The Study Of Proinflammation Factor IL-6 And VEGF From Fibroblast Like Synoviocyte Enhanse Progression Of Osteoarthritis

Part one Studies on the expression and significance of interleukin-6 and vascular endothelial growth factor in knee synovial membranes and synovial fluids of Patients with knee osteoarthritisObject To investigate the expression levels of interleukin-6 and vascular endothelial growth factor in knee synovial membranes and synovial fluids on different stages of osteoarthritis,clarify the role of synovial tissue played in the osteoarthritis,and assess its suitability as a marker of progressive osteoarthritis.Methods collect knee synovial membranes and synovial fluids of patients with osteoarthritis who underwent total knee arthroplasty or arthroscopy,and classify them into four groups according to the radiographic grading(K＆L-grade).Appoint K-L-0-grade patients suffered with menisci injuried only as controls.The levels of IL-6 and VEGF in synovial fluid were measured by enzyme-linked immunosorbent assay (ELISA) and IL-6-secreting cells and VEGF-secreting cells were identified by immunohistochemistry.Results The IL-6 levels in synovial fluids were signifieantly higher in the K＆LⅡandⅢgrade OA patients than those of controls with statistical difference(P＜0.01),but decreased in the K＆LⅣgrade patients. The VEGF levels in synovial fluids were increased accordingly with K-L grades,rose to the peak in the late stage of OA.There are inflammatory lesions in all the samples with different degree evaluated histologically on H＆E-stained sections.Synovial tissue inflammation can seen as synovial lining thickness,infiltration by inflammatory cells but also with proliferation of the deep synovium.VEGF expressed in the synovium linings and arounding blood vessel;IL-6 expressed in the synovium linings and under linings.Conclusions IL-6 and VEGF which is generated from synovial tissue play a role in the pathomechanism of OA,which might be the true reason responsible for the persistence of inflammation in the synovial tissue.High levels of VEGF in the synovial fluids can look as the marker of actived osteoarthritis.Part two The study of IL-6 impact on the biological activity of OA synovial fibroblast in vitroObjectives To evaluate the changes of biological activity of fibroblast like synoviocyte induced by IL-6,discuss the mechanism of synovial tissue participate in the cartilage destruction in osteoarthritis (OA).Methods Primary cultures of human synovial fibroblasts were obtained by enzymatic digestion of synovial tissue from end stage OA patients who underwent total knee arthroplasty.Cells were marked with anti-CD3/CD14/CD90 to identification phenotype of synovial fibroblasts by flow cytometry.Selected passages 3 to 5 synovial fibroblasts treated with IL-6,IL-1,or IL-1+IL-6.Superaatants were collected from confluent cultures,filtered,and stored at -70℃for enzyme-linked immunosorbent assay.Total RNA was extracted from synovial fibroblasts,and messenger RNA expression of VEGF and MMP-13 were analyzed using a reverse transcription- polymerase chain reaction(RT-PCR).Results Cells cultured from OA patients expressed CD90(95.62% positive cells),CD3(2.64%positive cells),CD14(3.44%positive cells),which consistent with fibroblast like synoviocyte.The results of ELISA and RT-PCR manifest:synovial broblasts induced by IL-6 only, expressed VEGF at moderate levels and MMP-13 was detected only slightly;when induced by IL-1,expression of VEGF was detected to increase markedly and MMP-13 was detected at moderate levels;when induced by IL-1 and IL-6 togather,the expression of VEGF and MMP-13 both increased significantly. Conclusions The ability of synthesis and secrete VEGF and MMP-13 by IL-6 induced fibroblast like synoviocyte was not strong;but IL-6 would augment the function of IL-1 markedly.IL-6 enhenced the expression of MMP-13 induced by IL-1 and participate in the cartilage destruction in osteoarthritis;IL-6 enhenced the expression of VEGF induced by IL-1 to urge synovial tissue inflammation to exist persistently.Part three p38MAPK inhibitor modulate the expression of VEGF and MMP-13 in OA synovial fibroblast induced by IL-6.Objectives To study the signal pathway of IL-6 participate in the cartilage destruction in osteoarthritis,and evaluate the possibility of p38MAPK inhibitor being used to therapy osteoarthritis.Methods Selected passages 3 to 5 synovial fibroblasts induced by IL-1+IL-6 treated with p38MAPK inhibitor SB220025.Total RNA was extracted from synovial broblasts,and messenger RNA expression of VEGF and MMP-13 were analyzed using a reverse transcription-polymerase chain reaction(RT-PCR).Total protein was extracted to perform Western-blot assay. Results Western-blot and RT-PCR manifest:synovial broblasts induced by IL-1 and IL-6 express VEGF and MMP-13 at different levels, were both down-regulated by p38MAPK inhibitor SB220025 dose-depedently.The inhibition on VEGF expression mediated by SB220025 is stronger than that of meloxicam and dexamethasone;the inhibition on MMP-13 expression mediated by SB220025 is much stronger than both meloxicam and dexamethasone.Conclusions Our results led to the identification of p38MAPK as being crucial for IL-1 and IL-6 induced VEGF and MMP-13 secretion by fibroblast like synoviocyte.p38MAPK inhibitor SB220025 can abolish the effect induced by IL-6 dose-depedently.Our data in vitro support p38MAPK inhibitor SB220025 as a new agent for OA therapy,which is stronger than meloxicam and dexamethasone.