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 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-α inhibitor in clinical treatments for patients with ONFH.

中文翻译：

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DNA适体通过RIP1/RIP3/MLKL通路减轻TNF-α介导的坏死性凋亡对股骨头坏死的保护作用

背景

肿瘤坏死因子α（TNF-α）介导的坏死性凋亡过程可能在股骨头坏死（ONFH）的发生和发展中起重要作用。骨微血管内皮细胞（BMECs）的功能障碍已被确定为类固醇诱导的ONFH病理过程的重要组成部分。适体是单链 DNA 或 RNA 寡核苷酸序列。以前的研究已经设计或筛选了各种可以与特定靶标或受体结合以阻断其作用的适体。

客观的

本研究有两个主要目的：1）在体外建立TNF-α诱导的人BMECs ONFH模型，2）验证TNF-α适体（AptTNF-α）对阻断TNF-α活性的作用在 ONFH 模型中。

方法

收集临床样品用于苏木精和伊红 (HE) 染色、免疫组织化学和进一步的 BMEC 分离。细胞培养和鉴定后，用细胞计数试剂盒8（CCK8）评估与TNF-α孵育后BMECs的细胞活力。通过TUNEL和Annexin V-FITC/PI染色检测BMECs的坏死性凋亡。AptTNF-α对TNF-α细胞毒性的减弱首先由CCK8评估。然后，通过定量实时聚合酶链反应和蛋白质印迹探索分子机制。

结果

ONFH患者骨组织中TNF-α的表达水平显着上调。通过 CD31 和 vWF 的高表达验证了 BMECs 的鉴定。CCK8、TUNEL 染色和 Annexin V-FITC/PI 测定的结果表明，在 TNF-α 刺激后，BMECs 的细胞活力降低，坏死性凋亡增加。进一步的研究表明，AptTNF-α 可以以剂量依赖性方式减弱 TNF-α 的细胞毒性。ONFH 模型中由 TNF-α 介导的坏死性凋亡受受体相互作用蛋白激酶 1 (RIPK1)/受体相互作用蛋白激酶 3 (RIPK3)/混合谱系激酶域样蛋白 (MLK​​L) 信号通路的调节。

结论

我们在体外建立了人 BMECs 中 TNF-α 诱导的 ONFH 模型。我们的研究还表明，AptTNF-α 可以通过抑制 RIP1/RIP3/MLKL 信号通路来保护 BMECs 免受坏死性凋亡。

本文的转化潜力：DNA适体提供的对细胞坏死性凋亡的有效保护证明了其作为新型TNF-α抑制剂在ONFH患者临床治疗中的转化潜力。