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Basic and translational research
Extended report
Enhancement of the synthesis of n-3 PUFAs in fat-1 transgenic mice inhibits mTORC1 signalling and delays surgically induced osteoarthritis in comparison with wild-type mice
  1. Min-jun Huang1,2,
  2. Liang Wang1,2,
  3. Da-di Jin1,2,
  4. Zhong-min Zhang1,2,
  5. Tian-yu Chen1,2,
  6. Chun-hong Jia3,
  7. Yan Wang4,
  8. Xiao-chen Zhen1,2,
  9. Bin Huang1,2,
  10. Bo Yan1,2,
  11. Yu-hui Chen1,2,
  12. Sheng-fa Li1,2,
  13. Jin-cheng Yang5,
  14. Yi-fan Dai4,
  15. Xiao-chun Bai2,3
  1. 1Department of Orthopedics, The Third Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong, PR China
  2. 2Academy of Orthopedics, Guangdong Province, Guangzhou, Guangdong, PR China
  3. 3Department of Cell Biology, School of Basic Medical Science, Southern Medical University, Guangzhou, Guangdong, PR China
  4. 4The Center of Metabolic Disease Research, Nanjing Medical University, Nanjing, Jiangsu, PR China
  5. 5Department of Orthopedics, Liu Hua Qiao Hospital, Guangzhou, Guangdong, PR China
  1. Correspondence to Dr Xiao-chun Bai, Department of Cell Biology, School of Basic Medical Science, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou 510515, China; baixc15{at}smu.edu.cn

Abstract

Background An exogenous supplement of n-3 polyunsaturated fatty acids (PUFAs) has been reported to prevent osteoarthritis (OA) through undefined mechanisms.

Objective This study investigated the effect of alterations in the composition of endogenous PUFAs on OA, and associations of PUFAs with mammalian target of rapamycin complex 1 (mTORC1) signalling, a critical autophagy pathway in fat-1 transgenic (TG) mice.

Methods fat-1 TG and wild-type mice were used to create an OA model by resecting the medial meniscus. The composition of the endogenous PUFAs in mouse tissues was analysed by gas chromatography, and the incidence of OA was evaluated by micro-computed tomography (micro-CT), scanning electron microscopy and histological methods. Additionally, primary chondrocytes were isolated and cultured. The effect of exogenous and endogenous PUFAs on mTORC1 activity and autophagy in chondrocytes was assessed.

Results The composition of endogenous PUFAs of TG mice was optimised both by increased n-3 PUFAs and decreased n-6 PUFAs, which significantly alleviated the articular cartilage destruction and osteophytosis in the OA model (p<0.01), decreased protein expression of matrix metalloproteinase-13 (MMP-13) and ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs) in the articular cartilage (p<0.01) and reduced chondrocyte number and loss of cartilage extracellular matrix. Both exogenous and endogenous n-3 PUFAs downregulated mTORC1 activity and promoted autophagy in articular chondrocytes. Conversely, mTORC1 pathway activation suppressed autophagy in articular chondrocytes.

Conclusions Enhancement of the synthesis of endogenous n-3 PUFAs from n-6 PUFAs can delay the incidence of OA, probably through inhibition of mTORC1, promotion of autophagy and cell survival in cartilage chondrocytes. Future investigation into the role of the endogenous n-6/n-3 PUFAs composition in OA prevention and treatment is warranted.

  • Chondrocytes
  • Osteoarthritis
  • Inflammation
  • Cytokines

https://doi.org/10.1136/annrheumdis-2013-203231

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    Introduction

    Osteoarthritis (OA) is a chronic degenerative disease characterised by a loss of articular cartilage and changes in the subchondral bone caused by the combined effects of biomechanical and biological factors.1 ,2 Healthy articular cartilage comprises a large amount of extracellular matrix (ECM) and few cellular components—only chondrocytes. Under normal physiological conditions, cartilage ECM maintains a steady dynamic equilibrium between synthesis and degradation. Sparsely distributed chondrocytes are crucial for maintaining the ECM homeostasis.3 An imbalance involving reduced synthesis and increased degradation of cartilage ECM by various factors leads to OA. Over recent decades, OA has been intensively studied and increasingly understood.4–6 However, its pathogenesis has not yet been fully elucidated. Moreover, an effective prevention strategy for OA is still limited in clinical practice. Most patients with severe OA eventually receive expensive, invasive and high-risk joint replacement surgery.7 ,8 Therefore, further exploration of OA pathogenesis is essential for the development of appropriate prevention and treatment in clinical practice.

    Polyunsaturated fatty acids (PUFAs), particularly n-6 PUFAs and n-3 PUFAs, are essential for mammals. In general, n-6 PUFAs exert proinflammatory effects, whereas n-3 PUFAs exhibit anti-inflammatory effects.9 Extensive in vivo and in vitro studies of the effects of PUFAs on OA have been conducted. Although the results of these studies suggest that exogenous supplementation of n-3 PUFAs might delay OA onset while high n-6 diets might accelerate OA progression,10–14 the underlying mechanisms have not been fully elucidated. Additionally, previous studies have primarily focused on the effects of exogenous PUFAs on OA, but studies on the association between endogenous PUFAs and OA, specifically the composition of endogenous n-6 and n-3 PUFAs in articular cartilage, have not been reported. It is widely known that the dietary content of n-6 and n-3 PUFAs differs significantly among mammals, owing to the complexity and diversity of the human diet. In addition, the biological metabolism of PUFAs is affected by genetic polymorphisms and enzyme activity that involves substrate competition.15 Consequently, additional research focusing on the composition of endogenous n-6 and n-3 PUFAs is needed.

    The mammalian target of rapamycin complex 1 (mTORC1) is critical for regulating cell growth, metabolism and autophagy.16–19 Recent studies have shown that autophagy is critical for ensuring normal articular chondrocyte function. Defective autophagy is one of the main mechanisms for the development of OA.20 Intragastric administration of rapamycin, an important inhibitor of mTORC1, to mice with OA induced by medial meniscus destabilisation can specifically inhibit the mTORC1 pathway activity and activate autophagy of articular chondrocytes, consequently significantly improving OA symptoms.21 These findings suggest that the mTORC1 pathway is involved in regulating OA pathogenesis. Furthermore, our previous studies showed that n-6 PUFAs effectively activated the mTORC1 signalling pathway.22 We propose that alterations of endogenous n-6 and n-3 PUFAs may affect OA incidence, partly through regulation of the mTORC1 signalling pathway.

    We have previously generated a transgenic (TG) mouse model expressing the fat-1 gene, which encode an n-3 fatty acid desaturase enzyme that can endogenously convert n-6 PUFAs to n-3 PUFAs.23 This conversion changes the composition of endogenous n-6 and n-3 PUFAs and decreases the ratio of n-6/n-3. Accordingly, we used the fat-1 TG mouse model to evaluate for the first time the effects of enhancing the synthesis of n-3 PUFAs on OA pathogenesis and the relationship between PUFAs and the mTORC1 signalling pathway in articular chondrocytes. The aim of this study was to provide objective, scientific evidence for the application of PUFAs for the clinical treatment of OA.

    Materials and methods

    Animal model and animal feeding

    Mice genotyping was conducted using a PCR kit (Takara, Dalian, China). C57BL/6 wild-type (WT) control mice were purchased from the Laboratory Animal Centre at the Southern Medical University. We selectively transected the medial meniscus for the OA model (destabilisation of the medial meniscus (DMM)), as previously described.24 Briefly, 24 WT and TG mice (8-week-old female) were used in each group. In the surgical group, the right medial collateral ligaments and anterior cruciate ligaments were dissected, followed by transection of the medial meniscus. In the sham-operated group, only the skin of the left knee joint was resected. At postoperative weeks 4 and 8, 12 WT and 12 TG mice from each group were randomly selected and killed for collection of serum and specimens of the bilateral knee joint. All experimental animals were provided with a standard diet (ResearchDiets Inc, #D10001) and were housed inside specific pathogen-free cages at constant temperature and humidity. The circadian rhythm was maintained at 12 h. Animal experiments were approved by the animal experimental ethics committee of Southern Medical University.

    Systematic assessment of OA

    Histological staining and OA scoring

    Bilateral knee joints were isolated, and the tissues fixed with 4% paraformaldehyde for 48 h and decalcified with 0.5 M EDTA at pH 7.4 for 3 weeks. After decalcification, the specimens were routinely embedded in paraffin, and 2 μm serial sections were obtained from sagittal sections through the medial side of the knee. After dewaxing, safranin O-fast green staining was carried out on the cartilage paraffin sections. Under high magnification imaging, three fields of tibial and femoral articular cartilage were randomly selected and the number of chondrocytes calculated to obtain a mean value. OA was evaluated using the modified Mankin scoring system,13 ,25 which is based on the following categories: articular cartilage structure, grade 0–11; tidemark duplication, grade 0–3; safranin O staining, grade 0–8; fibrocartilage, grade 0–2; chondrocyte cloning above the tidemark, grade 0–2; hypertrophic chondrocytes below the tidemark, grade 0–2; subchondral bone, grade 0–2. The calculation and score evaluation were independently performed by three researchers. Sections were stained with toluidine blue. The average thickness of the articular cartilage of the tibial plateau was measured using Image-Pro Plus V.6.0 software.

    Evaluation of the microstructure of cartilage surface by electron microscopy

    Articular cartilage of the tibial plateau was isolated 8 weeks after surgery and fixed with 2.5% glutaraldehyde (Sigma, USA) for 12 h. After tension-free drying, the specimens were coated in gold and analysed with a scanning electron microscope (Hitachi, S-3000N, Japan) under high-vacuum conditions (air pressure <1×10−7 mbar), with a 20 kV voltage and 18 mm working distance. A Philips XL 30 ESEM-FEG system (FEI Co, USA) was used to capture images at ×50 and ×500 magnification.

    Microtomography of osteophyte formation in the knee joint

    Tomography of fixed knee joint specimens was performed using a microtomographic (micro-computed tomography, micro-CT) imaging system (ZKKS-MCT-Sharp-III scanner, Caskaishen, China). A small field was selected for scanning and was corrected for the CT value, with a 70 kV scanning voltage, 30 W power, 429 μA current and 5 μm scan thickness. The 3D-MED 3.0 was used for three-dimensional knee reconstruction and image capture. The region of interest was selected from periarticular osteophytes using Mimics 5.0 and marked as red. The volume and surface area of osteophytes were calculated.

    In vitro determination of the composition of fatty acids by gas chromatography and immunohistochemical and immunofluorescence analysis

    See online supplementary text.

    Statistical analysis

    A Fisher test was first used to check the data homogeneity of variance by SPSS V.13.0 software before carrying out a Student t test. Once the variance was confirmed as equal, then the t test was used for data analysis. The results are presented as mean±SD and p values <0.05 were considered statistically significant.

    Results

    Optimisation of the composition of endogenous n-6 and n-3 PUFAs in fat-1 TG mice and change after DMM-induced OA

    The difference in the composition of n-6 and n-3 PUFAs in cartilage and serum was compared between TG and WT mice (figure 1A,B). TG mice showed an increased level of n-3 PUFAs and reduced level of n-6 PUFAs in comparison with WT mice. Additionally, the n-6/n-3 PUFA ratio was significantly decreased in TG mice compared with WT mice (see online supplementary tables S1 and S2). Interestingly, the level of n-3 PUFAs in articular cartilage of the DMM group was significantly increased compared with that in the sham group at 4 weeks after surgery, without significant alteration of n-6 PUFAs (figure 1C,D). In addition, the n-6/n-3 ratio was reduced significantly after surgical induction (see online supplementary table S3).

    Figure 1
    Figure 1

    Composition of n-3 and n-6 polyunsaturated fatty acids (PUFAs) in transgenic (TG) and wild-type (WT) mice with or without destabilisation of the medial meniscus (DMM). (A) In the cartilage of TG mice the percentage of n-3 PUFAs was significantly increased, and the percentage of n-6 PUFAs significantly reduced, in comparison with that in WT mice (n=6).  (B) In the serum of TG mice the percentage of n-3 PUFAs was significantly increased, and the percentage of n-6 PUFAs significantly reduced, in comparison with that in WT mice (n=6). (C) The percentage of n-3 PUFAs was significantly increased in both WT and TG groups 4 weeks postoperatively (n=6). (D) There was no significant change in the level of n-6 PUFAs in either the TG or the WT group 4 weeks postoperatively (n=6). ★ Comparison with the WT group, p<0.05; ▲ comparison with the sham group, p<0.05.

    These results suggested that the endogenous PUFAs composition of fat-1 TG mice was optimised by both increased n-3 PUFAs and decreased n-6 PUFAs. Additionally, the endogenous n-3 PUFAs were increased in response to surgically induced OA.

    Delayed progression of OA in TG mice after surgically induced OA

    To comprehensively evaluate the degradation and osteophytosis of knee joints after surgically induced OA, we virtually reconstructed the tissue using a micro-CT scan, then labelled the osteophytes and quantified them using Mimics V.5.0 software. The volume and surface area of periarticular osteophytes were significantly reduced at 4 and 8 weeks after surgery in TG compared with WT mice (figure 2A,B, see online supplementary figure S1). To further evaluate the microstructure of articular cartilage, we assessed the articular cartilage of the tibial plateau by scanning electron microscopy. The results showed that WT mice in the DMM group had a large area of stripped cartilage and exfoliation and exposed subchondral bone combined with a microfracture of subchondral bone in the weight-bearing portion of the cartilage (figure 2C). TG mice in the DMM group had only stripped cartilage and superficial avulsion in the weight-bearing cartilage (figure 2C). These results adequately indicated that enhancement of the synthesis of endogenous n-3 PUFAs from n-6 PUFAs could alleviate articular cartilage lesions, reduce development of osteophytes and delay OA pathogenesis.

    Figure 2
    Figure 2

    Evaluation of osteophyte formation and smoothness of the articular cartilage surface at 8 weeks postoperatively. (A) Micro-CT scan and three-dimensional reconstruction of the knee joint: the region of interest (ROI) is marked in red for periarticular osteophytes. DMM, destabilisation of the medial meniscus. (B) Calculation of the surface area and volume of periarticular osteophytes: both were smaller in transgenic (TG) mice than in wild-type (WT) mice. (C) Scanning electron microscopy of the articular cartilage of the tibial plateau: WT mice presented with stripped cartilage, a large area of exfoliation and exposed subchondral bone in the weight-bearing portion of cartilage; a microfracture of subchondral bone was also found (red arrows). TG mice presented with only mild stripped cartilage and a small area of avulsion. AC, articular cartilage; SB subchondral bone. ★ and ◆comparison with the sham group, p<0.05; ▲ and ●comparison with the WT group, p<0.05.

    The systemic effect of the composition of PUFAs on inflammatory cytokine in serum should also be taken into consideration. Interleukin (IL)-1β, tumour necrosis factor α (TNFα), hyaluronic acid and serum amyloid A protein in the serum of the TG group were significantly decreased in comparison with the WT group 8 weeks after the operation (see online supplementary figure S2). Furthermore, our in vitro study suggests that chondrocytes with an optimised composition of PUFAs from TG mice might reduce the release of IL-1β-induced inflammatory cytokines (see online supplementary figure S3A–C).

    Reduction in the loss of cartilage proteoglycan and chondrocytes in TG mice after surgically induced OA

    To confirm the effects of endogenous PUFAs on protecting joint cartilage and delaying OA pathogenesis, we carried out safranin O-fast green staining, calculated the chondrocyte number and used the modified Mankin scores to evaluate the cartilage. The results showed that the number of chondrocytes in WT mice was significantly reduced and the Mankin score was significantly increased compared with those of TG mice (figure 3A,C). High-magnification imaging showed that the tidemark was disarranged and had almost disappeared in WT mice, whereas it was not significantly changed in TG mice (figure 3B). Joint cartilage thickness was assessed by toluidine blue staining. The results showed that compared with TG mice, WT mice had severe abrasion, and the cartilage thickness was significantly reduced (figure 4A,B). These results indicated that enhancement of the synthesis of endogenous n-3 PUFAs from n-6 PUFAs reduced the loss of cartilage proteoglycan and chondrocytes after surgically induced OA.

    Figure 3
    Figure 3

    Histological staining and assessment after surgically induced osteoarthritis. (A) Safranin O-fast green staining (×100 magnification, scale bar=200 μm) for sagittal sections of the medial knee at 4 and 8 weeks postoperatively. Wild-type (WT) mice presented with typical fibrous hyperplasia at 4 weeks postoperatively, and a severe cartilage defect was shown at 8 weeks. Colour intensity was slightly reduced in transgenic (TG) mice. DMM, destabilisation of the medial meniscus. (B) Safranin O-fast green staining (×400 magnification, scale bar=50 μm) on the tibial plateau at 8 weeks postoperatively. The number of chondrocytes was slightly reduced in TG mice, with no significant changes of tidemark. The number of chondrocytes was significantly reduced, and the tidemark had nearly disappeared and was disarranged in WT mice. The arrows indicate the tidemark (×400, scale bar=50 μm). (C) Cartilage chondrocyte counts based on the staining results of (A). The number of chondrocytes in WT mice was significantly reduced compared with that in TG mice. The modified Mankin scoring based on the staining results of (A) was significantly lower in TG mice than in WT mice (n=6). ◆comparison with WT group, p<0.05; ▲ comparison with TG group, p<0.05; ★ comparison with sham group, p<0.05; NS, non-significant.

    Figure 4
    Figure 4

    Measurement of the thickness of cartilage at the knee joint (n=5). (A) Toluidine blue staining was performed on sagittal sections of the knee joints (×100 magnification, scale bar=200 μm). DMM, destabilisation of the medial meniscus. (B) Thickness of the cartilage of the tibial plateau based on the staining of (A): the loss of cartilage thickness on the tibial plateau was significantly less in transgenic (TG) mice than in wild-type (WT) mice. ▲comparison with TG group, p<0.05; ★ comparison with sham group at 8 weeks, p<0.05.

    Reduction in the proportion of ADAMTS-5- and MMP-13-positive cells in TG mice after surgically induced OA

    OA is accompanied by cartilage degradation, which has been identified as the direct cause of articular cartilage reduction. For immunohistochemistry assessment, we selected matrix metalloproteinase-13 (MMP-13) and ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs)—proteolytic enzymes that play a crucial role in cartilage ECM degradation. The former enzyme is primarily responsible for type II collagen degradation, and the latter is a major enzyme responsible for proteoglycan degradation.10 ,26 The results showed that the proportion of MMP-13- (figure 5A) and ADAMTS-5-positive cells (figure 5C) was significantly increased in WT mice compared with TG mice after surgically induced OA (figure 5B,D). In summary, these results indicated that enhancement of the synthesis of endogenous n-3 PUFAs from n-6 PUFAs decreased MMP-13 and ADAMTS-5 expression, leading to reduced cartilage ECM degradation.

    Figure 5
    Figure 5

    Immunohistochemistry and counting of positively stained cells at 8 weeks postoperatively (n=5). (A) Immunohistochemistry of the matrix metalloproteinase-13 (MMP-13) protein in sagittal sections of the knee joints. Black arrows indicate the positively stained cells (×200 magnification, scale bar=100 μm). (B) The proportion of positively stained cells was calculated based on the staining results. The proportion of MMP-13-positive cells was significantly reduced in transgenic (TG) mice compared with wild-type (WT) mice. (C) Immunohistochemistry of the ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs) protein in sagittal sections of the knee joint. Red arrows indicate positively stained cells (×200 magnification, scale bar=100 μm). (D) The proportion of positively stained cells was calculated based on the staining results. The proportion of the ADAMTS-5-positive cells was significantly reduced in TG mice compared with WT mice. ★ comparison with sham group, p<0.05; ▲comparison with TG group, p<0.05.

    PUFAs affect cartilage chondrocyte autophagy by regulating the mTORC1 signalling pathway

    Previous studies have shown that PUFAs mediate OA pathogenesis, but no research into the role of the mTORC1 signalling pathway has been reported. To investigate the relationship between PUFAs and the mTORC1 signalling pathway in OA pathogenesis, we isolated and identified primary chondrocytes for in vitro culture (see online supplementary figure S4). Before further experiments, the PUFAs composition of cultured chondrocytes was tested to imitate the same condition in vivo. The results suggested that the content of both n-3 and n-6 PUFAs was slightly decreased but the n-6/n-3 ratio was not significantly changed in comparison with the cartilage in vivo (see online supplementary figure S3D).

    mTORC1 is the nutrient (amino acids)-sensitive signalling pathway. Removal of amino acids eliminates mTORC1 activity while readdition of amino acids stimulates mTORC1 activity. Thus, amino acid starvation was performed to study the effect of exogenous PUFAs (docosahexaenoic acid (DHA) and arachidonic acid (AA)) on the mTORC1 signalling pathway and autophagy. The results showed that AA significantly promoted ribosomal S6 protein phosphorylation and inhibited the expression of LC3II. However, DHA significantly inhibited ribosomal S6 protein phosphorylation and enhanced the expression of LC3II. Moreover, DHA could inhibit the effect of AA (figure 6A,B). These results indicated that exogenous n-6 PUFAs inhibit cartilage autophagy by upregulating mTORC1 activity, while n-3 PUFAs have the opposite result and act in opposition to the n-6 PUFAs. To study the role of endogenous PUFAs in this process, we isolated cartilage chondrocytes from TG and WT mice and repeated the above experiments, with similar results (figure 6C,D).

    Figure 6
    Figure 6

    Effects of exogenous and endogenous polyunsaturated fatty acids on the mTORC1 signalling pathway and cartilage chondrocyte autophagy. (A) Arachidonic acid (AA) activated the mTORC1 signalling pathway independently of the amino acid availability (lane 1 vs lane 3), while docosahexaenoic acid (DHA) ameliorated amino acid and AA-induced activation of the mTORC1 signalling pathway (lane 2 vs lane 4). Additionally, DHA inhibited AA-induced activation of the mTORC1 signalling pathway (lane 3 vs lane 5). (B) Under amino acid starvation, AA inhibited the expression of the autophagic marker LC3II protein. DHA enhanced the expression of the LC3II protein. (C) AA significantly activated the mTORC1 signalling pathway of cartilage chondrocytes in wild-type (WT) mice, while cartilage chondrocytes in transgenic (TG) mice significantly blunted AA-induced activation of the mTORC1 signalling pathway. (D) Under amino acid starvation, expression of the LC3II protein in cartilage chondrocytes was increased in TG mice compared with WT mice. aa, amino acid starvation.

    Furthermore, we carried out immunofluorescence staining for LC3II protein to prove that endogenous n-3 PUFAs protected against chondrocyte autophagy in OA progression. The results showed that LC3II expression was reduced at OA onset, but expression of LC3II in TG mice significantly increased in comparison with that in WT mice (see online supplementary figure S5).

    In summary, enhancement of the synthesis of endogenous n-3 PUFAs from n-6 PUFAs protected against chondrocyte autophagy in OA progression, probably by downregulating the mTORC1 signalling pathway.

    Discussion

    Previous studies of PUFAs and OA can be divided into in vitro experiments and in vivo experiments involving dietary supplementation with PUFAs.10–14 However, few studies have examined the relationship between endogenous PUFAs and OA, specifically in relation to n-6 and n-3 PUFAs composition in tissues. Recently, the Multicenter OA Study (MOST) conducted by Baker et al11 reported an association between plasma PUFAs and synovitis. In addition, the recent review by Cleland and James12 also emphasised the association between the serum composition of n-6 and n-3 PUFAs and OA. In our study, we suggest that the optimised n-6 and n-3 PUFAs composition in articular cartilage, resulting from enhancement of the synthesis of endogenous n-3 PUFAs, plays a critical role in OA pathogenesis.

    This is the first study to investigate the effects of the composition of endogenous n-6 and n-3 PUFAs on OA in a TG animal model. Fortuitously, fat-1 TG mice can effectively convert endogenous n-6 PUFAs into n-3 PUFAs. Consequently, TG mice have both increased endogenous n-3 PUFAs and decreased n-6 PUFAs in serum and cartilage. Interestingly, this study found that the composition of n-3 PUFAs in knee cartilage was also significantly enhanced after surgically induced OA, possibly owing to the body's compensatory protective response. However, the primary cause of OA has not been determined. Therefore, this compensatory response cannot effectively delay OA progression, which is consistent with the previous concept that n-3 PUFAs are a protective factor while n-6 PUFAs are a risk factor for OA. In addition, this compensatory response may be different at different times—that is, n-6 and n-3 PUFAs composition may change during different stages of OA progression. Therefore, further studies are necessary to confirm this proposal.

    We demonstrated using an animal study that macroscopic and microscopic lesions were relatively mild after surgically induced OA in TG versus WT mice. In addition, we found that, unlike in humans, the non-treated adult mouse has a sesamoid bone on both the posterior medial and lateral femoral condyle, which can gradually increase with OA progression (data not shown). Therefore, we included this enlarged bone when calculating the volume and surface area of periarticular osteophytes to obtain more objective and accurate data (figure 2A). In addition, scanning electron microscopy analysis revealed the typical initial subchondral bone microfracture caused by stress on the subchondral bone. This subchondral bone microfracture could not be effectively reversed after the articular cartilage was stripped. This study also suggests that electron microscopy can be used for a sensitive evaluation of OA and may disclose more subtle lesions of the articular cartilage.

    Immunohistochemical and histological staining indicated that the expression of key ECM degradation enzymes (MMP-13 and ADAMTS-5) was significantly reduced in the articular cartilage of TG mice in comparison with WT mice (figure 5). Furthermore, the proteoglycan loss in TG mice was significantly improved compared with WT mice (figure 3). These results suggested that enhancement of the synthesis of endogenous n-3 PUFAs derived from n-6 PUFAs could suppress the expression of key proteolytic enzymes, thereby alleviating the cartilage matrix degradation, which is consistent with previous studies.10

    Cartilage chondrocyte counts and cartilage thickness measurements suggested that enhancement of the conversion of endogenous n-3 PUFAs could provide beneficial effects for the protection of cartilage chondrocytes and articular cartilage. We conducted in vitro experiments to study the possible mechanism. Our data showed that both addition of exogenous n-3 PUFAs and conversion of endogenous n-6 PUFAs to n-3 PUFAs downregulates the activity of the mTORC1 signalling pathway, thereby promoting cellular autophagy (figure 6). Conversely, addition of n-6 PUFAs produced the opposite effect. Autophagy, mainly regulated by the mTORC1 pathway, is considered to be a critical pathway for recycling cellular energy, which is essential for cellular survival in low-energy or stress situations.27 These results suggest that changes in the composition of endogenous n-6 and n-3 PUFAs may regulate cellular autophagy through the mTORC1 signalling pathway, thereby affecting the survival and activity of cartilage chondrocytes. In other words, conversion of endogenous PUFAs may be involved in the regulation of cartilage chondrocyte function and metabolism.

    Because the TG mice in this study systemically expressed the fat-1 gene, it is conceivable that other systemic factors, such as IL-1, TNFα and IL-6, are also involved in OA pathogenesis.23 ,28–30 On the other hand, we cannot ignore the possibility that enhancement of the synthesis of endogenous n-3 PUFAs might ameliorate systemic inflammation, which has been generally considered as a risk factor in OA progression.

    In summary, this is the first study to describe an association of the composition of endogenous n-6 and n-3 PUFAs with OA and the role of the mTORC1 pathway in this process. This study used TG mice expressing the fat-1 gene and produced the DMM-induced OA model, which is widely applied in OA studies. The results of this study require further validation using other OA models, such as the ageing OA mouse model or a fat-1 TG model in a large animal species, such as pig or rhesus monkey, to provide better guidance for clinical treatment.

    In conclusion, this study showed that enhancement of the synthesis of n-3 PUFAs in articular cartilage partially downregulates the activity of the mTORC1 signalling pathway, improves autophagy capability and promotes the survival and function of cartilage chondrocytes to delay OA development. These results suggest that more investigation of the role of the composition of endogenous n-6 and n-3 PUFAs in OA prevention and treatment is warranted.

    Acknowledgments

    We thank Lu Tang (Guangzhou Zhongke Kaisheng Medical Technology Co, Guangdong PR China) for excellent technical support with micro-CT.

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    Supplementary materials

    • Supplementary Data

      This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.

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    Footnotes

    • Handling editor Tore K Kvien

    • M-jH, LW and D-dJ contributed equally.

    • Contributors Design of the study: M-jH, LW, D-dJ and X-cB. Acquisition of data: M-jH, LW, Z-mZ, T-yC, C-hJ and YW. Interpretation of data: M-jH, LW, D-dJ, Z-mZ, T-yC, C-hJ, YW, X-cZ, BH, BY, Y-hC, S-fL, J-cY, Y-fD and X-cB. Manuscript preparation: M-jH, LW, D-dJ and X-cB.

    • Funding This work was supported by the State Key Development Program for Basic Research of China (2013CB945203), National Natural Sciences Foundation of China (91029727, 31271271, 81171746), Guangdong Natural Science Foundation (s2012010008209), Program for Changjiang Scholars and Innovative Research Team in University (IRT1142) and GDUPS (2011).

    • Competing interests None.

    • Ethics approval Ethical committee of Southern Medical University.

    • Provenance and peer review Not commissioned; externally peer reviewed.