The Expression Of AQP4 In Articular Cartilage Of Adjuvant-Induced Arthritis Rats And The Effects Of AQP4 SiRNA On IL-1β-induced Chondrocyte Apoptosis In Rats

Rheumatoid arthritis(RA)is a chronic autoimmune disease with high morbidity and disability rates,it is one of the major causes of people’s disability and labour’s loss.The pathogenesis of RA has not been fully elucidated,the results showed that there was a significant correlation between articular cartilage damage and irreversible disability of RA joint.So,to find the key pathological link of cartilage injury and protect the articular cartilage is one of the key points in the prevention and treatment of RA.As the only cell type in cartilage tissue,chondrocyte is involved in the regulation of cartilage formation,metabonism and remodeling processes.It is important for us to reveal the pathological mechanism of articular cartilage injury in RA by exploring the key links of the biological characeristics and the intrinsic molecular processes in articular chondrocyte.Aquaporins(AQPs)are members of a family of membrane transport proteins and involve in regulating the influx and outflow of water and small molecules.At least 13 mammalian AQPs(AQP0-12)have been studied in various tissues and organs.Some previous studies showed that the high expression of AQP1、AQP3 and AQP9 in joint diseases such as RA and osteoarthritis(OA),involved in the regulation of chondrocyte morphology,size and function.It is well known that AQP4 possess very high single channel water permeability(3-fold greater than that of AQP1),but the previous studies of AQP4 aim at nervous system diseases.So far,there is no report on the expression of AQP4 in mammals articular chondrocyte and the possible pathogenic role of AQP4 in RA articular cartilage injury is still unknown.So,we observed the AQP4 function ofarticular cartilage in adjuvant-induced arthritis(AIA)rats and explored the possible protective effect of AQP4 siRNA on articular chondrocyte injury(IL-1β induced)from cells proliferation and apoptosis.Therefore,experimental data were provided for the development of new therapeutic targets of RA through this study.The study is divided into two parts:1.The expression of AQP4 in articular chondrocytes of adjuvant-induced arthritis rats and its pathological significance.Objective: This study was aimed to investigate the expression of AQP4 in articular chondrocytes of AIA rats and its involvement in AIA development.Methods: Model rats were induced by intradermal injection of complete Freund’s adjuvant and evaluated by secondary paw swelling and histological assessments on ankle joint damage.Localization and protein level of AQP4 in articular cartilage were determined by immunohistochemistry and western blot.In vitro study,normal and AIA articular chondrocytes were isolated and cultured.AQP4 expression levels in cultured articular chondrocytes were measured by immunocytochemistry staining and western blot.Results: AQP4 localized at the membrane and cytoplasm of articular chondrocytes and showed higher protein levels in cartilage tissues of AIA rats than that of normal rats.The results of correlation analysis revealed that AQP4 protein levels in cartilage tissues of AIA rats remarkably correlated positively with secondary paw swelling on day 26 and the pathological scores of ankle joint damage.Similarly,AQP4 protein level in cultured AIA articular chondrocytes was higher than that in normal articular chondrocytes.Conclusions: Our data provide certain experimental evidence that AQP4 overexpression in articular chondrocytes aggravated AIA severity and might be a novel target for RA treatment.2.The effects of AQP4 siRNA on articular chondrocytes apoptosis induced by IL-1β in rats and its pathological mechanism.Objective: To observe the protective effect of AQP4 blockage by siRNA onIL-1β-induced chondrocyte apoptosis and explore the underlying mechanisms.Methods:AQP4 siRNA and SB203580 were used for the pretreatment of chondrocytes and then IL-1β were used to induce the apoptosis of chondrocytes in vitro.Cell proliferation was analyzed by MTT assay.Hoechst 33258 staining and flow cytometric assay were applied to detect chondrocyte apoptosis.Bcl-2,Bax,Caspase 3 mRNA levels and p38,phosphorylated-p38(P-p38)protein levels were assayed by real-time PCR and western blot.Results: Our findings indicated that AQP4 inhibition by siRNA protected the chondrocytes from IL-1β-induced apoptosis,evidenced by increased cell proliferation,few observations of apoptotic morphologic changes and decreased cell apoptosis rates.Additionally,AQP4 inhibition remarkably decreased Bax and Caspsese 3 mRNA levels and increased Bcl-2 mRNA level,accompanied by reducing P-p38 protein level,without affecting p38 protein.The aforementioned effects of AQP4 siRNA were similar to SB203580,a specific p38 inhibitor.Conclusions: Our results provide experimental evidence that AQP4 inhibition contributes to efficaciously decreasing chondrocyte apoptosis induced by IL-1β and provide a novel therapeutic target for RA,which might be related to down regulating anti-apoptotic factor,up regulating pro-apoptotic factors and inhibition the activation of p38 MAPK signal pathway.