Disused osteoporosis is a kind of osteoporosis, a common age-related disease. Neurological disorders are major risk factors for osteoporosis. Though there are many studies on disuse osteoporosis, the genetic mechanisms for the association between glutathione metabolism and ferroptosis in osteoblasts with disuse osteoporosis are still unclear. The purpose of this study is to explore the key genes and other related mechanism of ferroptosis and glutathione metabolism in osteoblast differentiation and disuse osteoporosis. By weighted gene coexpression network analysis (WGCNA), the process of osteoblast differentiation-related genes was studied in GSE30393. And the related functional pathways were found through the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. By combining GSE1367 and GSE100933 together, key genes which were separately bound up with glutathione metabolism and ferroptosis were located. The correlation of these key genes was analyzed by the Pearson correlation coefficient. GSTM1 targeted agonist glutathione (GSH) selected by connectivity map (CMap) analysis was used to interfere with the molding disused osteoporosis process in MC3T3-E1 cells. RT-PCR and intracellular reactive oxygen species (ROS) were performed. Two important pathways, glutathione metabolism and ferroptosis pathways, were found. GSTM1 and TFRC were thought as key genes in disuse osteoporosis osteoblasts with the two mechanisms. The two genes have a strong negative correlation. Our experiment results showed that the expression of TFRC was consistent with the negative correlation with the activation process of GSTM1. The strong relationship between the two genes was proved. Glutathione metabolism and ferroptosis are important in the normal differentiation of osteoblasts and the process of disuse osteoporosis. GSTM1 and TFRC were the key genes. The two genes interact with each other, which can be seen as a bridge between the two pathways. The two genes participate in the process of reducing ROS in disuse osteoporosis osteoblasts.

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Underlying pivotal pathways were identified to reveal potential key genes correlated with postmenopausal osteoporosis (PMOP). The pathways were enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) with genes intersection greater than 5 based on gene expression profile data, and the acquired pathways were then transformed to Markov chain (MC). Gibbs sampling was conducted to obtain a new MC. Moreover, the average probabilities of each pathway in normal and PMOP were computed via an MC Monte Carlo (MCMC) algorithm, and differential pathways were identified based on probabilities more than 0.7. In addition, frequencies of appearance of pathway genes were counted via MCMC and the hub genes were achieved with the probabilities of gene expression efficiencies in two states. Judging by the gene intersection more than 5, overall 280 pathways were determined. After Gibbs sampling, 2 differential pathways were obtained on the basis of probabilities more than 0.7. Moreover, the hub genes comprising TNNC1, MYL2, and TTN were achieved according to probabilities more than 0.7. The identified pathways and the three hub genes probably are useful for developing approaches for the diagnosis and treatment of PMOP in future preclinical and clinical applications.