Challenging yourself to achieve and enjoy healthy ageing through hormesis
Hormesis - an introduction (a 45-minute my webinar on Hormesis and Ageing can be seen by going to the university's podcast archives at: http://podcast.au.dk)
A critical component of the homeodynamic property of living systems is their capacity to respond to stress. In this context, the term “stress” is defined as a signal generated by any physical, chemical or biological factor (stressor), which in a living system initiates a series of events in order to counteract, adapt and survive. While successful and compensatory responses to low doses of stressors improve the overall homeodynamics of cells and organisms, an incomplete or failed homeodynamic response leads to the damaging and harmful effects of stress, including death.
Therefore, the idea is that if biological systems are intentionally exposed to mild stress, so that their homeodynamic pathways of maintenance and repair get challenged, and in response become activated, this should lead to achieving beneficial hormetic effects, including health- and longevity-promoting effects. Thus hormesis in aging is defined as the life supporting beneficial effects resulting from the cellular responses to single or multiple rounds of mild stress.
Using a regimen of repeated mild heat shock (RMHS) at 41°C, for 1 hr twice a week, given to normal human skin fibroblasts, keratinocytes and telomerase-immortalised bone marrow mesenchymal stem cells, we have reported a variety of hormetic effects. The choice of the above RMHS regimen was based on several pilot experiments performed for selecting conditions where 30% of the maximal HS response was elicited without affecting cell growth and survival. This does not imply that these are the ideal hormetic conditions for these cells. Other combinations of dose and duration may well have similar or even better effects, but that issue remains to be investigated. A summary of the results obtained and published previously in a series of papers between 1998 and 2009 is as follows:
(1) Anti-aging effects in fibroblasts and keratinocytes: Various anti-aging effects of RMHS were observed in human skin fibroblasts and keratinocytes, as determined by the established criteria of cellular aging in vitro. These effects are:
• a reduction in age-related alteration in cell morphology from a thin, long and spindle shape to irregularly enlarged and flattened shape ;
• a 10-15% increase in cellular replicative lifespan, as measured by cumulative population doublings;
• a 20-80% reduction in the accumulation of oxidatively damaged proteins, depending on the type of damage; and
• a 50-140% increase in intracellular antioxidative abilities including a significant increase in resistance to ethanol, hydrogen peroxide and UV-A irradiation .
The main mechanisms involved in bringing about the above anti-aging effects of RMHS included an increase in and maintenance of higher levels of various heat shock proteins (HSP) and a stimulation of proteasomal activities. Whereas the increased levels of HSP may be protective against protein denaturing, an increase in the proteasomal activities of RMHS-treated cells is responsible for the removal of damaged proteins. We had also reported that the anti-aging hormetic effects of RMHS was accompanied by the activation of stress kinases and an increase in the content and activity of Na,K-ATPase sodium pump. We are now extending these studies to find out the global changes in transcriptome (gene expression at the level of mRNA) and proteome (gene expression at the level of proteins), including post-translational modifications, to elucidate further the hormetic anti-aging effects and mechanisms of action of RMHS in human cells. The long term aim of these studies is to find effective hormetic treatments for delaying the onset and/or the prevention of age-related diseases related to replicative senescence, such as thinning of the skin, neurodegenerative diseases, immune deficiency, and muscle loss leading to sarcopenia.
(2) Enhanced differentiation in keratinocytes and bone marrow stem cells: RMHS enhanced the ability of keratinocytes to enter into differentiation in the presence of calcium, as measured by the levels of differentiation markers involucrin, p38 and Hsp27. Another cell type in which we have tested whether differentiation can be improved hormetically by RMHS is the telomerase-immortalized bone marrow mesenchymal stem cell-line, hTERT-MSC. We have reported that single or multiple exposures to mild HS significantly enhanced the vitamin-D-induced differentiation of hTERT-MSC into osteoblasts, as measured by determining the levels of osteoblastic markers alkaline phosphatase and mineralised matrix. Although the mechanistic aspects of the hormetic effects of HS on the differentiation of human cells are yet to be elucidated, such studies pave the way for developing novel means for the maintenance and improvement of differentiation abilities of various cell types and thus prevent age-related alterations leading to impairments such as thinning and excessive wrinkling of the skin, and a loss of bone mass leading to osteoporosis.
(3) Improved wound healing in vitro: We have observed a novel effect of mild HS-induced hormesis, in terms of enhanced wound healing in vitro, using the so-called "scratch assay".
Other observations that we have made with respect to the effects of HS conditioned medium on enhanced wound healing are summarized as follows:
• Enhanced wound healing was not due to an induction of cell proliferation in HSCM-treated cells, as determined by bromo-deoxyuridine (BrdU) labelling and autoradiography method.
• There was 68% increase in mobility and migration of HSCM-treated cells from the edge of the wound towards the wounded area, as determined by the colloidal gold particle assay .
• Increased migration of cells during wound healing was accompanied by about 54% elongation of cells in HSCM-treated cells, which was almost 5 times higher than elongation in SFCM-treated cells, measured by using the image analysis program.
• HSCM alleviated almost completely (more than 95%) the negative effects of 0.25 mM and 0.5 mM glyoxal on wound healing reported previously.
• HSCM could also stimulate wound healing in late passage senescent cells, but it was about 50% less than that in early passage or middle-aged cells. However, it must be pointed out that late passage senescent cells had their wound healing capacity already reduced significantly due to aging, but could still get some benefits from mild HS-induced hormesis.
The above studies indicate that mild HS induces the synthesis of one or more gene-products which are secreted by the cells in the culture medium. Furthermore, these molecules can stimulate wound healing either as direct stimulants or as inhibitors of the negative modulators of wound healing, such as the plasminogen activator inhibitor PAI-1. However, the full range of secreted proteins, including HSP which may be responsible for enhanced wound healing and other biological effects are yet to be identified.
(4) Enhanced angiogenesis by mild HS: Angiogenesis is a physiological process involving the growth of new blood vessels from pre-existing vessels, by the active participation of vascular and microvascular endothelial cells. Since there is an age-related decrease in angiogenesis, we have initiated studies to determine the effects of mild and severe stress on angiogenesis in vitro, using normal human umbilical vein endothelial cells (HUVEC) undergoing aging, and an immortal SV40-transformed human dermal microvascular cell line, HMEC-1.
HORMETINS AS MODULATORS OF AGEING, DIFFERENTIATION AND WOUND HEALING
Various natural or synthetic compounds which can bring about biologically beneficial hormetic effects by activating one or more pathways of stress response, are termed as hormetins. Actually, hormetin can be any condition which challenges one or more stress response pathways in the cells, and is potentially hormetic in strengh´thening the homeodynamic space. Three main categories of hormetins can be:
-Physical hormetins - exercise (running, walking, weight lifting etc), temperature (hot sauna or cold baths), irradiation (sunlight, solar-treatments)
- Nutritional hormetins - food restriction (fasting, low calorie diet), spices (turmeric, clove), zinger, garlic, onion, and micronutrients (zinc). There are many many more nutritional hormetins yet to be identified, and synthetic hormetins yet to be synthesized.
- Mental hormetins - mental activity (reading, puzzle solving, chess), public speaking/performance, focussed attention (meditation), "falling" in love....
Accordingly, several dietary components, such as resveratrol, vitamins, and trace elements and minerals including iron, iodine, fluorine, selenium and copper and zinc are potential hormetins. Other potential hormetins are the so-called antioxidants such as alpha lipoic acid and coenzyme Q10 which, owing to their pro-oxidant activities in producing hydrogen peroxide, induce antioxidative defensive responses and ultimately may bring about beneficial effects. Another hormetin is a plant growth factor kinetin, which we have snown to have several anti-ageing effects in human skin cells, promotes the differentiation of keratinocytes by inducing stress response pathways.
Similarly, some components of certain medicinal plants used frequently in the traditional Chinese medicine and in the Indian Ayurvedic system of medicine are claimed to have anti-aging effects, which appear to be achieved through hormetic pathways. For example, celasterols and paeoniflorin present in some medicinal herbs have cytoprotective effects and induce HSP in human cells. Leaf extracts of Ashwagandha (Withania somnifera) also induce HSP synthesis in mammalian cells, which may be one of the reasons for its observed beneficial medicinal effects.
Curcumin: Another potential hormetin being tested for its biological effects is curcumin or diferuloylmethane. It is the active component in the commonly used food spice turmeric, and is derived from the roots of Curcuma longa. Curcumin is a co-inducer of HSP and has wide ranging biological effects depending on its dosage. We have undertaken studies to check the hormetic effects of curcumin on human keratinocytes and fibroblasts. We have previously reported that at lower doses (0.3 and 1 μM) curcumin stimulates proteasome activity, enhances HSP induction after HS, and stimulates sodium pump activity. We are now extending these studies to find out long term effects of curcumin treatment on cellular aging, differentiation and angiogenesis.
Rosmarinic acid: Another potential hormetin that we are testing is the rosmarinic acid (RA), which is a phenolic compound present in several medicinal plants such as those from the Labiatae family, for example Salvia officinalis (common sage) and Rosmarinus officinalis. We are now in the process of testing further the short term and the long terms effects of the hormetin RA on aging, wound healing, angiogenesis and differentiation of human cells. Our preliminary results show that human skin fibroblast cultures grown continuously in the presence of 20 μM RA for 50 days have a significant (33%) reduction in the number of cells showing senescence-specific β-galactosidase marker. Furthermore, there was 50% reduction in the proportion of enlarged cell population with high levels of green autofluorescence in RA-treated cultures, as measured by flow cytometery.
In conclusion, using human fibroblasts, keratinocytes, endothelial cells and bone marrow mesenchymal stem cells we have provided evidence in support of the view that hormesis can be applied successfully to aging research and intervention. This includes the effects of mild HS on various parameters of cellular aging and other functional characteristics, such as differentiation, wound healing and angiogenesis.
At the mechanistic level, the induction of HSP as mediators of hormetic effects is only a partial explanation and cannot account for the wide ranging and long lasting biological effects. The same may apply to other stresses, such as mechanical stress, irradiation, intermittent fasting, and hormetins. Therefore, it is important to determine how various components of the homeodynamic machinery respond and interact during stress-induced hormesis, and how relatively small individual hormetic effects lead to a significant biological amplification that results in an overall improvement of the living system. Such understanding will be essential for developing effective means of slowing down ageing and preventing the onset of age-related diseases.
