Project description:Arnica m. effects were associated with a purported anti-inflammatory and tissue healing actions after trauma, bruises, or tissue injuries, but its cellular and molecular mechanisms are largely unknown. Here Arnica m. dilutions were tested using an in vitro model of macrophages polarized towards a “wound-healing” phenotype. The monocyte-macrophage human THP-1 cell line was cultured and differentiated with phorbol-myristate acetate and Interleukin-4, then exposed for 24 h to Arnica m. centesimal (c) dilutions 2c, 3c, 5c,9c, 15c or Control. None of these treatments affected cell viability. A total of 20 genes were differentially expressed comparing cells treated with Arnica m. 2c with those treated with Control only. Of these, 7 genes were up-regulated and 13 were down-regulated. Functional gene enrichment analysis showed that the most significantly upregulated function concerned 4 genes with a conserved site of EGF-like region (p<0.001) and three genes of proteinaceous extracellular matrix, including heparin sulphate proteoglycan 2 (HSPG2), fibrillin 2 (FBN2), and fibronectin (FN1) (p <0.01). Protein assay in supernatants confirmed a statistically significant increase of fibronectin production in Arnica m. 2c treated cells (p<0.05). Pooled extracts of cells treated with increasing dilutions of Arnica m. (3c, 5c, 15c) showed up-regulation of the same group of genes although with lower effect size. The down-regulated transcripts derive from mitochondrial genes coding for some components of electron transport chain. These findings provide new insights into the action of Arnica m. in tissue healing and repair, identifying increased fibronectin production by macrophages as a major therapeutic target.
Project description:Arnica m. effects were associated with a purported anti-inflammatory and tissue healing actions after trauma, bruises, or tissue injuries, but its cellular and molecular mechanisms are largely unknown. Here Arnica m. dilutions were tested using an in vitro model of macrophages polarized towards a “wound-healing” phenotype. The monocyte-macrophage human THP-1 cell line was cultured and differentiated with phorbol-myristate acetate and Interleukin-4, then exposed for 24 h to Arnica m. centesimal (c) dilutions 2c, 3c, 5c,9c, 15c or Control. None of these treatments affected cell viability. A total of 20 genes were differentially expressed comparing cells treated with Arnica m. 2c with those treated with Control only. Of these, 7 genes were up-regulated and 13 were down-regulated. Functional gene enrichment analysis showed that the most significantly upregulated function concerned 4 genes with a conserved site of EGF-like region (p<0.001) and three genes of proteinaceous extracellular matrix, including heparin sulphate proteoglycan 2 (HSPG2), fibrillin 2 (FBN2), and fibronectin (FN1) (p <0.01). Protein assay in supernatants confirmed a statistically significant increase of fibronectin production in Arnica m. 2c treated cells (p<0.05). Pooled extracts of cells treated with increasing dilutions of Arnica m. (3c, 5c, 15c) showed up-regulation of the same group of genes although with lower effect size. The down-regulated transcripts derive from mitochondrial genes coding for some components of electron transport chain. These findings provide new insights into the action of Arnica m. in tissue healing and repair, identifying increased fibronectin production by macrophages as a major therapeutic target.
Project description:In this study, differentially prepared Arnica extracts and the lead compounds Thymol and Helenalin were directly compared for their immunomodulatory potential using primary human T cells. Extracts from different plant parts (root or whole plant) and prepared by different manufacturing processes inhibited T cell activation and proliferation. nCounter gene expression analysis and subsequent gene set enrichment analysis showed that Arnica preparations intervene in T cell receptor (TCR) signaling. Interestingly, the three tested extracts were, however, found to mediate their effects via interfering with different TCR signaling pathways. The hydroethanolic root extract selectively inhibited the binding of NFκB to DNA, while the aqueous fermented whole plant extract preferentially inhibited NFAT-dependent gene expression. The hydroethanolic whole plant extract (by trend) diminished both, NFκB DNA-binding and NFAT-dependent gene expression.
Project description:A better understanding of secondary metabolites biosynthesis requires comprehensive research at the molecular level. Although the medicinal importance of secondary metabolites extracted from Arnica spp. has been well documented, the very plants themselves have been poorly studied. Characterization of biosynthetic pathways and their complex regulatory mechanisms may be enhanced via transcriptomic approach. To ensure the reliability of data, RT-qPCR based gene expression studies should be preceded by the reference genes selection step. Here, ten candidate reference genes were analyzed in terms of their expression stability in developing flowers of two arnica species, Arnica montana and Arnica chamissonis. After evaluating their expression stability with four distinct algorithms (geNorm, NormFinder, BestKeeper and delta Ct method), we found that SKIP16 and F-box were the most stable reference genes in A. montana, whereas SAND and F-box were best-performing genes in A. chamissonis. On the other hand, ACT1 in combination with F-box could be used as internal control for cross-species investigations. This study will aid in further research on molecular mechanisms underlying the processes of flowering and secondary metabolites production in medicinal plants.