Stigmasterol blocks cartilage degradation in rabbit model of osteoarthritis.

Stigmasterol has been shown exhibit anti-osteoarthritic properties in vitro studies. However, the in vivo effects of stigmasterol on cartilage are still unclear. This study investigated the anti-osteoarthritic properties of stigmasterol on cartilage degradation in a rabbit model of osteoarthritis (OA). Twenty rabbits underwent bilateral anterior cruciate ligament transection (ACLT) to induce OA. Five rabbits were used as normal control. Two weeks after operation, the rabbits were randomly divided into two groups. Each group of 10 rabbits received intra-articular injection with 0.3 ml of stigmasterol in left knees and vehicle in right knees, once weekly. Group 1 was killed 6 weeks after ACLT and 2 were sacrificed 9 weeks after ACLT. The knee joints were assessed by gross morphology, histology and gene expression analysis. We found that expression of genes encoding matrix metalloproteinases (MMPs) was significantly higher while tissue inhibitors of metalloproteinase (TIMP)-1 was significantly lower in the both joints of the two OA groups compared to normal controls. Stigmasterol reduced the cartilage degradation as assessed by histological analysis and markedly suppressed MMPs expression both in group 1 and group 2. Our results suggest that stigmasterol may be considered as a possible therapeutical agent in the treatment of OA.


INTRODUCTION
Osteoarthritis (OA) is a chronic joint disease manifested by cartilage degradation along with severe joint pain.Chondrocytes exposed to abnormal stimuli such as mechanical damage or proinflammatory stimuli including interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α), release matrix metalloproteinases (MMPs), the main matrix-degrading enzymes, leading to the breakdown of extracellular matrix (ECM) (Rowan et al., 2008).MMP-13 is well known as the most effective proteinase because of the ability to cleave collagen II which is the main component of ECM (Billinghurst et al., 1997).
Treatment of OA is still a problem.Although nonsteroidal anti-inflammatory drugs and selective cyclooxygenase-2 (COX-2) inhibitors are effective, they have severe side effects such as gastrointestinal bleeding and cardiovascular diseases (Farkouh et al., 2004;Lazzaroni et al., 2004).There is still a need to investigate new antiarthritic agents.
Phytosterols are plant lipids whose chemical structure is similar to that of cholesterol.Previous studies have shown that phytosterols exert various biological effects such as anti-cancer, anti-pyretic and immune-modulating activities (Gupta et al., 1980;Raicht et al., 1980;Bouic et al., 1996;Bouic et al., 1999).In addition, their antiinflammatory activities are also well demonstrated (Navarro et al., 2001;Okoye et al., 2010;Youssef et al., 2010).The role of phytosterols in rheumatoid arthritis (RA) has been investigated previously (Thiers, 1953).Recently, Gabay et al demonstrated that stigmasterol, a phytosterol, exerted anti-osteoarthritic properties in mouse chondrocytes and human chondrocytes (Gabay et al., 2010).That was the first study to report a possible role of phytosterols in preventing OA.However, the in vivo effects of stigmasterol on cartilage are still unclear.Therefore, in the present study, we investigated the in vivo effects of stigmasterol on cartilage in experimental OA.

Reagents.
Stigmasterol was purchased from Sigma-Aldrich (St.Louis, MO, USA).Stigmasterol stock solution at a concentration of 20 mg/ml was prepared in ethanol.Then, the solution was diluted with 50% polyethylene glycol (PEG).
OA induction and animal treatment.With the approval by the Institutional Animal Care and Use Committee of Zhejiang University (Hangzhou, China), twenty-five New Zealand White rabbits (male, 2.0-2.5 kg) were used in this study.Twenty rabbits underwent bilateral anterior cruciate ligament transection (ACLT) as described previously (Yudoh et al., 2007).Five rabbit were used as normal control.Two weeks after operation, the rabbits were randomly divided into two groups.Both groups of 10 rabbits were intra-articularly injected with 0.3 ml of stigmasterol solution (20 μg/ml in PEG) in left knees and vehicle in right knees, once weekly.Group 1 was killed 6 weeks after operation and 2 were sacrificed 9 weeks after operation.The knee joints were assessed by gross morphology, histology and gene expression analysis.
Macroscopic evaluation.Scoring of the articular cartilage of the rabbit knee joints was performed in a blind manner after using India ink.The criteria used were as follows: grade 1 (intact surface), grade 2 (minimal fibrillation), grade 3 (overt fibrillation), grade 4 (erosion) (Yoshioka et al., 1997).
Histological assessment.Femoral condyles from both knees were fixed in 10% neutral buffered formalin, decalcified with buffered ethylenediaminetetraacetate (EDTA) and dehydrated in a series of ethanol solutions.Then, the cartilage was embedded in paraffin, cut into 5-μm sections.The sections were stained with Safranin O-fast green.The OARSI assessment system with a combined score of grade (0-6) × stage (0-4) was used to evaluate the sections in a blind manner (Pritzker et al., 2006).
Gene expression analysis of cartilage.Cartilage was pulverized in liquid nitrogen, and total RNA was extracted using TRIzol reagent (Invitrogen).For the first strand cDNA synthesis, 1 μg of RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase cDNA synthesis kit (Promega, USA) at 37°C for 1 h.Gene expression of MMP-1, MMP-3, MMP-13 and tissue inhibitors of metalloproteinase (TIMP-1) was quantitated by real-time quantitative PCR with the iCycler apparatus system (Bio-Rad, USA).The primers used were shown in Table 2. 18S rRNA was used as an internal control.Levels of target gene expression were calculated as follows: n=100 × 2 − (ΔCT targeted gene -ΔCT 18S rRNA).
Statistical analysis.All data were expressed as mean ± standard deviation (S.D.).Statistical analyses were performed with, SPSS 12.0 software for Windows.Statistical analysis of the gross morphology was performed by nonparametric Mann-Whitney U test, histological and gene expression data was analyzed by a paired t-test.P values less than 0.05 were considered significant.

Macroscopic grading of cartilage
The articular cartilage showed various severities of lesions.Cartilage from OA animal treated with vehicle showed significant severe lesions compared to normal cartilage.Those lesions were more severe in cartilage 9 weeks after ACLT than 6 weeks after ACLT.The stigmasterol-treated animal showed less severe cartilage lesions compared with the vehicle-treated group, both in 6 and 9 weeks after ACLT, however, the difference was not significant (P > 0.05, data not shown).Figure 1 presents the cartilage lesions in different groups.

Histological evaluation
Representative histological pictures of control, vehicletreated OA and stigmasterol-treated OA cartilage are presented in Fig. 2, and the histological scores are shown in Table 3.In vehicle-treated knees, ACLT led to cartilage degradation.Surface irregularity and depletion were noted in the cartilage 6 weeks after ACLT.Severe degradation such as cartilage erosion was observed 9 weeks after ACLT.The cartilage degradation was significantly diminished by stigmasterol.

Gene expression in cartilage
There was a significant increase in the expression of MMP-1, MMP-3 and MMP-13 genes in the cartilage from vehicle-treated OA animal cartilage harvested 6 or 9 weeks after ACLT compared to normal cartilage.In contrast, expression of the TIMP-1 gene was decreased.In addition, the MMPs expression increased in parallel with the progression of OA.Furthermore, stigmasterol   significantly decreased the expression of the MMP-1, MMP-3 and MMP-13 genes, and increased the expression of TIMP-1 compared to the vehicle-treated group (Fig. 3).

DISCUSSION
Current treatment of OA is unsatisfactory and although symptom relief, no disease modifying OA drug (DMOAD) is yet available.There is still a need to find new agents for the treatment of OA.
Recently, Gabay et al. (2010) demonstrated that stigmasterol, a phytosterol, showed anti-osteoarthritic properties in both mouse chondrocytes and human chondrocytes in response to IL-1β.Little is known about the in vivo effects of stigmasterol on OA.Therefore, we design this study to investigate whether stigmasterol exerted in vivo effects on cartilage degradation in experimental OA.In the experimental rabbit OA model induced by ACLT, treatment with stigmasterol reduced the progression of cartilage damage as assessed by morphological non-sig-nificantly and histological evaluation.In addition, treatment with stigmasterol resulted in the down-regulation of expression of MMP-1, MMP-3, MMP-13 and up-regulation of TIMP-1.These findings confirmed the in vitro effects of stigmasterol reported by Gabay and coworkers.
It is well established that numerous proteinases such as MMPs, aggrecanases and cathepsins are implicated in the catabolism of OA cartilage (Dean et al., 1989;Konttinen et al., 2002;Tortorella et al., 2001).Among them, MMPs are believed to play pivotal roles in cartilage matrix degradation.MMP-13 has received extensive attention because it is mainly responsible for type II collagen degradation.An elevated MMP-13 expression was noted in arthritic cartilage (Aigner et al., 2001).In addition, a selective MMP-13 inhibitor has been shown to reduce cartilage degradation in experimental OA induced by ACLT in rabbits and in the rat model of mono-iodoacetate (MIA)-induced OA (Johnson et al., 2007;Baragi et al., 2009).In the present study, we focused on changes of gene expression of MMPs in cartilage due to their importance in OA.The present results revealed that the expression of MMP-1, MMP-3 and MMP-13 was significantly elevated by ALCT compared to normal cartilage and this up-regulation was related to the cartilage degradation in the progression of OA.Our results are partly consistent with the findings reported by Wu and coworkers (Wu et al., 2008), who demonstrated that the level of MMP-1 expression was correlated with the degree of cartilage degradation in ACLT rabbits.In addition, we found that the expression of TIMP-1 was decreased along with the cartilage degradation.Thus, the balance between MMPs and TIMPs was disturbed.Since the imbalance between MMPs and TIMPs in the present study increased with the progressive cartilage degradation as assessed by morphology and histology, our results confirmed the important role of MMPs/TIMPs in the process of OA.
In the present study, treatment with stigmasterol by intra-articular injection resulted in the down-regulation of the expression of MMP-1, MMP-3 and MMP-13 and up-regulation of TIMP-1 as compared to the vehicle-treated groups.These observations suggest that stigmasterol administration regulated the balance between MMPs and TIMPs during the progression of OA.This could be partly supported by the finding that stigmasterol inhibited the gene expression of MMP-3 and MMP-13 in mouse chondrocytes and human chondrocytes in vitro (Gabay et al., 2010).An inhibition of MMPs by phytosterol was also observed in another system as reported by Grether-Beck et al. (2008).In addition, a preparation rich in phytosterol, named avocado-soybean unsaponifiables (ASU), also has been shown to inhibit MMPs expression in experimental OA in dogs (Boileau et al., 2009).These observations suggest that phytosterol may exert inhibitory effects on MMPs in different systems.Additionally, many  agents showing inhibitory effects on MMPs have shown beneficial effects in experimental OA.Thus, we speculate that the beneficial effects of stigmasterol on cartilage may be partly associated with the regulation of the MMPs/TIMPs system.
The main limitations of our study are its duration (6 or 9 weeks after ACLT) and the single dosage in the study.Longer time could provide more information about the changes of gene expression of MMPs and TIMPs as well as the in vivo effects of stigmasterol in experimental OA.For instance, in earlier studies, three stage of OA was used to investigate the gene expression of catabolic mediators in ACLT rabbits (Mehraban et al., 1997;Bao et al., 2009).In addition, we only used one dosage in the experiment, maybe different dosage could better illustrate the effects of sigmasterol.Another limitation is that the mechanism by which stigmasterol regulated the MMPs/TIMPs in cartilage in vivo remains unclear.In vitro, stigmasterol has been shown to inhibit pro-inflammatory cytokines and matrix degradation mediators partly via inhibiting the nuclear factor-κB (NF-κB) pathway (Gabay et al., 2010).The effects of stigmasterol on another important signalling pathway, mitogen-activated protein kinase (MAPK) pathway which is implicated in MMPs regulation remains unknown (Yan & Boyd, 2007).In addition, cartilage degradation is partially dependent on MMPs activities, there are mechanisms involved in cartilage degradation indepent on MMPs, and the effects of sigmasterol on thes mechanisms are also unclear.Therefore, further studies are needed to get a better understanding about the mechanisms of action of stigmasterol in OA.
In summary, we first reported that stigmasterol effectively inhibits cartilage degradation in a rabbit ACLT model.Our results suggest that stigmasterol may be considered as a possible therapeutical agent in the treatment of OA.Further studies are needed to elucidate the action mechanism of the regulation of MMPs by stigmasterol.

Figure 1 .
Figure 1.Gross appearance of femoral condylesGross morphology of femoral condyles from normal (A) and ACLT rabbits 6 and 9 weeks after operation.Femoral condyles in vehicle-treated group showed more serious lesions during the development of OA, while the lesions in stigmasterol-treated cartilage was less severe 6 weeks (B compare with C) and 9 weeks (D compare with E) after ACLT.OA = osteoarthritis; ACLT = anterior cruciate ligament transection.

Figure 2 .
Figure 2. Safranin-O staining of articular cartilage Safranin O staining was performed in sections of cartilage.Typical cartilage lesions are shown (Original magnification ×50).

Table 3 . Histological assessment of articular cartilage using OAR- SI assessment system
Data are presented as mean ± S.D. *P<0.05.