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

Abstract

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.

Keywords: Aptamer; Bone microvascular endothelial cell; Necroptosis; Osteonecrosis of the femoral head; TNF-α.

Figure 1

Clinical samples for Hematoxylin and Eosin (HE) staining, immunohistochemistry and BMEC isolation. A, clinical sample of the femoral head in patient with ONFH. B, the bone debris for BMEC isolation which was extracted from the patients with femoral neck fracture. C, the HE staining demonstrated the existence of empty lacuna (white arrow), amorphous substance (black arrow) and trabecular fracture (blue arrow) in the ONFH group. D, the HE staining of the control group. E, Results of the immunohistochemistry in the necrotic regioin. F, Results of the immunohistochemistry in the normal region. G, The mean fluorescence intensity (MFI) of TNF-α (red) in the ONFH and control groups (n ​= ​6, each group consisted of six slices for analysis). ∗p ​< ​0.05. BMEC, bone microvascular endothelial cell; ONFH, osteonecrosis of the femoral head. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 2

Bone microvascular endothelial cell (BMEC) culture and identification. A,B, The typical morphologies of first passage (P1) and second passage (P2) of BMECs (50 x). C, BMEC identification by the marker of CD31. D, BMEC identification by the marker of vWF. E, the ratio of positive cells in CD31 and vWF staining (n ​= ​3, each group consisted of three repeated wells for analysis).

Figure 3

Reduced cell viability and increased necroptosis of BMECs after TNF-α stimulation. A, Cell Counting Kit-8 results of cell viability after incubation with TNF-α for 24 ​h (n ​= ​3, each group consisted of three repeated wells for analysis). B, the TUNEL staining results of the control group. C, the TUNEL staining results of the 100 ​ng/ml TNF-α group. D, the percentage of cell necroptosis from the TUNEL staining in the 100 ​ng/ml TNF-α and control group (n ​= ​3, each group consisted of three repeated wells for analysis). E, (1)–(3) the BMECs were stained with the Annexin V-FITC/propidium iodide (PI) in the control, 100 ​ng/ml TNF-α and 100 ​ng/ml TNF-α ​+ ​500 ​nM AptTNF-α group, respectively. The cells in the Q2(UR) and Q1(UL) stand for the percentage of necroptosis. (4) Quantitative data analyses of the Annexin V-FITC/PI results in these three groups (n ​= ​3, each group was repeated three times in six-well plate for analysis). ∗p ​< ​0.05. BMEC, bone microvascular endothelial cell. Apt 50, 50 ​nM TNF-α aptamer. Apt 100, 100 ​nM TNF-α aptamer. Apt 500, 500 ​nM TNF-α aptamer.

Figure 4

AptTNF-α attenuated TNF-α cytotoxicity in a dose-dependent manner. A, Cell Counting Kit-8 results of cell viability after incubation with TNF-α and AptTNF-α for 24 ​h (n ​= ​3, each group consisted of three repeated wells for analysis). B, the TUNEL staining results of the 100 ​ng/ml TNF-α + 100 ​nM AptTNF-α group. C, the TUNEL staining results of the 100 ​ng/ml TNF-α + 500 ​nM AptTNF-α group. D, the percentage of cell necroptosis from the TUNEL staining in 100 ​ng/ml TNF-α, 100 ​ng/ml TNF-α + 100 ​nM AptTNF-α, 100 ​ng/ml TNF-α + 500 ​nM AptTNF-α group (n ​= ​3, each group consisted of three repeated wells for analysis). E, (1)–(4) the BMECs were stained with the Annexin V-FITC/propidium iodide (PI) in the 100 ​ng/ml TNF-α, 100 ​ng/ml TNF-α ​+ ​500 ​nM AptTNF-α, 100 ​ng/ml TNF-α ​+ ​100 ​nM AptTNF-α and 100 ​ng/ml TNF-α ​+ ​50 ​nM AptTNF-α, respectively. The cells in the Q2(UR) and Q1(UL) stand for the percentage of necroptosis. (5) Quantitative data of the Annexin V-FITC/PI results in these four groups (n ​= ​3, each group was repeated three times in six-well plate for analysis). ∗p ​< ​0.05. BMEC, bone microvascular endothelial cell. AptTNF-α, TNF-α aptamer.

Figure 5

AptTNF-α regulated TNF-α-mediated necroptosis via RIP1/RIP3/MLKL signalling pathway. A,B,C, the mRNA levels of RIPK1, RIPK3 and MLKL gene were measured by RT-PCR after incubation with TNF-α and AptTNF-α for 24 ​h (n ​= ​3, each group was repeated from three batches of samples for analysis). ∗p ​< ​0.05. D, Expression level of RIPK1, RIPK3 and MLKL, along with their phosphorylated proteins in the BMECs examined by the Western blotting. E,F,G,H, the relative protein level of GAPDH, p-RIP1/RIP1, p-RIP3/RIP3 and p-MLKL/MLKL from the Western blotting analysis, respectively. The ratio of p-RIP1/RIP1, p-RIP3/RIP3 and p-MLKL/MLKL was normalized to GAPDH (n ​= ​3, each group was repeated from three batches of samples for analysis). ∗p ​< ​0.05. BMEC, bone microvascular endothelial cell. AptTNF-α, TNF-α aptamer. Apt 100, 100 ​nM TNF-α aptamer. Apt 500, 500 ​nM TNF-α aptamer.