Targeting the phosphorylation site of myristoylated alanine-rich C kinase substrate alleviates symptoms in a murine model of steroid-resistant asthma.

BACKGROUND AND PURPOSE: Myristoylated alanine-rich C kinase substrate (MARCKS), a PKC substrate, facilitates mucus production and neutrophil migration. However, the effects of therapeutic procedures targeting the phosphorylation site of MARCKS on steroid-resistant asthma and the mechanisms underlying such effects have not yet been investigated. We designed a peptide that targets the MARCKS phosphorylation site (MPS peptide) and assessed its therapeutic potential against steroid-resistant asthma. EXPERIMENTAL APPROACH: Mice were sensitized with ovalbumin (OVA), alum, and challenged with aerosolized OVA five times a week for 1 month. The mice were intratracheally administered MPS peptides three times a week, 1 hr before OVA challenge. Asthma symptoms and cell profiles in the bronchoalveolar lavage were assessed, and key proteins were analysed using Western blotting. KEY RESULTS: Phosphorylated (p)-MARCKS was highly expressed in inflammatory and bronchial epithelial cells in OVA-immunized mice. MPS peptide reduced eosinophils, neutrophils, mucus production, collagen deposition, and airway hyper-responsiveness. Dexamethasone (Dexa) did not alleviate steroid-resistant asthma symptoms. MPS peptide caused a decrease in p-MARCKS, nitrotyrosine and the expression of oxidative stress enzymes, NADPH oxidase dual oxidase 1 and inducible NOS, in lung tissues. Compared to Dexa, MPS peptides inhibited C5a production and attenuated IL-17A and KC production in the airway more effectively, thus suppressing asthma symptoms. CONCLUSIONS AND IMPLICATIONS: Our findings indicate that targeting MARCKS phosphorylation through MPS treatment may inhibit neutrophilic inflammation and relieve asthma symptoms, thereby highlighting its potential as a therapeutic agent for steroid-resistant asthma.

Baicalin Ameliorates Imiquimod-Induced Psoriasis-Like Inflammation in Mice.

Baicalin is the main flavonoid from the roots of an important medicinal plant, Scutellaria baicalensis, which shows a variety biological activities. Psoriasis is a chronic immune-mediated inflammatory disease that affects the skin. The unmet need of psoriasis is that many patients do not respond adequately to available clinical treatment. In this study, we found that baicalin showed inhibited dermal inflammation in a murine model of psoriasis via topical application of imiquimod. After a 5-day topical imiquimod application, baicalin or the control vehicle cream was to applied to the lesions of BALB/c mice for a further 4 days. The erythema, scaling, and thickness of the epidermal layer significantly improved in the baicalin-treated mice. The levels of interleukin-17A, interleukin-22, interleukin-23, and tumor necrosis factor in the skin significantly decreased after baicalin treatment. Baicalin also inhibited imiquimod-induced interleukin-17A production in skin draining lymph node cells. The infiltration of γδ T cells into the skin lesions induced by imiquimod was also suppressed after baicalin treatment. These results suggest that baicalin inhibited skin inflammation through the inhibition of the interleukin-17/interleukin-23 axis in a murine model of psoriasis.

Ovatodiolide suppresses allergic airway inflammation and hyperresponsiveness in a murine model of asthma.

Asthma, a complex pulmonary allergic disease, major therapy is applied of drugs to control the disease, but quickly recur after the drugs are stopped. In patients with severe asthma may show steroid resistance and would benefit from the development of novel therapeutic drugs. Ovatodiolide, a unique macrocyclic diterpenoid isolated from Anisomeles indica, showed therapeutic potential for the treatment of allergic asthma. As a model of allergic inflammation, we used ovalbumin (OVA)-immunized mice, which displayed T helper cell type 2 (TH2) cytokine expression in bronchoalveolar lavage fluid (BALF), as well as airway inflammation and hyperresponsiveness (AHR). The results showed that ovatodiolide suppressed TH2 activation, including cell proliferation and production of the TH2 related cytokines, interleukin (IL)-4, IL-5, IL-13, IL-33, eosinophil chemotactic protein (eotaxin), and also reduced airway hyperresponsiveness. In this study, ovatodiolide inhibited allergic asthma through downregulation of TH2 responses in a murine model of asthma.

High mobility group box 1-induced epithelial mesenchymal transition in human airway epithelial cells.

Epithelial-mesenchymal transition (EMT) is implicated in bronchial remodeling and loss of lung function in chronic inflammatory airway diseases. Previous studies showed the involvement of the high mobility group box 1 (HMGB1) protein in the pathology of chronic pulmonary inflammatory diseases. However, the role of HMGB1 in EMT of human airway epithelial cells is still unclear. In this study, we used RNA sequencing to show that HMGB1 treatment regulated EMT-related gene expression in human primary-airway epithelial cells. The top five upregulated genes were SNAI2, FGFBP1, VIM, SPARC (osteonectin), and SERPINE1, while the downregulated genes included OCLN, TJP1 (ZO-1), FZD7, CDH1 (E-cadherin), and LAMA5. We found that HMGB1 induced downregulation of E-cadherin and ZO-1, and upregulation of vimentin mRNA transcription and protein translation in a dose-dependent manner. Additionally, we observed that HMGB1 induced AKT phosphorylation, resulting in GSK3ß inactivation, cytoplasmic accumulation, and nuclear translocation of ß-catenin to induce EMT in human airway epithelial cells. Treatment with PI3K inhibitor (LY294006) and ß-catenin shRNA reversed HMGB1-induced EMT. Moreover, HMGB1 induced expression of receptor for advanced glycation products (RAGE), but not that of Toll-like receptor (TLR) 2 or TLR4, and RAGE shRNA inhibited HMGB1-induced EMT in human airway epithelial cells. In conclusion, we found that HMGB1 induced EMT through RAGE and the PI3K/AKT/GSK3ß/ß-catenin signaling pathway.

High mobility group box 1 induced human lung myofibroblasts differentiation and enhanced migration by activation of MMP-9.

High mobility group box 1 (HMGB1) is a nuclear protein that involves the binding with DNA and influences chromatin regulation and transcription. HMGB1 is also a cytokine that can activate monocytes and neutrophils involved in inflammation. In this study, we investigated the role of HMGB1 on cellular activation using human fibroblast cell line WI-38. After treatment with 1, 10, and 100 ng/mL of HMGB1 for 24 h, we did not find obviously cytotoxicity and cellular proliferation of WI-38 cells by MTT and BrdU incorporation assay, respectively. However, we found that treatment with 10 and 100 ng/mL of HMGB1 induced the differentiation of lung fibroblasts into myofibroblasts and myofibroblasts showed higher migration ability through activation of matrix metalloproteinase (MMP)-9 activation. To delineate the mechanism underlying HMGB1-induced cellular migration, we examined HMGB1-induced mitogen activated protein kinases (MAPKs), including extracellular signal related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen activated protein kinase (p38) phosphorylation, as well as nuclear factor (NF)-κB nuclear translocation. Using specific inhibitors and shRNAs of protein kinases, we observed that repression of ERK, JNK, p38, and NF-κB all inhibited HMGB1-induced cellular differentiation, migration and MMP-9 activation in WI-38 cells. In addition, knocking down of RAGE but not TLR2 and TLR4 by shRNAs attenuated HMGB1-induced myofibroblast differentiation and migration. In conclusion, our study demonstrated that HMGB1 induced lung fibroblasts' differentiation into myofibroblasts and enhanced cell migration through induction of MMP-9 activation and the RAGE-MAPK and NF-κB interaction signaling pathways. Targeting HMGB1 might be a potential therapeutic approach for alleviation of airway remodeling seen in chronic airway inflammatory diseases.

Inhibition of high-mobility group box 1 in lung reduced airway inflammation and remodeling in a mouse model of chronic asthma.

The role of high-mobility group box 1 (HMGB1) in chronic allergic asthma is currently unclear. Both airway neutrophilia and eosinophilia and increase in HMGB1 expression in the lungs in our murine model of chronic asthma. Inhibition of HMGB1 expression in lung in ovalbumin (OVA)-immunized mice decreased induced airway inflammation, mucus formation, and collagen deposition in lung tissues. Analysis of the numbers of CD4(+) T helper (Th) cells in the mediastinal lymph nodes and lungs revealed that Th17 showed greater increases than Th2 cells and Th1 cells in OVA-immunized mice; further, the numbers of Th1, Th2, and Th17 cells decreased in anti-HMGB1 antibody (Ab)-treated mice. In OVA-immunized mice, TLR-2 and TLR-4 expression, but not RAGE expression, was activated in the lungs and attenuated after anti-HMGB1 Ab treatment. The results showed that increase in HMGB1 release and expression in the lungs could be an important pathological mechanism underlying chronic allergic asthma and HMGB1 might a potential therapeutic target for chronic allergic asthma.

Antiallergic asthma properties of brazilin through inhibition of TH2 responses in T cells and in a murine model of asthma.

This study aimed to determine whether brazilin exhibits anti-inflammatory effects that inhibit T helper cell type II (T(H)2) responses and whether it suppresses allergic inflammation reactions in a murine model of asthma. We found that brazilin inhibited the mRNA and protein expression of interleukin (IL)-4 and IL-5 induced by phorbol myristate acetate (PMA) and cAMP in EL-4 T cells in a dose-dependent manner. Following the intratracheal instillation of brazilin in ovalbumin (OVA)-immunized mice, we found that brazilin-treated mice exhibited decreases in the release of IL-4, IL-5, IL-13, eotaxin-1, and tumor necrosis factor-α in bronchoalveolar lavage fluid (BALF); inhibited T(H)2 functioning via a decrease in IL-4 production; and exhibited attenuation of OVA-induced lung eosinophilia, airway hyperresponsiveness, and airway remodeling. These results suggest that brazilin exhibits anti-T(H)2 effects both in vitro and in vivo and may possess therapeutic potential for allergic diseases.

Shikonin inhibited mitogen-activated IL-4 and IL-5 production on EL-4 cells through downregulation of GATA-3 and c-Maf induction.

AIM: To investigate the effects of shikonin on phorbol myristate acetate (PMA) plus cyclic adenosine monophosphate (cAMP)-induced T helper (T(H)) 2 cell cytokine production, and the underlying mechanism. MAIN METHODS: We used activated EL-4 murine T-lymphoma cells, which produce interleukin (IL)-4 and IL-5, but not interferon (IFN)-γ, as T(H)2 cell-like cells and treated them with PMA+cAMP to investigate the effects of shikonin on T(H)2 cytokines, transcriptional factors, and the related mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB signaling pathway. KEY FINDINGS: The data show that shikonin inhibited the PMA+cAMP-induced mRNA and protein expression of IL-4 and IL-5 via the downregulation of GATA-binding protein-3 (GATA-3) and c-musculoaponeurotic fibrosarcoma (Maf) but not T-box expressed in T cells (T-bet). Moreover, shikonin suppressed the phosphorylation of p38, inhibitor of κB (IκB) kinase (IKK)-ß and IκB-α, and the subsequent IκB-α degradation induced by PMA+cAMP; however, the PMA+cAMP-induced phosphorylation of extracellular signal-related kinase (ERK), which resulted in minor inhibition and phosphorylation of c-Jun N-terminal kinase (JNK), seemed to be unaffected by shikonin treatment. SIGNIFICANCE: This study suggests that downregulation of GATA-3 and c-Maf via the suppression of p38, IKK-ß and IκB-α phosphorylation might contribute to the inhibitory effect of shikonin on mitogen-induced IL-4 and IL-5 production in EL-4T cells. Furthermore, shikonin is a potential drug for treating allergic diseases.

Shikonin inhibits maturation of bone marrow-derived dendritic cells and suppresses allergic airway inflammation in a murine model of asthma.

BACKGROUND AND PURPOSE: Shikonin exhibits a wide range of anti-inflammatory actions. Here, we assessed its effects on maturation of murine bone marrow-derived dendritic cells (BM-DCs) and on allergic reactions in a murine model of asthma. EXPERIMENTAL APPROACH: Cultured murine BM-DCs were used to investigate the effects of shikonin on expression of cell surface markers and their stimulation of T-cell proliferation and cytokine production. The therapeutic potential of shikonin was evaluated in a model of allergic airway disease. KEY RESULTS: Shikonin dose-dependently inhibited expression of major histocompatibility complex class II, CD80, CD86, CCR7 and OX40L on BM-DCs, induced by a mixture of ovalbumin (OVA; 100µg·mL(-1) ) and thymic stromal lymphopoietin (TSLP; 20ng·mL(-1) ). Shikonin-treated BM-DCs were poor stimulators of CD4(+) T lymphocyte and induced lower levels of interleukin (IL)-4, IL-5, IL-13 and tumour necrosis factor (TNF)-α release by responding T-cells. After intratracheal instillation of shikonin in OVA-immunized mice, OVA challenge induced lower IL-4, IL-5, IL-13, TNF-α and eotaxin release in bronchial alveolar lavage fluid, lower IL-4 and IL-5 production in lung cells and mediastinal lymph node cells and attenuated OVA-induced lung eosinophilia and airway hyperresponsiveness. CONCLUSION AND IMPLICATIONS: Shikonin effectively suppressed OVA + TSLP-induced BM-DC maturation in vitro and inhibited allergic inflammation and airway hyperresponsiveness in a murine model of asthma, showing good potential as a treatment for allergic asthma. Also, our model provides a novel platform for screening drugs for allergic diseases.