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      Baicalin Down-Regulates IL-1β-Stimulated Extracellular Matrix Production in Nasal Fibroblasts

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          Abstract

          Purpose

          Baicalin, a Chinese herbal medicine, has anti-fibrotic and anti-inflammatory effects. The aims of present study were to investigate the effects of baicalin on the myofibroblast differentiation, extracellular matrix production, migration, and collagen contraction of interleukin (IL)-1β-stimulated nasal fibroblasts and to determine the molecular mechanism of baicalin in nasal fibroblasts.

          Methods

          Nasal fibroblasts were isolated from the inferior turbinate of patients. Baicalin was used to treat IL-1β-stimulated nasal fibroblasts. To evaluate cytotoxicity, a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay was used. The expression levels of α-smooth muscle actin (SMA), fibronectin, phospho-mitogen-activated protein kinase (p-MAPK), p-Akt, p-p50, p-p65, and p-IκBα were measured by western blotting, reverse transcription-polymerase chain reaction (RT—PCR),or immunofluorescence staining. Fibroblast migration was analyzed with scratch assays and transwell migration assays. Total collagen was evaluated with the Sircol collagen assay. Contractile activity was measured with a collagen gel contraction assay.

          Results

          Baicalin (0–50 μM) had no significant cytotoxic effects in nasal fibroblasts. The expression of α SMA and fibronectin were significantly down-regulated in baicalin-treated nasal fibroblasts. Migration, collagen production, and contraction of IL-1β-stimulated nasal fibroblasts were significantly inhibited by baicalin treatment. Baicalin also significantly down-regulated p-MAPK, p-Akt, p-p50, p-p65, and p-IκBα in IL-1β-stimulated nasal fibroblasts.

          Conclusions

          We showed that baicalin down-regulated myofibroblast differentiation, extracellular matrix production, migration, and collagen contraction via the MAPK and Akt/ NF-κB pathways in IL-1β-stimulated nasal fibroblasts.

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          Most cited references21

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          Phosphorylation of NF-kappaB and IkappaB proteins: implications in cancer and inflammation.

          Nuclear factor-kappaB (NF-kappaB) is a transcription factor that has crucial roles in inflammation, immunity, cell proliferation and apoptosis. Activation of NF-kappaB mainly occurs via IkappaB kinase (IKK)-mediated phosphorylation of inhibitory molecules, including IkappaBalpha. Optimal induction of NF-kappaB target genes also requires phosphorylation of NF-kappaB proteins, such as p65, within their transactivation domain by a variety of kinases in response to distinct stimuli. Whether, and how, phosphorylation modulates the function of other NF-kappaB and IkappaB proteins, such as B-cell lymphoma 3, remains unclear. The identification and characterization of all the kinases known to phosphorylate NF-kappaB and IkappaB proteins are described here. Because deregulation of NF-kappaB and IkappaB phosphorylations is a hallmark of chronic inflammatory diseases and cancer, newly designed drugs targeting these constitutively activated signalling pathways represent promising therapeutic tools.
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            Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis.

            IL-1beta is one of a family of proinflammatory cytokines thought to be involved in many acute and chronic diseases. Although it is considered to participate in wound repair, no major role has been attributed to IL-1beta in tissue fibrosis. We used adenoviral gene transfer to transiently overexpress IL-1beta in rat lungs after intratracheal administration. The high expression of IL-1beta in the first week after injection was accompanied by local increase of the proinflammatory cytokines IL-6 and TNF-alpha and a vigorous acute inflammatory tissue response with evidence of tissue injury. The profibrotic cytokines PDGF and TGF-beta1 were increased in lung fluid samples 1 week after peak expression of IL-1beta. Although PDGF returned to baseline in the third week, TGF-beta1 showed increased concentrations in bronchoalveolar lavage fluid for up to 60 days. This was associated with severe progressive tissue fibrosis in the lung, as shown by the presence of myofibroblasts, fibroblast foci, and significant extracellular accumulations of collagen and fibronectin. These data directly demonstrate how acute tissue injury in the lung, initiated by a highly proinflammatory cytokine, IL-1beta, converts to progressive fibrotic changes. IL-1beta should be considered a valid target for therapeutic intervention in diseases associated with fibrosis and tissue remodeling.
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              Interleukin-1 receptor type I signaling critically regulates infarct healing and cardiac remodeling.

              The proinflammatory cytokine interleukin (IL)-1 signals exclusively through the type I IL-1 receptor (IL-1RI). IL-1 expression is markedly induced in the infarcted heart; however, its role in cardiac injury and repair remains controversial. We examined the effects of disrupted IL-1 signaling on infarct healing and cardiac remodeling using IL-1RI(-/-) mice. After reperfused infarction IL-1RI-null mice exhibited decreased infiltration of the infarcted myocardium with neutrophils and macrophages and reduced chemokine and cytokine expression. In the absence of IL-1 signaling, suppressed inflammation was followed by an attenuated fibrotic response. Infarcted IL-1RI(-/-) mice had decreased myofibroblast infiltration and reduced collagen deposition in the infarcted and remodeling myocardium. IL-1RI deficiency protected against the development of adverse remodeling; however, infarct size was comparable between groups suggesting that the beneficial effects of IL-1RI gene disruption were not attributable to decreased cardiomyocyte injury. Reduced chamber dilation in IL-1RI-null animals was associated with decreased collagen deposition and attenuated matrix metalloproteinase (MMP)-2 and MMP-3 expression in the peri-infarct area, suggesting decreased fibrotic remodeling of the noninfarcted heart. IL-1beta stimulated MMP mRNA synthesis in wild-type, but not in IL-1RI-null cardiac fibroblasts. In conclusion, IL-1 signaling is essential for activation of inflammatory and fibrogenic pathways in the healing infarct, playing an important role in the pathogenesis of remodeling after infarction. Thus, interventional therapeutics targeting the IL-1 system may have great benefits in myocardial infarction.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                21 December 2016
                2016
                : 11
                : 12
                : e0168195
                Affiliations
                [1 ]Department of Otorhinolaryngology-Head and Neck Surgery, Korea University College of Medicine, Seoul, South Korea
                [2 ]Division of Brain Korea 21 Program for Biomedical Science, Korea University College of Medicine, Seoul, South Korea
                [3 ]Institute for Korea University Medical Devices Support Center, Korea University College of Medicine, Seoul, South Korea
                Medical University of South Carolina, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: HML.

                • Data curation: JMS JHK.

                • Formal analysis: JMS SAL.

                • Funding acquisition: HML.

                • Investigation: JMS SAL HML.

                • Methodology: JMS JHK SAL.

                • Project administration: JMS.

                • Resources: HML.

                • Software: JHK SAL.

                • Supervision: HML IHP.

                • Validation: JHK JMS.

                • Visualization: JHK JMS.

                • Writing – original draft: JMS.

                • Writing – review & editing: IHP SAL HML JMS.

                Article
                PONE-D-16-22755
                10.1371/journal.pone.0168195
                5176301
                28002421
                d418b429-5da2-4db4-9401-923d83e6c497
                © 2016 Shin et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 6 June 2016
                : 28 November 2016
                Page count
                Figures: 5, Tables: 0, Pages: 14
                Funding
                This study was supported by a grant from the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI15C1512 and HI13C1501). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Connective Tissue Cells
                Fibroblasts
                Biology and Life Sciences
                Anatomy
                Biological Tissue
                Connective Tissue
                Connective Tissue Cells
                Fibroblasts
                Medicine and Health Sciences
                Anatomy
                Biological Tissue
                Connective Tissue
                Connective Tissue Cells
                Fibroblasts
                Biology and Life Sciences
                Biochemistry
                Proteins
                Collagens
                Biology and life sciences
                Cell biology
                Signal transduction
                Cell signaling
                Signaling cascades
                MAPK signaling cascades
                Biology and Life Sciences
                Physiology
                Muscle Physiology
                Muscle Contraction
                Medicine and Health Sciences
                Physiology
                Muscle Physiology
                Muscle Contraction
                Medicine and Health Sciences
                Inflammatory Diseases
                Research and analysis methods
                Bioassays and physiological analysis
                Biochemical analysis
                Colorimetric assays
                MTT assay
                Research and analysis methods
                Bioassays and physiological analysis
                Biochemical analysis
                Enzyme assays
                MTT assay
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Extracellular Matrix
                Physical Sciences
                Chemistry
                Chemical Compounds
                Bromides
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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