Nobiletin Inhibits Expression Of Inflammatory Mediators And Regulates JNK/ERK/p38 MAPK And PI3K/Akt Pathways In Human Osteoarthritic Chondrocytes

Purpose:Osteoarthritis(OA),a chronic degenerative painful disease of synovial joints,eventually leads to disability and reduces the quality of life.Inflammatory cytokines have been well documented in pathogenesis of OA.Recent research has focused on anti-inflammatory properties of plant-derived compounds.This study.aimed to investigate the anti-inflammatory effects of nobiletin on human osteoarthritic chondrocytes and also the possible molecular events.Methods:Isolated human osteoarthritic chondrocytes were stimulated with IL-1?.The effect of nobiletin on chondrocyte viability was assessed.Cell apoptosis was measured by annexin V-FITC and PI staining.Apoptosis was detected using apoptosis detection kit.The effect on NO production was determined using Griess reagent and the levels of IL-6 and PGE2 were assessed by enzyme linked immunosorbent assay(ELISA).Real-time PCR was used to detect the expression of iNOS and COX-2.Total RNA was extracted using TriZol(Invitrogen)according to the manufacturer's instructions and quantified.The influence of nobiletin on the expression of COX-2,iNOS and proteins of PI3/Akt,NF-?B and MAPK cascades were also assessed.Proteins extracted from chondrocytes were subjected to western blotting.The cells were subjected to lysis buffer and the nuclear and cytoplasmic proteins were extracted from the cells using NE-PER Mammalian Protein Extraction Reagent(Thermo).Concentration of the isolated protein was determined using BCA protein assay kit.The immunoreactive bands were detected by enhanced chemiluminescence.The data obtained from all the experiments are represented as mean ± SD,taken from 3 or 6 independent experiments.Statistical significance between the means of various experimental groups were assessed by one-way analysis of variance(ANOVA)followed by Duncan's Multiple Range Test as post-hoc analysis using SPSS 19.0.Statistical differences at p<0.05 are considered significant.Results:Nobiletin(75pg/ml,150p.g/ml and 300?gg/ml)significantly(p<0.05)improved the viability of chondrocytes,and remarkably reduced the levels of NO,IL-6 and PGE2.The expression levels of COX-2 and iNOS both at mRNA and protein levels were strikingly reduced by nobiletin,in a dose-dependent way.In addition,nobiletin caused marked(p<0.05)down-regulation of the NF-?B signalling pathway.IL-1?-induced activation of PI3/Akt,and JNK,ERK and p38 MAPK cascades were significantly(p<0.05)inhibited by nobiletin with 300 ?g/ml dose exhibiting maximum effects.Conclusion:Inflammatory cytokines are critically involved in the pathogenesis of OA.Significant suppression of expression of inflammatory cytokines and modulation of JNK/ERK/p38 MAPK and PI3K/Akt signalling cascades by nobiletin suggests its potent anti-inflammatory and anti-osteoarthritic effects,and nobiletin may be a new drug for the treatment of osteoarthritis.