The Effect And Mechanism Of Halofuginone And Gemigliptin On Osteoarthritis,Viewing From The Perspective Of TGF-? And NF/?B Signalling Pathway

Objective: To explore the role and mechanism of the interventional therapy for osteoarthritis by the TGF-1/Smad pathway and NF/kappa B pathway.1)by establishing beagle model of osteoarthritis anterior cruciate ligament,and followed by the local injection of Halofuginone into subchondral bone.The role of halofuginone on osteoarthritis and the role of TGF-1 was discussed from the perspective of cartilage and subchondral bone.2)Through the analysis of the cultivation of the human articular cartilage cells in knee joint tibial platform and gemigliptin of drug intervention,preliminary explore gemigliptin of interleukin 1 beta inhibition of statins,thus inhibiting the degradation of type II collagen.Methods: 1)By establishing beagle model of osteoarthritis anterior cruciate ligament,and followed by the local injection of Halofuginone into subchondral bone.At the point in time of 4 weeks,8 weeks and 12 weeks after ACLT and local injection of Halofuginone,score the lameness of dog models by Lameness Score System.Kellegren&Lawrence radiology classification was used for comparison osteoarthritis grade and MRI examination were performed to observe the bone marrow lesion in the knee joint of dogs.At the same time,the expression of TGF-beta 1 was measured by immunofluorescence assay of peripheral blood.2)12 weeks after ACLT local injection of halofuginone,euthanasia was performed in all dogs and the microscopic structure of the subchondral bone was observed by micro-ct.and to analyze the expression of TGF-?1.3)Chondrocytes culture andgemigliptin drug intervention for osteoarthritis of the humen knee joint specimens of tibia platform,followed by immunohistochemical and immunofluorescence analysis method for determining cartilage related biomarkers;Results: 1)All 12 dogs had no significant physical disability before the experiment.In ACLT OA group(N=6)and halofuginonegroup(N=6).The changes of gait were observed at 4 weeks,8 weeks and 12 weeks after ACLT and local injection of Halofuginone by application of LSS.There was no statistically significant difference in group B compared with group C in group C,and in the 8 and 12 weeks,the lameness score of the OA group was significantly higher than that of the HF administration group(P<0.001).The T2 Wl inhibition sequence of the HF group was not seen in the anterior cruciate ligament signal,and the T2 Wl antilipid sequence of articular cartilage did not show the local plate slightly positive signal,and the joint surface was smooth.The subchondral bone signal was uniform,and the anterior cruciate ligament was absent in the HF group model.The coronal surface showed no signs of bone marrow lesion.2)OARSI scores were observed in three groups of beagle cartilage HE staining,and there was significant statistical difference between the groups(P<0.01).Among them,the Sham group had the lowest score,and the OA group was higher than the administration group.Matrix metalloproteinase 13 expression was higher in the cartilage specimens of the OA group.At the same time,transforming growth factor beta 1 expression in the subchondral bone quantity were also different(P<0.01).The expression of TGF-?1 AC(articular catilage)was more than that of intact(P<0.01).Compared with macroscopic view the complete specimen of articular cartilage,articular cartilage of the wear of specimens,the subchondral bone of the bone volume fraction(BV/TV)increased,the thickness of the subchondral bone plate(TB.Th)increased,and the trabecular bone pattern factor(TB)Pf)reduced,the Structure model of index(Structure model index,SMI)is reduced,bone trabecular number(TB.N),has statistically difference two groups(P < 0.01);3)Interlukin-1? initiates the activation of inflammation related signaling pathways and stimulates the expression of matrix metalloproteinases(MMPs),which results in type IIcollagen degradation in chondrocytes;The treatment intervention group with three different concentrations of gemigliptin 10,25,50?M,all significantly reduced the degradation of type II collagen in chondrocytes;IL-1?induced gene expression inmmPs.The drug intervention of the three different concentrations of gemigliptin significantly inhibited the gene expression and protein expression level ofmmp-1,mmp-3,mmp-13 induced by IL-1?.Luciferase reporter assay displays that IL-1? severely increased NF-?B luciferase activity,which was remarkably inhibited by gemigliptin treatment;Conclusions: 1)Local injection of halofuginone can improve the function of knee joint and reduce the degree of lameness in beagle osteoarthritis model.Reduce stage of osteoarthritis in the radiology classification and prevent the production of local bone marrow lesion in the knee joint.There was no specific high expression of TGF-?1 in peripheral blood of the beagle OA model.2)ACLT beagle dog model was detected and degeneration of articular cartilage.Halofuginone,administered by local injection,inhibited the elevated activity of TGF-?1 coupled bone remodeling 3): Gemigliptin inhibited the degradation of collagen type ?in human chondrocytes;Gemigliptin suppressed the expression ofmmP-1,mmP-3,andmmP-13;Gemigliptin inhibited the IKK/I?B?/NF-?B pathway and the activation of p38.