Table 1.
Nucleotide sequences of primers used in real-time qRT-PCR.
Fig 1.
The molecular structure of ursodeoxycholic acid (UDCA) and the effect on the cell viability of RAW 264.7 macrophages.
(A) The molecular structure of UDCA. The macrophages were treated with various concentrations of UDCA (0, 0.5, 1, 2, or 5 mM) for 24 h. (B) Cell viability was measured using a CCK-8 assay and (C) a live/dead staining kit. Results are mean ± SD of triplicate experiments: **p < 0.01, significant difference as compared to the control and to each other.
Fig 2.
Nitric oxide (NO) secretion in RAW 264.7 macrophages treated with various concentrations of UDCA, lipopolysaccharide (LPS) or LPS containing various concentrations of UDCA.
(A) The macrophages were treated with 0.5, 1, or 2 mM UDCA for 1, 6, or 24 h. (B) The macrophages of LPS alone group were treated with 1 μg/mL LPS for 1, 6, or 24 h. The macrophages of the LPS containing UDCA groups were pre-treated with 0.5, 1, or 2 mM UDCA for 1 h. Then treated with LPS (1 μg/mL) containing equal concentration of UDCA for an additional 1, 6, or 24 h. The collected supernatants were reacted with Griess reagent, and the absorbance level was measured at 548 nm. Results are mean ± SD of triplicate experiments: *p < 0.05 and **p < 0.01, significant difference as compared to the control and to each other at the time point of 1, 6, or 24 h.
Fig 3.
The mRNA expression in RAW 264.7 macrophages treated with UDCA (1 mM), LPS (1 μg/mL), or LPS (1 μg/mL) containing 1 mM UDCA for 1, 6, or 24 h.
The macrophages in the UDCA with or without LPS group were pretreated with 1 mM UDCA for 1 h before the zero time point. Then, the UDCA alone group treated with 1 mM UDCA for an additional 1, 6, or 24 h and UDCA containing LPS group treated with LPS (1 μg/mL) containing 1 mM UDCA for an additional 1, 6, or 24 h. The macrophages of LPS alone group were treated with 1 μg/mL LPS for 1, 6, or 24 h. Cell pellets were subjected to a qRT-PCR analysis to detect the expression levels of (A) TNF-α, (B) IL-1α, (C) IL-1β (D) IL-6, and (E) IL-10 mRNA. The levels of each mRNA expression were normalized to the expression of GAPDH mRNA. The fold ratio of the 1 h control group was set at 1-fold and fold change was relatively calculated. Results are mean ± SD of triplicate experiments: *p < 0.05 and **p < 0.01, significant difference as compared to the control and to each other at the time point of 1, 6, or 24 h.
Fig 4.
The protein expression in RAW 264.7 macrophages treated with UDCA (1 mM), LPS (1 μg/mL), or LPS (1 μg/mL) containing 1 mM UDCA for 1, 6, or 24 h.
The macrophages in the UDCA with or without LPS group were pretreated with 1 mM UDCA for 1 h before the zero time point. Then, the UDCA alone group treated with 1 mM UDCA for an additional 1, 6, or 24 h and UDCA containing LPS group treated with LPS (1 μg/mL) containing 1 mM UDCA for an additional 1, 6, or 24 h. The macrophages of LPS alone group were treated with 1 μg/mL LPS for 1, 6, or 24 h. The collected supernatants were subjected to the plate of ELISA kit to detect the protein of (A) TNF-α, (B) IL-1α, (C) IL-1β (D) IL-6, and (E) IL-10. Results are mean ± SD of triplicate experiments: *p < 0.05 and **p < 0.01, significant difference as compared to the control and to each other at the time point of 1, 6, or 24 h.
Fig 5.
Effect of UDCA on the phosphorylation of ERK, JNK, p38 and IκBα in LPS-stimulated RAW 264.7 macrophages.
The macrophages in the UDCA with or without LPS group were pretreated with 1 mM UDCA for 1 h before the zero time point. Then, the UDCA alone group treated with 1 mM UDCA for an additional 24 h and UDCA containing LPS group treated with LPS (1 μg/mL) containing 1 mM UDCA for an additional 24 h. The macrophages of LPS alone group were treated with 1 μg/mL LPS for 24 h. Immunoblotting was used to detect the phosphorylation form or the total form of ERK, JNK, p38, and IκBα in lysates prepared from the macrophages. β-actin was used as an internal control. The phosphorylation/total form (p/t form) volumes were calculated. The p/t form volume at control group was set at 1-fold and the ratio of the normalized fold change was relatively calculated and quantified. Results are mean ± SD of triplicate experiments: *p < 0.05 and **p < 0.01, significant difference as compared to the control and to each other for 24 h.