Interleukin‐17‐induced expression of monocyte chemoattractant protein‐1 in cardiac myocytes requires nuclear factor κB through the phosphorylation of p65

IL‐17 plays a key role in a variety of autoimmune diseases. MCP‐1 is involved in the infiltration of mononuclear cells of myocardium in VMC. However, the relationship between IL‐17 and MCP‐1 in myocardial injury remains unclear. In this study, expression of MCP‐1 mRNA and protein in cardiac myocytes was detected with qRT‐PCR and ELISA, respectively. It was found that IL‐17A induced MCP‐1 expression in a dose‐ and time‐dependent manner in cardiac myocytes, which could be blocked by IL‐17A and IL‐17RA neutralizing antibodies. NF‐κB p65 and p‐p65 protein expression in cardiac myocytes was studied with western blotting. Rates of p‐p65 in whole lysates and in nuclear lysates all increased in the first 15 min. Meanwhile, the amount of NF‐κB p65 in whole lysates did not change, but the amount of NF‐κB p65 in nuclear lysates increased in the first 15 min. Then the optimal sequence and concentration of NF‐κB p65 siRNAs was selected. After transfection of 10 nM siRNA‐2 of NF‐κB p65 into cardiac myocytes before stimulation by IL‐17A, expression of MCP‐1 mRNA and protein obviously decreased. In conclusion, expression of MCP‐1 induced by IL‐17 requires NF‐κB through the phosphorylation of p65 in cardiac myocytes, which is meaningful to study the onset of chronic viral myocarditis and will provide a new target for the treatment of viral myocarditis.

     

NF‐κB regulates a cassette of immune/inflammatory genes in human pregnant myometrium at term

The onset of human labour resembles inflammation with increased synthesis of prostaglandins and cytokines. There is evidence from rodent models for an important role for nuclear factor‐κB (NF‐κB) activity in myometrium which both up‐regulates contraction‐associated proteins and antagonizes the relaxatory effects of progesterone. Here we show that in the human, although there are no differences in expression of NF‐κB p65, or IκB‐α between upper‐ or lower‐segment myometrium or before or after labour, there is nuclear localization of serine‐256‐phospho‐p65 and serine‐536‐phospho‐p65 in both upper‐ and lower‐segment myometrium both before and after the onset of labour at term. This shows that NF‐κB is active in both upper and lower segment prior to the onset of labour at term. To identify the range of genes regulated by NF‐κB we overexpressed p65 in myocytes in culture. This led to NF‐κB activation identical to that seen following interleukin (IL)‐1β stimulation, including phosphorylation and nuclear translocation of p65 and p50. cDNA microarray analysis showed that NF‐κB increased expression of 38 genes principally related to immunity and inflammation. IL‐1β stimulation also resulted in an increase in the expression of the same genes. Transfection with siRNA against p65 abolished the response to IL‐1β proving a central role for NF‐κB. We conclude that NF‐κB is active in myocytes in both the upper and lower segment of the uterus prior to the onset of labour at term and principally regulates a group of immune/inflammation associated genes, demonstrating that myocytes can act as immune as well as contractile cells.     

MiR‐643 inhibits lipopolysaccharide‐induced endometritis progression by targeting TRAF6

Endometritis is a prevalent disease with inflammation of uterus endangering women reproductive health. MicroRNAs (miRNAs) play important roles in inflammatory disorders, including endometritis. However, the role and mechanism of miR‐643 in endometritis development remain unclear. This study aimed to investigate the effect of miR‐643 on lipopolysaccharide (LPS)‐induced inflammatory response and clarify the potential mechanism. LPS‐treated human endometrial epithelial cells (HEECs) were cultured to investigate the role of miR‐643 in vitro. The expression levels of miR‐643 and tumor necrosis factor receptor‐associated factor 6 (TRAF6) were measured via quantitative real‐time polymerase chain reaction and western blot, respectively. LPS‐induced inflammatory response was assessed by inflammatory cytokines secretion via enzyme‐linked immunosorbent assay. The activation of nuclear factor‐κB (NF‐κB) pathway was investigated by western blot. The interaction between miR‐643 and TRAF6 was validated by bioinformatics analysis, luciferase reporter assay, and RNA immunoprecipitation. The expression of miR‐643 was decreased and TRAF6 protein level was enhanced in LPS‐treated HEECs. The overexpression of miR‐643 suppressed LPS‐induced secretion of inflammatory cytokines (tumor necrosis factor‐α, interleukin‐1β [IL‐1β], and IL‐6) and activation of NF‐κB pathway. The knockdown of TRAF6 inhibited LPS‐induced inflammatory response in HEECs. TRAF6 was validated as a target of miR‐643 and TRAF6 restoration reversed the effect of miR‐643 on inflammation response in LPS‐treated HEECs. Collectively, miR‐643 attenuated LPS‐induced inflammatory response by targeting TRAF6, indicating a novel avenue for the treatment of endometritis.     

NF‐κB and estrogen receptor α interactions: Differential function in estrogen receptor‐negative and ‐positive hormone‐independent breast cancer cells

Estrogen receptor (ER)‐positive breast cancer cells have low levels of constitutive NF‐κB activity while ER negative (?) cells and hormone‐independent cells have relatively high constitutive levels of NF‐κB activity. In this study, we have examined the aspects of mutual repression between the ERα and NF‐κB proteins in ER+ and ER? hormone‐independent cells. Ectopic expression of the ERα reduced cell numbers in ER+ and ER? breast cancer cell lines while NF‐κB‐binding activity and the expression of several NF‐κB‐regulated proteins were reduced in ER? cells. ER overexpression in ER+/E2‐independent LCC1 cells only weakly inhibited the predominant p50 NF‐κB. GST‐ERα fusion protein pull downs and in vivo co‐immunoprecipitations of NF‐κB:ERα complexes showed that the ERα interacts with p50 and p65 in vitro and in vivo. Inhibition of NF‐κB increased the expression of diverse E2‐regulated proteins. p50 differentially associated directly with the ER:ERE complex in LCC1 and MCF‐7 cells by supershift analysis while p65 antibody reduced ERα:ERE complexes in the absence of a supershift. ChIP analysis demonstrated that NF‐κB proteins are present on an endogenous ERE. Together these results demonstrate that the ER and NF‐κB undergo mutual repression, which may explain, in part, why expression of the ERα in ER? cells does not confer growth signaling. Secondly, the acquisition of E2‐independence in ER+ cells is associated with predominantly p50:p50 NF‐κB, which may reflect alterations in the ER in these cells. Since the p50 homodimer is less sensitive to the presence of the ER, this may allow for the activation of both pathways in the same cell. J. Cell. Biochem. 107: 448–459, 2009. © 2009 Wiley‐Liss, Inc.     

Activation and induction of cytosolic phospholipase A2 by IL‐1β in human tracheal smooth muscle cells: Role of MAPKs/p300 and NF‐κB

Cytosolic phospholipase A₂ (cPLA₂) plays a pivotal role in mediating agonist‐induced arachidonic acid (AA) release for prostaglandin (PG) synthesis during inflammation triggered by IL‐1β. However, the mechanisms underlying IL‐1β‐induced cPLA₂ expression and PGE₂ synthesis in human tracheal smooth muscle cells (HTSMCs) remain unknown. IL‐1β‐induced cPLA₂ protein and mRNA expression, PGE₂ production, or phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK1/2, which was attenuated by pretreatment with the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK1/2 (SP600125) or transfection with siRNAs of MEK1, p42, p38, and JNK2. IL‐1β‐induced cPLA₂ expression was also inhibited by pretreatment with a NF‐κB inhibitor, helenalin or transfection with siRNA of NIK, IKKα, or IKKβ. IL‐β‐induced NF‐κB translocation was blocked by pretreatment with helenalin, but not U0126, SB202190, and SP600125. In addition, transfection with p300 siRNA blocked cPLA₂ expression induced by IL‐1β. Moreover, p300 was associated with the cPLA₂ promoter, which was dynamically linked to histone H4 acetylation stimulated by IL‐1β. These results suggest that in HTSMCs, activation of MAPKs, NF‐κB, and p300 are essential for IL‐1β‐induced cPLA₂ expression and PGE₂ secretion. J. Cell. Biochem. 109: 1045–1056, 2010. © 2010 Wiley‐Liss, Inc.     

Bilobalide alleviates IL‐17‐induced inflammatory injury in ATDC5 cells by downregulation of microRNA‐125a

Ankylosing spondylitis (AS) is a high disability and greatly destructive disease. In this study, we preliminarily studied the function and mechanism of bilobalide (BIL) on interleukin (IL)‐17‐induced inflammatory injury in ATDC5 cells. CCK‐8 and migration assays were used to detect the functions of IL‐7, BIL, and microRNA (miR)‐125a on cell viability and migration. The miR‐125a level was changed by transfection, and tested by real‐time quantitative polymerase chain reaction. Additionally, Western blot tested the levels of inflammatory factors (IL‐6 and tumor necrosis factor‐α), matrix metalloproteinases (MMPs), and pathway‐related proteins. Moreover, the enzyme‐linked immunosorbent assay also was used to detect inflammatory factor levels. IL‐7 was used to construct an inflammatory injury model in ATDC5 cells. Based on this, BIL inhibited IL‐17‐induced cell viability, migration, and expressions of inflammatory factors and MMPs. Furthermore, we found BIL negatively regulated miR‐125a, and the miR‐125a mimic could partly reverse the effects of BIL on IL‐17‐injury. Finally, we showed that BIL inhibited the c‐Jun N‐terminal kinase (JNK) and nuclear factor kappa B (NF‐κB) pathways, and the miR‐125a mimic had the opposite effect. BIL inhibited IL‐17‐induced inflammatory injury in ATDC5 cells by downregulation of miR‐125a via JNK and NF‐κB signaling pathways.     

Baicalin effects on rats with spinal cord injury by anti‐inflammatory and regulating the serum metabolic disorder

Baicalin had neuroprotective effects on inhibiting neuronal cell apoptosis induced by spinal cord ischemic injury. This study aimed to explore the protective effects of Baicalin on rats with spinal cord injury (SCI) and its mechanism of action. The recovery of spinal cord nerve function in rats was evaluated by the Basso, Beattie, and Bresnahan (BBB) score and the combine behavioral score (CBS). The expressions of cytokines tumor necrosis factor α (TNF‐α), interleukin‐1β (IL‐1β), and IL‐6 were detected by the enzyme‐linked immunosorbent assay method. Expressions of inflammation‐related proteins were detected by Western blot. Multivariate statistical analysis was performed for serum metabolites. The BBB and CBS score results showed that Baicalin had a certain improvement on rats with SCI. SCI symptoms were significantly improved in low‐dose and high‐dose groups. The levels of TNF‐α, IL‐1β, and IL‐6 in the SCI group were significantly increased. The expressions of NF‐κB p65, NF‐κB p50, p‐IκBα, and IKKα in the SCI group showed the opposite trend compared with the low‐dose and high‐dose groups. Compared with the sham group, glutamine, levels of 3‐OH‐butyrate, N‐acetylaspartate, and glutathione were significantly reduced, and the levels of glutamate and betaine were significantly increased in the SCI group. When Baicalin was administered, the contents of glutamine synthase (GS) and glutaminase (GLS) were significantly reduced, indicating that Baicalin had the effect of improving GS and GLS. Baicalin has protective effects on improving SCI and lower extremity motor function, has a significant anti‐inflammatory effect, and regulates the serum metabolic disorder caused by SCI in rats.     

Gastrokine 1 regulates NF‐κB signaling pathway and cytokine expression in gastric cancers

Gastrokine 1 (GKN1) plays an important role in the gastric mucosal defense mechanism and also acts as a functional gastric tumor suppressor. In this study, we examined the effect of GKN1 on the expression of inflammatory mediators, including NF‐κB, COX‐2, and cytokines in GKN1‐transfected AGS cells and shGKN1‐transfected HFE‐145 cells. Lymphocyte migration and cell viability were also analyzed after treatment with GKN1 and inflammatory cytokines in AGS cells by transwell chemotaxis and an MTT assay, respectively. In GKN1‐transfected AGS cells, we observed inactivation and reduced expression of NF‐κB and COX‐2, whereas shGKN1‐transfected HFE‐145 cells showed activation and increased expression of NF‐κB and COX‐2. GKN1 expression induced production of inflammatory cytokines including IL‐8 and ‐17A, but decreased expression of IL‐6 and ‐10. We also found IL‐17A expression in 9 (13.6%) out of 166 gastric cancer tissues and its expression was closely associated with GKN1 expression. GKN1 also acted as a chemoattractant for the migration of Jurkat T cells and peripheral B lymphocytes in the transwell assay. In addition, GKN1 significantly reduced cell viability in both AGS and HFE‐145 cells. These data suggest that the GKN1 gene may inhibit progression of gastric epithelial cells to cancer cells by regulating NF‐κB signaling pathway and cytokine expression. J. Cell. Biochem. 114: 1800–1809, 2013. © 2013 Wiley Periodicals, Inc.     

The interplay of LncRNA ANRIL and miR‐181b on the inflammation‐relevant coronary artery disease through mediating NF‐κB signalling pathway

This study was designed to investigate whether ANRIL affected the aetiology of coronary artery disease (CAD) by acting on downstream miR‐181b and NF‐κB signalling. Altogether 327 CAD patients diagnosed by angiography were included, and mice models of CAD were established. Human coronary endothelial cells (HCAECs) and human umbilical vein endothelial cells (HUVECs) were also purchased. In addition, shRNA‐ANRIL, shRNA‐NC, pcDNA3.1‐ANRIL, miR‐181b mimic, miR‐181b inhibitor and miR‐NC were transfected into the cells. The lipopolysaccharides (LPS) and pyrrolidine dithiocarbamate (PDTC) were also added to activate or deactivate NF‐κB signalling. Both highly expressed ANRIL and lowly expressed miR‐181b were associated with CAD population aged over 60 years old, with smoking history, with hypertension and hyperlipidemia, with CHOL H 4.34 mmol/L, TG ≥ 1.93 mmol/L and Hcy ≥ 16.8 μmol/L (all P < 0.05). Besides, IL‐6, IL‐8, NF‐κB, TNF‐α, iNOS, ICAM‐1, VCAM‐1 and COX‐2 expressions observed within AD mice models were all beyond those within NC and sham‐operated groups (P < 0.05). Also VEGF and HSP 70 were highly expressed within AD mice models than within NC and sham‐operated mice (P < 0.05). Transfection of either pcDNA‐ANRIL or miR‐181b inhibitor could significantly fortify HCAECs’ viability and put on their survival rate. At the meantime, the inflammatory factors and vascular‐protective parameters were released to a greater level (P < 0.05). Finally, highly expressed ANRIL also notably bring down miR‐181b expression and raise p50/p65 expressions within HCAECs (P < 0.05). The joint role of ANRIL, miR‐181b and NF‐κB signalling could aid in further treating and diagnosing CAD.     

Vibration activates the actin/NF‐κB axis and upregulates IL‐6 and IL‐8 expression in human periodontal ligament cells

We previously reported that mechanical vibration‐induced proinflammatory cytokines, interleukin‐6 (IL‐6) and IL‐8, expression in human periodontal ligament (hPDL) cells, however, the underlying mechanism remained unclear. Mechanical stimuli are able to activate cellular responses by inducing the activation of several signaling pathways including cytoskeletal changes and inflammation. The actin cytoskeleton is a highly dynamic network and plays many important roles in intracellular events. Here, we aimed to investigate the involvement of a pivotal mediator of inflammatory responses, nuclear factor‐κB (NF‐κB), and actin polymerization in vibration‐induced upregulation of IL‐6 and IL‐8 expression in hPDL cells. hPDL cells were pretreated with the NF‐κB inhibitor BAY 11‐7082 or cytochalasin D, respectively, before exposure to vibration. IL‐6 and IL‐8 messenger RNA (mRNA) and protein expression were quantified by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assays, respectively. Subcellular localization of the NF‐κB p65 subunit was visualized by immunofluorescent staining. We found an increase in NF‐κB nuclear translocation in vibrated cells compared with control cells. Pretreatment with BAY 11‐7082 significantly inhibited vibration‐induced IL‐6 and IL‐8 mRNA and protein expression in hPDL cells. Moreover, pretreatment with cytochalasin D inhibited NF‐κB nuclear translocation and attenuated upregulation of IL‐6 and IL‐8 mRNA and protein in vibrated cells. Therefore, modulation of actin cytoskeletal polymerization in response to vibration may activate the NF‐κB signaling pathway and subsequently upregulate IL‐6 and IL‐8 expression in hPDL cells.     

Embelin impact on paraquat‐induced lung injury through suppressing oxidative stress,inflammatory cascade,and MAPK/NF‐κB signaling pathway

The current examination was intended to observe the defensive impacts of embelin against paraquat‐incited lung damage in relationship with its antioxidant and anti‐inflammatory action. Oxidative stress marker, like malondialdehyde (MDA), antioxidative enzymes, for example, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH Px), inflammatory cytokines, such as interleukin‐1β (IL‐1β), tumor necrosis factor‐α, and IL‐6, histological examination, and nuclear factor kappa B/mitogen‐activated protein kinase (NF‐κB/MAPK) gene expression were evaluated in lung tissue. Embelin treatment significantly decreased MDA and increased SOD, CAT, and GSH Px. Embelin significantly reduced levels of inflammatory cytokines in paraquat‐administered and paraquat‐intoxicated rats. In addition, embelin suggestively decreased relative protein expression of nuclear NF‐κB p65, p‐NF‐κBp65, p38 MAPK, and p‐p38 MAPKs in paraquat‐intoxicated rats. The outcomes show the impact of embelin inhibitory action on NF‐κB and MAPK and inflammatory cytokines release, and the decrease of lung tissue damage caused by paraquat.