Digoxin Targets Low Density Lipoprotein Receptor-related Protein 4 And Protects Against Osteoarthritis

Backgrounds:Dysregulated chondrocyte metabolism is closely associated with the pathogenesis of osteoarthritis(OA).It is increasingly understood that OA is an active dynamic alteration arising from an imbalance between the repair and destruction of joint tissues,and not a passive degeneration or so-called wear-and-tear disease as commonly described.Suppressing chondrocyte catabolism to restore cartilage homeostasis has been extensively explored,whereas far less effort has been invested toward enhancing chondrocyte anabolism.To date,there is no safe treatment available that can halt OA progression.Considering the huge costs in terms of time and money associated with drug development,identification of new uses for old drugs is desirable.Objectives:This study aimed to repurpose clinically approved drugs as potential stimulators of chondrocyte anabolism in treating OA.Methods:Screening of a Food and Drug Administration-approved drug library;Assays for examining the chondroprotective effects of Digoxin in vitro;Assays for defining the therapeutic effects of Digoxin using a surgicallyinduced OA model;A propensity-score matched cohort study using The Health Improvement Network to examine the relationship between Digoxin use and the risk of joint OA-associated replacement among patients with atrial fibrillation;Identification and characterization of the binding of Digoxin to low density lipoprotein receptor-related protein 4(LRP4);Various assays,including use of CRISPR-Cas9 genome editing to delete LRP4 in human chondrocytes,for examining the dependence on LRP4 of Digoxin regulation of chondrocytes.Results:Three rounds of screening of a drug library containing 1046 approved drugs using mesenchymal stem cells and chondrocytes revealed that cardenolides represented by ouabain and digoxin up-regulated COL2A1 m RNA expression in both mesenchymal stem cells and chondrocytes.Through further in vitro cell studies,we confirmed that ouabain and digoxin can promote chondrocyte differentiation,promote chondrocyte anabolism and inhibit inflammation-related chondrocyte catabolism.We established a model of OA in mice by destabilization of the medial meniscus(DMM)surgery,and the mice were injected with ouabain or digoxin into the knee joints starting 3 days after surgery.Using histopathological scores,immunohistochemistry,microcomputed tomography(μCT)and analyses of pain-related behaviors,we found that ouabain and digoxin delayed the progression of OA and alleviated OArelated pain in vivo.We then conducted a cohort study with sequential propensity score matching of 56,794 patients using data from THIN and found that the rate difference(RD)in knee or hip OA-associated joint replacement between Digoxin initiators and non-users was-0.9(95%confidence interval [CI]:-1.5 to-0.3)per 1000 person-years and the hazard ratio(HR)was 0.85(95% CI: 0.77 to 0.93).Finally,we found in vitro that the chondroprotective effect of ouabain and digoxin is mediated by ERK,AKT and NF-κB signaling pathway.LRP4 was identified as the binding target of ouabain and digoxin in chondrocytes by a series of techniques,and Glu-343 was the key amino acid of LRP targeted binding.Conclusions:These findings not only provide new insights into the understanding of Digoxin’s chondroprotective action and underlying mechanisms,but also present new evidence for repurposing Digoxin for OA.