The Effect And Mechanism Of Novel Pro-resolving Mediator Maresin-1 For The Therapy Of Osteoarthritis In Exercise

Objectives: Osteoarthritis(OA)is a chronic,age-related,progressive disease mostly reported in the knee joints.Its main pathological feature is the inflammatory manifestations and degenerative changes in various tissue structures within the joints,including articular cartilage degeneration,subchondral bone erosion,joint effusion,and inflammatory changes of the synovium.The main clinical symptoms of OA include increasing pain as the pathological features progress,stiffness,and immobility.The aging population has increasing possibility of OA.At present,its pathogenesis is not clear,but it is certain that its pathogenesis is a combination of multiple factors.There are many causative factors of OA,such as traumas,mechanical load,nutrients,genetic heredity,metabolic abnormalities,etc.It is a disease caused by excessively strong inflammatory reaction Chronic inflammation is a necessary process for tissue repair.In healthy joints,macrophage-like synovial cells and the specialized pro-inflammatory mediators(SPM)keep the entire homeostasis of the joint an important role,and the repair process of chronic inflammation also depends on whether this repair response is excessive.The biological control of inflammation procession and resolution runs through the development of OA.Recent studies have shown that in the process of active resolution of inflammation,SPM plays a vital role.Therefore,research on active resolution of inflammation is very important to promote the treatment of OA.The specialized pro-resolving mediator,Maresin-1,has been shown that in M2 macrophages[1],it,with DHA as a precursor,was catalyzed by lipoxygenase 12 / 15-LOX to form Small molecule fatty acid [2].Maresin1 can inhibit neutrophil infiltration,enhance macrophage phagocytosis of apoptotic neutrophils,down-regulate proinflammatory mediators production,inhibit activation of nuclear factor ?B(NF-?B)and increase of de novo synthesis of regulatory T cells and increase of intracellular cyclic adenosine monophosphate levetiracetam level to reduce inflammation,and also serve to reduce inflammatory pain and promote tissue regeneration.The anti-inflammatory effect of Maresin1 has been confirmed in a variety of diseases related to inflammatory reactions in organs,tissues,but little have not been reported in OA-related research.For other inflammatory diseases,Maresin-1 inhibits the activation of NF-?B and other pathways,and the activation of these two pathways can promote the synthesis and release of inflammatory factors like MMPs and IL-1? in synovial fibroblasts.It is found in outher studies that Maresin-1 can activate the PI3 K / AKT pathway and inhibit the expression of inflammatory factors,which also enables Maresin-1 as an antiinflammatory mechanism in OA.Based on the previous research by the research team,we investigated in this study the changes in Maresin-1 in joint lavage fluid under different exercise loads by training of treadmill running on rats with OA,and explore the anti-inflammatory effect and mechanism of Maresin-1 in synovial fibroblasts.Further,we explored the mechanism of sports therapy for OA,hoping to provide a theoretical basis for scientifically formulating a treatment plan for OA.Methods:1.To study the change trend of Maresin-1 level in the joint lavage fluid at different time points after different training of treadmill running for normal rats at a single time: SD rats were selected as subjects for training with different intensity at various time points.After the training,the rats were sacrificed the extracted joint lavage fluid at different time points;and the changes of Maresin-1 in joint lavage fluid were detected by ELISA method.2.To prepare the SD rat model of knee OA: 0.5 mg of monosodium iodoacetate(MIA)in 25 ?l of sterile water was injected into each knee joint cavity of the rats,after which the rats were set for free activity.3.Treat rat model of OA by injecting Maresin-1 into knee joint cavities,of which each was injected with 25 ?L of sterile saline with 10 ng of Maresin-1 twice a week for 3 weeks.After the course of treatment,rats were were sacrificed for tissues.4.Histological scoring was performed on gross sections,and the expressions of Type II collages and MMP13,and other related indicators of cartilage and synovium of rats with OA in each group were detected by western blot and immunohistochemistry.5.To prepare primary culture of chondrocytes and synovial fibroblasts,and simulate the inflammatory stimulation of OA in vitro.6.Detection of the effect of Maresin-1 on IL-1?-induced synovial fibroblast synovial fibroblasts and proteins in related pathways: Chondrocytes and synovial fibroblasts of chondrocytes from 3-5 generations of primary culture were selected to be induced by IL-1? with appropriate concentration selected by MTT,and then Maresin-1 was injected to observe its effect on chondrocytes and synovial fibroblasts to detect proteins in NF-?B and PI3 K / AKT,during which,LY294002 was applied to observe the effect of Maresin-1.Results:1.Trend of changes in maresin-1 levels of joint lavage fluid at different time points after treadmill training with different intensity in normal rats at a time:(1)It is found in ELISA test that the levels of Maresin-1 in joint lavage fluid in SD rats with the same exercise intensity at different time(0h,2h and 4h)increased,and it was peaked at 2h and dropped down to pre-exercise levels at 4h.(2)Through the ELISA test,no significant difference was shown in the levels of Maresin-1 in the joint lavage fluid in SD rats groped under different exercise intensity(lower,medium moderate and high intensity)at the same time after exercise.2.Evaluation of animal model of knee OA(1)Genera assessment of the knee joints of the rats: It was found that the knee joint cartilage surface in the control group was smooth,bright in color,and non-destructive;while in the model with OA,the cartilage surface was dark in color,the cartilage was damaged and cartilage volume decreased,osteophytes formed,synovial hyperplasia detected,joint effusion increased,and what's more,as time elapsed,all these OA performance including chondral defects deteriorated.(2)Results from HE and toluidine blue staining: the knee cartilage in normal rats was intact,the cartilage tangent was continuous and intact,the cytoplasmic matrix was uniformly distributed and arranged regularly,the radiation layer and calcified layer were complete and clear,and the chondrocytes in good growth.In the model of OA,cartilage was found damaged by joint calcification,cracked on the uneven surface of the cartilage tangent;toluidine blue staining showed inconsistent staining,and it became more obvious after injection.After OARSI and Mankin scoring,it was shown significant statistical differences between the OA model group and the normal group.3.Therapeutic effect of knee injection of Maresin-1 on model rats with OA(1)Maresin-1(10ng,twice a week for three weeks)showed a significant efficacy on rat model of OA.In the HE and toluidine blue staining,compared with OA group,the treatment group was kept more cartilage volume and intact structure in certain alignment,toluidine blue staining less faded;and the OARSI score and Mankin score indicated that there was a significant statistical difference between the treatment group and the OA group.(2)IHC staining showed that after treatment with Maresin-1(10ng twice a week for three weeks),compared with OA group,type II collagen in the large cartilage matrix of the treatment group was significantly increased,and cartilage was significantly increased.MMP13 in chondrocytes was significantly reduced,showing significant statistical differences.4.Detection of effect of Mareisn-1 on IL-1?-induced synovial fibroblast synovial fibroblasts and proteins in related pathway:(1)Maresin-1(1nM,10 nM,100nM)acts on synovial fibroblasts induced by IL-1?(1ng / ml),through which it was found by western blotting that Maresin-1 can reduce the protein expression of MMP-13.And there were significant statistical differences between the groups under different concentrations of maresin-1.(2)Maresin-1(1nM,10 nM,100nM)acts on synovial fibroblasts induced by IL-1?(1ng / ml).It was found by western blot that maresin-1 can activate the PI3 K / AKT and inhibit NF-?B p65 pathway,and different concentrations of Maresin-1 were indicated statistically significantly.(3)After adding Maresin-1(10nM)and then the PI3 K / AKT inhibitor,LY294002,it was found that inhibiting the PI3 K / AKT can increase the expression of MMP13.Conclusion:1.After a single-time treadmill running,the levels of Maresin-1 in knee joint lavage fluid of rats increased significantly at 2h,and dropped to normal level at 4h,indicating that Maresin-1 represented quick effect in production and metabolism.With the same exercise intensity,as the exercise time passed,the levels of Maresin-1 varying at different time points were not significantly different;while with the same exercise time,the levels of Maresin-1 gradually increased with different exercise intensity till to a certain degree of exercise intensity,no significant difference was showed in the level of maresin-1 at different time points.2.After treatment with knee injection of maresin-1,knee OA progressed significantly in rat model of OA,suggesting the anti-inflammatory effect of maresin-1 in OA.3.Maresin-1 suppressed inflammation in IL-1?-induced synovial fibroblasts and inhibited the expression of MMP13 by activating the PI3 K / AKT and inhibiting the NF-?B p65.