The protective effect of DNA aptamer on osteonecrosis of the femoral head by alleviating TNF-α-mediated necroptosis via RIP1/RIP3/MLKL pathway

Background: The process of necroptosis mediated by tumor necrosis factor alpha (TNF-alpha) might play an important role in the onset and development of the osteonecrosis of the femoral head (ONFH). The dysfunctions of bone microvascular endothelial cells (BMECs) have been identified as an important part of pathological processes in the steroid-induced ONFH. An aptamer is a single-stranded DNA or RNA oligonucleotide sequence. Previous studies have designed or screened various aptamers that could bind to specific targets or receptors in order to block their effects.Objective: There are two main objectives in this study: 1) to establish a TNF-alpha-induced ONFH model in human BMECs in vitro, 2) to verify the effects of the TNF-alpha aptamer (AptTNF-alpha) on blocking TNF-alpha activity in the ONFH model.Methods: Clinical samples were collected for Hematoxylin and Eosin (HE) staining, immunohistochemistry and further BMEC isolation. After cell culture and identification, the cell viability of BMECs after incubation with TNF-alpha was assessed by Cell Counting Kit-8 (CCK8). The necroptosis of BMECs was detected by the TUNEL and Annexin V-FITC/PI staining. The attenuation of TNF-alpha cytotoxicity by AptTNF-alpha was evaluated by CCK8 at first. Then, the molecular mechanism was explored by the quantitative real-time polymerase chain reaction and western blotting.Results: The expression level of TNF-alpha was significantly up-regulated in bone tissues of ONFH patients. The identification of BMECs was verified by the high expressions of CD31 and vWF. Results from CCK8, TUNEL staining and Annexin V-FITC/PI assay demonstrated reduced cell viability and increased necroptosis of BMECs after TNF-alpha stimulation. Further investigations showed that TNF-alpha cytotoxicity could be attenuated by the AptTNF-alpha in a dose-dependent manner. Necroptosis mediated by TNF-alpha in the ONFH model was regulated by the receptor-interacting protein kinase 1 (RIPK1)/receptor-interacting protein kinase 3 (RIPK3)/mixed lineage kinase domain-like protein (MLKL) signalling pathway.Conclusion: We established a TNF-alpha-induced ONFH model in human BMECs in vitro. Our study also demonstrated that the AptTNF-alpha could protect BMECs from necroptosis by inhibiting the RIP1/RIP3/MLKL signalling pathway. The Translational Potential of this Article: The effective protection from cell necroptosis provided by the DNA aptamer demonstrated its translational potential as a new type of TNF-alpha inhibitor in clinical treatments for pa-tients with ONFH.