Hypoxia inhibits intervertebral disc degeneration by maintaining autophagy circadian rhythm via the HIF1α-PER2-mTOR pathway.

INTRODUCTION

Disruption of the circadian rhythm (CR) and autophagy in intervertebral discs contributes to intervertebral disc degeneration (IDD) progression. However, the circadian regulation of autophagy requires further investigation.

OBJECTIVES

We observed the expression of circadian proteins and autophagic markers of nucleus pulposus (NP) cells followed a diurnal rhythmic pattern in vivo and in vitro.

METHODS

NP tissues were collected from light/dark cycle-shifted rats and IDD patients of varying severity. CR and ECM-related proteins were analyzed by immunohistochemistry and western blotting. Primary rat NP cells were treated with hypoxia or CoCl2, followed by western blotting for CR proteins, HIF-1α, and autophagy markers. siRNA knockdown of PER2 or HIF-1α was performed to assess their roles in regulating autophagy, ECM, and CR-associated proteins.

RESULTS

The silencing of clock gene PER2 disrupted the rhythmic expression of autophagic markers, by contrast PER2 overexpression inhibited mTOR pathway and enhanced autophagic levels. Co-IP analysis demonstrated the PER2-mTOR interaction, linking CR and autophagy rhythm. The inflammatory stimulator dampened the CR and autophagy rhythm, however intermittent hypoxia and cobalt chloride (CoCl2) re-synchronized the rhythm. Mechanistically, HIF-1α-mediated regulation of PER2 by hypoxia was involved in the re-synchronization, which was further demonstrated by the loss of CR and autophagy rhythm after silencing of PER2 or HIF-1α under hypoxia. Furthermore, the rhythm of oxygen level and HIF-1α was proved in living healthy NP tissue, confirming the hypoxia as a Zeitgeber. CR disruption and autophagy dysfunction led to catabolism of extracellular matrix (ECM), but the hypoxia, CoCl2 and autophagic stimulator could promote the rebalance of ECM metabolism.

CONCLUSION

Our study demonstrates that hypoxia maintains the intrinsic CR and autophagy rhythm through the HIF-1α/PER2/mTOR pathway to prevent IDD.