Effect Of Nitric Oxide On Expression Of Cytokines In Human Ostearthritis Chondrocytes

BackgroundsOsteoarthritis(OA) is one of the four biggest senile diseases of our country,and make the older disable.The pathogenesis isn't clear up to now.The current research show that the pathogenesis of OA is subjected to regulation of various genes,involves kinds of cytokines,which form cascade network system,make chondrocytes cluster, cartilage matrix degrade,and result in vicious cycle.The expression of cytokines of matrix metalloproteinase(MMPs) and interleukins(ILs) is more conspicuous in OA chondrocytes comparing with normal chondrocytes by immunohistochemistry.Nitric oxide(NO) is one of the most importance mediators in the pathogenesis of OA,and promote the pro-inflammation,which can break the balance between pro-inflammantion and anti-inflammation and degrade extracellular matrix and cartilage.Inflammatory factor(IL-1β,LPS) can improve the level of NO in chondrocytes,and effect other cytokines as a mediators of inflammation.Inducible nitric oxide synthase(iNOS) inhibitor can depress the leves of NO in chondrocytes.Animal experiment have certified iNOS inhibitor can improve cartilage of OA,and cultured chondrocytes in vitro have certificate that iNOS inhibitor can depress some of cytokines,so iNOS inhibitor can prevent cartilage from degrading and pathological proceeding.The cytokines invovled in OA pathology don's work by single way but interacting,form cascade reaction.So it is important to study kinds of cytokines expression simultaneously.The application of proteinome array technique provide convenient instrument to study the much cytokines expression simultaneously. Liquichip workstation system is a new platform to proteinome study.Transversal compareing these cytokines relationship,help to find out the key cytokine that result in OA.And detecting the different cytokines in the present and absent iNOS inhibitor can help to approaching mechanism of iNOS inhibitor in pathology.ObjectivesDetecting chondrocytes supernatant with the inflammatory factor(IL-1β) which can induce chondrocytes to express NO and(or) aminoguanidine(AG) which can inhibit NO as iNOS inhibitor by liquichip workstation to find out how the cytokines(MMP-1,3,9,IL-6,TGF-βand VEGF) express,approach the cytokines machanism in OA pathology,further study the effect of NO to the key cytokirles,which can help to understanding iNOS inhibitor's effect to OA pathology.Methods1.Culturing normal chondrocytes(from the patients with comminuted tiblal plateau fracture cartilage avulsion and tumors) and OA chondrocytes(from the patients with TKA) in the present IL-1βand(or) AG,detecting NO expression from supernatant.2.Detecting the cytokines(MMP-1,3,9,IL-6,TGF-βand VEGF) in the supematants by liquichip workstation in the present or absent IL-1βand(or) AG,and find out the key cytokines.Comparing the diffenert cytokines between them,find out the key cytokines,and approach the effect of NO on the cytokines to understand its application in OA pathology.3.Data presented by(?)±S,statistics done with SPSS13.0.Comparing the different by Two-Way ANOVA of factorial design and LSD test of multi-group mean comparing.Analysing the experiment group and control group by Independent-samples T Test,if equal variances isn's assumed,2 independent samples Nonparametric Test can be carried out.P=0.05 presents statistics different.Results1.There isn's different in morphology between the normal chondrocytes and OA chondrocytes.The time of OA chondrocytes in the form of adherence and primary culture is longer than the normal chondrocytes.The chondrocytes occur dedifferentiation after passage 4.The normal chondrocytes' body is round,nucleus is round or ellipse,coloration of nucleus is moderate,there are bulks of dilated rough endoplasmic reticulum(RER) around nucleus,more glycogenosome lie in intracytoplasm.The OA chondrocytes' body is small,with much reductus.The nucleus is larger and its coloration is deeper,with polygon because of indentation.Less RER and glycogenosome lie in intracytoplasm.Cytoplasm's coloretue of the postive chondrocytes(with one-antibody) is deeper,with brunneus. The negative(without one-antibody) is tasteless.2.The level of NO without IL-1βis lower than with IL-1βin chondrocytes supernatant in vitro,and the level of NO in OA chondrocytes is higher than the normal chondrocytes.The inducion of IL-1βon NO can be inhibited by AG.3.The level of MMP-1,MMP-6,IL-6 and VEGF in OA chondrocytes is higher than in normal chondrocytes by liguichip workstation,and the cytokines can be induced by IL-1β,especially in OA chondrocyes,but inhibited by AG.The level of MMP-9 is no significant difference between the OA and normal chondrocytes,even with IL-1β.The level of TGF-βin OA chondrocytes is lower than in the normal chondrocytes,and can be inhibited by IL-1β.Conclusions1.The morphology different between normal chondrocytes and OA chondrocytes aren't conspicuous,but the funtion of OA chondrocytes in synthesing protein and glycogenosome decrease by transmission electron(TEM).2.Chondrocytes culturee in vitro have only little NO expression,after IL-1βstimulation,chondrocytes can express a lot of NO,and the inducing can be chocked by AG..OA chondrocytes can be induced easily.3.The cytokines of MMP-1.3,IL-6 and VEGF in chondrocytes play a important role in OA pathology,and aggravate cartilage.The distribution of MMP-9 in articular cartilage is different by layer.TGF-βmay be a kind of protection factor to OA,and AG can promote TGF-βexpression by inhibiting NO.4.NO is an important mediator of inflammation to OA,inhibitiong NO can postpone OA pathological proceeding,iNOS inhibitor can prevent cartilage from subjecting to some of proinflammatory,it help to healing OA clinically.5.Liguichio workstation is new detecting system with high-flux and hypersensitivity, help to study proteome.