Study On Prevention Of Steroid-induced Necrosis Of Femoral Head At The Early Stage By Regulating The Immune Microenvironment With Curcumin

Objective: Steroid-induced necrosis of femoral head is a disease with a high disability rate,which develops progressively.Early diagnosis and treatment is the key to preventing the progress of the disease.Exploring the mechanism and preventive measures of early steroid-induced necrosis of femoral head is the focus of orthopaedic research.Local inflammatory macrophage infiltration of the femoral head is an essential pathological sign of steroid-induced necrosis of femoral head.Curcumin,which has great potential in preventing and treating various macrophage-related diseases,has a prospect in treating steroid-induced necrosis of femoral head.Therefore,the purpose of this study is to explore the effect and mechanism of curcumin on macrophage polarization and to explore the preventive effect of curcumin on early steroid-induced necrosis of femoral head.Methods: In the in vitro experiment,immunofluorescence staining,flow cytometry and enzyme-linked immunosorbent assays were used to verify the effect of lipopolysaccharide combined with interferon-? on the polarization of RAW264.7 cells to the M1 phenotype.The effect of curcumin on the polarization of RAW264.7 cells was investigated by flow cytometry and enzyme-linked immunosorbent assay.The expressions of JAK1,p-JAK1,JAK2,p-JAK2,STAT1 and p-STAT1 of macrophages were detected by western blot assay.The effects of conditioned medium of different macrophages groups and different concentrations of curcumin on osteocyte apoptosis were detected by flow cytometry.In the in vivo experiment,we selected 4-month-old male C57BL/6 mice to establish the model of steroid-induced necrosis of femoral head.The mice were injected with lipopolysaccharide and glucocorticoid,forced to stand up to get food and run for 2 hours per day.Hematoxylin-eosin staining was used to observe the pathological changes of the femoral heads,and the macrophage infiltration of the femoral head was detected by immunofluorescence staining.The mice were divided into control group,model group and curcumin intervention group.Four weeks after the experiment,the bone mass of the femoral head was detected by Micro-CT,the macrophage infiltration in the bone marrow of the femoral head was detected by immunofluorescence staining,the apoptosis of osteocytes was detected by TUNEL staining,and the degree of osteonecrosis was evaluated by hematoxylin-eosin staining.Results: In vitro experiments showed that RAW264.7 was mainly composed of M0 macrophages.Lipopolysaccharide and interferon-? could effectively induce RAW264.7 to polarize to M1 phenotype and then secrete more inflammatory factors such as tumor necrosis factor-? and interleukin-1?,which could promote osteocyte apoptosis.Curcumin can reduce the phenotypic polarization of macrophages to M1 by inhibiting the JAK1/2-STAT1 signal pathway,and indirectly reduce osteocyte apoptosis induced by inflammation.The in vivo experiments showed that the modelling method we used was effective,and the pathological signs of early steroid-induced necrosis of femoral head could be seen in mice after four weeks of modelling.At four weeks,the femoral head's local part is infiltrated by pro-inflammatory phenotype M1 macrophages.Curcumin could inhibit the infiltration of M1 macrophages,decrease the expression of local inflammatory factor tumor necrosis factor-?,reduce the apoptosis of femoral head osteocytes and bone loss in the model mice,and prevent the occurrence of early steroid-induced necrosis of femoral head to a certain extent.Conclusion: Curcumin inhibits M1 polarization through the JAK1/2-STAT1 pathway and reduces osteocyte apoptosis mediated by inflammation,and prevents early steroid-induced necrosis of femoral head to a certain extent.