Fibronectin Gene Up-regulation by Arnica montana in Human Macrophages: Validation by Real-Time Polymerase Chain Reaction Assay

Abstract

Background and Aim Arnica montana L. (Arnica m.) is a popular traditional medicine, used for its therapeutic properties in healing traumas, but little is known about its biological action on tissue formation and repair. This new work tested the effects of Arnica m. homeopathic dilutions on human macrophages, key cells in tissue defence and repair.

Materials and Methods Macrophages derived from the THP-1 cell line were differentiated with interleukin-4 to induce a ‘wound-healing’-like phenotype, and treated with various dilutions of Arnica m. centesimal (100 times) dilutions (2c, 3c, 5c, 9c, and 15c) or control solvent for 24 hours. RNA samples from cultured cells were analysed by real-time quantitative polymerase chain reaction in five separate experiments.

Results Arnica montana at the 2c dilution (final concentration of sesquiterpene lactones in cell culture = 10⁻⁸ mol/L) significantly stimulated the expression of three genes which code for regulatory proteins of the extracellular matrix, namely FN1 (fibronectin 1, % increase of 21.8 ± standard error of the mean 4.6), low-density lipoprotein-receptor-related protein 1 (% increase of 33.4 ± 6.1) and heparan sulphate proteoglycan 2 (% increase of 21.6 ± 9.1). Among these genes, the most quantitatively expressed was FN1. In addition, FN1, unlike other candidate genes, was upregulated in cells treated with higher dilutions/dynamisations (3c, 5c, and 15c) of Arnica m.

Conclusion The results support evidence that the extracellular matrix is a potential therapeutic target of Arnica m., with positive effects on cell adhesion and migration during tissue development and healing.

Keywords

Arnica montana - macrophage - homeopathic medicines - wound healing - gene expression - inflammation - extracellular matrix - fibronectin

Highlights

• Macrophages derived from the THP-1 cell line were differentiated with IL-4 to induce a ‘wound-healing’-like phenotype.

• Measurements using RT-qPCR assay indicated that macrophages are a pharmacological target of Arnica m.

• A slight but statistically significant up-regulation of three ECM genes was observed.

• FN1 (fibronectin 1) is the major gene whose expression was stimulated by Arnica m.

• We conclude that Arnica montana impacts a network of biological functions including inflammation and extracellular matrix regulation.

• References

• 1 Brinkhaus B, Wilkens JM, Lüdtke R, Hunger J, Witt CM, Willich SN. Homeopathic arnica therapy in patients receiving knee surgery: results of three randomised double-blind trials. Complement Ther Med 2006; 14: 237-246
• 2 Karow JH, Abt HP, Fröhling M, Ackermann H. Efficacy of Arnica montana D4 for healing of wounds after Hallux valgus surgery compared to diclofenac. J Altern Complement Med 2008; 14: 17-25
• 3 Reddy KK, Grossman L, Rogers GS. Common complementary and alternative therapies with potential use in dermatologic surgery: risks and benefits. J Am Acad Dermatol 2013; 68: e127-e135
• 4 Iannitti T, Morales-Medina JC, Bellavite P, Rottigni V, Palmieri B. Effectiveness and safety of Arnica montana in post-surgical setting, pain and inflammation. Am J Ther 2016; 23: e184-e197
• 5 Lussignoli S, Bertani S, Metelmann H, Bellavite P, Conforti A. Effect of Traumeel S, a homeopathic formulation, on blood-induced inflammation in rats. Complement Ther Med 1999; 7: 225-230
• 6 Castro FC, Magre A, Cherpinski R. , et al. Effects of microcurrent application alone or in combination with topical Hypericum perforatum L. and Arnica montana L. on surgically induced wound healing in Wistar rats. Homeopathy 2012; 101: 147-153
• 7 Sharma S, Arif M, Nirala RK, Gupta R, Thakur SC. Cumulative therapeutic effects of phytochemicals in Arnica montana flower extract alleviated collagen-induced arthritis: inhibition of both pro-inflammatory mediators and oxidative stress. J Sci Food Agric 2016; 96: 1500-1510
• 8 Verma N, Tripathi SK, Sahu D, Das HR, Das RH. Evaluation of inhibitory activities of plant extracts on production of LPS-stimulated pro-inflammatory mediators in J774 murine macrophages. Mol Cell Biochem 2010; 336: 127-135
• 9 Kawakami AP, Sato C, Cardoso TN, Bonamin LV. Inflammatory process modulation by homeopathic Arnica montana 6CH: the role of individual variation. Evid Based Complement Alternat Med 2011; 2011: 917541
• 10 Hostanska K, Rostock M, Melzer J. , et al. A homeopathic remedy from arnica, marigold, St. John inverted question marks wort and comfrey accelerates in vitro wound scratch closure of NIH 3T3 fibroblasts. BMC Complement Altern Med 2012; 12: 100
• 11 Olioso D, Marzotto M, Bonafini C, Brizzi M, Bellavite P. Arnica montana effects on gene expression in a human macrophage cell line. Evaluation by quantitative real-time PCR. Homeopathy 2016; 105: 131-147
• 12 Marzotto M, Bonafini C, Olioso D. , et al. Arnica montana stimulates extracellular matrix gene expression in a macrophage cell line differentiated to wound-healing phenotype. PLoS One 2016; 11: e0166340 . (Retracted by PLoS ONE, June 20, 2019)
• 13 Olioso D, Marzotto M, Bonafini C, Brizzi M, Bellavite P. Response to commentary: Arnica montana effects on gene expression in a human macrophage cell Line. Evaluation by quantitative Real-Time PCR. Front Immunol 2016; 7: 320
• 14 Marzotto M, Bonafini C, Olioso D. , et al. Study of the action mechanisms of Arnica montana effects on macrophages. Int J High Dilution Res 2016; 15: 32-35
• 15 Qin L, Crews FT. NADPH oxidase and reactive oxygen species contribute to alcohol-induced microglial activation and neurodegeneration. J Neuroinflammation 2012; 9: 5
• 16 He L, Marneros AG. Macrophages are essential for the early wound healing response and the formation of a fibrovascular scar. Am J Pathol 2013; 182: 2407-2417
• 17 Bellavite P, Marzotto M. Comments on the retraction by PLoS ONE of a laboratory study on Arnica montana . Homeopathy 2019; 108: 291-293
• 18 Monograph: Arnica montana (whole plant for homeopathic preparation. Assay 2.2.29); in Agence Francaise de Sécurité Sanitaire, ed. Pharmacopée Homéopathique Française - X édition. Saint-Denis Cedex; 2008
• 19 Staneva J, Denkova P, Todorova M, Evstatieva L. Quantitative analysis of sesquiterpene lactones in extract of Arnica montana L. by 1H NMR spectroscopy. J Pharm Biomed Anal 2011; 54: 94-99
• 20 Ramakers C, Ruijter JM, Deprez RH, Moorman AF. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 2003; 339: 62-66
• 21 Muller PY, Janovjak H, Miserez AR, Dobbie Z. Processing of gene expression data generated by quantitative real-time RT-PCR. Biotechniques 2002; 32: 1372-1374 , 1376, 1378–1379
• 22 Yu G, Wang LG, Han Y, He QY. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 2012; 16: 284-287
• 23 Gratchev A, Guillot P, Hakiy N. , et al. Alternatively activated macrophages differentially express fibronectin and its splice variants and the extracellular matrix protein betaIG-H3. Scand J Immunol 2001; 53: 386-392
• 24 Robert C, Watson M. Errors in RNA-Seq quantification affect genes of relevance to human disease. Genome Biol 2015; 16: 177
• 25 Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods 2017; 14: 417-419
• 26 Ganzera M, Egger C, Zidorn C, Stuppner H. Quantitative analysis of flavonoids and phenolic acids in Arnica montana L. by micellar electrokinetic capillary chromatography. Anal Chim Acta 2008; 614: 196-200
• 27 de Oliveira CC, de Oliveira SM, Goes VM, Probst CM, Krieger MA, Buchi DdeF. Gene expression profiling of macrophages following mice treatment with an immunomodulator medication. J Cell Biochem 2008; 104: 1364-1377
• 28 Marzotto M, Olioso D, Brizzi M, Tononi P, Cristofoletti M, Bellavite P. Extreme sensitivity of gene expression in human SH-SY5Y neurocytes to ultra-low doses of Gelsemium sempervirens. BMC Complement Altern Med 2014; 14: 104
• 29 Bigagli E, Luceri C, Dei A, Bernardini S, Dolara P. Effects of extreme dilutions of Apis mellifica preparations on gene expression profiles of human cells. Dose Response 2016; 14: 1559325815626685
• 30 Olsen S. Effects of ultra-high dilutions of sodium butyrate on viability and gene expression in HEK 293 cells. Homeopathy 2017; 106: 32-36
• 31 Marotti I, Betti L, Bregola V. , et al. Transcriptome profiling of wheat seedlings following treatment with ultrahigh diluted arsenic trioxide. Evid Based Complement Alternat Med 2014; 2014: 851263
• 32 Saha SK, Das S, Khuda-Bukhsh AR. Phenotypic evidence of ultra-highly diluted homeopathic remedies acting at gene expression level: a novel probe on experimental phage infectivity in bacteria. J Chin Integr Med 2012; 10: 462-470
• 33 Preethi K, Ellanghiyil S, Kuttan G, Kuttan R. Induction of apoptosis of tumor cells by some potentiated homeopathic drugs: implications on mechanism of action. Integr Cancer Ther 2012; 11: 172-182
• 34 Khuda-Bukhsh AR. Current trends in high dilution research with particular reference to gene regulatory hypothesis. Nucleus 2014; 57: 3-17
• 35 Dei A, Bernardini S. Hormetic effects of extremely diluted solutions on gene expression. Homeopathy 2015; 104: 116-122
• 36 Saha SK, Roy S, Khuda-Bukhsh AR. Ultra-highly diluted plant extracts of Hydrastis canadensis and Marsdenia condurango induce epigenetic modifications and alter gene expression profiles in HeLa cells in vitro. J Integr Med 2015; 13: 400-411
• 37 Marzotto M, Olioso D, Bellavite P. Gene expression and highly diluted molecules. Front Pharmacol 2014; 5: 237
• 38 Bellavite P, Signorini A, Marzotto M, Moratti E, Bonafini C, Olioso D. Cell sensitivity, non-linearity and inverse effects. Homeopathy 2015; 104: 139-160
• 39 Bell IR. The complexity of the homeopathic healing response part 2: the role of the homeopathic simillimum as a complex system in initiating recovery from disease. Homeopathy 2020; 109: 51-64