| Rheumatoid arthritis(RA)is a class of autoimmune diseases.It is mainly characterized by chronic synovial inflammation,cartilage and bone destruction.It is one of the major causes of disability and labor loss in the population.At present,the pathogenesis of RA has not been fully elucidated.Studies have shown that synovial tissue hyperplasia and articular cartilage injury are directly related to irreversible RA disability.It is of great significance to actively explore possible mechanisms of RA synovial hyperplasia and cartilage injury and innovative drugs.Aquaporins(AQPs)are membrane transporters associated with water permeability.Thirteen types of AQPs(AQP 0-12)have been cloned and identified in mammals.There are p H-sensitive regions in the AQPs structure.And osmotic response elements,in the pathogenesis of RA,synovitis leads to the local organization of the joint acidification,synovial fluid p H value decreased,a large number of cartilage matrix degradation into the joint cavity caused by osmotic pressure changes,these factors may lead to synovial tissue AQPs are activated.Previous literature has shown that abnormal expression of AQP1 in RA synovium tissue is closely related to RA synovial inflammation,hydrosalpinx,and cartilage/bone damage.Intervention with AQP1 may be a key link and effective measure to curb RA synovitis and joint injury.Acetazolamide(AZ)acts as an inhibitor of glycosidase to inhibit AQP1-mediated water permeability.It also directly inhibits AQP1 protein expression and has the ability to inhibit tumor metastasis and angiogenesis.In this study,we used AZ as an appropriate AQP1 inhibitor to observe the therapeutic effect of AZ on adjuvant arthritis in rats and explore its possible mechanisms,providing new targets and new ideas for the prevention and treatment of RA.This study is mainly divided into the following two aspects:1.Therapeutic effect of acetazolamide,an aquaporin 1inhibitor,adjuvant-induced arthritis in rats by inhibiting NF-κB signal pathwayObjectives:Previous studies have shown that aquaporin 1(AQP1)is up-regulated insynovium and cartilage of rheumatoid arthritis(RA)patients and that AQP1 may beinvolved in joint swelling and synovial inflammation.This study was aimed toinvestigate the potential therapeutic effect of acetazolamide(AZ,an AQP1inhibitor)on rat adjuvant-induced arthritis(AIA)and explore its related mechanisms.Materials and Methods: Rat AIA was induced by complete Freund’s adjuvant.Theeffect of AZ on rat AIA was evaluated by secondary hind paw swelling,arthritis index,TNF-αand IL-1β serum levels and histological examination of ankle joint.Proteoglycans expression and m RNA levels of type II collagen(COII)and aggrecanin cartilage were measured by alcian blue staining and real-time PCR,respectively.The protein levels of AQP1,IκBα,phospho-IκBα(p-IκBα),NF-κB p65 andphospho-NF-κB p65(p-NF-κB p65)in synovial tissues were detected by western blot.Results:AZ treatment could inhibit secondary hind paw swelling and arthritis index,reduce serum levels of TNF-αand IL-1β,and ameliorate pathological changes ofankle joint in AIA rats.AZ increased proteoglycans production and m RNA levels of COII and aggrecan in cartilage tissues.Moreover,AZ decreased AQP1 protein leveland suppressed the activation of NF-κB pathway in synovium,indicated by inhibitingthe degradation and phosphorylation of IκBα and reducing p-NF-κB p65 protein level.2.Effects and mechanisms of acetazolamide on IL-1β-induced apoptosis of rat articular chondrocytes.Objective:To investigate the effects and mechanisms of acetazolamide(AZ),an aquaporin 1(AQP1)inhibitor,on IL-1β-induced apoptosis of rat articular chondrocytes.Methods:Articular chondrocytes were isolated and cultured by trypsin and collagenase digestion method.IL-1β was used to inducechondrocytesapoptosisin vitro.Different doses of AZ were added to IL-1β-treated chondrocytes.The cell proliferation was detected by MTT assay.The cell apoptosis was evaluated by hoechst 33342 staining,flow cytometry analysis and rhodamine-123 staining.Protein levels of AQP1,Bcl-2,Bax,Caspase 3,NF-κB p65,p-NF-κB p65,IκBα and p-IκBα were assayed by western blot.Results:AZ(12.5,25,50 μmol/L)dose-dependently increased the proliferation of IL-1β-inducedchondrocytes.AZ protected the chondrocytes from IL-1β-induced apoptosis,evidenced byfew observations of apoptotic morphologic changes,decreased cell apoptosis rates and increased mitochondrial membrane potential.Compared with normal group,increased AQP1 protein level,reduced Bcl-2 protein level and increased protein levelsof Bax and Caspase3 were observed in IL-1β-induced group.In addition,IκBprotein level of IL-1β-induced group was lower than normal group,whereas p-IκBαand p-NF-κB p65 protein levels of IL-1β-induced group were higher than normal group.Compared with IL-1β-induced group,AZ decreased AQP1 expression,increased Bcl-2 expression and down-regulated the expressions of Bax and Caspase3.Moreover,AZ inhibited the degradation and phosphorylation of IκBα and reduced p-NF-κB p65 protein level.Conclusion : AQP1 inhibition by AZcan decrease chondrocytes apoptosis induced by IL-1β,which might be related to down-regulating anti-apoptotic gene Bcl-2,up-regulating pro-apoptotic genes Bax and Caspase3,and inhibiting the activation of NF-κB signal pathway. |
