| Osteoarthritis(OA)is a chronic degenerative joint disease,which is caused by various reasons and characterized by articular cartilage destruction,subchondral osteosclerosis and synovitis.With the development of the disease,it eventually leads to irreversible destruction of the joint.The incidence rate of OA is high.The prevalence rate of OA is over 50% in aged people(>65 years old).With the gradual aging of the population,the incidence rate of OA will increase year by year.This will become an important cause of disabili ty in the elderly.Despite a large number of studies on OA,the pathogenesis of OA is still unclear.At present,it is generally agreed that the inflammatory environment in the joint plays an important role in the occurrence and development of OA.As an important inflammatory factor in the progression of OA,IL-1 β can not only increase the secretion of a variety of proinflammatory factors,but also promote nitric oxide(NO),thrombospondin motifs(ADAMTS),matrix metalloproteinases(MMPs),prostaglandin E2(prostaglandin,PGE2)and other catabolic factors causing the destruction of cartilage extracellular matrix.In addition,we found that IL-1 β can also activate the Rho A signaling of chondrocytes,and significantly increase the F-actin in chondrocytes,resulting in the increase of chondrocyte stiffness,the change of cell morphology and phenotype.At present,the etiology and pathogenesis of OA are still unclear,so there is no specific drug for the treatment of OA.The main treatment method is to choose appropriate methods to alleviate symptoms according to the severity of OA.At present,the treatment of OA is mainly divided into non drug treatment,drug treatment and surgical treatment.For patients with mild to moderate OA,drug therapy is mainly used,with NSAIDs being the most commonly used drugs.Ibuprofen is one of the NSAIDs drugs,which is commonly used in the treatment of OA.Its mechanism is to reduce the synthesis of PGE2 by inhibiting cyclooxygenase-2(COX-2),so as to reduce the pain of patients.In addition,ibuprofen has been reported to have the ability to inhibit Rho A signaling pathway in nerve cells.However,whether ibuprofen has the ability to inhibit Rho A signaling pathway and has not been clearly reported.Our previous studies have shown that IL-1 β can also activate Rho A signaling pathway of chondrocytes,increase the F-actin in chondrocytes,increase the rigidity of chondrocytes,and change the cell morphology and phenotype.Therefore,the main purpose of this study is to explore whether ibuprofen can attenuate some biological effects caused by IL-1 β by inhibiting Rho A signaling pathway.Research methods and results(1)The apoptosis of chondrocytes treated with different concentrations of ibuprofen and the activity of chondrocytes treated with different concentrations of ibuprofen were observed by phase contrast Microscopy.It was found that the apoptosis of chondrocytes treated with ibuprofen at 750 μ m and 1000 μ m was more than that of chondrocytes treated with ibuprofen at 0,125,250 and 500 μ m(P < 0.05),and there was no significant difference between the apoptosis of chondrocytes treated with ibuprofen at 0,125,250 and 500 μ m(P > 0.05).CCK-8 showed that the activity of chondrocytes treated with 750 μ m and 1000 μ m was lower than that of chondrocytes treated with 0,125,250 and 500 μ m(P < 0.05),while the activity of chondrocytes treated with 0,125,250 and 500 μ m had no significant difference(P > 0.05),which was consistent with the results of inverted microscope.Therefore,ibuprofen concentration of 500 μ m was selected in our subsequent experiments.(2)Griess test and ELISA were used to detect the content of NO and PGE2 in different cell culture media.The results showed that compared with the blank control group,the content of NO and PGE2 in chondrocytes treated with IL-1 β increased significantly(P < 0.01).The content of NO and PGE2 in chondrocytes pretreated with ibuprofen 2 h and then added with IL-1 β(IL-1 β + ibuprofen group)or treated with IL-1 β alone(IL-1 β group)decreased significantly(P < 0.01).(3)The cytoskeleton cells in different groups were observed by phalloidin staining.The results showed that compared with the control group,F-actin of chondrocytes treated with IL-1 β was more compact,denser,stronger fluorescence intensity,and stress fiber could be seen clearly,while the above situation did not occur in IL-1 β + ibuprofen group.We used Image J image processing software to detect the average fluorescence density of F-actin.The results showed that the average fluorescence density of F-actin in the IL-1 β group was significantly higher than that in the control group(P < 0.01),and the averag e fluorescence intensity of F-actin in the IL-1 β + ibuprofen group was lower than that in the IL-1 β group(P < 0.05).(4)The morphology of chondrocytes in each group was observed under phase contrast microscopy.The results showed that: compared with the control group,the number of the chondrocytes in the IL-1 β group became longer(length: width > 3:1),lost the original polygonal shape,and fibroblast like chondrocytes was significantly higher than that in the control group(P < 0.05).The chondrocytes in the IL-1 β + ibuprofen group were compared with those in the control group,there was no significant difference in the number of the above morphological changes of chondrocytes(P > 0.05).(5)Western blot was used to detect the ratio of F-actin to G-actin in chondrocytes(F-actin/G-actin).The results showed that compared with the control group,the F-actin / G-actin ratio of chondrocytes in the IL-1 β group was significantly higher than that in the control group,with an increase of nearly 50%(P < 0.05).Compared with the IL-1 β group,IL-1 β + ibuprofen group decreased by 30%(P < 0.05),and there was no significant difference between the IL-1 β + ibuprofen and the control group(P > 0.05).(6)Extracellular glycosaminoglycans(GAGs)were detected by alcian blue staining.The results showed that compared with the control group,the alcian blue staining of IL-1 β group was lighter.Compared with the IL-1 β + ibuprofen group,the alcian blue staining of the IL-1 β + ibuprofen group was significantly deeper than that of IL-1 β group,and even there was no difference between the control group and the IL-1 β+ ibuprofen group.Furthermore,we found that compared with the control group,the absorbance of IL-1 β group decreased by about 25%(P < 0.05).The absorbance of IL-1 β + ibuprofen group was increased by 33%(P < 0.05)compared with that of IL-1 β group,and there was no statistical difference between the IL-1 β + ibuprofen group and the control group(P > 0.05).(7)Quantitative real-time polymerase chain reaction(q RT-PCR)was used to detect the expression of type collagen II,aggrecan and SOX9 gene in chondrocytes.The results showed that compared with the control group,the expression of collagen II,aggrecan and SOX9 gene in the IL-1 β group decreased significantly(P < 0.01).Compared with the IL-1 β group,the expression of collagen II,aggrecan and SOX9 gene in the IL-1 β+ibuprofen group was significantly higher than that in the IL-1β group(P < 0.01).It is suggested that IL-1 β can change the phenotype of chondrocytes,while ibuprofen can inhibit the biological effect of IL-1 β.(8)The migration ability of chondrocytes was detected by scratch test.We compared the area of scratch reduction between 0 h and 24 h in each group.We found that the area of scratch reduction in IL-1 β treated group was significantly smaller than that in the control group(P < 0.05).Compared with IL-1 β + ibuprofen group,the scratch area decreased significantly in the IL-1β group(P < 0.05),and there was no significant difference between the IL-1 β + ibuprofen group and the control group(P > 0.05).These results indicate that IL-1 β causes the decrease of chondrocyte migration,while ibuprofen inhibits the effect of IL-1 β.(9)Pull down test was used to detect the activated Rho A in chondrocytes.The results showed that the activation of Rho A in chondrocytes of IL-1 β group was significantly higher than that of the control group(P < 0.05).Compared with IL-1 β group,ibuprofen + IL-1 β group decreased 50%(P < 0.05).There was no significant difference between the two groups(P > 0.05).There was no significant difference between ibuprofen + IL-1 β group and Y-27632 + IL-1 β group(P > 0.05).It is suggested that ibuprofen attenuates the increase of F-actin in chondrocytes induced by IL-1 β by inhibiting Rho A signaling pathway.ConclusionIL-1 β can increase the content of F-actin in chondrocytes by activating the Rho A signal pathway in chondrocytes,thus reducing the morphological changes,phenotype changes and cell migration ability of chondrocytes.While ibuprofen pretreatment to chondrocytes can inhibit this series of biological effects caused by IL-1 β. |
PDF Full Text Request