Effect of vitamin D3 and β-sitosterol on immune function of macrophages
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Introduction. Besides calcium homeostasis, Vitamin D has other functions in human body, including its roles in immune function, cell proliferation, cell differentiation, and apoptosis. Consumption of natural food sources alone may not be enough to meet the daily dietary requirements. Some people like those with lactose intolerance, vegetarians, breast feeding mothers, or the elderly have to resort to vitamin D dietary supplements to meet daily requirements. Excessive intake of vitamin D supplements can however, increase serum calcium levels resulting in kidney stones, heart diseases or impaired mental status. Currently, efforts are focused on finding a safe alternative molecule that can augment the effects of vitamin D and avoid any complications associated with toxicity. One such molecule tested here is the phytosterol. Eating a healthy diet with lots of vegetables and dried nuts should provide sufficient amount of phytosterols in the diet. Also phytosterols are known to have no side-effects in excess amounts. Hence, it would be of tremendous interest to see if phytosterols would act synergistically with vitamin D and enhance its effects in lower doses. This study is an effort to understand the effect of phytosterols in conjunction with vitamin D with regards to its role in immune function. Objective. Inflammation is a major component of many disease conditions. Previous studies have shown individual treatments with vitamin D3 (VD3) and B-Sitosterol (SIT) to be immuno-modulatory. The present study was designed to investigate if the combined treatment with VD3 and SIT might offer better protection against inflammation compared with their individual treatments. Methods. J774A.1 macrophages were grown to confluency and stimulated with LPS. The stimulated cells were then incubated with SIT (8uM) and VD (80nM), individually, and in combination. The control was LPS-treated cells with no added treatment agents After 24 hours of incubation, macrophage proliferation was assessed using cell proliferation assay. Griess assay was done to detect nitric oxide (NO) released from the macrophage. Released cytokines (TNF-α, IL-6 and IL-10) and MCP-1 were measured using ELISA kits. Fast Activated Cell-based ELISA (FACE) kit was used to assess NF-κB phosphorylation. Results. SIT (8μM) was found to reduce cell proliferation by 62% while VD at physiological levels was found to be not effective. In combination, SIT and VD reduced cell proliferation by 75%. The amount of NO released, as influenced by 8μM SIT nor 80nM VD3 treatments, was not significantly different from control. Combining SIT and VD3, resulted in a 220% more increase in NO release compared to control. The SIT + VD3 treatment brought about significant increase in all the cytokine release, regardless of whether they were pro- or anti-inflammatory. Conclusion. Using physiological levels of VD3 and SIT, we have demonstrated that SIT, the main phytosterol in our diet, augments the action of VD3 on the immune function of macrophages. Although a lot more work is necessary to understand the underlying mechanism, we could conclude from the present study that SIT boosts the immune function of VD3. This ability of SIT to enhance the vitamin D activity could be beneficial to lot of people with vitamin D deficiency or with autoimmune diseases like multiple sclerosis.