The Effect And Mechanism Of 15-HETE On The Therapy Of Osteoarthritis In Exercise

Objective:Osteoarthritis(OA)is the most common joint disease having an increasing prevalence[1].OA is characterized by articular cartilage degradation,subchondral bone formation,and joint swelling and causes j oint pain,stiffness,and even disability.However,OA is a poorly understood disease.Joint injury,gender,age,obesity,and occupational factors increase the risk of OA.During OA,chondrocytes secrete inflammatory mediators including interleukin(IL)-1?,a disintegrin and metalloproteinase with thrombospondin motif(ADAMTS)-5,matrix metalloproteinase(MMP3),and MMP-13,which contribute to OA progression.Exercise therapy has beneficial clinical effects on OA[2].Indeed,exercise prevents subchondral cyst formation and cartilage matrix loss and promotes cartilage glucosaminoglycan content,but improper exercise can aggravate OA[3][4].Since the mechanism of effect of exercise therapy on OA is not clearly understood,the optimal exercise type,frequency,duration,and intensity remain unclear,which possibly limits the beneficial effects of exercise therapy.15-hydroxyeicosatetraenoic acid(15-HETE)is metabolized from arachidonic acid by 15-lipoxygenase(15-LO),and has an anti-inflammatory effect.A previous study showed that chondrocytes express 15-LO[5].Further,15-HETE levels were increased in skeletal muscle and decreased in plasma after exercise in an OA model[6][7]).Several studies indicate that 15-LO and its metabolites have immunomodulatory and anti-inflammatory properties.15-HETE was found to inhibit leukotriene formation in experimental OA and neutrophil chemotaxis in chronic bronchitis[8,9].Recent studies indicate that the effect of 15-HETE is closely linked to the phosphoinositide 3-kinase(Pi3k)-protein kinase B(Akt)pathway[10-12].However,the effect of 15-HETE on exercise therapy during OA and its underlying mechanism remain unclear.In this study,we had that following aims:1)investigate the therapeutic effect of 15-HETE on OA,in vivo and in vitro;2)investigate whether the Pi3k-Akt pathway participates in the effect of 15-HETE;3)explore the possible mechanism of effect of exercise therapy on OA,which may related to the increased 15-HETE production in knee joint synovial fluid.Material and methods:1 Testing the amount of 15-HETE in joint after exerciseTo study the change of 15-HETE level in the joint lavage fluid at different time points after different training of treadmill running for normal rats and OA 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 to obtain the extracted joint lavage fluid at different time points;and the changes of 15-HETE in joint lavage fluid were detected by ELISA method,the concentration of protein was also tested to control the unequal of dilution.2 Testing the effect of 15-HETE on a rat OA model.To prepare the knee OA rat model:1 mg of monosodium iodoacetate(MIA)in 50?l of normal saline was injected into each knee joint cavity of the rats.To test the effect of 15-HETE on OA model,15-HETE were injected into knee joint cavity of the rats,twice a week for 4 weeks.Rats were then sacrificed,joints were then fixed in 4%formaldehyde for one week and decalcified with 20%ethylenediaminetetraacetic acid disodium salt.Serial 5-?m sagittal sections were collected for histological examination.HE and toluidine blue staining and Histological scoring was performed on gross sections,and the expressions of Type ? collages and MMP13 were detected by immunohi stochemi stry.3 Testing the effect of 15-HETE on rat primary chondrocytes.Primary chondrocytes of rats were obtained from knee joint of rats.IL-1? were applied to induce inflammation,15-HETE and LY294002 were applied to test the effect of 15-HETE and the function of Pi3k/Akt signal pathway.Western blot and Rt-PCR were used to test the expression of COL2,MMP13,MMP3,ADAMTS5,Akt and p-Akt.Results:Various 15-HETE concentrations were tested via ELISA after a single session of treadmill exercise of healthy rats.There was no significant difference in 15-HETE at 0 h at any exercise intensity.15-HETE levels in IALF in the low-,medium-,and high-intensity exercise groups were significantly elevated 2 h post-exercise and returned to nearly basal levels after 4 h.However,there was no significant difference between the low-,medium-,and high-intensity exercise groups at any time point.We then tested 15-HETE concentrations in IALF of MIA-induced OA rats after a single session of medium-intensity treadmill exercise.15-HETE concentrations in the 1-,2-,3-and 4-week OA groups were significantly elevated compared with the CG group.15-HETE concentrations at 2 hours after medium-intensity exercise in the 1-,2-,3-and 4-week exercise groups were significantly elevated compared with their respective non-exercising OA groups.2.We observed histological changes via X-ray images,as well as haematoxylin and eosin and toluidine blue staining.Joint damage was scored using the OARSI and Mankin systems.When we compared the CG,15-HETE,and MIA groups,we observed that MIA caused serious cartilage damage.The significant differences of OARSI and Mankin scores we detected between the MIA and MIA+15-HETE groups suggest that 15-HETE decreases cartilage damage.MMP-13 and COL2 expression in chondrocytes was also tested to confirm the effects of 15-HETE on OA.The expression of MMP-13 was significantly inhibited,whereas COL2 expression was significantly elevated by 15-HETE in the MIA-induced OA.3.Western blot and Rt-PCR were used to test the expression of COL2,MMP13,MMP3,ADAMTS5,Akt and p-Akt.IL-1? significantly increased the expression of MMP-13,MMP-3,and ADAMTS-5 and decreased COL2 expression.These pro-inflammatory effects were inhibited by 15-HETE in a dose-dependent manner.Indeed,30 nM 15-HETE significantly inhibited MMP-13,MMP-3,and ADAMTS-5 expression and increased COL2 expression.To investigate whether the Pi3k-Akt pathway is involved in the effect of 15-HETE,we used the Pi3k inhibitor LY294002 to block Pi3k-Akt signalling,and evaluated Akt and p-Akt using western blotting.LY294002 could block the downregulation of MMP-13,MMP-3,and ADAMTS-5.LY294002 also blocked COL2 upregulation.15-HETE significantly increased p-Akt,indicating that Pi3k-Akt signalling is involved in 15-HETE effects during OA.Conclusion:1.After a single-time treadmill running of both healthy and OA rats,the levels of 15-HETE in knee joint lavage fluid of rats increased significantly at 2h,and dropped to normal level at 4h on healthy rats,indicating that 15-HETE represented quick effect in production and metabolism.With the same exercise intensity,the levels of 15-HETE at different time points were not significantly different;suggesting that the different effect of different exercise intensities is not related to the synthesis of 15-HETE,but because of the high mechanic loading of high intensity exercise.2.After treatment with knee injection of 15-HETE,knee OA progressed significantly in rat model of OA,suggesting the anti-inflammatory effect of 15-HETE in OA.3.15-HETE suppressed inflammation in IL-1?-induced chondrocytes,inhibited the expression of MMP13,MMP3 and ADAMTS5,promote the expression of COL2,by activating the Pi3k/Akt.