Integrin‐linked kinase is involved in TNF‐α‐induced inducible nitric‐oxide synthase expression in myoblasts

Tumor necrosis factor‐α (TNF‐α) is a pleiotropic cytokine produced by activated macrophages. Nitric oxide (NO) is a highly reactive nitrogen radical implicated in inflammatory responses. We investigated the signaling pathway involved in inducible nitric oxide synthase (iNOS) expression and NO production stimulated by TNF‐α in cultured myoblasts. TNF‐α stimulation caused iNOS expression and NO production in myoblasts (G7 cells). TNF‐α‐mediated iNOS expression was attenuated by integrin‐linked kinase (ILK) inhibitor (KP392) and siRNA. Pretreatment with Akt inhibitor, mammalian target of rapamycin (mTOR) inhibitor (rapamycin), NF‐κB inhibitor (PDTC), and IκB protease inhibitor (TPCK) also inhibited the potentiating action of TNF‐α. Stimulation of cells with TNF‐α increased ILK kinase activity. TNF‐α also increased the Akt and mTOR phosphorylation. TNF‐α mediated an increase of NF‐κB‐specific DNA–protein complex formation, p65 translocation into nucleus, NF‐κB‐luciferase activity was inhibited by KP392, Akt inhibitor, and rapamycin. Our results suggest that TNF‐α increased iNOS expression and NO production in myoblasts via the ILK/Akt/mTOR and NF‐κB signaling pathway. J. Cell. Biochem. 109: 1244–1253, 2010. © 2010 Wiley‐Liss, Inc.     

FAK activation is required for TNF‐α‐induced IL‐6 production in myoblasts

Tumor necrosis factor‐α (TNF‐α) is a pleiotropic cytokine produced by activated macrophages. IL‐6 is a multifunctional cytokine that plays a central role in both innate and acquired immune responses. We investigated the signaling pathway involved in IL‐6 production stimulated by TNF‐α in cultured myoblasts. TNF‐α caused concentration‐dependent increases in IL‐6 production. TNF‐α‐mediated IL‐6 production was attenuated by focal adhesion kinase (FAK) mutant and siRNA. Pretreatment with phosphatidylinositol 3‐kinase inhibitor (PI3K; Ly294002 and wortmannin), Akt inhibitor, NF‐κB inhibitor (pyrrolidine dithiocarbamate, PDTC), and IκB protease inhibitor (L ‐1‐tosylamido‐2‐phenyl phenylethyl chloromethyl ketone, TPCK) also inhibited the potentiating action of TNF‐α. TNF‐α increased the FAK, PI3K, and Akt phosphorylation. Stimulation of myoblasts with TNF‐α activated IκB kinase α/β (IKKα/β), IκBα phosphorylation, p65 phosphorylation, and κB‐luciferase activity. TNF‐α mediated an increase of κB‐luciferase activity which was inhibited by Ly294002, wortmannin, Akt inhibitor, PDTC and TPCK or FAK, PI3K, and Akt mutant. Our results suggest that TNF‐α increased IL‐6 production in myoblasts via the FAK/PI3K/Akt and NF‐κB signaling pathway. J. Cell. Physiol. 223: 389–396, 2010. © 2010 Wiley‐Liss, Inc.     

Qiliqiangxin protects against anoxic injury in cardiac microvascular endothelial cells via NRG‐1/ErbB‐PI3K/Akt/mTOR pathway

Cardiac microvascular endothelial cells (CMECs) are important angiogenic components and are injured rapidly after cardiac ischaemia and anoxia. Cardioprotective effects of Qiliqiangxin (QL), a traditional Chinese medicine, have been displayed recently. This study aims to investigate whether QL could protect CMECs against anoxic injury and to explore related signalling mechanisms. CMECs were successfully cultured from Sprague‐Dawley rats and exposed to anoxia for 12 hrs in the absence and presence of QL. Cell migration assay and capillary‐like tube formation assay on Matrigel were performed, and cell apoptosis was determined by TUNEL assay and caspase‐3 activity. Neuregulin‐1 (NRG‐1) siRNA and LY294002 were administrated to block NRG‐1/ErbB and PI3K/Akt signalling, respectively. As a result, anoxia inhibited cell migration, capillary‐like tube formation and angiogenesis, and increased cell apoptosis. QL significantly reversed these anoxia‐induced injuries and up‐regulated expressions of NRG‐1, phospho‐ErbB2, phospho‐ErbB4, phospho‐Akt, phospho‐mammalian target of rapamycin (mTOR), hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF) in CMECs, while NRG‐1 knockdown abolished the protective effects of QL with suppressed NRG‐1, phospho‐ErbB2, phospho‐ErbB4, phospho‐Akt, phospho‐mTOR, HIF‐1α and VEGF expressions. Similarly, LY294002 interrupted the beneficial effects of QL with down‐regulated phospho‐Akt, phospho‐mTOR, HIF‐1α and VEGF expressions. However, it had no impact on NRG‐1/ErbB signalling. Our data indicated that QL could attenuate anoxia‐induced injuries in CMECs via NRG‐1/ErbB signalling which was most probably dependent on PI3K/Akt/mTOR pathway.     

Eriodictyol ameliorates lipopolysaccharide‐induced acute lung injury by suppressing the inflammatory COX‐2/NLRP3/NF‐κB pathway in mice

The purpose of this paper is to observe the protective action and its effective mechanism of eriodictyol on lipopolysaccharide (LPS)‐induced acute lung injury (ALI). In this study, our results indicated that eriodictyol could dramatically suppress the inflammatory mediators, including interleukin‐6 (IL‐6), IL‐1β, prostaglandin E2, and tumor necrosis factor‐α in bronchoalveolar lavage fluid of LPS‐challenged mice. Eriodictyol also alleviated the wet/dry ratio and improved pathological changes of the lung. In addition, eriodictyol significantly decreased myeloperoxidase activity and malondialdehyde content as well as increased superoxide dismutase activity. Moreover, eriodictyol inhibited the COX‐2/NLRP3/NF‐κB signaling pathway in the lung tissues of ALI mice. In conclusion, our observations validated that eriodictyol processed the protective effects on ALI mice, which was related to the regulation of the COX‐2/NLRP3/NF‐κB signaling pathway.     

Pinitol attenuates LPS‐induced pneumonia in experimental animals: Possible role via inhibition of the TLR‐4 and NF‐κB/IκBα signaling cascade pathway

Pneumonia is a chronic disorder of the respiratory system associated with worsening quality of life and a significant economic burden. Pinitol, a plant cyclic polyol, has been documented for immune‐inflammatory potential. The aim of present investigation was to evaluate the potential and possible mechanism of action of pinitol against lipopolysaccharide (LPS)‐induced pneumonia in the experimental animal model. Pneumonia was induced in Sprague‐Dawley rats by intratracheal administration of LPS (2 mg/kg). Animals were treated with either vehicle or dexamethasone or pinitol (5 or 10 or 20 mg/kg). Potential of pinitol against LPS‐induced pulmonary insult was assessed based on behavioral, biochemical, molecular, and ultrastructural studies. Intratracheal instillation of LPS induced significant (P < .05) inflammatory infiltration in bronchoalveolar lavage fluid (BALF) and lung tissue reflected by elevated pleural effusion volume, lung edema, BALF polymorphonuclear leukocytes count and lung myeloperoxidase levels, which was attenuated by pinitol (10 and 20 mg/kg) administration. Pinitol also markedly (P < .05) inhibited LPS‐induced alterations in electrocardiographic, hemodynamic changes, right ventricular, and lung function tests. The LPS‐induced downregulated nuclear factor erythroid 2–related factor 2 (Nrf‐2) and heme oxygenase‐1 (HO‐1), whereas upregulated transforming growth factor‐β (TGF‐β), tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), IL‐6, NOD‐, LRR‐, and pyrin domain‐containing protein 3 (NLRP3), and inducible nitric oxide synthase (iNOs) lung messenger RNA expressions were significantly (P < .05) inhibited by pinitol. Western blot analysis suggested pinitol markedly (P < .05) decreased nuclear factor‐κB (NF‐κB), inhibitor of nuclear factor κB (IkBα), toll‐like receptor 4 (TLR‐4), and cyclooxygenase‐II (COX‐II) protein expressions in the lung. These findings were further supported by histological and ultrastructural analyses of lung tissue that show pinitol significantly (P < .05) ameliorates LPS‐induced aberrations in lung tissue. In conclusion, pinitol attenuated LPS‐induced pneumonia via inhibition of TLR‐4 to downregulate the NF‐κB/IκBα signaling cascade and thus ameliorated the production of proinflammatory cytokines (TNF‐α, ILs, NLRP3, and TGF‐β), inflammatory mediators (COX‐II and iNOs) and elevated oxidative stress (Nrf‐2 and HO‐1).     

Dioscorealide B suppresses LPS‐induced nitric oxide production and inflammatory cytokine expression in RAW 264.7 macrophages: The inhibition of NF‐κB and ERK1/2 activation

Dioscorealide B (DB), a naphthofuranoxepin has been purified from an ethanolic extract of the rhizome of Dioscorea membranacea Pierre ex Prain & Burkill which has been used to treat inflammation and cancer in Thai Traditional Medicine. Previously, DB has been reported to have anti‐inflammatory activities through reducing nitric oxide (NO) and tumor necrosis factor‐α (TNF‐α) production in lipopolysaccharides (LPS)‐induced RAW 264.7 macrophage cells. In this study, the mechanisms of DB on LPS‐induced NO production and cytokine expression through the activation of nuclear factor‐κB (NF‐κB) and ERK1/2 are demonstrated in RAW 264.7 cells. Through measurement with Griess's reagent, DB reduced NO level with an IC₅₀ value of 2.85 ± 0.62 µM that was due to the significant suppression of LPS‐induced iNOS mRNA expression as well as IL‐1β, IL‐6, and IL‐10 mRNA at a concentration of 6 µM. At the signal transduction level, DB significantly inhibited NF‐κB binding activity, as determined using pNFκB‐Luciferase reporter system, which action resulted from the prevention of IκBα degradation. In addition, DB in the range of 1.5–6 µM significantly suppressed the activation of the ERK1/2 protein. In conclusion, the molecular mechanisms of DB on the inhibition of NO production and mRNA expression of iNOS, IL‐1β, IL‐6, and IL‐10 were due to the inhibition of the upstream kinases activation, which further alleviated the NF‐κB and MAPK/ERK signaling pathway in LPS‐induced RAW264.7 macrophage cells. J. Cell. Biochem. 109: 1057–1063, 2010. © 2010 Wiley‐Liss, Inc.     

PMC,a potent hydrophilic α‐tocopherol derivative,inhibits NF‐κB activation via PP2A but not IκBα‐dependent signals in vascular smooth muscle cells

The hydrophilic α‐tocopherol derivative, 2,2,5,7,8‐pentamethyl‐6‐hydroxychromane (PMC), is a promising alternative to vitamin E in clinical applications. Critical vascular inflammation leads to vascular dysfunction and vascular diseases, including atherosclerosis, hypertension and abdominal aortic aneurysms. In this study, we investigated the mechanisms of the inhibitory effects of PMC in vascular smooth muscle cells (VSMCs) exposed to pro‐inflammatory stimuli, lipopolysaccharide (LPS) combined with interferon (IFN)‐γ. Treatment of LPS/IFN‐γ‐stimulated VSMCs with PMC suppressed the expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase‐9 in a concentration‐dependent manner. A reduction in LPS/IFN‐γ‐induced nuclear factor (NF)‐κB activation was also observed in PMC‐treated VSMCs. The translocation and phosphorylation of p65, protein phosphatase 2A (PP2A) inactivation and the formation of reactive oxygen species (ROS) were significantly inhibited by PMC in LPS/IFN‐γ‐activated VSMCs. However, neither IκBα degradation nor IκB kinase (IKK) or ribosomal s6 kinase‐1 phosphorylation was affected by PMC under these conditions. Both treatments with okadaic acid, a PP2A‐selective inhibitor, and transfection with PP2A siRNA markedly reversed the PMC‐mediated inhibition of iNOS expression, NF‐κB‐promoter activity and p65 phosphorylation. Immunoprecipitation analysis of the cellular extracts of LPS/IFN‐γ‐stimulated VSMCs revealed that p65 colocalizes with PP2A. In addition, p65 phosphorylation and PP2A inactivation were induced in VSMCs by treatment with H₂O₂, but neither IκBα degradation nor IKK phosphorylation was observed. These results collectively indicate that the PMC‐mediated inhibition of NF‐κB activity in LPS/IFN‐γ‐stimulated VSMCs occurs through the ROS‐PP2A‐p65 signalling cascade, an IKK‐IκBα‐independent mechanism. Therapeutic interventions using PMC may therefore be beneficial for the treatment of vascular inflammatory diseases.     

Aromatic‐turmerone attenuates invasion and expression of MMP‐9 and COX‐2 through inhibition of NF‐κB activation in TPA‐induced breast cancer cells

Recent evidence suggests that breast cancer is one of the most common forms of malignancy in females, and metastasis from the primary cancer site is the main cause of death. Aromatic (ar)‐turmerone is present in Curcuma longa and is a common remedy and food. In the present study, we investigated the inhibitory effects of ar‐turmerone on expression and enzymatic activity levels of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced matrix metalloproteinase (MMP)‐9 and cyclooxygenaase‐2 (COX‐2) in breast cancer cells. Our data indicated that ar‐turmerone treatment significantly inhibited enzymatic activity and expression of MMP‐9 and COX‐2 at non‐cytotoxic concentrations. However, the expression of tissue inhibitor of metalloproteinase (TIMP)‐1, TIMP‐2, MMP‐2, and COX‐1 did not change upon ar‐turmerone treatment. We found that ar‐turmerone inhibited the activation of NF‐κB, whereas it did not affect AP‐1 activation. Moreover, The ChIP assay revealed that in vivo binding activities of NF‐κB to the MMP‐9 and COX‐2 promoter were significantly inhibited by ar‐turmerone. Our data showed that ar‐turmerone reduced the phosphorylation of PI3K/Akt and ERK1/2 signaling, whereas it did not affect phosphorylation of JNK or p38 MAPK. Thus, transfection of breast cancer cells with PI3K/Akt and ERK1/2 siRNAs significantly decreased TPA‐induced MMP‐9 and COX‐2 expression. These results suggest that ar‐turmerone suppressed the TPA‐induced up‐regulation of MMP‐9 and COX‐2 expression by blocking NF‐κB, PI3K/Akt, and ERK1/2 signaling in human breast cancer cells. Furthermore, ar‐turmerone significantly inhibited TPA‐induced invasion, migration, and colony formation in human breast cancer cells. J. Cell. Biochem. 113: 3653–3662, 2012. © 2012 Wiley Periodicals, Inc.     

Overexpression of microRNA‐138 alleviates human coronary artery endothelial cell injury and inflammatory response by inhibiting the PI3K/Akt/eNOS pathway

This study aimed to investigate the role of miR‐138 in human coronary artery endothelial cell (HCAEC) injury and inflammatory response and the involvement of the PI3K/Akt/eNOS signalling pathway. Oxidized low‐density lipoprotein (OX‐LDL)‐induced HCAEC injury models were established and assigned to blank, miR‐138 mimic, miR‐138 inhibitor, LY294002 (an inhibitor of the PI3K/Akt/eNOS pathway), miR‐138 inhibitor + LY294002 and negative control (NC) groups. qRT‐PCR and Western blotting were performed to detect the miR‐138, PI3K, Akt and eNOS levels and the protein expressions of PI3K, Akt, eNOS, p‐Akt, p‐eNOS, Bcl‐2, Bax and caspase‐3. ELISAs were employed to measure the expressions of TNF‐α, IL‐4, IL‐6, IL‐8, IL‐10 and nitric oxide (NO) and the activities of lactate dehydrogenase (LDH) and eNOS. MTT and flow cytometry were performed to assess the proliferation and apoptosis of HCAECs. Compared to the blank group, PI3K, Akt and eNOS were down‐regulated in the miR‐138 mimic and LY294002 groups but were up‐regulated in the miR‐138 inhibitor group. The miR‐138 mimic and LY294002 groups showed decreased concentrations of TNF‐α, IL‐6, IL‐8 and NO and reduced activities of LDH and eNOS, while opposite trends were observed in the miR‐138 inhibitor group. The concentrations of IL‐4 and IL‐10 increased in the miR‐138 mimic and LY294002 groups but decreased in the miR‐138 inhibitor group. The miR‐138 mimic and LY294002 groups had significantly decreased cell proliferation and increased cell apoptosis compared to the blank group. These findings indicate that up‐regulation of miR‐138 alleviates HCAEC injury and inflammatory response by inhibiting the PI3K/Akt/eNOS signalling pathway.     

A proteomics approach to identifying novel protein targets involved in erinacine A–mediated inhibition of colorectal cancer cells’ aggressiveness

Erinacine A, a major active component of a diterpenoid derivative isolated from Hericium erinaceus mycelium, has been demonstrated to exert anticancer effects. Herein, we present an investigation of the molecular mechanism of erinacine A induction associated with cancer cells’ aggressive status and death. A proteomic approach was used to purify and identify the differentially expressed proteins following erinacine A treatment and the mechanism of its action in apoptotic and the targets of erinacine A. Our results demonstrate that erinacine A treatment of HCT‐116 and DLD‐1 cells increased cell cytotoxicity and reactive oxygen species (ROS) production as well as decreased cell proliferation and invasiveness. Ten differentially displayed proteins were determined and validated in vitro and in vivo between the erinacine A‐treated and untreated groups. In addition, erinacine A time‐dependent induction of cell death and inhibitory invasiveness was associated with sustained phosphorylation of the PI3K/mTOR/p70S6K and ROCK1/LIMK2/Cofilin pathways. Furthermore, we demonstrated that erinacine A–induced HCT‐116 and DLD‐1 cells viability and anti‐invasion properties by up‐regulating the activation of PI3K/mTOR/p70S6K and production of ROS. Experiments involving specific inhibitors demonstrated that the differential expression of cofilin‐1 (COFL1) and profilin‐1 (PROF1) during erinacine A treatment could be involved in the mechanisms of HCT‐116 and DLD‐1 cells death and decreased aggressiveness, which occurred via ROCK1/LIMK2/Cofilin expression, with activation of the PI3K/mTOR/p70S6K signalling pathway. These findings elucidate the mechanism of erinacine A inhibiting the aggressive status of cells by activating PI3K/mTOR/p70S6K downstream signalling and the novel protein targets COF1 and PROF1; this could be a good molecular strategy to limit the aggressiveness of CRC cells.     

Simvastatin abolishes nitric oxide‐ and reactive oxygen species‐induced cyclooxygenase‐2 expression by blocking the nuclear factor κB pathway in rabbit articular chondrocytes

Nitric oxide (NO) and reactive oxygen species (ROS) have been shown to be linked with numerous diseases, including osteoarthritis (OA). Our study aimed to examine the effect of simvastatin on NO‐ or ROS‐induced cyclooxygenase‐2 (COX‐2) expression in OA. Simvastatin has attracted considerable attention since the discovery of its pharmacological effects on different pathogenic processes, including inflammation. Here, we report that simvastatin treatment blocked sodium nitroprusside (SNP)‐ and interleukin 1 beta (IL‐1β)‐induced COX‐2 production. In addition, simvastatin attenuated SNP‐induced NO production and IL‐1β‐induced ROS generation. Treatment with simvastatin prevented SNP‐ and IL‐1β‐induced nuclear factor kappa B (NF‐κB) activity. Inhibiting NO production and ROS generation using N‐acetylcysteine (NAC) and NG‐monomethyl‐ l ‐arginine ( l ‐NMMA), respectively, accelerated the influence of simvastatin on NF‐κB activity. In addition, NAC blocked SNP and simvastatin‐mediated COX‐2 production and NF‐κB activity but did not alter IL‐1β and simvastatin‐mediated COX‐2 expression. l ‐NMMA treatment also abolished IL‐1β‐mediated COX‐2 expression and NF‐κB activation, whereas SNP and simvastatin‐mediated COX‐2 expression were not altered compared with the levels in the SNP and simvastatin‐treated cells. Our findings suggested that simvastatin blocks COX‐2 expression by inhibiting SNP‐induced NO production and IL‐1β‐induced ROS generation by blocking the NF‐κB pathway.     

Acetate reduces microglia inflammatory signaling in vitro

Acetate supplementation increases brain acetyl‐CoA and histone acetylation and reduces lipopolysaccharide (LPS)‐induced neuroglial activation and interleukin (IL)‐1β expression in vivo. To determine how acetate imparts these properties, we tested the hypothesis that acetate metabolism reduces inflammatory signaling in microglia. To test this, we measured the effect acetate treatment had on cytokine expression, mitogen‐activated protein kinase (MAPK) signaling, histone H3 at lysine 9 acetylation, and alterations of nuclear factor‐kappa B (NF‐κB) in primary and BV‐2 cultured microglia. We found that treatment induced H3K9 hyperacetylation and reversed LPS‐induced H3K9 hypoacetylation similar to that found in vivo. LPS also increased IL‐1β, IL‐6, and tumor necrosis factor‐alpha (TNF‐α) mRNA and protein, whereas treatment returned the protein to control levels and only partially attenuated IL‐6 mRNA. In contrast, treatment increased mRNA levels of transforming growth factor‐β1 (TGF‐β1) and both IL‐4 mRNA and protein. LPS increased p38 MAPK and JNK phosphorylation at 4 and 2–4 h, respectively, whereas treatment reduced p38 MAPK and JNK phosphorylation only at 2 h. In addition, treatment reversed the LPS‐induced elevation of NF‐κB p65 protein and phosphorylation at serine 468 and induced acetylation at lysine 310. These data suggest that acetate metabolism reduces inflammatory signaling and alters histone and non‐histone protein acetylation.     

IL‐17A‐producing T cells exacerbate fine particulate matter‐induced lung inflammation and fibrosis by inhibiting PI3K/Akt/mTOR‐mediated autophagy

Fine particulate matter (PM2.5) is the primary air pollutant that is able to induce airway injury. Compelling evidence has shown the involvement of IL‐17A in lung injury, while its contribution to PM2.5‐induced lung injury remains largely unknown. Here, we probed into the possible role of IL‐17A in mouse models of PM2.5‐induced lung injury. Mice were instilled with PM2.5 to construct a lung injury model. Flow cytometry was carried out to isolate γδT and Th17 cells. ELISA was adopted to detect the expression of inflammatory factors in the supernatant of lavage fluid. Primary bronchial epithelial cells (mBECs) were extracted, and the expression of TGF signalling pathway‐, autophagy‐ and PI3K/Akt/mTOR signalling pathway‐related proteins in mBECs was detected by immunofluorescence assay and Western blot analysis. The mitochondrial function was also evaluated. PM2.5 aggravated the inflammatory response through enhancing the secretion of IL‐17A by γδT/Th17 cells. Meanwhile, PM2.5 activated the TGF signalling pathway and induced EMT progression in bronchial epithelial cells, thereby contributing to pulmonary fibrosis. Besides, PM2.5 suppressed autophagy of bronchial epithelial cells by up‐regulating IL‐17A, which in turn activated the PI3K/Akt/mTOR signalling pathway. Furthermore, IL‐17A impaired the energy metabolism of airway epithelial cells in the PM2.5‐induced models. This study suggested that PM2.5 could inhibit autophagy of bronchial epithelial cells and promote pulmonary inflammation and fibrosis by inducing the secretion of IL‐17A in γδT and Th17 cells and regulating the PI3K/Akt/mTOR signalling pathway.     

Lancemaside A inhibits lipopolysaccharide‐induced inflammation by targeting LPS/TLR4 complex

In our previous study, lancemaside A isolated from Codonopsis lanceolata (family Campanulaceae) ameliorated colitis in mice. In this study, the anti‐inflammatory effects of lancemaside A was investigated in lipopolysaccharide (LPS)‐stimulated mice and their peritoneal macrophage cells. Lancemaside A suppressed the production of pro‐inflammatory cytokines, TNF‐α and IL‐1β, in vitro and in vivo. Lancemaside A also down‐regulated inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2), as well as the inflammatory mediators, nitric oxide (NO), and PGE₂. Lancemaside A also inhibited the expression of IL‐1 receptor‐associated kinase‐4 (IRAK‐4), the phosphorylation of IKK‐β and IκB‐α, the nuclear translocation of NF‐κB and the activation of mitogen‐activated protein kinases in LPS‐stimulated peritoneal macrophages. Furthermore, lancemaisde A inhibited the interaction between LPS and TLR4, as well as IRAK‐4 expression in peritoneal macrophages. Based on these findings, lancemaside A expressed anti‐inflammatory effects by regulating both the binding of LPS to TLR4 on macrophages. J. Cell. Biochem. 111: 865–871, 2010. © 2010 Wiley‐Liss, Inc.     

Estrogen‐related receptor alpha triggers the proliferation and migration of human non‐small cell lung cancer via interleukin‐6

Human non‐small cell lung cancer (NSCLC) is one of the leading causes of cancer deaths worldwide. Estrogenic signals have been suggested to be important for the growth and metastasis of NSCLC cells. Our present data showed that estrogen‐related receptor alpha (ERRα), while not ERRβ or ERRγ, was significantly elevated in NSCLC cell lines as compared with that in normal bronchial epithelial cell line BEAS‐2B. The expression of ERRα in clinical NSCLC tissues was significantly greater than that in their matched normal adjacent tissues. Over expression of ERRα can trigger the proliferation, migration, and invasion of NSCLC cells, while si‐ERRα or ERRα inhibitor showed opposite effects. ERRα can increase the mRNA and protein expression of IL‐6, while not IL‐8, IL‐10, IL‐22, VEGF, TGF‐β, or TNF‐α, in NSCLC cells. Silence of IL‐6 attenuated ERRα induced proliferation and cell invasion. Furthermore, our data revealed the inhibition of NF‐κB, while not ERK1/2 or PI3K/Akt, abolished ERRα induced production of IL‐6. This might be due to that overexpression of ERRα can increase the expression and nuclear translocation of p65 in NSCLC cells. Collectively, our data showed that activation of NF‐κB/IL‐6 is involved in ERRα induced migration and invasion of NSCLC cells. It suggested that ERRα might be a potential target for NSCLC treatment.     

Hemoglobin induces the expression of indoleamine 2,3‐dioxygenase in dendritic cells through the activation of PI3K,PKC, and NF‐κB and the generation of reactive oxygen species

Indoleamine 2,3‐dioxygenase (IDO) is the rate‐limiting enzyme in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism. IDO is immunosuppressive and is induced by inflammation in macrophages and dendritic cells (DCs). Previous studies have shown the serum Kyn/Trp levels in patients with hemolytic anemia to be notably high. In the present study, we demonstrated that hemoglobin (Hb), but not hemin or heme‐free globin (Apo Hb), induced IDO expression in bone marrow‐derived myeloid DCs (BMDCs). Hb induced the phosphorylation and degradation of IκBα. Hb‐induced IDO expression was inhibited by inhibitors of PI3‐kinase (PI3K), PKC and nuclear factor (NF)‐κB. Hb translocated both RelA and p52 from the cytosol to the nucleus and induced the intracellular generation of reactive oxygen species (ROS). Hb‐induced IDO expression was inhibited by anti‐oxidant N‐acetyl‐L ‐cysteine (NAC) or mixtures of SOD and catalase, however, IDO expression was enhanced by 3‐amino‐1,2,4‐triazole, an inhibitor of catalase, suggesting that the generation of ROS such as O, H₂O₂, and hydroxyl radical is required for the induction of IDO expression. The generation of ROS was inhibited by a PKC inhibitor, and this action was further enhanced by addition of a PI3K inhibitor. Hb induced Akt phosphorylation, which was inhibited by a PI3K inhibitor and enhanced by a PKC inhibitor. These results suggest that the activation of NF‐κB through the PI3K‐PKC‐ROS and PI3K‐Akt pathways is required for the Hb‐induced IDO expression in BMDCs. J. Cell. Biochem. 108: 716–725, 2009. © 2009 Wiley‐Liss, Inc.     

Penehyclidine hydrochloride protects against anoxia/reoxygenation injury in cardiomyocytes through ATP‐sensitive potassium channels,and the Akt/GSK‐3β and Akt/mTOR signaling pathways

Penehyclidine hydrochloride (PHC) can protect against myocardial ischemia/reperfusion (I/R) injury. However, the possible mechanisms of PHC in anoxia/reoxygenation (A/R)‐induced injury in H9c2 cells remain unclear. In the present study, H9c2 cells were pretreated with PI3K/Akt inhibitor LY294002, ATP‐sensitive K⁺ (KATP) channel blocker 5‐hydroxydecanoate (5‐HD), PHC, or KATP channel opener diazoxide (DZ) before subjecting to A/R injury. Cell viability and cell apoptosis were determined by cell counting kit‐8 assay and annexin V/PI assay, respectively. Myocardial injury was evaluated by measuring creatine kinase (CK) and lactate dehydrogenase (LDH) activities. Intracellular Ca²⁺ levels, reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨ_m), and mitochondrial permeability transition pore (mPTP) were measured. The levels of cytoplasmic/mitochondrial cytochrome c (Cyt‐C), Bax, Bcl‐2, cleaved caspase‐3, KATP channel subunits (Kir6.2 and SUR2A), and the members of the Akt/GSK‐3β and Akt/mTOR signaling pathways were determined by western blotting. We found that PHC preconditioning alleviated A/R‐induced cell injury by increasing cell viability, reducing CK and LDH activities, and inhibiting cell apoptosis. In addition, PHC preconditioning ameliorated intracellular Ca²⁺ overload and ROS production, accompanied by inhibition of both mPTP opening and Cyt‐C release into cytoplasm, and maintenance of ΔΨ_m. Moreover, PHC preconditioning activated mitochondrial KATP channels, and modulated the Akt/GSK‐3β and Akt/mTOR signaling pathways. Similar effects were observed upon treatment with DZ. Pretreatment with LY294002 or 5‐HD blocked the beneficial effects of PHC. These results suggest that the protective effects of PHC preconditioning on A/R injury may be related to mitochondrial KATP channels, as well as the Akt/GSK‐3β and Akt/mTOR signaling pathways.