BDKRB2 +9/-9bp Gene Polymorphisms Influence The Proinflammatory Cytokines In Knee Osteoarthritis

BACKGROUNDOsteoarthritis(OA)is a degenerative joint disease that leads to the gradual loss of joint function,disabling and seriously affecting the quality of life of the patients.Osteoarthritis etiology is not yet clear,generally considered factors such as heredity,obesity,age,immune factors and mechanical damage.Recently genetic factors,as an important pathogenesis of osteoarthritis,were paid attention by more and more Experts and scholars.Bradykinin B2 receptor(BDKRB2)is widely found in various tissues,including joint tissues,to mediate most inflammatory responses caused by bradykinin.Although the role of BDKRB2 in inflammation has been elucidated,there are relatively few studies on the relationship between BDKRB2 gene polymorphism and inflammation.Prior researches have found that the cytokines plays an important role in the process of OA,Toll-like receptor(TLR)-2 and TLR-4 were up-regulated in the injury cartilage cells of OA,and BDKRB2+9/-9bp gene polymorphism was proved to be associated with the susceptibility and severity of OA.All of these suggest BDKRB2+9/-9bp gene polymorphism may be related to the expression of inflammatory cytokines and the effect of TLRs in OA,which may be a genetic markers of OA,and become the important clinical screening indicator.OBJECTIVEIn this study,gene sequencing and molecular biology were used to explore the relationship between BDKRB2+9/-9bp gene polymorphism and the levels of OA inflammatory cytokines.The molecular mechanism of OA development will be clarified from the perspective of genetic polymorphism,and new ideas and new targets will be provided for the diagnosis and treatment of osteoarthritis.METHODSA total of 156 patients with OA and 121 normal controls were included in the study.Peripheral venous blood were taken respectively and genotypes were determined by using Polymerase Chain Reaction-Single Strand Conformation PolymorPhism(PCR-SSCP)technology.Each group was divided into three of-9/-9bp,+9/-9bp and +9/+9bp.The inflammatory cytokine levels in serum and synovial fluid samples is measured by enzyme linked immunosorbent assay(ELISA),including tumor necrosis factor(TNF)-?,interleukin(IL)-1?,IL-6,IL-8.The levels of inflammatory cytokines in two groups were compared.The mRNA levels of the toll-like receptor TLR-2 and TLR-4 were measured by Quantitative real-time PCR,which the levels were compared.Synovial cells of OA patients are isolated and cultured,which respectively were added MEN16132 B2 receptor inhibitor,TLR-2 siRNA,scrambled siRNA,as well as the blank control group,four groups respectively were cultivated in BK(1?M)and normal cultivation conditions.Finally,the levels of pro-inflammatory cytokines in the culture matrix were measured and compared.RESULTS1.The TNF-?,IL-6,IL-8 and IL-1? proinflammatory cytokines of serum and synovial fluid in OA patients group were significantly higher than that in normal control group(P < 0.01).TNF-? and IL-8 of synovial fluid and serum were significantly higher in-9/-9bp group than in +9/+9bp group(P < 0.05);IL-6 levels in-9/-9bp and +9/-9bp groups were significantly higher than that in +9/+9bp group(P < 0.05).However,there was no significant difference in the level of IL-1? in each group.In normal control groups,there were no statistically significant differences in the level of proinflammatory cytokines2.Compared with the control group,the expression of TLR-2 and TLR-4 were up-regulated(P < 0.01)in OA patients group.In OA patients,the level of TLR-2 mRNA in +9/+9bp group was significantly lower than that in the other two groups-9/-9bp and +9/-9bp(P < 0.01),but there was no significant difference in TLR-4 mRNA level.In the control group there was no statistically significant difference between the gene groups of TLR-2 and TLR-4 mRNA levels.3.BK can stimulate the secretion of IL-6 and IL-8 in human OA synovial cells.MEN16132 bradykinin receptor antagonist inhibits the effect of BK and TLR-2 siRNA also significantly inhibited the secretion of il-6 and il-8 induced by the basal and bradykinin.However,there was no significant change in the secretion of TNF-? in each treatment group.CONCLUSIONThe results of this study indicated that BDKRB2+9/-9bp gene polymorphism affects the level of proinflammatory cytokines in serum and synovial fluid in patients with knee OA.BDKRB2+9/-9bp gene polymorphism is closely related to TLR-2 expression,which is in lower expression can inhibit the level of inflammatory cytokines in knee OA patients.This further proves that BDKRB2+9/-9bp gene polymorphism can affect the level of pro-inflammatory cytokines in knee OA patients by altering TLR-2 expression.