Inflammation plays a key role in the progression of many diseases, including rheumatoid arthritis, cancer, atherosclerosis and other cardiovascular diseases, therefore inflammation regulation and inhibition have been found as key therapeutic approaches to these disease conditions. The mechanism of inflammation formation is complex, involving various cellular signaling processes and paths. In the last decades, a number of inflammation markers have been discovered as targets for different anti-inflammatory approaches. Each marker has its own mechanism in inducing or regulating inflammation, with its function often cross relating to functions of other markers. Therefore the impact of a material on inflammation is better understood via multiple marker evaluation.
For nutraceutical investigation in this area, impact of an interested material on key markers for inflammation regulation would be good starting point. In our laboratory, we take effective human cells-based cellular investigation and enzymatic investigation approaches to evaluate inhibition potential of an interested material on inflammation from below five key areas. Multiple marker investigation builds a good understanding of anti-inflammatory potential of the test material, and provides foundation for further development in this functional area.
1. Cytokines (TNF-alpha, interleukins) inhibition in cells
Tumor necrosis factor (TNF)-alpha is a proinflammatory cytokine (small proteins impacting cell signaling) that triggers downstream cellular feedback loops governing inflammation. TNF-alpha has been identified as an inflammation trigger and precursor. In many anti-inflammation investigation, TNF-alpha inhibitors are sought to function as anti-inflammatory agents that has potential treatment effect for rheumatoid arthritis (RA) and other inflammatory diseases. At present, clinically proven TNF-alpha inhibitors are protein-based drugs such as infliximab and etanercept for inflammation diseases treatment. However, these protein-based drugs have potential devastating side effects such as heart failure and neurological changes. Natural plants have therefore become of investigative interests to explore their role as less toxic TNF-alpha inhibitors for inflammation treatment, with natural flavonoids such as quercetin and catechin being shown as TNF-alpha inhibitors without significant known side effects.
In our investigation, we focus on TNF-alpha inhibition evaluation of a nutraceutical material for dietary functional effect in anti-inflammation area. Here, human cells are first treated with or without a test material to allow for natural absorption of the material into the cells. Then, the cells will be stressed with a TNF-alpha inducer, which would normally stimulate TNF-alpha production then further develop into inflammation through series of cellular signaling. If a TNF-alpha inhibitor presents in the cellular environment, the material inhibits TNF-alpha production and the degree of inhibition can be assessed by level of decreased TNF-alpha production. TNF-alpha production level of the human cells, treated with and without a test material, under the stressed condition will be monitored and compared to assess the TNF-alpha inhibition effect of the test material.
Similar to TNF-alpha, interleukins are inflammatory cytokines that are also of keen interests to be investigated as biomarkers for inflammation. Cellular inhibition of interleukins can be also assessed in this cellular investigation if requested.
2. NF-κB inhibition in cells
NF-κB (Nuclear Factor kappa B), a protein complex that is involved in cellular responses to stimuli such as stress and free radicals, ultraviolet irradiation, oxidized LDL, and bacterial or viral antigens. It plays a key role in regulating the immune response to infection. Suppression of NF-κB limits the production of proinflammatory gene expression and reduces the level of inflammation, therefore NF-κB has been studied as a biomarker for inflammation, and inhibition of NF-κB has been used as an indicator for anti-inflammatory potential. A classic example of anti-inflammatory material via NF-κB inhibition are natural compounds such as curcumin and green tea polyphenols that have been demonstrated to inhibit inflammation via NF-κB inhibition.
In this analysis, human cells are first treated with or without a test material to allow for natural absorption of the material into the cells. Then, a proinflammatory cytokine is introduced to the human cells to mimic cellular stimulation which would normally induce NF-κB activation leading to inflammation. If an NFkB inhibiting material presents in the cellular environment, the material inhibits NFkB activation and the degree of inhibition can be monitored via NFkB expression. NFkB expression level of the human cells, treated with and without the test material, under the stressed condition are therefore monitored and compared to assess the NFkB inhibition effect of a material.
3. Cycloooxygenase (COX) Inhibition
Cyclooxygenases inhibitors are among the important targets for treatment of inflammation related diseases. COX has two well-known isoforms, COX-1 and COX-2, which are similar in their amino-acid sequences and identity. COX-2 predominates at sites of inflammation, and COX-1 is constitutively expressed in most tissues including gastrointestinal tract. It is reported that selective COX-2 inhibitors can target inflammation and pain with reduced risk of chronic ulceration and acute injury, where COX-1 inhibitors typically possess anti-inflammation effect but with gastric side-effects (e.g., hemorrhagic side effect).
A classic example of anti-inflammatory material via COX inhibition is aspirin, a salicylates class of drug that has been demonstrated to counter inflammation via COX inhibition. This treatment scheme aligns with an ancient Greek remedy which uses willow bark extract (now known as a rich source of salicylic acid) for pain relief.
In our investigation, we focus on evaluating COX inhibition effect of nutraceutical materials for potential anti-inflammation function. Through this investigation, we concentrate on assessing the COX inhibition capability of a material by monitoring its impact on the activity of a COX enzymes. Inhibition potential of two main COX enzymes, COX-1 and COX-2, can be investigated.
4. Lipooxygenase (LOX) Inhibition
Lipoxygenases (LOXs) are a family of nonheme iron-containing dioxygenases enzymes distributed in animals, plants, and fungi. These enzymes catalyze distinct cellular reactions and produce fatty acid hydroperoxides throughout the reaction processes. These fatty acid hydroperoxides products have been identified as mediators of a series of inflammatory diseases including rheumatoid arthritis, inflammatory bowel disease, atherosclerosis and certain types of cancer. Therefore LOX inhibitors have been discovered as beneficial to inflammation treatment. Another significance in LOX inhibition is that LOX 5 inhibitors have been identified to potentially counteract the gastric damage associated with the COX-1 inhibitors. This makes LOX 5 inhibitors potential side effect removers for many COX-1 inhibitor anti-inflammatory drugs including aspirin.
Through Lipooxygenase (LOX) Inhibition investigation we determine the inhibition potential of a material on LOXs activity via enzymatic analysis. Inhibition of two main LOX enzymes, LOX-5 and LOX-15, can be investigated via these analyses.
5. Secretory Phospholipases A2 (sPLA2) Inhibition
Secretory Phospholipases A2 (sPLA2) are a subfamily of Phospholipases A2 enzymes that catalyze the hydrolysis of phospholipids yielding precursors of pro-inflammatory lipid mediators including bioactive eicosanoids and platelet-activating factor (PAF). sPLA2 inhibitors hold an established role in inflammation treatment, since inhibition of sPLA2 in theory would prevent the formation of inflammatory eicosanoids prior to the cyclooxygenase (COX) reaction. Therefore, theoretically, sPLA2 inhibitors eliminate the need for COX inhibitors in anti-inflammatory therapeutics.
Through sPLA2 inhibition investigation we determine the inhibition potential of a material on sPLA2 activity via enzymatic analysis.
Plants are sources of medicines that have been discovered and used throughout thousands of years of civilization. Some 150,000 plant compounds have been explored in medicinal use with many more are expected to be discovered. Natural plants is a great source for screening for anti-inflammatory agents and we are still at the beginning of this nutraceutical age.