Obtusifolin, an Anthraquinone Extracted from Senna obtusifolia (L.) H.S.Irwin & Barneby, Reduces Inflammation in a Mouse Osteoarthritis Model

Abstract

Osteoarthritis (OA) is an age-related degenerative disease that causes cartilage dysfunction and inflammation. Obtusifolin, an anthraquinone extracted from Senna obtusifolia (L.) H.S.Irwin & Barneby seeds, has anti-inflammatory functions; it could be used as a drug component to relieve OA symptoms. In this study, we investigated the effects of obtusifolin on OA inflammation. In vitro, interleukin (IL)-1β (1 ng/mL)-treated mouse chondrocytes were co-treated with obtusifolin at different concentrations. The expression of matrix metalloproteinase (Mmp) 3, Mmp13, cyclooxygenase 2 (Cox2), and signaling proteins was measured by polymerase chain reaction and Western blotting; collagenase activity and the PGE₂ level were also determined. In vivo, OA-induced C57BL/6 mice were administered obtusifolin, and their cartilage was stained with Safranin O to observe damage. Obtusifolin inhibited Mmp3, Mmp13, and Cox2 expression to levels similar to or more than those after treatment with celecoxib. Additionally, obtusifolin decreased collagenase activity and the PGE₂ level. Furthermore, obtusifolin regulated OA via the NF-κB signaling pathway. In surgically induced OA mouse models, the cartilage destruction decreased when obtusifolin was administered orally. Taken together, our results show that obtusifolin effectively reduces cartilage damage via the regulation of MMPs and Cox2 expression. Hence, we suggest that obtusifolin could be a component of another OA symptom reliever.

Keywords: NF-κB; inflammation; obtusifolin; osteoarthritis.

Figure 1

Viability of chondrocytes after obtusifolin treatment. The structure of obtusifolin (A). Chondrocytes were treated with obtusifolin for 24 h in a dose-dependent manner, and viability was measured using the lactate dehydrogenase assay (B). Data were analyzed using the one-way ANOVA, and values of each group (n = 3) are shown as mean ± SEM; N.S, nonsignificant.

Figure 2

Obtusifolin downregulated Mmp3, Mmp13, and Cox2 expression in chondrocytes. Chondrocytes treated with IL-1β (1 ng/mL) for 24 h were co-treated with obtusifolin at the indicated concentrations and celecoxib (50 μM/mL) (A–C). The expression levels of Mmp3, Mmp13, and Cox2 were determined by RT-PCR (A) and qRT-PCR (B). Protein levels were determined by Western blotting (C, upper) and densitometry (C, lower). Collagenase activity and PGE₂ level were measured at each concentration (D). Data were analyzed using the one-way ANOVA. The values of each group (n = 5) are shown as mean ± SEM; ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 3

Obtusifolin regulates IL-1β-induced NF-κB phosphorylation. Chondrocytes were treated with IL-1β (1 ng/mL) for the indicated time. After harvesting, signaling proteins were quantified by Western blotting (A). Chondrocytes were treated with obtusifolin at the indicated concentrations for 24 h, and IL-1β (1 ng/mL) was added 10 min before harvest. pp65 was detected by Western blotting (B) and densitometry (C). Erk was used as the loading control in Western blotting. Data of each group (n = 5) are shown as mean ± SEM; * p < 0.05 and **** p < 0.0001.

Figure 4

Oral administration of obtusifolin inhibits cartilage degradation. DMM(Destabilization of the medial meniscus)-induced osteoarthritic (OA) mice were administered PBS or obtusifolin (10, 50, and 100 mg/kg) daily for 6 weeks after 4 weeks of surgery (A). Safranin O staining shows the degree of cartilage destruction (B). Scale bar: 50 μm (left) and 100 μm (right). Cartilage destruction was scored by Osteoarthritis Research Society International (OARSI), subchondral bone thickness, and osteophyte formation (C). A graphical summary of the inhibition mechanism of obtusifolin (D). Data were analyzed using the one-way ANOVA and Student’s t-test. Values of each group (n = 5) are shown as mean ± SEM; * p < 0.05, ** p < 0.01, and *** p < 0.001.