Naringenin pretreatment attenuates hepatic ischemia-reperfusion injury by activating the PI3K-Akt signaling pathway

Abstract

BACKGROUND

Hepatic ischemia-reperfusion injury (IRI) is a major cause of liver dysfunction following hepatic surgeries such as liver transplantation and liver tumor resection. Its pathogenesis involves multiple processes, including programmed hepatocyte death, oxidative stress, and inflammatory responses. However, there is still a lack of effective therapeutic drugs for liver IRI in clinical practice. Naringenin (NAR), a natural flavanone abundantly found in citrus fruits, exhibits various pharmacological activities. Yet, its specific protective effects and underlying molecular mechanisms in hepatic IRI remain to be systematically elucidated.

AIM

To systematically explore the therapeutic potential of NAR in hepatic IRI, clarify its regulatory effects on hepatocyte apoptosis, oxidative stress response and inflammatory factor release induced by hepatic IRI, and further systematically elucidate the potential molecular targets and signaling pathways of NAR in exerting its anti-hepatic IRI effects by means of network pharmacology.

METHODS

This study comprehensively evaluated the effect of NAR on hepatic IRI using in vivo, in vitro, and network pharmacology approaches. For the in vivo study, a mouse model of hepatic IRI (1 hour ischemia/6 hours reperfusion) was established. Mice were pretreated with NAR (25 or 50 mg/kg) via oral gavage for 7 consecutive days before modeling. For the in vitro study, a hypoxia/reoxygenation model was constructed using AML12 hepatocytes (12 hours hypoxia/6 hours reoxygenation). Liver injury was assessed by measuring serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and evaluating histopathological changes (hematoxylin and eosin staining and Suzuki scoring). Hepatocyte apoptosis was examined by TUNEL staining and western blot analysis of Bax and Bcl-2 expression. Inflammatory response was evaluated by immunofluorescence detection of CD11b+ cell infiltration and quantitative polymerase chain reaction analysis of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α mRNA levels. Oxidative stress was assessed by measuring superoxide dismutase activity, malondialdehyde content, and reduced glutathione level. Furthermore, network pharmacology was employed to identify common targets of NAR and hepatic IRI, construct a protein-protein interaction network, and perform Gene Ontology/Kyoto Encyclopedia of Genes and Genomes enrichment analysis to predict key signaling pathways. The role of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway was verified using the PI3K inhibitor LY294002.

RESULTS

In vivo, NAR pretreatment (especially at 50 mg/kg) significantly reduced serum ALT and AST levels, alleviated hepatic necrosis and structural damage and suppressed hepatocyte apoptosis in IRI mice. Additionally, NAR markedly reduced CD11b+ immune cell infiltration and pro-inflammatory cytokine (IL-1β, IL-6, tumor necrosis factor-α) mRNA expression, while enhancing superoxide dismutase activity and reduced glutathione content and reducing malondialdehyde levels, demonstrating anti-inflammatory and antioxidant effects. Network pharmacology analysis identified 35 common targets of NAR and hepatic IRI, and Kyoto Encyclopedia of Genes and Genomes enrichment analysis highlighted the PI3K-Akt signaling pathway as a potential key mechanism. Western blot results showed that NAR significantly upregulated the expression of p-PI3K and p-Akt in liver tissues. In vitro experiments further confirmed that 100 μM NAR significantly improved the viability of hypoxia/reoxygenation-induced AML12 cells, reduced reactive oxygen species generation and apoptosis rate, and activated the PI3K-Akt pathway. Critically, the PI3K inhibitor LY294002 completely abolished the protective effects of NAR, as evidenced by restored serum ALT, AST levels, aggravated liver histopathological injury, and suppressed upregulation of p-PI3K and p-Akt.

CONCLUSION

NAR alleviates hepatic IRI by activating the PI3K-Akt signaling pathway, thereby inhibiting oxidative stress, inflammatory response, and hepatocyte apoptosis. This study systematically elucidates the protective mechanism of NAR against hepatic IRI and provides preclinical evidence supporting its potential clinical application for the treatment of hepatic IRI.

Keywords: Hepatic ischemia-reperfusion injury; Naringenin; Phosphatidylinositol 3-kinase-protein kinase B signaling pathway; Oxidative stress; Apoptosis; Inflammatory response

Core Tip: The specific protective effects and underlying molecular mechanisms in hepatic ischemia-reperfusion injury remain to be systematically elucidated. This study demonstrates that naringenin protects the liver from ischemia-reperfusion injury by inhibiting oxidative stress, inflammatory response, and hepatocyte apoptosis, with the phosphatidylinositol 3-kinase-protein kinase B signaling pathway serving as a key regulatory axis.