Study On Protective Effects And Related Mechanisms Of α-bisabolol On Osteoarthritis

Background:Osteoarthritis(OA)is a progressive disease of joint dysfunction and degeneration of articular cartilage,which seriously affects the physical and mental health and quality of life of the elderly.The incidence of osteoarthritis increases with age,and is expected to increase year by year as China gradually enters the aging society.Patients with osteoarthritis are characterized by persistent joint pain,and the early main symptoms are knee pain and tenderness.In the late stage,joint movement may be limited,muscular atrophy and so on.For the treatment of osteoarthritis,the clinical practice is usually to control pain and eliminate joint swelling symptoms to delay the progress of osteoarthritis,so as to achieve the goal of improving the quality of life of patients.At present,many drugs have been developed to treat osteoarthritis and have been put into clinical use.For example,nonsteroidal anti-inflammatory drugs(NSAIDs),glucosamine drugs,sodium hyaluronate and chondroitin sulfate drugs.Although there are many osteoarthritis drugs on the market,most of them can only relieve the clinical symptoms of osteoarthritis,but cannot inhibit the osteoarthritis development.α-Bisabolol(BISA)is a kind of fat-soluble sesquiterpenoids,which widely exists in the essential oils of frankincense and chamomile.Previous studies found that it possesses many biological properties,such as anti-inflammatory,antibacterial,antioxidant and anti-parasite.However,the application of BISA in the treatment of osteoarthritis has not been reported.In this paper,we mainly confirmed whether BISA has therapeutic effect of osteoarthritis and discussed its potential mechanism.This study is divided into three parts:Part 1:BIS A delayed O A progression in DMM-induced mouse OA modelObjective:Investigate the therapeutic effects of α-Bisabolol on mice osteoarthritis model.Methods:In this study,we established a mouse osteoarthritis model by removing the mouse meniscus ligament and causing the destabilization of the medial meniscus(DMM).Forty-five 8-week-old mice(C57/BL6)were randomly divided into 3 groups with 15 mice in each group.1.Blank control group:mice were treated with sham surgery;2.Osteoarthritis modeling group:DMM modeling;3.BISA treatment group:administered BISA 30mg/kg by oral gavage once a day for eight consecutive weeks after surgery.The whole experimental animals were sacrificed after eight weeks post-surgery,and collected mouse knee joint tissue sample for histological and X-ray analysis.Results:In the DMM group,the knee joint space was abnormally narrowed and the cartilage surface density increased.However,cartilage surface calcification was decreased and joint space was wider after BISA treatment,compare to the DMM group.Base on S-O staining,we observed the erosion of superficial articular cartilage,the decrease of chondrocyte number and the loss of proteoglycan in the DMM group at 8 weeks after the operation.In contrast,the DMM+BISA group showed more complete cartilage surfaces and more proteoglycan and Collagen Ⅱ.The results of OARSI score were basically consistent with the results of S-O staining,and the OARSI score of mice in the DMM+BISA treatment group was decreased than the DMM group.In addition,compared with the DMM group,the serum IL-1β,TNF-α,and IL-6 levels of mice in the DMM+α-bisabolol group decreased.Conclusions:BISA has therapeutic effect on mice osteoarthritisPart 2:BISA alleviates inflammatory response and regulates ECM catabolism in AGEs-exposed human chondrocytesObjective:Investigate the anti-inflammatory and ECM regulatory effect of α-Bisabolol on AGEs-exposed human chondrocytesMethods:Human articular cartilage specimen was obtained from seven OA patients to extracted the primary chondrocytes.The AGEs was used to induce the inflammatory response to simulate the occurrence and development of osteoarthritis in vitro.In this study,the cytotoxicity of BISA and AGEs on chondrocytes was determined by using CCK-8 cell viability kit.The protein expression levels of TNF-α,PGE2,Nitrite,IL-6,collagen Ⅱ,aggrecan,MMP13 and ADAMTS5 in human chondrocytes were detected and analyzed by ELISA and Griess reagent kit.The expression levels of iNOS and COX-2 protein in chondrocytes were detected by Western blot.Changes in collagen II and aggrecan protein expression in chondrocytes were detected by immunofluorescence.Results:In this experiment,we found that BISA can inhibit the secretion of TNF-α,PGE2,Nitrite,IL-6,iNOS,COX-2,MMP-13,ADAMTS5 and promote the production of collagen II and aggrecan.Conclusions:BISA alleviates inflammatory response and regulates ECM catabolism in AGEs-exposed human chondrocytesPart 3:BISA inhibits AGEs-medicated activation of NF-κB pathway and MAPK signaling in human chondrocytesObjective:Investigate the potential mechanism of α-Bisabolol-induced protectionMethods:Human articular cartilage specimen was obtained from seven OA patients to extracted the primary chondrocytes.The AGEs was used to induce the inflammatory response to simulate the occurrence and development of osteoarthritis in vitro.The expression levels of IκBα and p65 protein in chondrocyte cytoplasm and nucleus were detected by western blot.Immunofluorescence was applied to detect the distribution of p65 inside and outside the nucleus of chondrocytes.The expression levels of p38 and JNK proteins in chondrocytes were detected by western blot.Molecular docking analysis was employed to simulate and detect the binding ability of BISA to p65,p38 and JNK proteins.Results:In this experiment,we found that BISA significantly inhibited the degradation of IκBα and reduced the nucleation of p65.In addition,we performed immunofluorescence staining on p65.In human chondrocytes,immunofluorescence staining showed that most of p65 was located in the cytoplasm.After stimulation with AGEs,p65 was transferred to the nucleus.However,the treatment with BISA blocked the entry of p65 into the nucleus.At the same time,we found that BISA inhibited phosphorylation of p3 8 and JNK proteins in human chondrocytes induced by AGEs.Molecular docking analysis indicated that BISA could form hydrogen bonds with p65,p38 and JNK proteins,and has high affinity of-3.3,-6.6 and-6.3 kcal mol-1,respectively.Conclusions:BIS A alleviates inflammatory response and regulates ECM catabolism in AGEs-exposed human chondrocytes.