Scientific Reports. 2024; 14: 745.
Arsenic album 30C exhibits crystalline nano structure of arsenic trioxide and modulates innate immune markers in murine macrophage cell linesSuvasmita Rath et al.
Various institutions in India
Abstract
Macrophages are associated with innate immune response and M1-polarized macrophages exhibit pro-inflammatory functions. Nanoparticles of natural or synthetic compounds are potential triggers of innate immunity. As2O3 is the major component of the homeopathic drug, Arsenic album 30C.This has been claimed to have immune-boosting activities, however, has not been validated experimentally. Here we elucidated the underlying mechanism of Ars. alb 30C-mediated immune priming in murine macrophage cell line. Transmission Electron Microscopy (TEM) and X-ray diffraction (XRD) used for the structural analysis of the drug reveals the presence of crystalline As2O3 nanoparticles of cubic structure. Similarly, signatures of M1-macrophage polarization were observed by surface enhanced Raman scattering (SERS) in RAW 264.7 cells with concomitant over expression of M1 cell surface marker, CD80 and transcription factor, NF-kappa B, respectively. We also observed a significant increase in pro-inflammatory cytokines like iNOS, TNF-a, IL-6, and COX-2 expression with unaltered ROS and apoptosis in drug-treated cells. Enhanced expression of Toll-like receptors 3 and 7 were observed both in transcriptional and translational levels after the drug treatment. In sum, our findings for the first time indicated the presence of crystalline As2O3 cubic nanostructure in Ars. alb 30C which facilitates modulation of innate immunity by activating macrophage polarization.
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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10772077/Excerpts:
“Various preclinical studies demonstrate immunomodulatory, anti-oxidative, and therapeutic applications of naturally derived homeopathic drugs in a dose response manner25,26. Arsenic album (Ars. alb) is a homeopathic drug prepared from arsenic trioxide and has a significant anticancer properties25. It has been reported that Ars. alb ameliorates the toxic effect of arsenic trioxide in ultra-diluted form27. Recently, Ars. alb 30C has also been reported to have preventive role against COVID-19 infection as evident from community-based and parallel cohort study in a contaminant zone of the Delhi region in India28,29. Ye et al., demonstrated the immunomodulatory effect of trivalent arsenic, As (III) at a non-toxic concentration30. Several other studies demonstrated the anticancer properties of As2O3 nanoparticles and their application in cancer immunotherapy31,32. Although priming of innate immunity has been hypothesized behind the protective role of Ars. alb 30C described above, its function as an immune booster and detailed mechanism of its action is not been experimentally validated to date.”
“Hypothesis of the study is that the immune priming potential of Ars. alb 30C is associated with the induction of innate immunity. Through our integrated experimental approaches, we have validated that Ars. alb 30C enhances innate immune signatures through upregulated expression of pro-inflammatory cytokines and concomitant polarization of M1 macrophages, as a part of its immune function. To gain insight into the mode of action of Ars. alb 30C, we have analysed its structure in Transmission Electron Microscope (TEM), X-ray diffraction (XRD) and M1 macrophage phenotype by Surface Enhanced Raman Scattering (SERS). For the first time, we have reported the presence of arsenic trioxide nanoforms in Ars. alb 30C and correlated such nano structure behind its mechanism of immunomodulation. Our findings are in line with the previous reports which clearly supports nanostructure-driven innate immunity through macrophage polarization33,34.”
"Results"
“Structural analysis of Ars. alb 30C was performed by transmission electron microscopy (TEM) and X-ray diffraction (XRD). Figure1a,1a, b showed the bright field low magnification TEM image of the ultra-high-diluted Ars. alb 30C sample and corresponding Selected Area Electron Diffraction (SAED) taken along [111] zone-axis. It clearly shows various planes...that corresponds d-spacings of 0.466 nm of cubic As2O3 structure. Similarly, (42 2¯)and (22 4¯) planes correspond to 0.268 nm, while (40 4¯)and (04 2¯) planes correspond to 0.235 nm. Lastly, (42 6¯) and (24 6¯) planes correspond to 0.176 nm of cubic As2O3 structure...Figure1c1c shows the High-Resolution TEM (HRTEM) image of the samples having nanoparticles. It indicates the formation of highly crystalline nanoparticles of an average size ~ of 15 nm. In Fig.1d, the HRTEM image of a single nanoparticles and corresponding Fast Fourier Transform (FFT) image (inset) clearly shows that the nanoparticles are highly crystalline in nature and (040) (440), (004) planes (along [100] zone-axis) confirm the presence of cubic As2O3 structure...To further characterize, XRD scans were acquired from the drop-cast sample of Ars. alb 30C on 300 nm SiO2/Silicon substrate to investigate the crystallinity of As2O3 nanoparticles. In supplementary figure (Fig. S2), XRD pattern were analyzed, indexed and the peak position at 63.63° (2 theta value) coincide with the (731) plane of As2O3 structure...Both TEM and XRD results confirmed the formation of highly crystalline nanoparticles of cubic As2O3 structure and their aggregates in the sample.”
“The RT-PCR data (n = 3) confirmed induced expression of TLR-3 and TLR-7 (P = 0.05) in transcript level after Ars. alb 30C treatment (Fig. 4a, b). We also observed induced expression of TLR-3 and TLR-7 adaptor molecules TRIF and MyD88, respectively in Ars. alb 30C-treated cells (Fig. 4c, d).”
“Representative western blot results (n = 3) showed that there is induced expression of TLR-3 and TLR-7 in Ars. alb 30C-treated cells as compared to the vehicle control and untreated cells (Fig. 4f, g).” Induced MyD88 expression was validated in Ars. alb 30C-treated cells (Fig. 4h).
“To study the expression pattern of innate immune markers TLR-3 and TLR-7, we also performed confocal experiment by staining macrophage cells with TLR-3 and TRL-7 specific antibodies tagged with Alexa fluor 488. We observed induced expression of both TLR-3 and 7 in Ars. alb 30 C-treated cells (Fig. 5a, b).”
“As TLR signalling pathways activate downstream transcription factor NF-kappa B, so we further validated expression of NF-kappa B in transcript and translate levels. Our findings showed induced expression of NF-kappa B in Ars. alb 30 C-treated cells (Fig. 6a, b).”
“Further, expression of pro-inflammatory cytokines TNF-a, COX-2, and iNOS at the transcript level was evaluated after Ars. alb 30C treatment. Our results confirmed the up regulation of these pro-inflammatory effector molecules in Ars. alb 30C-treated cells as compared to the untreated and alcohol-treated samples (Fig. 7a–c). The up-regulation of pro-inflammatory effector molecules like IL-6 and TNF-a after 30C treatment was confirmed by ELISA results (Fig. 7d, e).”
"Discussion"
"Research on nanoparticles (NPs) is a rapidly developing area of biomedicine and has made a significant contribution to health and the cure of diseases...Due to larger surface area, a higher degree of surface charge, and proton exchangeability, nanoparticles show significant immunomodulatory activity38,39. Various engineered and naturally-derived nanoparticles like cell membrane-derived nanoparticles (CMDNs) and silver nanoparticles (AgNPs), are reported to have immunomodulatory activity40,41. Although, homeopathic drugs are prepared by ultrahigh dilutions; the possibility of formation of nanoparticles cannot be avoided42,43. For the first time, we have reported the existence of crystalline As2O3 nanoparticles of cubic structure with an average size of ~ 15 nm and their aggregates in Ars. alb 30C by TEM & XRD...Nanoparticles can modulate the activity of innate immune cells by inducing inflammatory response46...M1 macrophages are associated with TLR signalling pathways and induce the expression of pro-inflammatory effectors like TNF-a, COX-2, IL-6, and iNOS49. TLR-3 and TLR-7 are markers of innate immune response expressed in the endosome of macrophage cells and are associated with pro-inflammatory pathways50,51...Our findings indicate induced expression of TLR-3 and TLR-7 in murine macrophages after Ars. alb 30C treatment along with their adaptor molecules TRIF and MyD88, respectively. This strongly supports the immunomodulatory potential of Ars. alb 30C and substantiates its preventive role against COVID-19 as proposed in previous studies28,29. As TLR-3 and TLR-7 are mostly double-stranded and single-stranded RNA sensors, respectively, their induced expression might provide further antiviral responses in Ars. alb 30C-treated cells...We have further reported an unaltered total ROS level in Ars. alb 30C-treated cells, which suggests that the drug does not contribute to additional ROS production. No change in cell viability was observed after Ars. alb 30C treatment which indicated no significant cell death after drug treatment. Our findings are consistent with previous studies which also reported that Ars. alb 30C could promote antioxidant defence and upregulation of glutathione (GSH) with a concomitant reduction of total ROS level in yeast and mammals, which supports its therapeutic potential against oxidative stress-associated pathogenesis25,56,57.
"Taken together, this study, for the first time, reports the existence of a crystalline nanostructure of As2O3 in Ars. alb 30C dilution, with a clear evidence of drug-dependent macrophage polarization, upregulation of TLR pathways, and a consequent immune effector function with distinct signature molecules."
"Immunomodulation which promotes the innate protective mechanism or surveillance in the host, is increasingly receiving attention as an alternative to vaccination. This is due to the rapid and unpredictable mutation of microbial pathogens which can evade the immune barrier58. An emerging strategy to combat infectious disease is the synergistic approach of vaccination and innate immune modulator (against TLR modulation)...This work, using in vitro model, established the mechanism of action of Ars. alb 30C as an immunomodulator. For the first-time our findings revealed the presence of As2O3 crystalline nanostructures in Ars. alb 30C, providing compelling evidence to explain the causal mechanism. Furthermore, the expression of innate immune markers and pro-inflammatory cytokines related to M1 macrophages is evident in Ars. alb 30C-treated murine macrophage cells.
"As homeopathic drugs are less costly and have minimal side-effects, Ars. alb 30C can be a prospective alternative medicine to boost innate immunity, with significant clinical applications. However, a major challenge to the acceptance of homeopathic medications is the lack of pharmacological validation. In this context, our findings provide considerable evidence in support of its efficacy and reveal a novel mechanism of Ars. alb 30C-mediated M1 macrophage polarization and induction of innate immune response. Ars. alb 30C can be used as innate immune modulator, particularly as TLR-3 and TLR-7 agonist, which can provide host-dependent long lasting innate immunity in general, and against the invading pathogens or infectious diseases, in particular. Nevertheless, further study with animal models and human trials needs to be carried out before translational intervention."
"Materials and methods"
"Ars. alb 30C was procured from Dr. Reckeweg.co, Germany and was used at a dilution of 10^–4 with complete media by serial dilution. Treatment was done for 24 h as per the standardised protocol. Since the drug was prepared in alcohol (90% ethanol), we included a solvent control (SC) as 90% ethanol which was also diluted to 10^–4 with complete media60."
"Acknowledgements
"Authors would like to thank Central Council for Research in Homoeopathy, CCRH, Ministry of AYUSH, Government of India for a collaborative research grant and supporting open access publication."
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Comments:
As usual, there is no mention of the fact that homeopathy is inconsistent with scientific knowledge, or even that it is controversial.
The product tested is Ars. alb 30C, which means that theoretically a solution of arsenic trioxide has been diluted by a factor of 10^60, and no arsenic should remain. However, if we believe the results, there are detectable As2O3 crystals in the preparation. Therefore this cannot be a correctly prepared homeopathic product.
Can a homeopathic preparation be considered to be adulterated if it contains the source material in amounts larger than should be there according to the potency?
If we consider the remaining experiments, and assume that the results are as described, these indicate that small amounts of arsenic when applied directly to cells in culture can have a variety of effects related to immune responses. This could be due to particles of the crystal, perhaps interacting at the cell surface, or to arsenic ions in solution. This does not mean that the homeopathic remedy Ars. alb 30C, even if it contains measurable amounts of arsenic trioxide, would have similar effects in the body. The arsenic would need to be taken up in the digestive system, then distributed through the circulation to a target tissue.
Papers like this are dangerous insofar as they encourage the use of homeopathic drugs as an alternative to vaccination.
