| Objective: Osteoarthritis(OA)is a major source of pain and disability,with high socioeconomic cost.OA was estimated to affect 250 million by the end of 2019.The pathological changes of OA involve structural changes in articular cartilage,subchondral bone,ligament,joint capsule,synovial membrane and periarticular muscles.Its symptoms are mainly chronic pain,joint stiffness and limited function.Current clinical treatment methods for OA include surgical treatment and non-surgical treatment.However,both of them can only relieve symptoms but not reverse the course of OA.And according to the latest OARSI guidelines for the non-surgical management of knee OA,physical exercise is strongly recommended as the first-line of treatment regardless of OA level;ESCEO agrees with this recommendation.However,the intrinsic mechanism that helps relieve OA through physical exercise remains unclear.OA is currently considered to be a disease characterized by low-grade inflammation and high clinical heterogeneity.In particular,abnormal chondrocyte metabolism caused by changes of inflammatory microenvironment in the joint cavity may play a key role in cartilage degeneration and OA development.David J Hunter figured out that during the occurrence and development of OA,cartilage components would change and thus lose their integrity.In order to repair cartilage,hypertrophic chondrocytes would show higher synthetic activity,but would therefore produce more matrix degradation products and pro-inflammatory mediators.This results in decreased cartilage regulation and stimulates proliferation and inflammation of adjacent synovium.Overexpression of pro-inflammatory factors such as IL-1β and IL-6 has also been shown to cause progressive joint destruction and remodeling by stimulating matrix metalloproteinases.Many literatures suggest that the relieving effect of exercise on OA is to improve knee arthropathy by inducing the levels of various antiinflammatory mediators in the body and thereby antagonizing inflammation.In recent years,many exercise-related muscle factors have attracted wide attention due to their involvement in a variety of low-grade inflammation-related diseases.Understanding the correlation between these muscle factors and OA may contribute to the generation of new therapeutic strategies.Meteorin-like(Metrnl),as a novel adipomyokine,is overexpressed in the skeletal muscle after resistance exercise and downhill running,then released into circulation.Metrnl has an anti-inflammatory effect in multiple metabolic diseases,especially type2 diabetes mellitus,atherosclerosis,and rheumatoid arthritis.Clinical evidence indicated faster deterioration of OA joints with the coexistence of these diseases.Some studies have reported that metrnl is involved in arthritis and is a potential mediator and therapeutic target in OA,but sufficient evidence to prove the correlation and pathogenesis in OA is lacking.Therefore,we studied the effect and mechanism of exercise-induced Metrnl on OA articular cartilage.Inflammation is crucial to the occurrence and development of OA.The nuclear factor kappa B(NF-κB)plays a critical role in the pathogenesis of OA.NF-κB signaling pathway deeply participates in the occurrence an development of OA by affecting cartilage matrix remodeling,chondrocyte apoptosis,synovial inflammatory response and indirectly stimulating downstream regulators of chondrocyte terminal differentiation.Metrnl was reported reducing NF-κB nuclear transposition and IκB phosphorylation in skeletal muscle of mice induced by high fat diet.PI3K/Akt activates NF-κB and promotes its translocation into the nuclei through phosphorylation.The PI3K/Akt/NF-κB signal cascade has been reported to cause chondrocyte inflammation,with an increase in matrix metalloproteinases(MMPs)and a disintegrin and metalloproteinase with thrombospondin motifs 5(ADAMTS5)and a decrease in collagen II.Therefore,we investigated whether motion-induced Metrnl acts on the PI3K/Akt/NF-κB cascade to inhibit inflammation.Meanwhile,as a new type of programmed cell death,pyrodeath is also involved in the pathological process of OA.Activation of NF-κB has also been shown to activate the NLRP3 inflammasome(consisting of NLRP3,Caspase-1,and ASC)to promote the activation of Gasdermin D(GSDMD)drilling protein,leading to the release of pro-inflammatory mediators.We also explored the correlation between Metrnl and pyroptosis in OA cartilage,and discussed the role and mechanism of inflammation and pyroptosis.In this study,we used Sprague-Dawley(SD)rats to construct OA model,artificially by monosodium iodoacetate(MIA).We observed the effects of different exercise intensities on cartilage and the relationship between exercise intensities and Metrnl levels.In addition,we treated primary chondrocytes with IL-1β and recombinant Metrnl to investigate the anti-inflammatory and anti-pyroptotic effects.To this end,we targeted the PI3K/Akt/NF-κB and NLRP3/caspase-1/GSDMD pathways in chondrocytes.Methods:1.The OA model(OAG)was obstructed by injecting MIA(0.5mg/50 μL sterile saline)into the knee cavity of normal SD rats,and different exercise intensity groups were designed(low intensity exercise group OAL:15.9 m/s,60 min/day,5days/week,4 weeks;The OAM of moderate intensity exercise group was 19.2m/s,60 min/day,5 days/week,4 weeks;Higher intensity exercise group OAH: 26.8m/s,60 min/day,5 days/week,4 weeks.).ELISA was used to observe the changes of Metrnl levels in serum and joint lavage fluid of control group,OAG group and different intensities group.2.Rats knee joint evaluation: 1)X-ray imaging of rat knee joints;2)The gross knee joint of rats was photographed and observed;3)The knee sections of rats were stained with HE and toluidine blue,and the results were evaluated by OARSI and modified Mankin score.3.Immunohistochemical analysis for knee sections: 1)The expression differences of Metrnl,collagen 2,MMP 13,ADAMTS5 were observed using immunohistochemistry to explore the effects of different intensities of exercise on OA cartilage and the correlation between exercise and Metrnl level;2)The expression level of p-Akt and NF-κB was deter mined by immunohistochemistry to verify the anti-inflammatory mechanism of exercise on OA;3)The expression levels of NLRP3,Caspase-1 and GSDMD were detected to verify the antipyroptosis effect and mechanism of exercise on OA.4.Primary SD rat chondrocytes were isolated to simulate the inflammatory phenotype of OA in vitro.5.The adherent cells were identified by immunofluorescence using collagen 2 and the appropriate IL-1β induction concentration was deter mined by CCK8.6.Deter mination of Metrnl’s effect on IL-1β-induced primary chondrocytes: western blotting and q RT-PCR were used to detect changes in protein and m RNA levels of cartilage specific indicators(Collagen 2,MMP 13,ADAMTS5,NF-κB).7.Mechanism of Metrnl protecting IL-1β-induced primary chondrocytes: 1)Western blotting and immunofluorescence were used to investigate the anti-inflammatory mechanism of Metrnl by observing the changes in the protein levels of p-PI3 K,PI3K,p-Akt,Akt,p-NF-κB,NF-κB and the nuclear translocation of NF-κB were deter mined.2)Flow cytometry,cell electron microscopy,and western blotting(NLRP3,caspase-1,GSDMD)were used to investigate the anti-pyroptotic effect of Metrnl on IL-1β-induced primary chondrocytes.8.The correlation between anti-inflammatory and anti-pyroptotic effects of Metrnl on primary chondrocytes: 1)The inhibition of inflammation-related pyroptosis of chondrocytes by Metrnl was verified by inhibiting PI3K/Akt/NF-κB pathway using the overexpressed NF-κB plasmid.2)NF-κB p65 si RNA was used to investigate whether the anti-pyroptotic effect of Metrnl was completely dependent on the inhibition of NF-κB.Results:1.After treadmill training of SD rats in different exercise groups,Metrnl concentrations in serum and lavage fluid in knee cavity were changed: compared with the control group,the concentration of Metrnl in OAG group was decreased,while the concentration of Metrnl in all exercise groups were significantly increased.2.Rat model evaluation:(1)X-ray imaging: OAG and OAH group showed obvious joint space stenosis,joint surface sclerosis and joint neoplasm compared with CG group.The pathogenetic changes were alleviated in OAL and OAM group,especially OAM group.(2)Gross imaging of rat knee joints: In CG group,the surface of cartilage was smooth with bright color;And that in OAG and OAH group was obviously rough,fibrosis,cracks,defects with subchondral bone esosion and pale color.While the cartilage damage in OAL and OAM group was significantly reduced.The score suggested that OAM group could best relieve OA cartilage injury.(3)HE and toluidine blue staining: The articular cartilage layer(tidal line)of CG group was intact;And that of OAG group was broken with uneven surface defects;While OAM group could effectively relieve OA cartilage destruction and repaired cartilage defects.OARSI and Mankin scores showed that OAG group score was significantly higher than that of CG group,and the OAM group score was lowest among the groups.(4)Immunohistochemical staining: OAL,OAM and OAH group significantly increased the expression of Metrnl;The expression of collagen 2 in OAG group was significantly decreased compared with CG group,which was recoverd most in OAM group;And compared with CG group,the expressions of MMP13,ADAMTS5,NF-κB p65,p-Akt,NLRP3,caspase-1 and GSDMD in OAG group were significantly increased,while in OAM group the increase were reversed.3.Effects of Metrnl on IL-1β-induced primary chondrocytes:(1)The immunofluorescence of collagen 2 showed that the adherent cells expressed collagen 2 which identified to be chondrocytes combining the cell morphology.(2)CCK8 assay showed that 24 h of recombinant Metrnl ad ministration had no effect on chondrocyte viability within the range of physiological concentration measured by ELISA;And 24 h of IL-1βad ministration in 10 ng/ m L had the most appropriate effect on chondrocytes to construct chondrocyte inflammation model.(3)Western blotting showed that the expression level of collagen 2 in IL-1β group was significantly decreased compared with the control group,and recombinant Metrnl effectively recovered the expression of collagen 2.The expression levels of MMP 13 and ADAMTS5 in the IL-1β group were significantly higher than those in the control group,and the recombinant Metrnl reversed the change.(4)q RT-PCR was used to detect the m RNA changes of collagen 2,MMP 13,ADAMTS5 and NF-κB.The m RNA expression level of collagen 2 in IL-1βgroup was significantly decreased compared with that in control group,which was improved by Metrnl treatment;And the m RNA expression levels of MMP13,ADAMTS5 and NF-κB in IL-1β group were significantly increased compared with control group.Samely,Metrnl ad ministration reversed the change.4.The effect and mechanism of Metrnl on IL-1β induced inflammation of Primary chondrocytes:(1)We used western blotting to detect inflammatory proteins to evaluate whether Metrnl exerts its anti-inflammatory effect through inhibiting PI3K/Akt/NF-κB pathway.Metrnl ad ministration suppressed the phosphorylation of PI3 K,Akt and NF-κB facilitated by IL-1β.(2)Immunofluorescence analysis of NF-κB p65 showed that the translocation of NF-κB into the nuclei increased significantly in the IL-1β group,while that decreased in the Metrnl treatment group.5.The effect and mechanism of Metrnl on IL-1β induced pyroptosis of Primary chondrocytes:(1)Caspase-1 fluorescence inhibition probe FLICA 660-VVAD-FMK was used to label chondrocytes,and flow cytometry indicated that the double positive percentage of chondrocytes in IL-1β treatment group was significantly higher than that in control group.However,the percentage of double positive cells in Metrnl treatment group decreased gradually.(2)Transmission electron microscopy of chondrocytes showed that chondrocytes in IL-1β treated group showed cytoplasmic edema,cell membrane swelling,cell nucleus shrinkage,and organelle vacuolation under the microscope.Metrnl pre-incubated group significantly improved cell morphology.(3)The expression levels of pyroptosis proteins were detected using western blotting.The expression levels of NLRP3,caspase-1,and GSDMD in IL-1βgroup were significantly increased compared with the control group,while Metrnl pre-incubated group decreased the levels.6.Correlation between the anti-inflammatory and anti-pyroptotic effects of Metrnl on primary chondrocytes:(1)Chondrocytes were transfected with NF-κB p65 overexpression plasmid to observe whether the overexpression of NF-κB p65 blocked the inhibitory effect of Metrnl on IL-1β-treated chondrocytes.The results showed that the expression levels of NLRP3,Caspase-1 and GSDMD in NF-κB p65 overexpression group increased compared with that in Metrnl(25 ng/ m L)preincubated group.(2)Chondrocytes were transfected with NF-κB p65 si RNA to investigate whether NF-κB p65 was the necessary mediator in the anti-pyroptotic effect of Metrnl.The results showed that NLRP3,GSDMD and caspase-1 levels in the Metrnl pre-incubated group were still decreased after NF-κB p65 si RNA knockout.Conclusion:1.The serum and lavage fluid concentration of Metrnl can be induced by exercise.The concentration of Metrnl increased with the increase of exercise intensity,which suggests that Metrnl may play a role in exercise protecting OA cartilage.2.Among different exercise intensity groups,the OAM group can best alleviate SD rats OA severity.3.Among different exercise intensity groups,the OAM group can inhibit chondrocytes inflammation and pyroptosis to alleviate SD rat joint cartilage injury most.4.The recombinant Metrnl had no significant influence on the activity of primary chondrocytes within the concentration in serum and articular lavage fluid measured by ELISA.5.Recombinant Metrnl pre-incubation inhibits IL-1β-induced primary chondrocyte inflammation by inhibiting the PI3K/Akt/NF-κB pathway.6.Recombinant Metrnl pre-incubation inhibits IL-1β-induced primary chondrocyte inflammation by inhibiting the NLRP3/caspase-1/GSDMD pathway. |
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