The Anti-inflammatory Effect And Intracellular Signaling Pathway Mechanism Study Of Nootkatone(NTK)in Osteoarthritis

Research background and objectives:Osteoarthritis is the most common joint degenerative disease worldwide and a major cause of the disability in middle-aged and elderly people.As yet,due to the lack of understanding of disease pathogenesis and etiology,there is no effective etiological treatment for early osteoarthritis currently.Therefore,further study of the etiology and pathogenesis of osteoarthritis and further understanding of the relationship between osteoarthritis molecular regulatory mechanisms and articular tissue are significant to find new therapeutic methods for the prevention and treatment of osteoarthritis.The pro inflammatory cytokine interleukin-1 is the inflammatory mediator secreted in early osteoarthritis.IL-1β plays an important role in pathogenesis of OA,which can affect articular chondrocytes and the extracellular cartilage matrix.The pathological role of IL-1β in chondrocytes and cartilage is caused by the activation of a variety of proinflammatory signaling pathways,among which nuclear factor κB(NF-κB)signaling pathway is one of the classic inflammatory signaling pathways.Activated NF-κB can trigger the osteoarthritis related inflammatory cytokines and catabolism factor gene expression,which eventually lead to osteoarthritis.Nootkatone(NTK)is a ketone derivative of the sesquiterpene compound Valenciene.Its various biological activities have been constantly researched and developed by scholars at home and abroad.At present,its anti-inflammatory properties have attracted particular attention,the newest research abroad reported that nootkatone can have an effect on the NF-κB,p38MAPK and PI3K/Akt signal pathway,and consequently inhibit the expression of IL-1β,TNF-α,iNOS,COX-2 and other inflammatory catabolic factors.However,NF-κB signaling pathway is one of the main catabolic signaling pathways in the pathogenesis of osteoarthritis,thereby we speculate that nootkatone inevitably play an important role in OA development.In this study,the OA model in vitro which was established by IL-1β-induced inflammatory injury murine chondrocytes was used to study the therapeutic effect of nootkatone on inflammation in OA model in vitro and the OA model in vivo induced by ACLT surgery in mice was used to study the therapeutic effect of nootkatone on inflammation in OA model in vivo.Finally,we studied the mechanism of nootkatone’s anti-inflammatory intracellular signaling pathway in osteoarthritis taking Western Blot technique to detect the activity of NF-κB signaling pathway.Research methods:In the OA model in vitro study.Firstly,LAL endotoxin assay and CCK8 assay were used to verify the biocompatibility of nootkatone.Then,we took the Griess assay to measure the effects of NO content affected by nootkatone.QRT-PCR,Western Blot and ELISA techniques were used to detect the effects of nootkatone on the expression levels of TNF-α,IL-6,iNOS,COX-2,ADAMTS-5,MMP-13,Aggrecan and Collagen Ⅱ produced by IL-1β-induced chondrocytes.In the OA model in vivo study.Firstly,anterior cruciate ligament transection(ACLT)induced OA models were established,meanwhile,control studies were also set up.Mice in the treatment group was intraperitoneal administered with NTK(10 mg/kg).Knee joint of all groups were collected for further experiments 6 weeks after treatment.Through the treatment and specimen preparation of the mouse articular cartilage tissue,HE staining and Safranin OFast Green staining were performed.Then,QRT-PCR,Western Blot and immunohistochemistry techniques were used to detect the effects of nootkatone on the expression levels of iNOS,COX-2,IL-1β,TNF-α,IL-6 and the extracellular cartilage matrix degrading enzymes ADAMTS-5 and MMP-13 in articular cartilage after ACLT surgery.Finally,we used the aforementioned OA model in vitro treated by nootkatone,taking Western Blot technique to detect the effects of nootkatone on the protein expression and phosphorylation levels of signature proteins IκB-α and P65 in NF-κB signaling pathway.Results:Study on OA model in vitro:1.LAL and CCK8 assay results:The working drug concentration of nootkatone should be less than 200μM in OA model in vitro established by extracting murine chondrocytes.At this time,the drug did not produce toxicity to mouse chondrocytes and the endotoxin contamination of the drug was lower than 0.01EU/mL.Nootkatone exhibited good biocompatibility in murine chondrocytes and could be used in subsequent experiments.2.Griess assay results:IL-1β(10ng/mL)could significantly stimulate murine chondrocytes to produce massive inflammatory mediator NO,while nootkatone(10,50,100,200μM)could significantly inhibit the production of NO in murine chondrocytes(P<0.05),and the inhibitory effect of nootkatone on the production of NO was gradually enhanced with the increase of nootkatone concentration.3.QRT-PCR results:Nootkatone could significantly inhibit the gene expression levels of inflammatory mediators TNF-α,IL-6,iNOS,COX-2 and extracellular cartilage matrix degrading enzymes ADAMTS-5 and MMP-13 which were all produced by IL-1β-induced murine chondrocytes(P<0.05).4.Western Blot and ELISA results:Nootkatone could significantly inhibit the protein expression levels of IL-1β-induced murine chondrocytes producing inflammatory mediators TNF-α,IL-6,iNOS,COX-2 and extracellular cartilage matrix degrading enzymes ADAMTS-5 and MMP-13,which were consistent with the effect on gene expression levels mentioned before.Study on OA model in vivo:1.Results of knee joint histology HE staining and Safranin O-Fast Green staining:Through comparative analysis,firstly,it was confirmed that the cartilage of OA model in vivo induced by ACLT surgery in mice was obviously damaged after operation,showing typical osteoarthritis changes,and the successful establishment of OA model in vivo induced by ACLT surgery in mice was verified.Secondly,it has been confirmed that nootkatone can significantly inhibit the inflammatory response caused by ACLT surgery,and significantly inhibit cartilage degradation and delay the disease progression of OA.2.QRT-PCR results:Nootkatone could significantly inhibit the gene expression levels of inflammatory mediators iNOS,COX-2,IL-1β,TNF-α,IL-6 and extracellular cartilage matrix degrading enzymes AD AMTS-5 and MMP-13 which were all produced by the articular cartilage of OA model in vivo induced by ACLT surgery in mice(P<0.05).3.Immunohistochemical and Western Blot results:Nootkatone could significantly inhibit the protein expression levels of inflammatory mediators iNOS,COX-2 and extracellular cartilage matrix degrading enzymes ADAMTS-5 and MMP-13 which were both produced by ACLT-induced articular cartilage.These were consistent with the effect on gene expression levels mentioned before.Study on intracellular signaling pathway mechanism of nootkatone in anti-inflammatory effect:Nootkatone could significantly inhibit the phosphorylation levels of IκB-α and P65 in the NF-κB signaling pathway.Conclusion:Nootkatone can significantly inhibit the expression levels of inflammatory mediators and extracellular cartilage matrix degrading enzymes in the OA model of mice in vitro and vivo,which can play an therapeutic role in anti-inflammation and inhibiting cartilage degradation in vitro and vivo.Nootkatone can significantly inhibit the activation of cellular NF-κB signaling pathway in murine chondrocytes,thereby inhibiting the expression of a variety of inflammatory mediators,and delay the disease progression of OA.