Thymol mitigates lipopolysaccharide-induced endometritis by regulating the TLR4- and ROS-mediated NF-κB signaling pathways

Abstract

The purpose of this study was to investigate the effects of thymol on lipopolysaccharide (LPS)-induced inflammatory responses and to clarify the potential mechanism of these effects. LPS-induced mouse endometritis was used to confirm the anti-inflammatory action of thymol in vivo. RAW264.7 cells were used to examine the molecular mechanism and targets of thymol in vitro. In vivo, thymol markedly alleviated LPS-induced pathological injury, myeloperoxidase (MPO) activity, and the production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in mice. Further studies were performed to examine the expression of the Toll-like receptor 4 (TLR4) -mediated nuclear factor-κB (NF-κB) pathway. These results showed that the expression of the TLR4-mediated NF-κB pathway was inhibited by thymol treatment. In vitro, we observed that thymol dose-dependently inhibited the expression of TNF-α, IL-1β, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW264.7 cells. Moreover, the results obtained from immunofluorescence assays also indicated that thymol dose-dependently suppressed LPS-induced reactive oxygen species (ROS) production. Silencing of TLR4 inhibited NF-κB pathway activation. Furthermore, H2O2 treatment increased the phosphorylation of p65 and IκBα, which were decreased when treated with N-acetyl cysteine or thymol. In conclusion, the anti-inflammatory effects of thymol are associated with activation of the TLR4 or ROS signaling pathways, contributing to NF-κB activation, thereby alleviating LPS-induced oxidative and inflammatory responses.

Keywords: TLR4; inflammation; nuclear factor-κB; reactive oxygen species; thymol.

Figure 1. A. Chemical structure of thymol, B. HPLC chromatogram of thymol

Figure 2. Effects of thymol on LPS-stimulated uterine injury

A. Morphology of the uterus. B. LPS group, C, D, E. LPS + Thymol (10, 20, and 40 mg/kg, respectively) treatment groups, F. Control group. CG indicates the control group. LPS indicates the LPS-stimulated group. The red arrow indicates the tissue lesion area. Data represent the mean ± S.E.M. of three independent experiments. ^#p<0.01 vs. Control group. *p<0.05 vs. LPS group. **p<0.01 vs. LPS group.

Figure 3. Effects of thymol on MPO activity and cytokine expression

A. MPO activity. B. Expression of TNF-α and IL-1β mRNA in tissues. GAPDH serves as the control. CG indicates the control group. LPS indicates the LPS-stimulated group. Data represent the mean ± S.E.M. of three independent experiments. ^#p<0.01 vs. Control group. *p<0.05 vs. LPS group, **p<0.01 vs. LPS group.

Figure 4. Effects of thymol on TLR4 expression and NF-κB pathway activation

A. TLR4 protein expression levels in uterine tissues. B. Expression of the p65 and IκBα proteins in uterine tissues. Phosphorylation of p65 and IκBα was analyzed using phospho-specific antibodies. β-actin served as an internal control. CG indicates the control group. LPS indicates the LPS-stimulated group. Data represent the mean ± S.E.M. of three independent experiments. ^#p<0.01 vs. Control group. *p<0.05 vs. LPS group, **p<0.01 vs. LPS group.

Figure 5. Effects of thymol on NF-κB p65 translocation into the nucleus

Paraffin-embedded uterine tissue sections were used to detect p65 translocation to the nucleus using immunofluorescence. A. Control group, B. LPS group, C, D, E. LPS + Thymol (10, 20, and 40 mg/kg, respectively) treatment groups. The white arrow indicates the translocation of p65. Data represent the mean ± S.E.M. of three independent experiments. ^#p<0.01 vs. Control group. *p<0.05 vs. LPS group, **p<0.01 vs. LPS group.

Figure 6. A. Effect of thymol on the cell viability of RAW264.7 cells

B. Effects of thymol on the expression of TNF-α and IL-1β in LPS-stimulated RAW264.7 cells. C. Expression of iNOS and COX-2 mRNA in LPS-stimulated RAW264.7 cells. GAPDH served as the control. CG indicates the control group. LPS indicates the LPS-stimulated group. Data represent the mean ± S.E.M. of three independent experiments. ^#p<0.01 vs. Control group. *p<0.05 vs. LPS group, **p<0.01 vs. LPS group.

Figure 7. Effect of thymol on ROS production in LPS-stimulated RAW264.7 cells

Microphotographs of ROS levels in LPS-stimulated RAW264.7 cells were obtained using fluorescence microscopy after DCF-DA staining (400× magnification, scale bar = 50 μm).

Figure 8. Effects of thymol on TLR4-mediated NF-κB pathway activation

A. Expression of TLR4 in LPS-stimulated RAW264.7 cells. B. Expression of IκBα and p65 protein in LPS-stimulated RAW264.7 cells. Phosphorylation of IκBα and p65 were analyzed using phospho-specific antibodies. β-actin served as an internal control. CG indicates the control group. LPS indicates the LPS-stimulated group. Data represent the mean ± S.E.M. of three independent experiments. ^#p<0.01 vs. Control group. *p<0.05 vs. LPS group, **p<0.01 vs. LPS group.

Figure 9. Analysis of TLR4-mediated NF-κB pathway expression in LPS-stimulated RAW264.7 cells

A. The interfering efficiency of TLR4 siRNA was estimated using qPCR. GAPDH served as a control. B. Phosphorylation levels of p65 and IκBα were examined by western blotting after silencing of TLR4 using siRNA or thymol treatment in LPS-stimulated RAW264.7 cells. CG indicates the control group. LPS indicates the LPS-stimulated group. TLR4-si indicates the TLR4 siRNA group. si-NC indicates the siRNA-negative control group. Data represent the mean ± S.E.M. of three independent experiments. ^#p<0.01 vs. Control group. *p<0.05 vs. LPS group, **p<0.01 vs. LPS group.

Figure 10. A. Effects of H₂O₂ on NF-κB activation after silencing of TLR4 in RAW264.7 cells

Phosphorylation levels of p65 and IκBα were determined by western blotting after knockdown of TLR4 using siRNA or thymol, NAC pre-treatment. β-actin serves as an internal control. B. Translocation of NF-κB p65 in TLR4-si RAW264.7 cells challenged with LPS as assessed using immunofluorescence. C. Expression of the cytokines, IL-1β and TNF-α, was detected using qPCR. GAPDH served as a control. CG indicates the control group. LPS indicates the LPS-stimulated group. TLR4-si indicates the TLR4 siRNA group. NAC indicates the N-acetyl cysteine group. The white arrow indicates the translocation of p65. Data represent the mean ± S.E.M. of three independent experiments. ^#p<0.01 vs. Control group. *p<0.05 vs. LPS group, **p<0.01 vs. LPS group.