Skip to main content

In vitro analyses of the effects of Vitamin D and allicin in modulation of the inflammatory response as a nutraceutical approach for the aged

Principal Supervisor: Dr Cordula Stover - Department of Infection, Immunity and Inflammation

Co-supervisor: E Watson (postdoctoral Research Fellow, Muscle Physiologist); A Smith (Honorary Reader, Exercise Immunologist); R Saunders (postdoctoral Research Associate, Respiratory Scientist)

PhD project title: In vitro analyses of the effects of Vitamin D and allicin in modulation of the inflammatory response as a nutraceutical approach for the aged

University of Registration: University of Leicester

Project outline:

The term “inflammaging” describes a chronic inflammatory condition in the aging population[1]. An increase in prevalence of being overweight or obese contributes to elevated fatty acids[2] , circulating endotoxins[3] and inflammatory levels in the aged. This results in added burden on health system resources[4] because chronicity of inflammation significantly contributes to muscle wasting/impaired muscle repair[5], airway hypersensitivity[6] and increased gut epithelial permeability[7].

This project draws on joint cell culture records to investigate interventionist modulation of “inflammaging”. We will use standard hydroxyurea treatment during multiple population doublings to produce replicational stress and thereby mimic aging in culture[8,9] for the cell types of interest: skeletal muscle cells, airway smooth muscle cells, macrophages, and gut epithelial cells.

Using hepatocytes and macrophages, we showed that Vitamin D or docosahexaenoic acid, an omega-3 fatty acid, reduced the inflammatory reactions induced by endotoxin, a pathogen associated molecular pattern (PAMP), while free fatty acids, damage associated molecular patterns (DAMPs), were pro-inflammatory. Our work was published as part of a Frontiers Research Topic “Lipids and the Immune system”[10]. Drs Watson and Smith used exercise as a therapy to reduce age-related loss of muscle mass[11] and quantified a reduction in cytokine expression potentially linked to decreased intramuscular inflammation[12]. Dr Saunders showed that oxidative stress, which is elevated in obesity and contributes to the aging process[13], and the cell stress DAMP HMGB1 increase hypercontractility in airway smooth muscle cells from asthmatics [14,15].

Initially we will characterise the anti-inflammatory response following incubation of target cells with Vitamin D after induction of senescence. This part of the study has the potential to reveal a role for Vitamin D in modulating inflammatory responses in senescent cells by interfering with processes of “inflammaging” and will provide a benchmark via which to assess the efficacy of allicin. Allicin, a key active component of garlic, was proposed as a potential nutraceutical -a dietary component with benefit to health- in obesity associated disease[16].

Recent pilot data demonstrated an anti-inflammatory role of allicin in vitro. The normal TNFa mRNA response elicited by stimulation of macrophages J774 with LPS E.coli 0111B:4 (100ng/ml, 4h) was significantly blunted when cells were preconditioned with 30 M allicin for one day (80 fold reduction).


Preconditioning of cells with allicin for one day led to a significant reduction of secreted pro-inflammatory cytokine in the supernatant of J774 stimulated with LPS for 24h (unpaired t-test, p<0.05).

We will determine a dose response for allicin and, like for our Vitamin D study, LPS and fatty acids will be used as disease relevant PAMPs and DAMPs to interrogate the potency of allicin to revert the pathological profile to normal.

The mode of action of allicin is likely to be the result of a direct cytoplasmic action with thiol containing proteins[17,18] and will be investigated by proteomic analysis. We will conduct functional analyses of specific cell activities: foam cell production (macrophages), contractility assays (smooth muscle cells), creatine kinase activity (skeletal muscle cells), trans-epithelial permeability assay (gut epithelial cells).

In summary, this project analyses in parallel target cells that are relevant to “inflammaging” in an aged population that is overweight or obese. It investigates the potential of dietary supplements (Vitamin D and allicin) to alleviate inflammatory responses and downstream events that are detrimental to health. On completion, the project will provide comprehensive data with which to formulate a justification for taking forward a possible interventionist approach for this increasing group of society.


  1. Franceschi C, Campisi J. Chronic Inflammation (Inflammaging) and Its Potential Contribution to Age-Associated Diseases. J Gerontol A Biol Sci Med Sci 2014; 69 (Suppl 1): S4-S9. doi: 10.1093/gerona/glu057
  2. Arner P, Rydén M. Fatty Acids, Obesity and Insulin Resistance. Obes Facts 2015;8:147-155 DOI:10.1159/000381224
  3. Pussinen P, Sundvall J, Havulinna A, Salomaa V, Lehto M. Endotoxemia Is Associated With an Increased Risk of Incident Diabetes. Diabetes Care 34:392–397, 2011
  4. Baker C, Bate A. Obesity Statistics. Briefing paper House of Commons Library. Number 3336, 2 February 2016
  5. Akhmedov D, Berdeaux R. The effects of obesity on skeletal muscle regeneration. Frontiers Pysiol 2013; 4: 371. doi: 10.3389/fphys.2013.00371
  6. Kim JY, Sohn JH, Lee JH, Park JW. Obesity increases airway hyperresponsiveness via the TNF-α pathway and treating obesity induces recovery. PLoS One. 2015;10(2):e0116540. doi: 10.1371/journal.pone.0116540.
  7. Man AL, Bertelli E, Rentini S, Regoli M, Briars G, Marini M, Watson AJ, Nicoletti C. Age-associated modifications of intestinal permeability and innate immunity in human small intestine. Clin Sci (Lond). 2015;129:515-27. doi: 10.1042/CS20150046
  8. Dong CM, Wang XL, Wang GM, Zhang WJ, Zhu L, Gao S, Yang DJ, Qin Y, Liang QJ, Chen YL, Deng HT, Ning K, Liang AB, Gao ZL and Xu J. A stress-induced cellular aging model with postnatal neural stem cells. Cell death and disease (2014) 5, e1116; doi:10.1038/cddis.2014.82.
  9. Sharples AP, Al-Shanti N, Lewis MP, Stewart CE. Reduction of myoblast differentiation following multiple population doublings in mouse C2 C12 cells: A model to investigate ageing? J Cell Biochem. 2011;112:3773-3785.
  10. Kheder RK, Hobkirk J, Stover CM. In vitro Modulation of the LPS-Induced Proinflammatory Profile of Hepatocytes and Macrophages- Approaches for Intervention in Obesity? Front Cell Dev Biol. 2016;4:61. doi: 10.3389/fcell.2016.00061.
  11. Watson EL, Greening NJ, Viana JL, Aulakh J, Bodicaot DH, Barratt J, Feehally J, Smith AC. (2014). Progressive Resistance Exercise Training in Chronic Kidney Disease: A Feasibility Study. AJKD, 66: 249-57.
  12. Viana JL, Watson EL, Greening N, Barratt J, Smith AC. Skeletal muscle cytokine expression after resistance exercise in chronic kidney disease. Presented as an oral presentation at the ASN, Philadelphia, 2014
  13. Bonomini F, Fabrizio Rodella L, Rezzani R. Metabolic Syndrome, Aging and Involvement of Oxidative Stress. Aging Dis 2015; 6: 109-120
  14. Sutcliffe A*, Hollins F*, Gomez E*, Saunders R, Doe C, Cooke MS, Challiss RAJ and Brightling CE. Increased NOX4 expression mediates intrinsic airway smooth muscle hyper-contractility in asthma. Am J Respir Crit Care Med. 2012;185:267-74. *These authors contributed equally to this work.
  15. Di Candia L, Gomez E, Venereau E, Bianchi ME, Challiss RAJ*, Brightling CE* and Saunders R*. HMGB1 is upregulated in the airways in asthma and mediates airway smooth muscle contraction via TLR4. Under revision following peer review at J Allergy Clin Immunol. *Co-senior authors.
  16. Hosseini A, Hosseinzadeh H. A review on the effects of Allium sativum (Garlic) in metabolic syndrome. J Endocrinol Invest. 2015;38:1147-57. doi: 10.1007/s40618-015-0313-8.
  17. Miron T, Rabinkov A, Mirelman D, Wilchek M, Weiner L. The mode of action of allicin: its ready permeability through phospholipid membranes may contribute to its biological activity. Biochim Biophys Acta. 2000;1463:20-30.
  18. Rabinkov A, Miron T, Konstantinovski L, Wilchek M, Mirelman D, Weiner L. The mode of action of allicin: trapping of radicals and interaction with thiol containing proteins. Biochim Biophys Acta. 1998;1379:233-44.

BBSRC Strategic Research Priority: Molecules, cells and systems

  • Techniques that will be undertaken during the project:
  • Cell culture and stimulation
  • ELISA, qPCR, Proteome profile arrays, densitometry, cell signalling via Western blotting
  • Microscopy
  • Specialist techniques: collagen gel contraction assay to assess smooth muscle contractility, creatine kinase assays for skeletal muscle function, trans-epithelial permeability (TEP) assay
  • Statistical evaluations

Contact: Dr Cordula Stover, University of Leicester