Modafinil exerts anti-inflammatory and anti-fibrotic effects by upregulating adenosine A2A and A2B receptors.

Adenosine receptor (AR) suppresses inflammation and fibrosis by activating cyclic adenosine monophosphate (cAMP) signaling. We investigated whether altered AR expression contributes to the development of fibrotic diseases and whether A2AAR and A2BAR upregulation inhibits fibrotic responses. Primary human lung fibroblasts (HLFs) from normal (NHLFs) or patients with idiopathic pulmonary fibrosis (DHLF) were used for in vitro testing. Murine models of fibrotic liver or pulmonary disease were developed by injecting thioacetamide intraperitoneally, by feeding a high-fat diet, or by intratracheal instillation of bleomycin. Modafinil, which activates cAMP signaling via A2AAR and A2BAR, was administered orally. The protein amounts of A2AAR, A2BAR, and exchange protein directly activated by cAMP (Epac) were reduced, while collagen and α-smooth muscle actin (α-SMA) were elevated in DHLFs compared to NHLFs. In liver or lung tissue from murine models of fibrotic diseases, A2AAR and A2BAR were downregulated, but A1AR and A3AR were not. Epac amounts decreased, and amounts of collagen, α-SMA, KCa2.3, and KCa3.1 increased compared to the control. Modafinil restored the amounts of A2AAR, A2BAR, and Epac, and reduced collagen, α-SMA, KCa2.3, and KCa3.1 in murine models of fibrotic diseases. Transforming growth factor-ß reduced the amounts of A2AAR, A2BAR, and Epac, and elevated collagen, α-SMA, KCa2.3, and KCa3.1 in NHLFs; however, these alterations were inhibited by modafinil. Our investigation revealed that A2AAR and A2BAR downregulation induced liver and lung fibrotic diseases while upregulation attenuated fibrotic responses, suggesting that A2AAR and A2BAR-upregulating agents, such as modafinil, may serve as novel therapies for fibrotic diseases.

Anti-inflammatory and anti-fibrotic effects of modafinil in nonalcoholic liver disease.

Small- and intermediate-conductance Ca2+-activated K+ channels, KCa2.3 and KCa3.1, are involved in cellular signaling processes associated with inflammation and fibrosis. KCa2.3 and KCa3.1 are upregulated by proinflammatory cytokines and profibrotic growth factors. Cyclic AMP, which downregulates KCa2.3 and KCa3.1, is elevated by modafinil in cells; accordingly, we investigated whether modafinil exerts anti-inflammatory and anti-fibrotic responses via KCa2.3- and KCa3.1-mediated pathways in high-fat diet (HFD)- or thioacetamide-induced liver disease models in mice. Modafinil was administered orally in the form of a racemate, (R)-isomer, or (S)-isomer. We also determined whether the treatment targeted the profibrotic activity of hepatic stellate cells using immortalized human hepatic stellate cells (LX-2 cells). Modafinil improved HFD- or thioacetamide-induced changes compared to the control, leading to a reduced inflammatory response, collagen deposition, and α-smooth muscle actin expression both in vivo and in vitro. However, modafinil did not relieve HFD-induced steatosis. There were no significant differences in the effects of the (R)- and (S)-isomers of modafinil. KCa2.3 and KCa3.1 were upregulated and catalase was downregulated in liver tissues from thioacetamide- or HFD-induced liver disease models or in TGF-ß-treated LX-2 cells. TGF-ß-induced upregulation of KCa2.3, KCa3.1, collagen, and α-smooth muscle actin and downregulation of catalase were reversed by modafinil, polyethylene glycol catalase, N-acetylcysteine, siRNA against KCa2.3 or KCa3.1, and Epac inhibitors. Our investigation revealed that modafinil attenuated inflammatory and fibrotic progression via KCa2.3- and KCa3.1-mediated pathways in nonalcoholic hepatitis, suggesting that inhibiting KCa2.3- and KCa3.1-mediated signaling may serve as a novel therapeutic approach for inflammatory and fibrotic liver diseases.

Toxic effects of dietary copper and EGCG on bioaccumulation, antioxidant enzyme and immune response of Korean bullhead, Pseudobagrus fulvidraco.

There are few reports of dietary Cu (copper) toxicity to Korean bullhead, Pseudobagrus fulvidraco, and little is known about recovery from dietary Cu exposure. In this study, P. fulvidraco (mean length 16.9 ± 1.38 cm, and mean weight 53.2 ± 1.22 g) were exposed for 4 weeks to dietary Cu concentration of 0 (control), 700, 900, and 1100 mg Cu kg-1 dry feed to establish maximum tolerable levels of dietary Cu. All fish were then fed the dietary EGCG (Epigallocatechin gallate) concentration of 100 and 500 mg EGCG kg-1 dry feed for a further 2 weeks to assess recovery. We were measured bioaccumulation (in the intestine, liver, and gill tissue), antioxidant enzymes (SOD and CAT) and immune responses (lysozyme and phagocytosis). The Cu exposure induced a significant accumulation in the intestine, liver, and gill tissues and the highest accumulation was observed in intestinal tissues (17-34 fold), but dietary EGCG exposure decreased (about 0.8-fold) Cu concentration in each tissue (ANOVA, P < 0.05). In antioxidant enzymes, SOD and CAT significantly increased by approximately 1.6-fold by dietary Cu exposure in the liver and gill tissue, respectively, but dietary EGCG exposure decreased SOD and CAT by about 1.1-fold, respectively (ANOVA, P < 0.05). For immune responses, lysozyme and phagocytosis in the blood significantly were decreased by approximately 1.5-fold, respectively, by dietary Cu exposure, but dietary EGCG exposure increased lysozyme and phagocytosis by about 1.1-fold, respectively (ANOVA, P < 0.05). During recovery period, bioaccumulation, antioxidant enzymes (SOD and CAT activity), and immune response (lysozyme and phagocytosis activity) tended to alleviate the significant changes by Cu exposure, and the tendency to return normal state was observed in high level of EGCG. The result of this study indicate that Cu exposure to P. fulvidraco affects bioaccumulation, antioxidant enzymes, and immune responses, and high level of EGCG were effective to alleviate the toxic effects of Cu exposure.

Effect of Influenza Virus Infection in a Murine Model of Asthma.

Respiratory virus infection is a major cause of asthma exacerbation. However, the underlying mechanisms of this exacerbation are unknown. Therefore, to determine the mechanisms, we examined the effect of influenza infection in a murine model of asthma. Mice were divided into four groups: the phosphate-buffered saline (PBS), house dust mite(HDM), influenza, and HDM/influenza groups. The influenza group and the HDM/influenza group were infected with influenza A virus. We measured airway resistance (Penh value), examined the lung tissue for pathology, and analyzed the cells and cytokines in bronchoalveolar lavage fluid (BALF) by ELISA. At 50 mg/mL methacholine, the HDM/influenza group showed a significantly higher Penh value than the PBS, HDM, and influenza groups. The number of neutrophils in BALF was higher in the HDM/influenza group than in the HDM group. A significantly greater number of lymphocytes and macrophages were detected in the HDM/influenza group than in the HDM group. IFN-γ and IL-1ß levels were higher in the HDM/influenza group than in the HDM group. IL-5 levels did not vary between the HDM and HDM/influenza groups, IL-10 was significantly lower in the HDM/influenza than in the HDM group. Chemokine (C-X-C motif) ligand 1 (CXCL1) and regulated upon activation, normal T cell expressed and secreted (RANTES) were higher in the HDM/influenza group than in the HDM group. In a murine model of asthma, influenza-induced airway inflammation appeared to be caused by simultaneous activation of neutrophilic and eosinophilic inflammation.

The effect of rhinovirus on airway inflammation in a murine asthma model.

PURPOSE: The aim of the present study was to investigate the differences in lower airway inflammatory immune responses, including cellular responses and responses in terms of inflammatory mediators in bronchoalveolar lavage fluid (BALF) and the airway, to rhinovirus (RV) infection on asthma exacerbation by comparing a control and a murine asthma model, with or without RV infection. METHODS: BALB/c mice were intraperitoneally injected with a crude extract of Dermatophagoides farinae (Df) or phosphate buffered saline (PBS) and were subsequently intranasally treated with a crude extract of Df or PBS. Airway responsiveness and cell infiltration, differential cell counts in BALF, and cytokine and chemokine concentrations in BALF were measured 24 hours after intranasal RV1B infection. RESULTS: RV infection increased the enhanced pause (Penh) in both the Df sensitized and challenged mice (Df mice) and PBS-treated mice (PBS mice) (P<0.05). Airway eosinophil infiltration increased in Df mice after RV infection (P<0.05). The levels of interleukin (IL) 13, tumor necrosis factor alpha, and regulated on activation, normal T cells expressed and secreted (RANTES) increased in response to RV infection in Df mice, but not in PBS mice (P<0.05). The level of IL-10 significantly decreased following RV infection in Df mice (P<0.05). CONCLUSION: Our findings suggest that the augmented induction of proinflammatory cytokines, Th2 cytokines, and chemokines that mediate an eosinophil response and the decreased induction of regulatory cytokines after RV infection may be important manifestations leading to airway inflammation with eosinophil infiltration and changes in airway responsiveness in the asthma model.

Rhinovirus-Infected Epithelial Cells Produce More IL-8 and RANTES Compared With Other Respiratory Viruses.

PURPOSE: The environmental factors human rhinoviruses (HRVs) and house dust mites (HDMs) are the most common causes of acute exacerbations of asthma. The aim of this study was to compare the chemokine production induced by HRVs in airway epithelial cells with that induced by other respiratory viruses, and to investigate synergistic interactions between HRVs and HDMs on the induction of inflammatory chemokines in vitro. METHODS: A549 human airway epithelial cells were infected with either rhinovirus serotype 7, respiratory syncytial virus (RSV)-A2 strain, or adenovirus serotype 3 and analyzed for interleukin (IL)-8 and regulated on activation, normal T-cell expressed and secreted (RANTES) release and mRNA expression. Additionally, activation of nuclear factor (NF)-κB and activator protein (AP)-1 were evaluated. The release of IL-8 and RANTES was also measured in cells stimulated simultaneously with a virus and the HDM allergen, Der f1. RESULTS: HRV caused greater IL-8 and RANTES release and mRNA expression compared with either RSV or adenovirus. NF-κB and AP-1 were activated in these processes. Cells incubated with a virus and Der f1 showed an increased IL-8 release. However, compared with cells incubated with virus alone as the stimulator, only HRV with Der f1 showed a statistically significant increase. CONCLUSIONS: IL-8 and RANTES were induced to a greater extent by HRV compared with other viruses, and only HRV with Der f1 acted synergistically to induce bronchial epithelial IL-8 release. These findings may correspond with the fact that rhinoviruses are identified more frequently than other viruses in cases of acute exacerbation of asthma.

Sodium sulfite enhances rhinovirus-induced chemokine production in airway epithelial cells.

We investigated the effects of sodium sulfite (Na(2)SO(3)) on rhinovirus (RV)-induced chemokine production in A549 airway epithelial cells. Our results demonstrated that the treatment of A549 cells with 2,500 µM Na(2)SO(3) enhanced the mRNA expression of RV-induced interleukin (IL)-8 1.8 fold (p = 0.025); and regulated on activation, normal T cell expressed and secreted (RANTES), 2.9 fold (p = 0.025). Moreover, the secretion of IL-8, RANTES, and interferon-γ-inducible protein (IP)-10 was increased in a statistically significant manner without affecting cell viability and RV replication. Our results suggest that Na(2)SO(3) may potentiate RV infection by enhancing chemokine production.

Effect of respiratory syncytial virus infection on regulated on activation, normal T-cells expressed and secreted production in a murine model of asthma.

PURPOSE: Synthesis of regulated on activation, normal T-cells expressed and secreted (RANTES) in the airway has previously been shown to be elevated after respiratory syncytial virus (RSV) infection. However, since few studies have examined whether RSV-infected asthma patients express a higher level of RANTES than do normal individuals, we used a murine model of asthma to address this question. METHODS: We prepared Dermatophagoides farinae-sensitized mice as an asthma model, and then infected them with RSV and analyzed the changes in airway responsiveness and the cell populations and cytokine levels of bronchoalveolar lavage fluid. RESULTS: RANTES synthesis increased in response to RSV infection in both control mice and in asthma model (D. farinae) mice. However, there was no significant difference in the amount of RANTES produced following RSV infection between control and D. farinae mice. RSV infection affected neither interferon-γsynthesis nor airway responsiveness in either control or D. farinae mice. CONCLUSION: RSV infection did not induce more RANTES in a murine model of asthma than in control mice.