Comprehensive analysis of pivotal biomarkers, immune cell infiltration and therapeutic drugs for steroid-induced osteonecrosis of the femoral head

Figures & data

Figure 1. Identification of DEGs in SONFH. (a) Hierarchical clustering heat map of SONFH samples and control samples based on identified DEGs (50 genes shown). (b) Volcano plot of the DEGs between SONFH samples and control samples

Table 1. The six most significant MCODE components were extracted from the PPI

MCODE_ALL	R-HSA-373076 Class A/1 (Rhodopsin-like receptors); R-HSA-500792 GPCR ligand binding; GO:0002274 myeloid leukocyte activation
MCODE_1	GO:0042026 protein refolding; GO:0019320 hexose catabolic process; hsa00010 Glycolysis/Gluconeogenesis
MCODE_2	R-HSA-373076 Class A/1 (Rhodopsin-like receptors); R-HSA-418594 G alpha (i) signaling events; R-HSA-500792 GPCR ligand binding
MCODE_3	GO:0002755 MyD88-dependent toll-like receptor signaling pathway; GO:0032757 positive regulation of interleukin-8 production; GO:0032677 regulation of interleukin-8 production
MCODE_4	R-HSA-416476 G alpha (q) signaling events; R-HSA-373076 Class A/1 (Rhodopsin-like receptors); R-HSA-500792 GPCR ligand binding
MCODE_5	GO:0008285 negative regulation of cell proliferation
MCODE_6	GO:0045669 positive regulation of osteoblast differentiation; GO:0045778 positive regulation of ossification; GO:0045667 regulation of osteoblast differentiation

Figure 2. Function enrichment analysis and PPI network construction. (a) GO functional enrichment analysis of BP. (b) GO functional enrichment analysis of CC. (c) GO functional enrichment analysis of MF. (d) KEGG pathway analyses results of the DEGs. (e) The PPI network of the DEGs. (f) The six most significant modules were obtained from the PPI network

Figure 3. The differences in immune cell infiltration between SONFH and normal controls. (a) The relative percentage of 24 subpopulations of immune cells in 40 samples from the GSE123568 dataset. (b) Correlation analyses of the 24 subpopulations of immune cells estimated in 40 samples from GSE123568. (c) Heat map of the 24 subpopulations of immune cells estimated in 40 samples from GSE123568. (d) The differences in immune cell infiltration between SONFH patients and normal controls (*p values < 0.05 were considered statistically significant)

Figure 4. The 10 most highly expressed hub genes in the PPI network as confirmed with RT-PCR. (a) The 10 most highly expressed hub genes in the PPI network as determined by cytoHubba. (b) RT-PCR validation of the hub genes in SONFH and normal controls. All experiments were performed in triplicate, and the results are presented as the means ± SD. (*p < 0.05)

Table 2. The top 10 compounds with activity against SONFH as predicted via connectivity map

Score	Name	Description	Target
−99.68	Phylloquinone	Vitamin K	BGLAP, GGCX
−99.58	cholic-acid	Bile acid	ADH1C, CES1, COX4I1, COX5A, COX5B, COX6A2, COX6B1, COX6C, COX7A1, COX7B, COX7C, COX8A, ESRRG, FABP6, FECH, GPBAR1, MT-CO1, MT-CO2, MT-CO3, PLA2G1B
−99.58	MRS-1220	Adenosine receptor antagonist	ADORA2B, ADORA3
−99.54	Bucladesine	Adenosine receptor agonist	PRKACA
−99.51	Isotretinoin	Retinoid receptor agonist	CYP2B6, CYP2C19, CYP2C8, CYP3A5, CYP3A7, NR2C2, PPARD, RARA, RARB, RARG, RORB
−99.22	Doxercalciferol	Vitamin D receptor agonist	VDR
−98.8	Betahistine	Histamine receptor agonist	HRH1, HRH3
−98.8	Aspirin	Cyclooxygenase inhibitor	AKR1C1, ASIC3, EDNRA, HSPA5, IKBKB, NFKB1, NFKB2, NFKBIA, PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3, PTGS1, PTGS2, RPS6KA3, TP53
−98.73	Maraviroc	CC chemokine receptor antagonist	CCR5, CYP3A5
−98.38	SR-27897	CCK receptor antagonist	CCKAR

Figure 5. Identification of the therapeutic effect of aspirin in GC-induced ONFH. (a) Schematic diagram of the treatment of different groups of mice. H&E staining of representative femoral heads in each group. (b) Bone volume/total volume ration (BV/TV) in each group. (c) Empty lacunae rate in each group. (d-f) H&E staining of representative femoral heads in each group (scale bar = 100 μm). (g-i) TRAP staining of representative femoral heads in each group (scale bar = 100 μm). (j-l) Relative Runx2, RANKL and PTGS2 mRNA expression in femoral heads in each group. All experiments were performed in triplicate, and the results are presented as the means ± SD. (*p < 0.05 versus the control; #p < 0.05 versus the GC)

Data availability statement

All the data in our study will be made available by the authors upon reasonable request. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE123568