| BACKGROUNDSOsteoarthritis (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. 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 low level of a steady dynamic equilibrium between synthesis and degradation. Sparsely distributed chondrocytes are crucial for maintaining the ECM homeostasis An imbalance involving reduced synthesis and increased degradation of cartilage ECM by various factors leads to OA. Over the past several decades, OA has been intensively studied and increasingly understood. Unfortunately, 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 an expensive, invasive and high-risk joint replacement surgery. 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 pro-inflammatory effects whereas n-3 PUFAs exhibit, on balance, anti-inflammatory effects. Extensive in vivo and in vitro studies regarding the effects of PUFAs on OA have been conducted. Although the results of these studies generally suggest that exogenous supplementation of n-3 PUFAs could delay OA onset while high n-6 diets could accelerate OA progression, the underlying mechanisms have not been fully elucidated. Additionally, previous studies have primarily focused on the effects of exogenous PUFAs on OA, however, studies on the association between endogenous PUFAs and OA, specifically the endogenous n-6 and n-3 PUFAs composition 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, especially considering 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. Consequently, additional research focus on the endogenous n-6 and n-3 PUFAs composition is warranted.Mammalian target of rapamycin complex 1 (mTORC1) is critical for regulating cell growth, metabolism and autophagy. 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. Intragastric administration of rapamycin, an important inhibitor of mTORC1, into mice with medial meniscus destabilisation-induced OA can specifically inhibit the mTORC1 pathway activity and activate autophagy of articular chondrocytes, consequently significantly improving the OA symptoms. 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. Collectively, we propose alterations of endogenous n-6 and n-3 PUFAs ingredient may affect OA incidence partially through regulating the mTORC1 signalling pathway.We have previously generated a transgenic 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. This conversion can change the endogenous n-6 and n-3 PUFAs composition. Accordingly, we used the fat-1 transgenic mouse model to evaluate for the first time the effects of constitutive n-3 PUFAs synthesis enhancement on OA pathogenesis and the relationship between PUFAs and the mTORC1 signalling pathway in articular chondrocytes. The aim of this study was to provide additional objective and scientific evidence for the application of PUFAs for the clinical treatment of OA.OBJECTIVESExogenous supplement of n-3 polyunsaturated fatty acids (PUFAs) have been reported to prevent osteoarthritis (OA) through undefined mechanisms. This study aimed to investigate alterations of endogenous PUFAs composition on OA and associations of endogenous PUFAs with mammalian target of rapamycin complex 1 (mTORC1) signalling pathway regulation, a critical autophagy pathway in fat-1 transgenic mice, and to provide further evidences for the clinical using of n-3 PUFAs in the prevention and treatment of OA.METHODSUsing 24 mice (female,8 weeks old) with transgenic (TG) and wildtype (WT) backgrounds, an OA model was created by destabilization of the medial meniscus. The endogenous PUFAs composition in mouse various tissues was analysed by gas chromatography, and OA incidence was evaluated by microtomography (micro-CT), scanning electron microscopy, histological and histochemical methods. The level of systematic pro-inflammatory cytokines and OA-associated proteins were detected by ELISA assay kits. Additionally, primary chondrocytes were isolated and cultured. The effect of exogenous PUFAs and changes in endogenous PUFAs composition on mTORC1 pathway signalling and autophagy in chondrocytes were assessed by western blot, and the IL-1β induced in vitro inflammation model were conducted to observe the influence of alteration of PUFAs composition on the expression of pro-inflammatory cytokines. The resluts were presented as mean±SD and analyzied by SPSS 13.0 software. The means of two independent sample were compared with Independent Sample Test, while the means of paired sample were analyzed with Paired Sample Test, P values<0.05 were considered statistically significant.RESULTS1、Endogenous n-6 and n-3 PUFAs composition were optimized in TG mice and changed after DMM-induced OAThe difference in n-6 and n-3 PUFAs composition in cartilage, serum and tail were compared between TG and WT mice. TG mice presented increased n-3 PUFAs level and reduced n-6 PUFAs level versus WT mice. Additionally, the n-6/n-3 PUFA ratio was significantly decreased in TG mice versus WT mice (P<0.001). Interestingly, the n-3 PUFAs level in articular cartilage of the DMM group was significantly increased compared to the sham group at 4 weeks after surgery (P<0.001), with slightly and significant increased n-6 PUFAs alteration (P=0.009, 0.014). In addition, the n-6/n-3 ratio was reduced significantly after surgical induction. These results suggested that the endogenous PUFAs composition of TG mice was optimized by both increased n-3 PUFAs and decreased n-6 PUFAs. Additionally, the endogenous n-3 PUFAs were increased with response to surgically induced OA.2、The systemic anti-inflammatory effect of optimized endogenous PUFAs composition in TG mice during OA progression compared to WT mice. The TG and WT mice were sacrificed and the serum was collected after 8 weeks post-operative. Serum samples collected from age matched mice, which did not received any surgical treatment, were selected as negative control. Serum IL-1β, TNF-a, HA and SAA were detected with ELISA assay kit. The results suggested that serum levels of IL-1β、TNF-α, HA and SAA were significantly increased, respectively, in both WT and TG group after 8 weeks post-operation than the non-operative group. However, the increasing rate of serum IL-β, TNF-α and HA levels were significantly decreased in TG group in comparision of WT group respectively, while there was no significant difference in the serum SAA level between the TG group and the WT group after 8 weeks post-operation (P=0.843).3、Delayed progression of osteoarthritis in TG mice after surgically induced OATo 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 using Mimics 5.0 software. The volume and surface area of periarticular osteophytes were significantly reduced at 4 and 8 weeks after surgery in TG versus WT mice. 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 presented with 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. TG mice in the DMM group only presented with stripped cartilage and superficial avulsion in the weight-bearing cartilage. These results adequately indicated that endogenous n-3 PUFAs synthesis enhancement from n-6 PUFAs could alleviate articular cartilage lesions, reduce development of osteophytes and delay OA pathogenesis.Notably, the systemic effect of PUFAs composition on inflammatory cytokine in serum should also be taken into consideration. The IL-1β, TNF-α, hyaluronic acid (HA) and serum amyloid A protein (S AA) in serum of TG group was significantly decreased compared to WT group 8 weeks postoperation. Furthermore, it has been suggested that chondrocytes with optimized PUFAs composition from TG mice could alleviate IL-1β-induced inflammatory cytokines release from our in vitro study.4、Alleviation of the loss of cartilage proteoglycan and chondrocytes in TG mice after surgically induced OATo confirm the effects of endogenous PUFAs on protecting joint cartilage and delaying OA pathogenesis, we performed safranin O-fast green staining, calculated chondrocyte number and applied Mankin scores for evaluating 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 (P<0.001). High magnification imaging revealed that the tidemark was disarranged and had nearly disappeared in WT mice, while the tidemark was not significantly changed in TG mice. The joint cartilage thickness was assessed by toluidine blue staining. The results showed that compared with TG mice, WT mice presented with severe abrasion, and the cartilage thickness was significantly reduced (P<0.001). These results indicated that endogenous n-3 PUFAs synthesis enhancement from n-6 PUFAs could alleviate the loss of cartilage proteoglycan and chondrocytes after surgery-induced OA.5、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. We selected MMP-13 and ADAMTS-5 for IHC assesessment, which are 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. The results showed that the proportion of MMP-13- and ADAMTS-5-positive cells was significantly increased in WT mice compared with TG mice after surgically induced OA (P<0.001). In summary, these results indicated that endogenous n-3 PUFAs synthesis enhancement from n-6 PUFAs could decrease MMP-13 and ADAMTS-5 expression, leading to alleviated cartilage ECM degradation.6、Protecting effects of endogenous n-3 PUFAs on chondrocyte autophagy during OA progression in TG mice compared to WT mice.The knee paraffin sections were conducted with Immunofluorescence staining for LC3-Ⅱ. The images were captured using laser scanning confocal microscope and representative images were shown. The nucleus is stained with DAPI and is shown in blue, and LC3-Ⅱ is shown in red. The results showed that LC3-Ⅱ expression was gradually decreased during OA development, which indicated the role of autophagy deficit in the progression of OA. However, the TG mice could ameliorate autophagy inactivation compared with WT mice during OA progression.7、PUFAs affect cartilage chondrocyte autophagy by regulating the mTORCl signalling pathwayPrevious studies have demonstrated that PUFAs could mediate OA pathogenesis, but no relevant research on 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. Before further experiments, PUFAs composition of cultured chondrocyte has also been tested to imitate the same condition in vivo. The results suggested the content of both n-3 and n-6 PUFAs was slightly decreased but the n-6/n-3 ratio was not changed significantly compared with the cartilage in vivo.Amino acid starvation was performed to study the effect of exogenous PUFAs (DHA and AA) on the mTORC1 signalling pathway and autophagy. The results showed that AA significantly promoted ribosomal S6 protein phosphorylation and inhibited the expression of LC3-Ⅱ. However, DHA significantly inhibited ribosomal S6 protein phosphorylation and enhanced the expression of LC3-Ⅱ. Moreover, DHA could antagonise the effect of AA. These results indicated that exogenous n-6 PUFAs inhibit cartilage autophagy by upregulating mTORC1 activity, while n-3 PUFAs could induce the opposite result and antagonise the effect of 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.Futhermore, we conducted immunofluorescence staining for LC3-Ⅱ protein to prove the chondrocyte autophagy protecting effects of endogenous n-3 PUFAs in OA progression. The results showed that LC3-Ⅱ expression was reduced in OA onset, but there was an significantly increased expression of LC3-Ⅱ in TG mice versus WT.In summary, endogenous n-3 PUFAs synthesis enhancement from n-6 PUFAs could exhibit an autophagy protecting effects of articular chondrocytes in OA progression. probably through downregulating the mTORCl signalling pathway.8、PUFAs composition of in vitro cultured chondrocytes was similar with the cartilage in vivo and IL-1β induced inflammatory cytokines release were reduced in TG mice chondrocyte versus WTThe 2nd generation chondrocyte samples were collected and the total fatty acids were extracted, PUFAs composition was detected with Gas Chromatography. The results showed that n-3 and n-6 PUFAs was slightly decreased but the n-6/n-3 ratio was not changed significantly in cultured chondrocytes compared with the cartilage in vivo (P<0.001). For the in vitro inflammatory model, the chondrocytes were cultured in serum-free medium with or without lOng/ml IL-1β for 48hs. Then the supernatant were collected and detected with ELISA assay kit. The result suggested that IL-1β induced TNF-α, IL-6, PGE2 release was significantly reduced in TG group versus WT group.Conclusions:In summary, this is the first study to describe an association of the endogenous n-6 and n-3 PUFAs composition with OA and the role of the mTORC1 pathway in this process. This study used transgenic mice expressing the fat-1 gene and produced the DMM-induced OA model, which is widely applied in OA studies. In conclusion, this study demonstrated that a constitutive enhancement of n-3 PUFAs synthesis in articular cartilage could partially downregulate the activity of the mTORC1 signalling pathway, improve autophagy capability and promote the survival and function of cartilage chondrocytes to delay OA development. These results suggest that more attention regarding the role of the endogenous n-6 and n-3 PUFAs composition in OA prevention and treatment is warranted. |
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