Published online Sep 7, 2026. doi: 10.3748/wjg.117979
Revised: February 4, 2026
Accepted: April 27, 2026
Published online: September 7, 2026
Processing time: 233 Days and 17.8 Hours
Hepatic ischemia-reperfusion injury (IRI) is a major cause of liver dysfunction following hepatic surgeries such as liver transplantation and liver tumor re
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.
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 path
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
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.
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
- Citation: Lu JS, Liang HL, Chen DC, Pan H, Han T, Zhang WY, Fan M, Hou W, Shen ZY. Naringenin pretreatment attenuates hepatic ischemia-reperfusion injury by activating the PI3K-Akt signaling pathway. World J Gastroenterol 2026; 32(33): 117979
- URL: https://www.wjgnet.com/1007-9327/full/v32/i33/117979.htm
- DOI: https://dx.doi.org/10.3748/wjg.117979
Hepatic ischemia-reperfusion injury (IRI) is the main cause of liver function impairment after liver surgeries including liver transplantation, liver tumor resection, and other liver-related operations[1]. Hepatic IRI is a complex process with multiple injury mechanisms involved, including cell necrosis, apoptosis, ferroptosis, inflammatory response, autophagy, oxidative stress, and metabolic disorders[2,3]. Due to the complex mechanisms, there is currently a lack of effective drugs to alleviate hepatic IRI and improve the prognosis of liver transplant patients[4].
Naringenin (NAR) [5,7-dihydroxy-2-(4-hydroxyphenyl)-2,3-dihydrochromen-4-one] is a flavanone that is readily soluble in organic solvents such as ethanol, dimethylformamide, and dimethyl sulfoxide, with a melting point of 208-
Network pharmacology, as an interdisciplinary research field, ingeniously integrates pharmacological methods with biological networks and drug action networks, transcending the limitations of a single target. It conducts comprehensive network analysis through a multi-target research strategy to achieve a comprehensive study of drug effects. This research will help us understand the complex relationship between drug components and disease targets, and provide an intuitive analysis method[13]. This study predicts that the mechanism by which NAR alleviates hepatic IRI is related to the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway through the method of network pharmacology. The PI3K-Akt signaling pathway is involved in regulating various physiological and pathological processes and is a signaling pathway with multiple biological functions[14]. Activating this pathway can alleviate hepatic IRI by inhibiting oxidative stress, reducing inflammatory responses, and reducing apoptosis[15-20].
This experiment adopts the method of clamping the portal vein and hepatic artery of mice in vivo to block the blood flow to establish a mouse liver IRI model. It is intended to clarify the role of NAR in mouse liver IRI, and to explore the effects of NAR on inflammatory response and cell apoptosis. Network pharmacology is applied to predict the target of NAR in reducing liver IRI, and to further study whether its effect is mediated by the PI3K-Akt signaling pathway, providing theoretical support for the clinical treatment of drugs related to liver IRI.
Male C57BL/6 mice (8-10 weeks old, weighing 22 ± 2 g) were purchased from Vital River Company (Beijing, China) and housed in the Animal Experiment Center of the Institute of Transplant Medicine at Nankai University. The housing environment was maintained at 22 ± 1 °C and 50% ± 10% relative humidity under a 12 hours/12 hours light-dark cycle. Food and water were provided ad libitum. All mouse experiments were performed in strict accordance with the experimental protocol approved by the Animal Ethics Committee of Nankai University, No. 2024-SYDWLL-000215.
NAR (IN0350, Solarbio, China) was dissolved in DMSO as a stock solution. For animal experiments, the stock solution was further diluted with PEG400, Tween-80, and normal saline. Mice were administered the solution via oral gavage daily for 7 consecutive days before modeling. The experimental groups were as follows: Sham-operated group (Sham), Sham + solvent control group, Sham + high-dose NAR group (50 mg/kg) IRI model group, IRI + low-dose NAR group (25 mg/kg), and IRI + high-dose NAR group (50 mg/kg). LY294002 (a PI3K inhibitor, GC15485, GLPBIO, CA, United States) was injected at a dose of 1.5 mg/kg 0.5 hour before surgery. For cell experiments, the stock solution was diluted to the required concentration and applied to cells 12 hours before hypoxia induction.
Hepatic IRI model was established as previously described[21]. After fasting for 12 hours with free access to water, mice were anesthetized using isoflurane inhalation. Following anesthesia, mice were fixed on a dedicated animal operating table, and the abdominal skin was disinfected with iodophor. A midline laparotomy was performed. The intestines were gently wrapped with sterile wet gauze and retracted to one side to fully expose the hepatic portal region. The left and median liver lobes (approximately 70% of total liver volume) were clamped using a non-invasive arterial clip. After 1 hour of ischemia, the clip was removed to restore blood flow. The intestines were then returned to their original position, and the abdomen was closed layer by layer. Postoperative mice were placed on a heating pad to maintain body temperature. After 6 hours of reperfusion, the animals were euthanized, and serum and liver tissues were collected for subsequent analysis. Six animal samples per group were used for serological index detection and histopathological hematoxylin and eosin staining, and 3 animals were randomly selected per group for liver protein detection and immunofluorescence assays.
Serum was separated from peripheral blood by centrifugation, and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using an automated biochemical analyzer.
Liver tissues were fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Morphological changes were observed under a light microscope, and the degree of liver injury was semi-quantitatively assessed according to the Suzuki scoring criteria.
Apoptosis in liver paraffin sections was detected using a one-step TUNEL in situ apoptosis detection kit (E-CK-A320, Elabscience, China) according to the manufacturer’s instructions. Images were captured under a fluorescence microscope, and TUNEL-positive cells were counted.
After antigen retrieval, liver paraffin sections were subjected to immunofluorescence staining. The primary antibody against CD11b (ab133357, Abcam, Cambridge, United States) was applied and incubated overnight at 4 °C. The following day, the corresponding fluorescent secondary antibody was added and incubated in the dark. Nuclei were counterstained with DAPI. CD11b+ cell infiltration was observed and recorded under a fluorescence microscope, and positive cells were quantified.
The levels of superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and reduced glutathione (GSH) in liver tissues were determined using respective assay kits: SOD Activity Assay Kit (BC0175, Solarbio, China), MDA Content Assay Kit (BC0025, Solarbio, China), and GSH Assay Kit (BC1175, Solarbio, China). All procedures were performed in strict accordance with the manufacturers’ instructions.
To identify the potential targets of NAR, we performed target prediction using the SwissTargetPrediction Database. A total of 96 potential NAR-related targets were obtained.
Targets associated with hepatic IRI were retrieved from the GeneCards Database (https://www.genecards.org/) using the keyword “hepatic ischemia-reperfusion injury”, yielding 971 IRI-related targets. A Venn diagram was then plotted to identify the common targets between NAR and hepatic IRI.
The common targets were imported into the STRING database (https://string-db.org/) to obtain protein-protein interaction information.
Gene Ontology functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed on the common targets of NAR and hepatic IRI using the DAVID database (https://david.ncifcrf.gov). These analyses aimed to elucidate the underlying biological processes, molecular functions, cellular components, and signaling pathways involved.
Total RNA was extracted from liver tissues using RNA-easy Isolation Reagent (R701-01, Vazyme, China), and RNA concentration was measured. Complementary DNA was synthesized using HiScript II Q Select RT SuperMix (R233-01, Vazyme, China). Target gene expression levels were detected by real-time quantitative polymerase chain reaction with Taq Pro Universal SYBR qPCR Master Mix (Q711-02, Vazyme, China). Primer sequences are listed in Supplementary Table 1.
Total protein was extracted from liver tissues using a highly efficient tissue lysis buffer (R0010, Solarbio, China) and protein concentration was determined using a bicinchoninic acid assay kit (PC0020, Solarbio, China). Proteins were separated by sodium-dodecyl sulfate gel electrophoresis and transferred to polyvinylidene fluoride membranes (Merck Millipore, Germany). The membranes were blocked with 5% skim milk (D8340, Solarbio, China) and incubated with primary antibodies Bax (50599-2-Ig, Proteintech, China), Bcl-2 (68103-1-Ig, Proteintech, China), p-Akt (YP0006, Immunoway, United States), Akt (YT0185, Immunoway, United States), p-PI3K (YP0765, Immunoway, United States), PI3K (YM3503, Immunoway, United States), β-actin (66009-1-Ig, Proteintech, China) at 4 °C overnight. After washing with Tris-buffered saline with Tween, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 hour. Protein bands were visualized using an enhanced chemiluminescence reagent and imaged with a Bio Rad imaging system (Hercules, United States). Grayscale values were quantified using Image J 7.0 software.
The AML12 cell hypoxia/reoxygenation (H/R) injury model was established as previously described[21]. Cells were routinely cultured at 37 °C under 5% CO2. Before hypoxia induction, the medium was replaced with serum-free medium. The cells were then placed in a tri-gas incubator (37 °C, 1% O2, 94% N2, 5% CO2) for 12 hours to simulate hypoxia. Subsequently, the medium was replaced with complete medium, and the cells were returned to a normoxic incubator
Cell viability was assessed using the cell counting kit-8 assay (GK10001, GlPBIO, CA, United States). AML12 cells were seeded in 96-well plates. After adherence, cells were treated with different concentrations of NAR for 12 hours. Then,
Apoptosis was analyzed using an Annexin V-FITC/propidium iodide Apoptosis Detection Kit (CA1020, Solarbio, China). After treatment, AML12 cells were collected, resuspended in buffer, and adjusted to a concentration of 5 × 106/mL. A
Intracellular reactive oxygen species (ROS) levels were measured using a ROS Assay Kit (CA1410, Solarbio, China). After treatment, AML12 cells were incubated with DCFH-DA probe diluted 1:5000 in serum-free medium for 30 minutes. The probe solution was then removed, and the cells were washed three times with serum-free medium. ROS levels were observed and images were captured under a fluorescence microscope.
Unless otherwise stated, all experimental data are expressed as the mean ± SD. Statistical analyses were performed using GraphPad Prism version 8. Comparisons among multiple groups were analyzed by one-way analysis of variance, followed by Tukey’s post hoc test. A P value of less than 0.05 was considered statistically significant.
To evaluate the protective effect of NAR on hepatic IRI in mice, animals received NAR (25 or 50 mg/kg) via oral gavage for seven consecutive days prior to IRI induction (Figure 1A and B). Serum biochemical analysis indicated markedly elevated levels of ALT and AST in the IRI group relative to the Sham group, confirming successful model establishment. In contrast, NAR pretreatment, especially at 50 mg/kg, significantly lowered ALT and AST levels compared with the IRI group. Moreover, treatment with 50 mg/kg NAR alone for 7 days did not induce significant changes in serum ALT or AST in healthy mice, demonstrating the absence of hepatotoxicity at this dosage (Figure 1C and D). Histopathological evaluation via H&E staining and Suzuki scoring further revealed that NAR pretreatment substantially ameliorated liver tissue damage compared to the IRI group (Figure 1E and F). Collectively, these findings suggest that pretreatment with 50 mg/kg NAR safely and effectively attenuates IRI-induced liver injury in mice, and we set 50 mg/kg as the dose regimen for the subsequent research.
Hepatocyte apoptosis represents a critical pathological component of hepatic IRI. To examine the influence of NAR on apoptosis following IRI, we initially evaluated apoptotic cells in liver tissues using TUNEL staining. Mice were pretreated with 50 mg/kg NAR for 7 days before IRI induction. The results indicated that NAR significantly diminished hepatocyte apoptosis (Figure 2A and B). Furthermore, western blot analysis showed that NAR pretreatment markedly suppressed the expression of the pro-apoptotic protein Bax and enhanced that of the anti-apoptotic protein Bcl-2 relative to the IRI group (Figure 2C-E). These data imply that NAR mitigates hepatic IRI-induced apoptosis.
Hepatic IRI is characterized by aggravated inflammatory responses and oxidative stress, which contribute to hepatic damage. Previous reports have indicated that NAR exhibits anti-inflammatory and antioxidant activities across multiple disease models. To further elucidate the effect of NAR on the inflammatory response after hepatic IRI, we evaluated CD11b+ immune cell infiltration in liver tissue via immunofluorescence staining. Prominent infiltration of CD11b+ cells was detected in the IRI group, while NAR pretreatment notably curtailed this infiltration (Figure 3A and B). Additionally, the mRNA expression levels of the inflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor-α were considerably lower in the NAR-pretreated group than in the IRI group (Figure 3C-E). To evaluate the impact of NAR on oxidative stress, we quantified the levels of MDA, SOD, and GSH in liver tissues. NAR pretreatment significantly enhanced SOD and GSH activities and decreased MDA content, an indicator of lipid peroxidation (Figure 3F-H). These results demonstrate that NAR effectively counteracts hepatic IRI-induced inflammatory response and oxidative stress.
To further investigate the mechanisms behind NAR-mediated protection against hepatic IRI, a network pharmacology-based strategy was adopted. Potential targets of NAR were acquired from the SwissTargetPrediction Databases, yielding 96 genes. Hepatic IRI-associated targets were sourced from the Genecards Databases, providing 971 genes. Intersection of both sets identified 35 common genes (Figure 4A). These common genes were utilized to generate a protein-protein interaction network via STRING (Figure 4B). Gene Ontology functional enrichment analysis revealed significant enrichment in biological processes including positive regulation of PI3K/Akt signal transduction and apoptosis (Figure 4C). Kyoto Encyclopedia of Genes and Genomes pathway analysis proposed that NAR may confer protection against hepatic IRI by regulating apoptosis and the PI3K-Akt signaling pathway (Figure 4D). To corroborate this, we assessed PI3K and Akt phosphorylation by western blotting. NAR pretreatment significantly elevated p-PI3K and p-Akt expression relative to the IRI group (Figure 4E-G), suggesting that NAR alleviates hepatic IRI via activation of the PI3K-Akt pathway.
To further validate the protective effect of NAR against IRI in vitro, we established a H/R model using AML12 cells to simulate the IRI process in vivo (Figure 5A). First, the cytotoxicity of NAR was evaluated by treating AML12 cells with various concentrations (0-400 μM) for 12 hours. The results showed that NAR did not significantly affect cell viability at concentrations ranging from 0 to 100 μM, whereas a notable reduction in viability was observed at 200 μM (Supplementary Figure 1). Therefore, 100 μM was selected as the maximum concentration for subsequent experiments. After determining the safe concentration range, AML12 cells were pretreated with 0-100 μM NAR for 12 hours followed by H/R induction (12 hours hypoxia and 6 hours reoxygenation). Cell viability assays revealed that compared with the control (Con) group, the H/R group exhibited significantly decreased cell viability. NAR pretreatment attenuated the
To ascertain whether NAR mitigates hepatic IRI through the PI3K-Akt pathway, the PI3K inhibitor LY294002 was administered alongside NAR before IRI. In vivo outcomes revealed that the IRI + NAR + LY294002 group exhibited significantly higher serum AST and ALT levels than the IRI + NAR group, implying that LY294002 abolished the protective effect of NAR (Figure 6A and B). This was further supported by histopathological evaluation using H&E staining (Figure 6C and D). Additional in vivo and in vitro western blot analyses indicated that LY294002 inhibited the NAR-induced upregulation of PI3K and Akt phosphorylation. Simultaneously, the reduction in Bax and elevation in Bcl-2 were likewise reversed (Figure 6E-H). These results confirm that NAR safeguards against hepatic IRI by activating the PI3K-Akt signaling pathway.
Hepatic IRI continues to represent a pivotal clinical challenge that demands urgent resolution in the context of liver transplantation and hepatobiliary surgeries[22]. Its pathological mechanisms are complex, involving multiple key processes such as oxidative stress, inflammatory infiltration, and hepatocyte apoptosis. Currently, effective pharmacological interventions for IRI are still limited[23]. Particularly in the field of liver transplantation, recent clinical studies have highlighted that despite advances in surgical techniques, conventional liver transplantation is still frequently associated with severe IRI-related complications, such as early allograft dysfunction and non-anastomotic biliary strictures[24]. This further underscores the urgent need for developing effective preventive strategies to alleviate hepatic IRI. This study systematically demonstrates that NAR significantly alleviates hepatic IRI. Through in vivo and in vitro experiments, NAR pretreatment exhibited substantial hepatoprotective effects: In a C57BL/6 mouse model of IRI, oral administration of 50 mg/kg NAR for 7 consecutive days markedly reduced serum levels of ALT and AST, attenuated histopathological damage in liver tissue, and effectively suppressed hepatocyte apoptosis. In vitro, treatment with 100 μM NAR significantly ameliorated H/R-induced injury in AML12 hepatocytes by enhancing cell viability, reducing ROS accumulation, and inhibiting apoptotic cell death. Further network pharmacological analysis confirmed that NAR modulates the PI3K-Akt signaling pathway. These findings not only underscore the potential of NAR as a therapeutic agent for hepatic IRI but also provide a foundation for elucidating its underlying molecular mechanisms.
NAR, a naturally occurring flavanone abundantly present in citrus fruits, exhibits a wide range of pharmacological activities[25]. Particularly in the field of liver diseases, a large body of research has confirmed its hepatoprotective effects[26]. It has been reported that NAR effectively alleviates isoniazid and rifampicin induced liver injury in mice, and this protective effect is directly associated with the inhibition of hepatocyte apoptosis[27]. This mechanism is highly consistent with our findings that NAR significantly reduces hepatocyte apoptosis induced by IRI, further supporting the notion that NAR exerts its hepatoprotective role through anti-apoptotic pathways. Regarding antioxidation, NAR exerts antioxidant effects by upregulating the activity of antioxidant enzymes and enhancing free radical scavenging capacity, thereby significantly alleviating lipid peroxidation damage in models of non-alcoholic fatty liver disease and hepatocellular carcinoma[28,29]. Consistent with these previous findings, in the hepatic IRI model of the present study, NAR pretreatment significantly increased the activity of SOD and the level of reduced GSH in liver tissues, while remarkably decreasing the content of MDA, a key marker of lipid peroxidation. Therefore, NAR can effectively restore redox homeostasis in liver tissues in the hepatic IRI model, providing important antioxidant mechanistic support for its hepatoprotective effects. In terms of inhibiting inflammatory responses, recent studies have confirmed that NAR can effectively suppress lipopolysaccharide induced macrophage activation and significantly reduce the release of pro-inflammatory cytokines in ex vivo human whole-blood models[7]. Moreover, studies in hepatic disease models have confirmed that NAR can significantly alleviate hepatic inflammatory responses[30,31]. The present study further validated this property in the hepatic IRI model: NAR significantly reduced the infiltration of CD11b+ immune cells in liver tissues, and simultaneously downregulated the mRNA expression levels of key pro-inflammatory cytokines (IL-1β, IL-6, and tumor necrosis factor-α) in liver tissues induced by IRI. This anti-inflammatory mechanism of NAR is highly consistent with previous research conclusions. However, the specific mechanism by which NAR regulates hepatic inflammation remains to be further elucidated. Besides the existing findings that NAR can directly regulate hepatic macrophages to modulate inflammation, recent studies have shown that liver-derived inflammatory factors (such as proprotein convertase subtilisin/kexin 9, PCSK9) can also participate in inflammatory regulation by regulating hepatic immune cells[32]. Therefore, further investigating the complete mechanism of NAR in regulating hepatic inflammatory responses is of great significance for expanding its application in the treatment of liver diseases. Collectively, the above findings indicate that NAR can significantly alleviate hepatic IRI through inhibiting hepatocyte apoptosis, reducing inflammatory responses, and mitigating oxidative stress.
Network pharmacology analysis serves as a powerful tool for deciphering the complex mechanisms of natural products. By integrating multi-target and multi-pathway analyses, it provides critical technical support for exploring the potential mechanisms by which NAR ameliorates hepatic IRI[33]. In this study, network pharmacological analysis identified the PI3K-Akt signaling pathway as a key pathway through which NAR alleviates hepatic IRI. This finding is highly consistent with its well-established roles in regulating cell survival, maintaining redox balance, and modulating pathological inflammatory responses in the liver[14,34], further suggesting that this pathway may represent a central hub for the hepatoprotective effects of NAR. The critical regulatory role of the PI3K-Akt pathway in the pathological process of hepatic IRI has been extensively demonstrated. For example, icariin and arsenic trioxide have been shown to attenuate hepatic IRI by modulating hepatocyte autophagy activity via targeted regulation of the PI3K-Akt pathway[20,35]. Pectolinarigenin significantly suppresses hepatocyte apoptosis, inflammatory response, and oxidative stress imbalance following IRI through the PI3K-Akt-Nrf2 signaling axis[36]. Furthermore, activation of the AXL receptor inhibits ferroptosis in hepatocytes via the PI3K-Akt pathway, thereby protecting the liver from IRI[19]. Moreover, activation of the PI3K-Akt pathway can significantly reduce liver fibrosis markers and improve liver function by inhibiting extracellular matrix synthesis and hepatic stellate cell activation[37], underscoring its pivotal role in liver diseases. Importantly, NAR has been demonstrated to regulate the PI3K-Akt signaling pathway in multiple disease models, providing cross-disease evidence supporting its mechanism of action in hepatic IRI. Previous studies have reported that in a dibutyl phthalate-induced cell injury model, NAR activates the PI3K-Akt pathway by downregulating phosphatase and tensin homolog expression, thereby counteracting dibutyl phthalate-induced oxidative stress and apoptosis[38]. In renal disease, NAR alleviates kidney inflammation by inhibiting PI3K-Akt signaling, downregulating glucose transporter 9 expression, and suppressing the Toll-like receptor 4-NF-κB pathway[39]. In the context of vascular protection, NAR activates Nrf2 through the PI3K-Akt and c-Jun N-terminal kinase pathways, effectively inhibiting high glucose - or free fatty acid-induced apoptosis in human umbilical vein endothelial cells[40]. In neuroprotection, NAR mitigates apoptosis and suppresses inflammatory responses by regulating the PI3K-Akt and NF-κB signaling pathways, thereby reducing isoflurane-induced neuronal damage[41]. This study further validated the association between NAR and the PI3K-Akt pathway in a hepatic IRI model. NAR pretreatment significantly upregulated the p-PI3K and p-Akt in both an in vivo hepatic IRI model and an in vitro H/R-induced AML12 hepatocyte model, directly demonstrating the activating effect of NAR on this pathway. More importantly, the protective effects of NAR, including reduced serum liver enzyme levels, improved liver histopathology, and inhibition of hepatocyte apoptosis, were completely abolished upon administration of the specific PI3K inhibitor LY294002. This result provides key evidence that the PI3K-Akt signaling pathway is indispensable for NAR-mediated protection against hepatic IRI.
Current studies indicate that NAR can significantly alleviate hepatocyte apoptosis, inflammatory response, and oxidative stress induced by hepatic IRI through activation of the PI3K-Akt signaling pathway. However, further improving the bioavailability and tissue targeting of NAR, as well as expanding its administration routes, may substantially broaden its therapeutic potential for a variety of diseases.
In summary, our study demonstrates that NAR protects the liver from IRI by inhibiting oxidative stress, inflammatory response, and hepatocyte apoptosis, with the PI3K-Akt signaling pathway serving as a key regulatory axis (Figure 7). These findings not only enhance our understanding of the pharmacological mechanisms of NAR but also provide compelling preclinical evidence supporting its potential as a therapeutic agent for hepatic IRI.
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