cimifugin and Inflammation

Cimifugin is one of the main bioactive components of Yu-Ping-Feng-San, a well-known traditional Chinese medicine, which can effectively relieve Allergic asthma (AA) and atopic dermatitis and reduce recurrence in clinic. However, the underlying mechanism of cimifugin on AA is still unknown.. In the present study, we aimed to investigate the effect and mechanism of cimifugin on AA.. In vivo and in vitro experimental studies were performed.. The effect of cimifugin on AA was demonstrated in vivo and in vitro. Sepiapterin reductase (SPR) was predicted as the most potent target of cimifugin in treating AA by reverse docking. Molecular docking and microscale thermophoresis (MST) were used to analyze the direct binding between cimifugin and SPR. Overexpression and interference of SPR were performed to verify whether targeting SPR is a key step of cimifugin in the treatment of AA. QM385, an inhibitor of SPR, was administrated in vivo and in vitro to evaluate the role of SPR in AA. Further, HPLC and cell-free direct hSPR enzyme activity assay were performed to research whether cimifugin regulated SPR by influencing the enzyme activity. Simultaneously, the inhibitors of protein degradation were used in vitro to explore the mechanism of cimifugin on SPR.. We found cimifugin effectively alleviated AA by reducing airway hyperresponsiveness, inhibiting type 2 cytokines-mediated airway inflammation, and restoring the expression of epithelial barrier proteins. Molecular docking predicted the direct binding ability of cimifugin to SPR, which was further verified by MST. Notably, the therapeutic effect of cimifugin on AA was dampened with SPR interfering, in contrast, the phenotypic features of AA were significantly alleviated with QM385 application both in vivo and in vitro. Interestingly, cimifugin showed no effect on the enzyme activity of SPR, as the level of its substrate sepiapterin was not affected with cimifugin treatment by cell-free enzyme activity assay. Furthermore, we found cimifugin could reduce SPR protein expression without affecting its mRNA expression probably through autophagosome pathway.. To our knowledge, we're reporting for the first time that cimifugin can suppresses type 2 airway inflammation to alleviate AA by directly binding to SPR and regulating its protein expression in a non-enzymatic manner.

Topics: Asthma; Cytokines; Humans; Inflammation; Molecular Docking Simulation; Surface Plasmon Resonance

BACKGROUND RAW264.7 cells are induced by lipopolysaccharide (LPS) as a rheumatoid arthritis (RA) model. The present study investigated the effect of cimifugin on the proliferation, migration, chemotaxis, and release of inflammation-related factors and inflammation-related signaling pathways of LPS-induced RAW264.7 cells. MATERIAL AND METHODS MTS assay was used to determine the proliferation of RAW264.7 cells. Transwell assay was employed to examine the migration and chemotaxis of the cells. ELISA was performed to measure the contents of chemotactic factors and inflammatory factors in cell culture supernatants. Western blotting was carried out to detect the expression of factors related with MAPKs and NF-κB signaling pathways. RESULTS Cimifugin (0-100 mg/L) had no cytotoxicity for RAW264.7 cells. LPS stimulation induced morphological differentiation of RAW264.7 cells, but intervention by cimifugin inhibited the activation effect by LPS by about 50%. Cimifugin (100 mg/L) decreased the migration and chemotaxis of RAW264.7 cells to 1/3 of that in control cells by decreasing the release of migration- and chemotaxis-associated factors by at least 30%. Cimifugin (100 mg/L) suppressed the release of inflammatory factors from RAW264.7 cells to less than 60% of that in the LPS group. In addition, cimifugin (100 mg/L) inhibited the activities of MAPKs and NF-κB signaling pathways. CONCLUSIONS The present study demonstrates that cimifugin reduces the migration and chemotaxis of RAW264.7 cells and inhibits the release of inflammatory factors and activation of related signaling pathways induced by LPS. Cimifugin may have potential pharmacological effects against RA.

Topics: Animals; Anti-Inflammatory Agents; Arthritis, Rheumatoid; Cell Movement; Chromones; Cyclooxygenase 2; Inflammation; Lipopolysaccharides; Macrophages; Mice; Models, Biological; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; RAW 264.7 Cells; Signal Transduction

Atopic dermatitis (AD) is a common allergic inflammatory skin disease, concomitant with a high relapse rate. Yu-Ping-Feng-San (YPFS), a well-known Chinese herbal decoction, reduces the AD relapse rate and recurring severity incidence. However, the underlying mechanism of YPFS on resisting AD recurrence is still unknown and further study is needed.. To evaluate the effects of YPFS on recurrent allergic inflammation of AD in a murine model and to investigate the underlying mechanisms in vivo and ex vivo.. A fluorescein isothiocyanate (FITC)-induced AD relapsing mouse model was established to study the effects of YPFS and three active components, claycosin, formononetin, and cimifugin, on recurrent allergic inflammation in vivo. Histological analyses of ear tissue inflammation were evaluated by hematoxylin and eosin staining. Production of interleukin (IL)-4, IL-5, IL-13, and interferon-gamma in mice ear tissues, IgE in serum, and thymic stromal lymphopoietin (TSLP) in cell cultures were measured by ELISAs. Tight junction (TJ) expression was detected by immunohistochemistry and western blots. Epithelial barrier integrity was observed with electron microscopy, transepithelial electric resistance (TER), and paracellular flux measurements. HaCaT cells were utilized for ex vivo cellular analyses.. In the recurrent phase of AD, YPFS exhibited both short- and long-term anti-allergic inflammatory efficacy with reduced ear tissue inflammation and decreased IL-4, IL-5, IL-13, and IgE production. The three active components, claycosin, formononetin, and cimifugin, showed similar effects as YPFS. Stimulus-induced decreased TER and increased FITC-dextran flux in air-liquid interface cultures of HaCaT cells were significantly repaired by YPFS and the three active components. Notably, the upregulated TJ (CLDN-1 and occludin) expression of epithelium was observed only with YPFS and the three components-treated mice as opposed to the result using conventional anti-allergy medicines. Restored TJ expression by YPFS three components was also detectable in the remission phase of AD. Moreover, decreased TJ expression influenced the effects of YPFS on epithelial cells-derived TSLP production.. YPFS ameliorated recurrent allergic inflammation of AD by repairing TJ defects of epithelial barriers. Intervening epithelial barrier functions could be a preventive and therapeutic approach for recurrent allergic inflammation of AD.

Topics: Animals; Anti-Allergic Agents; Chromones; Cytokines; Dermatitis, Atopic; Disease Models, Animal; Drugs, Chinese Herbal; Epithelium; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Inflammation; Isoflavones; Mice; Mice, Inbred BALB C; Recurrence; Thymic Stromal Lymphopoietin; Tight Junctions

Cimifugin is a bioactive component of Saposhnikovia divaricata, a Chinese herb for treating allergy. Our previous studies demonstrated that cimifugin inhibited allergic inflammation efficiently. This study aims to determine the mechanism of cimifugin on epithelial cells in allergic inflammation. Mice were sensitized and challenged with FITC to establish type 2 atopic dermatitis (AD) model. The initial stage of AD model, in which mice were just sensitized with FITC, was established in vivo and immortalized human epidermal (HaCaT) cells were utilized in vitro. Initiative key cytokines, TSLP and IL-33, were measured by ELISA, the junctions in ECs were observed by electron microscopy and TJs (CLDN-1, occludin and CLDND1) were assessed by Western blot, immunohistochemistry and immunofluorescence. The results showed that TSLP and IL-33 were inhibited significantly by cimifugin in the initial stage of AD model. Simultaneously, cimifugin reduced the separated gap among the epithelial cells and increased the expression of TJs. Similar effects on TSLP/IL-33 and TJs were obtained in vitro. The effect of cimifugin on TSLP decreased significantly when expression of CLDN1 was interfered with siRNA and this implied cimifugin inhibits initiative cytokines through restoring TJs. Furthermore, cimifugin administered only in the initial stage obviously attenuated the ultimate allergic inflammation, which indicate that impacts of cimifugin in the initial stage on TSLP/IL-33 and TJs are sufficient for suppressing allergic inflammation. This study not only revealed the mechanisms of cimifugin, but also indicated the possibility of initiative key cytokines and TJs as therapeutic targets.

Topics: Animals; Anti-Allergic Agents; Apiaceae; Cell Line, Transformed; Chromones; Claudin-1; Claudins; Dermatitis, Atopic; Disease Models, Animal; Fluorescein-5-isothiocyanate; Gene Expression Regulation; Humans; Immunoglobulins; Inflammation; Interleukin-33; Keratinocytes; Mice; Mice, Inbred BALB C; Occludin; Receptors, Cytokine; Signal Transduction; Tight Junctions