scholarly journals Dimethyl Fumarate Ameliorates Nucleus Pulposus Cell Dysfunction through Activating the Nrf2/HO-1 Pathway in Intervertebral Disc Degeneration

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Ruihong Wang ◽  
Dawei Luo ◽  
Zhiwei Li ◽  
Huimin Han
Keyword(s):  
Oxidative Stress ◽  
Intervertebral Disc ◽  
Western Blot ◽  
Er Stress ◽  
Nucleus Pulposus ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Dimethyl Fumarate ◽  
Matrix Composition ◽  
Type I

Background. Oxidative stress, inflammation, and nucleus pulposus cells (NPCs) apoptosis are involved in pathogenesis of intervertebral disc (IVD) degeneration (IVDD). Dimethyl fumarate (DMF) has been found to effectively depress oxidative stress and inflammation via the Nrf2 pathway. Hence, this project was designed to explore the underlying mechanisms of how DMF protects NPCs from damage by LPS challenge. Methods and Results. CCK8 assay and flow cytometry of apoptosis indicated that DMF treatment attenuated LPS-induced NPC damage. Western blot analysis demonstrated that DMF enhanced the expressions of nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in LPS-challenged NPCs. DMF treatment significantly decreased the accumulation of ROS, downregulated inflammatory cytokines (p-NF-κB, IL-1β, and TNF-α), and ER stress-associated apoptosis proteins (Bip, calpain-1, caspase-12, caspase-3, and Bax) in LPS-challenged NPCs. The level of antiapoptotic protein Bcl-2 was promoted by DMF treatment in LPS-challenged NPCs. Glutathione (GSH) assay showed that DMF treatment improved reduced to oxidized glutathione ratio in LPS-challenged NPCs. Furthermore, the results of western blot analysis indicated that in LPS-challenged NPCs, DMF treatment ameliorated the elevated levels of matrix degradation enzymes (MMP-13, aggrecanase 1) and type I collagen and the reduced levels of matrix composition (type II collagen and ACAN). However, Nrf2 knockdown abolished these protective effects of DMF. Conclusion. Our data suggested that treatment with DMF mitigated LPS-induced oxidative stress, inflammation, and ER stress-associated apoptosis in NPCs via the Nrf2/HO-1 signaling pathway, thus reliving LPS-induced dysfunction of NPCs, which offered a novel potential pharmacological treatment strategy for IVDD.

scholarly journals Kaempferol and Kaempferide Attenuate Oleic Acid-Induced Lipid Accumulation and Oxidative Stress in HepG2 Cells

2021 ◽  
Vol 22 (16) ◽  
pp. 8847
Author(s):  
Fangfang Tie ◽  
Jin Ding ◽  
Na Hu ◽  
Qi Dong ◽  
Zhi Chen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Oleic Acid ◽  
Lipid Metabolism ◽  
Western Blot ◽  
Blot Analysis ◽  
Lipid Accumulation ◽  
Hepg2 Cells ◽  
Western Blot Analysis ◽  
Heme Oxygenase 1 ◽  
And Oxidative Stress

Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases which lacks ideal treatment options. Kaempferol and kaempferide, two natural flavonol compounds isolated from Hippophae rhamnoides L., were reported to exhibit a strong regulatory effect on lipid metabolism, for which the mechanism is largely unknown. In the present study, we investigated the effects of kaempferol and kaempferide on oleic acid (OA)-treated HepG2 cells, a widely used in vitro model of NAFLD. The results indicated an increased accumulation of lipid droplets and triacylglycerol (TG) by OA, which was attenuated by kaempferol and kaempferide (5, 10 and 20 μM). Western blot analysis demonstrated that kaempferol and kaempferide reduced expression of lipogenesis-related proteins, including sterol regulatory element-binding protein 1 (SREBP1), fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD-1). Expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT enhancer binding proteins β (C/EBPβ), two adipogenic transcription factors, was also decreased by kaempferol and kaempferide treatment. In addition, western blot analysis also demonstrated that kaempferol and kaempferide reduced expression of heme oxygenase-1 (HO-1) and nuclear transcription factor-erythroid 2-related factor 2 (Nrf2). Molecular docking was performed to identify the direct molecular targets of kaempferol and kaempferide, and their binding to SCD-1, a critical regulator in lipid metabolism, was revealed. Taken together, our findings demonstrate that kaempferol and kaempferide could attenuate OA-induced lipid accumulation and oxidative stress in HepG2 cells, which might benefit the treatment of NAFLD.

scholarly journals Fucoxanthin Inhibits Myofibroblast Differentiation and Extracellular Matrix Production in Nasal Polyp-Derived Fibroblasts via Modulation of Smad-Dependent and Smad-Independent Signaling Pathways

Marine Drugs ◽  
2018 ◽  
Vol 16 (9) ◽  
pp. 323 ◽  
Author(s):  
Hyun Jung ◽  
Dae-Sung Lee ◽  
Seong Park ◽  
Jung Choi ◽  
Won-Kyo Jung ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathways ◽  
Nasal Polyp ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Type I ◽  
Molecular Signaling ◽  
Tgf Β1

Nasal polyps (NPs) are a multifactorial disorder associated with a chronic inflammatory state of the nasal mucosa. Fucoxanthin (Fx) is a characteristic orange carotenoid obtained from brown algae and has diverse immunological properties. The present study investigated whether Fx inhibits fibrosis-related effects in nasal polyp-derived fibroblasts (NPDFs) and elucidated the molecular signaling pathways involved. The production of collagen type I (Col-1) was investigated in NP tissue via immunohistochemistry and western blot analysis. NPDFs were treated with transforming growth factor (TGF)-β1 (1 ng/mL) in the presence or absence of Fx (5–30 µM). The levels of α-smooth muscle actin (α-SMA), Col-1, and phosphorylated (p)-Smad 2/3, signal protein-1 (SP-1), MAPKs (mitogen-activated protein kinases), and Akt were measured by western blot analysis. The expression of Col-1 was detected in NP tissues. TGF-β1 stimulated the production of α-SMA and Col-1, and stimulated the contraction of collagen gel. However, pretreatment with Fx attenuated these effects. Furthermore, these inhibitory effects were mediated through modulation of both Smad 2/3 and Akt/SP-1 signaling pathways in TGF-β1-induced NPDFs. The results from the present study suggest that Fx may be a novel anti-fibrotic agent for the treatment of NP formation.

scholarly journals Is Oxidative Stress in Mice Brain Regions Diminished by 2-[(2,6-Dichlorobenzylidene)amino]-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile?

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
A. C. Fortes ◽  
A. A. C. Almeida ◽  
G. A. L. Oliveira ◽  
P. S. Santos ◽  
W. De Lucca Junior ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Lipid Peroxidation ◽  
Superoxide Dismutase ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Brain Regions ◽  
Saline Control ◽  
Control Group ◽  
Nitrite Content

2-[(2,6-Dichlorobenzylidene)amino]-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile, 5TIO1, is a new 2-aminothiophene derivative with promising pharmacological activities. The aim of this study was to evaluate its antioxidant activity in different areas of mice central nervous system. Male Swiss adult mice were intraperitoneally treated with Tween 80 dissolved in 0.9% saline (control group) and 5TIO1 (0.1, 1, and 10 mg kg−1). Brain homogenates—hippocampus, striatum, frontal cortex, and cerebellum—were obtained after 24 h of observation. Superoxide dismutase and catalase activities, lipid peroxidation and nitrite content were measured using spectrophotometrical methods. To clarify the 5TIO1’s mechanism on oxidative stress, western blot analysis of superoxide dismutase and catalase was also performed. 5TIO1 decreased lipid peroxidation and nitrite content in all brain areas and increased the antioxidant enzymatic activities, specially, in cerebellum. The data of Western blot analysis did not demonstrate evidence of the upregulation of these enzymes after the administration of this compound. Our findings strongly support that 5TIO1 can protect the brain against neuronal damages regularly observed during neuropathologies.

scholarly journals Biochemical and Immuno-Histochemical Localization of Type IIA Procollagen in Annulus Fibrosus of Mature Bovine Intervertebral Disc

2021 ◽  
Author(s):  
Audrey McAlinden ◽  
David M Hudson ◽  
Aysel A Fernandes ◽  
Soumya Ravindran ◽  
Russell J Fernandes
Keyword(s):  
Intervertebral Disc ◽  
Nucleus Pulposus ◽  
Annulus Fibrosus ◽  
Small Diameter ◽  
Type Ii Collagen ◽  
Intervertebral Discs ◽  
Matrix Composition ◽  
Type I ◽  
Type Ii ◽  
Post Translational Modification

For next generation tissue-engineered constructs and regenerative medicine to succeed clinically, the basic biology and extracellular matrix composition of tissues that these repair techniques seek to restore have to be fully determined. Using the latest reagents coupled with tried and tested methodologies, we continue to uncover previously undetected structural proteins in mature intervertebral disc. In this study we show that the ″embryonic″ type IIA procollagen isoform (containing a cysteine-rich amino propeptide) was biochemically detectable in the annulus fibrosus of both calf and mature steer intervertebral discs, but not in the nucleus pulposus where the type IIB isoform was predominantly localized. Specifically, the triple-helical type IIA procollagen isoform immunolocalized in the outer margins of the inner annulus fibrosus. Triple helical processed type II collagen exclusively localized within the inter- lamellae regions and with type IIA procollagen in the intra-lamellae regions. Mass spectrometry of the a1(II) collagen chains from the region where type IIA procollagen localized showed high 3-hydroxylation of Proline-944, a post- translational modification that is correlated with thin collagen fibrils as in the nucleus pulposus. The findings implicate small diameter fibrils of type IIA procollagen in select regions of the annulus fibrosus where it likely contributes to the organization of collagen bundles and structural properties within the type I- type II collagen transition zone.

Apoptosis induction mediated through PI3-kinase/AKT/mTOR pathway using anti-ROR1 monoclonal antibody in chronic lymphocytic leukemia cells.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 7087-7087
Author(s):  
Amir Hossein Daneshmanesh ◽  
Mohammad Hojat Farsangi ◽  
Ali Moshfegh ◽  
Salam Khan ◽  
Anders Österborg ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mammalian Target Of Rapamycin ◽  
Signaling Molecules ◽  
Mtor Pathway ◽  
Lymphocytic Leukemia ◽  
Type I ◽  
Aberrant Expression ◽  
Downstream Signaling

7087 Background: The PI3K/AKT/mTOR is a central pathway activated in many types of cancer. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase regulating cell growth, proliferation and survival. In CLL cells PI3K pathway is constitutively activated leading to AKT activation with subsequent phosphorylation of other downstream signaling molecules. ROR1 is a type I transmembrane RTK, overexpressed and constitutively phosphorylated in CLL. A unique anti-ROR1 mAb directed against CRD region of ROR1 was capable of inducing direct apoptosis as well as dephosphorylating the ROR1 molecule. Here, we investigated the apoptotic effect of the anti-ROR1 mAb and effects on the PI3K/AKT/mTOR pathway using primary CLL cells. Methods: Apoptosis was detected by the MTT assay and Annexin V/PI methods in a 24 h assay. Antibody untreated and treated cell lysates were prepared and subjected to Western blot analysis for identification of the signaling molecules involved in apoptosis induced by the ROR1 mAb. We analysed total and phosphorylated levels of the following signaling proteins: AKT, p-AKT, PI3K, p-PI3K, mTOR, p-mTOR, ERK, p-ERK, PKC and p-PKC. Phosphoproteins were measured before incubation with the mAb and after 20 min-24 h. Results: ROR1 detection on surface of the CLL cells was 80-85% and apoptotic frequency 45-50%. Western blot analysis showed decreased levels of p-AKT, p85 isoform of p-PI3K and p-mTOR in treated compared to untreated samples. No changes in the phosphorylation levels of ERK and PKC proteins were seen. Conclusions: Incubation of CLL cells with the anti-ROR1 mAb induced apoptosis of CLL cells. Apoptosis was preceded by dephosphorylation of PI3K, AKT and mTOR proteins indicating deactivation of these proteins by the ROR1 mAb. In untreated CLL cells no effect was noted. Furthermore no dephosphorylation of PKC or ERK was seen. We suggest that activation of mTOR might occur via the PI3K/AKT pathway and may be a survival signal in CLL cells associated with the aberrant expression of ROR1. Further studies are warranted to understand better the signaling pathways associated with ROR1 and the downstream signaling effects of ROR1 targeting drugs.

Autophagic and apoptotic types of programmed cell death exhibit different fates of cytoskeletal filaments

2000 ◽  
Vol 113 (7) ◽  
pp. 1189-1198 ◽  
Author(s):  
W. Bursch ◽  
K. Hochegger ◽  
L. Torok ◽  
B. Marian ◽  
A. Ellinger ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Kinase Inhibitor ◽  
Autophagic Cell Death ◽  
Type I ◽  
Autophagic Vacuoles ◽  
Autophagic Degradation

Programmed cell death comprises several subtypes, as revealed by electron microscopy. Apoptosis or type I programmed cell death is characterized by condensation of cytoplasm and preservation of organelles, essentially without autophagic degradation. Autophagic cell death or type II programmed cell death exhibits extensive autophagic degradation of Golgi apparatus, polyribosomes and endoplasmatic reticulum, which precedes nuclear destruction. In the present study, we analysed the fate of cytokeratin and F-actin during autophagic cell death in the human mammary carcinoma cell line MCF-7 because recent studies suggest that an intact cytoskeleton is necessary for autophagocytosis. Programmed cell death was induced by 10(-)(6) M tamoxifen. For quantitative light microscopic analysis, autophagic vacuoles were visualized by monodansyl cadaverin, which stains autophagic vacuoles as distinct dot-like structures. In control cultures, the number of monodansylcadaverin-positive cells did not exceed 2%. Tamoxifen induced a dramatic increase 2–4 days after treatment to a maximum of 60% monodansylcadaverin-positive cells between days 5 and 7. Cell death, as indicated by nuclear condensation, increased more gradually to about 18% of all cells on day 7. In cells with pyknotic nuclei cytokeratin appeared disassembled but retained its immunoreactivity; actin was still polymerized to filaments, as demonstrated by its reaction with phalloidin. Western blot analysis showed no significant cleavage of the monomeric cytokeratin fraction. For comparison, apoptotic or type I cell death was studied using the human colon cancer cell HT29/HI1 treated with the tyrosine kinase inhibitor tyrphostin A25 as a model. Cleavage of cytokeratin was already detectable in early morphological stages of apoptosis. F-actin was found to depolymerize; its globular form could be detected by antibodies; western blot analysis revealed no products of proteolytic cleavage. In conclusion, in our model of apoptosis, early stages are associated with depolymerization of actin and degradation of intermediate filaments. In contrast, during autophagic cell death intermediate and microfilaments are redistributed, but largely preserved, even beyond the stage of nuclear collapse. The present data support the concept that autophagic cell death is a separate entity of programmed cell death that is distinctly different from apoptosis.

Abstract 447: Activation of IL-1β-Producing Inflammasomes Triggered by RNA Receptor RIG-I in Mouse Endothelial Cells

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Yang Zhang ◽  
Xiang Li ◽  
Xiao-Xue Li ◽  
Ashley L Pitzer ◽  
Pin-Lan Li
Keyword(s):  
Endothelial Cells ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammasome Activation ◽  
Type I ◽  
Mrna Levels ◽  
Cholesterol Crystals ◽  
Double Stranded Rna ◽  
Caspase 1

Retinoic acid-inducible gene-I (RIG-I) is a putative RNA helicase and recently identified as a cytosolic RNA receptor in mammalian cells. The role of RIG-I in the regulation of vascular function under physiological and pathological conditions is unknown. The present study tested whether RIG-I activation triggers inflammasome formation, turning on inflammation in mouse endothelial cells (EOMA cell line). By real time RT-PCR and Western blot analysis, transfection of mouse ECs with RIG-I specific agonist, 5’-triphosphate double-stranded RNA (3pRNA, 0.5 mg/L) increased RIG-I mRNA level by 106% and protein level by 81% compared to those in control double-stranded RNA (dsRNA) transfected ECs. ELISA analyses showed that 3pRNA significantly increased release of type I IFN alpha by 31 folds and IL-1 beta (a prototype cytokine from inflammasome activation) by 8 folds in these ECs. Proatherogenic stimulation of mouse ECs with cholesterol crystals or 7-ketocholesterol also markedly increased protein expression of RIG-I, but had no effect on RIG-I mRNA levels. Measurements of active caspase-1, an inflammasome activation marker using FLICA fluorescent probe that specifically binds to cleaved caspase-1, demonstrated that 3pRNA doubled FLICA positive cells compared to that in control dsRNA transfected ECs. Interestingly, cholesterol crystals significantly increased FLICA positive cells by 3 folds. This activation of caspase-1 in ECs by cholesterol crystals was further confirmed by increase in cleaved caspase-1 (p10) using Western blot analysis and by enhanced IL-1 beta release as detected by ELISA. In the presence of 3pRNA, cholesterol crystal-induced inflammasome activation was not further augmented. These data indicate that increased expression and activity of RIG-I activate IL-1 beta producing inflammasomes in ECs, which may represent an early molecular mechanism mediating vascular inflammation or injury upon atherogenic stimulations.

Rhamnazin Ameliorates Traumatic Brain Injury in Mice via Reduction in Apoptosis, Oxidative Stress, and Inflammation

2021 ◽  
pp. 1-8
Author(s):  
Boxiao Yang ◽  
Rui Zhang ◽  
Qire Sa ◽  
Yanli Du
Keyword(s):  
Oxidative Stress ◽  
Traumatic Brain Injury ◽  
Flow Cytometry ◽  
Brain Injury ◽  
Western Blot ◽  
Protective Effect ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Neuronal Degeneration ◽  
Flow Cytometry Analysis

<b><i>Background:</i></b> Traumatic brain injury (TBI) is posing serious health challenges for people across the globe due to high morbidity and mortality. However, none of the agents prevents or limits the damage caused by TBI because of its multifactorial etiology. Thus, the discovery of novel agents which can act via several pathways could serve the purpose and afford favorable consequence against TBI. Therefore, in the present article, we intended to investigate the protective effect of rhamnazin (RMZ), a dimethoxyflavone against experimentally induced TBI in mice. <b><i>Methods:</i></b> The effect of RMZ was investigated on cerebral edema and grip test score after induction of experimental brain injury in rats. The effect of RMZ was also investigated on neuronal degeneration in brain tissues of the experimental mice via Nissl staining and flow cytometry analysis. The expression of Bax and Bcl-2 was also quantified using Western blot analysis. The level of inflammatory cytokines (TNF-α and IL-1β) and oxidative stress markers (malondialdehyde, superoxide dismutase, and glutathione peroxidase) was also determined using enzyme-linked immunosorbent assay. <b><i>Results:</i></b> RMZ showed a significant reduction in edema and improved grip strength. It also prevented neuronal degeneration via inhibition of neuronal apoptosis as shown by flow cytometry analysis. RMZ showed an antiapoptotic effect via reduction of Bax and increased the expression of Bcl-2 in Western blot analysis. It also showed to inhibit oxidative stress and inflammation compared to the TBI group. <b><i>Conclusion:</i></b> Collectively, our study is first to demonstrate the protective effect of RMZ against experimentally induced TBI in rats.

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