Fermentation by Lactobacillus enhances anti-inflammatory effect of Oyaksungisan on LPS-stimulated RAW 264.7 mouse macrophage cells

Abstract

Background: Oyaksungisan (OY) has been used as a traditional drug in east-Asian countries. However, its effect on inflammation still remains unknown. In this study, to provide insight into the biological effects of OY and OY fermented by Lactobacillus, we investigated their effects on lipopolysaccharide (LPS)-mediated inflammation in the RAW 264.7 murine macrophage cells.

Methods: The investigation was focused on whether OY and fermented OYs could inhibit the production of pro-inflammatory mediators such as nitric oxide (NO) and prostaglandin (PG) E2 as well as the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, interleukin (IL)-6, nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) in LPS-stimulated RAW 264.7 cells.

Results: We found that OY inhibits a little LPS-induced NO, PGE2, TNF-α and IL-6 productions as well as the expressions of iNOS and COX-2. Interestingly, the fermentation significantly increased its inhibitory effect on the expression of all pro-inflammatory mediators. Furthermore, the fermented OYs exhibited elevated inhibition on the translocation of NF-κB p65 through reduced IκBα degradation as well as the phosphorylations of extracellular signal-regulated kinase (ERK), p38 and c-Jun NH2-terminal kinase (JNK) MAPKs than untreated control or original OY.

Conclusions: Finally, the fermentation by Lactobacillus potentiates the anti-inflammatory effect of OY by inhibiting NF-κB and MAPK activity in the macrophage cells.

Figure 1

Effect of OY and fermented OYs on LPS-induced (A) NO and (B) PGE₂ production. RAW 264.7 cells pretreated with the indicated each concentration of OY or fermented OYs for 30 min before being incubated with LPS (200 ng/mL) for 24 hours. The culture supernatant was analyzed for nitrite production. The data are mean ± SE values of duplicate determinations from three separate experiments. *p < 0.05 and **p < 0.005, compared with LPS-stimulated values.

Figure 2

Effect of OY and fermented OYs on LPS-induced (A) TNF-α cytokine and (B) mRNA expression. RAW 264.7 cells pretreated with the indicated concentrations of OY or fermented OYs for 30 min before being incubated with LPS (200 ng/mL) for (A) 24 hours and (B) 6 hours, respectively. The level of TNF-α was measured by ELISA and the mRNA level was assessed by RT-PCR. (A) The data are mean ± SE values of duplicate determinations from three independent experiments. *p < 0.05 and **p < 0.005, were calculated from comparing with LPS-stimulated values. (B) The values in the LPS-stimulated cells without OY treatment was set to 1.0 and the differences by treatment were quantitated. The experiments were repeated at least three times, and similar results were obtained.

Figure 3

Effect of OY and fermented OYs on LPS-induced (A) IL-6 cytokine and (B) mRNA expression. RAW 264.7 cells pretreated with the indicated each concentrations of OY or fermented OYs for 30 min were stimulated with LPS (200 ng/mL) for (A) 24 hours and (B) 6 hours, respectively. The cytokine and mRNA levels were evaluated by same methods as described in Figure 2.

Figure 4

Effect of OY and fermented OYs on LPS-induced (A) COX-2 and (B) iNOS expression. RAW 264.7 cells pretreated with OY or fermented OYs (500 μg/mL) for 30 min before being incubated with LPS (200 ng/mL) for 24 hours. Equal amounts of protein were used for immunoblot analysis for detection of COX-2 and iNOS. RT-PCR was used for COX-2 and iNOS mRNAs analysis.

Figure 5

Effect of OY and fermented OYs on LPS-induced (A) NF-κB translocation and (B) IκBα degradation. The cells treated with LPS (200 ng/mL) alone or with LPS and OY and fermented OYs (500 μg/mL) for 30 min (IκBα) or for 1 hour (NF-κB). The levels of IκBα and NF-κB (p65) in the nuclear and cytosol extracts were determined by Western blot analysis.

Figure 6

Effect of OY and fermented OYs on LPS-induced the phosphorylation of MAPKs. RAW 264.7 cells treated with OY or fermented OYs (500 μg/mL) for 30 min were activated with LPS (200 ng/mL) for 30 min. Whole-cell lysates were prepared for detection of MAP kinases in Western blot analysis.

Figure 7

The HPLC analysis chromatograms of OY, OY-A and OY-B. (A) 280 nm and (B) 335 nm; Increased peaks (1-5) were not identified; 1. t_R 4.11 min; 2. t_R 6.17 min; 3. t_R 9.13 min; 4. t_R 11.50 min; 5. t_R 12.03 min.

Figure 8

Chemical structures of five marker constituents in OY, OY-A and OY-B.

Figure 9

Assessment of OY and fermented OYs cytotoxicity in RAW 264.7 cells. The MTT assay was performed after incubation of RAW 264.7 cells treated with different doses (10, 100, 500 and 1000 μg/mL) of OY or fermented OYs for 48 h. Data are mean ± SE value of duplicate determinations from three separate experiments.