The hepatotoxicity of chemotherapeutic drugs has been noted to require close monitoring by oncologists during treatment, despite growing number of cancer survivors. However, many of these survivors still suffer from symptoms of liver because of their cancer therapy (Mudd and Guddati, 2021). One common first-line treatment for different types of cancer is doxorubicin (DOX), which has been found tocause hepatotoxicity. Several strategies have been developed to prevent liver damage caused by DOX, but additional strategies are needed to protect liver tissues from its harmful effects (Sirwi et al., 2021; Al-Qahtani et al., 2022; El-Said K. S. et al., 2023). Various processes, including apoptosis, inflammation, and oxidative stress, have been proposed to explain the mechanisms behind DOX-induced hepatotoxicity. Therefore, it is crucial to understand the mechanisms that protect liver tissue during DOX administration (Morsy et al., 2023). DOX may induce oxidative stress by blocking the transcription factor Nrf-2, which coordinates cellular redox homeostasis and regulates antioxidant and detoxifying responses (Jakobs et al., 2017). A recent study by Saleh et al. (2022) highlighted the protective role of Omega-3 in DOX-induced hepatotoxicity through Akt/GSK-3β axis modulation in rats. Additionally, Sirwi et al. (2021) demonstrated that Mokko lactone attenuated DOX-induced hepatotoxicity by regulating the Sirt-1/FOXO1/NF-κB axis in rats.

A major redox-sensitive protein kinase, glycogen synthase kinase (GSK)-3β is widely expressed in nearly every cell and plays a role in various intracellular and extracellular processes, including influencing cell development and apoptosis (Martelli et al., 2021). Akt could control the GSK-3β activity, and suppressing GSK-3β could modulate oxidative stress in hepatocytes by targeting Nrf-2 (Jiang et al., 2015). Importantly, GSK-3β signaling seems to be responsible for the apoptosis induction observed upon Akt inhibition (Li et al., 2020). The Akt/GSK-3β signaling pathway is crucial for cell survival and has been linked to the reduction of liver injury through anti-inflammatory and anti-apoptotic effects (Xing et al., 2024). Targeting GSK-3β may be a unique approach to mitigate the toxicity caused by DOX in multiple organs, according to evidence (Niringiyumukiza et al., 2019).

The development, differentiation, and cellular homeostasis of liver tissue rely on the complex Wnt signaling system. The Wnt pathway plays a significant role in liver metabolism, regeneration, and maintaining the normal function of liver (Hu and Monga, 2021). Abnormal regulation of Wnt/β-catenin signaling pathway is associated with various disorders making it a desirable target for disease treatment (Liu et al., 2022). Activation of Wnt/β-catenin signaling pathway is linked to the pathogenesis of liver fibrosis and protective role of natural compounds against liver fibrosis by inhibiting the Wnt/β-catenin pathway has been reported (El-Ashmawy et al., 2020).Phenolic compounds including, anthranilic acid amides, have multiple health promoting qualities, and were known for their antioxidant, anti-inflammatory, and anti-proliferative properties (Toma et al., 2020; Rahman et al., 2021; Mobasher et al., 2024). These anthranilic acid amides, or called avenanthramides (AVNS), are a class of N-cinnamoyl anthranilic acids that oat plants make as phytoalexins (Pretorius and Dubery, 2023). Oats contain AVN in a variety of forms, but AVN-C is the most common and has the strongest antioxidant effect (Perrelli et al., 2018). Additionally, AVNS have been shown to have potent biomedical potential both in vivo and in vitro through reducing oxidative stress-related disorders and cellular dysfunctions (Perrelli et al., 2018; Wankhede et al., 2023). Previous studies demonstrated the therapeutic effects of AVN-C against lung toxicity in rats, inflammation, and oxidative stress in human skin fibroblasts (Wang and Eskiw, 2019; Altwaijry et al., 2021).). AVNs have also been reported to reduce the risk of colon cancer by targeting apoptosis, which reduces cellular proliferation (Fu et al., 2019). Thisnovel study investigated for the first time the curative role, biochemical pathways, and molecular mechanisms of AVN-C treatment against DOX-induced hepatotoxicity in rats.

Cancer patient survival rates have gradually increased due to advancements in chemotherapy and treatment plans (Bluethmann et al., 2016). The traditional usage of DOX has been limited due to its adverse effects on various organs, including hepatotoxicity (Prasanna et al., 2020). The use of natural herbal constituents against hepatotoxicity mediated by DOX has been reviewed (Mahmoudi et al., 2023). The phenolic compounds (AVNs) exhibit high antioxidant properties, their efficiency against chemotherapy-induced toxicity has been reported in experimental animals (Umugire et al., 2022). A previous study reported that AVNs could decrease liver dysfunctions induced by cisplatin, in mice (El-Said K. et al., 2023). Therefore, this study aimed to evaluate the efficacy of AVN-C treatment versus hepatotoxicity in rats promoted by DOX through in silico studies, hematological, biochemical, molecular, and histopathological investigations. Furthermore, this study explored the mechanisms of AVN-C action on DOX-induced hepatotoxicity through antioxidant, anti-inflammatory, and modulation of Akt/GSK-3β and Wnt-4/β-Catenin pathways in rats.

Molecular docking studies revealed insights into the binding affinities and interaction patterns of AVN-C and DOX with key proteins involved in various cellular processes. Further investigation of protein-ligand interactions showed that both substances interacted with target proteins (Akt-1, GSK-3β, Wnt-4) through hydrogen bonds, hydrophobic contacts, and occasionally electrostatic interactions. These interactions suggest that AVN-C may modulate Akt signaling, potentially influencing cell survival and proliferation pathways. In Silico toxicity predictions provided insights into the safety profiles of AVN-C and DOX, withAVN-C predicted to be less toxic across several endpoints compared to DOX. This differential interaction helps explain the distinct effects observed when each compound is administered. Inhibition of p-Akt led to activation of GSK-3β, in silico studies revealed that the binding of DOX with GSK-3β does not suppress the vigorous activation of GSK-3β proteins, this could be due to the compensatory mechanisms of hepatocytes to overexpress GSK-3β for oxidative stress, inflammation, and apoptosis inductions, while co-treatment with DOX and AVN-C could reduce GSK-3β protein expression levels. The suppression of P-Akt levels by DOX in the in-vivo studies aligns with its strong interaction with Akt, indicating that DOX may inhibit Akt phosphorylation, leading to reduced cell survival signaling. This can contribute to increased apoptosis and liver damage. A previous study of Saleh et al. (2022) reported that suppression of hepatic GSK-3β levels upon treatment with omega-3 as compared to the DOX-treated group as well as restoration in hepatic levels of p-Akt, suggesting the GSK-3β inhibitor’s protective properties against DOX-induced oxidative stress. The upregulation of Wnt-4 and β-catenin expressions suggested also that DOX alters the balance of these pathways, promoting compensation processes in liver cells to overexpress these proteins that can lead to hepatocytes dysfunctions or death. Co-treatment with DOX + AVN-C led to an increase in P-Akt levels and the decrease in GSK-3β, Wnt-4, and β-catenin expressions suggest a protective effect of AVN-C. In Silico studies supports this by showing that AVN-C, having lower binding affinity, may act as a milder modulator of these pathways, but when co-treated and reacted with DOX in vivo counteracting the deleterious effects of DOX on the target proteins. These abilities of AVN-C to shift the expression patterns towards those observed in healthy liver function can be attributed to its potential to modulate the effects of DOX on these proteins.

The interaction of DOX with multiple targets points to a complex network of signaling pathways that are affected by its administration causes signaling pathway crosstalk. The in silico findings provide a mechanistic basis for understanding how changes in protein interactions could lead to the observed in vivo outcomes, particularly regarding the interplay between survival and apoptotic signals. The results suggest that AVN-C might mitigate some of the harmful effects of DOX by partially restoring the signaling balance, enhancing cell survival signals while dampening pro-apoptotic signals.

The hepatotoxicity prediction is particularly interesting, given DOX’s known clinical hepatotoxic effects and supports the idea that its stronger interactions with key proteins may contribute to this side effect. However, AVN-C is not entirely devoid of potential toxicity, albeit with lower probabilities compared to other endpoints. This suggests that while AVN-C may have a more favorable overall safety profile,. Interestingly, both compounds are predicted to be inactive for most Tox21 nuclear receptor signaling pathways and stress response pathways, suggesting that their mechanisms of action and potential toxicity may not be mediated through these specific pathways. The integration of molecular docking results with toxicity predictions paints a nuanced understanding of how these compounds might behave in biological systems. Doxorubicin’s stronger binding affinities across multiple proteins correlate with its predicted higher toxicity and known clinical effects. Its interactions with proteins like Akt, GSK-3β, and β-catenin suggest a multi-faceted impact on cell signaling, explaining both its therapeutic efficacy for hepatotoxicity and adverse effects induced by DOX.

Treatment with DOX led significant body weight loss and a substantial decrease in food intake, decreases in fat, skeletal muscle mass, and fatigue (de Lima Junior et al., 2016). In accordance with our study, DOX administration in rats caused significant body weight loss; this could be due to the toxic effects of DOX on metabolism and vital organs. The effect of DOX on the body weight of rats can be attributed to decreased appetite, reduced feed intake, disruption of basal metabolism, and inhibition of protein synthesis (El-Said K. S. et al., 2023). However, the treatment of DOX-injected rats with AVN-C resulted in a significant improvement in the % BW; this finding agreed with previous reports on the impacts of herbal products on the improvement of body weight loss induced by DOX injection (Koss-Mikołajczyk et al., 2021; Saleh et al., 2022; Alherz et al., 2023). Additionally, in response to liver damage caused by DOX, there were inflammatory responses and compensatory mechanisms that promote liver regeneration leading to an increase in liver weight. This increase in liver weight could also be attributed to cellular hypertrophy, surviving hepatocytes may increase in size due to hepatocytes vacuolation, degeneration of hepatocyte cords, bile duct hyperplasia and focal necrosis, as they attempt to compensate for lost functionality, leading to increased liver mass despite reduced protein content (Prasanna et al., 2020; Xu et al., 2022). Also, this could be due to altered protein turnover and protein metabolism might shift; although total protein content decreased, the liver might still be undergoing processes like protein degradation at a higher rate due to oxidative protein damage. While protein content decreased due to damage, the liver may still activate pathways to maintain its mass and function, such as increased glycogen storage or lipid accumulation. Additionally, the increase in liver weight after DOX treatment could be due to the accumulation of extracellular matrix components, which can alter liver structures (Jurj et al., 2022; Gedikli et al., 2023).

It has been reported that DOX treatment induced bone marrow and spleen immunosuppression in rats (Shaldoum et al., 2021). In this study, the outcomes reported that DOX injection in rats led to significant alterations in the hematological parameters, including RBCs, Hb, WBCs, and platelets. These reductions in the hematological parameters from DOX treatment could be due to damage in the hematopoietic system or the increased permeability of the cell membrane, which in turn caused osmotic swelling and erythrocyte hemolysis and the occurrence of systemic inflammation (Kameo et al., 2021). Additionally, hypoxia may potentially have been caused by the death of the mature cell, an increase in plasma volume, or a decrease in hemoglobin’s affinity for oxygen, which resulted in less oxygen being transported from the lungs to the blood and less oxygen being released from oxyhemoglobin to the tissues (Jagetia et al., 2006). Treating DOX-injected rats with AVN-C showed significant improvement in the previously mentioned hematological parameters as evidenced by restoration of RBCs, WBCs, and platelet counts and improvement of bone marrow thrombopoietin, megakaryocytopoiesis, and scavenging free radical-induced damage in cells (Fathy et al., 2018; El-Said et al., 2022). These results were in line with previous studies reporting the beneficial effects of natural constituents on improving the hematological parameter alterations that were induced by DOX in experimental animals (Afsar et al., 2017; Espírito Santo et al., 2023).

Our outcomes uncovered that DOX induced liver dysfunction as per elevations in liver transaminases (AST, ALT), serum ALP, and GGT enzymes, in addition to a significant decrease in the total protein concentrations. These findings could be attributed to the oxidative stress that was mediated by DOX injection, which attacked hepatocytes and consequently released hepatic enzymes from the damaged cells to the blood serum. They also might be due to alterations in protein synthesis and/or metabolic functions of hepatocytes. Decreasing the hepatic toxicity upon AVN-C treatment indicates that the AVNS has a therapeutic effect against liver dysfunction and cellular injury of the liver that were induced by DOX. Our data was in agreement with Tousson et al. (2022), who reported the potential curative role of AVNs against oxidative stress induced by acute hepatotoxicity in rats.

The present study found that DOX treatment resulted in elevation in MDA and depletions in GSH, SOD, and CAT in liver homogenates, consistent with previous studies demonstrating the effects of DOX on the imbalance of hepatic oxidants/antioxidants (Sirwi et al., 2021; Saleh et al., 2022; El-Said K. S. et al., 2023). Treating DOX-challenged rats with AVN-C significantly improved the reversal of changes in hepatic antioxidant/oxidant hemostasis of the DOX-injected rats, as it can inhibit lipid peroxidation and prevent oxidative stress. This suggested that AVN-C acts on DOX-promoted hepatotoxicity through its antioxidant properties, potentially by enhancing the antioxidant defense system in accordance with previous studies demonstrated the cytoprotective effects of AVN-C against oxidative damage and enhancement of antioxidant status (Wang and Eskiw, 2019). In the current investigation, the beneficial hepatoprotective effects of AVN-C could be due to the anti-inflammatory properties of AVN-C; it was found to reduce the key inflammatory mediators associated with DOX-induced liver injury. The current findings concur with previous reports that highlighted the potent in vivo anti-inflammatory efficacy of AVNs (Zhang et al., 2020; Tousson et al., 2022; Wankhede et al., 2023). A previous study reported that herbal compounds mitigated DOX-induced liver toxicity in rats by countering oxidative stress and inflammation (Ahmed et al., 2022). Our results revealed that AVN-C exert pronounced pharmacological actions as antioxidant, and anti-inflammatory, which indicated that AVS is a prospective therapeutic agent against DOX-induced toxicity.

The phosphorylated Akt triggers the activation of multiple downstream proteins that mediate signals encouraging cell proliferation, differentiation, and death. GSK-3β may be phosphorylated and its activity inhibited by the activated Akt (Saleh et al., 2022). Interestingly, the present study showed that GSK-3β inhibition provides guarding against DOX-prompted hepatic tissue damage accompanied by elevation of the Akt at the gene and protein expression levels. In accordance with a previous study by Abbas and Kabil (2017), who reported that liraglutide ameliorates DOX toxicity through the Akt/GSK-3β signaling pathway in rats. It has been reported that inhibition of the Wnt-4/β-catenin signaling pathway could be potential therapeutic targets in liver disorders. Our investigation showed the hepatoprotective effect of AVN-C was associated with the downregulation of the Wnt-4 and β-catenin expressions. Herein, attenuation of hepatotoxicity by DOX treatment through inhibition of GSK-3β and Wnt-4/β-Catenin might be a promising strategy of AVN-C to counteract DOX-induced hepatotoxicity. These findings were in line with previous studies that established the inhibition of the Wnt-4/β-Catenin signaling pathway by natural compounds in experimental animals (Li et al., 2014; Cao et al., 2022; Bakrania et al., 2023). Furthermore, a previous study reported the activation of WNT/β-catenin signaling pathway is involved in the pathogenesis of CCl₄-induced liver fibrosis and protective role of natural compounds against liver fibrosis by inhibiting the Wnt/β-catenin pathway (El-Ashmawy et al., 2020). Consistently, our histopathological investigations evidenced biochemical analysis indicating severe hepatocellular damages by DOX injection in rats. These results are consistent with recent findings about DOX-induced hepatotoxicity (AlAsmari et al., 2021; Saleh et al., 2022; El-Said K. S. et al., 2023). By significantly reducing central venous congestion, cell infiltration, degeneration, the number of hepatocytes with pyknotic, vacuolization, and sinusoidal narrowing, AVN-C treatment dramatically restores the histological abnormalities and increased hepatic enzyme activity seen in DOX-treated rats, suggesting protection against DOX-induced liver injury. A previous study reported that taurine protects DOX-induced hepatotoxicity via its membrane-stabilizing effect and improvement of histopathological changes in rats (Gedikli et al., 2023).

This study is the first to demonstrate that AVN-C has a significant attenuative effect against DOX-induced hepatotoxicity in male rats. This is achieved by enhancing the liver’s antioxidant capacity, inhibiting hepatic inflammatory cytokines, and modulating the Akt/GSK-3β and Wnt-4/β-Catenin pathways (Figure 10). The combination of AVN-C and DOX shows promise and effective chemotherapy strategy. Further research should explore the potential of AVN-C in ameliorating other toxicities induced by DOX and investigate the underlying mechanisms.