15-Lipoxygenase-1 Regulates TGF-?1 Expression In Osteoarthritis Subchondral Bone

Background: Osteoarthritis(OA)is a degenerative joint disease characterized by joint pain,deformity,and limited movement.Pathologically,it mainly involves in the destruction of articular cartilage,the hardening of subchondral bone,and the aseptic inflammation of surrounding synovial tissue.Although previous research focused on articular cartilage,recent research shows that the pathological changes of subchondral bone in osteoarthritis are earlier than that of articular cartilage.At the same time,the abnormal remodeling of bone and the osteosclerosis of subchondral bone will cause changes in the mechanical support characteristics of cartilage,affect the stability of articular cartilage covering its surface,make cartilage pathological changes,and eventually lead to osteoarthritis.Therefore,the pathological changes of subchondral bone play an important role in the pathogenesis of osteoarthritis.It is of great practical significance to study the pathological mechanism of subchondral bone.Osteoblasts account for the most of the cells in subchondral bone of osteoarthritis,and play the most important role in subchondral bone lesions.The overexpression of TGF-?1 in osteoblasts leads to abnormal changes in subchondral bone structure,eventually causing OA.Moreover,reduced the expression of TGF-?1 could obviously alleviate subchondral bone lesions in OA.Therefore,the study of TGF-?1 expression in OA subchondral bone could play an important role in the future treatment of OA.15-lipoxygenase-1(15-LOX-1)belongs to the lipoxygenase family involved in the inflammatory response and pathological process of various diseases,including the deterioration of cartilage in osteoarthritis.However,the pathogenesis of the disease is poorly defined,and the interaction between 15-LOX-1 and TGF-?1 in osteoblasts has not been evaluated in OA.Objective: In this study,the mechanism by which 15-LOX-1 regulates the expression of TGF-?1 and the role of 15-LOX-1 in OA subchondral bone was evaluated.Method: In the first part,identification of osteoblasts that extracted from OA subchondral bone were used by alizarin red staining and alkaline phosphatase staining.15-LOX-1 expression in osteoblasts of the subchondral bone of patients with OA was measured by immunohistochemistry,q RT-PCR,and western blotting.After the osteoblasts extracted from the subchondral bone of OA were transfected with 15-LOX-1 si RNA or 15-LOX-1 overexpression vector,the effect of 15-LOX-1 on the expression of TGF-?1 in OA osteoblasts was assessed by q RT-PCR and western blotting.The inhibitory effect of 15-LOX-1 on autophagy in OA osteoblasts was evaluated by q RT-PCR,western blotting,and transmission electron microscopy.The expression of TGF-?1 in osteoblasts was measured by q RT-PCR,and western blotting after treatment with autophagy activator or inhibitor.The effect of 15-LOX-1 on the expression of AMPK/m TORC1 pathway was measured by western blotting.In the second part,we used mice DMM model to explore the effect of 15-LOX-1 on OA subchondral bone.The expression of TGF-?1 and 15-LOX-1 was measured by immunohistochemistry in mice subchondral bone.The degree of pathological changes of subchondral bone in mice was measured by mirco CT and Safranin O-fast green staining.Results: The expression levels of 15-LOX-1 along with the osteoblast expression of TGF-?1 were higher in OA subchondral bone than that in non-OA subchondral bone.Inhibition of 15-LOX-1,which upregulated autophagy by activating AMPK following the inhibition of m TORC1,downregulated the osteoblast expression of TGF-?1 in osteoarthritis.Treatment with autophagy inhibitors significantly increased the expression levels of TGF-?1 in osteoblasts.Moreover,treatment with autophagy activator significantly decreased the expression levels of TGF-?1 in osteoblasts.Inhibition of 15-LOX-1 could also reduce the expression of TGF-?1 and alleviate the pathological changes in mice OA subchondral bone.Conclusion: In the present study,our results suggested that the expression of 15-LOX-1 on osteoblasts from the subchondral bone increased in OA.our findings suggested that inhibition of 15-lipoxygenase-1 in osteoblasts reduce TGF-?1 expression via upregulating autophagy by activating AMPK following the inhibition of m TORC1 in osteoarthritis.Moreover,inhibition of 15-LOX-1 could alleviate the pathological changes and reduce the expression of TGF-?1 in mice OA subchondral bone.These novel results suggested that reduced 15-lipoxygenase-1 might be a promising therapeutic target in OA.