# Hydrogen attenuates ischaemia–reperfusion injury in skeletal muscles post-limb replantation by activating the NRF2/HO-1 signalling pathway to reduce BAX expression

## TL;DR

Hydrogen pre-perfusion reduces skeletal muscle damage after limb replantation by activating a protective pathway that lowers cell death.

## Contribution

This study shows hydrogen-rich heparin sodium activates the NRF2/HO-1 pathway to reduce apoptosis in skeletal muscle IRI.

## Key findings

- Hydrogen-rich heparin sodium activates the NRF2/HO-1 pathway and reduces oxidative stress.
- NRF2 inhibition weakens hydrogen's protective effects and increases apoptosis.
- Hydrogen pre-perfusion improves skeletal muscle recovery post-limb replantation.

## Abstract

Ischaemia–reperfusion injury (IRI) is a critical complication post-limb replantation. The oxidative stress and cellular apoptosis due to IRI considerably hinder the healing process. This study aimed to investigate the modulatory effects of pre-perfusion with hydrogen-rich heparin sodium on the nuclear factor erythroid 2–related factor 2 (NRF2)/haeme oxygenase-1 (HO-1) pathway and its potential mechanisms in mitigating skeletal muscle IRI post-limb replantation.

Forty healthy Sprague–Dawley rats (250–300 g) were classified into five groups (n = 8 each): normal control, IRI + heparin sodium pre-perfusion (heparin group), IRI + hydrogen-rich heparin sodium pre-perfusion (hydrogen-rich heparin group), IRI + hydrogen-rich heparin sodium pre-perfusion + NRF2 inhibitor (hydrogen-rich heparin + all-trans retinoic acid [ATRA] group), and IRI + heparin sodium pre-perfusion + NRF2 inhibitor (heparin + ATRA group). The activation of the NRF2/HO-1 pathway in skeletal muscle IRI was evaluated based on HO-1 expression using western blotting and immunofluorescence. Furthermore, haematoxylin and eosin staining and transmission electron microscopy were employed to determine the histopathological characteristics. Additionally, superoxide dismutase and malondialdehyde levels in skeletal muscle tissue were measured to assess antioxidant capacity and the degree of oxidative stress damage. Tissue hypoxia was assessed based on hypoxia-inducible factor 1-alpha expression, whereas apoptosis markers BCL-2-associated X protein (BAX) and Caspase-3 in skeletal muscle tissues were analysed using western blotting with terminal deoxynucleotidyl transferase dUTP nick end labelling staining to quantify cell apoptosis.

Compared with the control group, the heparin group exhibited significant pathological changes, including inflammatory infiltration and cellular hypertrophy, with increased apoptosis and oxidative stress. Notably, NRF2 suppression aggravated these effects. However, hydrogen-rich heparin sodium prominently activated the NRF2/HO-1 pathway, enhancing antioxidant defence and reducing BAX/Caspase-3-mediated apoptosis, thereby mitigating IRI-induced damage. The use of an NRF2 inhibitor to inhibit NRF2 excitation by hydrogen-rich heparin sodium notably weakened NRF2 activation and the antioxidant response, resulting in a substantial increase in cellular apoptosis.

Pre-perfusion with hydrogen-rich heparin sodium markedly diminishes the BAX/Caspase-3-mediated apoptotic pathway in skeletal muscle tissues with IRI through the excitation of the NRF2/HO-1 pathway.

Mechanism underlying the molecular action of hydrogen.Image 1

•This study refines a model for limb reimplantation, improving blood flow obstruction and perfusion methods.•Hydrogen-rich infusion pre-perfusion demonstrates protective effects on the skeletal muscle of amputated limbs.•Hydrogen significantly mitigates oxidative stress injuries in the skeletal muscles of severed limbs.•Hydrogen activates the Nrf2/HO-1 signaling pathway in skeletal muscles within a limb reimplantation model.•The activation of the Nrf2/HO-1 pathway has an impact on Bax-mediated apoptosis.

This study refines a model for limb reimplantation, improving blood flow obstruction and perfusion methods.

Hydrogen-rich infusion pre-perfusion demonstrates protective effects on the skeletal muscle of amputated limbs.

Hydrogen significantly mitigates oxidative stress injuries in the skeletal muscles of severed limbs.

Hydrogen activates the Nrf2/HO-1 signaling pathway in skeletal muscles within a limb reimplantation model.

The activation of the Nrf2/HO-1 pathway has an impact on Bax-mediated apoptosis.