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

Wen Li; Jia Wang; Yilian Yang; Chunlei Duan; Bing Shao; Mingxiu Zhang; Jiapan Wang; Peifeng Li; Ye Yuan; Yan Zhang; Hongyu Ji; Xingda Li; Zhimin Du

doi:10.1515/fzm-2024-0017

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

doi: 10.1515/fzm-2024-0017

Wen Li^{1, #},
Jia Wang^{1, #},
Yilian Yang^{1, #},
Chunlei Duan¹,
Bing Shao¹,
Mingxiu Zhang¹,
Jiapan Wang¹,
Peifeng Li¹,
Ye Yuan¹,
Yan Zhang¹,
Hongyu Ji¹,
Xingda Li¹,
Zhimin Du^{1, 2
, ,}

1.
Institute of Clinical Pharmacy, the Second Affiliated Hospital of Harbin Medical University (State Key Laboratory of Frigid Zone Cardiovascular Diseases), Harbin 150086, China
2.
State Key Laboratory of Quality Research in Chinese Medicines, Macau University of Science and Technology, Macau 999078, China

Funds:

National Natural Science Foundation of China 82073838

National Natural Science Foundation of China 82273917

More Information

Corresponding author: Zhimin Du, E-mail: dzm1956@126.com

摘要

Abstract: Background and objective In northern China's cold regions, the prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) exceeds 50%, significantly higher than the national and global rates. MASLD is an important risk factor for cardiovascular and cerebrovascular diseases, including coronary heart disease, stroke, and tumors, with no specific therapeutic drugs currently available. The ethanol extract of cassia seed (CSEE) has shown promise in lowering blood lipids and improving hepatic steatosis, but its mechanism in treating MASLD remains underexplored. This study aims to investigate the therapeutic effects and mechanisms of CSEE. Methods MASLD models were established in male Wistar rats and golden hamsters using a high fat diet (HFD). CSEE (10, 50, 250 mg/kg) was administered via gavage for six weeks. Serum levels of total cholesterol (TC), triglyceride (TG), lowdensity lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), aspartate aminotransferase (AST), and alanine aminotransferase (ALT), as well as liver TC and TG, were measured using biochemical kits. Histopathological changes in the liver were evaluated using Oil Red O staining, Hematoxylin-eosin (H&E) staining, and transmission electron microscopy (TEM). HepG2 cell viability was assessed using the cell counting kit-8 (CCK8) and Calcein-AM/PI staining. Network pharmacology was used to analyze drug-disease targets, and western blotting was used to confirm these predictions. Results CSEE treatment significantly reduced serum levels of TC, TG, LDL-C, ALT, and AST, and improved liver weight, liver index, and hepatic lipid deposition in rats and golden hamsters. In addition, CSEE alleviated free fatty acid (FFA)-induced lipid deposition in HepG2 cells. Molecular biology experiments demonstrated that CSEE increased the protein levels of p-AMPK, p-ACC, PPARα, CPT1A, PI3K P110 and p-AKT, while decreasing the protein levels of SREBP1, FASN, C/EBPα, and PPARγ, thus improving hepatic lipid metabolism and reducing lipid deposition. The beneficial effects of CSEE were reversed by small molecule inhibitors of the signaling pathways in vitro. Conclusion CSEE improves liver lipid metabolism and reduces lipid droplet deposition in Wistar rats and golden hamsters with MASLD by activating hepatic AMPK, PPARα, and PI3K/AKT signaling pathways.
- cassia seed ethanol extract /
- metabolic dysfunction related fatty liver disease /
- network pharmacology
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Figure 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) lowdensity 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.

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Figure 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.

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Figure 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.

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Figure 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.

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Figure 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.

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Figure 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 α (PPARα), (H) fatty acid synthase (FASN), and (I) sterol regulatory element-binding protein 1 (SREBP1).

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Figure 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.

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Figure 8. Inhibition of AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptorα (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.

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