Mogrol Protects Against Hepatic Ischemia–Reperfusion Injury by Preserving Mitochondrial Function Through PI3K/AKT Signaling

Technical Summary: Mogrol Protects Against Hepatic Ischemia–Reperfusion Injury by Preserving Mitochondrial Function Through PI3K/AKT Signaling

Problem Statement
Hepatic ischemia–reperfusion injury (HIRI) is a critical complication in liver surgery, trauma, and transplantation that significantly impairs clinical outcomes. The pathogenesis of HIRI involves a complex cascade of events, including calcium overload, oxidative stress, inflammatory responses, mitochondrial dysfunction, and apoptosis. While strategies such as surgical preconditioning and gene therapy exist, effective pharmacological interventions remain limited. Mitochondria are central to this injury, where ischemia leads to ATP depletion and reperfusion triggers excessive reactive oxygen species (ROS) production, causing mitochondrial membrane permeability transition pore (mPTP) opening, loss of membrane potential, and subsequent cell death. Mogrol, a bioactive aglycone derived from Siraitia grosvenorii (Luo Han Guo), possesses known antioxidant and anti-inflammatory properties but its specific efficacy and mechanism regarding HIRI have not been previously established.

Methodology
The study employed a dual-model approach to investigate Mogrol's protective effects:

1. In Vivo Model: Male C57BL/6J mice were subjected to a hepatic I/R model involving 90 minutes of ischemia (occlusion of the left and median lobes) followed by 6 hours of reperfusion. Animals were divided into six groups: Sham, Mogrol-only, I/R, I/R+Mogrol, I/R+LY294002 (PI3K inhibitor), and I/R+LY294002+Mogrol. Mogrol (10 mg/kg) was administered via gavage for 7 days prior to surgery.
2. In Vitro Model: AML12 hepatocyte cells were subjected to a hypoxia/reoxygenation (H/R) model (12h hypoxia followed by 6h reoxygenation). Cells were pre-treated with Mogrol (40 μmol/L) for 24 hours.
3. Mechanistic Investigation:
   • Biochemical & Histological Analysis: Serum ALT/AST, H&E staining, TUNEL assays, and oxidative stress markers (MDA, SOD, GSH, ROS via DHE/DCFH-DA) were measured.
   • Mitochondrial Assessment: Transmission electron microscopy (TEM) evaluated ultrastructure; ATP levels, mitochondrial membrane potential (MMP), and mPTP opening were quantified.
   • Molecular Analysis: Western blotting assessed proteins related to apoptosis (Bcl-2, Bax, Cytochrome c, Cleaved caspase-3) and the PI3K/AKT pathway. RT-qPCR measured inflammatory cytokines (IL-1β, TNF-α, IL-6, IL-10).
   • Omics & Docking: RNA sequencing (RNA-seq) was performed to identify differentially expressed genes and KEGG pathways. Molecular docking simulations tested the binding affinity between Mogrol and the PI3K p85 subunit.
   • Pathway Validation: The PI3K inhibitor LY294002 was used to determine if Mogrol's effects were dependent on the PI3K/AKT signaling axis.

Key Results

• Protection Against Liver Injury: Mogrol pretreatment significantly reduced serum ALT and AST levels and attenuated hepatic necrosis in both mouse and cell models compared to untreated I/R/H/R groups.
• Mitigation of Oxidative Stress and Inflammation: Mogrol reduced ROS accumulation and MDA levels while restoring SOD activity and GSH content. It suppressed the infiltration of Ly6G-positive cells and downregulated pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) while upregulating the anti-inflammatory cytokine IL-10.
• Preservation of Mitochondrial Function: TEM revealed that Mogrol preserved mitochondrial ultrastructure, preventing the swelling and cristae disruption seen in I/R groups. Mogrol treatment stabilized mitochondrial membrane potential, inhibited mPTP opening, and significantly increased ATP production.
• Inhibition of Apoptosis: Mogrol reduced the number of TUNEL-positive apoptotic cells. Mechanistically, it increased the expression of the anti-apoptotic protein Bcl-2 and decreased the pro-apoptotic proteins Bax, Cytochrome c, and Cleaved caspase-3.
• Activation of PI3K/AKT Signaling: RNA-seq and KEGG analysis identified the PI3K/AKT pathway as a key target. Molecular docking confirmed a stable binding interaction between Mogrol and the PI3K p85 subunit (binding energy: -8.0 kcal/mol). Western blotting demonstrated that Mogrol significantly increased the phosphorylation of PI3K and AKT.
• Reversal by Inhibitor: The administration of LY294002 partially reversed the protective effects of Mogrol. In the presence of the inhibitor, Mogrol failed to fully restore liver function, reduce oxidative stress, or prevent mitochondrial apoptosis, confirming that the PI3K/AKT pathway is essential for Mogrol's mechanism of action.

Key Contributions
This study provides the first evidence that Mogrol exerts a protective effect against HIRI. It elucidates the mechanism by which Mogrol preserves mitochondrial function and suppresses apoptosis, specifically linking these outcomes to the activation of the PI3K/AKT signaling pathway. The research bridges the gap between the known pharmacological properties of Mogrol and its specific application in liver ischemia-reperfusion contexts, supported by both transcriptomic data and molecular docking simulations.

Significance
The authors conclude that Mogrol mitigates HIRI, at least in part, by activating the PI3K/AKT signaling pathway, which subsequently stabilizes mitochondrial function and inhibits apoptosis. This highlights Mogrol as a potential naturally derived therapeutic candidate for liver protection in clinical settings involving ischemia-reperfusion. The study suggests that targeting the PI3K/AKT axis with Mogrol could offer a novel strategy to improve outcomes in liver surgery and transplantation, though the authors maintain a focus on the experimental validation of this specific mechanism rather than broad clinical extrapolation.