Supervillin contributes to LPS-induced inflammatory response in THP-1 cell-derived macrophages

Abstract Supervillin (SVIL), the largest member of villin/gelsolin family, is an actin-binding and membrane-associated protein, that can also be localized to the nucleus. It has been reported that the mRNA expression of SVIL in neutrophils could be increased by lipopolysaccharide (LPS), but the underlying mechanisms remain unknown. Moreover, SVIL was also observed to be involved in the regulation of macrophages’ movement. However, it is not clear whether SVIL is involved in the LPS-induced inflammatory response in macrophages. This work was to investigate the underlying molecular mechanisms of LPS regulating SVIL expression in macrophages and hence the possible role of SVIL in LPS-induced inflammation. Our data showed that in THP-1-derived macrophages, LPS stimulation significantly increased SVIL mRNA and protein expression. Inhibition of TLR4 by Resatorvid (Res) completely reversed the expression of SVIL and inflammatory cytokines (IL-6, IL-1β and TNF-α) induced by LPS. Additionally, ERK1/2 and NF-κB inhibitors (U0126 and BAY) significantly reduced SVIL and IL-6, IL-1β & TNF-α expression. Furthermore, down-regulation of SVIL by SVIL-specific shRNA significantly attenuated the expression of IL-6, IL-1β & TNF-α induced by LPS. Taken together, as a downstream molecule of TLR4/NF-κB and ERK1/2, SVIL was involved in the inflammatory response of LPS-induced elevated IL-6, IL-1β and TNF-α in macrophages.

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Paraquat-induced inflammatory response of microglia through HSP60/TLR4 signaling

Human & Experimental Toxicology ◽

10.1177/0960327118758152 ◽

2018 ◽

Vol 37 (11) ◽

pp. 1161-1168 ◽

Cited By ~ 16

Author(s):

Y Sun ◽

J Zheng ◽

Y Xu ◽

X Zhang

Keyword(s):

Inflammatory Response ◽

Mrna Expression ◽

Real Time ◽

Inflammatory Cytokines ◽

Molecular Mechanisms ◽

Nuclear Factor Κb ◽

Heat Shock Protein 60 ◽

Tlr4 Signaling ◽

Tnf Α ◽

Pro Inflammatory Cytokines

Previous studies showed that paraquat (PQ) caused the apoptosis of dopaminergic neurons by inducing the generation of oxygen radical. The purpose of this study is to explore PQ-induced microglial inflammatory response and its underlying molecular mechanisms. The murine microglia BV2 cell line was used. After stimulation with PQ and lipopolysaccharides (positive control), the concentrations of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6) in the culture supernatant and mRNA expression of TNF-α and IL-1β were determined by ELISA and quantitative real-time Polymerase Chain Reaction (PCR), respectively. The protein expression of heat shock protein 60 (HSP60) and toll-like receptor 4 (TLR4), along with the mRNA expression of transcription factors of nuclear factor κB-p65 (NF-κB-p65) and activated protein 1 (AP1, c-fos, and c-jun dimer) were evaluated with western blot and quantitative real-time PCR, respectively. The results showed that PQ activated microglia, which was characterized by increasing the generation and upregulated mRNA expression of pro-inflammatory cytokines, TNF-α, IL-1β, and IL-6. In addition, PQ significantly enhanced the expressions of HSP60 and TLR4 proteins in BV2 cells, as well as NF-κB-p65, c-fos, and c-jun mRNA. These findings suggest that PQ can activate microglia and enhance the expression and secretion of pro-inflammatory cytokines in a HSP60/TLR4 signaling, leading to the inflammatory response.

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Abstract 124: Sam68 Impedes the Recovery of Arterial Injury by Augmenting Inflammatory Response

Circulation Research ◽

10.1161/res.117.suppl_1.124 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Shuling Han ◽

Junlan Zhou ◽

Baron T Arnone ◽

Dauren Biyashev ◽

Chan Boriboun ◽

...

Keyword(s):

Inflammatory Response ◽

Inflammatory Cytokines ◽

Molecular Mechanisms ◽

Mononuclear Cells ◽

Vascular Inflammation ◽

Critical Role ◽

Inflammatory Cells ◽

Peritoneal Macrophages ◽

Raw264.7 Macrophages ◽

Tnf Α

Background: The role of Src-associated in mitosis 68 kDa (Sam68) in cardiovascular biology has not been studied. A recent report suggests that Sam68 suppresses TNF-α-induced NF-κB activation. Since NF-κB plays a critical role in vascular inflammation and injury via generation of inflammatory cytokines and recruitment of inflammatory cells, we sought to dissect the mechanism by which Sam68 regulates NF-κB signaling and its functional significance during vascular injury. Methods & Results: The endothelial denudation injury was induced in the carotid arteries of Sam68-/- and WT mice. Sam68-/- mice displayed an accelerated re-endothelialization and attenuated neointima hyperplasia, which was associated with a reduced number of macrophages and lowered expression of pro-inflammatory cytokines (i.e., TNF-α, IL-1β and IL-6) in the injured vessels. Importantly, the ameliorated vascular remodeling was recapitulated in WT mice after transplantation of bone marrow (BM) from Sam68-/- mice, suggesting beneficial role was attributed largely to BM-derived inflammatory cells. In cultured Raw264.7 macrophages, knockdown of Sam68 resulted in a significant reduction in the TNF-α-induced expression of TNF-α, IL-1β, and IL-6 and in the level of nuclear phospho-p65, indicating an attenuated NF-κB activation. These results were confirmed in peritoneal macrophages and macrophages differentiated from BM mononuclear cells of Sam68-/- and WT mice. To identify molecular mechanisms, Raw264.7 cells were treated with TNF-α and Vehicle, followed by Sam68 co-immunoprecipitation and mass-spec identification of Sam68-interacting proteins. Specifically, TNF-α treatment results in altered interactions of Sam68 with Filamin A (FLNA), a cytoskeleton protein known to be involved in NF-κB activation. Loss- and gain-of-function of Sam68 and FLNA suggest their mutual dependence in NF-κB activation and pro-inflammatory cytokine expression, and Sam68 is required for TRAF2-FLNA interaction. Conclusions: Our results for the first time suggest that Sam68 promotes pro-inflammatory response in injured arteries and impedes recovery, and this effect is attributed, in part, to the exaggerated NF-κB activity via Sam68-FLNA interaction and consequent TRAF2 stabilization.

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Angiotensin II AT1 blockade reduces the lipopolysaccharide-induced innate immune response in rat spleen

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.90962.2008 ◽

2009 ◽

Vol 296 (5) ◽

pp. R1376-R1384 ◽

Cited By ~ 32

Author(s):

Enrique Sánchez-Lemus ◽

Julius Benicky ◽

Jaroslav Pavel ◽

Ignacio M. Larrayoz ◽

Jin Zhou ◽

...

Keyword(s):

Gene Expression ◽

Immune Response ◽

Inflammatory Response ◽

Protein Expression ◽

Innate Immune Response ◽

Innate Immune ◽

Bacterial Endotoxin ◽

Ang Ii ◽

Tnf Α ◽

Mrna And Protein Expression

ANG II AT1 receptor blockade reduces inflammation in hypertension. To determine whether ANG II AT1 receptor blockers (ARBs) influence the innate immune inflammatory response in normotensive rats, we studied rat plasma and spleen after a 3-day subcutaneous pretreatment with the ARB candesartan followed by a single dose of the bacterial endotoxin LPS (50 μg/kg ip). Peripheral administration of LPS to rodents produced a generalized inflammatory response with increased release of TNF-α, IL-1β, and IL-6 into the circulation. Candesartan pretreatment reduced the LPS-induced release of TNF-α, IL-1β, and IL-6 into the circulation. The red pulp of rat spleen expressed large numbers of AT1 receptors and the LPS receptors Toll-like receptor 4 and CD14. Candesartan administration significantly blocked AT1 receptors. The ARB reduced the LPS-induced upregulation of CD14 gene expression; expression of TNF-α and IL-6 mRNA and protein; expression of IL-1β and IκB-α mRNA; COX-2 mRNA and protein expression and PGE2 concentration; inducible nitric oxide synthase (iNOS) gene and protein expression and iNOS activity; and Nox2 gene expression and 8-isoprostane levels. In addition, candesartan reduced the CD14 protein expression in saline- and LPS-treated rats. Our results suggest that AT1 receptors are essential for the development of the full innate immune response to bacterial endotoxin. The ARB decreased the general peripheral inflammatory reaction to LPS and partially decreased the inflammatory response in the spleen. An unrestricted innate immune response to the bacterial endotoxin may have deleterious effects for the organism and may lead to development of chronic inflammatory disease. We postulate that ARBs may have therapeutic effects on inflammatory conditions.

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Effects of TNFα receptor TNF-Rp55- or TNF-Rp75- deficiency on corneal neovascularization and lymphangiogenesis in the mouse

PLoS ONE ◽

10.1371/journal.pone.0245143 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0245143

Author(s):

Anna-Karina B. Maier ◽

Nadine Reichhart ◽

Johannes Gonnermann ◽

Norbert Kociok ◽

Aline I. Riechardt ◽

...

Keyword(s):

Mouse Model ◽

Mrna Expression ◽

Lymphatic Vessels ◽

Corneal Neovascularization ◽

Wild Type ◽

Delayed Effect ◽

Corneal Inflammation ◽

Tnf Α ◽

Mrna And Protein Expression

Tumor necrosis factor (TNF)α is an inflammatory cytokine likely to be involved in the process of corneal inflammation and neovascularization. In the present study we evaluate the role of the two receptors, TNF-receptor (TNF-R)p55 and TNF-Rp75, in the mouse model of suture-induced corneal neovascularization and lymphangiogenesis. Corneal neovascularization and lymphangiogenesis were induced by three 11–0 intrastromal corneal sutures in wild-type (WT) C57BL/6J mice and TNF-Rp55-deficient (TNF-Rp55d) and TNF-Rp75-deficient (TNF-Rp75d) mice. The mRNA expression of VEGF-A, VEGF-C, Lyve-1 and TNFα and its receptors was quantified by qPCR. The area covered with blood- or lymphatic vessels, respectively, was analyzed by immunohistochemistry of corneal flatmounts. Expression and localization of TNFα and its receptors was assessed by immunohistochemistry of sagittal sections and Western Blot. Both receptors are expressed in the murine cornea and are not differentially regulated by the genetic alteration. Both TNF-Rp55d and TNF-Rp75d mice showed a decrease in vascularized area compared to wild-type mice 14 days after suture treatment. After 21 days there were no differences detectable between the groups. The number of VEGF-A-expressing macrophages did not differ when comparing WT to TNF-Rp55d and TNF-Rp75d. The mRNA expression of lymphangiogenic markers VEGF-C or LYVE-1 does not increase after suture in all 3 groups and lymphangiogenesis showed a delayed effect only for TNF-Rp75d. TNFα mRNA and protein expression increased after suture treatment but showed no difference between the three groups. In the suture-induced mouse model, TNFα and its ligands TNF-Rp55 and TNF-Rp75 do not play a significant role in the pathogenesis of neovascularisation and lymphangiogenesis.

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Treatment with Radix Euphorbiae Ebracteolatae Significantly Decreases the Expression of E6 and L1, and Increases the Expression of p53 and Rb in HPV18-infected Human Foreskin Keratinocytes

Current Molecular Medicine ◽

10.2174/1566524019666190226102713 ◽

2019 ◽

Vol 19 (1) ◽

pp. 20-31

Author(s):

Xiang He ◽

Xufeng He ◽

Ping Xu ◽

Lili Yang ◽

Xin Ma ◽

...

Keyword(s):

Cell Proliferation ◽

Protein Expression ◽

Mrna Expression ◽

Molecular Mechanisms ◽

Control Group ◽

Inhibition Of Proliferation ◽

Tnf Α ◽

Ree Group ◽

Vitamin A Acid ◽

Plantar Warts

Background:Radix Euphorbiae Ebracteolatae (REE) was recently reported to be significantly superior to vitamin A acid ointment in treating multiple plantar warts. However, the effects of REE on HPV18 remain unclear. Therefore, the current study aimed to investigate the effects of REE on the proliferation of HPV18, and explore possible molecular mechanisms underlying the effects.Methods:HFK and HFK-HPV18 were treated with water-extracted single or compound REE, ethanol-extracted single or compound REE, TNF-α and IFN for 3 days, respectively. In addition, the organotypic rafts containing HFK-HPV18 and HFK were treated with REE, IFN and TNF-α for 7 days, respectively. Cell proliferation rates were measured with Brdu. mRNA expression of E6, L1, p53 and Rb was detected by qPCR. Protein expression of p53, Rb and L1 was detected by Western blot.Results:Compared to HFK group, HFK-HPV18 group had significantly higher expression of E6 and L1. Compared to the control group, HFK-HPV18 treated with REE, TNF-α and IFN displayed significantly lower proliferation rates. The mRNA expression of E6 was markedly lower, and mRNA expression of p53 and Rb was significantly higher after treatment of REE in HFK-HPV18 or in organotypic rafts containing HFK-HPV18. Treatment with REE markedly increased the protein expression of p53 and Rb, and decreased the protein expression of L1 in HFK-HPV18 or in organotypic rafts containing HFK-HPV18. Among all formula of REE, the inhibition of proliferation rates and expression of E6 and L1, and the increase in expression of p53 and Rb in HFK-HPV18 was highest in ethanol-extracted compound REE group.Conclusions:The proliferation rates are significantly lower in HFK-HPV18 treated with REE. The expression of E6 and L1 is markedly lower, and expression of p53 and Rb is significantly higher after REE treatment in HFK-HPV18 or organotypic rafts containing HFK-HPV18. Among all formula of REE, ethanol-extracted compound REE displays the highest protection against HPV18.

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Collecting duct (pro)renin receptor targets ENaC to mediate angiotensin II-induced hypertension

AJP Renal Physiology ◽

10.1152/ajprenal.00178.2016 ◽

2017 ◽

Vol 312 (2) ◽

pp. F245-F253 ◽

Cited By ~ 31

Author(s):

Kexin Peng ◽

Xiaohan Lu ◽

Fei Wang ◽

Adam Nau ◽

Ren Chen ◽

...

Keyword(s):

Angiotensin Ii ◽

Protein Expression ◽

Mrna Expression ◽

Collecting Duct ◽

Underlying Mechanism ◽

Ang Ii ◽

Induced Hypertension ◽

Mrna And Protein Expression ◽

Receptor Targets

The (pro)renin receptor (PRR) is abundantly expressed in the collecting duct (CD) and the expression is further induced by angiotensin II (ANG II). The present study was conducted to investigate the role of CD PRR during ANG II-induced hypertension and to further explore the underlying mechanism. Radiotelemetry demonstrated that a 1-wk ANG II infusion gradually and significantly induced hypertensive response in floxed mice and this response was significantly attenuated in mice lacking PRR in the CD (termed CD PRR KO). ANG II infusion in floxed mice increased urinary renin activity and selectively induced renal medullary α-epithelial sodium channel (α-ENaC) mRNA and protein expression, all of which were blunted in the null mice. In cultured mpkCCD cells grown in Transwells, transepithelial Na+ transport as measured by using a volt-ohmmeter was transiently stimulated by acute ANG II treatment, which was abolished by a PRR antagonist, PRO20. In a chronic setting, ANG II treatment induced α-ENaC mRNA expression in mpkCCD cells, which was similarly blocked by PRO20. Chronic intramedullary infusion of an ENaC inhibitor amiloride in rats significantly attenuated ANG II-induced hypertension. Overall, the present study suggests that CD PRR contributes to ANG II-induced hypertension at least partially via activation of renal medullary ENaC.

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Protective effects of baicalin on LPS-induced injury in intestinal epithelial cells and intercellular tight junctions

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2014-0262 ◽

2015 ◽

Vol 93 (4) ◽

pp. 233-237 ◽

Cited By ~ 24

Author(s):

Jian Chen ◽

Ren Zhang ◽

Jian Wang ◽

Peng Yu ◽

Quan Liu ◽

...

Keyword(s):

Epithelial Cells ◽

Protein Expression ◽

Tight Junctions ◽

Inflammatory Cytokines ◽

Intestinal Epithelial Cells ◽

Expression Level ◽

Protective Effects ◽

Intestinal Epithelial ◽

Tnf Α ◽

Mrna And Protein Expression

Aims: To investigate the protective effects and mechanisms of baicalin on lipopolysaccharide (LPS)-induced injury in intestinal epithelial cells and intercellular tight junctions. Methods: IEC-6 cells were stimulated with LPS (1.0 μg/mL), with or without baicalin, for 24 h. The levels of the inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α were determined using ELISA. Quantitative real-time PCR was used for determining the mRNA expression level of claudin-3, occludin, and ZO-1; Western blot and immunofluorescence analysis were used for analyzing the expression level and the distribution patterns of ZO-1 protein. Results: Pretreatment with baicalin (10.0 μg/mL) improved LPS-stimulated cell viability and repressed IL-6 and TNF-α levels. In addition, pretreatment with baicalin up-regulated mRNA and protein expression levels of ZO-1 and kept the protein intact in IEC-6 cells injured with LPS. Conclusion: Baicalin has the capacity to protect IEC-6 cells and the intercellular tight junctions from LPS-induced injury. The mechanisms may be associated with inhibiting the production of inflammatory cytokines, and up-regulating the mRNA and protein expression of ZO-1.

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Expression and role of ABIN1 in sepsis: In vitro and in vivo studies

Open Medicine ◽

10.1515/med-2021-0008 ◽

2020 ◽

Vol 16 (1) ◽

pp. 033-040

Author(s):

Haolan Li ◽

Aichen Sun ◽

Taocheng Meng ◽

Yan Zhu

Keyword(s):

Inflammatory Response ◽

Molecular Mechanisms ◽

Organ Injury ◽

In Vivo Studies ◽

Inflammatory Factors ◽

Septic Mouse ◽

Raw264.7 Macrophages ◽

Tnf Α

AbstractIn this research, we attempted to explain the effect and the related molecular mechanisms of ABIN1 in lipopolysaccharide (LPS)-induced septic mice or RAW264.7 macrophages. LPS was adopted to treat RAW264.7 macrophages for 4 h, and the levels of inflammatory factors were assessed by ELISA. Besides, ABIN1 expression was measured by quantitative reverse transcription polymerase chain reaction. Apparently, LPS enhanced immunoreaction, suggested by increased expression of IL-1β, tumor necrosis factor (TNF)-α, and IL-6. ABIN1 levels were obviously reduced compared to the control. Furthermore, we evaluated the roles of ABIN1-plasmid in immunoreaction and nuclear factor-κB (NF-κB) pathway. We found that ABIN1-plasmid significantly reduced the expression of IL-1β, TNF-α, and IL-6 in LPS-treated cells and inhibited NF-κB pathway activation. Meanwhile, a septic mouse mode was conducted to validate the role of ABIN1 in inflammatory response and organ damage in vivo. These data suggested that ABIN1-plasmid significantly inhibited the secretion of inflammatory cytokines and Cr, BUN, AST, and ALT levels in the serum of LPS-stimulated mice compared to LPS + control-plasmid group, reflecting the relieved inflammation and organ injury. In summary, the present findings indicated that ABIN1 alleviated sepsis by repressing inflammatory response through NF-κB signaling pathway, emphasizing the potential value of ABIN1 as therapeutic strategy for sepsis.

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Molecular Regulation of Ghrelin Expression by Pro-Inflammatory Cytokines TNF-α and IL-6 In Rat Pancreatic AR42J Cell Line

Journal of Biology and Life Science ◽

10.5296/jbls.v4i1.2306 ◽

2012 ◽

Vol 4 (1) ◽

Cited By ~ 1

Author(s):

Kah-Meng Lao ◽

Wyi-Sian Lim ◽

Di-Lin Ng ◽

Tengku-Sifzizul Tengku-Muhammad ◽

Quok-Cheong Choo ◽

...

Keyword(s):

Growth Hormone ◽

Protein Expression ◽

Cell Line ◽

Inflammatory Cytokines ◽

Pancreatic Cell ◽

Amino Acid Peptide ◽

Tnf Α ◽

Densitometry Analysis ◽

Mrna And Protein Expression ◽

Pro Inflammatory Cytokines

Ghrelin is a 28 amino acid peptide endogenous ligand for growth hormone secretatgogue receptor (GHS-R) that functions to stimulate growth hormone, regulate inflammation, appetite and energy balance. However, the regulation of ghrelin expression by pro-inflammatory cytokines during inflammation process has never been investigated systematically. This study was carried out to investigate the effects of major pro-inflammatory cytokines such as TNF-α and IL-6 on ghrelin expression using AR42J rat pancreatic cell line as model system. The cells were treated with different concentrations of the cytokines for 24 hours. Real-Time RT-PCR, Western blot and densitometry analysis were carried out to quantify ghrelin mRNA and protein expression, respectively. Although TNF-α and IL-6 stimulation resulted in a general down-regulatory pattern in both mRNA and protein expression of ghrelin, stimulation with 5 ng/ml of TNF-α and IL-6 slightly induced the expression of ghrelin expression. However, higher doses of the cytokines ranging from 10-50 ng/ml suppressed the ghrelin expression in a dose-dependant manner. These results indicate that ghrelin in AR42J pancreatic cell line is regulated by the pro-inflammatory cytokines and that the dosages of the cytokines play an important factor in the regulation of the expression.

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Downregulation of cGMP-dependent protein kinase expression by inflammatory cytokines in vascular smooth muscle cells

AJP Cell Physiology ◽

10.1152/ajpcell.00039.2004 ◽

2004 ◽

Vol 287 (1) ◽

pp. C88-C96 ◽

Cited By ~ 39

Author(s):

Natasha C. Browner ◽

Hassan Sellak ◽

Thomas M. Lincoln

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

Protein Expression ◽

Inflammatory Cytokines ◽

Inos Expression ◽

Dependent Protein Kinase ◽

Cgmp Dependent Protein Kinase ◽

Tnf Α ◽

Mrna And Protein Expression ◽

Dependent Protein

NO and cGMP have antigrowth and anti-inflammatory effects on the vessel wall in response to injury. It is well established that after vascular injury proinflammatory cytokines are involved in vascular wall remodeling. The purpose of this study was to ascertain the signaling mechanisms involved in cGMP-dependent protein kinase (PKG) suppression by inflammatory cytokines in primary bovine aortic vascular smooth muscle cells (VSMC). Interleukin (IL)-Iβ, tumor necrosis factor (TNF)-α, and LPS decreased the mRNA and protein levels of PKG in VSMC. IL-Iβ, TNF-α, and LPS increased inducible nitric oxide synthase (iNOS) expression and cGMP production. Treatment of cells with selective inhibitors of iNOS or soluble guanylate cyclase (sGC) reversed the downregulation of PKG expression induced by cytokines and LPS. The NO donor ( Z)-1-[2-(2-aminoethyl)- N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate) and 3-(5-hydroxymethyl-2-furyl)-1-benzylindazole (YC-1), a NO-independent sGC activator, decreased PKG mRNA and protein expression in bovine aortic VSMC. Cyclic nucleotide analogs [8-(4-chlorophenylthio)guanosine 3′,5′-cyclic monophosphate (CPT-cGMP) and 8-(4-chlorophenylthio)adenosine 3,5′-cyclic monophosphate (CPT-cAMP)] also suppressed PKG mRNA and protein expression. However, CPT-cAMP was more effective than CPT-cGMP in decreasing PKG mRNA levels. Selective inhibition of PKA with the Rp isomer of 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphorothioate (Rp-8p-CPT cAMPS) prevented the downregulation of PKG by LPS. In contrast, the Rp isomer of 8-(4-chlorophenylthio)guanosine 3,5′-cyclic monophosphorothioate (Rp-8p-CPT cGMPS; inhibitor of PKG) had no effect on LPS-induced inhibition of PKG mRNA and protein expression. These studies suggest that cross-activation of PKA in response to iNOS expression by inflammatory mediators downregulates PKG expression in bovine aortic VSMC.

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