Inhibition of insulin degrading enzyme suppresses osteoclast hyperactivity via enhancing Nrf2-dependent antioxidant response in glucocorticoid-induced osteonecrosis of the femoral head

Abstract

Background: Osteoclast hyperactivation due to the pathological overproduction of reactive oxygen species (ROS) stimulated by glucocorticoids (GCs) is one of the key drivers behind glucocorticoid-induced osteonecrosis of the femoral head (GIONFH). The insulin degrading enzyme (IDE), a conserved Zn²⁺ metallo-endopeptidase, facilitates the DNA binding of glucocorticoid receptor and plays a substantial role in steroid hormone-related signaling pathways. However, the potential role of IDE in the pathogenesis of GIONFH is yet undefined.

Methods: In this study, we employed network pharmacology and bioinformatics analysis to explore the impact of IDE inhibition on GIONFH with 6bK as an inhibitory agent. Further evidence was collected through in vitro osteoclastogenesis experiments and in vivo evaluations involving methylprednisolone (MPS)-induced GIONFH mouse model.

Results: Enrichment analysis indicated a potential role of 6bK in redox regulation amid GIONFH development. In vitro findings revealed that 6bK could attenuate GCs-stimulated overactivation of osteoclast differentiation by interfering with the transcription and expression of key osteoclastic genes (Traf6, Nfatc1, and Ctsk). The use of an H₂DCFDA probe and subsequent WB assays introduced the inhibitory effects of 6bK on osteoclastogenesis, linked with the activation of the nuclear factor erythroid-derived 2-like 2 (Nrf2)-mediated antioxidant system. Furthermore, Micro-CT scans validated that 6bK could alleviate GIONFH in MPS-induced mouse models.

Conclusions: Our findings suggest that 6bK suppresses osteoclast hyperactivity in GCs-rich environment. This is achieved by reducing the accumulation of intracellular ROS via promoting the Nrf2-mediated antioxidant system, thus implying that IDE could be a promising therapeutic target for GIONFH.

Keywords: Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH); Glucocorticoids (GCs); Insulin degrading enzyme (IDE); Osteoclast; Reactive oxygen species (ROS).

Fig. 1

IDE may play a positive role in osteoclast differentiation and maturation. (A) IDE expression profile in human tissues and organs downloaded from Human Protein Atlas (HPA). (B) The IDE transcriptional profiles of different cluster cell types in bone marrow. (C) Western blotting (WB) assay demonstrated increased IDE expression levels correlating with osteoclast differentiation and maturation. Quantitative analysis was performed on bands and normalized to β-actin. Data were shown as means ± SD (n = 3, *p < 0.05, **p < 0.01)

Fig. 2

IDE inhibitor 6bK may have a therapeutic intervention role in the progression of GIONFH. (A) The molecular docking of IDE with 6bK. (B) WB showed IDE protein levels in RANKL (Con) and RANKL + 6bK (40µM) groups. Quantitative analysis was performed on bands and normalized to β-actin. (C) Venn diagram identified 29 overlapping genes between 6bK-targets and gene clusters associated with GIONFH based on the terms of “femoral head necrosis” and “glucocorticoid” from GeneCards database. (D) Gene Ontology (GO) enrichment analysis based on the overlapping genes of (C) exhibited top 20 terms of biological characteristics of biological process (D), cell component (E), and molecular function (F). (G) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis based on the overlapping genes of (C) presented the top 20 enriched pathways. Data were presented as means ± SD (n = 3, ***p < 0.001)

Fig. 3

Dex promotes RANKL-induced osteoclastogenesis in vitro. (A) CCK-8 assay showed the viability of osteoclast precursor cells (OPCs) under Dex (0, 10^− 5, 10^− 6, 10^− 7, 10^− 8, 10^− 9, and 10^-10M) stimulation for 24 h. (B) TRAP staining exhibited osteoclastogenesis under Dex (0, 10^− 6, 10^− 7, 10^− 8, 10^− 9, and 10^-10M) treatment, and (C) the number of TRAP + osteoclasts with 3 or more nuclei were counted. Red arrow heads indicate typical TRAP + osteoclasts. Scale bar = 100 μm. Data were presented as means ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 4

IDE inhibitor 6bK impedes osteoclast differentiation and function under GIONFH condition. (A) CCK-8 assays detected the viability of bone marrow-derived macrophages (BMMs), or osteoclast precursor cells (OPCs) (B), or OPCs under Dex (10^-8M) stimulation (C) after incubated with 6bK in gradient concentration (0, 5, 10, 20, and 40µM) for 24 h. (D) TRAP staining of osteoclasts under different treatment, and (G) the number of TRAP + osteoclasts with different nuclei (n = 3, 6–9, and ≥ 10). Red arrow heads indicate typical TRAP + osteoclasts. Scale bar = 100 μm. (E) Acridine orange (AO) staining after osteoclast induction for 5 days under different treatment. White arrow heads indicate mature osteoclasts. Scale bar = 100 μm. (H) The ratio of fluorescence intensity (red to green) of AO staining was quantitatively measured by Image J software. (F) F-actin rings for osteoclasts under different treatment. The F-actin rings (red) and nuclei (blue) of osteoclasts were stained with phalloidin-iflour594 and DAPI, and captured by a fluorescence microscope. White arrow heads indicate typical mature osteoclasts with intact F-actin rings. Scale bar = 100 μm. (I) Quantitively analysis of osteoclasts with intact F-actin rings per group. Data were presented as means ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 5

IDE inhibitor 6bK suppresses the transcription and expression of osteoclastic marker genes under GIONFH conditions. (A-C) qPCR analysis of Traf6, Nfatc1 and Ctsk mRNA levels in different groups. Data were normalized to GAPDH. (D-G) WB presented protein levels of IDE, Nfatc1, and Ctsk in different groups. Data were normalized to β-actin. All data were shown as means ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 6

IDE inhibitor 6bK attenuates Dex-induced ROS accumulation by modulating the Nrf2/Keap1 pathway during osteoclast differentiation. (A) CCK-8 assays detected the viability of osteoclast precursor cells (OPCs), or OPCs under Dex (10^-8M) stimulation (B) after incubated with tBHQ (0, 2.25, 4.5, 9 and 18µM) for 24 h. (C) CCK-8 assays showed the viability of OPCs, or OPCs under Dex (10-8 M) stimulation (D) after incubated with NAC (0, 1.5, 3, 6 and 12mM) for 24 h. (E) and (G) TRAP staining of osteoclasts under different treatment, (F) and (H) the number of mature osteoclasts with different nuclei (n = 3, 6–9, and ≥ 10) were counted. Red arrow heads indicate typical TRAP + osteoclasts. Scale bar = 100 μm. (I) H₂DCFDA-labeled intracellular ROS in different groups (Scale bar = 100 μm), and (J) quantitative analysis of fluorescence intensity using Image J software. (K) WB exhibited the expression level of Nrf2 and Keap1 in different groups. (L-M) Quantitative analysis was normalized to β-actin. (N-P) qPCR results showed transcriptional levels of Sod1 (N), Gr (O) and Cat (P) in different groups. Quantitative analysis was normalized to GAPDH. Data were shown as means ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 7

The utilization of IDE inhibitor 6bK alleviates the progression of GIONFH. (A) Schematic illustration showed the establishment and intervention of MPS-induced GIONFH mouse model. (B) Representative micro-CT images of femoral heads (coronal, transverse, and sagittal images) from different groups. (C-F) Quantitative analysis of trabecular bone microstructure-related parameters of femoral head, including bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th) (n = 5 per group). Data were presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 8

A schematic diagram of 6bK in suppressing GCs stimulated osteoclast hyperactivation. 6bK attenuates IDE activity, which in turn upregulates Nrf2 expression and suppresses ROS overproduction induced by GCs, ultimately mitigating osteoclast hyperactivation under GCs stimulation. OPC: Osteoclast Precursor Cells; GCs: Glucocorticoids; IDE: Insulin Degrading Enzyme; Nrf2: Nuclear factor erythroid-derived 2-like 2; ROS: Reactive Oxygen Species; Nfatc1: Nuclear factor of activated T-cells 1