# From pathogenesis to treatment: the role of autophagic cell death in GONFH and its potential mitigation by naringenin

## TL;DR

This study explores how autophagic cell death contributes to a bone disease called GONFH and finds that naringenin can potentially treat it by targeting a specific protein.

## Contribution

The study identifies autophagy-dependent cell death as a key mechanism in GONFH and introduces naringenin as a treatment that modulates ULK1 phosphorylation.

## Key findings

- Autophagy, ferroptosis, and apoptosis are elevated in GONFH, while osteogenesis is reduced.
- Naringenin inhibits GC-induced autophagy and cell death by phosphorylating ULK1 at ser757.
- Pharmacological or genetic disruption of ULK1 phosphorylation negates naringenin's protective effects.

## Abstract

Glucocorticoid (GC)-associated osteonecrosis of the femoral head (GONFH) is an incurable orthopedic illness. Reduced osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is at the core of the pathogenesis of GONFH; however, its molecular mechanism remains unclear. The study aimed to explore the pathological mechanisms of GONFH and to investigate the efficacy and mechanism of naringenin (NAR) in treating GONFH.

RNA sequencing was conducted to investigate the pathogenesis of GONFH and identify the potential therapeutic mechanism of NAR. The levels of autophagy, ferroptosis, apoptosis, and osteogenesis were examined in clinical, animal, and BMSC samples. Moreover, the specific binding of NAR to ULK1 and its role in promoting ser757 phosphorylation of ULK1, leading to reduced autophagy-dependent cell death and increased osteogenic differentiation of BMSCs, were investigated using molecular dynamics simulations and systematic in vivo and in vitro experiments.

In clinical, animal, and BMSCs samples, autophagy, ferroptosis, and apoptosis were notably increased in the GONFH group, while osteogenesis was markedly decreased. In addition, the effects of rapamycin (RAPA, an autophagy agonist) and 3-methyladenine (3-MA, an autophagy inhibitor) were investigated to confirm that the GC-induced decrease in osteogenic differentiation of BMSCs is mediated through autophagy-dependent cell death. Additionally, NAR exhibits high affinity for ULK1, which increases its inhibitory phosphorylation at ser757. This particular communication inhibits GC-induced autophagy and subsequent cell death, thereby normalizing osteogenic differentiation of BMSCs. It is interesting to note that the protective effects of NAR were abolished by pharmacological (RAPA) and genetic (ULK1-S757A mutation) interventions.

Taken together, our work elucidates a pathogenic process involving autophagy-dependent cell death and defines NAR as a specific treatment that regulates ULK1 to halt this pathogenic cascade.