Single-Cell Transcriptomics Reveals Riluzole as an Osteoarthritis Candidate Drug via OB-NE Signaling Modulation and CTSS/NOS1 Inhibition

Kun Liu,1 Jia-Li Li,1 Yan Chen,2 Yu-Xin Li,1 Jeffrey D Deng,3 Yun Gong,4 Chong Cao,1 Qin Zeng,1 Zheng-Wu Xiao,5 Kai-Zhi Wen,5 Xiao-Chao Qu,1 Xiang-Ding Chen,1 Yun Deng,6 Hong-Wen Deng,4 Li-Jun Tan1 1Laboratory of Molecular and Statistical Genetics, College of Life Sciences, Hunan Normal University, Changsha, Hunan, 410081, People’s Republic of China; 2Xingtai Medical College, Xingtai City, Hebei, 054000, People’s Republic of China; 3Geisel School of Medicine, Dartmouth College, Hanover, NH, 03755, USA; 4Tulane Center of Biomedical Informatics and Genomics, Deming Department of Medicine, Tulane University School of Medicine, New Orleans, LA, 70112, USA; 5Center for System Biology, Data Sciences, and Reproductive Health, School of Basic Medical Science, Central South University, Changsha, Hunan, 410013, People’s Republic of China; 6Zebrafish Genetics Laboratory, College of Life Sciences, Hunan Normal University, Changsha, 410081, People’s Republic of ChinaCorrespondence: Li-Jun Tan, Laboratory of Molecular and Statistical Genetics, College of Life Sciences, Hunan Normal University, Changsha, Hunan, 410081, People’s Republic of China, Tel +86-731-8887-2791, Email ljtan@hunnu.edu.cn Hong-Wen Deng, Tulane Center of Biomedical Informatics and Genomics, Deming Department of Medicine, Tulane University School of Medicine, 1440 Canal Street Suite 1610, New Orleans, LA, 70112, USA, Tel +1 504-988-1310, Email hdeng2@tulane.eduIntroduction: Osteoarthritis (OA) is a degenerative joint disease involving multiple cell types, yet the role of osteoblast (OB)-immune cell interactions remains poorly understood.Methods: Single-cell RNA sequencing of human femoral head tissue was employed in this study, followed by integrated analysis using bioinformatics tools such as differential expression analysis, enrichment analysis and cell communication analysis. A network proximity-based drug repositioning approach was employed to identify potential drug candidates. Mendelian randomization (MR) analysis was used to explore the causal relationship between drug targets and OA. In vivo, a triclocarban (TCC)-induced zebrafish OA model was treated with selected drugs, and transcriptomic sequencing, network pharmacology and molecular docking were performed to identify potential drug targets.Results: We found OB populations as key OA-associated cells, differentially expressed genes in OBs were enriched in neutrophil (NE)-mediated immune pathways. Cell communication analysis revealed enhanced bidirectional OB-NE signaling in OA, primarily mediated through the CXCL12-CXCR4 and RETN-CAP1 pathways. Riluzole was identified as a therapeutic candidate through drug repositioning analysis. MR showed an inverse association between the expression of its target SLC7A11 and OA risk (OR=0.84, 95% CI=0.72– 0.98, P=0.027). In zebrafish OA models, riluzole treatment significantly reduced joint degeneration and downregulated CTSS, NOS1 and key components of the MAPK/ERK pathway. Molecular docking studies demonstrated strong binding affinity of riluzole to CTSS and NOS1, suggesting that inhibition of these targets may underlie its therapeutic action via suppression of MAPK/ERK suppression.Conclusion: This study highlights riluzole as a promising repositioned drug for OA. Limitations of this study include the relatively small sample size and the need for further translational validation in clinical settings.Keywords: osteoarthritis, single-cell transcriptome sequencing, drug repositioning, riluzole, osteoblasts