Simvastatin Retards Cartilage Degradation and Improves Subchondral Bone Microstructure in Osteoarthritis Mice Induced by High-fat Diet

Objectives This study aimed to evaluate the effects of simvastatin on the metabolism of cartilage and subchondral bone in a mice model of osteoarthritis (OA) induced by obesity. Methods We randomly assigned thirty C57BL/6J mice into 3 groups: the Control group received normal diet, the HFD group and HFD+S group received high-fat diet, HFD+S group were simultaneously treated with simvastatin (10 mg/kg/day) for 8 weeks. The pathology of OA was assessed by histomorphology analyses, immunohistochemistry, micro-computed tomography and enzyme-linked immunosorbent assay. Results Histomorphological analysis revealed that OA was signicantly exacerbated by the HFD-induced obesity and markedly alleviated by the simvastatin intervention. In details, simvastatin ameliorated the abnormal metabolic status and cartilage lesions, signicantly increased aggrecan and collagen-II expression and decreased the expression of MMP-13. Furthermore, the results of micro-computed tomography analysis revealed that the HFD+S group exhibited higher BMD, BV/TV, and Tb.N values but a lower Tb.Sp value than that of the HFD group. Serum COMP concentrations, the number of adipocytes in subchondral bone marrow and the number of osteoclasts on trabecular bone surface were signicantly correlated with OARSI score. abnormal metabolism was remarkably ameliorated by treatment with simvastatin: BMD, BV/TV, and Tb.N values signicantly increased, and Tb.Sp values signicantly decreased. These results demonstrate the inhibition of subchondral bone degeneration also exerts indirect protective effects on cartilage by avoiding secondary damage caused by abnormal mechanical stress and metabolism.


Introduction
Obesity is now one of the most urgent global healthcare challenges. Obesity reduces both life expectancy and quality of life [5], and is closely associated with the risk of developing metabolic, cardiovascular, muscle skeletal and many other diseases [7]. A signi cant contributor to obesity-induced disability is osteoarthritis (OA) [9], which is a worldwide public health problem that causes long-term pain and disability [19], and its disability-adjusted life years index rose by 34.8% between 2005 and 2015 [3].
OA is a whole-joint disorder involving the cartilage, subchondral bone, synovium, ligament and joint capsule. Many pathologies are related to OA, including cartilage degeneration, subchondral bone loss, and synovial in ammation. However, the potential mechanism of obesity-related OA is complex and controversial [36]. One contributing factor appears to be overweight-associated mechanical overload, which has long been pointed out to explain the link between obesity and osteoarthritis. Related clinical studies also showed positive correlations between BMI and the onset or progression of knee osteoarthritis [26]. In addition to the abnormal loading, obesity-related systemic metabolic factors may play an important role in the pathological process of OA. Various regular cellular processes are disturbed by systemic metabolic diseases, such as mitochondrial bioenergetics, nutrient sensing, and glycolysis.
Consistently, damaged cellular metabolism is the most important characteristics of chondrocytes in OA [6].
Current medical treatment strategies for OA are focused on pain relief and symptom control rather than disease improvement. Simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitor, has been widely used in the initial treatment of hyperlipidemia and prevention of cardiovascular disease.
Furthermore, simvastatin has been reported to have a protective effect on the cartilage in the knee joint, such as promoting the proliferation and inhibiting the apoptosis of chondrocytes [39], stimulating bone formation [10], and enhancing anti-in ammatory effects [30]. Previous in vivo studies have shown that application of statins reduces cartilage degradation in rabbits with experimental osteoarthritis and inhibits the expression of matrix metalloproteinase-3 (MMP-3) in a mechanically induced knee osteoarthritis [2,12]. Moreover, in vitro experiments have shown that statins up-regulate the mRNA levels of bone morphogenetic protein-2 (BMP-2), aggrecan, collagen-II and increase the synthesis of proteoglycan in rat chondrocytes [15]. These ndings suggest a possible effect of simvastatin for treating OA involving cartilage degeneration and subchondral bone loss.
The purpose of this study is to further explore the pathogenesis of OA induced by obesity in high-fat diet (HFD) mice and to investigate the therapeutic effects of simvastatin on knee OA.

Animals and treatments
All experimental procedures were approved by the Institutional Animal Care and Use Committee. Thirty C57BL/6J mice (age: 3 months old; Vital River Experimental Animal Technical Co., Ltd., China) were used in this research. All mice were randomized into three groups (n = 10/group): mice with normal diet + vehicle (distilled water) (Control), mice with High-fat diet + vehicle (HFD), mice with HFD + Simvastatin (HFD+S). Drug administration was initiated at the same day the HFD was given. Mice in the HFD+S group received oral gavage of Simvastatin (SL Pharmaceutical Co., Ltd. Beijing, China) at a dosage of 10 mg/kg/day, other groups were given distilled water. After 8 weeks of treatment, all mice were euthanized to collect blood and samples. For details see Supplementary Methods.

Macroscopic observation
After all mice were euthanized, the abdominal subcutaneous fat was photographed with a Canon 550D digital camera (Canon, Tokyo, Japan).
Body weight, Waist circumference and Body mass index (BMI) Body weight was measured weekly and waist circumference was measured before the macroscopic observation. Body mass index (BMI) was calculated using the formula: (Weight in kg/Body length in m 2 ) [18].

Histological assessments
In the mice, perirenal adipose tissue and knee joints were harvested and sequentially xed in 10% neutralbuffered formalin, dehydrated in a graded series of ethanols and embedded in para n wax (bone tissue needs decalci cation), and nally cut into 5-μm sections. Adipose tissues were stained with hematoxylin and eosin (H&E) to observe the surface area of fat cells. Bone tissues sections were stained for light microscopy with H&E to evaluate the pathological changes in subchondral bone marrow fat cells, cartilage degradation was assessed using Safranin-O and fast green staining and scored according to the Osteoarthritis Research Society International (OARSI) scoring system (Supplementary Table 1). We also performed tartrate-resistant acid phosphatase (TRAP) staining to evaluate TRAP + osteoclasts within the subchondral bone following a standard protocol (CK20203, MultiSciences Co., Ltd., Zhejiang, China). All histological evaluation was performed by 2 independent researchers in a blinded manner. Biomarker assays Serum concentrations of cartilage oligomer protein (COMP) (CUSABIO Co., Ltd., Wuhan, China), CTX-II (Abbexa Co., Ltd., Cambridge, UK) and IL-6 (Abbexa Co., Ltd., Cambridge, UK) were determined using enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions. The data were measured using an iMARK Reader (BioRad Laboratories Inc., USA).

Data analysis and statistics
All data are presented as mean ± standard deviation or mean with 95% con dence interval and were analyzed using SPSS version 22.0 (IBM Corp., Armonk, NY, USA). Non-Gaussian distributed data were compared using Kruskal-Wallis and Mann-Whitney non-parametric analysis. One-way analysis of variance (ANOVA) followed by Tukey HSD or Dunnett's T3 test were used for pairwise comparison of data with a Gaussian distribution and homogeneity of variance. Spearman's rank correlation analyses were used to investigate correlations between the concentration of serum COMP, CTX-II, IL-6, the number of adipocytes in subchondral bone marrow, the number of osteoclasts on trabecular bone surface and OARSI score. The level of statistical signi cance was established at P < 0.05.

Macroscopic ndings of abdominal subcutaneous fat
In HFD-treated mice, macroscopic images showed evident abnormal accumulation of subcutaneous fat in the abdomen, while simvastatin treatment reversed the abnormality in the HFD+S group ( Figure 1A).

Changes in body weight, waist circumference and BMI index
The weight of HFD mice was signi cantly higher than that of the Control group from the second week (P < 0.05). Simvastatin down-regulates this abnormal weight gain, and the weight of HFD+S group increased only slightly compared to Control group. At the end, the body weight of HFD mice was signi cantly higher than Control mice (P < 0.05). The intervention of simvastatin signi cantly reduced the weight of mice fed a HFD (P < 0.05) ( Figure 1B). The waist circumference and BMI index of mice in HFD group substantially increased compared with the Control group at 8 weeks (P < 0.001 and P < 0.01). Compared with HFD mice, the waist circumference and BMI index of HFD+S mice was signi cantly reduced after treatment (P < 0.001, and P < 0.05) ( Figure 1C, E). There was no statistical difference in the body length of mice in each group ( Figure 1D).

Histological evaluation of adipose tissue
The mean surface area of adipocytes observed in the HFD group was signi cantly increased compared to the control group (P < 0.001), whereas after simvastatin treatment, mice in the HFD+S group showed a signi cantly lower area than that in the HFD group (P < 0.001) (Figure 2A, B). The high-fat diet greatly promoted the differentiation of adipocytes in the subchondral bone marrow of mice ( Figure 2C). The number of adipocytes in subchondral bone marrow was signi cantly higher in the HFD group than in the Control group (P < 0.001). The effects were reversed by gavage of simvastatin. However, there was still signi cant difference between HFD+S group and Control group (P < 0.001) ( Figure 2D).

Histological changes in articular cartilage
In the Control group, the cartilage surface and the structures of chondrocytes were normal, Safranin-O staining of extracellular matrix (ECM) was evenly distributed. With the induction of high-fat diet, the articular cartilage of HFD group exhibited extensive cartilage lesions, and Safranin-O staining area was signi cantly reduced. The administration of simvastatin alleviated the severity of cartilage degeneration ( Figure 2E). The HFD group presented a signi cantly higher OARSI score than the Control group (P < 0.001), and the score was signi cantly decreased after simvastatin intervention (P < 0.001) ( Figure 2F).

Simvastatin improve cartilage metabolism
Aggrecan and collagen-II expression was signi cantly lower in the HFD group than in the Control group (P < 0.001), whereas these two proteins expression was signi cantly higher in the HFD+S group than in the HFD group (P < 0.001). (Figure 3A-D). Signi cantly higher MMP-13 and caspase-3 expression level was observed in the HFD group than in the Control group (P < 0.001 and P < 0.01). After simvastatin intervention, the expression level of MMP-13 but caspase-3 in the HFD+S group was signi cantly reduced (P < 0.001) ( Figure 3G-J). There was no signi cant difference in the expression level of ADAMTS-4 among the groups. (Figure 3E, F).
Simvastatin improve subchondral bone metabolism OCN immunohistochemical staining and TRAP staining showed a signi cantly increased number of osteoblasts and osteoclasts on trabecular bone surface in the HFD mice compared with Control mice (P < 0.001). However, the intervention of simvastatin signi cantly decreased the number of osteoblasts and osteoclasts in HFD+S group (P < 0.01 and P < 0.001) (Figure 4)

Micro-CT parameters of subchondral bone
The Micro-CT image of the femur and tibial subchondral bone of mice in each group are shown in Figure  5A. Quantitative analysis of the subchondral bone of the tibia showed that HFD mice exhibited signi cantly lower BMD, BV/TV, Tb.N, Tb.Th, and signi cantly higher Tb.Sp than that in Control mice (P < 0.001, P < 0.001, P < 0.001, P < 0.01, and P < 0.001, respectively). Compared with those of the HFD group, the BMD, BV/TV, and Tb.N values of the HFD+S group were signi cantly higher, but the Tb.Sp value was signi cantly lower (P < 0.001, P < 0.01, P < 0.05, and P < 0.001, respectively) ( Figure 5B-G).

Biomarker analysis
The concentration of serum COMP and IL-6 was signi cantly higher in the HFD group than in the Control group (P < 0.001 and P < 0.05, respectively), after the intervention of simvastatin, the COMP and IL-6 concentrations of mice in the HFD+S group decreased, but there was no signi cant difference compared with the HFD group ( Figure 6A, C). The serum CTX-II concentration was not signi cantly different among groups ( Figure 6B).

Discussion
The results of the present study demonstrate that HFD-induced obesity aggravated the degradation of cartilage matrix and the destruction of subchondral bone in mice. All these degenerative changes in the OA model were retarded by simvastatin inhibiting overactivated subchondral bone remodeling and cartilage catabolic metabolism.
OA etiology studies generally suggested cartilage lesions are major pathologic changes of OA joints [13,20]. The extracellular matrix (ECM) homeostasis imbalanced in OA cartilage [24], high-magnitude mechanical stress and high levels of in ammatory cytokines, which all exacerbate the in ammatory responses of OA [37]. In our study, the HFD group displayed distinct cartilage degeneration and a higher histological score than in the Control group. In order to further explore the underlying mechanism of cartilage impairment in obese mice, we detected the proteins related to cartilage metabolism and chondrocyte apoptosis. Aggrecan and collagen-II are components of chondrocyte ECM, which plays an important role in maintaining the proper function of cartilage [14,27]. MMP-13 and ADAMTS-4, two matrix-degrading enzymes, involved in the process of degrading aggrecan and collagen-II and usually highly expressed in OA cartilage [28,34], and the expression of caspase-3 re ects the degree of apoptosis of chondrocytes [38]. Immunohistochemical results indicated that the expression levels of MMP-13 and caspase-3 were signi cantly increased, whereas the expression of aggrecan and collagen-II was signi cantly decreased in HFD mice. These ndings indicate that HFD-induced obesity leads to enhanced catabolism of cartilage and apoptosis of chondrocytes, which may be the primary cause of cartilage impairment in the model used in the present study.
Previous studies have pointed out that in OA, subchondral bone undergoes an abnormal remodeling process with the regulation of both mechanical and biological signals [17]. The overactive osteoblast metabolism might be responsible for the aberrant mineralization of subchondral bone in OA [25]. In our study, OCN immunohistochemical staining showed a signi cantly higher number of osteoblasts on trabecular bone surface in the HFD mice compared with Control mice. Besides, TRAP staining indicates a signi cant increase the osteoclast number in obesity-induced OA. This elevated number and distribution of osteoclasts adjacent to the trabecular bone is also considered a representative phenomenon in subchondral bone marrow lesions [35].
Micro-CT analysis of the subchondral bone microstructure is another way to assess the level of detail regarding the pathological changes present in subchondral bone [29]. previously showed that the abnormalities of subchondral bone were present in OA with high bone turnover and subsequent loss of bone mass [8]. In our study, the BMD, BV/TV, Tb.N and Tb.Th values of the subchondral bone in HFD mice were signi cantly reduced, but the Tb.Sp value was signi cantly increased, indicating that the subchondral bone of obesity-induced OA mice was abnormal bone remodeling. Deleterious alterations of subchondral bone may transmit abnormal stress to articular cartilage and cause the deterioration of cartilage [23]. These ndings suggest that subchondral bone plays a critical role in the progression of OA.
Statins are clinically used for the treatment of dyslipidemia currently. With the deepening of research, it has been discovered that statins, in addition to reducing blood lipids, also have therapeutic effects on many other diseases. There have been studies discussing the treatment of osteoarthritis with statins, which is found to be able to promote the proliferation of chondrocytes in the knee joint and inhibit their apoptosis, as well as delay the degradation of cartilage matrix [4,22]. These reports revealed the therapeutic effect of OA after simvastatin administration. In this study, we con rmed that simvastatin had a protective effect on the impaired OA cartilage and subchondral bone.
From the data, the intervention of simvastatin signi cantly reduced the body weight, the accumulation of neutral lipid in visceral adipose tissue and the subchondral bone marrow adipocytes of mice with HFD. The weight loss effect of simvastatin has been con rmed in clinical studies [31]. Although it is not a weight loss drug in essence, simvastatin has a bene cial effect on the regulation of abnormal lipid metabolism to retard the progression of OA induced by obesity. Then, we found that simvastatin exhibits a signi cant cartilage-protective effect, which have been reported in previous in vitro studies [32] and in vivo studies [2] of knee OA. Our research showed similar results, simvastatin therapy signi cantly decreased the expression levels of MMP-13 in the OA cartilage of obesity mice and signi cantly increased the expression of aggrecan and collagen-II. Not only cartilage metabolism, the subchondral bone is also closely associated with the development and progression of OA [11]. In the present study, the deterioration of subchondral bone micro-architecture and abnormal metabolism was remarkably ameliorated by treatment with simvastatin: BMD, BV/TV, and Tb.N values signi cantly increased, and Tb.Sp values signi cantly decreased. These results demonstrate the inhibition of subchondral bone degeneration also exerts indirect protective effects on cartilage by avoiding secondary damage caused by abnormal mechanical stress and metabolism.
COMP is an important component of the cartilage ECM [1]. Many studies have shown that serum COMP is a potential diagnostic marker for the occurrence and progression of OA [16,33]. In our study, we found that the serum COMP concentration in the HFD group was signi cantly higher than that in the Control group. After administration of simvastatin, the serum COMP concentration of obesity mice was reduced, although there was no statistical difference. In addition, the serum IL-6 levels of mice in the HFD group were signi cantly increased. As a systemic in ammatory factor, the elevated level of IL-6 could facilitate in ammatory response and further aggravate the degeneration of the knee joint [21]. Moreover, serum COMP concentration, the number of adipocytes in subchondral bone marrow and the number of osteoclasts on trabecular bone surface were signi cantly correlated with OARSI score. Thus, we believed that abnormalities in the subchondral microenvironment such as overactive adipogenic differentiation of bone marrow mesenchymal cells instead of osteogenic differentiation, may be another cause of exacerbate joint damage.

Conclusion
In summary, obesity aggravates articular degeneration, characterized by enhanced cartilage catabolism and the abnormal metabolic pathology and remodeling of subchondral bone. Simvastatin have a potential therapeutic effect on obesity-induced OA. For OA patient with obesity, simvastatin may be used to treat hyperlipidemia or weight control and also have protective effect on the knee joint. But for OA patient with no obesity, the side effects of simvastatin should also be noted. Further research is needed before it can be put into clinical practice.