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-α) 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-α -induced ONFH model in human BMECs in vitro, 2) to verify the effects of the TNF-α aptamer (AptTNF-α) on blocking TNF-α 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-α 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-α cytotoxicity by AptTNF-α 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-α 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-α stimulation. Further investigations showed that TNF-α cytotoxicity could be attenuated by the AptTNF-α in a dose-dependent manner. Necroptosis mediated by TNF-α 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-α-induced ONFH model in human BMECs in vitro. Our study also demonstrated that the AptTNF-α could protect BMECs from necroptosis by inhibiting the RIP1/RIP3/MLKL signalling pathway.: The effective protection from cell necroptosis provided by the DNA aptamer demonstrated its translational potential as a new type of TNF-α inhibitor in clinical treatments for patients with ONFH.