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. 2017 Dec;21(12):3231-3243.
doi: 10.1111/jcmm.13227. Epub 2017 Jun 23.

Anemonin attenuates osteoarthritis progression through inhibiting the activation of IL-1β/NF-κB pathway

Zuqiang Wang  1 , Junlan Huang  1 , Siru Zhou  1 , Fengtao Luo  1 , Wei Xu  1 , Quan Wang  1 , Qiaoyan Tan  1 , Liang Chen  1 , Jun Wang  1 , Hangang Chen  1 , Lin Chen  1 , Yangli Xie  1 , Xiaolan Du  1
Affiliations

Anemonin attenuates osteoarthritis progression through inhibiting the activation of IL-1β/NF-κB pathway

Zuqiang Wang et al. J Cell Mol Med. 2017 Dec.

Abstract

The osteoarthritis (OA) progression is now considered to be related to inflammation. Anemonin (ANE) is a small natural molecule extracted from various kinds of Chinese traditional herbs and has been shown to inhibiting inflammation response. In this study, we examined whether ANE could attenuate the progression of OA via suppression of IL-1β/NF-κB pathway activation. Destabilization of the medial meniscus (DMM) was performed in 10-week-old male C57BL/6J mice. ANE was then intra-articularly injected into joint capsule for 8 and 12 weeks. Human articular chondrocytes and cartilage explants challenged with interleukin-1β (IL-1β) were treated with ANE. We found that ANE delayed articular cartilage degeneration in vitro and in vivo. In particular, proteoglycan loss and chondrocyte hypertrophy were significantly decreased in ANE -treated mice compared with vehicle-treated mice. ANE decreased the expressions of matrix metalloproteinase-13 (MMP13), A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), collagen X (Col X) while increasing Aggrecan level in murine with DMM surgery. ANE treatment also attenuated proteoglycan loss in human cartilage explants treated with IL-1β ex vivo. ANE is a potent protective molecule for OA; it delays OA progression by suppressing ECM loss and chondrocyte hypertrophy partially by suppressing IL-1β/NF-κB pathway activation.

Keywords: NF-κB; anemonin; cartilage; osteoarthritis.

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Figures

Figure 1
Figure 1
Effects of anemonin on cartilage degradation in mouse articular cartilage at 8 and 12 weeks after DMM. After DMM surgery or sham operation, mice were intra‐articularly injected with vehicle (50% 2‐hydroxypropyl‐β‐cyclodextrin) or anemonin (2 mg/kg bodyweight) during following 8 weeks and 12 weeks (n = 14 per group). (A–P) Knee joints were harvested at 8 weeks and 12 weeks after surgery and analysed histologically by Safranin O–fast green staining. Representative images were shown. (Q–X) Summed and maximal histologic scores for cartilage structure damage were evaluated by OARSI‐recommended scoring system in mice with or without anemonin treatment after DMM surgery at 8 weeks and 12 weeks. MFC: medial femoral condyle; MTP: medial tibial plateau. Scale bar: 200 μm (A–D and I–L); 100 μm (E–F and M–P). Data are expressed as the mean (symbols) ± 95% confidence intervals (error bar).
Figure 2
Figure 2
Micro‐CT analysis of ANE effects on tibia after DMM surgery. (A–D) Contrast agent‐enhanced tomography of articular cartilage in mice after DMM surgery. (E–H) Micro‐CT 2D reconstructions of tibial subchondral bone in mice after DMM surgery or sham operation.(I) Bone volume/total volume (BV/TV), (J) trabecular thickness (Tb.Th) and (K) trabecular separation (Tb.Sp) were measured in the subchondral bone of tibia of vehicle‐treated and anemonin‐treated mice at 8 weeks after DMM surgery or sham operation (n = 7 per group). Data are expressed as the mean (symbols) ± 95% confidence intervals (error bar).
Figure 3
Figure 3
The effects of anemonin treatment on the homoeostasis of articular cartilage after DMM surgery. Sections of articular cartilage from the mice with or without anemonin treatment after 8 weeks of DMM surgery and sham operation were analysed by immunohistochemical staining (n = 5 per group). IHC stainings of (A–D) MMP13, (E–H) ADAMTS5, (I–L) collagen X, (M–P) Aggrecan and (Q–T) cleaved caspase‐3 were performed in mice after DMM surgery. (U–X) The ratios of immunoreactive positive cells, MMP13 (U), ADAMTS5 (V), collagen X (W) and cleaved caspase‐3 (X) were analysed. Scale bar: 50 μm (A–T). Data are expressed as the mean (symbols) ± 95% confidence intervals (error bar).
Figure 4
Figure 4
Effects of anemonin on human articular cartilage ex vivo. The full‐thickness human cartilage explants were obtained from adult human joint tissues (n = 10, Mankin score 0–2). Cartilage explants were cultured in the absence or presence of IL‐1β (10 ng/ml) and anemonin (10 μM) for 4 days. (A–D) 5‐μm paraffin sections were stained with Safranin O–fast green to examine the cartilage proteoglycan loss. (E–H) Immunohistochemical staining for collagen II, (I–L) collagen X and (M–P) MMP13 in human articular cartilage. (R) Collagen X‐ and (S) MMP13‐positive cells in human articular cartilage were counted. (Q) Culture medium were collected, and the amount of GAG release into the medium was quantified by DMMB assay. GAG released into the medium was normalized as mass of GAG per millilitre (ml) of culture medium. Scale bar: 200 μm (A–P). Data are expressed as the mean (symbols) ± 95% confidence intervals (error bar).
Figure 5
Figure 5
Effects of anemonin on the mRNA levels of catabolic event genes in IL‐1β‐stimulated human articular chondrocytes. (A–H) Human articular chondrocytes were cultured without serum for 24 hrs and then treated with anemonin (10 μM) for 1 hr, followed by treatment of IL‐1β (10 ng/ml) for 24 hrs. Total RNA was isolated, and levels of mRNAs for the chondrocytes catabolic event markers (mmp3,mmp13 and collagen X), articular chondrocyte marker (Aggrecan) and pro‐inflammatory cytokines (Il‐1, Il‐6 and Il‐8) were detected by RT‐PCR. (I) Cell lysates were analysed by Western blot with antibodies specific for MMP13, Aggrecan and IL‐1β. β‐actin was used as a loading control. Data are expressed as the normalized fold expression relative to controls. (J–L) The signal intensities of Aggrecan, IL‐1β and MMP 13 were quantified using software ImageJ (version 1.47) (n = 5). Data are expressed as the mean (symbols) ± 95% confidence intervals (error bar). P‐values between groups with * are <0.05.
Figure 6
Figure 6
Effects of anemonin on NF‐κB signalling in IL‐1β‐induced human chondrocytes and DMM mouse model. (A and C) Western blot analysis of the phosphorylation of IKKα/β (A) and p65 (C) in human chondrocytes treated with the combination of recombinant human IL‐1β and decreasing doses of anemonin for 4 h. (B and D) The relative expression of protein level of (B) p‐IKKα/β and (D) p‐P‐65 with anemonin treatment in vitro. The knee joints were immunohistochemically stained for (E–H) phospho‐P65 at 8 weeks after DMM with intra‐articular injection of anemonin or vehicle. The ratios of immunoreactive positive cells. (I) phospho‐P65‐positive cells were quantified (n = 5 per group). Scale bar: 50 μm (D–G). Data are expressed as the mean (symbols) ± 95% confidence intervals (error bar). P‐values between groups with * are <0.05.
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