Genome-wide DNA methylation profiling after Ayurveda intervention to bronchial asthmatics identifies differential methylation in several transcription factors with immune process related function.

BACKGROUND: The Indian traditional medicinal system, Ayurveda, describes several lifestyle practices, processes and medicines as an intervention to treat asthma. Rasayana therapy is one of them and although these treatment modules show improvement in bronchial asthma, their mechanism of action, particularly the effect on DNA methylation, is largely understudied. OBJECTIVES: Our study aimed at identifying the contribution of DNA methylation changes in modulating bronchial asthma phenotype upon Ayurveda intervention. MATERIALS AND METHODS: In this study, genome-wide methylation profiling in peripheral blood DNA of healthy controls and bronchial asthmatics before (BT) and after (AT) Ayurveda treatment was performed using array-based profiling of reference-independent methylation status (aPRIMES) coupled to microarray technique. RESULTS: We identified 4820 treatment-associated DNA methylation signatures (TADS) and 11,643 asthma-associated DNA methylation signatures (AADS), differentially methylated [FDR (≤0.1) adjusted p-values] in AT and HC groups respectively, compared to BT group. Neurotrophin TRK receptor signaling pathway was significantly enriched for differentially methylated genes in bronchial asthmatics, compared to AT and HC subjects. Additionally, we identified over 100 differentially methylated immune-related genes located in the promoter/5'-UTR regions of TADS and AADS. Various immediate-early response and immune regulatory genes with functions such as transcription factor activity (FOXD1, FOXD2, GATA6, HOXA3, HOXA5, MZF1, NFATC1, NKX2-2, NKX2-3, RUNX1, KLF11), G-protein coupled receptor activity (CXCR4, PTGER4), G-protein coupled receptor binding (UCN), DNA binding (JARID2, EBF2, SOX9), SNARE binding (CAPN10), transmembrane signaling receptor activity (GP1BB), integrin binding (ITGA6), calcium ion binding (PCDHGA12), actin binding (TRPM7, PANX1, TPM1), receptor tyrosine kinase binding (PIK3R2), receptor activity (GDNF), histone methyltransferase activity (MLL5), and catalytic activity (TSTA3) were found to show consistent methylation status between AT and HC group in microarray data. CONCLUSIONS: Our study reports the DNA methylation-regulated genes in bronchial asthmatics showing improvement in symptoms after Ayurveda intervention. DNA methylation regulation in the identified genes and pathways represents the Ayurveda intervention responsive genes and may be further explored as diagnostic, prognostic, and therapeutic biomarkers for bronchial asthma in peripheral blood.

Dosha phenotype specific Ayurveda intervention ameliorates asthma symptoms through cytokine modulations: Results of whole system clinical trial.

ETHNOPHARMACOLOGICAL RELEVANCE: Over the past few decades, there have been significant scientific advances leading to improved understanding of asthma as a disease and treatment providing immediate relief. However, prevention of recurrent attacks, exacerbations and disease cure remains a challenge. Ayurveda refers to bronchial asthma as Tamaka Swasa and it is well explained in Charaka Samhita. Management of asthma in Ayurveda includes removal of vitiated Kapha through Shodhana, Shamana procedures, herbal and herbomineral formulations in addition to advising a healthy lifestyle and diet. Several clinical trials on Ayurvedic formulations for treatment of asthma are reported, however, whole system management of asthma has rarely been studied in the manner in which it is actually being practiced. Ayurveda therapeutics provides Dosha specific approaches, which needs biological investigation. AIM OF THE STUDY: The objective of our study was to investigate lung functions and cytokine changes in Asthmatic individuals in response to Ayurvedic intervention. METHODS: The study design was approved by the Institutional Ethics Committee of Tilak Ayurveda Mahavidyalaya (TAMV) & Sheth Tarachand Ramnath Charitable Ayurveda Hospital and followed guidelines of the Declaration of Helsinki and Tokyo for humans. It was conducted as a whole system individualized pragmatic clinical trial and written consent of patients was collected before enrollment. One hundred and fifteen patients with mild-to-moderate asthma were divided into 2 sub-groups depending on their disease subsets and administered phenotype specific ayurvedic interventions. Seventy six asthma patients completed the treatment. Serum IgE levels, blood eosinophil counts, spirometry and blood cytokine levels were measured before the start of treatment and six months at the end of treatment. Age and sex matched healthy participants (n=69) were recruited in the study for comparison of cytokines levels. RESULTS: Significant improvements in FEV1(% predicted) (p<0.0001) and FVC (% predicted) (p=0.0001) was observed in asthmatic patients who underwent Ayurvedic treatment. Circulating levels of IgE (p<0.03) and eosinophil numbers (p=0.001) reduced significantly in the asthmatics after Ayurvedic treatment. This was associated with significant reduction in levels of circulating cytokines. Levels of Th2, Th1 and inflammatory cytokines in the peripheral blood were higher than healthy control participants at baseline (p values <0.0001) and reduced significantly after ayurvedic intervention. CONCLUSION: This proof of concept study highlights the potential benefits and possible mechanism of Ayurvedic interventions in patients with mild-to-moderate asthma. The interventions significantly reduced IgE and eosinophil count, also improved lung function and reduced levels of circulating Th2 cytokines.

Positive Selection on a Regulatory Insertion-Deletion Polymorphism in FADS2 Influences Apparent Endogenous Synthesis of Arachidonic Acid.

Long chain polyunsaturated fatty acids (LCPUFA) are bioactive components of membrane phospholipids and serve as substrates for signaling molecules. LCPUFA can be obtained directly from animal foods or synthesized endogenously from 18 carbon precursors via the FADS2 coded enzyme. Vegans rely almost exclusively on endogenous synthesis to generate LCPUFA and we hypothesized that an adaptive genetic polymorphism would confer advantage. The rs66698963 polymorphism, a 22-bp insertion-deletion within FADS2, is associated with basal FADS1 expression, and coordinated induction of FADS1 and FADS2 in vitro. Here, we determined rs66698963 genotype frequencies from 234 individuals of a primarily vegetarian Indian population and 311 individuals from the US. A much higher I/I genotype frequency was found in Indians (68%) than in the US (18%). Analysis using 1000 Genomes Project data confirmed our observation, revealing a global I/I genotype of 70% in South Asians, 53% in Africans, 29% in East Asians, and 17% in Europeans. Tests based on population divergence, site frequency spectrum, and long-range haplotype consistently point to positive selection encompassing rs66698963 in South Asian, African, and some East Asian populations. Basal plasma phospholipid arachidonic acid (ARA) status was 8% greater in I/I compared with D/D individuals. The biochemical pathway product-precursor difference, ARA minus linoleic acid, was 31% and 13% greater for I/I and I/D compared with D/D, respectively. This study is consistent with previous in vitro data suggesting that the insertion allele enhances n-6 LCPUFA synthesis and may confer an adaptive advantage in South Asians because of the traditional plant-based diet practice.

Association of homocysteine with global DNA methylation in vegetarian Indian pregnant women and neonatal birth anthropometrics.

OBJECTIVE: The present study was designed to evaluate if plasma maternal folate, vitamin B-12 and homocysteine levels had an effect on maternal global DNA methylation and neonatal anthropometrics in Indian pregnant women. METHODS: A total of 49 participants having completed ≥36 weeks of pregnancy were enrolled in the study. Estimation of folate was by Ion capture assay, vitamin B-12 by microparticle enzyme immunoassay, total homocysteine by fluorescence polarization immunoassay and global DNA methylation using Cayman's DNA methylation enzyme immunoassay (EIA) kit. RESULTS: Folate and vitamin B-12 were inversely correlated to homocysteine in pregnant women consuming vegetarian and non-vegetarian diet. No difference in global DNA methylation was found between the vegetarian and non-vegetarian pregnant women. Folate and vitamin B-12 did not show association with global DNA methylation, however plasma total homocysteine of the vegetarian group showed significant correlation to global DNA methylation (r(2 )= 0.49, p = 0.011). Plasma total homocysteine was inversely related to tricep skinfold (r(2 )= -0.484, p = 0.01) and chest circumference (r(2 )= -0.104, p = 0.04) of neonates in vegetarian group. CONCLUSION: Moderate vitamin B-12 deficiency in vegetarian pregnant women might be the cause of hyperhomocystinemia, hypermethylation when compared to vitamin B-12 sufficient non-vegetarian group.