| Inflammation occurs as a result of activation of the immune system. Inflammatory processes can either be acute or chronic, and may be triggered by pathogenic organisms, foreign bodies, or autoimmune diseases. Pharmacological therapy is used to manage and treat the symptoms of acute and chronic inflammation, but research into alternative and complementary medicine (CAM) may show added benefits without adverse side effects. The use of antioxidants has been shown to be beneficial in stimulating immune cells to increase phagocytosis and up-regulating the cellular processes to limit inflammation. In this study, we investigated the effects of 2 potent plant-derived antioxidants, epigallocatechin-3-gallate (EGCG) and thymoquinone (TQ) on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The goals of this study were to examine the role of these antioxidants in a model of inflammation and to understand the mechanisms of action of these substances.;It was determined that varying concentrations (0.01microg, 0.1microg, 1.0microg) of LPS affect macrophage function and viability for 2, 24 and 48 hours. As the concentration increased, cell numbers and glutathione (GSH) levels decreased and malondialdehyde (MDA) and nitric oxide (NO) increased. The exact concentration of LPS needed to elicit an inflammatory response is questionable, but in this study, we determined that amount to be 0.01 microg/mL. When 0.01microg/mL was delivered to macrophages continuously for a prolonged period of time (72, 96 and 120 hours), macrophages continued to display a robust inflammatory response as indicated by increases in IL-1beta, IL-6, and TNF-alpha.;The effects of EGCG and TQ individually on macrophages may be dose dependent. Low concentrations of EGCG (≤10muM) and TQ (≤10microM) caused a decrease in cellular membrane damage and NO, but high concentrations of EGCG (≤50microM) and TQ (≤50microM) began to display oxidant qualities. EGCG (10microM) and TQ (microM) were also added to macrophages before or after stimulation with LPS to determine if antioxidants could suppress the effects of LPS. Western Blot analysis demonstrated that EGCG and TQ partially inhibited NF-kappaB translocation into the nucleus when given before or after LPS stimulation. Pre-treatment with EGCG and TQ individually showed the most inhibition in comparison to cells treated with EGCG and/or TQ after LPS-stimulation. This suggests that EGCG alone may be more effective in comparison to TQ with respect to decreasing NF-kappaB translocation.;To investigate the effects of sustained delivery, tri-calcium phosphate (TCP) capsules were loaded with EGCG and/or TQ before being added to macrophages stimulated daily (72, 96 and 120 hours) with LPS. Cell numbers showed an overall decline, but by 120 hours numbers began to climb to levels similar to the control. Groups treated with EGCG had the most membrane damage, but the damage did decline over time indicating that there may have been some protection occurring. NO levels were elevated in all treatment groups, which was probably a result of exposure to LPS. The TQ treated groups had the least NO, whereas EGCG had the highest levels. Sustained delivery of antioxidants did not completely abolish the effects of prolonged LPS stimulation. Although NO levels returned to normal by 120 hours, but cytokine levels remained variable.;In conclusion, this study demonstrated that EGCG and TQ may be able to reduce inflammation in the presence of a stimulus such as LPS. Although EGCG enters the cell differently than TQ, the mechanisms of action may be similar once they have reached the cytosol. This study demonstrated that EGCG and TQ may decrease NF-kappaB translocation, possibly by blocking activation of a MAPK pathway. This study also indicated that sustained delivery of antioxidants may not completely reduce the effects of continuous LPS stimulation. The continuous stimulation of LPS may be too overwhelming for the macrophages to recover. Further studies are suggested to understand these mechanisms. Overall, EGCG and TQ may be useful in the prevention and treatment of inflammation. |
