Bilobalide alleviates ischemia-reperfusion injury during liver transplantation by regulating macrophage polarization through inhibiting NF-κB pathway.

BACKGROUND: Hepatic ischemia-reperfusion injury (IRI) causes donor graft dysfunction and increases mortality after liver transplantation (LT). Bilobalide (BB), a natural sesquiterpene lactone extracted from Ginkgo biloba leaves, exhibits anti-inflammatory and anti-necrotic bioactivities. However, there is no study currently on the use of bilobalide in IRI therapy for liver transplant. This study aimed to investigate whether bilobalide protects grafted livers from IRI and to explore the underlying mechanisms.

METHODS: Donor livers preserved in histidine-tryptophan-ketoglutarate (HTK) solution for 5 h underwent orthotopic liver transplantation in Sprague-Dawley rats. Following LT, rats were administered intraperitoneal injections of bilobalide (30 mg/kg, LT + BB group), saline (LT group), or N-acetylcysteine (150 mg/kg, LT + NAC group) at two-time points: immediately after surgery and again at 12 h post-transplant. Serum liver enzymes levels at 24 h after transplantation were measured. Liver tissues underwent hematoxylin and eosin (H&E) staining to confirm the injury. To examine hepatocyte apoptosis, both TUNEL staining and Western blot analysis were employed. Inflammatory responses in liver tissue were examined using immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Potential signaling pathways were identified through transcriptome sequencing (RNA-seq) and subsequently validated by Western blot analysis. In addition, macrophage polarization was evaluated using immunofluorescence and Western blot.

RESULTS: The LT + BB group exhibited significantly milder liver injury and hepatocyte apoptosis compared to the LT group. The LT + NAC group also showed reduced liver injury and apoptosis, although the protective effects were less pronounced than those observed in the LT + BB group. Bilobalide treatment significantly reduced inflammatory cell infiltration and suppressed pro-inflammatory cytokine expression. RNA-seq and Western blot analyses demonstrated that bilobalide inhibited the activation of the nuclear factor Kappa B (NF-κB) signaling pathway in transplanted liver tissue. Immunofluorescence and Western blot results further revealed that bilobalide suppressed macrophage polarization toward the M1 phenotype.

CONCLUSIONS: Bilobalide protects the transplanted liver from IRI by regulating macrophage polarization through NF-κB signaling pathway.