Ethanol extract of cassia seed alleviates metabolic dysfunction-associated steatotic liver disease by acting on multiple lipid metabolism-related pathways

2.1 Chemicals and reagents

Cassia extract was provided by the Department of Natural Medicinal Chemistry, Harbin Medical University. The extraction process involved initial extraction with 70% ethanol, followed by treatment with microporous resin and elution with 90% ethanol. The content of total anthraquinone compounds in the extract is detailed in Supplementary Table 1. The high-fat diet (HFD) used in this study was supplied by Huafukang Bio-technology Co., Ltd (Beijing, China) under license No. SCXK (Beijing) 2019-0008. The HFD composition includes Casein 25.84%, L-cystine 0.39%, maltodextrin 16.15%, sucrose 8.89%, cellulose 6.46%, soybean oil 3.23%, lard 31.66%, minerals 1.29%, DiCalcium phosphate 1.68%, calcium carbonate 0.72%, potassium citrate 2.13%, choline bitartrate 0.26%, vitamins 1.29%, and dye 0.01%.

2.2 Animals maintenance and experiments

All experimental animal protocols were approved by the Ethics Committee of the Second Affiliated Hospital of Harbin Medical University (SYDW2019-258). Specific pathogen-free (SPF) male wistar rats (180-200 g) and male golden Syrian hamsters (110130g) were purchased from Changsheng Biotechnology (Liaoning, China) under license number SCXK (Liao) 2020-0001. Animals were housed in a standard environment (temperature 23 ± 1°C, humidity of 55 ± 5%) with a 12:12-hour light-dark cycle, and were provided with a normal diet (ND) and free access to water. After one week of acclimatization, they were randomly assigned to experimental groups. Animal models of MASLD were constructed by feeding the rats a HFD for four weeks and the hamsters for 25 days^[23-24]. The HFD-fed animals were then randomly divided into a model group and ethanol extract of cassia seed (CSEE) treatment groups (10, 50, 250 mg/kg), with treatments administered via gavage for six weeks.

2.3 Serum and liver biochemical indicators assay

After successfully constructing the MASLD model, 1-1.5 ml of blood was collected via orbital blood sampling. Blood samples were placed on ice for 30 min, then centrifuged at 3000 rpm for 10 min at 4°C. The serum was transferred to new centrifuge tubes and analyzed immediately or stored at -80°C for later use. Serum total cholesterol (TC, A111-1-1), triglyceride (TG, A110-1-1), and low-density lipoprotein cholesterol (LDL-C, A113-1-1) were measured at 500 nm and 600 nm, respectively, while alanine aminotransferase (ALT, C009-2-1) and aspartate aminotransferase (AST, C010-2-1) were measured at 510 nm using biochemical kits from Jiancheng Biotechnology Co., China..

Liver tissues were rapidly dissected and frozen in liquid nitrogen. About 20 mg of liver tissue from the same lobe was homogenized with lysate, centrifuged at 2500 rpm for 10 min, and the supernatant was used to determine TC and TG content. The samples were either analyzed immediately or stored at -80°C for future use.

2.4 Histopathological analysis

Liver tissues were fixed in 4% paraformaldehyde for 24 h. Subsequently, the tissues were dehydrated through a gradient of ethanol and xylene, embedded in paraffin wax, and sectioned into 5-μm slices. Morphological changes in the liver were examined using Hematoxylin and eosin (H&E) staining (G1120, Solarbio, Beijing, China). Additionally, another portion of the liver was dehydrated using a 30% sucrose solution, embedded in optimal cutting temperature (O.C.T.) compound (SAKURA Tissue-Tek®), and sectioned into 10-μm slices. Lipid deposition of the liver was assessed using Oil Red O staining (G1261, Solarbio, Beijing, China).

2.5 Transmission electron microscopy (TEM)

Tissue blocks of approximately 1 mm3 were extracted from the same liver location in each experimental group and fixed d in 2.5% glutaraldehyde solution for 48 h. The blocks were then stained with 4% cobalt acetate solution for 30 min, followed by dehydration through a gradient of ethanol. The samples were embedded in a mixture of anhydrous acetone andf embedding agent for 2 h, then polymerized in an oven at 37°C for 24 h and at 60°C for 48 h. Finally, the samples were stained with uranyl acetate and lead citrate, and the tissue ultrastructure was examined using TEM.

2.6 Cell culture and treatment

HepG2 cells were cultured in Dulbecco modified Eagle medium (DMEM, Sigma, USA) supplemented with 10% fetal bovine serum (FBS, Bioind, Israel) and 5% penicillin and streptomycin (Seven, China). The cells were maintained in a water-saturated atmosphere of 95% air and 5% CO2 at 37°C. For the experiments, the cells were exposed to free fatty acid (FFA) at a concentration of 250 μmol/L for 24 h. Subsequently, the cells were treated with CSEE at concentrations of 0.1, 1, 10 μg/mL for 24 h. Additionally, specific signal pathway inhibitors (Compound C/GW6471/LY294002, MCE, USA) were pre-treated 4 h before the CSEE (10 μg/mL) treatment to evaluate the effect of CSEE. At the end of the experiments, the cells were harvested for further analysis.

2.7 Cell viability assay

Cell counting kit-8 (CCK8, Seven, China) and a Calcein-AM/ propidium iodide (Calcein-AM/PI) cell activity and cytotoxicity assay kit were used to assess the effect of CSEE on cell activity. HepG2 cells, grown to an appropriate density in 96-well plates, were treated with CSEE for 24 h. After treatment, 10 μL of CCK8 solution was added to each well, and the cells were incubated in the culture incubator for approximately 2 h. Absorbance was measured at 450 nm using a Tecan Infinite M200PRO Multimode Plate Reader (Switzerland) to calculate cell viability. For the Calcein AM/PI assay, the working solution was prepared according to the instructions and added to the cell culture plates. The samples were incubated at 37°C for 30 min, protected from light. Images were acquired using a laser scanning confocal microscope (Olympus, Fluoview1000, Tokyo, Japan), and the ratio of dead to live cells was determined based on fluorescence intensity.

2.8 Nile red staining

Cells were fixed with 4% paraformaldehyde for 20 min at room temperature. Following fixation, cells were washed with PBS buffer. Nile red dye working solution was then added according to the manufacturer’s instructions and incubated for 10 min at room temperature, protected from light. DAPI staining solution was subsequently added to each well and incubated for 10 min, also protected from light. Intracellular lipid deposition was observed and quantified using a laser scanning confocal microscope, with analysis based on fluorescence intensity.

2.9 Screening of the targets of MASLD

The Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) was used to identify gene expression datasets related to MASLD. The search criteria included “MASLD” and “NAFLD”^[25]. The dataset GSE135251, which comprises 206 patient samples and 10 control samples, was selected for analysis. GEO2R was used to visualize the data with volcano plots and heat maps. The Bioinformatics website (http://www.bioinformatics.com.cn/) was employed to further enhance these visualizations. Potential therapeutic targets for MASLD were predicted using several databases: GeneCards (https://www.genecards.org/), DisGeNET (https://www.disgenet.org/), MalaCards (https://previous.malacards.org/pages/info), the Comparative Toxicogenomics Database (CTD) (https://ctdbase.org/), and OMIM (https://www.omim.org/). The predicted therapeutic targets were then cross-referenced with the dataset GSE135251. Targets that appeared at least three times were selected for further study and were also standardized using the UniProt database.

2.10 Screening and analysis of drug-disease common targets

Livers from hamsters in the HFD group and those gavage-administered CSEE (250 mg/kg) were subjected to high-throughput sequencing to identify differential genes. Overlapping differential genes with \ MASLD therapeutic targets were analyzed using Venn diagrams on the bioinformatics website (http://www.bioinformatics.com.cn/). The 30 selected targets were uploaded to the STRING 12.0 (https://cn.string-db.org/) to construct protein-protein interaction (PPI) networks. Data visualization and network topology analysis were performed using Cytoscape 3.10.1, with hub genes further identified using Cytohubba and MCODE plugins.

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses for the 30 targets were visualized on the bioinformatics website (http://www.bioinformatics.com.cn/). GO analysis covered three ontologies: biological process (BP), molecular function (MF), and cellular component (CC). The top 20 GO terms and top 50 KEGG pathways were displayed.

2.11 Molecular docking

The SDF structure of CSEE active ingredient was obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/) and converted to mol2 format using OpenBabel 2.4.1. Hydrogenation was performed in Autodock Vina, and the file was exported to pdpqt format. The PDB structure of the receptor protein, was sourced from UniProt, filtered based on resolution and publication date. Dehydration and hydrogenation of the receptor were done in Autodock Vina, and the resulting pdbqt files were prepared. Both ligand and receptor pdbqt files were then imported into Autodock Vina for molecular docking.

2.12 Western blot

Total protein samples from liver tissues of rats and hamsters were extracted using RIPA buffer (Beyotime, China) supplemented with protease inhibitors (Roche, Germany). Protein concentrations were measured using a BCA protein detection kit (Beyotime, Shanghai, China). The proteins were then separated by 7.5% or 10% SDS-PAGE and transferred to nitrocellulose membranes. After blocking with a rapid blocking solution, the membranes were incubated with primary antibodies at 4°C overnight. The following primary antibodies were used: β-Actin (TA-09, 1 : 1000, ZSGB Co. Ltd, China), PPARα (A18252, 1 : 1000, ABclonal, China), PPARγ (16643-1-AP, 1 : 1000, Proteintech, China), Carnitine Palmitoyltransferase 1A (CPT1A) (15184-1-AP, 1 : 1000, Proteintech, China), Fatty Acid Synthase (FASN) (10624-2-AP, 1 : 1000, Proteintech, China), CCAAT/Enhancer Binding Protein α (C/EBPα) (18311-1-AP, 1 : 1000, Proteintech, China), p-AMPKα (CY5608, 1 : 1000, Abways, China), AMPKα1 (bsm-33338M, 1 : 1000, Bioss, China), p-Acetyl CoA Carboxylase (p-ACC) (11818T, 1 : 1000, Cell Signaling Technology, USA), ACC (3676T, 1 : 1000, Cell Signaling Technology, USA), PI3K/AKT (AF5112, 1 : 1000, Affinity, China), p-AKT (AF0016, 1 : 1000, Affinity, China), AKT (AF6261, 1 : 1000, Affinity, China), and Sterol Regulatory Element-Binding Protein 1 (SREBP1) (ab28481, 1 : 1000, Abcam, UK).

Following primary antibody incubation, the membranes were washed with PBS-Tween (0.5%) and then incubated with secondary antibodies at room temperature for 1 hour. The secondary antibodies used were goat anti-mouse IgG (P/N 926-32210, LI-COR Biosciences, USA) and goat anti-rabbit IgG (P/N 926-32211, LI-COR Biosciences, USA). Finally, blot band images were captured using an imaging system (LI-COR Biosciences, USA).

2.13 Real-time PCR (RT-PCR)

Total RNA was extracted from liver tissues of rats and hamsters using TRIZOL reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. RNA concentration was measured with a Nanodrop 2000 microspectrophotometer (Thermo Fisher Scientific), and then reverse-transcribed into complementary DNA using the ReverTra Ace qPCR RT Kit (Toyobo, Japan). Quantitative mRNA expression was assessed with an LC480 Real-time PCR system (Roche, USA) and SYBR Green PCR Master Mix (Roche, USA). The 18S rRNA gene was used as an internal control, and gene expression levels were calculated using the 2-ΔΔCt method. The primers used in this study are listed in Supplementary Table 2.

2.14 Statistical analysis

GraphPad Prism 9.0 was used for data analysis. The experimental data are presented as the mean ± SEM. Statistical comparisons were made using one-way analysis of variance (ANOVA) followed by a post hoc Tukey test for multiple group comparisons. Statistical significance was defined as a P-value < 0.05.

3.1 CSEE inhibits HFD-induced elevation of serum lipid and transaminase levels in rats with MASLD

An animal model of HFD-induced MASLD was established, and CSEE was administered orally for 6 weeks. The experimental flow is illustrated in Fig. 1A. Serum lipid changes in rats fed an HFD for four weeks are shown in Supplementary Fig. 1. Following successful model establishment, the effects of CSEE (10, 50, and 250 mg/kg) on serum lipid and transaminase levels were evaluated. As shown in Fig. 1B, CSEE (50 and 250 mg/kg) effectively inhibited chylomicronemia induced by HFD. Figures 1C, D, E, and F demonstrate that CSEE (50 and 250 mg/kg) significantly reduced serum levels of TC, TG, and LDL-C but had no effect on HDL-C. In addition, Fig. 1G and 1H indicates that CSEE (50 and 250 mg/kg) significantly inhibited the elevation of serum ALT and AST levels induced by HFD.

Fig. 1

Cassia seed ethanol extract (CSEE) reduces elevated blood lipids and serum transaminase in rats fed with high-fat diet (HFD)

(A) Diagram illustrating the rat-feeding timeline. (B) Appearance of rat serum samples, N = 3. (C-H) Serum levels of (C) total cholesterol (TC), (D) triglyceride (TG), (E) low-density lipoprotein cholesterol (LDL-C), (F) high-density lipoprotein cholesterol (HDL-C), (G) alanine aminotransferase (ALT), and (H) aspartate aminotransferase (AST), measured in rats after CSEE oral administration for 6 weeks, N = 8 per group. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. ND: Normal Diet.

3.2 CSEE inhibits HFD-induced hepatic steatosis in rats with MASLD

Hepatic steatosis is a key clinical feature of MASLD^[26]. Therefore, we investigated the effects of CSEE on liver lipid deposition in MASLD rats. Firstly, we assessed the morphological changes in liver samples from each group. Fig. 2A shows that livers from the ND group had a dark red color, smooth surface, and elastic texture. In contrast, livers from the HFD group were noticeably paler, larger, and slightly firmer. Treatment with CSEE (50 and 250 mg/kg) ameliorated the morphological changes induced by HFD, suppressing the increase in liver volume. We also examined the effects of CSEE on liver weight and liver index (the weight ratio of liver and body). Fig. 2B, C, and D shows that rats in the HFD group had significantly higher body weight, liver weight, and liver index compared to the ND group. Although CSEE administration did not affect the body weight of HFD-induced rats, it did reduce liver weight and liver index in MASLD rats at doses of 50 and 250 mg/kg. Additionally, we assessed the lipid content in the livers of rats from each experimental group. Fig. 2E and F demonstrate that CSEE (50 and 250 mg/kg) effectively inhibited the increase in liver TC and TG levels induced by HFD.

Fig. 2

Cassia seed ethanol extract (CSEE) prevents high-fat-diet (HFD) feeding-induced hepatic morphological changes and lipid deposition in rats

(A) Representative images of rat livers from various groups. (B) Body weight, N = 8 per group. (C) Liver weight, N = 8 per group. (D) The weight ratio of liver and body, N = 8 per group. (E-F) Liver (E) total cholesterol (TC), and (F) triglyceride (TG) contents of rats from various groups, N = 8 per group. (G) Hematoxylin-eosin (H&E) staining of rat liver tissues. (H) Oil Red O staining of rat liver tissues. Scale bar: 100 μm; 50 μm, N = 8 per group. (I) Representative electron microscopic images of rat liver tissue slices from various groups. Scale bar: 5 μm. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. ND: Normal diet.

Histopathological staining was used to observe lipid changes in the live. H&E staining in results Fig. 2G revealed that hepatocytes in the ND group were regularly arranged with normal structure, showing no apparent inflammatory infiltration or lipid droplet accumulation. In contrast, the liver tissue from the HFD group exhibited severe steatosis, characterized by numerous irregular fat vacuoles within the cytoplasm, swollen hepatocytes, and nuclei displaced to the cell peripheries. These pathological changes were significantly ameliorated by CSEE (50 and 250 mg/kg). Oil Red O staining results in Fig. 2H corroborated the findings from H&E staining. The HFD group displayed pronounced red lipid droplet aggregation, whereas CSEE (50 and 250mg/kg) improved lipid droplet accumulation in MASLD rats. These observations were further validated by TEM in Fig. 2I, which shows that CSEE (50 and 250 mg/kg) inhibited hepatic lipid deposition and reduced both the volume and number of lipid droplets in the livers of MASLD rats.

3.3 CSEE inhibits HFD-induced elevation of serum lipid and transaminase levels in golden Syrian hamsters with MASLD

We have confirmed that CSEE has therapeutic effects on HFD-induced hyperlipidemia and hepatic steatosis in rats with MASLD.

To ensure the reliability and reproducibility of these effects, as outlined in the technical guidelines for new drug studies, it is important to evaluate CSEE in a different animal model. Thus, we investigated the effects of CSEE in golden Syrian hamsters with MASLD. Previous studies established that feeding a HFD for 25 days leads to hyperlipidemia and hepatic steatosis in hamsters^[24]. Fig. 3A provides a schematic of the hamster feeding process, and blood lipid levels at 25 days are shown in Supplementary Fig. 2. Fig. 3B-F indicates that continuous HFD feeding induces hyperlipidemia in hamsters. However, CSEE administered by gavage at doses of 10, 50, and 250 mg/kg for six weeks improved HFD-induced alterations in serum lipid levels, in hamsters, although it effectively reduced serum ALT and AST levels. The 50 mg/kg dose of CSEE did not affect serum TG, ALT, or AST levels in hamsters, which may be attributed to e differences in lipid metabolism between rats and hamsters. This suggests that a higher dose of CSEE may be required to effectively treat hyperlipidemia in MASLD hamsters.

Fig. 3

Cassia seed ethanol extract (CSEE) reduces elevated blood lipids and serum transaminase in hamsters fed with high fat diet (HFD)

(A) Diagram illustrating the feeding timeline for hamsters. (B) Appearance of hamster serum samples, N = 3. (C-H) Serum levels of (C) total cholesterol (TC), (D) triglyceride (TG), (E) low-density lipoprotein cholesterol (LDL-C), (F) high-density lipoprotein cholesterol (HDL-C), (G) alanine aminotransferase (ALT), and (H) aspartate aminotransferase (AST), measured in hamsters after CSEE oral administration for 6 weeks, N = 8 per group. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. ND: Normal diet.

3.4 CSEE inhibits HFD-induced hepatic steatosis in golden Syrian hamsters with MASLD

Next, we went on to evaluate the effect of CSEE on hepatic steatosis in hamsters. The livers from the HFD group appeared lighter in color and slightly larger than those from the ND group. CSEE (250 mg/kg) alleviated these HFD-induced morphological alterations in the liver of hamsters (Fig. 4A). Unlike the results observed in rats, there was no significant change in the body weight of hamsters after HFD feeding (Fig. 4B). This lack of change might be attributed to differences in intake and lipid metabolism between hamsters and rats, consistent with previous findings^[24]. Liver weight and liver index were significantly higher in the HFD group compared with the ND group (Fig. 4C and D). Treatment with CSEE (50, 250 mg/kg) for six weeks reduced liver weight and liver index in hamsters. We further analyzed liver lipid content across all groups. Fig. 4E and F show that CSEE (250 mg/kg) effectively inhibited the HFD-induced increase in TC and TG levels in the liver. However, the 50 mg/kg dose of CSEE did not significantly improve hepatic lipid deposition, which may be related to the more advanced progression of MASLD in hamsters compared to rats.

Fig. 4

Cassia seed ethanol extract (CSEE) prevents high-fat-diet (HFD) feeding-induced hepatic morphological changes and lipid deposition in hamsters

(A) Representative images of hamster livers from different groups. (B) Body weight, N = 8 per group. (C) Liver weight, N = 8 per group. (D) The weight ratio of liver and body, N = 8 per group. (E-F) Liver (E) total cholesterol (TC), and (F) triglyceride (TG) contents in hamsters of different groups, N = 8 per group. (G) Hematoxylin-eosin (H&E) staining of hamster liver tissues. (H) Oil Red O staining of hamster liver tissues. Scale bar: 100 μm; 50 μm, N = 8 per group. (I) Representative electron microscopic images of hamster livers of various groups. Scale bar: 2 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ND: Normal diet.

We then analyzed the histopathological staining of hamster liver tissues from each group. H&E staining results in Fig. 4G indicated that CSEE (250 mg/kg) improved HFD-induced hepatic tissue steatosis by reducing hepatocyte swelling and the number of fat vacuoles. Similarly, Oil Red O staining in Fig. 4H demonstrated that CSEE (250 mg/kg) effectively inhibited the accumulation of lipid droplets in the liver. These findings were further corroborated by the observation of liver ultrastructure in Fig. 4I, which was consistent with the above results.

3.5 CSEE inhibits FFA-induced lipid accumulation in HepG2 cells

To further elucidate the role of CSEE, we conducted in vitro studies using HepG2 cells. The effects of CSEE on cell proliferation and viability were determined using Calcein-AM/PI staining and CCK8 assay. Fig. 5A and 5B demonstrates that CSEE concentrations ranging from 0.1 to 10 μg/mL did not affect HepG2 cell viability, whereas a concentration of 100 μg/mL of CSEE induced apoptosis in vitro. To avoid potential interference with cell viability in subsequent experiments, we selected CSEE doses of 0.1-10 μg/mL to investigate its effects on cellular lipid deposition.

Fig. 5

Cassia seed ethanol extract (CSEE) prevents free fatty acid (FFA)-induced increase in lipid content in HepG2 cells

(A) Representative images of Calcein acetoxymethyl ester/Propidium Iodide (Calcein-AM/PI) staining in HepG2 cells after CSEE treatment. Scale bar: 50 μm, N = 6 per group. (B) Cell viability of HepG2 measured by cell counting kit-8 (CCK8). N = 5 per group. (C-D) The contents of (C) total cholesterol (TC) and (D) triglyceride (TG) in HepG2 after CSEE treatment. N = 8 per group. (E) Representative images of Oil Red O staining in HepG2. N = 6 per group. (F) Representative images of Nile Red staining in HepG2 cells. N = 6 per group. ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. DMSO: Dimethyl sulfoxide.

We used FFA at a concentration of 250 μmol/L to induce intracellular lipid deposition in HepG2 cells and treated the cells with CSEE for 24 h to observe changes in intracellular lipid levels. Fig. 5C and 5D reveals that FFA treatment significantly increased intracellular levels of TC and TG. However, treatment with 10 μg/mL of CSEE reduced the TC and TG content in the cell.

Finally, Oil Red O staining was used to assess the deposition of intracellular lipid droplets in HepG2 cells. Fig. 5E shows that lipid droplets were significantly increased after 24 h of FFA treatment. However, 10 μg/mL of CSEE reduced the aggregation of these lipid droplets, while the other two concentrations of CSEE had no effect on lipid droplet formation. Additionally, Nile Red staining was used to further evaluate intracellular lipid deposition. The results, shown in Fig. 5F, were consistent with those obtained from Oil Red O staining, confirming the inhibitory effect of 10 μg/mL of CSEE on lipid droplet accumulation.

3.6 Prediction and analysis of targets related to MASLD and CSEE

To further explore the mechanism underlying the pharmacological effects of CSEE, we used network pharmacology for prediction and analysis. Initially, we identified therapeutic targets for MASLD. Given that MASLD is a relatively recent reclassification of NAFLD, data related to NAFLD remains relevant for MASLD research^[25]. Consequently, we used “NAFLD” as a keyword to search the GEO database. We selected the GSE135251 dataset, which includes 10 normal liver tissues and 206 liver tissues from patients. We predicted the disease-related targets using several databases: GeneCards, DisGeNET, MalaCards, CTD, and OMIM. These predicted targets were then compared with differentially expressed genes (DEGs) from the GSE135251 dataset. The overlap of these targets was visualized using venn diagrams, as shown in Fig. 6A. Targets that appeared in at least three of the sources were selected, resulting in 673 targets for further analysis. High-throughput sequencing was conducted to analyze liver samples from the HFD group and the CSEE (250 mg/kg) group in hamsters. As shown in the volcano plot in Fig. 6B, the threshold of fold change and P-value were set at 1.5 and 0.05, respectively. This analysis identified a total of 668 DEGs, comprising 363 down-regulated genes and 305 up-regulated genes. To facilitate the understanding of the functions and biological processes associated with these DEGs, we performed GO enrichment and KEGG pathway analysis. The results of these analyses are presented in Supplementary Fig. 3A-B. To identify targets associated with MASLD, we intersected the 668 DEGs with disease-associated targets, yielding thirty common targets for further study, as shown in Fig. 6C. These targets were analyzed using the String 12.0 for PPI network analysis and visualised by Cytoscape 3.10.1. Fig. 6D illustrates a network with 30 nodes and 98 edges, where nodes like STEAP4, SLC13A5, NNMT, and SOCS2 were isolated without interactions. The node size and color intensity reflect their correlation with the drug’s therapeutic mechanism. We calculated the Degree value of nodes using the CytoHubba plugin and reclassified the nodes with the M-CODE plugin, as shown in Supplementary Fig. 3C-D. GO enrichment and KEGG pathway analyses of core targets revealed their involvement in biological processes like nutrient response, lipid metabolism, and cellular responses to external stimuli (Fig. 6E). Key cellular components include pigment granules, melanosomes, endoplasmic reticulum chaperone complexes, and lipid droplets. Molecular functions involve unfolded protein binding and phosphatase binding. KEGG pathway analysis (Fig. 6F) highlighted pathways related to endoplasmic reticulum protein processing, non-alcoholic fatty liver disease, lipid metabolism, and atherosclerosis, with key signaling pathways including PPAR, PI3K/AKT, AMPK, and MAPK. Lastly, Autodock Vina was used to predict the binding modes of total anthraquinone components from CSEE—aloe emodin, rhein, edomin, chrysophanol, emodin 1-O-β-D-glucoside, emodin 8-O-β-D-glucoside, and physcion 8-O-β-D-glucoside—to key targets. Fifty docking simulations were performed, and the results were visualized in Fig. 6G-I, highlighting the complementary spatial structures and energy minimization of the ligand-receptor interactions.

Fig. 6

Prediction and analysis of disease-drug targets

(A) Venn diagram of disease-related targets in five databases and GSE135251 dataset. (B) Volcano plot of differentially expressed genes (DEGs) in golden hamster livers between the high-fat-diet (HFD) group and the HFD + Cassia seed ethanol extract (CSEE) (250 mg/kg) group. (C) Venn diagram of DEGs. (D) Visualization of proteinprotein interaction (PPI) network analysis. (E) Gene ontology (GO) enrichment analysis of targets (Top 20 terms of biological process, molecular function and cellular component). (F) Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of targets (Top 50 pathways). (G-I) Molecular docking analysis of CSEE and (G) Peroxisome proliferator-activated receptor a (PPARα), (H) fatty acid synthase (FASN), and (I) sterol regulatory element-binding protein 1 (SREBP1).

3.7 CSEE alleviates HFD-induced hepatic steatosis in MASLD rats and hamsters by acting on lipid metabolism-related pathways

Based on the results from network pharmacology and high-throughput sequencing, we assessed the protein levels of AMPK, PPAR, PI3K/AKT, and their downstream lipid metabolism related proteins using Western blotting. Fig. 7A and 7B illustrates that a 10-week HFD significantly decreased the protein levels of p-AMPKα and p-ACC, while CSEE treatment increased their phosphorylation levels. This suggests that CSEE activates the AMPK signaling pathway, which helps alleviate hepatic lipid deposition in both rats and hamsters with MASLD. AMPK, when activated, reduces hepatic fat synthesis by influencing fatty acid β-oxidation pathways, with CPT1A being a key enzyme in this process. In contrast, PPARγ and C/EBPα are crucial regulators of adipocyte formation. The data (Fig. 7A-B) show that, compared to the ND group, the HFD group had decreased levels of PPARα and CPT1A, and increased expression of C/EBPα and PPARγ. CSEE treatment, however, promoted PPARα and CPT1A expression while inhibiting liver C/EBPα and PPARγ levels. This indicates that CSEE enhances hepatic fatty acid oxidation and reduces adipocyte formation.

Fig. 7

Cassia seed ethanol extract (CSEE) alleviates high-fat-diet (HFD)-induced hepatic steatosis by activating multiple signalling pathways

(A-B) Relative protein levels of the AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor (PPAR), and phosphoinositide 3-kinase (PI3K)/AKT signalling pathways in (A) rat and (B) hamster livers, determined by Western blotting. N = 6 per group. (C-D) mRNA levels of the genes related to fatty acid synthesis, oxidation, uptake, and adipogenesis, as quantified by qRT-PCR. N = 8 per group. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. ND: Normal diet.

Additionally, the PI3K/AKT signaling pathway can promote fatty acid synthesis and lipid droplet formation through its effect on SREBP1c^[27]. The results in Fig. 7A-B showed that hepatic steatosis inhibited the expression of PI3K P110 and p-AKT, whereas CSEE administration activated the PI3K P110/AKT pathway and inhibited hepatic lipid droplet formation.

We also examined the expression of genes involved in lipid synthesis and metabolism in the livers of rats and hamsters using qRT-PCR. The results presented in Figures 7C and 7D demonstrated that MASLD was associated with increased expression of lipid synthesis genes (PPARγ, CEBPA) and fatty acid uptake genes (FABP1, ACACA, ACACB, SREBF1, FASN, and ACLY), alongside a decrease in the expression of fatty acid oxidation-related genes (PPARα, CPT1A, and ACOX1). Treatment with CSEE reversed these HFD-induced changes in gene expression. Specifically, CSEE inhibited the upregulation of lipid synthesis and fatty acid uptake genes and promoted the expression of fatty acid oxidation genes. These results suggest that CSEE effectively suppresses lipid synthesis and fatty acid uptake while enhancing fatty acid oxidation, thereby contributing to the amelioration of MASLD.

3.8 Inhibition of the AMPK, PPARα, and PI3K/AKT signaling pathways reversed the lipid-lowering effect of CSEE

To further validate the roles of the AMPK, PPARα, and PI3K/ AKT signaling pathways in the treatment of MASLD with CSEE, we performed in vitro experiments using specific small molecule inhibitors. The results from biochemical assays (Fig. 8A and 8B) demonstrated that CSEE effectively reduced the FFA-induced elevations in TC and TG levels. However, these beneficial effects of CSEE were reversed when cells were co-treated with a mixture of inhibitors targeting AMPK, PPARα, and PI3K/AKT pathways. Additionally, Oil Red O staining (Fig. 8C) and Nile Red staining (Fig. 8D) corroborated these findings, showing that the reduction in intracellular lipid droplets induced by CSEE was mitigated in the presence of the inhibitors. These results confirm that the therapeutic effects of CSEE are, at least in part, mediated through the AMPK, PPARα, and PI3K/AKT signaling pathways.

Fig. 8

Inhibition of AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptora (PPARα), and phosphoinositide 3-kinase (PI3K)/AKT reverses the beneficial effects of Cassia seed ethanol extract (CSEE) on lipid contents

(A-B) The contents of (A) total cholesterol (TC) and (B) triglyceride (TG) in HepG2 cells. N = 6 per group. (C) Representative images of Oil Red O staining in HepG2 cells. N = 6 per group. (D) Representative images of Nile Red staining in HepG2 cells. N = 6 per group. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. DMSO: Dimethyl sulfoxide. FFA: Free fatty acid.