Echinacoside alleviates glucocorticoid induce osteonecrosis of femoral head in rats through PI3K/AKT/FOXO1 pathway.

Steroid-induced osteonecrosis of the femoral head (SONFH), caused by glucocorticoid (GC) administration, is known to exhibit a high incidence worldwide. Although osteoblast apoptosis has been reported as an important cytological basis of SONFH, the precise mechanism remains elusive. Echinacoside (Ech), a natural phenylethanoid glycoside, exerts multiple beneficial effects, such as facilitation of cell proliferation and anti-inflammatory and anticancer activities. Herein, we aimed to explore the regulatory mechanism underlying glucocorticoid-induced osteoblast apoptosis and determine the protective efficacy of Ech against SONFH. We comprehensively surveyed multiple public databases to identify SONFH-related genes. Using bioinformatics analysis, we identified that the PI3K/AKT/FOXO1 signaling pathway was most strongly associated with SONFH. We examined the protective effect of Ech against SONFH using in vivo and in vitro experiments. Specifically, dexamethasone (Dex) decreased p-PI3K and p-AKT levels, which were reversed following Ech addition. Validation of the PI3K inhibitor (LY294002) and molecular docking of Ech and PI3K/AKT further indicated that Ech could directly enhance PI3K/AKT activity to alleviate Dex-induced inhibition. Interestingly, Dex upregulated the expression of FOXO1, Bax, cleaved-caspase-9, and cleaved-caspase-3 and enhanced MC3T3-E1 apoptosis; application of Ech and siRNA-FOXO1 reversed these effects. In vitro, Ech decreased the number of empty osteocytic lacunae, reduced TUNEL and FOXO1 positive cells, and improved bone microarchitecture. Our results provide robust evidence that PI3K/AKT/FOXO1 plays a crucial role in the development of SONFH. Moreover, Ech may be a promising candidate drug for the treatment of SONFH.

Glucocorticoids induce osteonecrosis of the femoral head in rats via PI3K/AKT/FOXO1 signaling pathway.

Background: Steroid-induced osteonecrosis of the femoral head (SONFH) is a disorder that causes severe disability in patients and has a high incidence worldwide. Although glucocorticoid (GC)-induced apoptosis of osteoblasts is an important cytological basis of SONFH, the detailed mechanism underlying SONFH pathogenesis remains elusive. PI3K/AKT signaling pathway was reported to involve in cell survival and apoptosis. Objective: We explored the role of PI3K/AKT/FOXO1 signaling pathway and its downstream targets during glucocorticoid -induced osteonecrosis of the femoral head. Methods: We obtained gene expression profile of osteoblasts subjected to dexamethasone (Dex) treatment from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were screened out and functional enrichment analysis were conducted by bioinformatics analysis. In vitro, we analyzed Dex-induced apoptosis in MC3T3-E1 cells and explored the role of PI3K/AKT/FOXO1 signaling pathway in this phenomenon by employing siRNA-FOXO1 and IGF-1(PI3K/AKT agonist). Finally, we verified our results in a rat model of SONFH. Results: In Dex-treated osteoblasts, DEGs were mainly enriched in the FOXO signaling pathway. Dex inhibited MC3T3-E1 cell viability in a dose-dependent effect and induced apoptosis by increasing the expression levels of FOXO1, Bax, cleaved-Caspase-3, and cleaved-Caspase-9, while reducing the expression of Bcl-2. Notably, these results were reversed by siRNA-FOXO1 treatment. Dex inhibited PI3K/AKT signaling pathway, upregulated FOXO1 expression and increased FOXO1 nuclear translocation, which were reversed by IGF-1. Compared to normal rats, the femoral head of SONFH showed increased expression of FOXO1, increased number of apoptotic cells, and empty osteocytic lacunas, as well as decreased bone tissue content and femoral head integrity. Significantly, the effects of GC-induced SONFH were alleviated following IGF-1 treatment. Conclusion: Dex induces osteoblast apoptosis via the PI3K/AKT/FOXO1 signaling pathway. Our research offers new insights into the underlying molecular mechanisms of glucocorticoid-induced osteonecrosis in SONFH and proposes FOXO1 as a therapeutic target for this disease.

Dexamethasone induces ferroptosis via P53/SLC7A11/GPX4 pathway in glucocorticoid-induced osteonecrosis of the femoral head.

Recently, ferroptosis as new regulatory necrosis has attracted the scientific community. However, the study focused on the effect of ferroptosis on osteocytes in steroid (glucocorticoid)-induced osteonecrosis of the femoral head (SONFH) is still scarce. In this study, we use bioinformatic analysis to screen out differentially expressed genes (DEGs) in osteoblasts that treated by dexamethasone (Dex) in GSE10311 and found these DEGs are enriched in the ferroptosis signaling pathway. The results in vitro experiments show that Dex can induce MC3T3-E1 cells ferroptosis by down-regulating SLC7A11. Specifically, Dex inhibits the expression of SLC7A11/GPX4, decreases the activity of the intracellular antioxidant system such as intracellular glutathione (GSH), while increasing Malondialdehyde (MDA), reactive oxygen species (ROS), and lipid ROS, and reduces the volume of mitochondria, the mitochondrial ridges and a series of obvious ferroptosis features. The overexpression of SLC7A11 and the use of ferroptosis inhibitor (Fer-1) can reverse the Dex-induced MC3T3 ferroptosis. Dex can induce an increase in the expression of p53 and knocking down the expression of p53 by small interfering ribonucleic acid (siRNA) can reverse the suppression of SLC7A11 and GPX4 expression in MC3T3-E1 and MOLY4 cells, thereby reducing the production of ferroptosis. Thus, this study demonstrated that Dex induces MC3T3-E1cells ferroptosis via p53/SLC7A11/GPX4 pathway. The present finding offers novel insight to understand the underlying molecular mechanisms for glucocorticoid-induced osteonecrosis. Moreover, the suppression of ferroptosis may be a novel and promising treatment option for SONFH.