Casticin Attenuates Experimental Osteoarthritis-related Cartilage Degeneration By Inhibiting The ROS-mediated NF-?B Signaling Pathway In Vitro And In Vivo

Background:Osteoarthritis(OA)is an age-related or posttraumatic joint disease leading to a number of changes characterized by the loss of articular cartilage,subchondral bone remodeling,synovial inflammation and osteophyte formation.Knee arthritis contributes to a heavy economic burden as it is the leading cause of chronic pain and disability.However,there are no effective treatments that halt or reverse OA progression.There is a clear and urgent need to look for an effective alternative to those drugs.Inflammation is now generally accepted as an important contributor to OA joint pathology.There is sufficient evidence that anti-inflammatory treatment for OA may be a worthwhile strategy.Flavonoids are found in many plants,and the casticin that we studied was isolated from the fruit of Vitex trifolia.Several studies have revealed that the flavonoid casticin has regulatory effect on inflammation and oxidative stress.However,the effect of casticin on osteoarthritis remains unknown.Therefore,the present study investigated the effects of casticin on osteoarthritis in vitro and in vivo.Methods:For some experiments,the ADTC5 cells were treated with IL-1?(10 ng/mL)to induce inflammation.Casticin was dissolved in 0.1%DMSO.Cell viability was assessed using the CCK-8 assay.In brief,ADTC5 cells were pretreated with different concentrations of casticin.A cell viability assay was performed using a cell counting kit-8.Cells in confocal dishes were pretreated with control media or various concentrations of casticin for 2 h followed by stimulation with IL-1?(10 ng/ml).ROS levels were measured using a DCFH-DA probe kit.To determine the mechanism how casticin inhibits inflammation in ADTC5 cells stimulated with IL-1?,we assayed the activation state of each component in the NF-?B signaling pathway.We visualized NF-?B p65 by immunofluorescence in ADTC5 cells.Concentrations of COX-2,iNOS,I?B-?,p-I?B-?,and P65 were determined by Western blotting.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of proinflammatory cytokines in cell culture media,including TNF-?,IL-6 and PGE2 production.The effects of casticin on the expression of the antioxidant enzymes SOD,GSH and MDA were detected with kits.Mice were randomly separated into three groups.Sham,DMM-induced OA treated with vehicle,and DMM-induced OA treated with casticin.Starting one day after surgery,one group of animals was treated with 10 mg/kg casticin(dissolved in 100%DMSO)by intraperitoneal injection,and another group was treated with vehicle.Mice were killed at 4 or 8 weeks after surgery for collection of knee-joint specimens and blood.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of proinflammatory cytokines in serum.Joint tissues from the mice were fixed using 4%paraformaldehyde and then decalcified.Bones were embedded in paraffin and sectioned using a paraffin microtome.Immunohistochemical analysis of MMP-13,collagen ? and SOD2 levels were performed in an OA mouse model.Representative mid-sagittal sections were selected and stained with toluidine blue or Safranin-O/Fast green.Concentrations of COX-2,iNOS,I?B-?,p-I?B-?,and P65 in tissues were determined by Western blotting.First,we checked the data homogeneity of variance with a Fisher test before performing a Student's t test with SPSS V.13.0 software.When the variance was equal,Student's t test was used to assess statistically significant differences in the data between two groups.One-way analysis of variance(ANOVA)and Dennett's multiple comparison test were used to assess statistically significant differences in the data among three groupsResults:Our results show that casticin downregulated TNF-? and PGE2 protein expression in IL-1?-stimulated ADTC5.Casticin prevents the oxidative stress response in ADTC5 cells.Casticin inhibits the NF-?B signaling pathway in IL-1?-induced ADTC5 cells.Casticin treatment showed decreased cartilage erosion,increased subchondral plate thickness,reduced degradation of type II collagen and aggrecan,and decreased expression of MMP-13 following casticin treatment when compared to vehicle controls.Conclusion:Casticin prevents the oxidative stress response and inflammation in IL-1?-induced ADTC5 cells with inhibition of NF-?B signaling pathway.In addition,casticin attenuates experimental osteoarthritis-related cartilage degeneration by inhibiting the ROS-mediated NF-?B signaling in vivo.