Protective effects of Bushen Huoxue Formula on intervertebral disc degeneration and endplate microcirculation in rats via the SIRT1/CXCL12/CXCR4/CXCR7 axis in endothelial progenitor cells

Abstract

Objective

To investigate the efficacy of the Bushen Huoxue Formula (补肾活血方, BSHXF) in alleviating intervertebral disc degeneration (IVDD), improving endplate microcirculation, and enhancing endothelial progenitor cell (EPC) function, while investigating the potential role of the sirtuin 1 (SIRT1)/C-X-C motif chemokine ligand 12 (CXCL12)/C-X-C motif chemokine receptor 4 (CXCR4)/C-X-C motif chemokine receptor 7 (CXCR7) signaling pathway.

Methods

This study comprised both in vivo and in vitro experiments. In vivo, 24 male Sprague-Dawley (SD) rats (4 - 6 weeks old) were used, and allocated into four groups (n = 6 per group): Rat-Control, Rat-Model, Rat-BSHXF, and Rat-EX527 + BSHXF groups. IVDD was induced by applying continuous axial compression (approximately 11 N) to the caudal intervertebral discs for two weeks. Following model establishment, rats in the Rat-BSHXF group received oral BSHXF at 17.71 g/(kg·d) once daily, whereas those in the Rat-EX527 + BSHXF group received the SIRT1 inhibitor EX527 at 5 mg/kg by intraperitoneal injection once daily in addition to the same BSHXF regimen for two consecutive weeks. Disc degeneration was evaluated using magnetic resonance imaging (MRI), Pfirrmann grading, and hematoxylin and eosin (HE) staining. Endplate microvascular density was assessed via CD31 immunostaining. The mRNA expression levels of SIRT1, CXCL12, CXCR4, and CXCR7 in disc and endplate tissues were quantified using reverse transcription quantitative polymerase chain reaction (RT-qPCR), whereas their protein expression levels were assessed using Western blot and immunofluorescence staining. In vitro, rat bone marrow-derived EPCs were isolated from a separate cohort of six SD rats, cultured, and identified via CD34/CD133 immunofluorescence staining. The EPCs were allocated into Control, BSHXF, si-SIRT1, si-SIRT1 + BSHXF, and Celecoxib groups. Cells in the BSHXF-related groups were treated with 10% BSHXF-containing serum prepared using another separate cohort of six SD rats, and SIRT1 knockdown was achieved using SIRT1 targeting small interfering RNA (si-SIRT1). EPC viability, migration, and tube formation were evaluated using cell counting kit-8 (CCK-8), transwell migration, and Matrigel tube formation assays, respectively. Additionally, the mRNA and protein expression levels of SIRT1, CXCL12, CXCR4, and CXCR7 in EPCs were measured via RT-qPCR and Western blot, respectively.

Results

In vivo, axial compression induced significant disc degeneration, characterized by reduced T2 signal intensity, decreased disc height, and Pfirrmann grade IV – V changes. Compared with the Rat-Model and Rat-EX527 + BSHXF groups, the Rat-BSHXF group exhibited less severe histological degeneration and significantly increased endplate microvascular density (P < 0.05). Furthermore, BSHXF significantly upregulated the mRNA and protein expression levels of SIRT1, CXCL12, CXCR4, and CXCR7 in disc and endplate tissues compared with both the Rat-Model and Rat-EX527 + BSHXF groups (P < 0.05). These elevated expression levels in the Rat-BSHXF group were further corroborated by immunofluorescence analysis (P < 0.05). In vitro, treatment with BSHXF-containing serum significantly enhanced EPC viability, migration, total tube length, and branch point formation compared with the Control, si-SIRT1, and si-SIRT1 + BSHXF groups (P < 0.05). No significant differences in tube formation were observed between the BSHXF and Celecoxib groups (P > 0.05). Conversely, SIRT1 knockdown markedly impaired EPCs angiogenesis-related functions and abrogated the BSHXF-induced enhancements in EPCs viability, migration, and tube formation, with no significant differences observed between the si-SIRT1 and si-SIRT1 + BSHXF groups (P > 0.05).

Conclusion

BSHXF attenuated IVDD-related degenerative changes and increased endplate microvascular density in a rat model of caudal disc compression, while enhancing EPC viability, migration, and angiogenesis in vitro. These findings suggest that the effects of BSHXF on endplate microcirculation during IVDD may be associated with the SIRT1-related modulation of the CXCL12/CXCR4/CXCR7 axis in EPCs.