Inflammation plays an important role in affecting the bone-health. It can interfere with the body’s own natural ability to repair bone mass. Many of the bone disorders are mediated by inflammation as the common crucial factor. Rheumatoid arthritis (RA) is one of the common chronic autoimmune disorder categorized by systemic inflammation, persistent synovitis, and auto-antibodies. This inflammation of the joints leading to erosion of bone, prolonged deformities, and dysfunctional in the joints^[1]. About 1% of the worldwide population is affected and most commonly are the middle-aged women. The chronic inflammation mainly of the small joints and synovial fluid leads to damage of the juxta-articular bone and articular cartilage^[2]. As an autoimmune disease, its auto antigen has a wide range which would affect the specific immunomodulation and hampers the effective therapeutic approach. Increased secretion of inflammatory cytokines is a hallmark feature of RA. The transcriptional factors nuclear factor (NF)–kB and activator protein (AP)–1 are known to play an important role in gene regulation during inflammatory reactions, which regulates the transcription of inflammatory cytokines (interleukins), MMPs, and COX-2. Various pro-inflammatory cytokines have been reported with desirable roles such as synovial fluid interleukin-8 (IL-8) and neutrophil in RA^[3]. Besides, various chemo tactic factors released in the affected area are related to the employment and activation of leukocytes at the inflammation sites. Time course of leukocyte immigration and kind of cellular infiltration were determined by various triggering factors^[4]. IL-8, a chemo tactic cytokine that was found to have a high selectivity for neutrophils formed by the mononuclear phagocytes and different tissue cells, on stimulation with interleukin-1 (IL-1), tumor necrosis factor (TNF), and bacterial endotoxins^[5]. The SW982 (human synovial sarcoma cell line) cell is characterized by expression of inflammatory cytokines and matrix metalloproteinase (MMP) genes. The SW982 cells express genes encoding IL-1β, IL-6, cyclooxygenase (COX-2), and MMPs^[6,7]. Hence, in the present study, inhibition of cytokine secretion (IL-8) by SW982 cells against IL-1β stimulated levels suggested beneficial effects of Biofield Energy Treatment in DMEM against RA.

Biofield Therapy has been reported as one of the significant CAM (Complementary and Alternative Medicine) approach, which was reported in many scientific studies with outstanding results and alterations in living and non-living materials^[8]. Human has the ability to harness energy from the universal and can transmit it to any living organism(s) or nonliving object(s) around the globe. Biofield Energy Healing has been considered as Energy therapy, and was accepted by the National Center for Complementary and Alternative Medicine (NCCAM)^[9]. NCCAM recommend and accepted various types of Energy therapies under CAM due to several advantages in addition to other treatments, medicines and practices such as natural products, Qi Gong, deep breathing, yoga, Tai Chi, chiropractic/osteopathic manipulation, meditation, massage, special diets, homeopathy, progressive relaxation, acupressure, acupuncture, relaxation techniques, hypnotherapy, guided imagery, healing touch, movement therapy, Rolfing structural integration, mindfulness, Pilates, Reiki, Ayurvedic medicine, essential oils, traditional Chinese herbs and medicines, naturopathy, cranial sacral therapy, aromatherapy, and applied prayer (as is common in all religions, like Christianity, Hinduism, Buddhism and Judaism)^[10,11]. Bio field Energy Healing Treatment (The Trivedi Effect^®) contains putative bio energy, which is channeled by a renowned practitioner from a distance. Bio field Energy Healing as a CAM showed significant results in biological studies^[12]. However, the National Center for Complementary and Alternative Medicine (NCCAM), well-defined Bio field therapies in the subcategory of Energy Therapies^[13]. The Trivedi Effect^®- Consciousness Energy Healing Treatment has been reported with significant revolution in the physicochemical properties of metals, chemicals, ceramics and polymers^[14-16], improved agricultural crop yield, productivity, and quality^[17,18], transformed antimicrobial characteristics^[19-21], biotechnology^[22,23], improved bioavailability^[24-26], skin health^[27,28], nutraceuticals^[29,30], cancer research^[31,32], bone health^[33-35], human health and wellness.

From the significant outcomes of Bio field Treatment in numerous previous studies, authors intend to evaluate the impact of the Bio field Energy Healing Treatment (The Trivedi Effect^®) on DMEM as test sample for bone health activity with respect to the assessment of bone health parameter, inhibition of cytokine secretion (IL-8) against IL-1β stimulation using standard in vitro assaying SW982 cells.

Materials and Methods

Chemicals and Reagents

Fetal Bovine Serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Life Technology, USA. Ethylene Diaminetetra Acetic acid (EDTA), antibiotics solution like ‘Penicillin-Streptomycin’ was obtained from Hi Media, India however, 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium) (MTT), and Direct Red 80 were purchased from Sigma, USA.

Cell Culture

The SW982 (Synovial cell line) was used as a test system in the present study. The SW982 cell line was maintained in DMEM growth medium for routine culture supplemented with 10% FBS. Growth conditions were maintained at 37°C, 5% CO₂, and 95% humidity and sub cultured by trypsinization followed by splitting the cell suspension into fresh flasks and supplementing with fresh cell growth medium. Three days before the start of the experiment, the growth medium of near-confluent cells was replaced with fresh phenol-free DMEM, supplemented with 10% charcoal-dextran stripped FBS (CD-FBS) and 1% penicillin-streptomycin^[36].

Experimental Design

The experimental groups consisted of group 1 (G-I) with cells with the untreated DMEM. Group 2 (G-II) included one-time Bio field Energy Treated DMEM (BT-I) and group 3 (G-III) included the test item with two-times Biofield Energy Healing Treatment and denoted as BT-II.

Consciousness Energy Healing Treatment Strategies

The test sample (DMEM medium) was divided into three parts, first part received one-time Biofield Treatment by a renowned Consciousness Energy Healer, Alice Branton and was labeled as the one-time Biofield Energy Treated (BT-I) DMEM, while second part received the two-times the Biofield Energy Treatment and is denoted as BT-II DMEM. The third part did not receive any treatment and defined as the untreated DMEM groupA renowned Biofield Energy Healer, Alice Branton was provided this Bio field Energy Healing Treatment for ~5 minutes to the test items through the Healer’s unique Energy Transmission process. The Bio field Energy Healer was located in the USA, while the test items were located in the research laboratory of Dabur Research Foundation, New Delhi, India. Alice Branton in this study never visited the laboratory in person, nor had any contact with the test items. Further, the untreated DMEM group was treated with a “sham” healer for better comparative purposes. The “sham healer” did not have any knowledge about the Bio field Energy Treatment. After that, the Bio field Energy Treated and untreated samples were kept in similar sealed conditions for experimental study.

MTT Assay

MTT assay was used for the evaluation of cell viability in SW982 cells of the untreated and Biofield Energy Treated test items (DMEM). The details methodology of cell viability assay was followed by Lorraine et al. (2018) with few alterations^[37]. The cytotoxicity of each tested concentration of the test items was calculated with the help of Equation (1):

% Cytotoxicity = {(1-X)/R}*100…………… (1)

Where, X = Absorbance of the Biofield Treated DMEM group;

R = Absorbance of the untreated DMEM group

The percentage of cell viability corresponding to each treatment group was calculated by Equation (2):

% Cell Viability = (100 - % Cytotoxicity)…………… (2)

The concentration exhibiting ≥70% cell viability was defined as non-cytotoxic[38].

Assessment of IL-8 using ELISA

The SW982 cell suspension in DMEM medium containing 10% FBS were plated at a density of 0.1 X 106 cells/well/mL in 12-well plates. The cells were incubated in a CO₂ incubator for 24 hours at 37°C, 5% CO₂ and 95% humidity. The cells were sera starved by replacing the fresh DMEM medium with 1% FBS for 24 hours. After 24 hours of sera starvation, the medium was removed and treated with 900 µL of the test items to each well along with inflammatory stimulus IL-1β at a final concentration of 0.25 ng/mL. After treatments, cells were further incubated in a 5% CO₂ incubator for 24 hours. The level of a cytokine (IL-8) in culture supernatants of SW982 cells was determined using ELISA as per manufacturer’s instructions. The absorbance of each well was read at 450 nm using Synergy HT micro plate reader, BioTek, USA.

Statistical Analysis

All the values were represented as the mean ± standard error of the mean (SEM). For multiple groups comparison, one-way analysis of variance (ANOVA) was used followed by post-hoc analysis by Dunnett’s test. Statistically significant values were set at the level of p≤0.05.

Results and Discussion

Cell Viability Study Using MTT Assay

The percentage of cell viability in SW982 cell using MTT assay was performed among the Biofield Energy Treated test sample (DMEM medium). The data in term of percentage values are presented in Figure 1. The percentage of cell viability showed a significantly improved cell viability in the test items groups. The results showed that the test sample was found to have significant cell viability with more than 95%. The one-time Biofield Energy Treated (BT-I) group showed 95.8% and two-times Biofield Energy Treated (BT-II) group showed 104.6% cell viability (Figure 1). Overall, experimental MTT assay data suggested that the Biofield Energy Treated DMEM was found safe and non-toxic. Thus, the test sample was used to study the bone health parameter, cytokine IL-8 in SW982 cells.

Figure 1: Assessment of cell viability using MTT assay of the test items (DMEM) on SW982 cells. BT-I: One-time Biofield Energy Treated DMEM; BT-II: Two-times Biofield Energy Treated DMEM

Effect of the Test Items on IL-8 in SW982 cells

The effect of the test items on the secretion of IL-8 in SW982 cells against IL-1β stimulation was determined after 24 hours. Figure 1 demonstrated the level of IL-8 in SW982 cells. The untreated DMEM group showed 534.6 ± 13.32 pg/mL of IL-8. Besides, the level of IL-8 was significantly (p≤0.001) inhibited by 31.82% in the two-times Biofield Energy Treated DMEM (BT-II) group as compared to the untreated DMEM group; while 31.16% inhibited as compared to the one-time Biofield Energy Treated (BT-I) group after stimulation with the IL-1β in SW982 cells (Figure 2).

Figure 2: Effect of test item on the secretion of IL-8 in SW982 cells after stimulated with IL-1β.BT-I: One-time Biofield Energy Treated DMEM; BT-II: Two-times Biofield Energy Treated DMEM. ***p≤0.001 vs. untreated DMEM group.

The scientific studies showed the importance of inflammation in the synovial membrane, proliferation in its lining are one of the major changes occurred in rheumatoid arthritis (RA). These inflammation results in significant erosion of the cartilage and bone. Macrophage-like synoviocytes and the fibroblast-like synoviocytes play a serious role in the joint destructive process^[39]. The major mediators of joint inflammations are the IL-6 and IL-8^[40,41]. Pro-inflammatory mediator’s expression was promoted by the IL-1β produced by the synovial lining macrophages^[42]. Thus, the mechanisms of IL-8 and to reduce its expression are one the best approach against any joint inflammation disorders such as RA. Besides, the experimental model using SW982 cells demonstrated significant inhibition of IL-1β stimulated IL-8 secretion in SW982 cells by the test items, which would play a significant role in the bone-health and work as a best supplementation to treat various bone and age-related diseases such as osteoporosis. Overall, the data suggested that The Trivedi Effect^®-Energy of Consciousness Healing based DMEM could be used to improve the bone health and worked as an anti-RA activity in bone health.

Conclusion

MTT assay for cell viability showed significant improved cell viability with more than 95.8% among all the tested groups, which suggested that the test items were safe and nontoxic. In addition to, the effect of the two-times Biofield Energy Treated DMEM (BT-II) on IL-1β stimulated secretion of IL-8 in SW982 cells after 24 hours demonstrated a significant inhibition of IL-8 level by 31.82% as compared to the untreated DMEM group. Thus, the Biofield Energy Treated (The Trivedi Effect^®) DMEM was found to have significant impact on rheumatoid arthritis, which is very vital to combat against various bone-related disorders. Thus, with respect to the untreated DMEM, Biofield Energy Treated DMEM would be highly significant in the growth of SW982 cells on bone health. Therefore, the Consciousness Energy Healing based DMEM might be a suitable alternative media for cell growth. It can be useful for the management of rheumatoid arthritis and against various bone-related disorders such asosteoma, osteoporosis, Paget’s disease of bone, osteomalacia, rickets, deformed bones, etc. Apart from it could be useful for the management of hormonal balance, stress, aging, and other bone disorders that occurs due to poor nutrition, genetically predefined, etc. Besides, it might be useful various immune-related disease conditions such as Sjogren Syndrome, Ulcerative Colitis, Dermatomyositis, Alzheimer’s Disease, Myasthenia Gravis, Dermatitis, Irritable Bowel Syndrome, Graves’ Disease, Asthma, Hashimoto Thyroiditis, Pernicious Anemia, Multiple Sclerosis,Systemic Lupus Erythematosus, Aplastic Anemia, Hepatitis, Atherosclerosis, Diverticulitis, Diabetes, Parkinson’s Disease, etc.

Acknowledgements: Authors are grateful to Dabur Research Foundation, Trivedi Global, Inc., Trivedi Science, Trivedi Testimonials, and Trivedi Master Wellness for their support throughout the work.

Abbreviations

CAM: Complementary and Alternative Medicine; NCCAM: National Center for Complementary and Alternative Medicine; DMEM: Dulbecco’s Modified Eagle’s Medium; FBS: Fetal Bovine Serum.

References

1. 1. Scott, D.L., Wolfe, F., Huizinga, T.W. Rheumatoid arthritis. (2010) Lancet 376 (9746): 1094-1108.

   Pubmed | Crossref | Others

2. 2. Birch, J.T., Bhattacharya, S. Emerging trends in diagnosis and treatment of rheumatoid arthritis. (2010) Prim Care ٣٧(٤): 779-792. 

   Pubmed | Crossref | Others

3. 3. Feldmann, M., Brennan, F.M., Maini, R.N. Role of cytokines in rheumatoid arthritis. (1996) Annu Rev Immunol 14: 397-440.

   Pubmed | Crossref | Others

4. 4. Jin, R., Yang, G., Li, G. Inflammatory mechanisms in ischemic stroke: role of inflammatory cells. (2010) J Leukoc Biol 87(5): 779-789.

   Pubmed | Crossref | Others

5. 5. Clavel, G., Thiolat, A., Boissier, M.C. Interleukin newcomers creating new numbers in rheumatology: IL-34 to IL-38. (2013) Joint Bone Spine 80(5): 449-453.

   Pubmed | Crossref | Others

6. 6. Lee, Y.S., Choi, E.M. Myricetin inhibits IL-1beta-induced inflammatory mediators in SW982 human synovial sarcoma cells. (2010) Intl Immunopharmacol 10(7): 812-814.

   Pubmed | Crossref | Others

7. 7. Yamazaki, T., Tukiyama, T., Tokiwa, T. Effect of dexamethasone on binding activity of transcription factors nuclear factor-kappaB and activator protein-1 in SW982 human synovial sarcoma cells. (2005) In Vitro Cell Dev Biol Anim 41(3-4): 80-82.

   Pubmed | Crossref | Others

8. 8. Institute of Medicine (US) Committee on the Use of Complementary and Alternative Medicine by the American Public. Complementary and Alternative Medicine in the United States. Washington (DC): National Academies Press (US); 2005. 7, Integration of CAM and Conventional Medicine.

   Pubmed | Crossref | Others

9. 9. Tabish, S.A. Complementary and Alternative Healthcare: Is it Evidence-based? (2008) Int J Health Sci (Qassim) 2(1): V-IX.

   Pubmed | Crossref | Others

10. 10. Barnes, P.M., Bloom, B., Nahin, R.L. Complementary and alternative medicine use among adults and children: United States, 2007. (2008) Natl Health Stat Report 10(12): 1-23.

    Pubmed | Crossref | Others

11. 11. Jain, S., Hammerschlag, R., Mills, P., et al. Clinical studies of biofield therapies: Summary, methodological challenges, and recommendations. (2015) Glob Adv Health Med 4: 58-66.

    Pubmed | Crossref | Others

12. 12. Barnes, P.M., Powell-Griner, E., McFann, K., Complementary and alternative medicine use among adults: United States, 2002. (2004) Natl Center for Health Statistics 23(343): 1-19.

    Pubmed | Crossref | Others

13. 13. Frass, M., Strassl, R.P., Friehs, H., et al. Use and acceptance of complementary and alternative medicine among the general population and medical personnel: A systematic review. (2012) Ochsner J 12(1): 45-56.

    Pubmed | Crossref | Others

14. 14. Trivedi, M.K., Tallapragada, R.M. A transcendental to changing metal powder characteristics. (2008) Met Powder Rep 63(9): 22-28.

    Pubmed | Crossref | Others

15. 15. Trivedi, M.K., Nayak, G., Patil, S., et al. Studies of the atomic and crystalline characteristics of ceramic oxide nano powders after bio field treatment. (2015) Ind Eng Manage 4: 161.

    Pubmed | Crossref | Others

16. 16. Trivedi, M.K., Nayak, G., Patil, S., et al. Effect of biofield energy treatment on physical and structural properties of calcium carbide and praseodymium oxide. (2015) Int J Mat Sci Applicat 4(6): 390-395.

    Pubmed | Crossref | Others

17. 17. Trivedi, M.K., Branton, A., Trivedi, D., et al. Morphological characterization, quality, yield and DNA fingerprinting of biofield energy treated alphonso mango (Mangiferaindica L.). (2015) J Food Nutr Sci 3: 245-250.

    Pubmed | Crossref | Others

18. 18. Trivedi, M.K., Branton, A., Trivedi, D., et al. Evaluation of biochemical marker – Glutathione and DNA fingerprinting of biofield energy treated Oryza sativa. (2015) Am J Bio Sci 3(6): 243-248.

    Pubmed | Crossref | Others

19. 19. Trivedi, M.K., Branton, A., Trivedi, D., et al. Phenotyping and 16S rDNA analysis after biofield treatment on Citrobacter braakii: A urinary pathogen. (2015) J Clin Med Genom 3: 129.

    Pubmed | Crossref | Others

20. 20. Trivedi, M.K., Patil, S., Shettigar, H., et al. Evaluation of biofield modality on viral load of Hepatitis B and C viruses. (2015) J Antivir Antiretrovir 7(3): 083-088.

    Pubmed | Crossref | Others

21. 21. Trivedi, M.K., Patil, S., Shettigar, H., et al. An impact of biofield treatment: Antimycobacterial susceptibility potential using BACTEC 460/MGIT-TB System. (2015) Mycobact Dis 5: 189.

    Pubmed | Crossref | Others

22. 22. Trivedi, M.K., Patil, S., Shettigar, H., et al. Phenotypic and biotypic characterization of Klebsiella oxytoca: An impact of biofield treatment. (2015) J Microb Biochem Technol 7(4): 203-206.

    Pubmed | Crossref | Others

23. 23. Nayak, G., Altekar, N. Effect of biofield treatment on plant growth and adaptation. (2015) J Environ Health Sci 1(2): 1-9.

    Pubmed | Crossref | Others

24. 24. Branton, A., Jana, S. The influence of energy of consciousness healing treatment on low bioavailable resveratrol in male Sprague Dawley rats. (2017) Int J Clin Develop Anat 3: 9-15.

    Pubmed | Crossref | Others

25. 25. Branton, A., Jana, S. The use of novel and unique biofield energy healing treatment for the improvement of poorly bioavailable compound, berberine in male Sprague Dawley rats. (2017) Am J Clin Exp Med 5(4): 138-144.

    Pubmed | Crossref | Others

26. 26. Branton, A., Jana, S. Effect of The biofield energy healing treatment on the pharmacokinetics of 25-hydroxyvitamin D3 [25(OH)D3] in rats after a single oral dose of vitamin D3. (2017) Am J Pharmacol Phytother 2(1): 11-18.

    Pubmed | Crossref | Others

27. 27. Kinney, J.P., Trivedi, M.K., Branton, A., et al. Overall skin health potential of the biofield energy healing based herbomineral formulation using various skin parameters. (2017) Am J Life Sci 5(2): 65-74.

    Pubmed | Crossref | Others

28. 28. Singh, J., Trivedi, M.K., Branton, A., et al. Consciousness energy healing treatment based herbomineral formulation: A safe and effective approach for skin health. (2017) Am J Pharmacol Phytother 2(1): 1-10.

    Pubmed | Crossref | Others

29. 29. Trivedi, M.K., Branton, A., Trivedi, D., et al. A Systematic study of the biofield energy healing treatment on physicochemical, thermal, structural, and behavioral properties of magnesium gluconate. (2017) Intl J Bioorgan Chem 2(3): 135-145.

    Pubmed | Crossref | Others

30. 30. Trivedi, M.K., Branton, A., Trivedi, D., et al. Chromatographic and spectroscopic characterization of the consciousness energy healing treated Withania somnifera (ashwagandha) root extract. (2017) Eur J Biophysics 5(2): 38-47.

    Pubmed | Crossref | Others

31. 31. Trivedi, M.K., Patil, S., Shettigar, H., et al. The potential impact of biofield treatment on human brain tumor cells: A time-lapse video microscopy. (2015) J Integr Oncol 4: 141.

    Pubmed | Crossref | Others

32. 32. Trivedi, M.K., Patil, S., Shettigar, H., et al. In vitro evaluation of biofield treatment on cancer biomarkers involved in endometrial and prostate cancer cell lines. (2015) J Cancer Sci Ther 7: 253-257.

    Pubmed | Crossref | Others

33. 33. Anagnos, D., Trivedi, K., Branton, A., et al. Influence of biofield treated vitamin D3 on proliferation, differentiation, and maturation of bone-related parameters in MG-63 cell-line. (2018) Intl J Biomed Eng Clin Sci 4: 6-14.

    Pubmed | Crossref | Others

34. 34. Lee, A.C., Trivedi, K., Branton, A., et al. The potential benefits of biofield energy treated vitamin D3 on bone mineralization in human bone osteosarcoma cells (MG-63). (2018) Intl J Nutr Food Sci 7(1): 30-38.

    Pubmed | Crossref | Others

35. 35. Stutheit, M.E., Trivedi, K., Branton, A., et al. Biofield energy treated vitamin D3: Therapeutic implication on bone health using osteoblasts cells. (2018) Am J Life Sci 6(1): 13-21.

    Pubmed | Crossref | Others

36. 36. Czekanska, E.M., Stoddart, M.J., Richards, R.G., et al. In search of an osteoblast cell model for in vitro research. (2012) Eur Cells Mater 24: 1-17.

    Pubmed | Crossref | Others

37. 37. Lorraine, M.H., Mahendra, K.T., Alice, B., et al. Biofield energy enriched vitamin D3 versus vitamin D3 in preventing fractures and bone loss using MG-63 Cells. (2008) Ortho Res Online J 3(5): 1-7.

    Pubmed | Crossref | Others

38. 38. Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity (ISO 10993-5:2009), I.S.EN ISO, 10993-5:20093.

    Pubmed | Crossref | Others

39. 39. Burmester, G.R., Stuhlmüller, B., Keyszer, G., et al. Mononuclear phagocytes and rheumatoid synovitis. Mastermind or workhorse in arthritis. (1997) Arthritis Rheum 40(1): 5-18.

    Pubmed | Crossref | Others

40. 40. Miyazawa, K., Mori, A., Yamamoto, K., et al. Constitutive transcription of the human interleukin-6 gene by rheumatoid synoviocytes: Spontaneous activation of NF-kappaB and CBF1. (1998) Am J Pathol 152(3): 793-803.

    Pubmed | Crossref | Others

41. 41. Tan, P.L., Farmiloe, S., Yeoman, S., et al. Expression of the interleukin 6 gene in rheumatoid synovial fibroblasts. (1990) J Rheumatol 17(12): 1608-1612.

    Pubmed | Crossref | Others

42. 42. van den Berg, W.B. Joint inflammation and cartilage destruction may occur uncoupled. (1998) Springer Semin Immunopathol 20(1-2): 149-164.

    Pubmed | Crossref | Others