ACY-1215 Exhibits Anti-inflammatory And Chondroprotective Effects In Human Osteoarthritis Chondrocytes Via Inhibition Of STAT3 And NF-κB Signaling Pathways

Background:Osteoarthritis(OA)is one of the most common chronic bone and joint diseases in middle-aged and elderly people,and it is also the most common disabling chronic joint disease.With the increase of age,the incidence of osteoarthritis gradually increases.The clinical manifestations are often pain,joint stiffness,joint deformity,etc.,and the quality of life of patients will be greatly affected.The previous view is that osteoarthritis is mainly caused by cartilage wear,and sports injury or heavy physical labor is the main cause of its disease.With the deepening of basic medicine research on osteoarthritis,it is currently considered that inflammation is also osteoarthritis.One of the indispensable factors of the disease.Some inflammatory factors can promote the secretion of matrix metalloproteinases(MMPs)by chondrocytes,thereby accelerating the degradation of cartilage extracellular matrix,leading to cartilage degeneration.At present,early and mid-stage osteoarthritis often uses non-steroidal anti-inflammatory drugs for analgesic treatment,and advanced severe osteoarthritis often requires joint replacement surgery to relieve pain and improve joint function.Long-term use of non-steroidal anti-inflammatory drugs such as ibuprofen can cause gastrointestinal side effects,and joint replacement surgery is also a heavy economic burden for patients.Therefore,it is particularly important to explore osteoarthritis for the treatment of new and effective small molecule drugs.Objective:The aim of this study was to investigate whether ACY-1215,a highly selective inhibitor of histone deacetylase 6,has anti-inflammatory and cartilageprotective effects on human osteoarthritic chondrocytes and its mechanism of action.Methods:The cartilage specimens were obtained from OA patients who underwent surgery in the department of joint surgery,the First Affiliated Hospital of Anhui Medical University,form October 2017 to May 2018.The chondrocytes were identified by toluidine blue method and type II collagen immunofluorescence staining.In an in vitro environment,in order to simulate a model of inflammatory environment in chondrocytes,chondrocytes are stimulated with interleukin 1β(IL-1β)to obtain a chondrocyte model of osteoarthritis.Subsequently,chondrocytes were treated with different concentrations of ACY-1215,and the treated chondrocytes were assayed for viability using CCK8 method,thereby screening out the concentration range of ACY-1215 which is non-toxic to chondrocytes,and conveniently setting the concentration of anti-inflammatory against ACY-1215.And cartilage protection to explore.In the human chondrocyte experiment,a blank control group,a DMSO alone treatment group,an ACY-1215 treatment group,an IL-1β treatment group,and an IL-1β+ACY-1215 concentration group were added.The mRNA levels of IL-1β,IL-6,MMP-1,MMP-13 and COL2A1 were detected by Real-time PCR,and IL-1β,IL-6,MMP-1 and MMP-13 were detected by Western blot.COL2A1,p-STAT3,STAT3,p-IKK,IKK,p-NFκB P65,NF-κB P65,p-IKBα,IKBα were detected for protein levels.Results:The mRNA and protein expression levels of IL-1β,IL-6,MMP-1 and MMP-13 in the blank control group,DMSO alone treatment group and ACY-1215 treatment group were not significantly different(P>0.05).The expression levels of IL-1β,IL-6,MMP-1 and MMP-13 in IL-1β treatment group were significantly higher than those in the control group(P<0.05),and NF-κB and STAT3 signaling pathways were activated in IL-1β treatment group.Related proteins such as p-STAT3,p-NFκB P65,p-IKBα,and p-IKK were significantly higher than the control group(P<0.05),while the IL-βBsignaling pathway inhibitory protein IKBα was significantly lower in the IL-1β-treated group than in the control group.(P<0.05).The expression levels of IL-1β,IL-6,MMP-1and MMP-13 in IL-1β+ACY-1215 group were significantly lower than those in IL-1βalone group(P<0.05).The expressions of p-STAT3,p-NFκB P65,p-IKBα,p-IKK in IL-1β+ACY-1215 group were significantly lower than those in IL-1β alone.The level of IKBα protein was significantly higher than that of IL-1β alone.Group(P < 0.05).In addition,the expression of COL2A1 protein in IL-1β+ACY-1215 group was significantly higher than that in IL-1β alone group(P<0.05).Conclusion:ACY-1215 inhibits the expression of IL-1β,IL-6,MMP-1,and MMP-13 in human osteoarthritic chondrocytes and promotes the synthesis of COL2A1,thereby exerting anti-inflammatory and cartilage protective effects.Inhibition of STAT3 and NFκB signaling pathways.