Ghrelin attenuates neuronal apoptosis and oxidative stress in a rat model of neonatal HIE via the GHSR-1α/AMPK/Sirt1/PGC-1α/UCP2 pathway.
Background
Neuronal apoptosis driven by oxidative stress is a primary pathological process causing neurological impairment following hypoxic-ischemic encephalopathy (HIE), a severe form of brain injury in newborns due to oxygen deprivation. Current therapeutic options are limited. Ghrelin, the natural ligand for the growth hormone secretagogue receptor-1α (GHSR-1α), is known for its anti-apoptotic effects in the brain. However, its specific ability to protect against neuronal death by reducing oxidative stress after a hypoxic-ischemic event remained an open question, prompting this investigation into its potential as a neuroprotective agent for neonatal brain injury.
Study Design
Ten-day old unsexed rat pups were subjected to a hypoxic-ischemia (HI) injury model. The treatment group received exogenous recombinant human ghrelin (rh-Ghrelin) administered intranasally at 1 hour and 24 hours post-injury. To confirm the mechanism, specific inhibitors were used: [D-Lys3]-GHRP-6 (a selective GHSR-1α inhibitor) and Ex527 (a selective Sirt1 inhibitor) were given intranasally 1 hour before HI induction. Additionally, small interfering RNA (siRNA) for GHSR-1α was administered via intracerebroventricular injection 24 hours prior to HI. Neurological outcomes and cellular changes were assessed using immunofluorescence, MitoSox staining, Fluoro-Jade C staining, TUNEL staining, and western blot experiments.
Results
Ghrelin administration significantly improved neurobehavioral outcomes in the rat pups following HI injury. The treatment effectively reduced oxidative stress and attenuated neuronal apoptosis. Mechanistically, ghrelin treatment prompted a significant increase in the phosphorylation of AMPK and upregulated the expression of downstream proteins Sirt1, PGC-1α, and UCP2. This protective signaling cascade also led to an increased ratio of anti-apoptotic Bcl2 to pro-apoptotic Bax and downregulated levels of cleaved caspase-3, a key executioner of apoptosis.
The neuroprotective effects of ghrelin were confirmed to be dependent on its receptor and downstream pathway, as the benefits were reversed by pre-treatment with the
GHSR-1αinhibitor [D-Lys3]-GHRP-6,GHSR-1αsiRNA, or theSirt1inhibitor Ex527. This demonstrates that ghrelin exerts its effects, at least in part, via theGHSR-1α/AMPK/Sirt1/PGC-1α/UCP2signaling pathway.
Key Findings
- Ghrelin significantly improved neurobehavioral outcomes after hypoxic-ischemic injury in neonatal rats.
- Ghrelin treatment reduced oxidative stress and neuronal apoptosis post-injury.
- Ghrelin upregulated the expression of Sirt1, PGC-1α, and UCP2, and increased the Bcl2/Bax ratio.
- Ghrelin decreased levels of cleaved caspase-3, an indicator of apoptosis.
- The protective effects of ghrelin were reversed by a GHSR-1α inhibitor ([D-Lys3]-GHRP-6) and a Sirt1 inhibitor (Ex527).
Why It Matters
This study identifies ghrelin as a potent neuroprotective agent in a preclinical model of neonatal brain injury, operating through a clearly defined signaling pathway that combats oxidative stress and cell death. For clinicians and researchers, this positions ghrelin and the GHSR-1α receptor system as a promising therapeutic target for treating neonatal HIE. The intranasal route of administration is particularly notable, as it is non-invasive and could facilitate easier clinical translation. Ghrelin's ability to modulate the AMPK/Sirt1 pathway suggests it may offer a multi-faceted approach to neuroprotection, not just replacing growth hormone but actively shielding neurons from damage. While human trials are a distant prospect, these findings provide a strong rationale for further investigation into ghrelin-based therapies for acute brain injuries in newborns.
