Immunohistochemical expression of proinflammatory cytokines IL-1β, IL-6, TNF-α and involvement of COX-2, quantitatively confirmed by Western blot analysis, in Wernicke's encephalopathy

2011 ◽  
Vol 207 (10) ◽  
pp. 652-658 ◽  
Author(s):  
Margherita Neri ◽  
Santina Cantatore ◽  
Cristoforo Pomara ◽  
Irene Riezzo ◽  
Stefania Bello ◽  
...  
Keyword(s):  
Western Blot ◽  
Proinflammatory Cytokines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Wernicke’S Encephalopathy ◽  
Wernicke's Encephalopathy ◽  
Immunohistochemical Expression ◽  
Tnf Α ◽  
Cox 2

scholarly journals Clarithromycin Inhibits NF-κB Activation in Human Peripheral Blood Mononuclear Cells and Pulmonary Epithelial Cells

2001 ◽  
Vol 45 (1) ◽  
pp. 44-47 ◽  
Author(s):  
Takashi Ichiyama ◽  
Miki Nishikawa ◽  
Tomomi Yoshitomi ◽  
Shunji Hasegawa ◽  
Tomoyo Matsubara ◽  
...  
Keyword(s):  
Western Blot ◽  
Proinflammatory Cytokines ◽  
Peripheral Blood Mononuclear Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mononuclear Cells ◽  
A549 Cells ◽  
Peripheral Blood Mononuclear ◽  
Tnf Α ◽  
Blood Mononuclear Cells

ABSTRACT Macrolide antibiotics modulate the production of proinflammatory cytokines in vivo and in vitro. Transcription of the genes for these proinflammatory cytokines is regulated by nuclear factor κB (NF-κB). We examined whether or not clarithromycin inhibits the activation of NF-κB induced by tumor necrosis factor alpha (TNF-α) or staphylococcal enterotoxin A (SEA) in human monocytic U-937 cells, a T-cell line (Jurkat), a pulmonary epithelial cell line (A549), and peripheral blood mononuclear cells (PBMC). Flow cytometry revealed that clarithromycin suppresses NF-κB activation induced by TNF-α in U-937 and Jurkat cells in a concentration-related manner. Western blot analysis also demonstrated that clarithromycin inhibits NF-κB activation induced by TNF-α in U-937, Jurkat, and A549 cells and PBMC and by SEA in PBMC. Western blot analysis of cytoplasmic extracts of A549 cells revealed that this inhibition is not linked to preservation of expression of the IκBα protein. The chloramphenicol acetyltransferase assay indicated that NF-κB-dependent reporter gene expression is suppressed in U-937 cells pretreated with clarithromycin. These findings are consistent with the idea that clarithromycin suppresses the production of proinflammatory cytokines via inhibition of NF-κB activation.

scholarly journals Punicalagin Prevents Inflammation in LPS-Induced RAW264.7 Macrophages by Inhibiting FoxO3a/Autophagy Signaling Pathway

Nutrients ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2794 ◽  
Author(s):  
Cao ◽  
Chen ◽  
Ren ◽  
Zhang ◽  
Tan ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammatory Responses ◽  
Raw264.7 Macrophages ◽  
Tnf Α ◽  
Autophagy Inhibition ◽  
Pro Inflammatory Cytokines

Punicalagin, a hydrolysable tannin of pomegranate juice, exhibits multiple biological effects, including inhibiting production of pro-inflammatory cytokines in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In this study, we investigated the anti-inflammatory potential of punicalagin in lipopolysaccharide (LPS) induced RAW264.7 macrophages and uncovered the underlying mechanisms. Punicalagin significantly attenuated, in a concentration-dependent manner, LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 release at the highest concentration. We found that punicalagin inhibited NF-κB and MAPK activation in LPS-induced RAW264.7 macrophages. Western blot analysis revealed that punicalagin pre-treatment enhanced LC3II, p62 expression, and decreased Beclin1 expression in LPS-induced macrophages. MDC assays were used to determine the autophagic process and the results worked in concert with Western blot analysis. In addition, our observations indicated that LPS-induced releases of NO, TNF-α, and IL-6 were attenuated by treatment with autophagy inhibitor chloroquine, suggesting that autophagy inhibition participated in anti-inflammatory effect. We also found that punicalagin downregulated FoxO3a expression, resulting in autophagy inhibition. Overall these results suggested that punicalagin played an important role in the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages and that the mechanisms involved downregulation of the FoxO3a/autophagy signaling pathway.

scholarly journals The Effect and Mechanism of Celecoxib in Hypoxia-Induced Survivin Up-Regulation in HUVECs

2015 ◽  
Vol 37 (3) ◽  
pp. 991-1001 ◽  
Author(s):  
Ning-ning Liu ◽  
Ning Zhao ◽  
Na Cai
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Time Course ◽  
Umbilical Vein ◽  
Survivin Expression ◽  
Mrna Levels ◽  
Cytoplasmic Staining ◽  
Hypoxic Conditions ◽  
Cox 2

Background/Aims: To investigate the roles of hypoxia-inducible factor 1α (HIF-1α), cyclooxygenase-2 (Cox-2) and its product, Prostaglandin E2 (PGE2), in the mechanisms underlying hypoxia-induced survivin expression in human umbilical vein endothelial cells (HUVECs) and to examine the effect of celecoxib, a selective Cox-2 inhibitor, on survivin expression. Methods: HUVECs were exposed to a normal (95% O2) or hypoxic (3% O2) environment for 24 hrs. We observed the localized expression of survivin, Cox-2 and HIF-1α in HUVECs using immunocytochemistry and detected the inhibitory effects of celecoxib on the growth of HUVECs using an MTT assay. mRNA and protein levels of Cox-2, HIF-1α and survivin were determined by real-time PCR and Western blot analysis under hypoxic conditions for 0, 6, 12, or 24 hrs. The time course changes of HIF-1α and survivin protein expression induced by cobalt chloride (CoCl2) were studied using Western blot analysis. We then treated HUVECs under hypoxia for 24 hrs with celecoxib (a Cox-2 selective inhibitor), genistein (a HIF-1α inhibitor) or exogenous PGE2 to further investigate the changes in hypoxia-induced survivin expression. Results: Following 24 hrs of hypoxic treatment, cells exhibited strongly positive survivin, HIF-1α and Cox-2 cytoplasmic staining. Celecoxib (65 μM) effectively inhibited cell proliferation under hypoxic conditions. The protein and mRNA levels of Cox-2, HIF-1α and survivin were increased under hypoxia. The patterns of HIF-1α and survivin expression induced by CoCl2 were similar to those induced by exposure to hypoxia. Genistein partially blocked survivin expression. Celecoxib reversed the hypoxia-induced survivin expression, whereas the addition of PGE2 partially restored this effect. Conclusions: Hypoxia-induced survivin expression in HUVECs may be mediated by dual interdependent mechanisms directly involving HIF-1α and indirectly involving the Cox-2/PGE2 pathways. Celecoxib may offset hypoxia-induced survivin expression.

scholarly journals Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

2022 ◽  
Vol 12 ◽  
Author(s):  
Ahmed M. Abu El-Asrar ◽  
Ajmal Ahmad ◽  
Mohd Imtiaz Nawaz ◽  
Mohammad Mairaj Siddiquei ◽  
Alexandra De Zutter ◽  
...  
Keyword(s):  
Diabetic Retinopathy ◽  
Matrix Metalloproteinase ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Tissue Inhibitor ◽  
Matrix Metalloproteinase 3 ◽  
Tnf Α ◽  
Brb Breakdown

Purpose: Endogenous tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) has powerful regulatory effects on inflammation and angiogenesis. In this study, we investigated the role of TIMP-3 in regulating inflammation in the diabetic retina.Methods: Vitreous samples from patients with proliferative diabetic retinopathy (PDR) and non-diabetic patients were subjected to Western blot analysis. Streptozotocin-treated rats were used as a preclinical diabetic retinopathy (DR) model. Blood-retinal barrier (BRB) breakdown was assessed with fluorescein isothiocyanate (FITC)-conjugated dextran. Rat retinas, human retinal microvascular endothelial cells (HRMECs) and human retinal Müller glial cells were studied by Western blot analysis and ELISA. Adherence of human monocytes to HRMECs was assessed and in vitro angiogenesis assays were performed.Results: Tissue inhibitor of matrix metalloproteinase-3 in vitreous samples was largely glycosylated. Intravitreal injection of TIMP-3 attenuated diabetes-induced BRB breakdown. This effect was associated with downregulation of diabetes-induced upregulation of the p65 subunit of NF-κB, intercellular adhesion molecule-1 (ICAM-1), and vascular endothelial growth factor (VEGF), whereas phospho-ERK1/2 levels were not altered. In Müller cell cultures, TIMP-3 significantly attenuated VEGF upregulation induced by high-glucose (HG), the hypoxia mimetic agent cobalt chloride (CoCl2) and TNF-α and attenuated MCP-1 upregulation induced by CoCl2 and TNF-α, but not by HG. TIMP-3 attenuated HG-induced upregulation of phospho-ERK1/2, caspase-3 and the mature form of ADAM17, but not the levels of the p65 subunit of NF-κB and the proform of ADAM17 in Müller cells. TIMP-3 significantly downregulated TNF-α-induced upregulation of ICAM-1 and VCAM-1 in HRMECs. Accordingly, TIMP-3 significantly decreased spontaneous and TNF-α- and VEGF-induced adherence of monocytes to HRMECs. Finally, TIMP-3 significantly attenuated VEGF-induced migration, chemotaxis and proliferation of HRMECs.Conclusion:In vitro and in vivo data point to anti-inflammatory and anti-angiogenic effects of TIMP-3 and support further studies for its applications in the treatment of DR.

Adenosine monophosphate-activated protein kinase activator inhibits activation of fibroblast-like synoviocytes but promotes hyaluronan and proteoglycan link protein 1 secretion

2020 ◽  
Author(s):  
Yong Chen ◽  
Qiu Fujuan ◽  
Yu Beijia ◽  
Zuo Fangfang ◽  
Bi Yanan ◽  
...  
Keyword(s):  
Western Blot ◽  
Rna Sequencing ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Enrichment Analysis ◽  
Protective Mechanism ◽  
Sequencing Data ◽  
Positive Correlation ◽  
Tnf Α ◽  
Fibroblast Like Synoviocytes

Abstract Objectives: To determine whether any correlation exists between disease activity and AMPK levels in rheumatoid arthritis (RA) patients and investigate the effects of AMPK activator treatment on RA fibroblast-like synoviocytes (RA-FLS).Methods: Serum AMPK-α1, p-AMPK-α1, TNF-α and IL-17 levels between osteoarthritis (OA) and RA patients having different disease activities were compared by ELISA. Differentially expressed genes (DEGs) between RA and OA synovium from NCBI GEO Profiles (accession numbers: GSE1202112, GSE55235, GSE5545713) were identified and the genes intersecting in all the three datasets were selected for enrichment analysis. Immunohistochemical staining was done with synovium obtained from OA and RA patients for p-AMPK-α1. AMPK gene expression in synovium was semi-quantified by RT-qPCR. RNA sequencing of FLS was performed and DEGs were selected for KEGG enrichment analysis. AMPK activator, metformin, treated RA-FLS were tested for proliferation and migration by MTT and scratch test, respectively. Expression of IL-6, AMPK-α1, PKA-α, RAPTOR, mTOR, HAPLN1, RUNX1 and RUNX2 genes were determined by qPCR. Phosphorylated AMPK-α1 and HAPLN1 levels were determined by an automated electrophoresis-western blot analysis method.Results: In RA sera, a positive correlation between p-AMPK-α1 levels and DAS28 (r = 0.270, 95%CI: 0.142-0.492, p < 0.0001) as well as CRP levels (r = 0.259, 95%CI: 0.009-0.478, p < 0.05) was found. Similarly, a positive correlation was observed between AMPK-α1 and TNF-α levels (r = 0.460, 95% CI: 0.241-0.640, p = 0.0002). DEGs between OA and RA synovium from NCBI GEO profiles and our RNA sequencing data suggested activation of metabolic pathways specific to RA-FLS. AMPK-α1 was highly expressed in the synovium of RA but not OA patients. Metformin at higher concentrations inhibited RA-FLS proliferation in a dose dependant manner, however, at lower concentrations it has an opposite effect. On the other hand, AMPK inhibitor, dorsmophin, promoted the proliferation of RA-FLS significantly. Interestingly, both metformin and dorsmophin substantially inhibited the migration of RA-FLS. In FLS, relative expression level of IL-6 mRNA was significantly decreased after metformin treatment, while the expression of AMPK-α1, PKA-α and HAPLN1 genes were significantly increased. Western blot analysis confirmed increased expression of p-AMPK-α1 and HAPLN1 genes in the metformin treated FLS.Conclusions: Inflammatory stress in RA synovium leads to an increase in AMPK levels, possibly as a protective mechanism. AMPK activator but not metformin per se could be a potential therapeutic for RA by promoting HAPLN1 secretion to protect the joints.

scholarly journals Effects of Neuromedin S on the Proliferation of Splenic Lymphocytes and the Cytokine Secretion by Pulmonary Alveolar Macrophages in Pigs in vitro

2016 ◽  
Vol 19 (3) ◽  
pp. 485-494 ◽  
Author(s):  
R. Lin ◽  
Q. Wang ◽  
B. Qi ◽  
Y. Huang ◽  
G. Yang
Keyword(s):  
Immune Response ◽  
Western Blot ◽  
Alveolar Macrophages ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Splenic Lymphocytes ◽  
Rt Pcr ◽  
Tnf Α ◽  
Pulmonary Alveolar Macrophages

Abstract Neuromedin S (NMS), a 36-amino acid neuropeptide, has been found to be involved in the regulation of the endocrine activity. It has been also detected in immune tissues in mammals, what suggests that NMS may play an important role in the regulation of immune response. The aim of this study was to demonstrate the presence of NMS receptor 1 (NMU1R) and effect of NMS in pig splenic lymphocytes (SPLs) and pulmonary alveolar macrophages (PAMs). The presence of NMU1R in pig SPLs and PAMs was respectively confirmed by reverse transcription-polymerase chain reaction (RT-PCR), western blot analysis and immunocytochemical methods. Furthermore, SPL proliferation was analyzed using the 3-(4,5)-dimethyl-thiahiazo-(-2-yl)-3,5-di-phenytetrazoliumromide (MTT) method. Additionally, the secretion of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) in PAMs was all measured by enzyme-linked immunosorbent assay (ELISA) kits. In the present study, the results of RT-PCR and western blot analysis revealed that NMU1R mRNA and protein were both expressed in pig SPLs and PAMs, and the immunocytochemical investigations further revealed that the positive signal of NMU1R immunoreactivity was observed in plasma membranes of both SPLs and PAMs. In the in vitro study, we found that at concentrations of 0.001-1000 nM NMS alone or combined with lipopolysaccharide or phytohemagglutinin significantly increased SPL proliferation. Application of ELISA method showed that NMS could induce the secretion of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in PAMs. These results suggest that NMS can act as a potently positive pro-inflammatory factor and immunomodulatory agent that affects the immune response of immune cells by combining with its receptor NMU1R.

FGF-7 facilitates the process of psoriasis by inducing TNF-α expression in HaCaT cells

2019 ◽  
Vol 51 (10) ◽  
pp. 1056-1063 ◽  
Author(s):  
Jiaojiao Pu ◽  
Rui Wang ◽  
Guanglin Zhang ◽  
Ju Wang
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Specific Antibody ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Quantitative Polymerase Chain Reaction ◽  
Hacat Cells ◽  
Tnf Α

Abstract The purpose of this study was to uncover the mechanism of tumor necrosis factor (TNF)-α induction by fibroblast growth factor-7 (FGF-7) in human HaCaT cells and the potential role of FGF-7-specific antibody F-9 in psoriatic therapy. TNF-α expression in HaCaT cells induced by FGF-7 was analyzed by quantitative polymerase chain reaction, western blot analysis, and enzyme-linked immunosorbent assays. In vivo, the BALB/c mouse psoriasis model established by topical application of imiquimod (IMQ) was used to determine the role of FGF-7-specific antibody (F-9) in skin inflammation. We found that induction of TNF-α expression by FGF-7 in HaCaT cells was suppressed by FGF-7-specific antibody F-9. Western blot analysis results showed that FGF-7 induced TNF-α expression in HaCaT cells via the FGF receptor 2 (FGFR2)/AKT/NF-κB signaling pathway. In vivo, F-9 could significantly ameliorate the inflammations in a mouse psoriatic model evaluated by Psoriasis Area and Severity Index scores and ear thickness, which was consistent with the results of hematoxylin–eosin staining, immunohistochemistry assay, and western blot analysis. These results indicate that FGF-7 induces TNF-α expression in HaCaT cells and FGF-7 antibody F-9 alleviates IMQ-induced psoriasiform in mice. Therefore, FGF-7/FGFR2 signaling pathway is a potential target for psoriasis treatment.

scholarly journals Expression of cyclooxygenase-2 in urinary bladder in rats with cyclophosphamide-induced cystitis

2007 ◽  
Vol 293 (2) ◽  
pp. R677-R685 ◽  
Author(s):  
Mary Beth Klinger ◽  
Abbey Dattilio ◽  
Margaret A. Vizzard
Keyword(s):  
Smooth Muscle ◽  
Urinary Bladder ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammatory Cell ◽  
Nerve Fibers ◽  
Cyclooxygenase 2 ◽  
Detrusor Smooth Muscle ◽  
Cox 2

These studies examined the expression of cyclooxygenase-2 (COX-2) expression in the urothelium and suburothelial space and detrusor from rats treated with cyclophosphamide (CYP) to induce acute (4 h), intermediate (48 h), or chronic (10-day) cystitis. Western blot analysis and immunohistochemistry were used to demonstrate COX-2 expression. In whole mount preparations of urinary bladder, nerve fibers in the suburothelial plexus, and inflammatory cell infiltrates were characterized for COX-2 expression after CYP-induced cystitis. COX-2 expression significantly ( P ≤ 0.01) increased in the urothelium + suburothelium and detrusor smooth muscle with acute, intermediate, and chronic (10-day) CYP-induced cystitis, but expression in urothelium + suburothelium was significantly greater. CYP-induced upregulation of COX-2 showed by immunostaining in the urothelium + suburothelium was similar to that observed with Western blot analysis and also demonstrated COX-2 inflammatory cell infiltrates (CD86+) and nerve fibers (PGP+) in the suburothelial plexus. Although COX-2 expression was significantly ( P ≤ 0.01) increased in detrusor smooth muscle, immunohistochemistry failed to demonstrate an obvious change in COX-2-immunoreactivity (IR) in detrusor muscle, but COX-2 inflammatory infiltrates were present throughout the detrusor. COX-2-IR nerve fibers exhibited increased density in the suburothelial plexus with acute or chronic CYP-induced cystitis. COX-2-IR macrophages (CD86+) were present throughout the urinary bladder with acute and chronic CYP-induced cystitis. These studies demonstrate cellular targets in the urinary bladder where COX-2 inhibitors may act.

scholarly journals Sinoacutine inhibits inflammatory responses to attenuates acute lung injury by regulating NF-κB and JNK signaling pathways

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yuancui Zhao ◽  
Lili Cui ◽  
Xing Xin Yang ◽  
Xingqian Sun ◽  
Yunkuan Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nitric Oxide ◽  
Acute Lung Injury ◽  
Lung Injury ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Signalling Pathway ◽  
Tnf Α ◽  
Anti Inflammatory

Abstract Background Stephania yunnanensis H. S. Lo is widely used as an antipyretic, analgesic and anti-inflammatory herbal medicine in SouthWest China. In this study, we investigated the anti-inflammatory activity and mechanism of sinoacutine (sino), one of the primary components extracted from this plant. Methods A RAW264.7 cell model was established using lipopolysaccharide (LPS) induced for estimation of cytokines in vitro, qPCR was used to estimate gene expression, western blot analysis was used to estimate protein level and investigate the regulation of NF- κB, JNK and MAPK signal pathway. In addition, an acute lung injury model was established to determine lung index and levels of influencing factors. Results Using the RAW264.7 model, we found that sino reduced levels of nitric oxide (NO), tumour necrosis factor-α (TNF-α), interleukin (IL)-1β and prostaglandin E2 (PGE2) but increased levels of IL-6. qPCR analysis revealed that sino (50, 25 μg/ml) inhibited gene expression of nitric oxide synthase (iNOS). western blot analysis showed that sino significantly inhibited protein levels of both iNOS and COX-2. Further signalling pathway analysis validated that sino also inhibited phosphorylation of p65 in the NF-κB and c-Jun NH2 terminal kinase (JNK) signalling pathways but promoted the phosphorylation of extracellular signal regulated kinase (ERK) and p38 in the MAPK signalling pathway. In addition, in a mouse model induced by LPS, we determined that sino reduced the lung index and the levels of myeloperoxidase (MPO), NO, IL-6 and TNF-α in lung tissues and bronchoalveolar lavage fluid (BALF) in acute lung injury (ALI). Conclusion Taken together, our results demonstrate that sino is a promising drug to alleviate LPS-induced inflammatory reactions.

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