The Antagonism of Neuropeptide Y Type I Receptor (Y1R) Reserves the Viability of Bone Marrow Stromal Cells in the Milieu of Osteonecrosis of Femoral Head (ONFH)

Abstract

Neuropeptide Y (NPY)-Y1 receptor (Y1R) signaling is known to negatively affect bone anabolism. Our study aimed at investigating the impact of NPY-Y1R signaling in the pathogenesis of glucocorticoid-related osteonecrosis of the femoral head (ONFH). Femoral heads were retrieved from 20 patients with and without ONFH, respectively. The bone marrow stromal cells (BMSCs) from ONFH femoral heads were treated with Y1R agonists and antagonists for subsequent analysis. We showed that the local NPY expression level was lower in ONFH heads. The Y1R agonists and antagonists disturb and facilitate the survival of BMSCs. The transcription of stromal derived factor-1 (SDF-1) was enhanced by Y1R antagonists. Our study showed that the local NPY expression level was lower in ONFH heads. Y1R antagonists facilitate the survival of BMSCs and stimulate the transcription of SDF-1 by BMSCs. These findings shed light on the role of NPY-Y1R signaling in the pathogenesis of ONFH.

Keywords: BIBO3304; neuropeptide Y; osteonecrosis of femoral head (ONFH); stromal derived factor-1 (SDF-1).

Figure 1

The representative illustration of NPY IHC staining of the femoral head obtained from non-ONFH (A) and ONFH (B) patients, and the percentage of NPY positively stained cells between non-ONFH and ONFH groups (C) (*** p < 0.001). The red arrows indicate positively stained cells. The black dots and the black squares indicate the information concerning the percentage of NPY (+) cells for respective patients.

Figure 2

The serum NPY levels among non-ONFH and ONFH patients (*** p < 0.001).

Figure 3

The representative illustration of DAPI-TUNEL staining of the BMSCs harvested from ONFH heads treated with Y1R agonist (Leu 31, Pro 34) (A) and antagonist BIBO3304 (B) of graded concentrations.

Figure 4

The 570 nm optic density (OD) values of BMSCs treated in serial concentrations of Y1R agonist (Leu 31, Pro 34) (A) and antagonist BIBO3304 (B) in MTT assay. Respective p-values for post-hoc analysis: (A) NC-500 nM: 0.008; 50–500 nM: <0.001; (B) NC-1000 nM: 0.004; 100–1000 nM: 0.018; 500–1000 nM: 0.032 (* p < 0.05, ** p < 0.01, and *** p < 0.001). The black triangles/dots/squares indicate the information concerning the respective 570nm OD values for respective assays.

Figure 5

The illustration of the Annexin V-FITC apoptosis assay for the BMSCs treated by control (A), 1000 nM Y1R agonist (Leu 31, Pro 34) (B) and 1000 nM Y1R antagonist BIBO3304 (C). The P1 value quantified cellular necrosis, and the P2 value quantified cellular apoptosis.

Figure 5

The illustration of the Annexin V-FITC apoptosis assay for the BMSCs treated by control (A), 1000 nM Y1R agonist (Leu 31, Pro 34) (B) and 1000 nM Y1R antagonist BIBO3304 (C). The P1 value quantified cellular necrosis, and the P2 value quantified cellular apoptosis.

Figure 5

The illustration of the Annexin V-FITC apoptosis assay for the BMSCs treated by control (A), 1000 nM Y1R agonist (Leu 31, Pro 34) (B) and 1000 nM Y1R antagonist BIBO3304 (C). The P1 value quantified cellular necrosis, and the P2 value quantified cellular apoptosis.

Figure 6

The transcription levels of stromal cell-derived factor-1 (SDF-1) of BMSCs treated in a serial concentration of Y1R agonist (Leu 31, Pro 34) (A) and antagonist BIBO3304 (B). Respective p-values for post-hoc analysis: (B) 0–1000 nM: 0.045; 100–1000 nM: 0.024 (* p < 0.05). The black triangles/dots/squares indicate the information concerning the respective SDF-1 mRNA expression levels for respective assays.