| Objective: To determine whether the Kidney Tonifying And Blood Activating Method can interfere with the pathogenesis and possible mechanism of steroid-induced osteonecrosis of femoral head(SONFH)by regulating macrophage(Macrophage,M0)polarization.Methods: Construct a rat SONFH model for experimentation,and 100 SD rats were randomly divided into four groups: control group(Group A),GCs group(Group B),GCs+200ug/ml lowdose drug group(Group C),and GCs+400ug/ml high-dose drug group(Group D).Group A received routine feeding,while Group B constructed a rat SONFH model using an improved hormone method.Groups C and D were given kidney tonifying and blood activating drugs by gavage in addition to GCs.HE staining was used to evaluate the rate of empty bone lacunae and the incidence of bone necrosis.Micro-CT was used to observe whether the arrangement of bone trabeculae in each group was neat,the density of bone trabeculae,and quantitative analysis indicators included bone trabecular volume fraction,bone trabecular thickness,bone trabecular quantity,and bone trabecular dispersion;Multiple Immunohistochemistry(m IHC)was used to detect and compare the number of M1 type macrophages(CD68+,CD80+,CD206-)and M2 type macrophages(CD68+,CD80-,CD206+)in different groups of bone tissue.The expression levels of TLR-4,MyD88,NF-κB P65 in the classic inflammation related TLR4/MyD88/NF-κB signaling pathway were also evaluated;ELISA method was used to detect the expression levels of inflammatory cytokines IL-6,IL-1β,TNF-α,and IL-10 in serum.Using SPSS 29.0 statistical software,statistical analysis was conducted based on data categories,and P<0.05 was considered statistically significant;the pairwise comparison of the rates between the four groups was performed using chi square segmentation,with a P<0.0125 being considered statistically significant.Results: HE staining showed that there were no obvious empty bone lacunae in the trabeculae of group A specimens,and the nuclei were normal.Empty bone lacunae appeared in the trabeculae of specimens from Group B,Group C,and Group D,with the nucleus becoming smaller,atrophied,and moving to the cell edge,with Group B being the most prominent.The Micro-CT results showed that the bone trabeculae in Group A presented a porous grid structure with regular arrangement.The number and thickness of bone trabeculae were normal,while Group B showed a significant reduction,thinning,and sparsity of bone trabeculae compared to Group A.The arrangement was irregular,while Group D showed a significant thickening,thickening,denser,and more regular arrangement of bone trabeculae compared to Group B.Group C showed a performance between Group B and Group D.The m IHC results showed that compared with Group B,the Kidney Tonifying and Blood Activating Medicine groups(C and D)showed a significant decrease in M1 and an increase in M2 phenotype.The expression levels of TLR-4,NF-κB P65,and MyD88 in the TLR4/MyD88/NF-κB signaling pathway decreased,and the above changes were more pronounced in Group D compared to Group C.The ELISA results showed that compared with Group B,the expression levels of pro-inflammatory factors IL-6,IL-1β,and TNF-α in the serum of the kidney tonifying and blood activating drug groups(C and D groups)were significantly reduced,while the expression levels of anti-inflammatory factor IL-10 were significantly increased,and the inflammatory environment was significantly reduced.The above trend of changes was more evident in Group D compared to Group C.Conclusion: SONFH is closely related to chronic inflammatory response mediated by abnormal polarization of M1 macrophages.Kidney Tonifying And Blood Activating Method can inhibit TLR4/MyD88/NF-κB signal pathway can increase the polarization macrophages to M2 phenotype,regulating the activation state of macrophages can reduce the inflammatory environment and thereby treat the disease. |
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