Immunostimulatory Activity of Opuntia ficus-indica var. Saboten Cladodes Fermented by Lactobacillus plantarum and Bacillus subtilis in RAW 264.7 Macrophages

To increase the functionality of Opuntia ficus-indica var. saboten cladodes, it was fermented by Lactobacillus plantarum and Bacillus subtilis. Eighty percent methanol extracts were investigated for their effects on nitric oxide (NO) production, cytokine secretion, nuclear factor-kappa B (NF-kappa B) activity, and mitogen-activated protein kinase (MAPK) phosphorylation in RAW 264.7 cells. Methanol extracts of L. plantarum culture medium (LPCME) and B. subtilis culture medium (BSCME) did not affect lipopolysaccharide (LPS)-induced NO production but, at 500 mu g/mL, increased interferon (IFN)-gamma-induced NO production by 55.2 and 66.5 mu M, respectively, in RAW 264.7 cells. In RAW 264.7 cells not treated with LPS and IFN-gamma, LPCME did not affect NO production, but BSCME increased NO production significantly in a dose-dependent manner. In addition, BSCME induced the expression of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) in RAW 264.7 cells in a dose-dependent manner. BSCME at 500 mu g/mL increased TNF-alpha and IL-1 beta mRNA levels by 83.8% and 82.2%, respectively. BSCME increased NF-jB-dependent luciferase activity in a dose-dependent manner; 500 mu g/mL BSCME increased activity 9.1-fold compared with the control. BSCME induced the phosphorylation of p38, c-JUN NH2-terminal protein kinase (JNK), and extracellular signal-regulated kinase (ERK) in a dose-dependent manner, but did not affect total ERK levels. In conclusion, BSCME exerted immunostimulatory effects, which were mediated by MAPK phosphorylation and NF-kappa B activation, resulting in increased TNF-alpha and IL-1 beta gene expression in RAW 264.7 macrophages. Therefore, BSCM shows promise for use as an immunostimulatory therapeutic.