The Effect And Mechanism Of Lipoxin A₄ Via AMPK/NF-?B Signaling Pathway For The Therapy Of Osteoarthritis In Exercise

Objectives:Osteoarthritis?OA?,also known as degenerative disease,is a chronic progressive disease characterized by degeneration of articular cartilage,erosion of subchondral bone and synovitis.The main clinical symptoms included pain,stiffness and limited movement.With the aging of the population,the incidence of OA is increasing year by year.At present,the etiology and pathogenesis of OA are not clear,but it can be confirmed that the pathogenesis of OA is a complex pathological process with multiple factors.Mechanical stimulation is closely related to the biological activity of chondrocyte and can change the morphology and composition of cartilage.Chronic inflammation is one of the risk factors of osteoarthritis.In recent years,lipid factors produced by adipose tissue have been extensively studied in low-intensity inflammation.Studies have shown that inflammatory factors can cause further joint damage.Lipoxin A₄?LXA₄?is an endogenous decane produced in the process of cell-cell interaction,which is the termination signal of inflammation.As an anti-inflammatory mediator in recent years,it plays an important role in maintaining chronic inflammation in vivo.The progress of OA is closely related to chronic inflammation in joints.LXA₄ can activate human synovial fibroblasts,further inhibit the secretion of inflammatory cytokines and matrix metalloproteinases?MMPs?,thereby alleviating OA.Exercise not only acts on articular chondrocytes,but also changes in synovial fibroblasts.However,the mechanism of the effect of exercise on synovial fibroblasts is rarely studied.LXA₄ regulates intracellular Ca²⁺influx by activating the receptor.At the same time,appropriate pressure stimulation will consume ATP and increase AMP content.Both signal molecules activate AMPK?AMP-activated protein kinase?pathway in synovial fibroblasts.AMPK,as the main pathway of energy metabolism,can regulate energy balance.Intracellular energy metabolism can regulate inflammation,which regulates the energy metabolism of cells themselves.Activated AMPK can induce the degradation of the proteasome of NF-?B p65 and inhibit the NF-?B pathway.Studies have shown that AMPK pathway can inhibit the activation of NF-?B pathway induced by IL-1?and TNF-?,which inhibit the inflammation of synovium and the production of MMPs to protect chondrocytes.On the basis of previous studies,this study trained OA rats on the treadmill exercise to study the correlation between the increase of LXA₄ and cartilage of OA under different kind of treadmill exercise.To explore the effects of treadmill exercise on LXA₄ metabolism under different conditions.LXA₄ receptor antagonist?BOC-2?was used to clarify the role of LXA₄ in exercise therapy of OA.Under the condition of ensuring LXA₄ elevation,excessive stress stimulation can be reduced as far as possible,and more effective detection indicators can be provided for the formulation of various reasonable exercises.On this basis,LXA₄ is taken as the starting point to study the therapeutic mechanism of exercise on OA.In this study,synovial fibroblasts and chondrocytes were cultured.By adding LXA₄ and Flexcell stress loading system,the effects of LXA₄ on AMPK/NF-?B pathway in synovial fibroblasts and chondrocytes were observed.The pathways of LXA₄ generation,metabolism and regulation under exercise stimulation were revealed.The mechanism of exercise therapy for OA was elucidated,and the mechanism of exercise therapy for OA was further explored.Methods:1.SD rats were selected to study the changes of LXA₄ content in serum and intra-articular lavage fluid with different time after treadmill training of different intensity and duration respectively.2.Establishment of knee OA model in SD rats:SD male rats,8 weeks old,weight:220-240g,SPF.The rat model of OA was established by intra-articular injection of 50?L monosodium iodoacetate?MIA?containing 1.0 mg.3.According to the changing trend of LXA₄ under different exercise modes,different treadmill training is designed:?1?the same amount of exercise,moderate-intensity,different frequency of treadmill exercise;?2?the different intensity of treadmill training.4.Evaluation of OA of knee joint of rats in each group:?1?General observation and evaluation of knee joint of rats in each group;?2?HE and toluidine blue staining of knee joint tissue after section of rats in each group;OARSI and Mankin score were performed.5.The expression of related proteins in articular cartilage and synovial tissue of rats in each group was detected:?1?Western-blot was used to detect the expression of related proteins in cartilage and synovial tissue of each group;?2?The expression of related proteins in cartilage and synovial tissue of each group was detected by immunohistochemical method;?3?The expression of related proteins in cartilage and synovial tissue of each group was detected by qPCR.6.Primary cultures of chondrocytes and synovial fibroblasts were carried out to simulate the inflammation of OA in vitro.7.The effect of LXA₄ on IL-1?-induced synovial fibroblasts and the detection of related pathway proteins:3-5 passages of primary cultured synovial fibroblasts were selected,and MTT was used to select the appropriate concentration of IL-1?for induction.LXA₄ was used to detect the effects of IL-1?on synovial fibroblasts and the related pathway proteins.On this basis,BOC-2 was added to observe the effect of LXA₄.8.The effects of Flexcell stress loading system on IL-1?-induced AMPK/NF-?B pathway in chondrocyte:?1?The changes of ATP,ADP and AMP in chondrocyte were detected by high-spectral liquid chromatography;?2?The changes of ATP,ADP and AMP in chondrocyte were detected by fluo-3AM intracellular calcium staining,flow cytometry and immunofluorescence;?3?AMPK and p-AMPK were detected by Western-blot and qPCR.The expression of MMP-13,I?B-a,COL II and other related proteins and mRNAs.?4?The nuclear entry of NF-?B p65 was detected by immunofluorescence.Result:1.To study the changes of LXA₄ levels in serum and knee joint lavage fluid in normal rats at different time points after single treadmill training.?1?The level of LXA₄ in serum and joint lavage fluid of SD rats increased after treadmill training with the same intensity at different time?20,30 and 60 minutes?by ELISA.It increased within 2 hours of exercise and returned to normal level after 4hours,which indicated that it could rapidly generate and metabolize after exercise.?2?The levels of LXA₄ in serum and joint lavage fluid of SD rats after treadmill training of the same intensity at different time?20,30 and 60 minutes?were detected by ELISA.?3?The level of LXA₄ in serum and joint lavage fluid of SD rats increased after treadmill training at the same time with different intensity?low,medium and high intensity?detected by ELISA.There was significant difference between low and medium intensity,but there was no significant difference between moderate and high intensity.2.Evaluation of knee OA in each group?1?The general evaluation of the knee joint of the rats showed that the surface cartilage of the knee joint of the normal control group was smooth,bright and non-destructive.However,in the OA model?4-8 weeks after MIA injection?,the cartilage was gray and destroyed.With the prolongation of injection time,the cartilage defect became more serious.?2?HE and toluidine blue staining showed that the calcified cartilage?tidal line?in the normal group was intact,and the cartilage was relatively intact.In the OA model?4-8 weeks after MIA injection?,the cartilage with calcified joints was damaged,the surface was uneven,and the staining was stained with toluene ampere staining.With the prolongation of injection time,the staining was more obvious.The results of OARSI and Mankin scoring showed that there were significant statistical differences between the OA model group and the normal group.3.Designing different treadmill training according to the changing trend of LXA₄ under different sports modes.?1?The treadmill training of different periods is carried out under the condition that the total time of exercise is the same.Compared with OAE2?twice a day?and OAE3?three times a day?,OARSI and Mankin scores were significantly different,and the expression of cartilage-related proteins was consistent with the changes of tissue sections,and there was statistical significance.?2?The treadmill training with the same exercise time and different intensity.The results showed that moderate intensity treadmill training could alleviate cartilage damage and toluidine blue contamination in OA.Moreover,OARSI and Mankin scores showed that there were significant differences in moderate exercise intensity between OA group and OA group.?3?Moderate exercise intensity was selected and BOC-2?LXA₄ receptor antagonist?was intraperitoneally injected on this basis.OARSI and Mankin scores showed that there was significant difference between BOC-2 injection group and moderate intensity treadmill training group.Western-blot,qPCR and immunohistochemistry were used to detect the expression trend of related proteins,which was consistent with pathological sections,and the results were statistically different.4.Effect of LXA₄ on IL-1?-induced synovial fibroblasts and detection of related pathway proteins?1?Three to five generations of primary cultured synovial fibroblasts were selected.According to MTT test results,IL-1?using 1 ng/ml had statistical difference.?2?LXA₄?1nM?acts on synovial fibroblasts induced by IL-1??1ng/ml?.Western-blot showed that LXA₄ could reduce the expression of MMP-3 and MMP-13.?3?LXA₄?1nM?acts on synovial fibroblasts induced by IL-1??1ng/ml?.By immunofluorescence assay,LXA₄ can inhibit the nucleation of NF-?B p65 induced by IL-1?.?4?On the basis of adding LXA₄?1nM?and BOC-2?1nM?,we found that BOC-2 could increase the expression of MMP-3 and MMP-13 by Western-blot.?5?On the basis of adding LXA₄?1nM?and BOC-2?1nM?,it was found that BOC-2 could increase the nuclear entry of NF-?B p65 by immunofluorescence.5.Effect of Flexcell stress loading system on IL-1?induced AMPK/NF-?B pathway in chondrocytes?1?To study the effect of Flexcell stress loading system?CTS,0.5Hz,10%?on chondrocyte induced by IL-1beta at different time.The content of COL II detected by Western-blot showed that CTS at 4h and 8h had significant difference compared with chondrocyte induced by IL-1?,but there was no significant difference between CTS at 4h and 8h.?2?To study the effect of flexcell stress loading system?CTS,0.5Hz,10%?on normal chondrocytes at different duration.The content of COLII detected by western-blot showed that there was no significant difference among the groups of cells at 0,2h,4h,8h,16h and 24h.?3?The content of ATP,ADP and AMP in chondrocytes was detected by high-performance liquid chromatography?HPLC?after 4 hours of CTS.It was found that the content of AMP increased significantly and ATP decreased significantly after CTS.?4?The expression of AMPK,p-AMPK,MMP-13,I?B-a and NF-?B p65 in chondrocyte cytoplasm were detected by Western-blot after 4 h of CTS,and there were significant differences.?5?After CTS for 4 hours was detected by immunofluorescence,the nuclear of chondrocyte NF-?B p65 was detected.It was found that CTS could inhibit the nucleation of IL-1?-induced NF-?B p65.Conclusion:1.After treadmill training,the level of LXA₄ in serum and joint lavage fluid of rats increased significantly in 2 hours and returned to normal level after 4 hours,indicating that LXA₄ is the product of rapid production and rapid metabolism;In moderate exercise intensity,with the increase of exercise time,the level of LXA₄changes at different time points has no significant difference;in the same exercise time,with the increase of exercise intensity,the level of LXA₄ changes at different time points.The level of LXA₄ increased gradually,but there was no significant difference in the level of LXA₄ at different time points after exercise intensity reached a certain level.2.In treadmill exercise with the same amount of exercise,moderate intensity and different periods,treadmill training three times a day has better therapeutic effect.At the same time,according to the results of LXA₄ training with different treadmills,it is suggested that LXA₄ plays a certain role in it.3.In treadmill training with different intensity of exercise,moderate intensity has a better effect on alleviating the progress of OA in rats.BOC-2 can inhibit the therapeutic effect of moderate exercise intensity,which proves that LXA₄ plays an important role in exercise therapy of OA.4.LXA₄ alleviates the inflammatory response in synovial fibroblasts induced by IL-1beta by inhibiting the NF-?B signaling pathway.5.CTS at different time?0.5Hz,10%?acted on chondrocyte induced by IL-1?.It was found that CTS at 4H had the effect of alleviating inflammation.6.CTS?0.5Hz,10%?at 6.4h activates AMPK by increasing AMP/ATP and Ca²⁺influx,and further inhibits the nuclear translocation of NF-?B p65.7.Proper CTS did not change COLII content in normal chondrocytes,but decreased its response to IL-1?.