Effect And Relationship Of Nitric Oxide On Expression Of Cytokines In Osteoarthritis Synoviocytes

BackgroundsThe osteoarthritis (OA) is one of the four biggest senile diseases of ourcountry. The pathogenesis doesn't is clear up to now. The current research show thatthe pathogenesis of OA is subjected to regulation of various genes, involves to severalcytokines, and form network. The normal cartilages and synoviocytes secrete variousgrowth factors and cytokines. When cartilage appears pathological changes,synoviocyte's secretion of growth factors and cytokines is abnormality. Nitric oxide(NO) is an importance factor to the pathogenesis of OA. The cartilages andsynoviocytes are important sources of NO in patient of OA. Inducible nitric oxidesynthase (iNOS) inhibitor can improve cartilage of OA. Since these molecules do notto produce effect by single but interaction, it is important to study several cytokinesexpression simultaneously. The application of gene array technique providedpowerful instrument to study this several cytokines expression simultaneously.Liquichip workstation system is a new platform to protein's study. It can detectseveral cytokines in the same sample simultaneously. It can detect cytokines that OArelated efficiently. Through transversal compare these cytokines relationship, it canfind out the key cytokine that result in OA. In this study, we culture OA synoviocytesin vitro, we apply lipopolysaccharide (LPS) as stimulating factor induce synoviocytesexpress NO, apply iNOS inhibitor as interfere factor, study NO's influence to OA synoviocytes express related cytokines, get the message of the change andrelationship of these cytokines expression.ObjectivesLPS applied to induce synoviocytes express iNOS, through aminoguanidine (AG)inhibit the activity of iNOS, adjust synoviocytes produce NO, detect severalcytokines expression which under different NO concentration. To get the message ofseveral cytokines expression's change and analyze the relationship between cytokinesand NO.Methods1. OA synovium were collected and synoviocytes were cultured in vitro and identifiedby immunohistochemistry with anti-vimentin antibody. LPS concentration divideinto 4 group:0,1,10,100mg/L. 20 mg/L LPS add into 4 group AGconcentration:0,0.1,1,10mmol/L.Then culture supernatant was collected. The changeof iNOS concentration induced by difference concentration of LPS was detected. Thechange of NO concentration induced by 20 mg/L LPS and difference concentration ofAG.2. synoviocyte divide into 4 group: synoviocytes; synoviocytes+LPS 20mg/L;synoviocytes+AG lmmol/L; synoviocytes+ LPS 20mg/L+AG 1mmol/L。After addin LPS and AG to different groups, continue culture 48h, and collect supernatant. NOconcentration of each group was detected. MMP-1.3.9 was detected with liquid chiptechnology. The message of the influence of NO to MMP-1.3.9 expression was got.The relationship between MMP-1.3.9 and NO was presumed.3. synoviocyte divide into 4 group: synoviocytes; synoviocytes+LPS 20mg/L;synoviocytes+ AG 1mmol/L; synoviocytes+ LPS 20mg/L+AG 1mmol/L.After addLPS and AG to different groups, continue culture 48h, collect supernatant. NOconcentration of each group was detected.IL-6,TGF-β,VEGF were detected withliquid chip technology. The message of the influence of NO to IL-6,TGF-β,VEGF expression were got. The relationship between IL-6,TGF-β,VEGF and NO werepresumed..4. Data present by (?)±S, statistics done with SPSS10.0.The comparation of eachgroup data:first carry out homogeneity test for variance,if homogeneity test regularity,One-WayANOVA of completely random design and LSD test of multi-group mean compare wereadopted;if not, Kruskal-Wallis test of completely random design was adopted, P＜0.05 asstatistics different. Pearson correlation analysis was adopted to analyze correlation, P＜0.05 asstatistics different.Result1. Tissue primary culture 3d, a few synoviocytes were emit from edge of tissue, after2-3w from conferted cell monolayer pave full bottle floor, part district as cumulate orwhirlpool form grow. After 2-3 generation serial subcultivation, bulk arefibroblast-like cell,it can reach 95% identify by immunohistochemistry. In LPS freegroup, iNOS concentration is very low in supernatant, compare medium controlgroup have none obviously distinction (P＞0.05).After add in LPS, accompany LPSconcentration increase iNOS concentration also increase (P＜0.05). Synoviocytesinduced by LPS, add different concentration AG, accompany AG's concentrationincrease, NO concentration taper (P＜0.05).2. There is no significant difference between synoviocytes +AG group andsynoviocytes group on NO concentration (P=0.089). There is significant difference insynoviocytes group,synoviocytes +AG group and synoviocytes +LPS group,synoviocytes +LPS +AG group on NO concentration (P＜0.001). There is significantdifference in synoviocytes group, synoviocytes +AG group and synoviocytes +LPSgroup on MMP-1 concentration (P＞0.05).There is no significant difference betweenany other two groups on MMP-1 concentration (P＞0.05). There is significantdifference in synoviocytes group,synoviocytes +AG group and synoviocytes +LPSgroup, synoviocytes +LPS +AG group on MMP-1 concentration (P＜0.05). Correlatedrelationship analysis between NO and MMP-1 is done, r=0.782, P＜0.001, positive correlated relationship present. There is no significant difference betweensynoviocytes +AG group and synoviocytes group on MMP-3 concentration (P＞0.05).There is significant difference in synoviocytes group,synoviocytes +AG group andsynoviocytes +LPS group,synoviocytes +LPS +AG group on MMP-3 concentration(P＜0.05). Correlated relationship analysis between NO and MMP-3 is done, r=0.868,P＜0.001, positive correlated relationship present. There is no significant difference insynoviocytes group,synoviocytes +AG group and synoviocytes +LPS group,synoviocytes +LPS +AG group on MMP-9 concentration (F=0.105,P＞0.05).Correlated relationship analysis between NO and MMP-9 is done, r=0.126, P=0.596,no correlated relationship present.3. There is no significant difference between synoviocytes +AG group andsynoviocytes group on IL-6 concentration (P=0.497). There is significant differencein synoviocytes group,synoviocytes +AG group and synoviocytes +LPS group,synoviocytes +LPS +AG group on IL-6 concentration (P＜0.05). Correlatedrelationship analysis between NO and IL-6 is done, r=0.879, P＜0.001, positivecorrelated relationship present. There is no significant difference betweensynoviocytes +AG group and synoviocytes group on TGF-βconcentration (P=0.211).There is significant difference in synoviocytes group,synoviocytes +AG group andsynoviocytes +LPS group,synoviocytes +LPS +AG group on TGF-βconcentration(P＜0.05). Correlated relationship analysis between NO and TGF-βis done, r=-0.818,P＜0.001, negative correlated relationship present. There is no significant difference insynoviocytes group,synoviocytes +AG group and synoviocytes +LPS group,synoviocytes +LPS +AG group on VEGF concentration (F=0.0.685,P＞0.05).Correlated relationship analysis between NO and VEGF is done, r=0.165, P=0.488,no correlated relationship present.Conclusion1. Synoviocytes culture in vitro system have only little NO and iNOS expression innormally, after LPS stimulation, synoviocytes can express a lot of NO and iNOS,then add AG can effectively inhibit synoviocytes express NO and iNOS. AG as a kind ofiNOS not completes suppress NO generate.2. Synoviocytes culture system, there is positive correlation relationship among NO,MMP-1,3, and has not obviously relationship with MMP-9.3. Synoviocytes culture system, there is positive correlation relationship between NOand IL-6. There is negative correlation relationship between NO and TGF-β. TGF-βmay be a kind of protection factor. NO has no obviously relationship with VEGF.4. Synoviocytes increase NO expression under the stimulation of inflammatory factor andthen increase the expression of MMP-1.3,IL-6 promote the development of OA.It's can delay thedevelopment of OA by inhibit the expression of NO, decrease the expression ofMMP-1.3,IL-6.