Arnica montana Stimulates Extracellular Matrix Gene Expression in a Macrophage Cell Line Differentiated to Wound-Healing Phenotype

Abstract

Arnica montana (Arnica m.) is used for its purported anti-inflammatory and tissue healing actions after trauma, bruises, or tissue injuries, but its cellular and molecular mechanisms are largely unknown. This work tested Arnica m. effects on gene expression 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 24h to Arnica m. centesimal (c) dilutions 2c, 3c, 5c, 9c, 15c or Control. Total RNA was isolated and cDNA libraries were sequenced with a NextSeq500 sequencer. Genes with significantly positive (up-regulated) or negative (down-regulated) fold changes were defined as differentially expressed genes (DEGs). A total of 20 DEGs were identified in Arnica m. 2c treated cells. Of these, 7 genes were up-regulated and 13 were down-regulated. The most significantly up-regulated function concerned 4 genes with a conserved site of epidermal growth factor-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 confirmed a statistically significant increase of fibronectin production (p<0.05). The down-regulated transcripts derived from mitochondrial genes coding for some components of electron transport chain. The same groups of genes were also regulated by increasing dilutions of Arnica m. (3c, 5c, 9c, 15c), although with a lower effect size. We further tested the healing potential of Arnica m. 2c in a scratch model of wound closure based on the motility of bone marrow-derived macrophages and found evidence of an accelerating effect on cell migration in this system. The results of this work, taken together, provide new insights into the action of Arnica m. in tissue healing and repair, and identify extracellular matrix regulation by macrophages as a therapeutic target.

Fig 1. Absorption spectrum of Arnica m. 1c used as starting material.

Fig 2. Nanoparticle spectrum of Arnica m. 1c. used as starting material.

The line is the average of 10 replicate measurements and vertical bars indicate SD.

Fig 3. Cell viability of macrophages.

THP-1 macrophages in the resting state (diagonal bars) or after differentiation with IL-4 (crossed bars) were cultivated for 24 hours in the presence of Arnica m. at various dilutions or Control solvent. The histograms report mean values ± SE of 6 separate wells of a typical experiment. There are no significant differences between any Arnica m. dilution and Control (p>0.05)

Fig 4. Fibronectin detected in supernatants of cell cultures in the absence and in the presence of Arnica m. 2c.

Symbols indicate the fibronectin values of the same experiments in the two conditions of polarization. The reported values are percent effect as compared with Control of the same experiment.

Fig 5. Effects induced by increasing dilutions of Arnica m. on gene expression in THP-1 cells.

DEGs described in Table 1 were divided in the two groups as upregulated (red bars) and down-regulated (blue bars) genesets. Grey bars report the mean fold changes ± SE of the two genesets at each dilution tested. Panel A: Arnica m. 2c Log₂ fold change values calculated from 5 experiments; Panels B-F Arnica m. 2c, 3c, 5c, 9c, 15c Log₂ fold changes values of pooled RNAs of 5 experiments. P values of Wilcoxon signed-rank test statistics are reported near the mean of each geneset.

Fig 6. Wound closure effect of Arnica m.

Light microscope images of in vitro wound closure using a confluent monolayer of BMD macrophages. The microphotographs show one representative experiment of cell migration into the created wound area in the absence (A and C) and in the presence (B and D) of Arnica m. 2c. Images A and B show the wound area immediately after scratching, while C and D show the wound area after 4.5 h. Pictures were acquired by means of contrast phase microscopy with 100x original magnification. The bar charts (E and F) report the gap width of the wound area before and after cell migration. E and F: Time-course of wound occupancy in the absence (E) and in the presence (F) of 20 ng/ml IL-4. Gray bars: Control solvent, yellow bars: Arnica m. 2c. Means±SE of three replicate wells of an experiment representative of the three performed. The result of the Friedman test comparing the whole series of changes in cells treated with drug or with Control solvent is reported in the graph panels.