Commiphora Extract Mixture Ameliorates Monosodium Iodoacetate-Induced Osteoarthritis

Abstract

Osteoarthritis (OA) is a chronic inflammatory joint disease that affects millions of elderly people around the world. The conventional treatments for OA consisting of nonsteroidal anti-inflammatory drugs and steroid have negative health consequences, such as gastrointestinal, renal, and cardiac diseases. This study has evaluated the Commiphora extract mixture (HT083) on OA progression as an alternative treatment in animal models. The root of P. lactiflora and the gum resin of C. myrrha have been in use as traditional medicines against many health problems including bone disorders since ancient time. The extracts of P. lactiflora root and C. myrrha gum resin were mixed as 3:1 for their optimal effects. Male Sprague-Dawley rats were injected with monosodium iodoacetate (MIA) into the knee joints to induce the symptoms identical to human OA. HT083 substantially prevented the loss of weight-bearing inflicted with MIA in rats. The MIA-induced cartilage erosion as well as the subchondral bone damage in the rats was also reversed. In addition, the increase of serum IL-1β concentration, a crucial pro-inflammatory cytokine involved in OA progression was countered by HT083. Furthermore, HT083 significantly reduced the acetic acid-induced writhing response in mice. In vitro, HT083 has shown potent anti-inflammatory activities by inhibiting the production of NO and suppressing the interleukin -1β, interleukin -6, cyclooxygenase-2, and inducible nitric oxide synthase expression in lipopolysaccharide -stimulated RAW 264.7 cells. Given its potent analgesic and anti-inflammatory activities in MIA rats and acetic acid-induced writhing in mice, HT083 should be further studied in order to explain its mechanism of actions in alleviating OA pain and inflammation.

Keywords: Commiphora myrrha; Paeonia lactiflora; analgesic; anti-inflammatory; osteoarthritis.

Figure 1

3-D HPLC chromatogram of Paeonia lactiflora and Commiphora myrrha extracts. Detection was performed by using a photodiode array detector. X-axis is retention time; Y-axis is wavelength, and Z-axis is absorbance unit. Analytical conditions were as follows: column, C18 (4.6 × 250, 5 µm); mobile phase, solvent A (0.1% phosphoric acid) and solvent B (acetonitrile); flow rate, 1 mL/min; program, 20–20%; 15–15.1 min, 20–70%; 15.1–20 min, 70–70%; 20–20.1 min, 70–20%; 20.1–25 min, 20–20% solvent B for P. lactiflora extract (A). Additionally, 0–60 min, 0–100%; 60–65 min, 100–100%; 65–67 min, 100–0%; 67–72 min, 0–0% solvent B for C. myrrha extract (B).

Figure 2

Effects of HT083 on the change of weight-bearing in rats injected with monoiodoacetate (MIA). (A) Distribution ratio of right and left hind-limb weight-bearing of the MIA rats after 24 days of MIA injection. The positive control (PC) was indomethacin (10 mg/kg). (B) AUC, area under the curve; evaluated the right and left hind-limb weight-bearing in MIA rats after 24 days of MIA injection. The values were expressed as the mean ± S.E.M. (n = 9). ### p < 0.001 vs sham, * p < 0.05, and *** p < 0.001 vs control.

Figure 3

Effects of HT083 on the cartilage and subchondral bone damage. (A) Macroscopic images of the articular surfaces of tibia and femur of the sham and MIA rats (B) Axial Micro-computed tomography (Micro-CT) image of a rat tibia, in the medial (M) and lateral (L) subchondral bone compartment (C) Micro-CT images of the cartilage surface of the sham and MIA rats. (D,E) Histomorphometry parameters obtained by micro-CT of subchondral medial and lateral cortical thickness (Cor.Th). Each value is the mean ± SEM. The number of animals was nine in each group; * p < 0.001 vs. control and ^### p < 0.001 vs sham (one-way ANOVA, Dunnett’s post hoc test).

Figure 3

Effects of HT083 on the cartilage and subchondral bone damage. (A) Macroscopic images of the articular surfaces of tibia and femur of the sham and MIA rats (B) Axial Micro-computed tomography (Micro-CT) image of a rat tibia, in the medial (M) and lateral (L) subchondral bone compartment (C) Micro-CT images of the cartilage surface of the sham and MIA rats. (D,E) Histomorphometry parameters obtained by micro-CT of subchondral medial and lateral cortical thickness (Cor.Th). Each value is the mean ± SEM. The number of animals was nine in each group; * p < 0.001 vs. control and ^### p < 0.001 vs sham (one-way ANOVA, Dunnett’s post hoc test).

Figure 4

The change of serum IL-1β concentration in MIA rats after HT083 treatment. Each value is the mean ± SEM. The number of animals is nine per group; * p < 0.05, * p < 0.01 and * p < 0.001 vs. control (one-way ANOVA, Dunnett’s post hoc test).

Figure 5

Effects of HT083 on acetic acid-induced writhing response in rat. The number of writhes was measured after 0.7% acetic acid injection for 10 min. Each value is the mean ± SEM. The number of animals is eight per group; ** p < 0.01 and *** p < 0.001 vs. control (one-way ANOVA, Dunnett’s post hoc test).

Figure 6

Effects of HT083 on cell viability (A) and NO production (B) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Cells were pretreated with 10, 30, 100, and 300 μg /mL of HT083. Each value is the mean ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control; ^### p < 0.001 vs. no treatment (NT) (one-way ANOVA, Dunnett’s post hoc test). MTT; thiazolyl blue tetrazolium bromide, NO; nitric oxide.

Figure 7

Quantitative Real Time-PCR (qRT-PCR) analysis of IL-1β, IL-6, COX-2, and iNOS mRNA expression in LPS-stimulated RAW264.7 cells. Cells were pretreated with positive control, Dexmethason 1 μg/mL and HT083 (30, 100, 300 μg/mL), and then incubated with LPS for 24 h. The cells were lysed, and total RNA was subjected to quantitative Real Time-PCR with the primers IL-1β, IL-6, COX-2 and iNOS (A–D). Each experiment was repeated three times. Each value is the mean ± SEM. The number of animals is nine per group; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control; ^### p < 0.001 no treatment (NT) (one-way ANOVA, Dunnett’s post hoc test).

Figure 8

Effects of HT083 on the protein expression of inflammatory cytokines in LPS-stimulated RAW264.7 cells. (A–C) Multiplex analysis of the expression of TNFα, IL-6, and IL-1β. Cells were stimulated with PBS, trophozoite lysates or LPS for 24 h. Supernatants were harvested and inflammatory cytokine concentrations (pg/mL) were measured. * p < 0.05, *** p < 0.001 indicate statistically significant differences from the control group. ^### p < 0.001 indicates significant difference from the non-treated (NT) group. (D–G) Western blot image and analysis of the expression of COX-2, iNOS and IL-1β.