Rosmanol and Carnosol Synergistically Alleviate Rheumatoid Arthritis through Inhibiting TLR4/NF-κB/MAPK Pathway

Abstract

Callicarpalongissima has been used as a Yao folk medicine to treat arthritis for years in China, although its active anti-arthritic moieties have not been clarified so far. In this study, two natural phenolic diterpenoids with anti-rheumatoid arthritis (RA) effects, rosmanol and carnosol, isolated from the medicinal plant were reported on for the first time. In type II collagen-induced arthritis DBA/1 mice, both rosmanol (40 mg/kg/d) and carnosol (40 mg/kg/d) alone alleviated the RA symptoms, such as swelling, redness, and synovitis; decreased the arthritis index score; and downregulated the serum pro-inflammatory cytokine levels of interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), and tumor necrosis factor α (TNF-α). Additionally, they blocked the activation of the Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB)/c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways. Of particular interest was that when they were used in combination (20 mg/kg/d each), the anti-RA effect and inhibitory activity on the TLR4/NF-κB/MAPK pathway were significantly enhanced. The results demonstrated that rosmanol and carnosol synergistically alleviated RA by inhibiting inflammation through regulating the TLR4/NF-κB/MAPK pathway, meaning they have the potential to be developed into novel, safe natural combinations for the treatment of RA.

Keywords: Callicarpa longissima; TLR4/NF-κB/MAPK; carnosol; rheumatoid arthritis; rosmanol; synergistic effect.

Figure 1

Chemical structures of rosmanol (a) and carnosol (b).

Figure 2

Rosmanol, carnosol, and their combination alleviated RA severity in CIA mice. Schematic of establishment of CIA DBA/1 mouse model and the treatments of rosmanol (R), carnosol (C), and their combination (R + C) in the CIA mice (a). Representative pictures from the right hind limbs of the normal group (b), model group (c), rosmanol-treated group (d), carnosol-treated group (e), and combination-treated group (f) on day 42.

Figure 3

Effects of rosmanol (R), carnosol (C), and their combination (R + C) on arthritis index scores in CIA mice. Arthritis index scores were the cumulative scores for all four paws, which represented the overall disease severity. The arthritis index score for each group is presented as the mean ± SD (n = 6). The significance of differences was analyzed by unpaired t-test. Note: * p < 0.05; ** p < 0.01.

Figure 4

Rosmanol (R), carnosol (C), and their combination (R + C) alleviated synovitis in CIA mice. Representative H&E staining results from synovial tissue samples of the normal group (a), model group (b), rosmanol-treated group (c), carnosol-treated group (d), and combination-treated group (e) mice are shown (200×, n = 3).

Figure 5

The effects of rosmanol (R), carnosol (C), and their combination (R + C) on the production of TNF-α, MCP-1, and IL-6. The serum levels of TNF-α, MCP-1, and IL-6 of each group were determined by ELISA on day 42. Data are presented as the means ± SD (n = 6). The significance of differences was analyzed by unpaired t-test. Note: * p < 0.05; ** p < 0.01.

Figure 6

Rosmanol (R), carnosol (C), and their combination (R + C) inhibited the TLR4/NF-κB pathway. The relative transcription levels of TLR4 (a), MyD88 (b), and NF-κB p65 (c) in synovial tissue samples of each group were quantified by real-time PCR on day 42. The representative Western blot analysis results and protein relative expression levels for TLR4 (d), MyD88 (e), and NF-κB p-p65 (f) in synovial tissue samples of each group on day 42 are shown. GAPDH was used as an internal reference to quantify the expression levels of TLR4 and MyD88. The phosphorylation of p65 was expressed as p-p65/p65. Data are presented as the mean ± SD (n = 3). The significance of differences was analyzed by unpaired t-test. Note: *, p < 0.05; **, p < 0.01.

Figure 7

Rosmanol (R), carnosol (C), and their combination (R + C) inhibited the JNK and p38 MAPK pathways. The protein expression levels of JNK, p-JNK, p38, and p-p38 in synovial tissue samples of each group were examined by Western blot on day 42 (a) and the relative phosphorylation rates of JNK (b) and p38 (c) were calculated. GAPDH was used as the internal reference to quantify the expression levels of JNK, p-JNK, p38, and p-p38. The phosphorylation rates of JNK and p38 were expressed as p-JNK/JNK and p-p38/p38, respectively. Data are presented as the means ± SD (n = 3). The significance of differences was analyzed by unpaired t-test. Note: * p < 0.05; ** p < 0.01.

Figure 8

The signaling pathways that rosmanol (R), carnosol (C), and their combination (R + C) target in RA. The intensity of inhibition is proportional to the line width of the inhibition symbol.