Inhibition Of Interleukin-6 Expression Research Articles

The protective role of chemokines 12 and chemokines 4 by mediating interleukin-6 in delayed diabetic foot wound healing

Refractory diabetic foot ulcer is commonly encountered in clinical practice and it is hardly cured with a long duration of treatment and a high expense, as such problem involves multiple disciplines. Therefore, in case of refractory wounds, it is necessary to first analyze the causes, general or local. Immunohistochemistry (IHC) was conducted to determine α-SMA, chemokines 12 (CXCL12) and chemokines 4 (CXCR4) levels in pancreatic tissues and distant normal pancreatic tissues. Cells were treated with recombinant human CXCL12 (rhCXCL12) or the CXCR4 antagonist AMD3100. After treatment, Western blot determined FAK-AKT and ERK1/2 expression in islet cells, whilst ELISA detected the content of IL-6 and IL-8. rhCXCL12 increased the expression and secretion of Interleukin-6 (IL-6) time- and dose-dependently. But the advent of CXCR4 antagonist abrogated the protective effect of rhCXCL12. rhCXCL12 exhibited a protective effect on apoptosis, but this effect was abrogated by down-regulation of IL-6 with AMD3100. In addition, rhCXCL12 increased the phosphorylation of FAK, ERK1/2, AKT, and P38 and inhibition of FAK inhibited IL-6 expression. FAK inhibition almost completely blocked CXCL12-induced activation. This study demonstrates that CXCL12/CXCR4 pathway mediates the expression of IL-6 to enhance healing of refractory diabetic foot wounds by down-regulating the FAK pathway.

Lutein inhibits IL‑6 expression by inducing PPAR‑γ activation and SOCS3 expression in cerulein‑stimulated pancreatic acinar cells.

Acute pancreatitis is a severe inflammatory disease of the pancreas. In experimental acute pancreatitis, cerulein induces the expression of interleukin-6 (IL-6) by activating Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3 in pancreatic acinar cells. Ligands of peroxisome proliferator activated receptor-γ (PPAR-γ) and suppressor of cytokine signaling (SOCS) 3 inhibit IL-6 expression by suppressing JAK2/STAT3 in cerulein-stimulated pancreatic acinar AR42J cells. Lutein, an oxygenated carotenoid, upregulates and activates PPAR-γ to regulate inflammation in a renal injury model. The present study aimed to determine whether lutein activated PPAR-γ and induced SOCS3 expression in unstimulated AR42J cells, and whether lutein inhibited activation of JAK2/STAT3 and IL-6 expression via activation of PPAR-γ and SOCS3 expression in cerulein-stimulated AR42J cells. The anti-inflammatory mechanism of lutein was determined using reverse transcription-quantitative PCR, western blot analysis and enzyme-linked immunosorbent assay in AR42J cells stimulated with or without cerulein. In another experiment, cells were treated with lutein and the PPAR-γ antagonist GW9662 or the PPAR-γ agonist troglitazone prior to cerulein stimulation to determine the involvement of PPAR-γ activation. The results indicated that lutein increased PPAR-γ and SOCS3 levels in unstimulated cells. Cerulein increased phospho-specific forms of JAK2 and STAT3, and mRNA and protein expression of IL-6, but decreased SOCS3 levels in AR42J cells. Cerulein-induced alterations were suppressed by lutein or troglitazone. GW9662 alleviated the inhibitory effect of lutein on JAK2/STAT3 activation and IL-6 expression in cerulein-stimulated cells. In conclusion, lutein inhibited the activation of JAK2/STAT3 and reduced IL-6 levels via PPAR-γ-mediated SOCS3 expression in pancreatic acinar cells stimulated with cerulein.

Classical swine fever virus NS4B protein interacts with MAVS and inhibits IL-8 expression in PAMs

Classical swine fever virus (CSFV) infection causes a severe disease of pigs, resulting in significant economic losses. The CSFV NS4B protein is crucial for viral replication and pathogenicity. Interleukin 8 (IL-8), a main chemokine, is induced by multiple cell types and plays an essential role in host defense mechanisms against numerous viruses. It has been reported that NS4A of CSFV is involved in the induction of IL-8 expression in swine umbilical vein endothelial cells. However, the effect of CSFV NS4B on IL-8 expression is unknown. In this study, we showed that CSFV NS4B inhibited IL-8 expression in porcine alveolar macrophages (PAMs), and NS4B inhibited mitochondrial antiviral signaling protein (MAVS)-induced IL-8 expression. Moreover, CSFV NS4B interacted with MAVS. However, NS4B did not alter MAVS expression. Subsequently, we demonstrated that IRF3 knockdown or NF-κB inhibition reduced MAVS-induced IL-8 expression. Furthermore, the IRF3 and NF-κB pathways were activated by MAVS expression. However, CSFV NS4B inhibited MAVS-mediated NF-κB activation and IRF3 expression. Finally, CSFV NS4B inhibited IRF3 expression. Our findings reveal that CSFV NS4B interacts with MAVS and inhibits IL-8 expression by blocking the activation of IRF3 and NF-κB. Taken together, this study provides insights into the mechanism of NS4B-inhibited IL-8 expression.

Astaxanthin Inhibits Interleukin-6 Expression in Cerulein/Resistin-Stimulated Pancreatic Acinar Cells.

Acute pancreatitis is a common clinical condition with increasing the proinflammatory mediators, including interleukin-6 (IL-6). Obesity is a negative prognostic factor in acute pancreatitis. Obese patients with acute pancreatitis have a higher systemic inflammatory response rate. Levels of serum resistin, an adipocytokine secreted by fat tissues, increase with obesity. Cerulein, a cholecystokinin analog, induces calcium (Ca2+) overload, oxidative stress, and IL-6 expression in pancreatic acinar cells, which are hallmarks of acute pancreatitis. A recent study showed that resistin aggravates the expression of inflammatory cytokines in cerulein-stimulated pancreatic acinar cells. We aimed to investigate whether resistin amplifies cerulein-induced IL-6 expression and whether astaxanthin (ASX), an antioxidant carotenoid with anti-inflammatory properties, inhibits ceruelin/resistin-induced IL-6 expression in pancreatic acinar AR42J cells. We found that resistin enhanced intracellular Ca2+ levels, NADPH oxidase activity, intracellular reactive oxygen species (ROS) production, NF-κB activity, and IL-6 expression in cerulein-stimulated AR42J cells, which were inhibited by ASX in a dose-dependent manner. The calcium chelator BAPTA-AM inhibited cerulein/resistin-induced NADPH oxidase activation and ROS production. Antioxidant N-acetyl cysteine (NAC) and ML171, a specific NADPH oxidase 1 inhibitor, suppressed cerulein/resistin-induced ROS production, NF-κB activation, and IL-6 expression. In conclusion, ASX inhibits IL-6 expression, by reducing Ca2+ overload, NADPH oxidase-mediated ROS production, and NF-κB activity in cerulein/resistin-stimulated pancreatic acinar cells. Consumption of ASX-rich foods could be beneficial for preventing or delaying the incidence of obesity-associated acute pancreatitis.

Inhibitory Effect of Korean Red Ginseng Extract on Interleukin-8 Expression in Helicobacter pylori-Infected Gastric Epithelial Cells

ObjectivesInfection of Helicobacter pylori (H. pylori), a gram-negative bacterium, leads to various gastric diseases, such as gastritis, peptic ulcer and gastric cancer. H. pylori increases cytokine release and activates inflammatory mediators in gastric mucosa. Particularly, H. pylori upregulates the inflammatory chemokine interleukin-8 (IL-8), which are activated by oxidative stress. IL-8 can cause severe inflammation of the stomach and gastric cancer. Korean red ginseng is the steamed root of 6-year-old Korean ginseng (Panax ginseng Meyer). Ginsenosides, triterpene glycosides, are the active components of Korean red ginseng. Ginsenosides have antioxidant, anti-inflammatory, and antitumor activities. The present study is aimed at determining whether Korean red ginseng extract inhibits H. pylori-induced IL-8 expression in gastric epithelial cells. MethodsThe human gastric epithelial cell line AGS was used. Gastric epithelial AGS cells were treated with Korean red ginseng extract, and infected with H. pylori (NCTC 11,637). Reactive oxygen species (ROS) levels were determined using dichlorofluorescein fluorescence. NADPH oxidase activity was measured using lucigenin chemiluminescence. IL-8 mRNA expression was measured by using real-time PCR. NADPH oxidase subunits were determined in cytosolic extract and membrane extract by using Western blotting. ResultsH. pylori increased NADPH oxidase activity, ROS levels, and upregulated IL-8 expression in gastric epithelial cells. Korean red ginseng extract inhibited IL-8 expression by suppressing NADPH oxidase activity and reducing ROS levels in gastric epithelial cells. H. pylori induced translocation of NADPH oxidase cytosolic subunits to membrane, which is a marker of NADPH oxidase activation, in AGS cells. Korean red ginseng extract inhibited translocation of cytosolic subunits of NADPH oxidase to membrane in AGS cells. ConclusionsKorean red ginseng may be beneficial for preventing H. pylori-associated gastric inflammation by inhibiting oxidative stress and IL-8 expression. Funding SourcesThis study was supported by a Brain Korea 21 FOUR Project, Yonsei University, Seoul, Republic of Korea.

Ellagitannins from Rosa roxburghii suppress poly(I:C)-induced IL-8 production in human keratinocytes.

The anti-inflammatory effects of a 50% aqueous extract of Rosa roxburghii fruit (RRFE) and two ellagitannins (strictinin and casuarictin) isolated from the RRFE were evaluated in a cell model of skin inflammation induced by self-RNA released from epidermal cells damaged by UV ray (UVR) irradiation. The RRFE inhibited interleukin-8 (IL-8) mRNA expression in normal human epidermal keratinocytes (NHEKs) stimulated with polyinosinic:polycytidylic acid (poly(I:C)), a ligand of toll-like receptor-3 (TLR-3). The plant-derived anti-inflammatory agents, dipotassium glycyrrhizinate (GK2) and allantoin, had no influence on the IL-8 expression. The purified compounds, strictinin and casuarictin, inhibited the IL-8 mRNA expression and IL-8 release induced in NHEKs by poly(I:C). These ellagitannins were thus found to be responsible for the biological activity exhibited by the RRFE. This study demonstrates that RRFE and isolated RRFE compounds show promise as ingredients for products formulated to improve skin disorders induced by UVR irradiation.

Docosahexaenoic Acid Induces Expression of NAD(P)H: Quinone Oxidoreductase and Heme Oxygenase-1 through Activation of Nrf2 in Cerulein-Stimulated Pancreatic Acinar Cells.

Oxidative stress is a major risk factor for acute pancreatitis. Reactive oxygen species (ROS) mediate expression of inflammatory cytokines such as interleukin-6 (IL-6) which reflects the severity of acute pancreatitis. The nuclear factor erythroid-2-related factor 2 (Nrf2) pathway is activated to induce the expression of antioxidant enzymes such as NAD(P)H: quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) as a cytoprotective response to oxidative stress. In addition, binding of Kelch-like ECH-associated protein 1 (Keap1) to Nrf2 promotes degradation of Nrf2. Docosahexaenoic acid (DHA)—an omega-3 fatty acid—exerts anti-inflammatory and antioxidant effects. Oxidized omega-3 fatty acids react with Keap1 to induce Nrf2-regulated gene expression. In this study, we investigated whether DHA reduces ROS levels and inhibits IL-6 expression via Nrf2 signaling in pancreatic acinar (AR42J) cells stimulated with cerulein, as an in vitro model of acute pancreatitis. The cells were pretreated with or without DHA for 1 h and treated with cerulein (10−8 M) for 1 (ROS levels, protein levels of NQO1, HO-1, pNrf2, Nrf2, and Keap1), 6 (IL-6 mRNA expression), and 24 h (IL-6 protein level in the medium). Our results showed that DHA upregulates the expression of NQO1 and HO-1 in cerulein-stimulated AR42J cells by promoting phosphorylation and nuclear translocation of Nrf2. DHA increased interaction between Keap1 and Nrf2 in AR42J cells, which may increase Nrf2 activity by inhibiting Keap1-mediated sequestration of Nrf2. In addition, DHA-induced expression of NQO1 and HO-1 is related to reduction of ROS and IL-6 levels in cerulein-stimulated AR42J cells. In conclusion, DHA inhibits ROS-mediated IL-6 expression by upregulating Nrf2-mediated expression of NQO1 and HO-1 in cerulein-stimulated pancreatic acinar cells. DHA may exert positive modulatory effects on acute pancreatitis by inhibiting oxidative stress and inflammatory cytokine production by activating Nrf2 signaling in pancreatic acinar cells.

LncRNA DILC is downregulated in osteoarthritis and regulates IL‐6 expression in chondrocytes

Excessive production of interleukin 6 (IL-6) is involved in the pathogenesis of osteoarthritis. It has been reported that long noncoding RNA (lncRNA) DILC inhibited IL-6 to regulate liver cancer stem cells. Therefore, lncRNA DILC may also participate in osteoarthritis. We found that lncRNA DILC was downregulated, while IL-6 was upregulated in plasma of osteoarthritis patients comparing to the control group. Levels of plasma lncRNA DILC and IL-6 were significantly and inversely correlated only in patients with osteoarthritis. Downregulation of lncRNA DILC effectively distinguished patients with osteoarthritis from the control group. Overexpression of lncRNA DILC resulted in inhibited IL-6 expression in chondrocytes, while treatment with exogenous IL-6 did not affect lncRNA DILC expression. However, lncRNA DILC overexpression did not affect the proliferation and apoptosis of chondrocytes. Therefore, lncRNA DILC is downregulated in osteoarthritis and regulates IL-6 expression in chondrocytes.

Genetic Inactivation of ZCCHC6 Suppresses Interleukin-6 Expression and Reduces the Severity of Experimental Osteoarthritis in Mice.

Cytokine expression is tightly regulated posttranscriptionally, but high levels of interleukin-6 (IL-6) in patients with osteoarthritis (OA) indicate that regulatory mechanisms are disrupted in this disorder. The enzyme ZCCHC6 (zinc-finger CCHC domain-containing protein 6; TUT-7) has been implicated in posttranscriptional regulation of inflammatory cytokine expression, but its role in OA pathogenesis is unknown. The present study was undertaken to investigate whether ZCCHC6 directs the expression of IL-6 and influences OA pathogenesis in vivo. Human and mouse chondrocytes were stimulated with recombinant IL-1β. Expression of ZCCHC6 in human chondrocytes was knocked down using small interfering RNAs. IL-6 transcript stability was determined by actinomycin D chase, and 3'-uridylation of microRNAs was determined by deep sequencing. Zcchc6-/- mice were produced by gene targeting. OA was surgically induced in the knee joints of mice, and disease severity was scored using a semiquantitative grading system. ZCCHC6 was markedly up-regulated in damaged cartilage from human OA patients and from wild-type mice with surgically induced OA. Overexpression of ZCCHC6 induced the expression of IL-6, and its knockdown reduced IL-6 transcript stability and IL-1β-induced IL-6 expression in chondrocytes. Reintroduction of Zcchc6 in Zcchc6-/- mouse chondrocytes rescued the IL-1β-induced IL-6 expression. Knockdown of ZCCHC6 reduced the population of micro-RNA 26b (miR-26b) with 3'-uridylation by 60%. Zcchc6-/- mice with surgically induced OA produced low levels of IL-6 and exhibited reduced cartilage damage and synovitis in the joints. These findings indicate that ZCCHC6 enhances IL-6 expression in chondrocytes through transcript stabilization and by uridylating miR-26b, which abrogates repression of IL-6. Inhibition of IL-6 expression and significantly reduced OA severity in Zcchc6-/- mice identify ZCCHC6 as a novel therapeutic target to inhibit disease pathogenesis.

Cyclosporin A inhibits adipogenic differentiation and regulates immunomodulatory functions of murine mesenchymal stem cells

Aplastic anemia (AA) is generally considered as an immune-mediated bone marrow failure syndrome. Several studies show that bone marrow mesenchymal stem cells (BM-MSCs), as key cellular components of the bone marrow microenvironment, are also involved in the pathogenic mechanism of AA. Cyclosporin A (CsA) is a classic immunosuppressive drug for AA, and it specifically inhibits mammalian T cells by preventing activation of transcription factors involved in cytokine gene expression. However, little is known about the effect of CsA on the BM-MSCs. In this study, murine BM-MSCs were stimulated in the presence of CsA. Further, we found that CsA could inhibit murine BM-MSC proliferation and promote BM-MSC apoptosis, what's more CsA could inhibit adipogenic differentiation. Our study also showed that CsA could inhibit interleukin-6 expression in BM-MSCs, while promoting programmed death-ligand 2 expression. In conclusion, our results proposed that CsA may exert an effect on regulating the bone marrow environment by influencing BM-MSCs, which have a beneficial effect on treating AA.

Phenylmethimazole and a thiazole derivative of phenylmethimazole inhibit IL-6 expression by triple negative breast cancer cells

Inhibition of interleukin–6 (IL-6) holds significant promise as a therapeutic approach for triple negative breast cancer (TNBC). We previously reported that phenylmethimazole (C10) reduces IL-6 expression in several cancer cell lines. We have identified a more potent derivative of C10 termed COB-141. In the present work, we tested the hypothesis that C10 and COB-141 inhibit TNBC cell expressed IL-6 and investigated the potential for classical IL-6 pathway induced signaling within TNBC cells. A panel of TNBC cell lines (MDA-MB-231, Hs578T, MDA-MB-468) was used. Enzyme linked immunosorbent assays (ELISA) revealed that C10 and COB-141 inhibit MDA-MB-231 cell IL-6 secretion, with COB-141 being ~6.5 times more potent than C10. Therefore, the remainder of the study focused on COB-141 which inhibited IL-6 secretion, and was found, via quantitative real time polymerase chain reaction (QRT-PCR), to inhibit IL-6 mRNA in the TNBC panel. COB-141 had little, if any, effect on metabolic activity indicating that the IL-6 inhibition is not via a toxic effect. Flow cytometric analysis and QRT-PCR revealed that the TNBC cell lines do not express the IL-6 receptor (IL-6Rα). Trans-AM assays suggested that COB-141 exerts its inhibitory effect, at least in part, by reducing NF-κB (p65/p50) DNA binding. In summary, COB-141 is a potent inhibitor of TNBC cell expressed IL-6 and the inhibition does not appear to be due to non-specific toxicity. The TNBC cell lines do not have an intact classical IL-6 signaling pathway. COB-141's inhibitory effect may be due, at least in part, to reducing NF-κB (p65/p50) DNA binding.

Activation of GPER suppresses migration and angiogenesis of triple negative breast cancer via inhibition of NF-κB/IL-6 signals

Triple-negative breast cancer (TNBC) is characterized by high vascularity and frequent metastasis. Here, we found that activation of G protein-coupled estrogen receptor (GPER) by its specific agonist G-1 can significantly inhibit interleukin 6 (IL-6) and vascular endothelial growth factor A (VEGF-A). TNBC tissue microarrays from 100 TNBC patients revealed GPER is negatively associated with IL-6 levels and higher grade and stage. Activation of GPER or anti-IL-6 antibody can inhibit both in vitro tube formation of human umbilical vein endothelial cells (HUVECs) and migration of TNBC cells. While recombinant IL-6 supplementary can significantly reverse the inhibitory effects of G-1, suggesting the essential role of IL-6 in G-1 induced suppression of angiogenesis and invasiveness of TNBC cells. G-1 treatment decreased the phosphorylation, nuclear localization, transcriptional activities of NF-κB and suppressed its binding with IL-6 promoter. BAY11-7028, the inhibitor of NF-κB, can mimic the effect of G-1 to suppression of IL-6 and VEGF-A. While over expression of p65 can attenuate the inhibitory effects of G-1 on IL-6 and VEGF expression. The suppression of IL-6 by G-1 can further inhibit HIF-1α and STAT3 signals in TNBC cells by inhibition their expression, phosphorylation and/or nuclear localization. Moreover, G-1 also inhibited the in vivo NF-κB/IL-6 signals and angiogenesis and metastasis of MDA-MB-231 xenograft tumors. In conclusion, our study demonstrated that activation of GPER can suppress migration and angiogenesis of TNBC via inhibition of NF-κB/IL-6 signals, therefore it maybe act as an important target for TNBC treatment.

Nicorandil Inhibits Cyclic Strain-Induced Interleukin-8 Expression in Human Umbilical Vein Endothelial Cells

Background: Nicorandil, a mitochondrial adenosine triphosphate-sensitive potassium (mitoKATP) channel opener, exerts protective effects on the cardiovascular system. This study examined the effect of nicorandil on cyclic strain-induced interleukin-8 (IL-8) expression in human umbilical vein endothelial cells (HUVECs). Methods: Cultured HUVECs were exposed to cyclic strain in the presence or absence of nicorandil (1-10 μmol/l); we then analyzed IL-8 expression. We also assessed the effects of nicorandil on heme oxygenase-1 (HO-1) expression and cyclic strain-modulated IL-8 expression after HO-1 silencing in HUVECs. Summary: HUVECs exposed to cyclic strain showed increased IL-8 messenger RNA expression and protein secretion. Nicorandil (1-10 μmol/l) inhibited cyclic strain-induced IL-8 expression, whereas 5-hydroxydecanoate (100 μmol/l), a selective inhibitor of the mitoKATP channel, completely reversed the inhibitory effects of nicorandil on cyclic strain-induced IL-8 expression. We demonstrated that nicorandil increased HO-1 expression in HUVECs. In addition, cobalt protoporphyrin (10 μmol/l), an inducer of HO-1 expression, mimicked the effects of nicorandil and inhibited IL-8 expression under cyclic strain, whereas zinc protoporphyrin IX (10 μmol/l), an inhibitor of HO-1 expression, antagonized the effect of nicorandil. HO-1 silencing significantly abrogated the inhibitory effects of nicorandil on cyclic strain-induced IL-8 expression, suggesting that HO-1 plays a role in the mechanism of action of nicorandil. Key Messages: This study is the first to report that nicorandil inhibits cyclic strain-induced IL-8 expression through the induction of HO-1 expression in HUVECs. This finding provides valuable new insight into the molecular pathways contributing to the vasoprotective effects of nicorandil.

Oxidative stress and endocytosis are involved in upregulation of interleukin-8 expression in airway cells exposed to PM2.5.

Inhaled PM2.5 (particulate matter with an aerodynamic diameter of 2.5 μm or less) can induce lung inflammation through released inflammatory mediators from airway cells, such as interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α). However, the mechanisms underlying PM2.5-induced IL-8 gene expression have not been fully characterized. BEAS-2B cells (a human bronchial epithelial cell line) and THP-1 cells (a human macrophage-like cell line) were used as the in vitro models to investigate the underlying mechanism in this study. IL-8 expression was increased in the cells treated with PM2.5 in a dose-dependent manner. The water-soluble and insoluble fractions of PM2.5 suspension were both shown to induce IL-8 expression. PM2.5 exposure could obviously induce ROS (reactive oxygen species) generation, indicative of oxidative stress. Pretreatment with the antioxidant N-acetyl-l-cysteine (NAC) potently inhibited PM2.5-induced IL-8 expression. Employment of the transition metal chelators including TPEN (N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine) or DFO (desferrioxamine) inhibited IL-8 expression induced by PM2.5 by over 20% in BEAS-2B cells, but had minimal effect in THP-1 cells. Pretreatment with the endocytosis inhibitor CytD markedly blocked IL-8 expression induced by PM2.5 in both BEAS-2B and THP-1 cells. In summary, exposure to PM2.5 induced IL-8 gene expression through oxidative stress induction and endocytosis in airway cells. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1869-1878, 2016.

Growth inhibition effect of matrine on K562 cells mediated by IL-6/JAK/STAT3 signaling pathway

目的探讨JAK/STAT3介导的信号途径在苦参碱对K562细胞增殖抑制作用中的分子机制。方法以不同浓度苦参碱处理K562细胞，Western blot法分析JAK/STAT3通路分子JAK2、STAT3及其磷酸化蛋白表达的改变；荧光实时定量RT-PCR及Western blot法分析STAT3下游调控基因Bcl-xL、Cyclin D1、c-Myc的表达；荧光实时定量RT-PCR及ELISA法检测K562细胞IL-6表达的改变。以IL-6预处理K562细胞，Western blot法分析苦参碱对细胞内JAK2、STAT3及其磷酸化蛋白表达的作用。结果0.5 mg/ml苦参碱处理48 h, K562细胞内STAT3及JAK2总蛋白表达无明显变化，磷酸化STAT3(p-Tyr705 STAT3、p-Ser727 STAT3)和磷酸化JAK2 (p-JAK2）表达水平分别由处理前的0.690± 0.119、1.150±0.263和0.670±0.137下降至0.370±0.024、0.700±0.172和0.049±0.057。苦参碱对STAT3下游调控基因Bcl-xL、Cyclin D1及c-Myc的转录及蛋白表达均有抑制作用，并可显著抑制IL-6的表达和分泌，0.5和0.8 mg/ml苦参碱处理后细胞培养上清中IL-6浓度分别为(10.74±1.83) pg/ml和(8.66± 1.24) pg/ml，较处理前[(35.1±1.93) pg/ml]显著降低(P<0.05)。IL-6预处理K562细胞后，STAT3、JAK2、p-STAT3及p-JAK2表达均有增加，苦参碱可抑制IL-6对上述分子表达的上调。结论苦参碱抑制K562细胞增殖与JAK/STAT3介导的信号通路抑制有关，IL-6表达抑制可能是苦参碱调控JAK/STAT3通路活性的始动机制。

Taurine inhibits interleukin-6 expression and release induced by ultraviolet B exposure to human retinal pigment epithelium cells

Background: The massive uptake of compatible osmolytes is a self-protective response shared by retina exposed to hypertonic stress and ultraviolet stress.Objective: This study aimed to investigate the protective effects of taurine against ultraviolet damage in human retinal pigment epithelium cells.Methods: Real-time PCR, radioimmunoassay, ELISA and immunoassay were used to measure osmolyte uptake and IL-6 expression.Results: Compared with normotonic stress, hypertonic stress led to an induction of osmolyte uptake including betaine, myoinositol and taurine. UVB exposure upregulated osmolyte transporter mRNA expression and increased osmolyte uptake respectively. Especially, taurine suppressed UVB-induced IL-6 mRNA expression significantly. The accumulation of IL-6 in UVB-exposed human retinal pigment epithelial cells supernatant was much slower when the cells were preincubated with taurine. Moreover, taurine suppressed IL-6 concentration in aqueous humour.Conclusion: The effect of compatible osmolyte taurine on IL-6 expression and release may play an important role in cell resistance and adaption to UVB exposure.

Tristetraprolin is involved in the glucocorticoid-mediated interleukin 8 repression.

Glucocorticoids have been widely used in various inflammatory disorders, and the transcriptional repression of inflammatory mediators has been considered to be the main mechanism of action. However, a previous study showed that dexamethasone inhibited interleukin 8 (IL-8) expression by promoting IL-8 mRNA decay, which implies a posttranscriptional regulation. Nevertheless, by which mechanism dexamethasone destabilized IL-8 mRNA was unclear. Another study indicated that an RNA-binding protein, tristetraprolin (TTP), could be induced by dexamethasone. TTP can bind to AU-rich elements (ARE) in the 3'-untranslated region of target mRNAs and promotes mRNA degradation. So, we speculated that dexamethasone destabilized IL-8 mRNA by upregulating TTP expression. Here, we report that dexamethasone reduced IL-8 expression through destabilizing IL-8 mRNA in human pulmonary microvascular endothelial cells (HPMECs). Dexamethasone stimulation increased TTP mRNA and protein levels. TTP silencing led to mRNA stabilization and protein upregulation of IL-8. These results provide the evidence that the glucocorticoid, in HPMECs, inhibits IL-8 expression through TTP at the posttranscriptional level.

IL-1β Upregulates IL-8 Production in Human Müller Cells Through Activation of the p38 MAPK and ERK1/2 Signaling Pathways.

Diabetic retinopathy shares some similarity with chronic inflammation and Müller cells dysfunction may play an important role in its initiation and progression since these cells are thought to be a major source of inflammatory factors. The goal of this study was to examine the effect of cytokines on human retinal Müller cells and to understand the underlying signal transduction pathways regulating interleukin-8 (IL-8) expression. In this study, human MIO-M1 cells were treated with interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-8, vascular endothelial growth factor (VEGF), interferon-gamma (IFN-γ), glucose, or mannitol, followed by examination of their IL-8 protein and mRNA levels by Western blotting and PCR, respectively. After treatment with IL-1β, the levels of phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), Janus kinase 2 (JAK2), and signal transducer and activator of transcription 3 (STAT3) were measured. IL-8 was also measured by Western blotting and ELISA following Müller cell culture with IL-1β and specific inhibitors of the p38 MAPK, ERK1/2, JNK, or JAK2 pathways. The results showed that IL-1β was a potent inducer of IL-8 expression in MIO-M1 cells, although a relatively small increase was induced by TNF-α. IL-6, IL-8, VEGF, and IFN-γ did not modify IL-8 expression. Increase of IL-8 expression was accompanied by a significant increased phosphorylation of p38 MAPK, ERK, and JNK, but not of JAK2 and STAT3. Furthermore, inhibitors of p38 MAPK and MEK1/2, but not for JNK and JAK2, significantly inhibited IL-8 expression. In conclusion, IL-1β potently stimulates IL-8 expression in Müller cells mainly through the p38 MAPK and ERK1/2 pathways.

Inhibition of Interleukin-6 Expression by Curcumin in Rat Vascular Smooth Muscle Explants &lt;I&gt;In Vitro&lt;/I&gt;

In atherosclerotic plaques, infectious agents from microbes may release Lipopolysaccharide (LPS) and heat shock proteins that can stimulate the production of mediators, i.e., pro-inflammatory cytokines, by vascular endothelial cells and Smooth Muscle Cells (SMCs). The elevated level of Interleukin-6 (IL-6) is strongly associated with the development of atherosclerosis. Here, we investigated whether curcumin isolated from the rhizome of Curcuma longa affected the expression of IL-6 at protein and gene levels in rat smooth muscle cells treated with LPS in vitro by conducting ELISA and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assays. LPS at 2 µg mL-1 activated the expression of IL-6 protein and mRNA in SMCs. Curcumin (1-15 µM) caused the decreased levels of IL-6 protein and mRNA in the dose-dependent manner in LPS-induced SMCs, indicating its potential antiatherosclerotic effect for cardiovascular risk management. In addition, curcumin also partially blocked the activation of LPS-induced phosphorylation of MAP kinases, i.e., ERK1/2, p38 and JNK, in SMCs, suggesting it may inhibit IL-6 expression via attenuating MAP kinase signaling pathways in LPS-induced SMCs. These data may in part explain the molecular action of antiatherosclerotic effects of curcumin.

Prostaglandin E2 Induces IL-6 and IL-8 Production by the EP Receptors/Akt/NF-κB Pathways in Nasal Polyp-Derived Fibroblasts.

PurposeInterleukin 6 (IL-6) and IL-8 participate in the pathogenesis of chronic rhinosinusitis with nasal polyps, and their levels are increased by prostaglandin E2 (PGE2) in different cell types. The purposes of this study were to determine whether PGE2 has any effect on the increase in the levels of IL-6 and IL-8 in nasal polyp-derived fibroblasts (NPDFs) and subsequently investigate the possible mechanism of this effect.MethodsDifferent concentrations of PGE2 were used to stimulate NPDFs at different time intervals. NPDFs were treated with agonists and antagonists of E prostanoid (EP) receptors. To determine the signaling pathway for the expression of PGE2-induced IL-6 and IL-8, PGE2 was treated with Akt and NF-κB inhibitors in NPDFs. Reverse transcription-polymerase chain reaction for IL-6 and IL-8 mRNAs was performed. IL-6 and IL-8 levels were measured byenzyme-linked immunosorbent assay (ELISA). The activation of Akt and NF-κB was evaluated by western blot analysis.ResultsPGE2 significantly increased the mRNA and protein expression levels of IL-6 and IL-8 in NPDFs. The EP2 and EP4 agonists and antagonists induced and inhibited IL-6 expression. However, the EP4 agonist and antagonist were only observed to induce and inhibit IL-8 expression level. The Akt and NF-κB inhibitors significantly blocked PGE2-induced expression of IL-6 and IL-8.ConclusionsPGE2 increases IL-6 expression via EP2 and EP4 receptors, and IL-8 expression via the EP4 receptor in NPDFs. It also activates the Akt and NF-κB signal pathways for the production of IL-6 and IL-8 in NPDFs. These results suggest that signaling pathway for IL-6 and IL-8 expression induced by PGE2 might be a useful therapeutic target for the treatment of nasal polyposis.