| BackgroundOsteoarthritis(OA) is a slowly progressive degenerative disease characterized by gradual loss of articular cartilage,but the mechanism of OA pathology is still unknown.Biochemical and genetic factors contribute to the OA lesions in cartilage by disrupting chondrocyte-matrix associations and altering metabolic responses in the chondrocyte.OA is characterized by a progressive depletion of aggrecan from articular cartilage,with subsequent impairment of tissue function.The most abundant cartilage proteoglycan is aggrecan,comprising a protein core to which are attached many chondroitin sulphate(CS) and keratin sulphate(KS) chains.Monoclonal antibodies (Mab)which specifically against structural carbohydrate epitopes on the KS and CS side chains of aggrecan have been produced.The antibody 5D4 recognises KS neo-epitopes and has been used to estimate KS in body fluids as well as cultured media in vitro.Many studies found that the level KS epitopes are increased in OA patients.Certain neo-epitopes on CS chains recognised by the Mab 3B3 were also found to increase in OA patients.Although OA has been regarded primarily as a noninflammatory arthropathy, symptoms of local inflammation and synovitis are present in many patients and have been observed in animal models of OA.Proinflammatory cytokines,such as interleukin-1(IL-1),IL-17,IL-18 and tumor necrosis factor-α(TNF-α),are suspected of causing damage to OA cartilage and degradation of matrix components.The balance of synthesis and degradation of matrix components is disturbed,with both degradation and synthesis usually enhanced.The new insight of OA aetiology will be leading some new potential therapeutic applications.Extracts from plants of sesquiterpene lactones have been used as folk remedies for migraine,inflammatory arthritis,and tumors.Parthenolide(PAR),one of the major products from sesquiterpene lactones is an active ingredient responsible for the anti-inflammatory properies.Because of the key role of cytokines in the pathegenesis of OA,our interest in this field prompted us to hypothesize that PAR,which has been reported to be anti-inflammatory agent on several kinds of cells,might have anti-inflammatory ability to OA chondrocyte and protect degradation of aggrecan.Objective1.To evaluate the effects of TNF-α,IL-1βand LPS on aggrecan metabolism in cultured human chondrocytes with OA.2.To investigate whether these three cytokines can regulate the gene expresstions of 8 different cell ingredients.3.Further to study the possible inhibition of PAR in cartilage aggrecan depletion induced by three cytokines(TNF-α,IL-1βand LPS) and PAR may have potential therapeutic effect in OA.Methods1.Human Chondrocytes were cultured with different stimulated subjects for up to 8 days.The cytotoxic effect of the experimental substances was evaluated by a cell viability test based on the cleavage of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) by mitochondrial dehydrogenases of metabolically active cells.2.Cultured chondrocytes were classified into 8 groups;they are control,TNF-α, IL-1β,LPS,PAR,PAR+TNF-α,PAR+IL-1βand PAR+LPS.3.The content and release of aggrecan in chondrocyte and conditional culture media as sulfated glycosaminoglycan(GAG),which is primarily a measure of aggrecan content.GAG content was measured by using a modification of a 1,9-dimethylmethylene blue spectrophotometric assay(DMMB).GAG release into medium was represented as a percentage of the total cumulative GAG in the chondrocyte plus cumulative GAG release into medium.4.Aggrecan catabolic fragments in medium were measured by methods of ELISA and Western Blotting using anti-aggrecan monoclonal antibodies 3B3 and 5D4.5.RNA was isolated from chondrocyte with different culture condition and analyzed for expression of aggrecan,A disintegrin and metalloproteinase with thrombospondin motifs(ADAMTS-4),ADAMTS-5,TIMP-1,COX-Ⅱ,MAPK-1, TNF-α,IL-1βand 18S by reverse transcription-polymerase chain reaction (RT-PCR).Results1.The phenotypes of different conditional cultured chondrocyte were no difference in light microscope except LPS group and the cell viability rate were all more than 95%in 8 groups.2.Levels of GAG in medium samples of three groups(TNF-α,IL-1βand LPS) increased significantly comparing with control group.Fragments of 5D4 in medium of three groups also experienced significantly increases at the same time. There was significant decrease of 3B3 level in group LPS than control,whereas 3B3 fragments had no difference among TNF-α,IL-1βand control groups.3.TNF-α,IL-1βand LPS can alter gene expression of some cytokines in cultured chondrocyte.①Levels of aggrecan mRNA in three groups were lower than control,and the gene expression of ADAMTS-4,TNF-a,IL-1βand COX-Ⅱwas up-regulated in three groups comparing the control.②TNF-a and LPS up-regulated ADAMTS-5 and MAPK-1 gene expression while there was no effect of IL-1βon these two kinds of gene expression.③There was no difference of TIMP-1 gene expression among four groups.4.Levels of GAG in medium of PAR+TNF-α,PAR+IL-1βand PAR+LPS group decreased significantly comparing to TNF-α,IL-1βand LPS groups,respectively. PAR produced dose-dependent decreases of GAG release in OA chondrocyte induced by three studied cytokines.5.Fragments of 5D4 in medium of PAR+TNF-α,PAR+IL-1βand PAR+LPS group decreased significantly comparing to TNF-α,IL-1βand LPS groups,respectively. PAR can inhibit the degradation of KS chains in aggrecan stimulated by three cytokines.6.RT-PCR results showed:①PAR down-regulated gene expression of ADAMTS-5 and TNF-αin cultured chondrocyte stimulated by TNF-α,IL-1βand LPS,and up-regulated aggrecan gene expression at the same time.②PAR inhibited gene expression of ADAMTS-4 in chondrocyte induced by IL-1β,whereas there was no difference of ADAMTS-4 gene expression in chondrocyte when stimulated by combination of TNF-αand PAR,LPS and PAR,respectively.③IL-1βgene expression induced by TNF-αand IL-1βwas suppressed by PAR.④PAR inhibited COX-Ⅱand MAPK-1 gene expression of chondrocyte induced by TNF-αand LPS.Conclusion1.TNF-α,IL-1βand LPS can stimulate aggrecan catabolism in cultured human OA chondrocyte;especially the degradation of KS chains in core protein of aggrecan. These three cytokines may play an important role in pathogenesis of OA.2.TNF-a,IL-1βand LPS can up-regulate different kinds of pro-inflammatory cytokines to some degree,and suppress aggrecan gene expression.3.PAR can inhibit aggrecan catabolism of chondrocyte induced by TNF-a,IL-1βand LPS. 4.PAR can down-regulate gene expression of proinflammatory cytokines and aggrecanase,such as TNF-αand ADAMTS-5,in chondrocyte,and up-regulate aggrecan gene expression,indicating PAR may have chondrocyte protection in OA.5.PAR may have potential therapeutic effect in OA.
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