Impact of homeopathic remedies on the expression of lineage differentiation genes: an in vitro approach using embryonic stem cells.

BACKGROUND: Well-documented studies of the potential effects and safety of homeopathic medicines in pregnancy are required. In this study, specific genes were studied which could serve as biomarkers for specification of three lineages to predict the safety of homeopathic remedies using mouse embryonic stem (ES) cells. Thus, the present work was to study the effects of homeopathic remedies taken during pregnancy using ES cells as the model. METHODS: Mouse ES cells were exposed to 30C potency of Nux Vomica and Sepia, which are homeopathic medicines prescribed for the management of pregnancy related symptoms. Cytotoxicity studies were done using a modified Embryonic Stem cell test (EST). The expression levels of key genes and proteins were analyzed using real time polymerase chain reaction and immunocytochemistry, respectively. RESULTS: Homeopathic treatment led to modulations in the expression of certain lineage specific genes but this difference was not significant with respect to solvent control and showed normal differentiation as demonstrated by the expression of α/ß MHC and α-actinin proteins in the differentiated ES cells. CONCLUSIONS: Our study for the first time has shown the feasibility of using ES cells in the developmental toxicity testing of remedies. The results suggest that they are not associated with developmental toxicity.

Hypocapnia leads to enhanced expression of pluripotency and meso-endodermal differentiation genes in mouse embryonic stem cells.

The efficient utilization of embryonic stem cells for applications like cell based therapy, transplantation and drug discovery largely depends upon the culturing conditions of these cells. In this report, we have analyzed gene, protein expression and morphological changes of embryonic stem cells when subjected to lowered CO2 levels i.e. hypocapnia. We studied the quantitative expression of pluripotent genes, Oct3/4, Nanog and Sox2 and genes involved in the differentiation to the three lineages, under varying CO2 levels. Enhanced expression of these genes was seen at cultures maintained at 1.5% CO2 as compared to those maintained at 5% CO2. The cells exposed to hypocapnic conditions when subjected to immunocytochemical analysis stained positive for Oct-3/4, Nanog and Sox2 transcription factors. Flow cytometry and western blot further showed that the pluripotent proteins in the 1.5% CO2 maintained cultures have higher levels of expression as compared to the ES cells at 5% CO2. In addition, there was enhanced differentiation particularly towards the mesodermal and endodermal lineages at cultures maintained and differentiated at 1.5% CO2 at all the time periods analyzed i.e. day 10 (5+5d), 12 (5+7d) and day 15 (5+10d). These results, which we feel are the first of their kind, indicate that lowered CO2 levels seem to be preferred for the maintenance of pluripotency and the subsequent differentiation.

Anti-proliferative effects of homeopathic medicines on human kidney, colon and breast cancer cells.

OBJECTIVE: Homeopathy is controversial, due to the claims made for very high dilutions. Although several theories are proposed to understand the mechanisms of action, none are scientifically verified. This study aimed to investigate the efficacy of the selected homeopathic medicines in specific in vitro cancer models. METHODS: We assessed the cytotoxic activity of selected homeopathic medicines in mother tincture (MT), and ultramolecular dilution (30C, 200C, 1M and 10M) against cell lines deriving from tumors of particular organs, Sarsaparilla (Sars) on ACHN cells (human renal adenocarcinoma), Ruta graveolens (Ruta) on COLO-205 (human colorectal carcinoma), and Phytolacca decandra (Phyto) on MCF-7 (human breast carcinoma). Sars was also tested against Madin-Darby canine kidney (MDCK) cells (a non-malignant cell line). Cytotoxicity was measured using the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) method, anti-proliferative activity by trypan blue exclusion assay, apoptosis determined by dual staining the cells with ethidium bromide (EB) and acridine orange (AO) dyes. RESULTS: MTs and ultra-diluted preparations of the three homeopathic medicines had highly significant effects in the respective cancer cell lines, producing cytotoxicity and a decrease in cell proliferation. The effects were greatest with the MTs, but in all cases and persisted, although to a lesser degree in the ultra-diluted molecular preparations. Sars showed no effect on MDCK cells. In the homeopathic medicine treated cultures, hallmarks of apoptosis were evident including, cell shrinkage, chromatin condensation and DNA fragmentation. CONCLUSION: This study provides preliminary laboratory evidence indicating the ability of homeopathic medicines as anticancer agents. Further studies of the action of these homeopathic remedies are warranted.

Ayurveda Body-Mind Constitutional Types and Role of Yoga Intervention Among Type 2 Diabetes Mellitus Population of Chandigarh and Panchkula Regions.

BACKGROUND: Type 2 diabetes needs a better understanding of etiological factors and management strategies based on lifestyle and constitutional factors, given its high association rate with many cardiovascular, neurological disorders, and COVID-19 infection. PURPOSE: The present study was undertaken to investigate the effect of Diabetes-specific integrated Yoga lifestyle Protocol (DYP) on glycemic control and lipid profiles of diabetic adults. Along with the DYP intervention, the individuals residing in Chandigarh and Panchkula union territories in the northern part of India were assessed for Ayurveda-based body-mind constitutional type. Ayurveda describes body-mind constitution as "prakriti," which has been discussed from two angles, namely physiological and psychological as body and mind are correlated. METHODS: Cluster sampling of waitlist control study subjects was used as the sampling method for the study. A total of 1,215 registered subjects (81 diabetic) responded in randomly selected clusters in Chandigarh and Panchkula. Ayurveda physicians did Ayurveda body-mind constitutional assessment called prakriti assessment (physiological body-mind constitution assessment) in 35 participants (23 diabetic, 12 prediabetic) as a part of the study. RESULTS: A group of 50 subjects was randomly selected for yoga intervention out of 81 diabetes mellitus adults, and 31 subjects were enrolled as waitlist controls. A significant decrease in the glycosylated hemoglobin levels from 8.49 ± 1.94% to 7.97 ± 2.20% in the intervention group was noticed. The lipid profiles of the DYP intervention and control groups were monitored. Three-month follow-up results of lipid profile diagnostic tests in intervention and control groups showed a significant difference between the two groups (P < 0.05). Most diabetic and prediabetic individuals were found to have pitta dosha (pitta controls all heat, metabolism, and transformation in the mind and body) as dominant constitution type. CONCLUSION: The study results demonstrated significant positive effects of yoga in diabetic individuals. This study has indicated the evidence for the safety and efficacy of the validated DYP for community-level interventions to prevent maladies like brain damage and stroke.

Disruption of mitochondrial membrane potential coupled with alterations in cardiac biomarker expression as early cardiotoxic signatures in human ES cell-derived cardiac cells.

Cardiotoxicity is one of the most significant reasons of attrition in drug development. The present study assessed the sensitivity of various endpoints for early monitoring of drug-induced cardiotoxicity using human embryonic stem cell-derived cardiac cells, including precursors as well as mature cardiomyocytes, by correlating changes in cardiac biomarker expression. Directed differentiation was induced and cardiac progenitor cell (CPC) population were treated with cardiotoxic drugs, namely, doxorubicin (Dox) and paclitaxel (Pac), and with noncardiotoxic drug, namely penicillin G. To assess cardiac-specific toxicity, the changes in the expression of key markers of cardiac lineage, such as Nkx2.5, Tbx5, α-myosin heavy chain α-MHC, and cardiac troponin T, were studied using quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry (FC). The half-maximal inhibition in the expression of these cardiac markers was analyzed from the dose-response curves. We also assessed the half-maximal inhibition (IC50) in cardiac cells using propidium iodide dye (IC50 PI) and by measuring disruption in the mitochondrial membrane potential (IC50 MMP). We observed that the most sensitive marker was α-MHC in the case of both Dox and Pac, and the order of sensitivity of the various prediction assays was MMP > protein expression by FC > gene expression by qRT-PCR > cell viability by PI staining. The results could enrich the screening of drug-induced cardiotoxicity in vitro and propose disruption in MMP along with downregulation of α-MHC protein as a potential biomarker of predicting cardiotoxicity earlier during drug safety evaluation.

Chemical and physical factors influencing the dynamics of differentiation in embryonic stem cells.

Differentiating embryonic stem cells into a specific lineage or cell type is one of the most investigated areas of stem cell research; however it is wrought with hurdles. The differentiation process during embryogenesis is greatly influenced by physiochemical factors. They direct key genes for lineage commitment, which in turn are responsible for patterning into highly organized tissues resulting in organ formation. In this review, we have focused on the influence of physiochemical factors on the differentiation process in embryonic stem cells, which mimics, embryogenesis in vivo.