Objective:To investigate the efficacy of Saussurea involucrata injection (SII) in alleviating rheumatoid arthritis (RA) and to explore the mechanism of action of SII in alleviating RA through the Toll-like receptor 4 (TLR4)/nuclear factor-kappaBp65 (NF-κBp65) pathway-mediated M1 macrophage polarization.Methods:In vivo experiments were conducted using a collagen-induced arthritis (CIA) rat model. After successful modeling, the CIA rats were randomly assigned into five groups ( n=10 per group): CIA control group, MTX group, low-dose SII (L-SII) group, medium-dose SII (M-SII) group, and high-dose SII (H-SII) group. The efficacy of SII in alleviating RA was evaluated using arthritis index scores, histopathology, and ELISA to measure serum levels of nitric oxide (NO), interleukin 1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α). Subsequently, Western blot analysis was used to detect the expression of inducible nitric oxide synthase (iNOS) and CD86 proteins in synovial tissue. In vitro experiments involved first isolating and inducing rat bone marrow-derived macrophages (BMDMs). Then, BMDMs were polarized toward the M1 phenotype using lipopolysaccharide (LPS) combined with interferon-γ (IFN-γ). Concurrently, cells were treated with SII and the TLR4 inhibitor TAK242. Subsequently, ELISA was used to detect NO, IL-1β, TNF-α levels in the cell culture supernatant via ELISA. RT-qPCR was used to detect the expression of IL-1b, IL-6, and TNF-α genes in each group of cells. Western blot analysis was performed to detect the expression of iNOS, CD86, TLR4, myeloid differentiation primary response 88 (MyD88), and p-NF-κBp65/NF-κBp65 proteins in the cells. Data analysis between multiple groups was performed using one-way analysis of variance, and between pairs using LSD- t-tests. Results:In vivo experimental results showed that compared with the CIA group(7.90 ± 0.70), MTX and SII both improved the pathological symptoms of rats and reduced the ankle joint pathological score [MTX (4.40 ± 0.92), L-SII (7.00 ± 0.89), M-SII (5.10 ± 1.30), H-SII (4.90 ± 0.94), t=33.86, P<0.001; t=9.10, P<0.001; t=2.38, P=0.029; t=5.69, P<0.001; t=7.66, P<0.001], while downregulating serum levels of NO, IL-1β, IL-6, and TNF-α levels in serum, as well as iNOS [ t=30.01, P<0.001; t=6.17, P=0.003; t=10.86, P<0.001; t=28.95, P<0.001; t=19.03, P<0.001] and CD86 [ t=65.61, P<0.001; t=8.76, P<0.001; t=13.18, P<0.001; t=13.22, P<0.001; t=18.91, P<0.001] expression. In vitro experimental results showed that compared with BMDMs treated with LPS and IFN-γ, SII and TAK242 treatment reduced the levels of NO, IL-1β, IL-6, and TNF-α in the supernatant and decreased the expression of IL-1b, IL-6, and TNF-α genes. Additionally, SII and TAK242 treatment downregulated the expression of iNOS and CD86 proteins in cells, and simultaneously downregulated TLR4, MYD88, and p-NF-κBp65/NF-κBp65 expression ( t=35.84, P<0.001; t=15.69, P<0.001; t=21.99, P<0.001; t=23.64, P<0.001; t=22.50, P<0.001). Additionally, compared with the TAK242 group alone, TAK242 + H-SII showed no significant differences in the modulation of M1 macrophage polarization and TLR4/NF-κBp65 pathway-related indicators. Conclusion:SII exerts anti-inflammatory and anti-RA effects by inhibiting TLR4/NF-κBp65-mediated M1 macrophage polarization.

Objective:To investigate the efficacy of Saussurea involucrata injection (SII) in alleviating rheumatoid arthritis (RA) and to explore the mechanism of action of SII in alleviating RA through the Toll-like receptor 4 (TLR4)/nuclear factor-kappaBp65 (NF-κBp65) pathway-mediated M1 macrophage polarization.Methods:In vivo experiments were conducted using a collagen-induced arthritis (CIA) rat model. After successful modeling, the CIA rats were randomly assigned into five groups ( n=10 per group): CIA control group, MTX group, low-dose SII (L-SII) group, medium-dose SII (M-SII) group, and high-dose SII (H-SII) group. The efficacy of SII in alleviating RA was evaluated using arthritis index scores, histopathology, and ELISA to measure serum levels of nitric oxide (NO), interleukin 1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α). Subsequently, Western blot analysis was used to detect the expression of inducible nitric oxide synthase (iNOS) and CD86 proteins in synovial tissue. In vitro experiments involved first isolating and inducing rat bone marrow-derived macrophages (BMDMs). Then, BMDMs were polarized toward the M1 phenotype using lipopolysaccharide (LPS) combined with interferon-γ (IFN-γ). Concurrently, cells were treated with SII and the TLR4 inhibitor TAK242. Subsequently, ELISA was used to detect NO, IL-1β, TNF-α levels in the cell culture supernatant via ELISA. RT-qPCR was used to detect the expression of IL-1b, IL-6, and TNF-α genes in each group of cells. Western blot analysis was performed to detect the expression of iNOS, CD86, TLR4, myeloid differentiation primary response 88 (MyD88), and p-NF-κBp65/NF-κBp65 proteins in the cells. Data analysis between multiple groups was performed using one-way analysis of variance, and between pairs using LSD- t-tests. Results:In vivo experimental results showed that compared with the CIA group(7.90 ± 0.70), MTX and SII both improved the pathological symptoms of rats and reduced the ankle joint pathological score [MTX (4.40 ± 0.92), L-SII (7.00 ± 0.89), M-SII (5.10 ± 1.30), H-SII (4.90 ± 0.94), t=33.86, P<0.001; t=9.10, P<0.001; t=2.38, P=0.029; t=5.69, P<0.001; t=7.66, P<0.001], while downregulating serum levels of NO, IL-1β, IL-6, and TNF-α levels in serum, as well as iNOS [ t=30.01, P<0.001; t=6.17, P=0.003; t=10.86, P<0.001; t=28.95, P<0.001; t=19.03, P<0.001] and CD86 [ t=65.61, P<0.001; t=8.76, P<0.001; t=13.18, P<0.001; t=13.22, P<0.001; t=18.91, P<0.001] expression. In vitro experimental results showed that compared with BMDMs treated with LPS and IFN-γ, SII and TAK242 treatment reduced the levels of NO, IL-1β, IL-6, and TNF-α in the supernatant and decreased the expression of IL-1b, IL-6, and TNF-α genes. Additionally, SII and TAK242 treatment downregulated the expression of iNOS and CD86 proteins in cells, and simultaneously downregulated TLR4, MYD88, and p-NF-κBp65/NF-κBp65 expression ( t=35.84, P<0.001; t=15.69, P<0.001; t=21.99, P<0.001; t=23.64, P<0.001; t=22.50, P<0.001). Additionally, compared with the TAK242 group alone, TAK242 + H-SII showed no significant differences in the modulation of M1 macrophage polarization and TLR4/NF-κBp65 pathway-related indicators. Conclusion:SII exerts anti-inflammatory and anti-RA effects by inhibiting TLR4/NF-κBp65-mediated M1 macrophage polarization.