| Background Osteoarthritis(OA)has gradually increased the rate of joint disability for middle-aged and elderly people in worldwide,and it is the main chronic joint disease that affects the quality of life of these population.The previous view is that cartilage degradation caused by mechanical stress is the main factor in the pathogenesis of OA.However,with the in-depth understanding of the OA progression,synovial inflammation,abnormal bone remodeling of subchondral bone,bone marrow lesion,and osteophyte formation are also typical pathological features of OA.So far,clinical OA treatment strategies are still unsuccessful.The pain management and surgery are the main therapeutic methods,and most patients are in advanced OA at the time of consultation.Therefore,the study of the pathogenesis of OA is more urgent.The pathological changes of OA subchondral bone have been studied as early as the 1970 s,and this process involved a variety of changes in cellular metabolism and bone microstructure.In recent years,it has been found that the bone-cartilage functional unit plays a vital role in the OA development.A large number of vessels and nerves invade this interface of osteochondral unit,which are obviously related to articular cartilage degeneration.The angiogenesis and neurogenesis form the microchannels,which on the one hand transport small molecules produced from subchondral bone to articular cartilage and on the other hand disturb the normal metabolic microenvironment to facilitate the OA progression.Therefore,the study of the mechanism of microangiogenesis at the bone-cartilage interface may provide new therapeutic ideas for OA.Objective A previous study has shown that the increase of local bioavailability of3'3'5-triiodothyronine(T3)influenced the osteoarthritis onset.Based on the study backgroud,we aimed to investigate the role of thyroid hormone receptor(THRs)signaling for the microangiogenesis in OA subchondral bone and try to provide the new target for OA treatment.Materials and Methods1.Detection of differences in serum TT3 and TT4 levels between OA patients and normal participants;2.The osteoblasts were isolated and cultured from human OA subchondral bone specimens.Immunohistochemistry,immunofluorescence staining,western blot(total protein and nuclear protein)and RT-q PCR were used to detect the expression of THR?,THR? in subchondral bone tissue and osteoblasts.And comparasion was performed between the two groups;3.The angiogenesis-related factors(VEGF,HIF-1?)were examined in OA osteoblasts,after the concentration-dependent and time-dependent T3 treatment by ELISA and Western blot,and the role of PI3K/AKT signaling pathway in angiogenesis was explored.4.The hypoxia model of osteoblast was conducted,and PX-478 inhibited the expression of HIF-1? to verify its mediating role in VEGF expression;5.The small interfering RNAs(si RNAs)were used to knock down the genes expression of THR? and THR? in OA osteoblasts,and the expressions of VEGF and HIF-1? were detected by ELISA and Western blot.Then,the co-localization of THR and VEGF in osyeoblats by fluorescence in situ hybridization;6.The co-culture model of osteoblasts and chondrocytes was conducted and chondrocytes was stimulated using IL-1? to mimic the OA in vivo,and then VEGF expression was detected in chondrocytes when THR was knocked down in osteoblasts;7.The OA model mice were conducted to verify these findings in vitro.Immunohistochemistry was used to detect the angiogenic activity of subchondral bone and the MMPs(metalloproteinases)expression of articular cartilage in OA mice after different treatment.OARSI scores were assessed in different group.HE staining and Saffron O and fast green staining were used in articular cartilage and micro CT was employed to scan the subchondral bone.The sagittal medial tibial plateau region was captured to perform 3D reconstruction and bone microstructural parameters were collected for analysis.Results1.Serum thyroid hormone levels in the OA group were not different from those in the normal group,and the expressions of THR? and THR? in human OA subchondral bone tissue and osteoblasts were up-regulated.The results of western blot showed that nuclear translocation of THRs was elevated;2.The data of ELISA detection and wstern blot demonstrated that T3 upregulated the expression of VEGF and HIF-1? in OA osteoblasts through PI3K/AKT signaling pathway,and enhance THR? nuclear translocation after 24 h and 48 h culture,while high concentration T3(10-5 M)inhibited the VEGF and HIF-1? expression;3.The VEGF expression of OA osteoblasts was decreased when PX-478 inhibited the HIF-1? protein under hypoxic stimulation;4.The VEGF and HIF-1? expression was significantly decreased after knocking down THR? of OA osteoblasts,but the expression of them did not change after knocking down THR?.The result of fluorescence in situ hybridization indicated that THR?and VEGF were co-localized in OA osteoblasts;5.A series of changes were found after injection with si THR? in OA mice joint cavity,such as reduced OARSI scores of the knee joint,decreased angiogenic activity of subchondral bone,reduced the proteoglycan loss of cartilage and the decreased MMP9 and MMP13 expression of articular cartilage.Additionally,the thinned calcified cartilage and the increased BV/TV were also observed in 3D reconstruction of subchondral bone.Conclusions1.The increase of local T3 bioavailability was caused by the increased expression of THR? and THR? in OA subchondral bone;2.T3 up-regulates the expression of angiogenesis-related factors in OA osteoblasts through the PI3 K / AKT signaling pathway,and the enhanced nuclear translocation of THR? is involved in this process;3.The HIF-1? regulates VEGF expression in OA osteoblasts under hypoxic environment of subchondral bone;4.Abnormal THR? signaling in OA osteoblasts mainly regulates microvascularization of subchondral bone,and inhibition of THR? can alleviate OA progression. |
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