Effect of two homeopathic remedies at different degrees of dilutions on the wound closure of 3T3 fibroblasts in in vitro scratch assay
Keywords:wound healing, 3T3 fibroblasts, homeopathic remedy, arnica, calendula
AbstractBackground: Since ancient times, preparations from traditional medicinal plants e.g. Arnica montana, Calendula officinalis or Hypericum perforatum have been used for different wound healing purposes. The aim of this study was to investigate the efficacy of the commercial low dilution homeopathic remedy SimilasanÃƒâ€šÃ‚Â® Arnica plus Spray, a preparation of Arnica montana 4x, Calendula officinalis 4x, Hypericum perforatum 4x and Symphytum officinale 6x (0712-2) and medium diluted SIM WuS (Petroleum 15x, Arnica montana 15x, Calcium fluoratum 12x, Calendula officinalis 12x, Hepar sulfuris 12x and Mercurius solubilis 15x; 1101-4), on the wound healing in cultured NIH 3T3 fibroblasts. Both remedies were from Similasan AG (Jonen, Switzerland) and prepared according the German Homoeopathic Pharmacopoeia (GHP) following descriptions 4a for arnica, 3a for marigold and St. JohnÃƒÂ¢Ã¢â€šÂ¬Ã¢â€žÂ¢s wort, 2a for comfrey, 5a for petroleum, and 6 for calcium fluoride, hepar sulfuris and mercurius solubilis. Materials and Methods: Cell proliferation, migration and wound closure promoting effect of the preparations (0712-2, 1101- 4) and their succussed solvents (0712-1, 1101-3) were investigated on mouse NIH 3T3 fibroblasts. Cell viability was determined by WST-1 assay, cell growth using BrdU uptake, cell migration by chemotaxis assay and wound closure by CytoSelect ÃƒÂ¢Ã¢â‚¬Å¾Ã‚Â¢Wound Healing Assay Kit which generated a defined wound area. All assays were performed in three independent controlled experiments. In some experiments diluted unsuccussed alcohol (0712-3) was also investigated. Results: Preparations (0712-1), (0712-2), (0712-3), (1101-3) and (1101-4) were investigated at decimal dilution steps from 1x to 4x. Cell viabilty was not affected by any of the substances and (0712-1) and (0712-2) showed no stimulating effect on cell proliferation. Preparation (0712-2) exerted a stimulating effect on fibroblast migration (31.7%) vs 15% with succussed solvent (0712-1) at 1:100 dilutions (p<0.001). Unsuccussed solvent (0712-3) had no influence on cell migration (6.3%; p>0.05). Positive control 2 ng/ml EGF increased migratory activity of cells by 49.8%. Preparation (0712-2) at a dilution of 1:100 promoted in vitro wound closure by 59.5% and differed significantly (p<0.001) from succussed solvent (0712-1), which caused 22.1% wound closure. Medium diluted remedy (1101-4) exerted accelerating effect on wound closure after 14h of treatment. Wounded area was closed by 20% with (1101-4) and 13% by (1101-3) compared to untreated control. Succussed solvent (1101-3) caused about 23% and the remedy (1101-4) about 30% wound closure after 24h. Remedy (1101-4) and succussed solvent (1101-3) modestly stimulated cell growth at dilutions 1:100 and 1:1000 by about 25% and 15%, respectively. No statistically significant differences between preparations 1101-3 and 1101-4 could be detected. Conclusions: Our results demonstrate that the SimilasanÃƒâ€šÃ‚Â® Arnica plus low dilution homeopathic remedy exerted wound healing potential, which is a result of increased ability of fibroblasts to migrate without affecting cell proliferation. Medium diluted preparation SIM WuS exerted stimulating effect on the wound closure accompanied by a cell proliferating effect. Used in vitro wound closure test was sensitive enough for low dilutions preparation, however for medium diluted preparation despite of a trend, no significant differences could be detected.
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