cell ◽  
Neuronal Injury ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Human Neuroblastoma ◽  
Cord Injury ◽  
Induced Apoptosis

Object Immunosuppressive agents are believed to play a role in recovery from spinal cord injury, but the underlying mechanisms by which neuronal function is improved by these agents are poorly understood. In this study, the authors evaluate the effect of immunosuppressive medications on neurite outgrowth and cell survival after a pharmacologically induced injury. Methods Differentiated human neuroblastoma SH-SY5Y cells were injured using the calcium agonist thapsigargin. After cellular injury, neurite outgrowth in the presence or absence of immunosuppressive agents was measured. Apoptosis was quantified with the aid of a terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling assay. Neurite outgrowth was severely restricted following thapsigargin injury. Outgrowth was potentiated, however, by the addition of concentrations of 1 and 10 μM cyclosporin A in a dose-dependent fashion. Similarly, addition of 10 nM FK506 increased the percentage of neurites in the 20- to 40-micron range. A low dose (1 μM) of dexamethasone did not have a significant effect on neurite outgrowth, but a higher dose (10 μM) increased the percentage of neurites in the 10- to 45-micron range. These agents also lessened the degree of thapsigargin-induced apoptosis. Conclusions Immunosuppressive agents such as cyclosporin A, FK506, and dexamethasone can potentiate neurite outgrowth and protect against apoptotic cell death in a human postmitotic neuronal cell line. Such effects may have implications for lessening neuronal injury after neurotrauma, stroke, or neurodegeneration.

scholarly journals Overexpression of IκBα in cardiomyocytes alleviates hydrogen peroxide-induced apoptosis and autophagy through inhibiting NF-κB activation

2020 ◽  
Author(s):  
Min Han ◽  
Xiao-Cui Chen ◽  
Ming-Hui Sun ◽  
Min-Tao Gai ◽  
Yi-Ning Yang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Western Blot ◽  
Cell Viability ◽  
Signaling Pathway ◽  
Nuclear Translocation ◽  
Neonatal Rat ◽  
Cell Counting ◽  
Pyrrolidine Dithiocarbamate ◽  
Induced Apoptosis

Abstract Background: Inflammation and oxidative stress play a predominant role in the initiation and progression of ischemia/reperfusion (I/R) injury, of which nuclear factor kappa B (NF-κB) is a crucial mediator. Overexpression of the inhibitor of κB alpha (IκBα) gene is hypothesized to have protective effects against apoptosis and autophagy in cardiomyocytes subjected to hydrogen peroxide (H2O2) through inhibiting the NF-κB pathway.Methods: The IκBαS32A, S36A gene was transfected via adeno-associated virus serotype 9 (AAV9) delivery into neonatal rat ventricular cardiomyocytes (NRVMs) prior to H2O2 treatment. NRVMs were divided into control, H2O2, GFP +H2O2, IκBα+H2O2, and pyrrolidine dithiocarbamate (PDTC)+H2O2 groups. Nuclear translocation of NF-κB p65 subunit was evaluated by immunofluorescence and Western blot. Cell viability was assessed by Cell Counting Kit-8 assay. Supernatant lactate dehydrogenase (LDH) and intracellular malondialdehyde (MDA) were measured to identify H2O2-stimulated cytotoxicity. Apoptosis was determined by Annexin V-PE/7-AAD staining, and the mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining. Western blot was used to detect apoptosis- and autophagy-related proteins.Results: IκBα transfection significantly increased cell viability and ΔΨm, but decreased the supernatant LDH and cellular MDA levels in cardiomyocytes exposed to H2O2. Meanwhile, IκBα overexpression decreased H2O2-induced apoptosis by upregulating the Bcl-2/Bax ratio and reduced autophagy by downregulating the expression of Beclin-1 and the LC3-Ⅱ/LC3-Ⅰ ratio. These effects partly accounted for the ability of IκBα to inhibit the NF-κB signaling pathway, as evidenced by decreases in p65 phosphorylation and nuclear translocation. Indeed, the effects of inactivation of NF-κB signaling with the specific inhibitor, PDTC, resembled the cardioprotective effects of IκBα during H2O2 stimulation.Conclusion: IκBα overexpression can ameliorate H2O2-induced apoptosis, autophagy, oxidative injury, and ΔΨm loss through inhibition of the NF-κB signaling pathway. These findings suggest that IκBα transfection can successfully resist oxidative stress-induced damage through inhibiting NF-κB activation, which may provide a potential therapeutic target for prevention of myocardial I/R injury.

Protective Effect of SCUBE1 on Oxidative Stress-induced Apoptosis in Human Ovarian Granulosa Cells

2021 ◽  
Author(s):  
Huijiao Fu ◽  
Xuzi Cai ◽  
Qiwen Liu ◽  
Wei Yang ◽  
xuefeng wang
Keyword(s):  
Membrane Potential ◽  
Western Blot ◽  
Cell Viability ◽  
Mitochondrial Membrane Potential ◽  
Granulosa Cells ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Cell Counting ◽  
Ovarian Granulosa Cells ◽  
Induced Apoptosis

Abstract Background: Apoptosis of ovarian granulosa cells (GCs) is a sign of follicular atresia. This study aimed to explore the role and mechanism of signal peptide, CUB domain, epidermal growth factor-like protein1 (SCUBE1) in protecting GCs from apoptosis induced by hydrogen peroxide (H2O2). Methods: Firstly, the expression of SCUBE1 on the ovaries of humans and mice was analyzed by qRT-PCR, western blot and immunohistochemistry. Subsequently, the H2O2 treated GCs were pretreated with SCUBE1 recombinant protein, and their cell viability and proliferation were detected by Cell Counting Kit-8 (CCK-8) assay. The levels of reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) in the cells were determined by DCFH-DA and rhodamine 123, respectively. The percentage of apoptotic cells was analyzed by flow cytometry after staining with Annexin V/PI. The expression levels of pathway related proteins, such as Bcl-2, Bax, p53, caspase-3, were determined by western blot analysis. Finally, the pathogenicity of SCUBE1 (c.1169C>G, p.P390R) were analyzed based on the software.Results: SCUBE1 was expressed in women of all ages and had the highest expression level in the ovaries in multiple organs and tissues of KM mouse. In vitro cell experiments show that SCUBE1 pretreatment reduced H2O2-induced apoptosis and improved cell viability. SCUBE1 also blocked the production of ROS in cells and improved mitochondrial membrane potential. After SCUBE1 pretreatment, anti-apoptotic protein Bcl-2 expression was upregulated, whereas the expression of the pro-apoptotic proteins Bax, Bax/Bcl-2, Caspase-3, and p53 were downregulated. Analysis of the impact of SCUBE1 (c.1169C >G, p.P390R) mutation from the aspect of mutation pathogenicity; protein stability; and gene haplotype insufficiency, indicated that the p.P390R mutation is significantly pathogenic.Conclusions: This is the first time that the potential role of SCUBE1 in protecting GCs from H2O2-induced damage through the mitochondrial pathways, attributing to POI, is studied. SCUBE1 (c.1169C >G, p.P390R) mutation has significant pathogenicity but the specific harm needs to be confirmed by further studies. Trial registration: Not applicable.

scholarly journals Apoptosis of Rheumatoid Arthritis Fibroblast-Like Synoviocytes: Possible Roles of Nitric Oxide and the Thioredoxin 1

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Huili Li ◽  
Ajun Wan
Keyword(s):  
Rheumatoid Arthritis ◽  
Nitric Oxide ◽  
Molecular Mechanisms ◽  
Caspase 3 ◽  
Joint Destruction ◽  
Redox System ◽  
Chronic Inflammatory Disease ◽  
Thioredoxin 1 ◽  
Fibroblast Like Synoviocytes

Rheumatoid arthritis is a chronic inflammatory disease characterized by synovial hyperplasia and progressive joint destruction. The impaired apoptosis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) is pivotal in this process. However, the molecular mechanisms responsible for the reduced apoptosis are not fully understood. Both nitric oxide and thioredoxin 1 as two important mediators are widely investigated in the pathogenesis of rheumatoid arthritis. Interestingly, studies have showed that thioredoxin 1 may serve as a master regulator of S-nitrosylation of caspase-3 to fine-tune apoptosisin vivo. Thus, it is anticipated that further investigations on the role of thioredoxin 1 in the S-nitrosylation and denitrosylation of caspase-3 in RA-FLS will likely provide a novel understanding of mechanisms implicated in the impaired apoptosis of RA-FLS. In this paper, we will provide an overview on pathways involved in the reduced apoptosis of RA-FLS and then discuss specially the possible roles of nitric oxide and the thioredoxin 1 redox system associated with apoptosis of RA-FLS.

scholarly journals In situ apoptotic cell labeling by the TUNEL method: improvement and evaluation on cell preparations.

1996 ◽  
Vol 44 (9) ◽  
pp. 959-968 ◽  
Author(s):  
A Negoescu ◽  
P Lorimier ◽  
F Labat-Moleur ◽  
C Drouet ◽  
C Robert ◽  
...  
Keyword(s):  
Apoptotic Cell ◽  
Rapid Identification ◽  
Cell Labeling ◽  
Cell Fraction ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Current Debate ◽  
Double Labeling ◽  
Apoptotic Morphology ◽  
Induced Apoptosis

TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) is a method of choice for rapid identification and quantification of the apoptotic cell fraction in cultured cell preparations. However, TUNEL application has been restricted to a narrow spectrum of sample conditions, and only detergents have been proposed as labeling enhancers. This study was aimed at extending TUNEL to variously fixed cells and improving TUNEL sensitivity by optimized pretreatments, the specificity being assessed by reference to the apoptotic morphology. Comparative TUNEL was performed with three protocols on CEM-C7 cells, a model of glucocorticoid-induced apoptosis. Samples were submitted to six modalities of fixation and TUNEL was performed after each of the following conditions: no pretreatment; detergent permeabilization; proteolytic digestion; microwave irradiation; and a recently published combination of the latter two. The proportion of TUNEL-stained elements within the cell fraction, with and without apoptotic morphology, was quantified. Our results showed that: (a) with an adequate pretreatment, reliable TUNEL can be obtained after each fixative tested; (b) detergent was inefficient in improving sensitivity; (c) whatever the fixation, microwave pretreatment provided the best TUNEL sensitivity without notable loss of specificity; (d) under adaptive technical conditions, TUNEL can be associated with detection of various proteins by double labeling; and (e) the existence of a limited population of intensely TUNEL-positive cells that lacked apoptotic morphology contributes to the current debate about a preapoptotic state.

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