Explore of potential targets and mechanisms of hesperetin in treating metabolic dysfunction-associated steatosis liver disease via network pharmacology and in vitro experiments

doi:10.21203/rs.3.rs-5036368/v1

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Explore of potential targets and mechanisms of hesperetin in treating metabolic dysfunction-associated steatosis liver disease via network pharmacology and in vitro experiments

https://doi.org/10.21203/rs.3.rs-5036368/v1

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Metabolic dysfunction-associated steatosic liver disease (MASLD) is a serious public health issue globally; however, there is no specific drug treatment. Hesperetin, a flavonoid extracted from citrus, possesses multiple pharmacological properties. However, limited reports have elucidated the pharmacological targets of and molecular mechanisms underlying hesperetin on lipid metabolism disorders in MASLD. First, in vitro experiments confirmed the ameliorative effect of hesperetin on lipid accumulations. Second, putative target genes of the compounds were screened using public databases. MASLD-related targets were obtained through data mining of the GEO database. Third, a PPI network was constructed to screen for the core targets through the STRING database. Additionally, GO and KEGG enrichment analyses were performed on the key targets to identify the enriched genes with specific biological themes. We analyzed the binding mode of hesperetin to the key targets using molecular docking. Finally, the potential mechanism by which hesperetin affects MASLD was validated experimentally on an in vitro model. The current evidence suggested that hesperetin ameliorated lipid accumulation by inhibiting the IL-6-mediated STAT3-SOCS3 signaling pathway. Our findings provided novel insights into the underlying mechanisms and the clinical potential of hesperetin in MASLD management or prevention.

Hesperetin

MASLD

IL6-STAT3-SOCS3 signaling pathway

Lipid metabolism

Network pharmacology

Metabolic dysfunction-associated steatosic liver disease (MASLD) was previously termed non-alcoholic fatty liver disease (NAFLD). MASLD is the initial stage for developing a malignant cascade characterized by the pathologic accumulation of lipids in hepatocytes (Eslam et al., 2020). The worldwide prevalence of MASLD is > 25%; approximately 20% of the patients progress to non-alcoholic steatohepatitis (NASH) and cirrhosis, which eventually leads to hepatocellular carcinoma (HCC) (Friedman et al., 2018, Younossi et al., 2018). To date, limited reports have elucidated the mechanisms underlying MASLD pathology. Additionally, no satisfactory treatments are available, relying primarily on lifestyle changes to mitigate the pathologic process of MASLD. Fatty acid transport, synthesis, and β-oxidation processes in hepatocytes are influenced by the pathological conditions of MASLD, resulting in the incomplete breakdown and metabolism of excess fats, causing large amounts of fats. These phenomena trigger substantial fat accumulation fats in the hepatocytes, which ultimately leads to liver injury (Mato et al., 2019, Barbier-Torres et al., 2020, Yan et al., 2023). Consequently, reducing hepatic lipid deposition is an effective strategy for MASLD treatment.

Flavonoid compounds possess multiple medicinal properties, particularly decelerating cellular oxidative stress and inflammatory responses (Yang et al., 2019, Shao et al., 2023, Yang et al., 2023b, Yang et al., 2023c). Hesperetin (3′, 5′, 7′-trihydroxy-4-methoxyflavone) is a natural flavonoid derivative extracted from citrus. It is found in the form of hesperetin and is hydrolyzed by human intestinal flora to produce hesperetin. Studies have reported on multiple therapeutic effects, including antidiabetic, antiviral, antitumor and neuroprotective effects, of hesperetin (Hussain et al., 2022, Khezri et al., 2022, Yang et al., 2022, Song et al., 2023a). However, the effects of hesperetin on MASLD-associated hepatic lipid accumulation and the underlying regulatory mechanisms have not been elucidated completely. Network pharmacology is a systems biology approach; it integrates a comprehensive strategy of systems biology, bioinformatics and topological analysis to systematize the intricate mechanisms underlying small molecules and target proteins of human diseases systematically from a holistic perspective (Xu et al., 2022, Cao et al., 2024, Li et al., 2024). Hesperetin exerts substantial biological effects, such as anti-inflammatory and antioxidant effects; nonetheless, a few reports have demonstrated its effects and underlying molecular mechanisms in MASLD. This drawback warrants a network pharmacological assessment of the positive effects and underlying mechanisms of hesperetin on MASLD.

In the present study, we investigated the efficacy of hesperetin in ameliorating abnormal lipid accumulation in MASLD. Subsequently, we conducted a systematic analysis using several publicly accessible databases, enrichment tools, and bioinformatics resources, combined with comprehensive strategies such as molecular docking and in vitro experiments (Fig. 1). Overall, our study provided new scientific support for the therapeutic potential and new drug development of hesperetin in MASLD prevention and treatment.

2.1 Materials

Hesperetin (H107699) was purchased from Aladdin (Shanghai, China). NP-40 (P0013F) and MTT (ST1537) were purchased from Beyotime (Nanjing, China). The primary antibodies against interleukin- 6 (IL-6) (DF6087), p-JAK2 (AF3024), JAK2 (AF6022), p-STAT3 (AF3293) and STAT3 (AF6294) were purchased from Affinity Biosciences (OH, USA). The primary antibody against β-actin (PAC006) was purchased from Proteinbio (Nanjing, China). Phosphatase inhibitor cocktail (HY-K0021) was purchased from MCE (New Jersey, USA). Free fatty acid (FFA) was purchased from Kunchuang (Xi'an, China). Recombinant Human IL-6 (P5138) was purchased from Beyotime (Nanjing, China)

2.2 Network pharmacology Analysis

2.2.1 Data Preparation

First putative hesperetin targets were screened from seven databases, namely the Encyclopedia of Traditional Chinese Medicine (ETCM, http://www.tcmip.cn/ETCM/index.php/Home/), Bioinformatics Annotation Database for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM, http://bionet.ncpsb.org.cn/), Swiss Target Prediction Database (http://www.swisstargetprediction.ch), Traditional Chinese Medicine Systems Pharmacology (TCMSP, https://www.tcmsp-e.com/), Similarity Ensemble Approach Database (SEA, http://sea.bkslab.org), SymMap (http://www.symmap.org/), and Comparative toxicogenomics database (CTD, http://www.ctdbase.org) seven databases. After that, using the Uniprot database (https://www.uniprot.org/) to obtain the normalization target gene for hesperetin by selecting "Homo sapiens".

Then, the GSE89632 and GSE126848 datasets were retrieved from the Gene Expression Omnibus database (GEO, https://www.ncbi.nlm.nih.gov/). To identify MASLD-related genes, the differently expressed genes among patients with MASLD and healthy individuals were identified using the interactive web tool GEO2R. This step helped us to screen for the genes with substantial differential expression, based on adj.P.Val < 0.05 & |log FC| > 1. Additionally, Venn diagrams are drawn to represent the plotted disease-drug sharing targets.

2.2.2 Protein-Protein Interaction Network

Gene symbols of hesperetin treatment targets for MSALD were imported into the STRING online database (version 11.5, https://www.string-db.org/). “Homo sapiens” was selected as the organism species, to obtain protein interactions among the targets. Subsequently, the Protein-Protein Interaction (PPI) networks in tab-separated value (TSV) format files were imported into Cytoscape 3.7.1. The network topology was analyzed using the network analyzer plug-in; topological features were calculated for each node.

2.2.3 Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology Enrichment Analysis

The intersecting targets underwent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) enrichment analyses using the Metascape platform (https://metascape.org/gp/index.html). The threshold value was set at p < .05.

2.2.4 Molecular docking

The 2D structure of hesperetin was acquired in Spatial Data File (SDF) format from the NCBI PubChem (https://pubchem.ncbi.nlm.nih.gov/). Protein crystal structures of the potential targets (CEBPA, IL1B, IL6, TGFB1, SOCS3, and CYC3) were obtained from the Protein Data Bank (PDB) database (https://www.rcsb.org/). PDB IDs of CEBPA, IL1B, IL6, TGFB1, SOCS3, and CYC3 were 6DC0, 1I1B, 4O9H, 4KV5, 2JZ3 and 3NWV, respectively. Molecular docking of the ligand molecules and proteins was performed using the Discovery Studio 2019 client, with the results presented in visual formats (Hu et al., 2023).

2.3 Cell culture and treatment

HepG2 was kindly provided by Dr. Wang Zeng from Nanjing Medical University. The cells were cultured in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified incubator at 37°C with 5% CO₂. The cells were exposed to 1 mM free fatty acid (FFA), comprising of oleic acid (OA)/palmitic acid (PA) in a 2:1 ratio, consisting of 1% bovine serum albumin for 24 h to simulate the pathological state of MASLD. Subsequently, low (10 µM), medium (50 µM), and high (100 µM) doses of Hesperetin hesperetin (10, 50, and 100 µM) were added for 24 h.

2.4 Cell viability assay

HepG2 cells were incubated with hesperetin (0.1, 0.5, 1, 5, 10, 20, 25, 50, 100, 200 and 400 µM) for 24 h. Subsequently, cell viability was monitored using the 3-(4,5-dimethiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The percentage of cell viability was computed relative to the absorbance intensity of the control cells.

2.5 Western blot

HepG2 cells were lysed with NP-40 lysis buffer (Beyotime, China) and total protein was isolated. Protein concentration was determined using bicinchoninic acid protein assay reagent (Beyotime, China). Total proteins (40 µg) were resolved on 8% or 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis gel and transferred to nitrocellulose membranes. The membranes were blocked in Tris-buffered saline with 0.1% Tween® 20 detergent (TBST) consisting of 5% skim milk powder for 1 h at room temperature. Furthermore, they were incubated overnight with special antibodies, including IL-6, p-STAT3, STAT3, SOCS3 and β-actin (1:1000) at 4℃. After washing with TBST three times, the membranes were incubated with secondary antibodies (1:15,000) for 1 h at room temperature.

2.6 Cellular triglyceride and total cholesterol measurement

Triglyceride (TG) and total cholesterol (T-CHO) values in the cell samples were detected by commercial detection kits (Jiancheng Bioengineering Institute, Nanjing, China). After collecting the cells, appropriate amount of phosphate-buffered saline (PBS) was added. The cells were broken by ultrasonication under an ice-water bath. Subsequently, T-CHO and TG levels were detected directly. The absorbance was measured at 500 nm.

2.7 Detection of IL-6

The level of inflammatory cytokines IL-6 in cell culture medium was determined using commercial human immunoassay enzyme-linked immunosorbent assay kits (Neobioscience, Shenzhen, China), according to the manufacturer' s instructions.

2.8 Oil-red staining

Oil Red O stain was applied to assess lipid droplet formation in the HepG2 cells (Yang et al., 2023a). Briefly, HepG2 cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min. The cells were incubated with Oil Red O working solution for 30 min, briefly rinsed in 60% isopropanol, and counterstained with hematoxylin for 1 min. The images were acquired using a microscope (Olympus, Tokyo, Japan).

2.9 Glucose consumption assay

HepG2 cells were inoculated in 96-well plates. Glucose uptake was assayed as described previously (Hu et al., 2023).

2.10 Statistical analysis

Data from the three independent experiments are presented as means ± SD. Statistical analysis was conducted using GraphPad Prism software 6.0. P-values ≤ 0.05 indicated statistical significance.

3.1 Hesperetin alleviated lipid accumulation in FFA-induced HepG2 cells

We used the FFA-induced HepG2 cell model to evaluate the anti-MASLD effects of hesperetin. First, we evaluated the effects of different hesperetin concentrations on the activity of HepG2 cells by an MTT assay. At concentrations < 100 µM, hesperetin did not exert significant effects on cell viability (Fig. 2B). The cell viability was significantly inhibited as the concentration reached 200 or 400 µM. Additionally, we examined the effects of hesperetin on FFA-induced HepG2 cell viability. The cytotoxicity results suggested that hesperetin exerted no significant effect on FFA-induced HepG2 cell viability at concentrations < 100 µM. Thus, we used hesperetin concentrations of 10, 50, and 100 µM for subsequent experiments. Hesperetin treatment significantly reduced the TG and TCHO levels in FFA-induced HepG2 cells in a concentration-dependent manner (Fig. 2D, E). Oil Red O staining results suggested that hesperetin significantly reduced intracellular lipid accumulation in a dose-dependent manner, consistent with the results of intracellular TG and TC-HO levels (Fig. 2F). Collectively, the above results supported the potential of hesperetin to ameliorate hepatocyte lipid accumulation in MASLD. However, researchers should investigate the precise molecular mechanisms underlying this process.

3.2 Exploring hesperetin targets for the MASLD treatment by Network Pharmacology Analysis

Figure 1 depicted the workflow for integrating network pharmacology, molecular docking and experimental validation. Through the GEO database, we obtained 1,850 MASLD-associated disease genes from the GSE89632 and GSE126848 datasets, following |log FC| > 1 and adj.P.Val < 0.05 screening criteria (Fig. 3A, B). Subsequently, we screened 88, 54, 30, 8, 34, 95 and 60 targets of hesperetin targets from the Swiss target prediction, ETCM, BATMAN-TCM, TCMSP, SEA, CTD, and SymMap databases, respectively. Targets from these seven databases were merged and de-duplicated, resulting in 284 drug targets (Fig. 3C). Finally, we obtained 40 overlapping targets corresponding to “Hesperetin-MASLD” by further integration (Fig. 3D).

3.3 PPI analysis for targets of hesperetin against MASLD

We constructed a PPI network by importing 40 intersecting targets into the STRING database to explore the mechanism underlying the therapeutic effects of hesperetin on MASLD (Fig. 4A). It was visualized using the Cytoscape 3.7.2 software, followed by topological analysis (Fig. 4B). The center network was based on three topological parameters as follows: betweenness centrality, degree value and closeness centrality. Table 1 summarizes the topological parameter analysis structure. The leading 10 targets were as follows: MYC (Degree = 24), IL6 (Degree = 23), IL1B (Degree = 21), PTGS2 (Degree = 21), CYCS (Degree = 17), CCL2 (Degree = 16), TGFB1 (Degree = 16), CDKN1A (Degree = 16), and HMOX1 (Degree = 14).

Table 1

Degree value, betweenness centrality and closeness centrality of key targets in the PPI network
Gene Name	Degree value	Betweenness centrality	ClosenessCentrality
MYC	24	0.227508	0.75
IL6	23	0.091871	0.734694
IL1B	21	0.082048	0.705882
PTGS2	21	0.090734	0.705882
CYCS	17	0.084592	0.631579
CCL2	16	0.06862	0.631579
TGFB1	16	0.012414	0.642857
CDKN1A	16	0.026659	0.631579
HMOX1	14	0.034351	0.610169
NOTCH1	14	0.010395	0.62069
KDR	14	0.072083	0.610169
SERPINE1	13	0.061264	0.590164
SOCS3	12	0.033615	0.571429
IGF1R	12	0.051962	0.580645
CSF3	11	0.012691	0.571429
CEBPA	11	0.014722	0.545455

3.4 GO and KEGG pathway enrichment analyses

We performed KEGG pathway analysis on 40 intersecting targets to explore the various mechanisms by which hesperetin action contributes to MASLD treatment. The KEGG analysis examined the pathways central to MASLD pathogenesis. Figure 4C depicted the leading 10 enriched pathways with a false discovery rate (FDR) < 0.05. Among these pathways, Non-alcoholic fatty liver disease, JAK-STAT signaling pathway, and MAPK signaling pathway were highly enriched and met the FDR criteria. Table 2 summarizes the target genes enriched for each pathway.

Table 2

KEGG pathways with TOP10 enrichment degree and related gene targets.
KEGG pathway	Log10(P)	Target count	Targets
hsa05200: Pathways in cancer	-13.00	13	CCNA2, CDKN1A, CEBPA, HMOX1, IGF1R, IL6, MYC, NOTCH1, PIM1, PTGS2, TGFB1, WNT5A, CYCS
hsa04932: Non-alcoholic fatty liver disease	-8.89	7	CEBPA, DDIT3, IL1B, IL6, TGFB1, SOCS3, CYCS
hsa04218: Cellular senescence	-8.89	7	CCNA2, CCNB1, CDKN1A, IL6, MYC, SERPINE1, TGFB1
hsa05163: Human cytomegalovirus infection	-7.77	7	CDKN1A, IL1B, IL6, MYC, PTGS2, CCL2, CYCS
hsa04630: JAK-STAT signaling pathway	-7.09	6	CDKN1A, CSF3, IL6, MYC, PIM1, SOCS3
hsa05224: Breast cancer	-5.84	5	CDKN1A, IGF1R, MYC, NOTCH1, WNT5A
hsa05221: Acute myeloid leukemia	-5.73	4	CCNA2, CEBPA, MYC, PIM1
hsa04010: MAPK signaling pathway	-5.58	6	DDIT3, IGF1R, IL1B, KDR, MYC, TGFB1
hsa00591: Linoleic acid metabolism	-5.07	3	CYP1A2, CYP2C19, PLA2G10
hsa05418: Fluid shear stress and atherosclerosis	-4.47	4	HMOX1, IL1B, KDR, CCL2

Similar to KEGG analysis, we performed a GO enrichment analysis of the 40 intersecting targets; we analyzed biological processes (BP), cellular components (CC), and molecular functions (MF). The leading 10 enriched BP, MF, and CC terms are presented as bubble charts (Fig. 4D). The GO terms with high enrichment in biological processes consisted of the regulation of inflammatory response, cellular response to biotic stimulus, protein kinase complex, and cytokine activity.

3.5 Analysing of molecular docking results

Based on the PPI network and KEGG pathway analyses, we selected relevant targets, namely IL6, CYCS, TGFB1, IL1B, SOCS3, and CEPBA, enriched in the non-alcoholic fatty liver disease pathway for molecular docking. For identifying the interactions, the "LibDock" module in Discovery Studio was used and clusters with the highest LibDockScore were selected (Table 3). Figure 5 presents the corresponding structures. Notably, the LibDockScore for hesperetin-IL6 interaction was 113.311; it featured distance-dependent interactions that consisted of conventional hydrogen bond, carbon hydrogen bond and pi-alkyl interactions (Fig. 5A). For hesperetin-CYCS interaction, a LibDockScore was 101.249, accompanied by distance-dependent interactions encompassing conventional hydrogen bond, carbon hydrogen bond, pi-anion interactions, pi-alkyl interactions, pi-sigma interactions, and pi-cation interactions (Fig. 5B). In the case of hesperetin- TGFB1 interaction, the LibDockScore was 96.276. Moreover, the distance-dependent interactions comprised unfavorable bump, unfavorable donor-donor, conventional hydrogen bond, carbon hydrogen bond, pi-donor hydrogen bond and pi-alkyl interactions (Fig. 5C). The hesperetin-IL1B interaction demonstrated a LibDockScore of 77.722, with distance-dependent interactions encompassing carbon hydrogen bonds, conventional hydrogen bonds, pi-anion interactions, and pi-sigma interactions (Fig. 5D). The LibDockScore of hesperetin with SOCS interaction was 112.091; the distance-dependent interactions comprised conventional hydrogen bond, carbon hydrogen bonds, pi-donor hydrogen bond, pi-sigma interactions, pi-cation interactions, and pi-alkyl interactions (Fig. 5E). LibDockScore of hesperetin with CEBPA interaction was 83.923; the distance-dependent interactions encompassed unfavorable bump, unfavorable donor-donor, conventional hydrogen bond, carbon hydrogen bonds, pi-anion interactions, pi-cation interactions, and pi-pi t-shaped interactions (Fig. 5E). Thus, IL6 might be a possible key target of hesperetin intervention in MASLD.

Table 3

Binding energy and interactions of Hesperetin with target proteins
Targets	LibDockScore	Hydrogen Bonds and Other Interacting Residues
IL6	113.311	Ala130, Ala135, Asn132, Leu84, Leu133, Val85
IL1B	77.722	Glu25, Leu80, Leu134, Phe133, Pro78, Thr79
CYCS	101.249	Asp62, Arg91, Glu66, Glu66, Glu69, Lys88, Thr63
TGFB1	96.276	Arg25, Ile33, Lys26, Lys37, Phe24, Trp30
SOCS3	112.091	Lys104, Pro92, Pro96, Pro100, Ser94, Ser95
CEBPA	83.923	Asp205, Asp228, His230, His243, Lys207, Lys210

3.6 Hesperetin mitigated hepatocyte lipid accumulation and insulin resistance by inhibiting IL-6-STAT3-SOCS3 pathway in FFA-induced HepG2 cells

Network pharmacology and molecular docking are computer-based simulations; thus, further experimental validation is crucial. Network pharmacological analyses indicated that IL-6 was a possible an important target for the anti-MASLD effects of hesperetin. To validate the underlying mechanism, we examined the effects of hesperetin on IL-6 secretion in FFA-induced HepG2 cells. Hesperetin attenuated the IL-6 release from FFA-stimulated HepG2 cells in a dose-dependent manner (Fig. 6A). Western blot results corroborated this trend, displaying consistent changes in IL-6 protein expression (Fig. 6B). Furthermore, p-STAT3 and SOCS3 expressions were significantly increased in FFA-induced HepG2 cells than in controls. However, p-STAT3 and SOCS3 expression decreased significantly after hesperetin treatment than after no-treatment (Fig. 6C). Additionally, glucose consumption by FFA-induced HepG2 cells was significantly low. Compared with the low-dose group, the high-dose group demonstrated significant improvement in glucose consumption, suggesting that hesperetin reduced insulin resistance (IR) and promoted glucose uptake (Fig. 6D).

To further validate the critical role of hesperetin in the IL-6-mediated STAT3-SOCS3 pathway, the IL-6-stimulated HepG2 cells was carried out. The results of Western blot demonstrated that p-STAT3/STAT3 and SOCS3 levels were significantly elevated after IL-6 stimulation compared to the control. By contrast, the levels were significantly decreased after hesperetin treatment compared with IL-6 stimulation (Fig. 6E).

MASLD is one of the most prevalent types of liver disease worldwide; it is characterized by abnormal lipids accumulation in the liver, which can eventually lead to cirrhosis and HCC (Chen et al., 2023). No approved drugs are available for the treatment of MASLD. Lifestyle and dietary interventions remain the only recognized treatment options (Mitra et al., 2023). Hesperetin exerts therapeutic effects on numerous diseases as a natural product, characterized by multi-target and multi-pathway regulation; however, the exact mechanism by which it ameliorates hepatic abnormal lipid accumulation warrants further investigation (Khan et al., 2020, Evans et al., 2022, Salehi et al., 2022). Therefore, we utilized an integrated strategy of network pharmacology combined with molecular docking to identify the key targets of hesperetin against MASLD. Furthermore, we evaluated its potential mechanism using in vitro experiments, which provided a novel perspective on the mechanism by which hesperetin reduces lipid metabolism disorders in hepatocytes.

Hesperetin, a flavonoid extracted from citrus species with high bioavailability, has been extensively evaluated for its wide range of pharmacological activities, such as antioxidant, anti-inflammatory, anti-diabetes, and anti-cancer properties (Ferreira de Oliveira et al., 2020, Khan et al., 2020). Previously, hesperetin attenuated OA or PA-induced hepatotoxicity and oxidative stress; nonetheless, there are limited data on the in vitro hypolipidemic activity of hesperetin (Geng et al., 2020, Li et al., 2021). Therefore, we investigated the hypolipidemic activity of hesperetin in FFA-induced HepG2 cells. After determining the hesperetin dose, we investigated the effects of hesperetin on FFA-induced lipid accumulation in HepG2 cells. Oil-red O staining results and biochemical indices reflected that hesperetin significantly reduced aberrant intracellular lipid accumulation in the model group. Our findings elucidated the protective effects of hesperetin against MASLD-induced lipid accumulation, thus necessitating a better understanding of the underlying molecular mechanisms.

First, we screened 284 target genes related to hesperetin from seven online databases. Secnond, we obtained 1859 MASLD disease targets from the GEO datasets. Forty putative hesperetin-MASLD targets were identified. To explore the core targets, we constructed a PPI network expressing protein interactions by the STRING database and Cytoscape software; MYC, IL6, IL1B, PTGS2, CYCS, CCL2, CDKN1, TGFB1, HMOX1 and KDR might be the possible core targets warranting attention. These targets are closely related to oxidative stress, inflammatory response and lipid metabolism. MYC has the highest degree value and is central to hepatocyte apoptosis, liver fibrosis, and HCC in the MASLD mouse model (Porcu et al., 2018, Cheng et al., 2022). Additionally, IL6 and IL1B are the promoters of inflammatory responses; they are vital in numerous inflammatory diseases, and accelerate the MASLD progression (Stojsavljević et al., 2014). PTGS2, also termed cyclooxygenase 2 (COX-2), is expressed predominantly in inflammatory cells. It is substantially upregulated in chronic and acute inflammation, and, thus is a key target for several pharmacological inhibitors (Ferrer et al., 2019, Hu et al., 2023). GO and KEGG enrichment analyses were performed to explore the mechanism underlying hesperetin treatment for MASLD. GO enrichment analysis demonstrated that several biological processes are associated with the role of hesperetin in ameliorating MASLD. The regulation of inflammatory response, cellular response to biotic stimulus, regulation of stem cell proliferation, positive regulation of programmed cell death, and regulation of secretion were considerably enriched in biological processes. Additionally, the target genes were predominantly enriched in cytokine activity, phosphotransferase activity, and protein kinase complex, which demonstrated a potential relationship between hesperetin and the 40 MASLD targets. According to KEGG pathway analysis, significantly enriched pathways associated with MASLD encompassed cancer, non-alcoholic fatty liver disease, cellular senescence, human cytomegalovirus infection, and JAK-STAT signaling pathway. To explore the binding mode and ability of hesperetin to potential MASLD targets, we selected core targets enriched in the NAFLD signaling pathway for molecular docking with hesperetin based on KEGG analysis results. Results of molecular docking analysis indicated that hesperetin exhibited stable binding interactions with all five core targets, except DNA damage-inducible transcript 3 (DDIT3). Among these interactions, hesperetin demonstrated the highest binding score with IL6, suggesting that it may exhibit anti-MASLD lipid metabolism disorders by directly acting on IL6. However, in vitro experiments are still required to validate these predictions.

Based on the results of network pharmacology and molecular docking, hesperetin could directly regulate IL-6 expression and secretion in FFA-exposed HepG2 cells. Notably, hesperetin exerted a potent inhibitory effect on IL-6 in different disease models. Zhang et al. demonstrated that hesperetin significantly alleviated dextran sodium sulfate-induced colitis symptoms and inhibited IL-6 expression both in vivo and in vitro (Zhang et al., 2020). Additionally, it exerted anti-neuroinflammatory effects by inhibiting microglia activation and down-regulating the mRNA transcript levels of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and IL-6 (Jing et al., 2023). Consistent with our findings, Dr. Sun reported on the lipid-lowering ability of hesperetin and showed that hesperetin inhibited inflammatory responses in OA-induced HepG2 cells and high-fat diet (HFD)-induced mice (Li et al., 2021). The JAK/STAT pathway is vital for lipid metabolism and inflammation (Yu et al., 2009, Dodington et al., 2018). Dysregulation of the JAK/STAT signaling pathway leads to numerous diseases, including liver disease (Liu et al., 2023). Activation of the JAK/STAT signaling pathway in the liver by cytokines, including IL-6, promotes an increase in the STAT3 phosphorylation level; this in turn leads to IR and abnormal lipid accumulation (Shi et al., 2012). Furthermore, SOCS3 is closely related to IR development. Briefly, SOCS3 overexpression in the liver leads to IR; on the contrary, SOCS3 inhibition increases insulin sensitivity and ameliorates hepatic steatosis (Wójcik et al., 2014, Song et al., 2023b). Cytokines upregulate SOCS3 transcription, primarily through STAT3 activation (Rehman et al., 2017). To elucidate the mechanism by which hesperetin ameliorates steatosis, we examined p-STAT3/STAT3 and SOCS3 expression in FFA-induced HepG2 cells. Additionally, we used IL-6 to directly stimulate HepG2 cells. Hesperetin treatment significantly down-regulated p-STAT3/STAT3 levels, accompanied by SOCS3 down-regulation, both in FFA-treated and IL-6-treated HepG2 cells. Taken together, hesperetin attenuated hepatocellular steatosis by inhibiting IL6 expression and secretion, thereby blocking STAT3 phosphorylation and SOCS3 levels.

This study has several limitations that need to be addressed. First, the accuracy and reliability of retrieving data on compounds and disease targets from different databases remain challenging because these databases are not comprehensive and up-to-date. Meanwhile, it lacked in vivo and clinical experimental studies to directly validate the anti-MASLD effects and the related pathways of hesperetin. Previous studies have demonstrated the hepatoprotective effects of hesperidin in OA-induced HepG2 cells and HFD-induced NAFLD rat models (Li et al., 2021). In addition, studies in different disease models have explored the biological effects of hesperetin on IL-6 gene expression and STAT3 phosphorylation signaling pathway (Shirzad et al., 2017, Elhennawy et al., 2021). However, in-depth in vivo and clinical studies are indispensable. Additionally, more research is required to determine the optimal dose of hesperetin for clinical treatment, as well as the potential effects of long-term hesperetin treatment on other organs and tissues.

In summary, we systematically explored the pharmacological mechanism by which hesperetin alleviated MASLD using a comprehensive strategy of network-based computational pharmacology, along with bioinformatics analysis. We identified 40 targets on MASLD for hesperetin treatment, of which IL-6 was a possible core therapeutic target. Furthermore, the in vitro validation experiments confirmed that hesperetin significantly ameliorated FFA-induced lipid accumulation by inhibiting the IL-6-mediated STAT3-SOCS3 signaling pathway. Taken together, we hypothesized that hesperetin may be a potential therapeutic agent for MASLD.

BATMAN-TCM

bioinformatics annotation database for molecular mechanism of traditional Chinese medicine

biological processes

cellular components

COX-2

cyclooxygenase-2

CTD

comparative toxicogenomics database

DDIT3

DNA damage-inducible transcript 3

DSS

dextran sodium sulfate

ETCM

encyclopedia of traditional Chinese medicine

FDR

false discovery rate

FFA

free fatty acid

GEO

gene expression omnibus

gene ontology

HCC

hepatocellular carcinoma

HFD

high-fat diet

IL-6

interleukin- 6

insulin resistance

KEGG

kyoto encyclopedia of genes and genomes

MASLD

metabolic dysfunction-associated steatosic liver disease

molecular functions

MTT

3-(4,5-dimethiazol-2-yl)-2,5-diphenyl tetrazolium bromide

NAFLD

non-alcoholic fatty liver disease

NASH

non-alcoholic steatohepatitis

oleic acid

palmitic acid

PBS

phosphate-buffered saline

PDB

protein data bank

PPI

protein-protein interaction

SDF

spatial data file

SEA

similarity ensemble approach Database

TCHO

total cholesterol

TBST

Tris-buffered saline with 0.1% Tween® 20 detergent

TCMSP

traditional Chinese medicine systems pharmacology

triglyceride

TSV

tab-separated value

TNF-α

tumor necrosis factor-alpha.

Acknowledgments

The work was supported by the Applied Basic Research of Changzhou Science and Technology Program (CJ20220254, CJ2023066, CJ20239014), and the Advanced Training Program for Key Talents in Traditional Chinese Medicine of Jiangsu Province Administration of Traditional Chinese Medicine (2022No.11). We also thank Bullet Edits Limited for the linguistic editing and proofreading of the manuscript.

Conflict of interest

The authors declared that no conflict of interest.

Author's contributions

L-DT and HY designed the study. YW, YL, and DS performed the experiments. YW, and SX, analyzed the data. YL edited the figures and tables. HY wrote the paper. L-DT reviewed and edited the draft.

Data Availability

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

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No competing interests reported.

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Explore of potential targets and mechanisms of hesperetin in treating metabolic dysfunction-associated steatosis liver disease via network pharmacology and in vitro experiments

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Materials

2.2 Network pharmacology Analysis

2.2.1 Data Preparation

2.2.2 Protein-Protein Interaction Network

2.2.3 Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology Enrichment Analysis

2.2.4 Molecular docking

2.3 Cell culture and treatment

2.4 Cell viability assay

2.5 Western blot

2.6 Cellular triglyceride and total cholesterol measurement

2.7 Detection of IL-6

2.8 Oil-red staining

2.9 Glucose consumption assay

2.10 Statistical analysis

3. Results

3.1 Hesperetin alleviated lipid accumulation in FFA-induced HepG2 cells

3.2 Exploring hesperetin targets for the MASLD treatment by Network Pharmacology Analysis

3.3 PPI analysis for targets of hesperetin against MASLD

3.4 GO and KEGG pathway enrichment analyses

3.5 Analysing of molecular docking results

4. Discussion

5. Conclusion

Abbreviations

Declarations

Acknowledgments

Conflict of interest

Author's contributions

Data Availability

References

Additional Declarations

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