scholarly journals Suppression of lncRNA MALAT1 Reduces LPS- or IL-17A-Induced Inflammatory Response in Human Middle Ear Epithelial Cells via the NF-κB Signaling Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Xiangru Yang ◽  
Qinna Zhang ◽  
Hui Lu ◽  
Chenxin Wang ◽  
Lijun Xia
Keyword(s):  
Inflammatory Response ◽  
Western Blot ◽  
Middle Ear ◽  
Blot Analysis ◽  
Proinflammatory Cytokine ◽  
Western Blot Analysis ◽  
Cytokine Production ◽  
Proinflammatory Cytokine Production ◽  
Protein Levels ◽  
Human Middle Ear

Otitis media (OM) is a common inflammatory disease of the middle ear cavity and mainly occurs in children. As a critical regulator of inflammation response, the nuclear factor kappa B (NF-κB) pathway has been found to play an essential role in the pathogenesis of various human diseases. The aim of this study was to explore the potential mechanism under the inflammatory response of human middle ear epithelial cells (HMEECs). We established in vitro models of OM by treating HMEECs with lipopolysaccharide (LPS) or interleukin 17A (IL-17A). Enzyme-linked immunosorbent assay and western blot analysis were used to measure the inflammatory response of HMEECs under LPS or IL-17A stimulation. The results revealed that the concentrations of proinflammatory cytokines ( p < 0.001 ) and protein levels of mucin (MUC) (for MUC5AC, p = 0.002 , p = 0.004 ; for MUC8, p = 0.004 , p < 0.001 ) were significantly elevated by LPS or IL-17A stimulation in HMEECs. Moreover, we found that LPS or IL-17A treatment promoted the phosphorylation of IκBα (for p-IκBα, p = 0.018 , p = 0.002 ; for IκBα, p = 0.238 , p = 0.057 ) and the translocation of p65 from cytoplasm to nucleus in HMEECs (for nucleus p65, p = 0.01 ; for cytoplasm p65, p < 0.001 ). In addition, RT-qPCR analysis revealed that long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was verified to be upregulated in LPS- or IL-17A-stimulated HMEECs ( p < 0.001 ). Western blot analysis and immunofluorescence staining assay revealed that that MALAT1 knockdown significantly suppressed the activation of the NF-κB pathway by reducing phosphorylated IκBα levels and inhibiting the nuclear translocation of p65 ( p < 0.001 ) in LPS- or IL-17A-stimulated HMEECs (for p-IκBα, p < 0.001 ; for IκBα, p = 0.242 , p = 0.647 ). Silence of MALAT1 decreased the proinflammatory cytokine production and MUC protein levels ( p < 0.001 ). Furthermore, rescue assays revealed that the increase of proinflammatory cytokine production (for TNF-α, p = 0.002 , p = 0.015 ; for IL-1β, p < 0.001 , p = 0.006 ; for IL-6, p = 0.002 , p < 0.001 ) and MUC protein levels (for MUC5AC, p = 0.001 , p < 0.001 ; for MUC8, p < 0.001 , p = 0.001 ) induced by MALAT1 overexpression was neutralized by 4-N-[2-(4-phenoxyphenyl) ethyl] quinazoline-4, 6-diamine (QNZ) treatment in LPS- or IL-17A-stimulated HMEECs. In conclusion, MALAT1 promotes inflammatory response in LPS- or IL-17A- stimulated HMEECs via the NF-κB signaling pathway, which may provide a potential novel insight for the treatment of OM.

Visfatin facilitates gastric cancer malignancy by targeting snai1 via the NF-κB signaling

2021 ◽  
pp. 096032712110061
Author(s):  
D Cao ◽  
L Chu ◽  
Z Xu ◽  
J Gong ◽  
R Deng ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Migration ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Epithelial Mesenchymal Transition ◽  
Migration And Invasion ◽  
Mesenchymal Transition ◽  
Cell Migration And Invasion ◽  
Protein Levels

Background: Visfatin acts as an oncogenic factor in numerous tumors through a variety of cellular processes. Visfatin has been revealed to promote cell migration and invasion in gastric cancer (GC). Snai1 is a well-known regulator of EMT process in cancers. However, the relationship between visfatin and snai1 in GC remains unclear. The current study aimed to explore the role of visfatin in GC. Methods: The RT-qPCR and western blot analysis were used to measure RNA and protein levels, respectively. The cell migration and invasion were tested by Trans-well assays and western blot analysis. Results: Visfatin showed upregulation in GC cells. Additionally, Visfatin with increasing concentration facilitated epithelial-mesenchymal transition (EMT) process by increasing E-cadherin and reducing N-cadherin and Vimentin protein levels in GC cells. Moreover, endogenous overexpression and knockdown of visfatin promoted and inhibited migratory and invasive abilities of GC cells, respectively. Then, we found that snai1 protein level was positively regulated by visfatin in GC cells. In addition, visfatin activated the NF-κB signaling to modulate snai1 protein expression. Furthermore, the silencing of snai1 counteracted the promotive impact of visfatin on cell migration, invasion and EMT process in GC. Conclusion: Visfatin facilitates cell migration, invasion and EMT process by targeting snai1 via the NF-κB signaling, which provides a potential insight for the treatment of GC.

scholarly journals Implication of Hypoxia-Inducible Factor-1 (HIF-1) As a New Therapeutic Target in JAK2V617F Positive Myeloproliferative Neoplasms (MPN)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4318-4318 ◽  
Author(s):  
Julian Baumeister ◽  
Nicolas Chatain ◽  
Annika Hubrich ◽  
Caroline Küstermann ◽  
Stephanie Sontag ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Line ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Research Funding ◽  
Myeloproliferative Neoplasms ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Protein Levels

Abstract Myeloproliferative neoplasms (MPN) are a heterogeneous group of malignancies including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The JAK2V617F mutation can be found in 90% of PV and approximately 50% of ET and PMF patients. Hypoxia-inducible factors (HIFs) are master transcriptional regulators of the response to decreases in cellular oxygen levels. Unveiling the function of deregulated HIF-1 signaling in normal and malignant hematopoiesis was the aim of several recent publications, highlighting the importance of HIF-1 for the maintenance of leukemic stem cells (LSCs) in acute and chronic myeloid leukemia (AML/CML). In a JAK2V617F knock-in mouse model and in patients, JAK2V617F was shown to induce the accumulation of reactive oxygen species (ROS) in the hematopoietic stem cell compartment, leading to a stabilization of HIF-1α protein. Further, aberrant STAT5 and PI3K/AKT/mTOR signaling induced HIF-1α expression on the transcriptional and translational level. Ruxolitinib treatment inhibited growth and reduced the expression of HIF-1α and its target gene VEGF in the JAK2V617F human erythroleukemia cell line HEL. In several leukemic cell lines constitutive expression of HIF-1α was reported, even under normoxic conditions. However, it still remains unknown whether HIF-1α plays a role in JAK2V617F positive MPN. In this study, we investigated the role HIF-1α signaling in JAK2V617F positive MPN in vitro. We retrovirally transduced the murine bone marrow cell line 32D with JAK2V617F or JAK2WT. Western blot analysis revealed significant increases in HIF-1α protein levels in JAK2V617F positive cells compared to JAK2WT controls after cultivation in normoxic conditions and this effect was abrogated by treatment with the JAK1/JAK2 inhibitor ruxolitinib. Inhibition of HIF-1, binding to hypoxia response elements (HRE), by low doses of echinomycin (1 nM), significantly impaired proliferation and survival. Using an Annexin-V/7-AAD flow cytometry assay apoptosis was found to be selectively induced in JAK2V617F positive, but not JAK2WT cells after echinomycin treatment. Additionally, BrdU/7-AAD cell cycle analysis revealed that only JAK2V617F positive cells were significantly arrested in G0/1 phase. These findings were consistent with shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F transduced 32D cells in presence but not the absence of HIF-2 antagonist 2. Inhibition of HIF-2 was necessary due to a compensatory increase of HIF-2α protein levels, shown by Western Blot analysis, counteracting HIF-1α-KD mediated effects. We isolated PBMCs and BMMNCs from JAK2V617F positive patients or healthy controls using Ficoll density gradient centrifugation. Echinomycin significantly abrogated the colony formation ability alone and in combination with ruxolitinib. In vitro treatment with echinomycin significantly decreased cell number and viability of 8 JAK2V617F positive BMMNC samples (4 PV, 3 PMF, 1 preMF; p[1nM]=0.0169, p[5nM]=0.0009) and 7 PBMC samples (6 PV, 1 PMF; p[1nM]=0.0156, p[5nM]=0.0156) in a dose-dependent manner. In contrast, PBMCs from 6 healthy donors were unaffected by the treatment. The same effect was observed in heterozygous and homozygous iPS cell-derived progenitors from JAK2V617F positive PV patients, whereas JAK2WT cells were unaffected by the treatment. Collectively, our data indicate that targeting HIF-1 might represent a novel therapeutic approach in classical Philadelphia-chromosome-negative MPN. Disclosures Brümmendorf: Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Takeda: Consultancy.

Long non-coding RNA LINC01194 promotes the inflammatory response and apoptosis of LPS-treated MLE 12 cells through the miR-203a-3p /MIP-2 axis

2021 ◽  
Author(s):  
Yuyao Shen ◽  
Senwei Zhao ◽  
Minglei Hua
Keyword(s):  
Inflammatory Response ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Luciferase Reporter ◽  
Mouse Lung ◽  
Elisa Assay ◽  
Non Coding Rna ◽  
Potential Biomarker ◽  
Long Non Coding Rna

Acute lung injury (ALI) induced by bacteria LPS is characterized by the upregulation of the apoptosis rate of tissue cells and aggravation of inflammatory response. Although many studies have focused on the pathogenesis of this disease, its mechanism remains unknown. This study examined the regulatory role of long non-coding RNA (lncRNA) LINC01194 in the progression of ALI through various bioinformatics analyses and experimental work, including ELISA assay, dual-luciferase reporter assay, biotinylated RNA pull-down assay, and western blot analysis. The result showed that the LINC01194 was overexpressed in the ALI-induced mice model. We observed a significant upregulation of LINC01194 in LPS-treated Mouse lung epithelial type II cells (MLE-12 cells) after 24 hrs of induction. Bioinformatics analysis, Elisa assay, qRT-PCR analysis, Biotinylated RNA pull-down assay, apoptosis test, and western blot analysis demonstrated that the LINC01194 could act as a miR-203a-3p sponge to activate the inflammatory response in LPS-induced ALI model through post-transcriptional upregulation of MIP-2. We showed that LINC01194 regulates the inflammatory response and apoptosis of LPS-induced mice and MLE-12 cells via the miR-203a-3p/MIP-2 axis. LINC01194 could be a potential biomarker for early diagnosis and the treatment of ALI.

Functional overlap of the dictyostelium RasG, RasD and RasB proteins

2000 ◽  
Vol 113 (8) ◽  
pp. 1427-1434 ◽  
Author(s):  
M. Khosla ◽  
G.B. Spiegelman ◽  
R. Insall ◽  
G. Weeks
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ras Proteins ◽  
Wild Type ◽  
Vegetative Cells ◽  
Protein Levels ◽  
Ras Genes ◽  
Downstream Effector ◽  
In The Wild

Disruption of the rasG gene in Dictyostelium discoideum results in several distinct phenotypes: a defect in cytokinesis, reduced motility and reduced growth. Reintroduction of the rasG gene restores all of the properties of the rasG(-) cells to those of the wild type. To determine whether the defects are due to impaired interactions with a single or multiple downstream effectors, we tested the ability of the highly related but non identical Dictyostelium ras genes, rasD and rasB, to rescue the defects. Introduction of the rasD gene under the control of the rasG promoter into rasG null (rasG(-)) cells corrected all phenotypes except the motility defect, suggesting that motility is regulated by a RasG mediated pathway that is different to those regulating growth or cytokinesis. Western blot analysis of RasD protein levels revealed that vegetative rasG(-)cells contained considerably more protein than the parental AX-3 cells, suggesting that RasD protein levels are negatively regulated in vegetative cells by RasG. The level of RasD was enhanced when the rasD gene was introduced under the control of the rasG promoter, and this increase in protein is presumably responsible for the reversal of the growth and cytokinesis defects of the rasG(-)cells. Thus, RasD protein levels are controlled by the level of RasG, but not by the level of RasD. Introduction of the rasB gene under the control of the rasG promoter into rasG(-) cells produced a complex phenotype. The transformants were extremely small and mononucleate and exhibited enhanced motility. However, the growth of these cells was considerably slower than the growth of the rasG(-) cells, suggesting the possibility that high levels of RasB inhibit an essential process. This was confirmed by expressing rasB in wild-type cells; the resulting transformants exhibited severely impaired growth. When RasB protein levels were determined by western blot analysis, it was found that levels were higher in the rasG(-)cells than they were in the wild-type parental, suggesting that RasG also negatively regulates rasB expression in vegetative cells. Overexpression of rasB in the rasG(-)cells also reduced the level of RasD protein. In view of the fact that alternate Ras proteins correct some, but not all, of the defects exhibited by the rasG(-) cells, we propose that RasG interacts with more than one downstream effector. In addition, it is clear that the levels of the various Ras proteins are tightly regulated in vegetative cells and that overexpression can be deleterious.

scholarly journals Stimulation of iodide uptake by human chorionic gonadotropin in FRTL-5 cells: effects on sodium/iodide symporter gene and protein expression

2002 ◽  
pp. 655-661 ◽  
Author(s):  
F Arturi ◽  
I Presta ◽  
D Scarpelli ◽  
JM Bidart ◽  
M Schlumberger ◽  
...  
Keyword(s):  
Western Blot ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Iodide Uptake ◽  
Protein Levels

BACKGROUND: Various clinical and experimental findings support the concept that human chorionic gonadotropin (hCG) can stimulate iodide uptake in thyroid cells. DESIGN: We investigated the molecular mechanisms underlying the effects of hCG on iodide uptake, and particularly its action on the expression of Na+/I- symporter (NIS) mRNA and protein. METHODS: Iodide uptake was analyzed in FTRL-5 cells by measuring (125)I concentrations in cells after a 30-min exposure to 0.1 microCi carrier-free Na (125)I in the presence or absence of hCG or, for control purposes, TSH. Expression of NIS mRNA and NIS protein synthesis were evaluated, respectively, with a semiquantitative 'multiplex' RT-PCR method and Western blot analysis. RESULTS: Iodide uptake was increased by hCG in a dose- and time-dependent manner: maximal effects were observed after 72 h of stimulation. The effect was cAMP dependent and paralleled that of TSH, although it lacked the early cycloheximide-independent component seen with TSH, and its peak effect was lower. Semiquantitative multiplex RT-PCR revealed that hCG produced a significant increase in NIS mRNA levels that was detectable after 4 h and peaked after 48 h. In contrast, in TSH-stimulated FRTL-5 cells, maximum NIS mRNA expression was observed after 24 h of stimulation. Western blot analysis demonstrated that hCG also caused a 2.5-fold increase over basal values in NIS protein levels, which was similar to that observed after TSH stimulation although the peak effects of the latter hormone were less marked and occurred earlier. CONCLUSION: Our data demonstrated that hCG stimulates iodide uptake in FRTL-5 cells by increasing NIS mRNA and protein levels. Thus, the functional status of the thyroid may be influenced by hCG-dependent changes in NIS expression occurring during pregnancy.

scholarly journals Antioxidant/Anti-Inflammatory Effects of Caloric Restriction in an Aged and Obese Rat Model: The Role of Adiponectin

Biomedicines ◽  
2020 ◽  
Vol 8 (12) ◽  
pp. 532
Author(s):  
Daniele La Russa ◽  
Alessandro Marrone ◽  
Maurizio Mandalà ◽  
Rachele Macirella ◽  
Daniela Pellegrino
Keyword(s):  
Adipose Tissue ◽  
Western Blot ◽  
Caloric Restriction ◽  
Rat Model ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Normal Weight ◽  
Protein Levels ◽  
Oxidative Balance ◽  
Anti Inflammatory

Caloric restriction (CR) represents a powerful intervention for extending healthspan and lifespan in several animal models, from yeast to primates. Additionally, in humans, CR has been found to induce cardiometabolic adaptations associated with improved health. In this study, we evaluated in an aged and obese rat model the effect of long-term (6 months) caloric restriction (−40%) on the oxidative/inflammatory balance in order to investigate the underlining mechanisms. In plasma, we analyzed the oxidative balance by photometric tests and the adiponectin/tumor necrosis factor-α-induced gene/protein 6 (TSG-6) levels by Western blot analysis. In the white adipose tissue, we examined the protein levels of AdipoR1, pAMPK, NFκB, NRF-2, and glutathione S-tranferase P1 by Western blot analysis. Our results clearly showed that caloric restriction significantly improves the plasmatic oxidative/inflammatory balance in parallel with a major increase in circulating adiponectin levels. Additionally, at the level of adipose tissue, we found a positive modulation of both anti-inflammatory and antioxidant pathways. These adaptations, induced by caloric restriction, with the achievement of normal weight, suggest that inflammatory and redox imbalance in obese aged rats appear to be more linked to obesity than to aging.

scholarly journals Nobiletin, a hexamethoxyflavonoid from citrus pomace, attenuates G1 cell cycle arrest and apoptosis in hypoxia-induced human trophoblast cells of JEG-3 and BeWo via regulating the p53 signaling pathway

2021 ◽  
Author(s):  
Mengling Zhang ◽  
Jian Liu ◽  
Rui Zhang ◽  
Zengenni Liang ◽  
Shenghua Ding ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Blot Analysis ◽  
Cell Apoptosis ◽  
Western Blot Analysis ◽  
P53 Protein ◽  
Human Trophoblast ◽  
Protein Levels ◽  
Bewo Cells ◽  
Induced Apoptosis

Background: Hypoxia is associated with abnormal cell apoptosis in trophoblast cells, which causes fetal growth restriction and related placental pathologies. Few effective methods for the prevention and treatment of placenta-related diseases exist. Natural products and functional foods have always been a rich source of potential anti-apoptotic drugs. Nobiletin (NOB), a hexamethoxyflavonoid derived from the citrus pomace, shows an anti-apoptotic activity, which is a non-toxic constituent of dietary phytochemicals approved by the Food and Drug Administration. However, their effects on hypoxia-induced human trophoblast cells have not been fully studied. Objective: The aim of this study was to investigate the protective effects of NOB on hypoxia-induced apoptosis of human trophoblast JEG-3 and BeWo cells, and their underlying mechanisms. Design: First, the protective effect of NOB on hypoxia-induced apoptosis of JEG-3 and BeWo cells was studied. Cell viability and membrane integrity were determined by CCK-8 assay and lactate dehydrogenase activity, respectively. Real Time Quantitative PCR (RT-qPCR) and Western blot analysis were used to detect the mRNA and protein levels of HIF1α. Propidium iodide (PI)-labeled flow cytometry was used to detect cell cycle distribution. Cell apoptosis was detected by flow cytometry with Annexin V-FITC and PI double staining, and the expression of apoptosis marker protein cl-PARP was detected by Western blot analysis. Then, the molecular mechanism of NOB against apoptosis was investigated. Computer molecular docking and dynamics were used to simulate the interaction between NOB and p53 protein, and this interaction was verified in vitro by Ultraviolet and visible spectrum (UV-visible spectroscopy), fluorescence spectroscopy and circular dichroism. Furthermore, the changes in the expression of p53 signaling pathway genes and proteins were detected by RT-qPCR and Western blot analysis, respectively. Results: Hypoxia treatment resulted in a decreased cell viability and cell membrane integrity in JEG-3 and BeWo cell lines, and an increased expression of HIF1α, cell cycle arrest in the G1 phase, and massive cell apoptosis, which were alleviated after NOB treatment. Molecular docking and dynamics simulations found that NOB spontaneously bonded to human p53 protein, leading to the change of protein conformation. The intermolecular interaction between NOB and human p53 protein was further confirmed by UV-visible spectroscopy, fluorescence spectroscopy and circular dichroism. After the treatment of 100 μM NOB, a down-regulation of mRNA and protein levels of p53 and p21 and an up-regulation of BCL2/BAX mRNA and protein ratio were observed in JEG-3 cells; however, there was also a down-regulation of mRNA and protein levels observed for p53 and p21 in BeWo cells after the treatment of NOB. The BCL2/BAX ratio of BeWo cells did not change after the treatment of 100 μM NOB. Conclusion: NOB attenuated hypoxia-induced apoptosis in JEG-3 and BeWo cell lines and might be a potential functional ingredient to prevent pregnancy-related diseases caused by hypoxia-induced apoptosis. These findings would also suggest the exploration and utilization of citrus resources, and the development of citrus industry.

scholarly journals HMGB1 translocation and release mediate cigarette smoke–induced pulmonary inflammation in mice through a TLR4/MyD88-dependent signaling pathway

2017 ◽  
Vol 28 (1) ◽  
pp. 201-209 ◽  
Author(s):  
Yao Cheng ◽  
Dan Wang ◽  
Bin Wang ◽  
Huanan Li ◽  
Junjie Xiong ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Signaling Pathway ◽  
Cigarette Smoke ◽  
Proinflammatory Cytokine ◽  
Cytokine Production ◽  
Pulmonary Inflammation ◽  
High Mobility ◽  
Lung Epithelial Cells ◽  
Proinflammatory Cytokine Production ◽  
Tnf Α

We performed studies to determine the role of high-mobility group box 1 (HMGB1) in cigarette smoke (CS)–induced pulmonary inflammation. After mice were exposed to five cigarettes four times a day for 3 d, toll-like receptor 4 (TLR4) expression and TLR4-mediated signaling were significantly up-regulated, and HMGB1 had translocated from the nucleus to the cytoplasm in lung epithelial cells and then been released into the extracellular lung space. On CS exposure, inflammatory cell recruitment and proinflammatory cytokine production were significantly increased in lung tissue and bronchoalveolar lavage, and these effects depended on the TLR4 signaling pathway. HMGB1 inhibition decreased the CS-induced inflammatory response, whereas treatment with exogenous HMGB1 aggravated the damage and increased the phosphorylation of JNK, p38, and IκBα in the lungs of wild-type mice but not in TLR4-knockout mice. Blockade of TLR4 action or TLR4 knockout significantly inhibited HMGB1-induced proinflammatory cytokine production in mouse tracheal epithelial (MTE) cells and lung tissues. In addition, a MyD88 deficiency inhibited JNK, p38, and IκBα phosphorylation, and this effect was associated with the suppressed production of TNF-α and IL-1β in MTE cells and lung tissues in response to CS stimulation. Thus HMGB1 activates the NF-κB and JNK/p38 pathways through TLR4/MyD88-dependent signaling and induces an inflammatory response in lungs exposed to CS.

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