Published on March 10, 2014
Brief Background Authors: Manoj Bhasin, Jeffery Dusek, Bei-Hung Chang, Marie Joseph, John Denninger, Gregory Fricchione, Herbert Benson, Towia Libermann Published: May 2013 Grant Funding: H75/CCH123424 and R01 DP000339 from the Centers for Disease Control and Prevention (CDC) (HB), RO1 AT006464-01 from the National Center for Complementary and Alternative Medicine (NCCAM)(HB), M01 RR01032 from the NCRR, National Institutes of Health (The Harvard-Thorndike GCRC). These grant funders had “no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” Conflicts of Interest: None.
Relaxation Response (RR) This is the opposite response of the stress response (better known as “flight or fight” response) which produces specific psychological and physiological reactions in the mind and body. This response often activates the parasympathetic nervous system whereas the stress response activates the sympathetic nervous system. Over time, over heightened activity of the sympathetic nervous system can activate the inflammatory response and lead to chronic disease where as the activation of the parasympathetic response is responsible for helping to bring the body and mind into a state of equilibrium and health. The RR is activated by (1) focusing on a word, prayer, phrase, sound, or movement, and (2) “disregarding everyday thoughts” Examples of activities that activate RR: meditation, yoga, movement practices, biofeedback, breathing exercises, progressive muscle relaxation Physiological changes associated with RR activation: biochemical changes; < oxygen consumption, CO2 elimination, blood pressure, heart and respiratory rate, and norepinephrine responsiveness, >heart rate variability, and changes in cortical and subcortical brain regions
Previous Research, Purpose of This Study Previous Research Findings: Activation of the RR has shown to be an effective therapeutic intervention to counteract stress associated with hypertension, anxiety, aging, diabetes, rheumatoid arthritis, and insomnia; however, the cellular evidence for this effect has not been fully defined. Previous study by authors established changes in genetic expression with a similar design, specific with genes associated with oxygen phosphorylation, antigen processing/presentation, and apoptosis with short and long term practitioners when compared to novices This same previous study also found psychobiological changes only in long term practitioners during one session of RR What is unique for this Research? It is examining acute genetic changes within one RR session and how the length of previous practices affects this genetic expression.
Hypotheses of Study (1) One Relaxation Response session for both short and long-term practitioners would make specific changes in gene expression that would be linked specific biological pathways in comparison to a control group with no experience (2) More significant genetic changes would be found in long term practitioners of Relaxation Response sessions in comparison to short term practitioners of RR.
Study Sample Study enrolled 26 healthy subjects with no previous formal RR activation experience (ie. no formal practices of RR) which were considered the control group, Novices (N1). These subjects then took 8 weeks of RR training and then became the first comparison as Short-term Practitioners (N2) A second group of 26 healthy subjects with prior practices in RR (4-20 years experience) were to compared with the Novice (N1) group and Short-term Practitioners (N2). This group was named Long-term Practitioners (M). All subjects were recruited within Boston, MA
N1 (control group): Listened to a 20 minute Health Education CD on first visit N2 (control group which became N2) and M: Listened to 20 minute RR activating CD Blood samples (to be used for gene expression profiles) and biological measurements were taken before the session (T0), after the session (T1) and 15 minutes after the session was complete. Fractional exhaled nitric oxide (FeNO) were collected at the 3 time points, which helps to assess physiological effects of RR (such as reduction in blood pressure) and has shown that RR typical increases FeNO levels which can influence immune system responses. Methods
Techniques RNA was isolated from peripheral blood mononuclear cells (PBMCs) in blood samples Real-time FeNO (exhaled) was measured with a rapid response chemoluminescent Nitric Oxide Analyzer before each session Transcriptional Profiling: Done by Affymetrix human genome high throughput array plates. These plates contained 96 arrays and 22,000 transcripts. Array images were analyzed with dChip. Types of Gene Analyses: The purpose was to identify RR affected genes/sets of genes with a hierarchy of bioinformatic techniques including Individual Gene Analysis, Gene Ontology (GO) enrichment analysis, Gene Set Enrichment Analysis, Pathways and Interactive network analysis, Systems Biology analysis
Results Explained RR leads to qualitative and quantitative temporal transcriptome changes: Individual Gene Analysis RR elicits distinct temporal patterns of differential gene expression: Self- Organizing Map (SOM) Analysis RR progressively affected energy metabolism and inflammation pathways: Canonical pathways: Gene Set Enrichment Analysis (GSEA) Upregulated Progressive changes induced by RR are linked to energy production in mitochondria: Systems Biology Analysis Upregulated Long-term changes induced by RR are linked to telomerase stability and maintenance: Systems Biology Analysis Progressive and Long-term Downregulated gene expression changes induced by RR are linked to alteration of NF-kB-dependent pathways: Systems Biology Analysis Immune response and telomere maintenance related pathways are affected among Long-term RR practitioners: GSEA RR affected pathways are correlated with Fractional exhaled Nitric Oxide (FeNO) levels : Correlation Analysis
Results Across Group: At T (0): Greatest differentiated genes between N1 vs. N2 At T (1): Greatest differentiated genes between N1 vs. M At T (2): Greatest differentiated genes between N1 vs. M What about Within Groups, what is the greatest differentiation?
Results: Gene Regulation
Results: Gene Regulation ! Table S1: Gene-ontology enrichment analysis of progressive and long-term expression patterns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
Results: FeNO Gene Regulation Table S3: Correlation analysis of NO levels and Selected 10 pathways affected progressively or only in long term manner by RR (Bold). The correlation analysis was performed both by comparing FeNO and gene expression levels at particular time point (e.g. T0, T1, T2) as well as changes in gene expression and FeNO levels within a group. The significance of the correlation was determined on the basis of P value (P < 0.05) and FDR (<25%). The positive and negative correlations between FeNO and gene expression levels are indicated by red and green color respectively. !"#$%"&' !" !# !$ %&' ' () *+, ) +!+- (. &/+0!&/(123!&3456+6 %&' ' (. !32(6+' - 04+- ' (10!78 05 "9": "9#; "9""< "9"# "9"# "9#= %&' ' (>02/+0>(. ) 6>4&(>3- !20>!+3- %&' ' (0- !+' &- (123>&66+- ' (0- /(12&6&- !0!+3- "9"< "9"$ "9"< *+3>02!0(2&40(10!78 05 " "9"$ "9"= "9"< "9# *+3>02!0(+4; (10!78 05 "9""< "9""< *+3>02!0(- ?%*(10!78 05 *+3>02!0(. +!3>73- /2+0(10!78 05 *+3>02!0(6!2&66(10!78 05 " "9""< "9"@ "9"# "9#$ *+3>02!0(!- ?2$(10!78 05 "9"# "9#$ "9"< "9#@ 1ABCDE ?/2 1ABCDE ?/2 1ABCDE ?/2 1ABCDE ?/2 1ABCDE ?/2 1ABCDE ?/2 1ABCDE ?/2 1ABCDE?/2 !$F!# . GHIJDK 1GABCDE !#F!" - #GHIJDK- $GHIJDK !$F!" !$F!# !$F!" !$F!# !#F!" !$F!"
Results: Gene Hubs How is this explained?
Discussion: Inflammation Relationships Significant research has shown that activation of the RR can reduce chronic stress and promote wellness and these authors have provided some of the first evidence of prolonged gene level changes that are opposite of the transcriptional changes that accompany chronic stress. This research specifically looked at transient transcriptome changes in temporal analysis because it focuses on indentifying genes affected by RR at time points and reduces the likelihood of false positives. It is suspected that with upregulation of gene sets with RR for oxidation might improve efficiency of oxidation-reduction reactions and reduce oxidative stress. Upregulation of the ATP synthase pathway with RR can play an important role in understanding the biological supportive evidence of RR. Critical pro-inflammatory transcriptor factors were downregulated by RR thus having an influential effect on the inflammatory response, which can reduce oxidative stress, insulin resistance, and apoptosis. Psychological stress can cause chronic mitochondrial oxidative stress that can lead to metabolic syndrome and activate NF-kB (pro-inflammatory factor) which can make this worse. Understanding the NF-kB relationship in inflammation is important for understanding the molecular/cellular mechanisms for health benefits for RR.
Long term RR practice helped to upregulate pathways associated with gene stability especially with telemere packing, telemere maintenance, and tight junction interaction. Since telemere breakdown can affect mitochondria function and lead to apoptosis. The psychological stress is linked to deregulated immune system function and DNA repair that may be influenced by RR and thus RR may be able to reverse stress related transcriptome changes. RR practice may create mitochondrial resiliency or mitochondrial reserve capacity which can create cellular benefits in relationship to health and reducing psychological stress and chronic disease. Mitochondria are considered the energy powerhouse of a cell and also „master regulators of danger signaling‟ to help protect cellular health and life of an organism. Certain mitochondria also experience differential reserve capacity to help work with the different pathogenic effects of oxidative stress. Mitochondrial reserve capacity and resiliency begins to fail when there is high cellular metabolic demands, which contributes to high disease vulnerability. The Future of This Research: To continue to clinically define the in-depth cellular/molecular pathways and genetic connections related to RR with secondary biochemical testing. Discussion: Mitochondria Relationships
Reference Bhasin M., Dusek J., Chang B., Joseph M., Denninger J., Fricchione G., Benson H., Libermann T. (2013). Relaxation response induces temporal transcriptome changes in energy metabolism, insulin secretion, and inflammatory pathways. Retreived from PlosOne on March 6, 2014: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone. 0062817
4 RR3 predesigned TaqMan Gene Expression Assays, real-time PCR primers and probes.
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