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Journal of Chinese Integrative Medicine ›› 2012, Vol. 10 ›› Issue (3): 337-246.doi: 10.3736/jcim20120314

• Original Experimental Research • Previous Articles     Next Articles

Potential of the homeopathic remedy, Arnica Montana 30C, to reduce DNA damage in Escherichia coli exposed to ultraviolet irradiation through up-regulation of nucleotide excision repair genes

Das Sreemanti,Kumar Saha Santu,De Arnab,Das Durba,Rahman Khuda-Bukhsh Anisur()   

  1. Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, IndiaCytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India
  • Received:2011-11-06 Accepted:2011-11-21 Online:2012-03-20 Published:2018-10-10

OBJECTIVE: To examine to what degree an ultra-highly diluted homeopathic remedy, Arnica Montana 30C (AM-30C), used in the treatment of shock and injury, can modulate the expression of nucleotide excision repair genes in Escherichia coli exposed to ultraviolet (UV) irradiation.
METHODS: E. coli were cultured to their log phase in a standard Luria-Bertani medium and then exposed to sublethal doses of UV irradiation at 25 and 50 J/m 2 for 22.5 and 45 s, respectively. The UV-exposed bacteria were then supplemented with either AM-30C (drug) or placebo (P-30C). The drug-treated and placebo-treated bacteria were subjected to assay for DNA damage and oxidative stress 90 min after UV exposure. Several protocols like comet assay, gel electrophoresis for DNA ladder and intracellular reactive oxygen species (ROS) generation, and biomarker measurement like superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH) were conducted. The mRNA expressions of the excision repair genes like ultraviolet repair uvrA, B and C genes (or also known as excision repair genes) were estimated by reverse transcription-polymerase chain reaction method.
RESULTS: The UV-exposed bacteria showed DNA damage and oxidative stress, as revealed by an increase in ROS generation, and a decrease in SOD, CAT and GSH activities. As compared to placebo, the AM-30C-treated bacteria showed less DNA damage and oxidative stress as manifested by a decrease in ROS generation, and an increase in SOD, CAT and GSH activities. AM-30C also up-regulated the expression of repair genes as compared to the control.
CONCLUSION: AM-30C helped repair the DNA damage through up-regulation of repair genes and also ameliorated the oxidative stress through the reduction of ROS generation and suitable modulation of anti-oxidative stress enzymes.

Key words: Escherichia coli, ultraviolet rays, homeopathy, Arnica, reactive oxygen species, DNA damage, DNA repair

"

Primer name Primer sequence (5′-3′)
uvrA Forward: TAAGCTGCAAACGTTGATGG
Reverse: GTGCTCAATCACCACAATGG
uvrB Forward: GTCTGGGCGATCCTGATTTA
Reverse: CAATCGTTCCACTTCCTCGT
uvrC Forward: TCCGGGATCTTACTGTCGTC
Reverse: TGGAGTGCTTTGACATCAGC
G-3-PDH Forward: CCCACTAACATCAAATGGGG
Reverse: CCTTCCACAATGCAAAGTT

Figure 1

E.coli dose-response curve under different UV irradiation doses A: The dose-response curve; Bar graph showing the percentage of viable cells subjected to dose 1 (UV at 25 J/m2) and dose 2 (UV at 50 J/m2) are presented in B and C, respectively. Values were measured in three independent experiments done in triplicate. Mean±standard error of mean was plotted. **P<0.01, vs control; △P<0.05, vs dose 1 (25 J/m2); ▲P<0.05, vs dose 2 (50 J/m2). UV: ultraviolet; AM30C: Arnica Montana 30C; P30C: placebo-30C."

Figure 2

Intracellular reactive oxygen species generation in control and treatment groups A: Control; B: Dose 1-treated; C: Dose 1 plus Arnica Montana-30C-treated; D: Dose 1 plus placebo-30C-treated; E: Dose 2-treated; F: Dose 2 plus Arnica Montana-30C-treated; G: Dose 2 plus placebo-30C-treated."

Figure 3

Data of superoxide dismutase assay A bar graph showing specific activity of superoxide dismutase (μmol/(L·mg·min)) was plotted. Values were measured in three independent experiments carried out in triplicate and were expressed as mean±standard error of mean. **P<0.01, vs control; △△P<0.01, vs dose 1 (UV at 25 J/m2); ▲▲P<0.01, vs dose 2 (UV at 50 J/m2). AM30C: Arnica Montana 30C; P30C: placebo-30C."

Figure 4

Data of catalase assay A bar graph showing specific activity of catalase (μmol/(L·mg·min)) was plotted. Values were measured in three independent experiments carried out in triplicate and were expressed as mean±standard error of mean. **P<0.01, vs control; △△P<0.01, vs dose 1 (UV at 25 J/m2); ▲P<0.05, ▲▲P<0.01, vs dose 2 (UV at 50 J/m2). AM30C: Arnica Montana 30C; P30C: placebo-30C."

Figure 5

Data of glutathione assay Intracellular glutathione concentration in E.coli cells (μmol/L per 108 cells) was plotted. Values were measured in three independent experiments carried out in triplicate and were expressed as mean±standard error of mean. **P<0.01, vs control; △P<0.05 vs dose 1 (25 J/m2); ▲▲P<0.01, vs dose 2 (50 J/m2). AM30C: Arnica Montana 30C; P30C: placebo-30C."

Figure 6

DNA damage assay through gel electrophoresis Images of DNA gel after electrophoresis containing DNA samples of different treatments. A: Dose 1-treated; B: Dose 2-treated. Ln1: Control; Ln2: Ultraviolet-treated; Ln3: Ultraviolet plus Arnica Montana-30C-treated; Ln4: Ultraviolet plus placebo-30C-treated."

Figure 7

DNA damage analysis by comet assay A: Photographs showing the DNA damage by comet assay. B: Graphical representation of mean values of comet tail lengths (μm). Values were measured in three independent experiments carried out in triplicate and were expressed as mean ± standard error of mean. **P<0.01, vs control; △P<0.05, vs dose 1 (25 J/m2); ▲▲P<0.01, vs dose 2 (50 J/m2). (a) represents the ultraviolet-irradiated control cells, (b) and (e) represent ultraviolet doses 1- and 2-treated cells, respectively; (c) and (f) represent dose 1 plus Arnica Montana-30C-treated cells and dose 2 plus Arnica Montana-30C-treated cells, respectively; (d) and (g) represent dose 1 plus placebo-30C-treated cells and dose 2 plus placebo-30C-treated cells, respectively."

Figure 8

Gene expression analysis by RT-PCR Arbitrary band intensities of uvrA, B and C were analyzed against the house keeping gene G-3-PDH under different conditions. Ln1: Control; Ln2: Dose 1-treated; Ln3: Dose 1 plus Arnica Montana-30C-treated; Ln4: Dose 1 plus placebo-30C-treated; Ln5: Dose 2-treated; Ln6: Dose 2 plus Arnica Montana-30C-treated; Ln7: Dose 2 plus placebo-30C-treated. G-3-PDH: glyceraldehyde-3-phosphate dehydrogenase; RT-PCR: reverse transcription-polymerase chain reaction."

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