HDAC6 Regulates Substrate Stiffness-mediated Inflammation Response In Chondrocytes

Objective:Osteoarthritis(OA)is a common chronic disease of articular cartilage and difficult to cure,and it is rooted in the imbalance of cellular metabolic activity caused by abnormal mechanical microenvironment of cartilage extracellular matrix(ECM).Substrate stiffness,as a key physical factor in the cell microenvironment,is involved in modulating cell morphology,phenotype,and mechanical behavior.Chondrocytes are the only cell type in the articular cartilage,are slowly renewed,and are highly mechanosensitivity.Mechanical therapy has received attention as a therapeutic direction for osteoarthritis.However,how substrate stiffness affects the anti-inflammatory response and morphology of cells remains to be investigated.Inhibition of histone deacetylase 6(HDAC6)has been reported to contribute to osteoarthritis amelioration and modulate primary cilia length,but whether it plays the same role on different substrate stiffness remains unclear.In this study,we used polydimethylsiloxane(PDMS)to fabricate cell growth substrates with different elastic modulus to mimic different mechanical microenvironments,used IL-1β to mimic inflammatory environments,and used Tubastatin A(Tub A)to inhibit HDAC6.The following hypotheses are explored:(1)Whether substrate stiffness affects the response of chondrocytes to inflammation.(2)The effects of HDAC6 on the morphology and function of chondrocytes.(3)The influences of substrate stiffness,inflammation and HDAC6 in the mechanical properties of chondrocytes.Methods:1.Fabricate three PDMS substrate of different stiffness,which are stiff substrate,medium substrate and soft substrate.Primary chondrocytes from suckling mice of C57BL/6(5-7 days)were extracted and cells were seeded on three different substrate stiffness.The inflammatory environment was simulated by IL-1β at a concentration of 10ng/ml for 12 h.NO and PGE2 release were detected by NO and PGE2 assay kits in inflammatory and non-inflammatory environments on varying PDMS substrate,and the inflammatory response of chondrocytes on different substrate stiffness was compared.2.Western blot was used to detect the protein expression levels of COL2 and SOX9,the key indicators of matrix synthesis,and MMP13,one of the indicators of matrix degradation.To compare the response of matrix metabolism of chondrocytes to inflammatory and non-inflammatory conditions on substrate stiffness.3.HDAC6 was inhibited by Tub A,before and after HDAC6 inhibition,the level of matrix metabolism,NO and PGE2 release in response to inflammation on different substrate stiffness were detected.4.Cell immunofluorescence staining was used to stain the cytoskeketon with Phalloidin and primary cilia with Arl13 b.Before and after HDAC6 inhibition,chondrocyte size and morphology were compared between inflammatory and non-inflammatory environments on varying substrate stiffness,and the length of primary cilia were counted.Results:1.There was no significant difference in the release of NO and PGE2 on stiff,medium,and soft substrates without IL-1β treatment.After IL-1β treatment for 12 h at the concentration of 10ng/m L,the release of NO and PGE2 increased greatly,and significantly,the inflammatory signal on the stiff substrate was lower than that on soft substrate.2.Western blot results showed that COL2 and SOX9 protein expression were higher on stiff substrate,and MMP13 protein expression was higher on soft substrate.COL2 and SOX9 expression were significantly decreased during IL-1β treatment,and COL2 was lowest on soft substrate.MMP13 was significantly increased,and higher on soft substrate.3.HDAC6 activity differed on varying substrate stiffness without IL-1β treatment.On stiff substrate,HDAC6 activity was lower,while COL2 and SOX9 expression were higher and MMP13 expression was lower;On soft substrate,HDAC6 activity was higher,while COL2 and SOX9 expression were lower and MMP13 expression was higher.HDAC6 activity decreased when chondrocytes were treated with IL-1β,however,instead of causing an increase in COL2 and SOX9 protein levels and a decrease in MMP13 protein level,the decrease in HDAC6 activity showed the opposite result.These results suggested HDAC6 may play different roles in inflammatory and non-inflammatory environments.Interestingly,on stiff substrate,the inflammatory and non-inflammatory environments had little effect on HDAC6 activity,but had an obvious response to inflammatory mediators and COL2,SOX9,and MMP13,suggesting that as for stiff substrate,its responses to inflammation is not directly dependent on HDAC6 activity.When HDAC6 was inhibited by Tub A for 24 h at a concentration of 10μM,the increase of NO and PGE2 in inflammatory environment was inhibited,but the degree of inhibition on stiff and soft substrates had no difference,and the decrease of COL2 and SOX9 were reversed,and the degree of reversal on stiff substrate was less than on soft substrate.4.Using Image J software to count the cell and nuclear spreading area under different treatment conditions.The results showed that both the cell area and nuclear area on stiff substrate were larger than those on soft substrate.After IL-1β treatment,the cell area and nuclear area became larger,especially on soft substrate.When HDAC6 was inhibited by Tub A,the spreading area was also increased,however,unlike IL-1β treatment,chondrocytes treated with Tub A were more dendritic antennae.5.Primary cilia were longer on stiff substrate.When chondrocytes were treated with IL-1β,the length of primary cilia was significantly prolonged,and increasesd more on soft substrate than on stiff substrate.When inflammation was inhibited with Tub A,the effect of IL-1β elongating primary cilia was amplified.Conclusion:1.The response of chondrocytes to inflammation is substrate-dependent,and stiff substrate has better anti-inflammatory effect than soft substrate.2.Substrate stiffness affects the level of extracellular matrix(ECM)metabolism,stiff substrate is more conducive to matrix synthesis than soft substrate,and in an inflammatory environment,the metabolic level on stiff substrate is slightly better than that on soft substrate.3.HDAC6 may play different roles in inflammatory and non-inflammatory environments,and there is a certain correlation between HDAC6 enzyme activity and substrate stiffness and matrix metabolism levels in non-inflammatory environments,however,this correlation is destroyed by inflammatory stimuli.Inhibition of HDAC6 resists inflammatory stimuli,especially on soft substrate.4.The chondrocytes area spreading and the length of primary cilia are substrate-dependent,and the spreading of cells and the length of primary cilia on stiff substrate are significantly higher than that on soft substrate.Inflammatory stimulation increases the cell spreading area and prolongs the primary cilia,and HDAC6 inhibition synergistically amplifies this effect.However,after HDAC6 inhibition treatment,the morphology of chondrocytes also changed significantly,showing more dendritic-like antennae.