Robert Thomas ND Clinician Educator

Cell-LogicRobert - Professional Thomas Cell-Logic Education © 2018 Division - 2018 www.cell-logic.com.au 1 1.The role of Inflammation 2.Inflammation/ oxidative stress 3.Cellular defences - mechanisms of balance 4.Endothelial inflammation and human disease 5.Case study 6.Clinical studies

Robert Thomas - Cell-Logic 2018 Cell-Logic - AUSTRALIA 2 • Part of the body's immune response. • Self protection. • Attempt to isolate, exterminate and eliminate harmful stimuli • Beginning of the healing process. • Essential for infections, wounds, and any tissue damage to heal. • Chronic inflammation can cause several chronic diseases including: Cancer, Heart disease, Diabetes, Auto immune conditions, IBS, Depression, Anxiety, Autism, Alzheimer’s …… Robert Thomas Cell-Logic © 2018 www.cell-logic.com.au 4

Robert Thomas - Cell-Logic 2018 Cell-Logic - AUSTRALIA 6 Robert Thomas - Cell-Logic 2018 Cell-Logic - AUSTRALIA 7 Condition Impact of Inflammation Condition Impact of Inflammation Amenorrhea Increases Insulin resistance Increases Avolition Increases Interleukin-6 serum levels (example of a plasma Increases Body temperature and sweating Increases cytokine) Libido, erectile function (loss of activity of the HPG Decreases Bone loss Increases axis) Cachexia, cachectic obesity Increases Numbness Increases Circadian rhythms of symptoms Become apparent Hypertension/fluid retention Often Physical activity Decreases Coagulation system Activated Disposition to pain (skeletal muscle, joints, other) Increases Proinflammatory high density lipoproteins (HDL), Increases dyslipidemia

Erythrocyte sedimentation rate Increases Protein in the urine Increases Serum albumin Decreases Fatigue Increases Sleeping problems Increases Food intake, appetite, malnutrition Decreases Stress negatively influences inflammation Becomes apparent Headache Increases Heart rate, sympathetic nervous tone Increases Symptoms of depression Increases Vertigo Increases Inflammation-related anaemia Decrease Weakness Increases

2016: Straub RH, Schradin C: Chronic inflammatory systemic diseases: An evolutionary trade-off between acutely beneficial but chronically harmful programs. Evol Med Public Health. Robert Thomas Cell-Logic © 2018 www.cell-logic.com.au 9 Cytoplasm OXIDATIVE Signals STRESS detected H2O2 Superoxide

Nucleus

↑ Expression of Pro-Inflammatory Genes

Tissue Damage Activated NF-kB, STAT-3 and others generates ROS

Inflammatory Cytokines, Chemokines, Lipid Mediators, and others eg IL-1, IL-6, TNF-alpha

Cytoprotective Compounds Produced within the Cell

ANTIOXIDANT ENZYMES NON-ENZYME Quench millions of ROS per minute • Glutathione • Coenzyme Q10 1. Superoxide dismutase • Metallothionein 2. Glutathione peroxidase • Thioredoxin 3. Catalase • BRCA 1 & BRCA 2 • Ferritin DETOXIFICATION ENZYMES • Vitamin D Receptor 1. Gluathione-S-transferase • …... 2,000 others 2. Quinone reductase • Uric acid 3. UDP-glucuronosyl transferase • Bilirubin • and others OTHER ENZYMES...... Haemoxygenase-1 Glutathione reductase Others…..

Robert Thomas- Cell-Logic © 2018 13 Non-Enzyme antioxidants Glutathione, Lipoic acid, Co enzyme Q10 & others Superoxide Dismutase (SOD) Glutathione Peroxidase (GPx) Catalase ‘One For One’ ONLY (Cat) . Neutralize over one million free radicals per molecule Secondary antioxidants Vitamin E, C, Carotenoids, per minute Polyphenols from plants, Minerals, Proteins, Bilirubin, Uric acid … Robert Thomas- Cell-Logic © 2018 14 Pathway 1. .- O2 SOD Superoxide (Mn++ Zn++ Cu++) H2O2 Hydrogen peroxide Pathway 3. Pathway 2. Glutathione peroxidase (Se++)

+ + Fe++ & other Catalase (Fe++) NO metal ions (Nitric Oxide)

OH- - ONOO Hydroxyl H2O + O2 Peroxynitrite free radical

Robert Thomas - Cell-Logic 2018 Cell-Logic - AUSTRALIA 15 F. Santilli, et al. Oxidative stress in chronic vascular disease: from prediction to prevention. Vascul.Pharmacol.(2015) Robert Thomas- Cell-Logic © 2018 5000 4500 4000 3500 3000 CONTROL 2500 2000 GliSODin 1500 1000 500 0 ANTIOXIDANT SOD GPx Catalase STATUS x (Units/gram (Units/gram (Units/gram 1000 Hb) Hb) Hb) x 50

Supplemented over 28 days

Vouldoukis I et al. Supplementation with Gliadin-combined Plant Superoxide Dismutase extract Promotes Antioxidant Defences and Protects Against Oxidative Stress. Phytother. Res. 18, 957–962 (2004) Robert Thomas - Cell-Logic 2018 Cell-Logic - AUSTRALIA 17 Cytoprotective Compounds Produced within the Cell

ANTIOXIDANT ENZYMES NON-ENZYME Quench millions of ROS per minute • Glutathione 1. Superoxide dismutase • Coenzyme Q10 2. Glutathione peroxidase • Metallothionein 3. Catalase • Thioredoxin • BRCA 1 & BRCA 2 DETOXIFICATION ENZYMES • Ferritin 1. Gluathione-S-transferase • Vitamin D Receptor 2. Quinone reductase • …... 2,000 others 3. UDP-glucuronosyl transferase • Uric acid • Bilirubin OTHER ENZYMES...... • and others Heme oxygenase-1

Nrf2 activates > 2000 cytoprotective genes, including: Quinone reductase, Thioredoxin & Heme oxygenase 1. Robert Thomas- Cell-Logic © 2018 19 Nrf2 …increases highly-coordinated 2015 antioxidant producing activities:

•  anti-inflammatory changes •  mitochondrial biogenesis • Removes toxic protein aggregates

Raising Nrf2 directly prevents or treats numerous inflammatory diseases including: • CVD • Kidney Disease • Lung Diseases • Toxic liver damage • Diabetes/metabolic syndrome • Sepsis • Autoimmune diseases, inflammatory bowel disease

Pall ML, Levine S. Acta Physiologica Sinica, February 25, 2015, 67(1): 1–18

Robert Thomas- Cell-Logic © 2017 20 Helping break the Oxidative Stress /Inflammation Cycle • Phase 2 Detoxification incl. estrogen metabolism • Preventing DNA adducts • Preventing Dopamine oxidation • Preventing Adrenaline oxidation • Inactivation of toxins eg. benzene • Recycling • Vit. E • Recycling CoQ10 • Activating Vitamin K (QR inhibited by warfarin) - the Oxidative Stress-Inflammation Connection

…. increased levels of oxidative stress and inflammation in Major Depressive Disorders

Robert Thomas - Cell-Logic 2018 Cell-Logic - AUSTRALIA 22 & Atherosclerosis

Atherosclerosis … characterised by infiltration of lipids & inflammatory cells into the vascular wall

…. The vasculature has a plethora of protective recourses against oxidative stress and inflammation…..

Heme oxygenase-1 (HO1) is an Nrf2-regulated gene that plays a critical role in the prevention of vascular inflammation. HO-1 recognised to have immunomodulatory and anti-inflammatory properties.

…. provides a negative feedback which could modulate the inflammatory Robert Thomas - Cell-Logic 2018 Cell-Logic - AUSTRALIA response. 24 … endowed with potent anti- diabetic and insulin sensitising effect ..

Shown to suppress visceral & subcutaneous obesity The HO system suppresses inflammatory/oxidative mediators and pro-inflammatory cytokines to enhance insulin signalling and improve Glucose metabolism.

Robert Thomas - Cell-Logic 2018 Cell-Logic - AUSTRALIA 26 Thioredoxin effective in a wide variety of inflammatory diseases including pneumonia, gastric ulcer, dermatitis, rheumatoid arthritis, hepatitis, food allergies. Thioredoxin is an antioxidant … involved in protecting against … inflammation and cardiovascular disease • Cancer • Diabetes • Atheroscerosis • Viral infection

Alterations of endothelial cells play a vital role in … the most dreadful human diseases… 2015: Santilli F, Damiano D, Giovanni D: Oxidative Stress in chronic vascular disease: From prediction to prevention. Vascular Pharmacology

HISTORY: Hypertension, mild food and chemical sensitivities, muscle soreness, fatigue, insomnia, very disciplined with diet and rigorous gym program (4-5 times per week). PRIOR FINDINGS: Blood lipids slightly elevated, PSA rise 2 years ago PRIOR THERAPY: Coversyl (24 months) NOTE: Relationship between SOD, NADPH-CYBA and eNOS-2

H2O + O

+ Endothelial Nitric Oxide

PEROXYNITRITE ➢ Superoxide + Nitric Oxide  Peroxynitrite

Ceriello A. New Insights on Oxidative Stress and Diabetic Complications may lead to a ‘causal’ antioxidant therapy. Diabetes Care 26:1589-1596, 2003 Robert Thomas- Cell-Logic © 2017 34 5000 4500 4000 3500 3000 CONTROL 2500 2000 GliSODin 1500 1000 500 0 ANTIOXIDANT SOD GPx Catalase STATUS x (Units/gram (Units/gram (Units/gram 1000 Hb) Hb) Hb) x 50 Sulforaphane the most Potent Natural Upregulator of Nrf2 expression.

Fahey JW Kensler TW Role of Dietary Supplements/ Nutraceuticals in Chemoprevention through Induction of Cytoprotective Enzymes Chem Res Toxicol. 20(4): 2007;573

Houghton CA et al. Sulforaphane and Other Nutrigenomic Nrf2 Activators: Can the Clinician’s Expectation Be Matched by the Reality? Oxidative Medicine and Cellular RobertLongevity. Thomas Volume -2016,Cell- ArticleLogic 2018ID 7857186, 17 pages Cell-Logic - AUSTRALIA 36 Use corrected version of the graph Table 1: Clinically-trialled Sulforaphane Doses per Condition Protective Effect of Sulforaphane on Liver Hepatic TNF-alpha (pg/mg tissue)

Control Valproate 180 160 140

120 Negative Control

alpha - 100 SFN Control 80 60 Valproate Control

Hepatic TNF 40 SFN + Valproate 20 0 Sulforaphane + Negative SFN Valproate SFN + Sulforaphane + Control Control Control Valproate Valproate Valproate Nazmy EA et al. Sulforaphane protects against Valproate-induced acute liver injury. Can. J. Physiol. 95: 420-426. (2017) Hepatic Haemoxygenase (ng/mg tissue) 35

1 1 25 - Negative Control Conclusion: Sulforaphane can offer significant 20 SFN Control protection against sodium valproate-induced toxicity 15 Valproate Control primarily through modulating its anti-inflammatory and HO Hepatic 10 SFN + Valproate immune-modulating antioxidant properties. 5

0 Negative SFN Control Valproate SFN + PROFESSIONAL EDUCATION DIVISION Control Control Valproate www.cell-logic.com.au 39 Conclusion: Sulforaphane proved to be an effective in vivo Protective Effect of Sulforaphane on Fatty Liver inhibitor of acute ethanol–induced fatty liver in mice. Control Ethanol 70 Effect of SFN ± Ethanol on Glutathione 60

50 Control 40 Ethanol

30 SFN Glutathione Sulforaphane Ethanol + Sulforaphane 20 Ethanol + SFN 10

0 Control Ethanol SFN Ethanol + SFN

Zhou R. et al. Sulforaphane Induces Nrf2 and Protects Against CYP2E1- dependent Binge Alcohol –induced Liver Steatosis. Biochim Biophys Acta. 2014 January; 1840(1)

70 Effect of SFN ± Ethanol on Total Cholesterol 180 Effect of SFN ± Ethanol on Triglycerides 60 160 140 50 Control 120 40 Control 100 Ethanol Ethanol 30 80 SFN SFN 20 60 TotalCholesterol Ethanol + SFN Ethanol + SFN 10 Triglycerides 40 0 20 Control Ethanol SFN Ethanol + 0 SFN Control Ethanol SFN Ethanol + SFN 40 Specialists in Nutrigenomics, Nutraceuticals & Functional Foods

Robert Thomas - Cell-Logic 2018 Cell-Logic - AUSTRALIA 41