Identification of key ferroptosis-related biomarkers in steroid-induced osteonecrosis of the femoral head based on machine learning.

BACKGROUND: This study was aimed to identify key ferroptosis-related biomarkers in steroid-induced osteonecrosis of the femoral head (SONFH) based on machine learning algorithm. METHODS: The SONFH dataset GSE123568 (including 30 SONFH patients and 10 controls) was used in this study. The differentially expressed genes (DEGs) were selected between SONFH and control groups, which were subjected to WGCNA. Ferroptosis-related genes were downloaded from FerrDb V2, which were then compared with DEGs and module genes. Two machine learning algorithms were utilized to identify key ferroptosis-related genes, and the underlying mechanisms were analyzed by GSEA. Correlation analysis between key ferroptosis-related genes and immune cells was analyzed by Spearman method. The drug-gene relationships were predicted in CTD. RESULTS: Total 2030 DEGs were obtained. WGCNA identified two key modules and obtained 1561 module genes. Finally, 43 intersection genes were identified as disease-related ferroptosis-related genes. After LASSO regression and RFE-SVM algorithms, 4 intersection genes (AKT1S1, BACH1, MGST1 and SETD1B) were considered as key ferroptosis-related gene. The 4 genes were correlated with osteoclast differentiation pathway. Twenty immune cells with significant differences were obtained between the groups, and the 4 key ferroptosis-related genes were correlated with most immune cells. In CTD, 41 drug-gene relationship pairs were finally obtained. CONCLUSIONS: The 4 key ferroptosis-related genes, AKT1S1, BACH1, MGST1 and SETD1B, were identified to play a critical role in SONFH progression through osteoclast differentiation and immunologic mechanisms. Additionally, all the 4 genes had good disease prediction effect and could act as biomarkers for the diagnosis and treatment of SONFH.

[REVERSED ARTHROSC OPIC SUBACROMIAL DEC OMPRESSION FOR TREATMENT OF ROTATOR CUFF TEARS].

OBJECTIVE: To investigate the effectiveness of reversed arthroscopic subacromial decompression in the treatment of rotator cuff tears. METHODS: Between November 2012 and January 2015, 53 patients with rotator cuff tears were treated with reversed arthroscopic subacromial decompression and rotator cuff repair. Of 53 patients, 38 were male and 15 were female, with the age of 47-61 years (mean, 53.4 years). The injury was caused by falling in 36 cases and other in 17 cases without an obvious history of trauma. All patients had shoulder pain and limited shoulder movement before operation. Visual analogue scale (VAS) was 6.4±0.9, and University of California at Los Angeles (UCLA) score was 16.3±1.9. MRI showed that distal supraspinatus tear was observed in 41 cases and distal infraspinatus tear in 12 cases; partial-thickness rotator cuff tear was observed in 9 cases and full-thickness tear in 44 cases. And the tear size was from 1 to 3 cm (mean, 1.9 cm). Combined injuries included tendinitis of long head of biceps brachii in 31 cases, Bankart lesion in 5 cases, and superior labrum anterior and posterior lesion in 2 cases. RESULTS: Incision healed by first intention in all patients; no infection or nerve injury occurred. Forty-nine cases were followed up from 12 to 35 months (mean, 22.8 months). After operation, shoulder pain relief was achieved in 42 cases; 7 cases had anterior shoulder pain at 3 months after operation, which was relieved after symptomatic treatment. At last follow-up, VAS score was significantly decreased to 0.5±0.6 (t=40.565, P=0.000). UCLA score was significantly increased to 33.8±1.7 (t=-79.799, P=0.000). The results were excellent in 42 cases, good in 6 cases, and fair in 1 case; the excellent and good rate was 98.0%. CONCLUSIONS: Reversed arthroscopic subacromial decompression can avoid coracoacromial arch injury and achieve good recovery of joint function, so it can be used in rotator cuff tears procedure.

Effect of hepcidin on intracellular calcium in human osteoblasts.

Hepcidin is known to increase intracellular iron through binding to and degrading ferroportin, which is a transmembrane protein that transports iron from the intracellular to the outside. However, it is not clear whether hepcidin has a similar effect on intracellular calcium. Here, we investigated the influence of hepcidin on intracellular calcium in human osteoblasts, with or without high environmental iron concentrations. Our data showed that hepcidin (<100 nmol/L) could increase intracellular calcium, and this effect was more significant when cells were exposed to high environmental iron concentrations. To further explore its underlying mechanisms, we pretreated human osteoblasts with Nimodipine, a L-type calcium channel blocker, and Dantrolene, a ryanodine receptor antagonist to inhibit abnormal calcium release from the sarco-endoplasmic reticulum. These treatments had not resulted in any alteration of intracellular calcium in human osteoblasts. Thus, these findings indicate that the increase of intracellular calcium induced by hepcidin is probably due to calcium release from endoplasmic reticulum, which is triggered by calcium influx.

Hepcidin increases intracellular Ca2+ of osteoblast hFOB1.19 through L-type Ca2+ channels.

Hepcidin is a key player in the regulation of iron homeostasis. Several pathological conditions associated with iron overload are attributed to the depressed expression of hepcidin and are often associated with bone diseases including osteoporosis. Hepcidin was suggested to have anti-osteoporosis effects by preventing iron overload. We recently observed that hepcidin could increase intracellular calcium concentration in cultured osteoblast cells. The present study was designed to elucidate the source of the increased intracellular calcium following hepcidin activation. Cultured hFOB1.19 cells were used to test whether there was a dose dependent effect of hepcidin on increasing intracellular calcium. After finding the optimal concentration in increasing intracellular calcium, Cultured hFOB1.19 cells were then divided into three groups: (1) control group, (2) and (3) groups pretreated with either nimodipine (2 × 10(-5)mol/L) or EDTA (2 × 10(-3)mol/L) for 10 min before incubation with hepcidin (100 nmol/L). All cells were stimulated with hepcidin for 60 min and then stained with fluo-3/AM for 40 min before the intracellular calcium was observed using flow cytometry (FCM). As compared with controls, hepcidin treatment significantly increased intracellular calcium concentration. This effect was blocked by nimodipine and EDTA pretreatments which suggested that hepcidin-mediated calcium inflow was mainly through L-type Ca(2+) channels and that the release of intracellular calcium store was not significant. Hepcidin increases of intracellular calcium may be related to its anti-osteoporosis effect but this hypothesis needs further investigation.

Increased intracellular iron and mineralization of cultured hFOB 1.19 cells following hepcidin activation through ferroportin-1.

OBJECTIVE: To address whether hepcidin functions in bone metabolism. METHODS: This study was carried out in the Laboratory of Radiation Medicine and Public Health of Soochow University, and the Laboratory of the Second Affiliated Hospital of Soochow University, Suzhou, China, from September 2009 to July 2010. The positive expression of ferroportin-1 (Fpn-1) was detected by reverse transcriptase-polymerase chain reaction. After the treatment with distilled water (control group) and hepcidin (25noml/L, 50noml/L, 100noml/L), the fluorescence intensity related to intracellular iron concentration of a human fetal osteoblast cell line (hFOB 1.19) was measured by a confocal laser scanning microscope. A 3-(4,5- dimethylthiazol-2-yl) -2-5-diphenyltetrazolium bromide assay, and Von Kossa staining was performed to evaluate cell proliferation and mineralization in cultured hFOB 1.19 cells. RESULTS: This study revealed a high level expression of Fpn-1 in hFOB 1.19. On the basis of which, it was found that 25noml/L, 50noml/L, 100noml/L hepcidin could promote the fluorescence intensity related to intracellular iron concentration and mineralization in hFOB 1.19 in a dose-dependent manner (p<0.05), but hepcidin had no effect on FOB 1.19 proliferation (p>0.05). CONCLUSION: The hepcidin-ferroportin signal pathway may function in the osteoblast cell line of hFOB 1.19 cells. It is also suggested that cross-talk between iron and calcium homeostasis may play a role in bone metabolism in responding to hepcidin activation.