| Osteoarthritis, also be called as degenerative joint disease, is the most common reason of joint pain in the elderly. Medicine is the primary means of early osteoarthritic treatment, and the medicine what can improve the structure of cartilage is considered as the most promising drug to treat Osteoarthritis.But there are very few medicine avaible,and the effect has not be confirmed.carboxymethyl chitosan (CM-chitosan) is a water-soluble chitosan derivatives, the structure and physicochemical properties are similar to the extracellular proteoglycan of the hyaline cartilage, such as hyaluronic acid, chondroitin sulfate and keratin sulfate and so on. Our previous studies have shown that carboxymethyl chitosan has a protective effect for rabbit's osteoarthritis, but the specific mechanism has not entirely be clarified.The pathogenesis of osteoarthritis are not well understood, but nitric oxide has been confirmed (NO) what may play an important role in the development of osteoarthritis. NO can inhibit the proliferation of chondrocytes and induced chondrocytes apoptosis,inhibit the synthesis ofⅡcollagen and proteoglycan by chondrocyte,and promote the decomposition of cartilage extracellular matrix. In osteoarthritis, the nitric oxide synthase (iNOS) is a key enzyme to induce the NO production.Nuclear factor KB (NF-κB) is a sort of specific nuclear factor what can specally bonding withκB sequence of the immunoglobulin K-chain gene enhancer.it is the central transcription factor involved in inflammatory and immune responses.It can be activated by interleukin-1 (3 (IL-1β) and tumor necrosis factor a (TNF-a) and other cytokines.Then it can rapidly induce the immune and inflammation responses through a series of reactions.So it can enhance the expression of multiple genes, including cytokines (IL-1, IL-6, IL-12, TNF-a) inflammatory enzymes (NOS-2, COX-2),matrix metalloproteinases (MMPs) and so on. The results show that the NF-κB signaling pathway is activated at the chondrocyte in the rat animal model of osteoarthritis.According to previous research we suggest that the protective effect of CM-chitosan on chondrocytes may be related to NF-κB signal transduction pathway.The similar research has not been reported at home and abroad. This study aimed to further explore the effect of CM-chitosan on rat osteoarthritis cartilage iNOS expression and NF-κB signal transduction pathway.Part 1:The Efffect of CM-chitosan on NF-κB signalling pathway and iNOS expression in IL-1β-induced rat chondrocytesObjective:To investigate the Efffect of CM-chitosan on NF-κB activation and iNOS expression in IL-1β-induced rat chondrocytes, and explore the mechanism.Methods:The chondrocytes derived from knee joint of rats were primary Cultured in vitro,10ng/ml IL-1βwith or without carboxmethyl-chitosan (CM-chitosan) of varied concentrations (100,200,300μg/ml) was added into culture medium and Co-cultured for 24 hours.Then expression of NF-κBn and NF-κBe as well as I-κB were examined by western blotting. Inducible NO synthase (iNOS) were examined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and western blotting;Results:CM-chitosan could significantly decrease iNOS expression of IL-1β-induced rat chondrocytes, Degradation of I-κB and NF-κB nuclear translocation were reversed by carboxmethyl-chitosan.Conclusion:The study demonstrated that CM-chitosan could inhibits the synthesis of proinflammatory mediators in rat chondrocytes stimulated with IL-1βthrough a NF-κB-dependent mechanism. Part2:The Efffect of intra-articular injection of CM-chitosan on nuclear factorκB (NF-κB) activation and nitric oxide synthase (iNOS) expression in rat cartilageObjective:To observe the efffect of intra-articular injection of CM-chitosan on NF-κB activation and nitric synthase expression in rat osteoarthritis cartilage, and to explore the mechanism of inhibition of joint destruction.Methods:36 SD rats were randomly divided into three groups A,B,C,12 in each group, A group for the sham group, B, C group rats were cut off the medial collateral ligament and removal a part of medial meniscus to establish osteoarthristis model, C group rats were injected 3%carboxymethyl chitosan intra-articular of 0.15mg.Kg-1after 5 weeks,and repeated injection at every1 week, animals were sacrificed in 11 weeks after surgery, the rats generally change of cartilage and the expression of NF-KBn (P65) were compared by immunohistochemistry, the protein expression of I-κB and P65 in nucleus were detecttd by Western blot, induced nitric oxide synthase (iNOS) mRNA and protein expression were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot.Results:C group (intra-articular injection of CM-chitosan group) cartilage degeneration scores were significantly better than the B group, the protein expression of NF-KBn by the articular cartilage in C group significantly lower than B group, C group was significantly inhibited the I-κB degradation, and the expression of iNOSmRNA and iNOS protein by arthritis rat chondrocytes were reduced in OA articular cartilage, it has protectted the articular cartilage.Conclusion:CMC may inhibit NF-κB signaling pathway by inhibiting the degradation of I-κB in cartilage, which reduced iNOSmRNA and protein expression in rat osteoarthritis cartilage, thereby protecting rat osteoarthritis cartilage cells.
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