Region-Dependent Expression and Function of Integrin α5β1 in Protecting against Disc Degeneration via Autophagy Promotion: An Ex Vivo Organ Culture Model under Dynamic Mechanical Loading

Mingbin Zhan^1,2Zhen Li³Shuai Chen^1,2Hongkun Chen^4,5Shaozheng Lin^1,6Wentao Sun^1,6ZEMIN LING^4,5Peiqiang Su^1,2Shangbin Cui^1,2*Xuenong Zou^1,2*

• ¹The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
• ²Guangdong Provincial Key Laboratory of Orthopedics and Traumatology/Department of Spinal Surgery, Guangzhou, China
• ³AO Research Institute Davos, Davos, Switzerland
• ⁴The Seventh Affiliated Hospital Sun Yat-sen University, Shenzhen, China
• ⁵Shenzhen Key Laboratory of Bone Tissue Repair and Translational Research, Department of Orthopaedic Surgery, Shenzhen, China
• ⁶Guangdong Provincial Key Laboratory of Orthopedics and Traumatology/Department of Spinal Surgery, Guangdong, China

Introduction: Abnormal mechanical loading is a significant pathogenic factor in intervertebral disc degeneration (IVDD), yet the underlying mechanotransduction mechanisms remain incompletely elucidated. This study aimed to investigate the role of integrin α5β1 as a key mechanosensor in regulating the autophagy-apoptosis balance under mechanically induced IVDD. Methods: Bovine intervertebral discs (IVDs) with intact endplates were cultured in a bioreactor and subjected to dynamic mechanical loading, including physiological loading (PL: 0.02-0.2 MPa, 0.2 Hz) and degenerative loading (DL: 0.32-0.5 MPa, 5 Hz) for 3 and 7 days. Interventions involved the autophagy inhibitor 3-Methyladenine (3-MA), integrin α5β1-specific inhibitory peptide RGD (Arg-Gly-Asp), and the autophagy activator rapamycin. A systematic evaluation was performed, assessing disc height, histomorphology, cell viability, gene/protein expression, autophagy levels, and apoptosis. Results: Degenerative loading induced progressive IVD degeneration, characterized by irreversible disc height loss, structural disruption, decreased cell viability, and extracellular matrix (ECM) metabolic imbalance. Treatment with 3-MA exacerbated these degenerative changes, confirming the protective role of autophagy. Integrin α5β1 exhibited distinct spatial distribution patterns: its expression was significantly upregulated in the nucleus pulposus (NP) and inner annulus fibrosus (IAF) under degenerative loading, whereas only the β1 subunit was increased in the outer annulus fibrosus (OAF). Functional experiments demonstrated that competitive inhibition of integrin α5β1 by RGD peptide significantly suppressed autophagy activity, exacerbated apoptosis, and promoted ECM degradation. Conversely, rapamycin alleviated degeneration by restoring autophagic flux. Mechanistically, degenerative loading suppressed the FAK/PI3K/AKT/mTOR pathway while upregulating ULK1, and these effects were partially reversed by RGD inhibition. Discussion: The autophagy-apoptosis balance plays a critical regulatory role in IVDD progression, with integrin α5β1 serving as a crucial upstream mechanosensor that may exert its protective function through modulating the FAK/PI3K/AKT/mTOR pathway. The region-specific distribution of integrin subtypes determines the specificity of mechanotransduction across different disc areas. Targeting the integrin-autophagy axis and its associated signaling pathways may represent a potential therapeutic strategy for mitigating mechanically induced IVDD.