A herbal formula comprising Rosae Multiflorae Fructus and Lonicerae Japonicae Flos, attenuates collagen-induced arthritis and inhibits TLR4 signalling in rats

Abstract

RL, a traditional remedy for Rheumatoid arthritis (RA), comprises two edible herbs, Rosae Multiflorae Fructus and Lonicerae Japonicae Flos. We have reported that RL could inhibit the production of inflammatory mediators in immune cells. Here we investigated the effects and the mechanism of action of RL in collagen-induced arthritis (CIA) rats. RL significantly increased food intake and weight gain of CIA rats without any observable adverse effect; ameliorated joint erythema and swelling; inhibited immune cell infiltration, bone erosion and osteophyte formation in joints; reduced joint protein expression levels of TLR4, phospho-TAK1, phospho-NF-κB p65, phospho-c-Jun and phospho-IRF3; lowered levels of inflammatory factors (TNF-α, IL-6, IL-1β, IL-17A and MCP-1 in sera and TNF-α, IL-6, IL-1β and IL-17A in joints); elevated serum IL-10 level; reinvigorated activities of antioxidant SOD, CAT and GSH-Px in the liver and serum; reduced Th17 cell proportions in splenocytes; inhibited splenocyte proliferation and activation; and lowered serum IgG level. In conclusion, RL at nontoxic doses inhibited TLR4 signaling and potently improved clinical conditions of CIA rats. These findings provide further pharmacological justifications for the traditional use of RL in RA management.

Figure 1. Effects of RL on disease progression in CIA rats.

Male Wistar rats were immunized on day 0 and day 7 with bovine type II collagen for CIA or with vehicle. CIA rats were intragastrically (i.g.) given vehicle (saline), indomethacin (Indo), low dose of RL (RL-L), middle dose of RL (RL-M) or high dose of RL (RL-H) (n = 8 for each group) daily from days 14 to 56. (A) Average volumes of hind paws. (B) Mean arthritic score. (C) Representative photographs showing the gross features of hind paws at day 57. (D) Average daily food intake during the experimental period. (E) Changes in body weight over the experimental period. Values are the mean ± SEM (n = 8). *P < 0.05, **P < 0.01 compared with the immunized control (saline).

Figure 2. Effects of RL on the radiographs of CIA rats.

(A) Representative radiographs of the hind limbs showing the talocrural joints at day 57. (B) Mean radiological score of the radiographs. Values are the mean ± SEM (n = 8). *P < 0.05, **P < 0.01 compared with the immunized control (saline).

Figure 3. Effects of RL on histological parameters in the talocrural joints of CIA rats.

(A) Representative histological observation from light microscope of the talocrural joint sections stained with H&E for inflammatory cell influx and bone destruction (magnification ×100). (B) Mean score of the histological observation. Values are the mean ± SEM (n = 8). *P < 0.05, **P < 0.01 compared with the immunized control (saline).

Figure 4. Effects of RL on TLR4 signalling in joint tissues of CIA rats.

(A) Representative western blots showing the protein expression of TLR4, phospho-TAK1, phospho-NF-κB p65, phospho-c-Jun, phospho-IRF3 and β-actin (as loading control) in joint tissues. (B) Relative intensity values from band densitometry. The values are the mean ± SEM of the band density normalized to the loading control in relation to mean value of untreated control (control) normalized to the loading control (n = 8). (C) Levels of cytokines from joint tissue lysates determined by Milliplex MAP Rat Cytokine/Chemokine Panel or ELISA at the end of the experiment. Values are the mean ± SEM (n = 8). *P < 0.05, **P < 0.01 compared with the immunized control (saline).

Figure 5. Effects of RL on serum levels of cytokines and chemokines in CIA rats.

Serum levels of cytokines and chemokines were determined by Milliplex MAP Rat Cytokine/Chemokine Panel or ELISA at different time points. (A) Changes in serum TNF-α levels. (B) Changes in serum IL-1β levels. (C) Changes in serum IL-10 levels. (D) Changes in serum IL-6 levels. (E) Changes in serum MCP-1 levels. Values are the mean ± SEM (n = 8). *P < 0.05, **P < 0.01 compared with the immunized control (saline).

Figure 6. Effects of RL on the activities of antioxidant enzymes in CIA.

(A) Antioxidant enzyme (SOD, CAT, and GSH-Px) activities in sera collected on day 49 after first immunization. (B) Antioxidant enzyme (SOD, CAT, and GSH-Px) activities in homogenized liver tissues at the end of the experiment. Values are the mean ± SEM (n = 8). *P < 0.05, **P < 0.01 compared with the immunized control (saline).

Figure 7. Effects of RL on IL-17 and Th17 cells in CIA rats.

(A) Levels of IL-17A from joint tissue lysates determined by Milliplex MAP Rat Cytokine/Chemokine Panel at the end of the experiment. (B) Serum levels of IL-17 determined by Milliplex MAP Rat Cytokine/Chemokine Panel at different time points. (C) Isolated splenocytes were stimulated with PMA (0.05 μg/ml) and ionomycin (1 μg/ml) for 4 h and then BFA (5 μg/ml) for additional 2 h. Cells were extracellularly stained with FITC-conjugated anti-CD4, fixed, permeabilized and labelled with PE-conjugated anti-IL-17A. Representative graphs showing the percentages of positive-stained Th17 in CD4⁺ cells analyzed by flow cytometry. (D) The mean percentages of positive-stained Th17 in CD4⁺ cells. (E) Concentration of IL-17A determined by ELISA in the supernatants of isolated splenocytes stimulated with PMA (0.05 μg/ml) and ionomycin (1 μg/ml). Values are the mean ± SEM (n = 8). *P < 0.05, **P < 0.01 compared with the immunized control (saline).

Figure 8. Effects of RL on immune responses in CIA rats.

(A) Splenocytes isolated from rats were stimulated with CII (50 μg/ml) and cultured for 72 h. Cell proliferation was measured by the MTT assay and expressed as mean percentage of control cells. (B) Antibody concentrations in the sera were measured by ELISA. Values are the mean ± SEM (n = 8). *P < 0.05, **P < 0.01 compared with the immunized control (saline).

Figure 9. A schematic diagram showing the effects of RL in CIA rats.