| Objective: Osteoarthritis(OA)is a progressive degenerative joint disease,which is the most common in the elderly and can occur in both men and women.Osteoarthritis is the most common disease of the knee joint,other weight-bearing hip joint,spine and other parts can also occur,in addition,the hand joint is also a common site of the disease.So far,the cause is still unknown.Its main pathological features are articular cartilage degeneration,periarticular hyperosteogeny with osteophyte formation,subchondral sclerosis remodeling,chronic inflammation of synovium and so on.the main related factors include age,sex,race,heredity,weight,living habits,chronic strain,trauma,primary or secondary joint deformities and so on.It is related to chronic pain,dysfunction and disability of joints,leading to a decline in the quality of life and a serious socio-economic burden,and is one of the major health problems worldwide.With the development trend of aging and the increasing number of obese people,there is an urgent need to increase medical and health services to deal with the growing chronic disease of osteoarthritis.Throughout the world,osteoarthritis is a chronic joint disease that seriously threatens the health of middle-aged and elderly people and reduces the quality of life.It is a huge health problem,economic problem and social problem.So far,all research and efforts have not been able to determine the exact cause.At present,osteoarthritis is still lack of effective preventive measures,intervention and treatment.The current concept of treatment is mainly focused on the relief of symptoms and the improvement of joint disability,rather than changing the progression of the disease.Joint replacement is an effective treatment for end-stage osteoarthritis,and there is no reasonable treatment for early and middle stage OA except palliative pain control and physiotherapy.There is no treatment that can satisfactorily stop or delay the progression of OA or provide effective and lasting symptom relief.Therefore,the dilemma of prevention,diagnosis and treatment of osteoarthritis in clinical treatment makes researchers begin to study how to avoid the occurrence of osteoarthritis,slow down the development of osteoarthritis or reverse the destruction of articular cartilage structure.and then to prevent the occurrence of advanced osteoarthritis,reduce the social and economic burden,and improve the quality of life of the people.The development of new treatments for the destruction of articular cartilage or the apoptosis of chondrocytes,subchondral bone remodeling or chronic inflammatory changes of the synovium,there is a need for a deeper understanding of how these interventions play a role in these pathological changes.Since Nusse and Varmus first discovered Wnt in 1982,with the study of Wnt gene family,the members of Wnt gene family have been recognized by us gradually.Some previous studies have fully proved that members of the Wnt family are involved in cell proliferation,cell migration,a series of complex metabolic pathways and so on.Other studies have shown that abnormal expression of Wnt signal transduction pathway is associated with various malignant tumors and other diseases,such as OA.Wnt inhibitor1(WIF1),as an important antagonist,can bind to Wnt-related proteins in the extracellular space,and the binding between them will inhibit the upstream of Wnt/ ?-catenin signal pathway.Some studies have shown that WIF1 is down-regulated in many human malignant tumors,such as gliomas and astrocytomas.In addition,previous studies have shown that WIF1 can inhibit Wnt-3a-mediated inhibition of cartilage formation.Therefore,the first part mainly studies the expression of WIF1 in human normal chondrocytes and OA chondrocytes.The current view is that osteoarthritis occurs in all joint tissues,such as meniscus,subchondral bone,periarticular muscles and synovium,all of which contain inflammatory factors.Related studies have shown that OA is related to chondrocyte hypertrophy and calcified cartilage injury,and cartilage calcification promotes cartilage degeneration.Recent studies have shown that the progress of OA is closely related to oxidative stress and reactive oxygen species(ROS).In normal chondrocytes,moderate ROS participates in many normal physiological processes as an indispensable second messenger.However,in the pathological condition of OA,excessive ROS in chondrocytes triggers cell oxidative damage through some mechanism,oxidizing cell lipids,proteins,carbohydrates and DNAs,to induce apoptosis.In articular chondrocytes,ROS acts as a signal intermediate in a variety of signaling pathways,including those initiated by cytokines,growth factors and extracellular matrix(ECM)proteins.The production of ROS in chondrocytes and the level of intracellular antioxidants are kept in a relative balance,in which the redox state of cells determines the response of cells to cytokines and growth factors.This balance is subtly regulated by exogenous factors,such as oxygen content or cytokines.Under pathological conditions,the redox state can be changed,and the cell response to biochemical factors can be completely changed.Matrix metalloproteinases(MMPs)secreted by OA cells,degrades cartilage and ECM.The structure and function of cartilage and ECM are affected by chondrocytes,especially MMP-1,MMP-3 and MMP-13.The decomposing enzymes of chondrocytes can realize the tolerance to mechanical stress and the elasticity of articular cartilage,but these properties are impaired in OA.Because the repair and regeneration ability of damaged cartilage is very poor,it can inhibit the development of osteoarthritis by enhancing the proliferation of chondrocytes.Therefore,the proliferation ability of chondrocytes is an important factor in delaying cartilage degeneration,and can increase the secretion of MMP by reducing the level of ROS.Recent studies have shown that there may be a linkage relationship among ROS,WIF1 and Wnt/ ?-catenin signaling pathways,which can lead to apoptosis.Therefore,in the second part,we speculate that WIF1 may regulate ROS production and Wnt/ ?-catenin signal pathway in chondrocytes.Methods: Osteoarthritis cartilage tissue samples were collected from 10 OA patients(4 males,6 females,age 55–78,average age 68.5 years)who received sodium hyaluronate injection.Normal cartilage samples were collected from 10 traumatic knee joint fracture patients without OA(5 males,5 females,age 47–69,average 58.2 years).All the patients received knee arthroplasty in the People's Hospital of China Medical University during the period between December 2015 and October 2016.All procedures of this research were approved by the Ethics Committee of the People's Hospital of China Medical University.Cartilage tissue fragments were washed and digested.The scattered cells were collected and cultured in 10% fetal bovine serum.After 2 generations,chondrocytes could be used for subsequent experiments.Wi F1 c DNA plasmid osteoarthritis chondrocytes were treated with human interleukin 1?(IL-1?),antioxidant acetylcysteine(NAC)and ?-catenin inhibitor XAV-939.The osteoarthritis chondrocytes were treated with interleukin-1?(IL-1?)to simulate inflammation.The expression of WIF-1 in OA chondrocytes was detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and Western blot.MTT assay and flow cytometry were used to detect cell proliferation and apoptosis.ROS content was determined by flow cytometry,and Wnt/?-catenin signaling pathway activity was determined by immunofluorescence,Western blot and luciferase reporter gene assay.Western blot and enzyme-linked immunosorbent assay(ELISA)were used to detect the expression of apoptosis-related proteins and the secretion of matrix metalloproteinases(MMPs).Results: 1.WIF1 was low-expressed in human OA chondrocytes.The OA chondrocytes were treated with IL-1? to simulate the inflammatory condition.We then detected the differential expressed gene WIF1 in normal and OA chondrocytes.Both qRT-PCR and western blot showed that WIF1 expression of human OA chondrocytes was significantly lower than that in normal chondrocytes(p < 0.01),while that of IL-1? treated OA chondrocytes was lower than OA chondrocytes(p < 0.05),indicating that WIF1 was negatively related to chondrocyte inflammation.2.WIF1 down-regulated ROS level in OA chondrocytes.The DCF fluorescence intensity of OA chondrocytes decreased significantly compared with normal chondrocytes,while that of IL-1? treated OA chondrocytes was even higher than OA chondrocytes,indicating that the ROS level was up-regulated in OA(p < 0.01,Figure2 A).Then IL-1? treated OA chondrocytes were incubated with NAC(1/2/5 m M)for12 h,and ROS level was gradually decreased with the increase of NAC concentration,showing the efficiency of NAC in ROS elimination(p < 0.001,Figure 2 B).After WIF1 c DNA transfection,ROS level of IL-1? treated OA chondrocytes was significantly decreased(p < 0.01,Figure 2 C),revealing that WIF1 could suppress ROS production.3.WIF1 promoted proliferation and reduced apoptosis of OA chondrocytes through suppressing ROS production.We transfected OA chondrocytes with WIF1 c DNA and observed the results of qRT-PCR,which demonstrated that WIF1 c DNA could remarkably enhance the expression of WIF1(p < 0.01),suggesting that we succeeded in over expressing WIF1.Cellular experiments were then performed to test the effect of up-regulated WIF1.MTT assay revealed that both up-regulated WIF1 and NAC(5m M)would promote the proliferation of IL-1? treated OA chondrocytes(p < 0.01);according to flow cytometry results,the apoptosis rate of IL-1? treated OA chondrocytes was significantly reduced by WIF1 and NAC(p < 0.01).Western blot was performed to detect apoptosis-related proteins caspase-3,PARP,Bax and Bcl-2.The expression of cleaved caspase-3,cleaved PARP and Bax was significantly reduced,while expression of anti-apoptotic protein Bcl-2 was increased after WIF1 c DNA transfection or NAC treatment,indicating that WIF1 or NAC could suppress cell apoptosis of OA chondrocytes(p < 0.01).All these results revealed that WIF1 would protect the viability of chondrocytes via reducing ROS production.4.WIF1 blocked Wnt/?-catenin signaling pathway and therefore reduced MMP secretion.Activity of ?-catenin was detected by western blot,immunofluorescence and luciferase reporter assay.Western blot results revealed enhanced nuclear and total ?-catenin expression as well as decreased cytoplasmic ?-catenin expression in IL-1?treated OA chondrocytes compared with OA chondrocytes(negative control),while overexpressing WIF1 or the presence of ?-catenin inhibitor XAV-939 showed an inverse result(p < 0.01).Immunofluorescence demonstrated the parallel result that the fluorescence intensity of ?-catenin in the IL-1? + WIF1 and IL-1? + XAV939 group was distinctly lower compared with the IL-1? group(p < 0.05).Luciferase reporter assay confirmed that the transcriptional activity of ?-catenin,which was enhanced by IL-1? stimulation,could be significantly reduced by WIF1 or XAV-939(p < 0.01).Moreover,we detected the secretion of MMP-1,MMP-3 and MMP-13 in the cell culture supernatant.All of them were remarkably suppressed by WIF1 and XAV-939(p< 0.01).All these results confirmed that up-regulated WIF1 blocked the Wnt/?-catenin pathway.Conclusion: 1.WIF1 was low-expressed in human OA chondrocytes;2.WIF1 downregulated ROS level in OA chondrocytes;3.WIF1 promoted proliferation and reduced apoptosis of OA chondrocytes through suppressing ROS production;4.WIF1 blocked Wnt/?-catenin signaling pathway and therefore reduced MMP secretion. |
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