Protective Effects of Schizochytrium Microalgal Fatty Acids on Alcoholic Liver Disease: A Network Pharmacology and In Vivo Study

Abstract

This study aimed to elucidate the hepatoprotective mechanisms of microalgal fatty acids (MFA) from Schizochytrium against alcoholic liver disease (ALD) through network pharmacology and in vivo analysis. Network pharmacology and molecular docking methodologies were employed to predict the potential mechanisms of MFA against ALD. To substantiate these predictions, an acute alcoholic liver injury mouse model was utilized to assess the impact of MFA on serum levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), total protein (TP), and albumin (ALB). Additionally, liver histopathology and the expression levels of phosphatidylinositol 3 kinase (PI3K) and protein kinase B (AKT) protein were evaluated. Seven active ingredients and 53 potential targets (including 7 core targets) for ALD treatment were identified in MFA. Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that these seven core targets are implicated in various biological pathways, notably those associated with cancer, viral infections, and the PI3K/AKT signaling pathway. Furthermore, molecular docking studies demonstrated that docosahexaenoic acid and docosapentaenoic acid in MFA exhibited strong binding affinity for these seven crucial targets. Animal experiments demonstrated that administration of MFA significantly decreased the levels of AST, ALT, and ALP, while increasing the levels of ALB and TP in mice with acute alcoholic liver injury. Moreover, MFA ameliorated liver tissue pathology and markedly down-regulated the expression of PI3K and AKT proteins in the liver. These results suggest that MFA may possess therapeutic potential for ALD by targeting multiple pathways, with its mechanisms likely involving the inhibition of the PI3K/AKT signaling pathway.

INTRODUCTION

Alcoholic liver disease (ALD), resulting from the abuse of alcohol products, has emerged as a significant global public health issue. The progression of ALD unfolds through various stages, from alcohol-induced hepatitis and steatosis to fibrosis and cirrhosis, potentially culminating in liver cancer.¹ Despite advancements in prolonging lives of patients with ALD, effective clinical treatments remain elusive.² Therefore, prioritizing the prevention of ALD progression during its early stages is considered more efficacious than addressing advanced conditions.

The beneficial effects of ω-3 polyunsaturated fatty acids (ω-3 PUFAs), specifically docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and eicosapentaenoic acid (EPA), have been extensively documented concerning various diseases, including cardiovascular diseases, metabolic disorders, inflammation-associated diseases, and liver disease.³ Clinical studies have shown that ω-3 PUFAs supplementation in patients with ALD can mitigate disease severity.⁴ The study conducted by Lagarde et al. demonstrated that ω-3 PUFAs possess the potential to mitigate alcohol-induced liver injury, as evidenced by the reversal of elevated circulating aspartate transaminase (AST) and alanine transaminase (ALT) activities resulting from alcohol exposure.⁵

Traditionally, ω-3 PUFAs were obtained from deep-sea fish oil. However, overfishing of deep-sea species has led to a diminishing supply of fish oil, causing a steady price increase.⁶ Recently, artificial cultivation of marine microalgae has emerged as a promising alternative for obtaining ω-3 PUFA-rich oils.⁷ Compared with traditional fish oil, microalgal PUFA-rich oil offers several advantages, including independence from seasonal fluctuations, absence of fishy odor, reduced risk of marine pollutant contamination, lower processing costs, and decreased accumulation of toxic organic pollutants.⁸ Among the investigated microalgae, Schizochytriumis emerges as a superior candidate for research on ω-3 PUFA production, attributed to its easy cultivation, rapid growth rate, high DHA content, and straightforward fatty acid profile. PUFAs-rich oil derived from Schizochytrium is a complex mixture predominantly consisting of DHA, DPA, and EPA. Each component of this marine microalgae oil exhibits distinct properties, exerting unique or synergistic effects in the treatment of liver diseases.⁹ However, the hepatoprotective mechanism of marine microalgae oil has not been systematically elucidated, and active compounds and potential targets have not been fully identified.

Network pharmacology, grounded in systems biology theory, enables the analysis of biological systems networks and the identification of specific signal nodes for designing multi-target drug molecules.¹⁰ This study utilizes virtual screening and network prediction methodologies, leveraging visualization software and algorithms, and integrates extensive databases encompassing gene, protein, disease, and drug networks. Through this approach, it systematically investigates the effects of pharmacological agents on diseases at the network level.¹¹ Given this context, the objectives of this study are as follows: (1) to utilize the network pharmacology approach in exploring hub genes and key pathways of microalgal fatty acids (MFA) from Schizochytrium in the treatment of ALD and (2) to validate the effects of MFA on ALD and elucidate the underlying mechanisms through in vivo experiments. The primary aim of this research is to furnish essential scientific insights that will inform future pharmacological investigations and clinical applications of MFA in the treatment of ALD.

MATERIALS AND METHODS

Extraction and Analysis of Chemical Composition from Microalgae Oil

The dry biomass of marine microalgae Schizochytrium sp. was obtained from King Dnarmsa Spirulina Co., LTD (Fujian, China). Extraction and chemical composition analysis of MFA from Schizochytrium sp. were conducted following methodology reported by He et al.¹² Lipinski’s rules (https://admetmesh.scbdd.com) were utilized for screening, adhering to criteria including molecular weight (MW <500), the logarithm of the octanol/water partition coefficient (log P < 5), the number of hydrogen bond donors (H _don < 5), and the number of hydrogen bond acceptors (H _acc < 10). A single deviation from these properties is deemed permissible, whereas two or more deviations indicate potential issues with absorption or permeability.¹³

Network Pharmacology

Potential targets of MFA were identified using the Swiss Target Prediction database (http://swisstargetprediction.ch/), as previously described.¹⁴ Briefly, the isomeric Simplified Molecular Input Line Entry System (SMILES) representations of eight constituent molecules in MFA were obtained from the PubChem database. For each query molecule, the species of origin was specified as human, and the SMILES string was individually entered into the designated text box on the Swiss Target Prediction platform. Upon entering a query molecule, the “Predict targets” button is activated, indicated by a change to red. Upon activation by clicking, the computational process commences, involving the chemical processing of the input structure. The potential targets for eight molecules within the MFA framework were individually predicted and subsequently downloaded. The comprehensive potential targets for MFA were then determined by integrating these eight predictive datasets. To identify protein targets associated with ALD, “alcoholic liver disease” was used as a keyword to search for ALD-related targets from Genecard (https://www.genecards.org/), Comparative Toxicogenomics Database (CTD) (http://ctdbase.org/), Disease Gene Network (DisGeNET) (https://www.disgenet.org/), Online Mendelian Inheritance in Man (OMIM) (https://omim.org/), and National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/).¹⁵

The redundant targets obtained from MFA and ALD were deduplicated and subsequently integrated. A Venn diagram analysis was employed to discern potential targets common to both MFA and ALD. The intersected targets were subsequently imported into the Search Tool for the Retrieval of Interaction Genes/Proteins (STRING) 11.5 platform (https://www.string-db.org/), with the “Organism” parameter specified as “Homo sapiens.” Upon obtaining the results, the confidence level was set to “highest confidence 0.900,” and the free nodes within the hidden network diagram were selected, while all other settings were maintained at their default values.¹⁶ Network topology data were analyzed using the Network Analyzer plugin in Cytoscape v3.8.2 (http://cytoscape.org). Key targets were selected based on degree values, with the upper threshold set at the maximum degree value in the topology data, and the lower threshold set at twice the median degree of freedom.¹⁷ Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted using the KEGG Orthology Based Annotation System (KOBAS) online tool (http://kobas.cbi.pku.edu.cn).

Molecular Docking

The structure of DHA, DPA, palmitic acid, eicosatetraenoic acid, eicosatrienoic acid, eicosenoic acid, pentadecanoic acid and tetradecanoic acid were obtained from the PubChem (https://pubchem.ncbi.nlm.nih.gov). For molecular docking, the protein crystal structures of key targets, including interleukin-6 (IL-6), peroxisome proliferator-activated receptor alpha (PPARα), peroxisome proliferator-activated receptor gamma (PPARγ), mitogen-activated protein kinase 3 (MAPK3), prostaglandin-endoperoxide synthase 2 (PTGS2), tumor protein 53 (TP53), and estrogen receptor 1 (ESR1), were obtained from the Protein Data Bank database (https://www.rcsb.org). Molecular docking analyses were conducted utilizing the ligand fit feature of the GLIDE software from Schrodinger (http://www.schrodinger.com/).

Animal Experiment

Male Institute of Cancer Research (ICR) mice, weighing 19–21 g, were procured from Beijing HFK Bioscience Co. Ltd. (Beijing, China) [license number: SCXK (Jing) 2019–0008, SPF], for utilization in this study. The mice were housed individually in cages maintained at a temperature of 18–22°C and relative humidity of 40–60%, under a 12-h dark/light cycle, with ad libitum access to water. All animal experiments were performed in compliance with the ethical guidelines set by the Experimental Animal Ethical Committee of Quanzhou Medical College, China (QZMC No. 2021010).

Following a 7-day acclimatization period, 40 mice were randomly allocated into four groups of 10: the normal control (NC), model control (MC), positive control (PC), and MFA groups. The NC and MC groups were administered ddH₂O (0.2 mL), whereas the PC group received silymarin at a dosage of 100 mg/kg/day, following the protocol outlined by Yi et al.¹⁸ Based on the findings from our previous research, the optimal dosage of MFA for treating alcohol-induced liver injury in mice was determined to be 200 mg/kg.¹⁹ Consequently, the MFA group was administered 200 mg/kg of MFA intragastrically daily for 15 days. Three hours after the final administration, MC, PC, and MFA groups were administered a 50% ethanol solution (12 mL/kg) intragastrically, whereas the NC group received an equivalent volume of ddH₂O.²⁰ After 18 h of fasting, during which water was provided ad libitum, the mice were anesthetized with ethyl ether for blood sampling via eyeball extirpation. Following dissection, liver tissues were partially fixed in formalin for histological analysis, with the remaining portion stored at −80°C for future experiments.

Serum Biochemical Analysis

The collected blood was centrifuged at 10,000×g for 5 min, and the supernatant was collected. Levels of AST, alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin (ALB), and total protein (TP) were measured using an automatic hematology analyzer (Sysmex xe-2100, TOA Medical Electronics, Kobe, Japan).

Histological Analyses

Liver tissue sections, 4 μm thick, were prepared and mounted on slides. These sections were then stained with hematoxylin-eosin (H&E) according to a previously described protocol.²¹ The inflammation grade (G0–G4) of hepatic necro inflammatory activity was assessed using a modified Scheuer scoring system.²² Specifically, G0 (score of 0) indicates no inflammation; G1 (score of 1) represents portal inflammation and lobular inflammation without necrosis; G2 (score of 2) corresponds to portal mild piecemeal necrosis and lobular focal necrosis or acidophil bodies; G3 (score of 3) denotes portal moderate piecemeal necrosis and severe focal cell damage in lobular; and G4 (score of 4) is characterized by portal severe piecemeal necrosis and lobular damage including bridging necrosis.

Western Blotting

Western blotting analysis was performed as previously described.²³ Briefly, TPs were extracted from mouse liver tissue samples, and protein concentration was determined using a BCA protein assay kit (Beyotime, Shanghai, China). Proteins were initially separated by 10% sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, USA). The membranes were blocked with 5% Bovine Serum Albumin (BSA) (Millipore) for 1 h at room temperature, then washed three times with tris-buffered saline containing 0.1% tween-20 (TBST) (Beyotime). Membranes were incubated overnight at 4°C with primary antibodies (ABclonal, Wuhan, China). Following this, they were washed three times with TBST at room temperature and subsequently incubated with an horseradish peroxidase (HRP)-conjugated secondary antibody (Beyotime) for 2 h at room temperature. Protein bands were visualized using the Immobilon Western Chemiluminescent horseradish peroxidase substrate (Millipore) and quantified using Image J software (version 1.46).

Statistical Analysis

All data are expressed as the mean ± standard deviation. Statistical analyses were conducted using one-way analysis of variance, followed by Dunnett’s multiple comparison test. Differences were considered statistically significant at p < 0.05.

RESULTS

Active Compound-Target Analysis

Gas chromatography results revealed the fatty acid composition of MFA as follows: DHA of 69.67%, DPA of 17.06%, palmitic acid of 10.00%, eicosenoic acid of 1.10%, eicosatrienoic acid of 0.53%, eicosatetraenoic acid of 0.56%, pentadecanoic acid of 0.57%, and tetradecanoic acid of 0.50%. The computed molecular descriptors demonstrated that all eight fatty acid molecules adhere to Lipinski’s rules, indicating drug-like structural properties (Table 1). Utilizing the Swiss Target Prediction database, 255 potential targets for MFA were identified after deduplication, based on the structures of the eight fatty acids (Fig. 1A). Simultaneously, 613 disease-related targets associated with ALD were identified from the GeneCards, CTD, DisGeNET, OMIM, and NCBI databases following credibility screening (Fig. 1B). The intersection of these datasets revealed 53 genes as potential targets for MFA action against ALD (Fig. 1C). A compound-target network was developed to visualize the interactions between MFA and ALD targets (Fig. 2). This network consisted of 61 nodes (8 compounds, 53 targets) and interconnected by 185 edges. On average, each active MFA compound was associated with 23.13 targets (185/8), whereas each target was linked to 3.49 compounds (185/53). The three compounds exhibiting the highest degree of activity within this network were DHA, DPA, and eicosatrienoic acid (Fig. 2, shown in dark color).

Table 1.

The Composition of MFA

Name	Content (%)	Molecular weight	Log P	H _don	H _acc	Lipinski rule
Tetradecanoic acid (C_14:0)	0.50	228.21	5.820	1	2	Accepted
Pentadecanoic acid (C_15:1 _n ₋₅)	0.57	242.22	6.283	1	2	Accepted
Palmitic acid (C_16:0)	10.00	256.42	6.732	1	2	Accepted
Eicosenoic acid (C_20:1)	1.10	310.29	8.071	1	2	Accepted
Eicosatrienoic acid (C_20:3 _n ₋₆)	0.53	306.26	4.104	1	2	Accepted
Eicosatetraenoic acid (C_20:4 _n ₋₆)	0.56	304.24	5.534	1	2	Accepted
Docosapentaenoic acid (C_22:5 _n ₋₆)	17.06	330.26	1.983	1	2	Accepted
Docosahexaenoic acid (C_22:6 _n ₋₃)	69.67	328.24	0.669	1	2	Accepted

H _acc, number of hydrogen bond acceptors; H _don, number of hydrogen bond donors; Log P, logarithm of the octanol/water partition coefficient; MFA, microalgal fatty acids.

Protein–Protein Interaction Network Analysis and Core Target Identification

To analyze protein–protein interactions (PPIs) among the 53 intersection targets, we utilized the STRING database, specifying a minimum interaction score threshold of 0.700 and opting for a high confidence level. The resulting PPI network consisted of 53 protein nodes and 282 interaction edges (Fig. 3A). Target genes were ranked based on their degree values, with a median degree value of 10 and a maximum degree value of 36. To identify optimal core targets, we set the inclusion criteria for hub target screening, selecting nodes with degree values between 20 and 36. This analysis yielded seven optimal core targets including IL-6, PPARα, PPARγ, MAPK3, PTGS2, TP53, and ESR1 (Fig. 3B).

Functional Annotation and Classification

The bioinformatics analysis identified seven core targets (IL-6, PPARα, PPARγ, MAPK3, PTGS2, TP53, ESR1) that potentially operate through multi-target mechanisms in the treatment of ALD using MFA (Fig. 3A). These core targets were subjected to functional classification using the Database for Annotation, Visualization and Integrated Discovery (DAVID) database, focusing on biological processes (BP), molecular functions (MF), cellular components (CC), and KEGG pathways. The analysis indicated that these targets are implicated in BPs such as the positive regulation of transcription by RNA polymerase II, the positive regulation of DNA-templated transcription, and the negative regulation of triglyceride sequestration. In terms of MF, the targets were predominantly linked to transcription factor binding, enzyme binding, and murine double minute (MDM2)/MDM4 family protein binding. Predominant CCs identified were protein-containing complexes, nuclear receptors activity, and caveola (Fig. 4A). Additionally, the KEGG pathway enrichment analysis yielded 150 enriched pathways, encompassing those cancer-related pathways, Kaposi sarcoma-associated herpesvirus infection, human cytomegalovirus infection, phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) signaling pathway, and thyroid cancer (Fig. 4B).

Molecular Docking

The effects of eight active compounds from MFA on the seven key targets were verified through molecular docking, with the docking scores depicted in Figure 5. Theoretically, a lower ligand-receptor binding energy corresponds to a more stable conformation. A docking binding energy below −1.2 kcal/mol (−5.0 kJ/mol) is generally considered indicative of strong binding.²⁴ As illustrated in Figure 5, DHA and DPA exhibited binding energies less than −3.0 kcal/mol with all seven key targets. The low binding energy indicates a strong affinity between these two compounds and the targets. The strong binding affinity of DHA and DPA to the key targets suggests that these two compounds may be the primary active ingredients in MFA for combating ALD.

Effect of MFA on Serum Levels of Biochemistry Parameters

To verify the accuracy of molecular docking predictions and network pharmacology results, we designed a study to evaluate the hepatoprotective effects of MFA on ALD using a mouse model of acute alcohol-induced liver injury. Consistent with our expectations, alcohol administration in mice led to a significant elevation in the levels of ALT, AST, and ALP, alongside a marked reduction in TP and ALB levels. Nevertheless, treatment with silymarin in the PC group resulted in improvements in all the aforementioned parameters compared with the NC group. Notably, treatment with MFA demonstrated comparable therapeutic effects on ALT, AST, ALP, TP, and ALB levels as observed in the PC group. In comparison to the MC group, the MFA-treated groups showed significant reductions in ALT, AST, and ALP levels, accompanied by substantial increases in TP and ALB levels (Fig. 6A).

Effects of MFA on Liver Pathology

As depicted in Fig. 6C, the liver cells of NC mice displayed a regular arrangement, with large, round hepatocyte nuclei, indicating normal liver function. In contrast, the liver cells of the MC group exhibited larger spaces, irregular arrangements, and extensive cell necrosis, reflecting significant liver damage. The administration of silymarin, a commercially available liver medication, in the PC group demonstrated a hepatoprotective effect. Interestingly, administration with MFA also significantly reduced liver damage, which was similar to the PC group. The liver histopathological scores indicated that mice in the model group exhibited significantly elevated scores compared with those in the normal group. Conversely, the scores for mice in both the PC group and the MFA-treated groups were significantly reduced relative to the model group. These findings suggest that MFA confers a protective effect on the liver (Fig. 6B).

Effects of MFA on PI3K and AKT Expression Levels

Analysis using the DAVID database (Fig. 6) revealed that MFA exerts therapeutic effects on ALD through several pathways, with the PI3K/AKT signaling pathway being the most significant and playing a crucial role in ALD. Herein, this study comprehensively analyzed the effects of MFA on the expression levels of PI3K and AKT in mice. As shown in Figure 7, the MC group demonstrated significantly elevated protein levels relative to the NC group, indicating that alcohol consumption enhances the expression of PI3K and AKT proteins in mice. In contrast, following intragastric administration of silymarin in the PC group, PI3K and AKT protein levels markedly decreased, returning to normal levels. Comparable outcomes were observed in the MFA group, where PI3K and AKT protein levels also significantly diminished compared with the MC group.

DISCUSSION

The pharmacophore, which represents the spatial arrangement of essential features for ligand-receptor interaction, is crucial in understanding drug mechanisms. Identifying active ingredients and gene targets is the primary step in drug research to understand their intrinsic mechanism for disease treatment.²⁵ In this study, we employed the Swiss Target Prediction database to ascertain the active constituents within MFA derived from Schizochytrium sp. and their potential targets. A total of eight compounds were identified in the MFA, and these compounds were associated with 255 potential targets. By intersecting these MFA targets with 613 targets associated with ALD, we identified 53 candidate targets. Among these, DHA, DPA, and eicosatrienoic acid exhibited the highest degrees of activity in MFA against ALD.

The analysis of the PPI network showed that IL-6, PPARα, PPARγ, MAPK3, PTGS2, TP53, and ESR1 were the predicted core targets of MFA in the treatment of ALD. Excessive alcohol consumption has been shown to down-regulate PPAR-α, thereby inhibiting the β-oxidation process of fatty acids. Treatment with PPAR-α agonists not only aids in preventing the development of steatosis and mitigates hepatic inflammation but also serves as a preventive strategy against alcohol-induced hepatic insulin resistance.²⁶ PTGS2, an isozyme of PTGS, is a critical enzyme in prostaglandin biosynthesis.²⁷ Ethanol has been demonstrated to intensify PTGS2-mediated lipid peroxidation and elevate the expression of inflammatory cytokines, such as IL-6 and tumor necrosis factor α (TNFα), in rat models. Conversely, in rats supplemented with ω-3 PUFAs, the progression of ALD can be mitigated through the downregulation of PTGS2-mediated free radical production.²⁸ MAPKs are responsible for intracellular signaling pathways that regulate various cellular activities.²⁹ The activation of the MAPK pathway is frequently implicated as a mediator of hepatocyte injury caused by various stimuli, such as alcohol, Lipopolysaccharide (LPS)/TNFα, bile acids, and acetaminophen, and increased MAPK pathway gene expression has been found in both in vivo and in vitro models of ALD.³⁰ ESR1 serves as the primary receptor in the liver, with estrogen signaling via ESR1 being pivotal in the regulation of hepatic lipid metabolism.³¹ TP53 activation is crucial in the etiopathogenesis of ALD, contributing to disease progression through various mechanisms. TP53 can modulate ethanol-induced hepatocyte apoptosis, and its genetic ablation has been shown to abrogate ethanol-derived liver injury.³²

Given that ALT, AST, and ALP are three main sensitive indicators of hepatocyte damage,³³ and that TP and ALB levels typically decrease in hepatotoxic conditions due to impaired protein biosynthesis in the liver.³⁴ Hepatocellular injury in mice induced by alcohol was evidenced by increased serum levels of ALT, AST, ALP, TP, and ALB, along with liver histological evaluations, which are clinical indicators of ALD.³⁵ Consistent with the anticipated outcomes from network pharmacology, the administration of MFA demonstrated a hepatoprotective effect in ALD mice. Specifically, MFA significantly reduced alcohol-induced serum markers and mitigated hepatocellular damage. This suggests that MFA has a protective effect in ALD mice, similar to the findings reported by Byun et al.³⁶ In recent years, the utilization of marine microalgae extracts, including fatty acids and polysaccharides, has garnered significant attention in the context of liver disease prevention and treatment. MFA have demonstrated the capacity to reduce triglyceride and cholesterol levels, thereby preventing the onset of fatty liver disease in murine models. For individuals with pre-existing chronic liver damage, these fatty acids may aid in ameliorating the condition by reducing inflammation and fibrosis within hepatic tissue.³⁷ Furthermore, MFA exhibit antioxidant and anti-inflammatory properties, which can decrease oxidative stress and the inflammatory response in the liver, thereby safeguarding it from further damage.³⁸ A randomized, 12-month study has demonstrated the advantageous effects of a diet supplemented with MFA in patients with non-alcoholic fatty liver disease, in comparison to those receiving a placebo-supplemented diet. Patients treated with MFA exhibited reductions in fasting insulin, triglyceride, ALT, AST levels, and homeostasis model assessment of insulin resistance values. Furthermore, significant improvements were observed in the hepatic ultrasonographic pattern following this intervention.³⁹ In alignment with these findings, the present study indicates that MFA has the potential to alleviate alcohol-induced liver injury in mice, suggesting its promise as a therapeutic agent for the prevention and treatment of liver disease.

Based on molecular docking, the compounds of DHA and DPA exhibited strong affinity with the seven key targets (Fig. 5), suggesting that DHA and DPA may be the primary active ingredients of MFA in treating ALD. These findings align with previous research demonstrating the beneficial effects of supplementation with PUFAs enriched in DHA and DPA.⁴⁰ Furthermore, the functional classification of these seven key targets using the DAVID database revealed that MFA exerts therapeutic effects on ALD by regulating multiple pathways, including those related to cancer, viral infections, PI3K/AKT pathway, IL-17 signaling pathway, and TNF signaling pathway. There is a strong association between the IL-17 signaling pathway and ALD. Long-term alcohol consumption increases the expression of pro-inflammatory IL-17A and its receptor IL-17RA in the liver, thereby activating downstream signaling processes such as nuclear factor kappa-B (NF-κB) and MAPK, which play a key role in the onset and progression of ALD.⁴¹ IL-17 signaling pathway not only promotes the inflammatory response and fibrosis process of the liver but also affects the occurrence and development of hepatocellular carcinoma. Studies have shown that loss of the IL-17RA gene can inhibit the development of hepatocellular carcinoma in alcohol-fed mice, suggesting that the IL-17 signaling pathway plays an important role in the pathogenesis of hepatocellular carcinoma.⁴² TNF is an important regulator of inflammation and regulates cytokine production in immune cells. TNF-induced cell death is an important component of inflammatory injury in the liver and other tissues, particularly when inflammation is prolonged.⁴³ Ethanol consumption can activate immune cells, prompt the release of TNF-α, and initiate a cascade of intricate signal transduction processes. These processes not only facilitate the infiltration of inflammatory cells and the release of pro-inflammatory mediators but may also directly induce hepatocyte damage and death.⁴⁴ Furthermore, the pleiotropic biological effects of TNF may contribute to the onset and progression of liver fibrosis, thereby expediting the advancement of ALD to more severe stages, such as cirrhosis.⁴⁵ Chronic alcohol consumption induces persistent damage to hepatocytes, subsequently initiating the cellular aging process.⁴⁶ Cellular senescence exacerbates hepatocyte injury and impairs the liver’s regenerative and reparative capacities, leading to a progressive deterioration in hepatic function. This process further promotes inflammatory responses and fibrotic progression within the liver.⁴⁷ The PI3K/AKT pathway is the most significant pathway of MFA on alcohol-induced liver injury in mice. A previous study showed that the administration of Qinggan Huoxue Recipe could mitigate alcohol-induced liver injury by suppressing the PI3K/AKT signaling pathway.⁴⁸ Notably, activation of the AKT pathway in response to alcohol exposure is a key determinant of the molecular mechanisms underlying alcohol-drinking behaviors. Therefore, inhibiting the PI3K/AKT signaling pathway is a promising therapeutic strategy for addressing alcohol-related diseases.⁴⁹ The findings of this study align with previous reports, as evidenced by the significantly elevated protein levels of PI3K and AKT in the MC group compared with the NC group. However, administration of MFA restored these levels to normal, demonstrating the potential of MFA supplementation in effectively mitigating alcohol-induced liver injury through modulation of the PI3K/AKT pathway.

CONCLUSION

The findings demonstrated that MFA improves ALD through a multi-pathway and multi-target approach. DHA and DPA as the primary bioactive compounds in MFA against ALD. MFA effectively protected against alcohol-induced liver injury in mice by modulating the PI3K/AKT signal pathway. Nevertheless, the current study is subject to certain limitations. Firstly, network pharmacology relies heavily on comprehensive biomolecular data; however, the existing volume of such data is inadequate, which may undermine the accuracy and thoroughness of investigations into drug mechanisms. Secondly, although the mouse model can replicate some pathological processes pertinent to human ALD, physiological and metabolic discrepancies between mice and humans limit the model’s capacity to accurately represent only the initial stages of the disease. As a result, the study does not adequately capture the long-term progression of ALD, thereby limiting the generalizability of its findings to human conditions. To obtain more comprehensive and precise results, subsequent studies should be combined with clinical research, epidemiological investigations, and other methodological approaches. Additionally, given that algal oil is a complex mixture, further research is needed to elucidate the mechanisms by which its specific chemical constituents, such as DHA and DPA, contribute to the prevention of ALD.

Footnotes

AUTHORS’ CONTRIBUTIONS

C.L.: Conceptualization, formal analysis, methodology, investigation, writing, funding acquisition. L.T.: Investigation, methodology, formal analysis. Y.W.: Conceptualization, validation, methodology, formal analysis, writing, supervision, editing. Z.Z.: Methodology, formal analysis.

AVAILABILITY OF DATA

The data used to support the findings of this study are available from the corresponding author upon request.

DISCLOSURE STATEMENT

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

FUNDING INFORMATION

This work was supported by the Key Project of Quanzhou Medical College (XJK2207A), Science and Technology Development Projects of Quanzhou (2024NY096).

Abbreviations Used

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