Flavonoid-Rich mulberry leaf extract modulate lipid metabolism, antioxidant capacity, and gut microbiota in high-fat diet-induced obesity: potential roles of FGF21 and SOCS2
Guangxi Key Laboratory of Environmental Exposure Omics and Life Cycle Health, College of Public Health, Guilin Medical University, Guilin 541199, China
Guangxi Key Laboratory of Drug Discovery and Optimization, College of Pharmacy, Guilin Medical University, Guilin 541199, China
Guangxi Eco-engineering Vocational and Technical College, School of Tourism and Traffic Management, Liuzhou 545004, China
Liuzhou Institute of Technology, Liuzhou Special Food Flavor and Quality Control Research Center of Engineering Technology, Liuzhou 545616, China
†These authors contributed equally to this work.
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Highlights
(1) Network pharmacology analysis identified key targets such as IL6, STAT3, FXR, PPAR, and CYP family, revealing the potential mechanisms of mulberry leaves in NAFLD treatment.
(2) Mulberry leaf aqueous extract (MLQE) intervention significantly reduced body weight, improved lipid profiles, and alleviated hepatic steatosis in high-fat diet-induced obese mice, with notable histological and metabolic improvements.
(3) MLQE enhanced hepatic and plasma antioxidant activities, elevating catalase (CAT), glutathione peroxidase (GSH-Px), and HDL-C levels, while reducing oxidative stress markers.
(4) RNA-seq and Western Blot analyses demonstrated that MLQE normalized metabolic gene expressions, upregulating FXR and SHP, and correcting FGF21 and SOCS2 downregulation, with positive modulation of gut microbiota composition.
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Mulberry leaves are a rich source of active ingredients exhibiting notable antioxidant activity and have been demonstrated to possess hypolipidemic and hypoglycemic effects. Non-alcoholic fatty liver disease (NAFLD), the most prevalent chronic liver disease, is increasingly prevalent worldwide, with a strong correlation between NAFLD occurrence and obesity. We aimed to investigate the association between the active ingredient targets in mulberry leaves and stems and the disease targets of NAFLD/NASH through network pharmacology, and designed animal experiments to verify our findings. In addition to the normal and model groups, three intervention groups were established for the experiment: the mulberry leaf aqueous extract (MLQE), Orlistat, and swimming exercise groups. The intervention effect of MLQE on NAFLD was observed by comparing it with widely recognized weight loss drugs and exercise interventions. The results demonstrated that all three interventions effectively reduced body weight and body fat, exhibited robust antioxidant capacity, and significantly improved the dysbiosis of gut microbiota induced by a high-fat diet. Histological analysis also supported the conclusions of lipid-lowering, anti-inflammatory, and amelioration of liver injury. Through RNA-seq analysis, we identified the key genes FGF21 and SOCS2 that may be associated with the effective intervention of mulberry leaves in NAFLD, and validated the genes and proteins related to NAFLD and lipid metabolism by qPCR and WB analysis. Our study found that MLQE has an anti-NAFLD effect not inferior to orlistat and swimming exercise, indicating that it is a promising candidate for the treatment of NAFLD. Meanwhile, FGF21 and SOCS2 may be key targets for the treatment of NAFLD, and more studies are needed to verify this.
Abstract
Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease in clinical practice. This study examined the effects of mulberry leaf aqueous extract (MLQE) on lipid metabolism and gut microbiota in mice with high-fat diet-induced NAFLD. Using network pharmacology, we identified key targets of mulberry leaf and stem’s constituents for NAFLD regulation and validated their ameliorative effects through animal experiments. Our results demonstrated that interventions with orlistat (HFO), swimming exercise (HFS), and MLQE (HFM) significantly reduced weight gain, lipid accumulation, and oxidative stress in mace compared to the high-fat control (HFC) group. Histopathological analysis revealed improvements in liver steatosis, hepatocyte arrangement, inflammation, and adipocyte deformation. Molecular analyses indicated decreased mRNA levels of lipid synthesis genes (sterol regulatory element-binding protein 1 (SREBP1), bile salt export pump (BSEP), small heterodimer partner (SHP), and stearoyl-CoA desaturase-1 (SCD1)) and increased protein levels of farnesoid X receptor (FXR) and SHP in the intervention groups, consistent with RNA-seq findings. Additionally, RNA-seq analysis showed upregulation of fibroblast growth factor 21 (FGF21) and suppressor of cytokine signaling 2 (SOCS2), aligning with protein level observations. The interventions also modulated gut microbiota composition, reducing the Firmicutes-to-Bacteroidetes (F/B) ratio and adjusting the abundance of Acetatifactor, Roseburia, and Parasutterella. These findings suggest MLQE as a promising therapeutic approach for NAFLD, with lipid-lowering, antioxidative, and gut microbiota-regulating effects. FGF21 and SOCS2 may be key targets in NAFLD treatment.
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References
[1]
Salna, M., Fried, J., Kaku, Y., et al. Obesity is not a contraindication to veno-arterial extracorporeal life support. European Journal of Cardio-Thoracic Surgery, 2021, 60: 831–838. https://doi.org/10.1093/ejcts/ezab165
Zhou, M. The shifting income-obesity relationship: conditioning effects from economic development and globalization. SSM-Population Health, 2021, 15: 100849. https://doi.org/10.1016/j.ssmph.2021.100849
Grabia, M., Markiewicz-Żukowska, R., Socha, K., et al. Prevalence of metabolic syndrome in relation to cardiovascular biomarkers and dietary factors among adolescents with type 1 diabetes mellitus. Nutrients, 2022, 14: 2435. https://doi.org/10.3390/nu14122435
Rühle, A., Billeter, A. T., Müller-Stich, B. P. Metabolic surgery: paradigm shift in metabolic syndrome/diabetes therapy. Visceral Medicine, 2022, 38: 56–62. https://doi.org/10.1159/000521707
Guan, Q., Wang, Z., Cao, J., et al. Mechanisms of melatonin in obesity: a review. International Journal of Molecular Sciences, 2021, 23: 218. https://doi.org/10.3390/ijms23010218
Lugo, R., Avila-Nava, A., Pech-Aguilar, A. G., et al. Relationship between lipid accumulation product and oxidative biomarkers by gender in adults from Yucatan, Mexico. Scientific Reports, 2022, 12: 14338. https://doi.org/10.1038/s41598-022-18705-8
Stefan, N., Häring, H. U., Schulze, M. B. Metabolically healthy obesity: the low-hanging fruit in obesity treatment? The Lancet Diabetes & Endocrinology, 2018 , 6: 249–258. https://doi.org/10.1016/S2213-8587(17)1730292-9
Hashemi Kani, A., Alavian, S. M., Haghighatdoost, F., et al. Diet macronutrients composition in nonalcoholic fatty liver disease: a review on the related documents. Hepatitis Monthly, 2014, 14: e10939. https://doi.org/10.5812/hepatmon.10939
Deng, W., Fan, W., Tang, T., et al. Farnesoid x receptor deficiency induces hepatic lipid and glucose metabolism disorder via regulation of pyruvate dehydrogenase kinase 4. Oxidative Medicine and Cellular Longevity, 2022, 2022: 3589525. https://doi.org/10.1155/2022/3589525
Yin, X., Guo, X., Liu, Z., et al. Advances in the diagnosis and treatment of non-alcoholic fatty liver disease. International Journal of Molecular Sciences, 2023, 24: 2844. https://doi.org/10.3390/ijms24032844
Khalafi, M., Hossein Sakhaei, M., Kheradmand, S., et al. the impact of exercise and dietary interventions on circulating leptin and adiponectin in individuals who are overweight and those with obesity: a systematic review and meta-analysis. Advances in Nutrition, 2023 , 14: 128–146. https://doi.org/10.1016/j.advnut.2022.10.001
Zang, L., Shimada, Y., Nakayama, H., et al. Preventive effects of green tea extract against obesity development in zebrafish. Molecules, 2021, 26: 2627. https://doi.org/10.3390/molecules26092627
Abolghasemi, J., Farboodniay Jahromi, M. A., Hossein Sharifi, M., et al. Effects of Zataria oxymel on obesity, insulin resistance and lipid profile: a randomized, controlled, triple-blind trial. Journal of Integrative Medicine, 2020, 18: 401–408. https://doi.org/10.1016/j.joim.2020.06.003
Son, G. H., Lee, H. J., Na, Y. G., et al. Formulation and statistical analysis of an herbal medicine tablet containing Morus alba leaf extracts. Journal of Pharmaceutical Investigation, 2019, 49: 625–634. https://doi.org/10.1007/s40005-018-00417-9
Thaipitakwong, T., Supasyndh, O., Rasmi, Y., et al. A randomized controlled study of dose-finding, efficacy, and safety of mulberry leaves on glycemic profiles in obese persons with borderline diabetes. Complementary Therapies in Medicine, 2020, 49: 102292. https://doi.org/10.1016/j.ctim.2019.102292
Du, Y., Li, D., Lu, D., et al. Amelioration of lipid accumulations and metabolism disorders in differentiation and development of 3T3-L1 adipocytes through mulberry leaf water extract. Phytomedicine, 2022, 98: 153959. https://doi.org/10.1016/j.phymed.2022.153959
Shivangi, S., Dorairaj, D., Negi, P. S., et al. Development and characterisation of a pectin-based edible film that contains mulberry leaf extract and its bio-active components. Food Hydrocolloids, 2021, 121: 107046. https://doi.org/10.1016/j.foodhyd.2021.107046
Ji, S., Zhu, C., Gao, S., et al. Morus alba leaves ethanol extract protects pancreatic islet cells against dysfunction and death by inducing autophagy in type 2 diabetes. Phytomedicine, 2021 , 83: 153478. https://doi.org/10.1016/j.phymed.2021.153478
Zhu, Y., Zhou, X., Ling, N., et al. The effect of Guisangyou tea on abnormal lipid metabolism in mice induced by high-fat diet. Foods, 2023, 12: 2171. https://doi.org/10.3390/foods12112171
Chang, Y. C., Yu, M. H., Huang, H. P., et al. Mulberry leaf extract inhibits obesity and protects against diethylnitrosamine-induced hepatocellular carcinoma in rats. Journal of Traditional and Complementary Medicine, 2024, 14: 266–275. https://doi.org/10.1016/j.jtcme.2024.01.007
Bagnall, A., Radley, D., Jones, R., et al. Whole systems approaches to obesity and other complex public health challenges: a systematic review. European Journal of Public Health, 2019, 29: ckz185.472. https://doi.org/10.1093/eurpub/ckz185.472
Tabassum, S., Misrani, A., Yang, L. exploiting common aspects of obesity and Alzheimer’s disease. Frontiers in Human Neuroscience, 2020 , 14: 602360. https://doi.org/10.3389/fnhum.2020.602360
Yuan, Q., Xie, Y., Wang, W., et al. Extraction optimization, characterization and antioxidant activity in vitro of polysaccharides from mulberry ( Morus alba L.) leaves. Carbohydrate Polymers, 2015, 128: 52–62. https://doi.org/10.1016/j.carbpol.2015.04.028
Wang, G., Dong, J. Network pharmacology approach to evaluate the therapeutic effects of mulberry leaf components for obesity. Experimental and Therapeutic Medicine, 2021, 23: 56. https://doi.org/10.3892/etm.2021.10978
Yu, R., Gu, Y., Zheng, L., et al. Naringenin prevents NAFLD in the diet-induced C57BL/6J obesity model by regulating the intestinal barrier function and microbiota. Journal of Functional Foods, 2023, 105: 105578. https://doi.org/10.1016/j.jff.2023.105578
Filali-Mouncef, Y., Hunter, C., Roccio, F., et al. The ménage à trois of autophagy, lipid droplets and liver disease. Autophagy, 2022, 18: 50–72. https://doi.org/10.1080/15548627.2021.1895658
Clifford, B. L., Sedgeman, L. R., Williams, K. J., et al. FXR activation protects against NAFLD via bile-acid-dependent reductions in lipid absorption. Cell Metabolism, 2021, 33: 1671–1684. https://doi.org/10.1016/j.cmet.2021.06.012
Fang, X., Miao, R., Wei, J., et al. Advances in multi-omics study of biomarkers of glycolipid metabolism disorder. Computational and Structural Biotechnology Journal, 2022, 20: 5935–5951. https://doi.org/10.1016/j.csbj.2022.10.030
Wu, C., Lin, F., Qiu, S., et al. The characterization of obese polycystic ovary syndrome rat model suitable for exercise intervention. PLoS ONE, 2014 , 9: e99155. https://doi.org/10.1371/journal.pone.0099155
Tamaki, N., Kurosaki, M., Yasui, Y., et al. Attenuation coefficient (ATT) measurement for liver fat quantification in chronic liver disease. Journal of Medical Ultrasonics, 2021, 48: 481–487. https://doi.org/10.1007/s10396-021-01103-4
Kankara, I. A., Paulina, G. A., Aliyu, M. Hypoglycaemic and hypolipidemic effects of Treculia africana aqueous leaves extract in alloxan induced diabetic rats. Asian Journal of Biochemistry, Genetics and Molecular Biology, 2018, 4: 1–9. https://doi.org/10.9734/ajbgmb/2018/v1i1433
Lee, Y., Lee, E., Lee, M. S., et al. Hypolipidemic effect of mulberry leaf extract in rats fed a high-cholesterol diet (P06-014-19). Current Developments in Nutrition, 2019 , 3: nzz031.P06-014-19. https://doi.org/10.1093/cdn/nzz031.P06-014-19
Du, H., Li, C., Wang, Z., et al. Effects of Danhong injection on dyslipidemia and cholesterol metabolism in high-fat diets fed rats. Journal of Ethnopharmacology, 2021, 274: 114058. https://doi.org/10.1016/j.jep.2021.114058
Mashhad, I., Ghaeni Pasavei, A., Mohebbati, R., et al. Anti-hypolipidemic and anti-oxidative effects of hydroalcoholic extract of Origanum majorana on the hepatosteatosis induced with high-fat diet in rats. Malaysian Journal of Medical Sciences, 2020, 27: 57–69. https://doi.org/10.21315/mjms2020.27.1.6
Labban, R. S. M., Alfawaz, H. A., Almnaizel, A. T., et al. Garcinia mangostana extract and curcumin ameliorate oxidative stress, dyslipidemia, and hyperglycemia in high fat diet-induced obese Wistar albino rats. Scientific Reports, 2021, 11: 7278. https://doi.org/10.1038/s41598-021-86545-z
Zhong, Y., Pan, Y., Liu, L., et al. Effects of high fat diet on lipid accumulation, oxidative stress and autophagy in the liver of Chinese softshell turtle ( Pelodiscus sinensis). Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2020, 240: 110331. https://doi.org/10.1016/j.cbpb.2019.110331
Liao, S., Long, X., Zou, Y., et al. Mulberry leaf phenolics and fiber exert anti-obesity through the gut microbiota-host metabolism pathway. Journal of Food Science, 2021, 86: 1432–1447. https://doi.org/10.1111/1750-3841.15679
Huang, P., Hao, M., Gao, Q., et al. Constituents of Morus alba Var. multicaulis leaf improve lipid metabolism by activating the AMPK signaling pathway in HepG2 cells. Journal of Natural Medicines, 2022, 76: 200–209. https://doi.org/10.1007/s11418-021-01581-3
Du, Y., Li, D., Lu, D., et al. Lipid metabolism disorders and lipid mediator changes of mice in response to long-term exposure to high-fat and high sucrose diets and ameliorative effects of mulberry leaves. Food & Function, 2022, 13: 4576–4591. https://doi.org/10.1039/D1FO04146K
Wang, J., Yang, N., Xu, Y. Natural products in the modulation of farnesoid X receptor against nonalcoholic fatty liver disease. The American journal of Chinese Medicine, 2024, 52: 291–314. https://doi.org/10.1142/S0192415X24500137
Geng, L., Lam, K. S. L., Xu, A. The therapeutic potential of FGF21 in metabolic diseases: from bench to clinic. Nature Reviews Endocrinology, 2020, 16: 654–667. https://doi.org/10.1038/s41574-020-0386-0
Zarei, M., Pizarro-Delgado, J., Barroso, E., et al. Targeting FGF21 for the treatment of nonalcoholic steatohepatitis. Trends in Pharmacological Sciences, 2020, 41: 199–208. https://doi.org/10.1016/j.tips.2019.12.005
Schumacher, J. D., Guo, G. L. Pharmacologic modulation of bile acid-FXR-FGF15/FGF19 pathway for the treatment of nonalcoholic steatohepatitis. Handbook of Experimental Pharmacology, 2019, 256: 325–357. https://doi.org/10.1007/164_2019_228
Val, C. H., de Oliveira, M. C., Lacerda, D. R., et al. SOCS2 modulates adipose tissue inflammation and expansion in mice. The Journal of Nutritional Biochemistry, 2020, 76: 108304. https://doi.org/10.1016/j.jnutbio.2019.108304
Li, S., Han, S., Jin, K., et al. SOCS2 suppresses inflammation and apoptosis during NASH progression through limiting NF-κB activation in macrophages. International Journal of Biological Sciences, 2021, 17: 4165–4175. https://doi.org/10.7150/ijbs.63889
Hedl, M., Sun, R., Huang, C., et al. STAT3 and STAT5 signaling thresholds determine distinct regulation for innate receptor-induced inflammatory cytokines, and STAT3/stat5 disease variants modulate these outcomes. Journal of Immunology, 2019, 203: 3325–3338. https://doi.org/10.4049/jimmunol.1900031
Gui, L., Wang, S., Wang, J., et al. Effects of forsythin extract in Forsythia leaves on intestinal microbiota and short-chain fatty acids in rats fed a high-fat diet. Food Science and Human Wellness, 2023, 12: 1–18. https://doi.org/10.26599/FSHW.2022.9250055
Ou-Yang, J., Li, X., Liu, C., et al. Junshanyinzhen tea extract prevents obesity by regulating gut microbiota and metabolic endotoxemia in high-fat diet fed rats. Food Science and Human Wellness, 2024, 13: 2036–2047. https://doi.org/10.26599/FSHW.2022.9250169
Ye, J., Zhao, Y., Chen, X., et al. Pu-erh tea ameliorates obesity and modulates gut microbiota in high fat diet fed mice. Food Research International, 2021, 144: 110360. https://doi.org/10.1016/j.foodres.2021.110360
Xie, C., Zhang, Z., Yang, M., et al. Lactiplantibacillus plantarum AR113 exhibit accelerated liver regeneration by regulating gut microbiota and plasma glycerophospholipid. Frontiers in Microbiology, 2021, 12: 800470. https://doi.org/10.3389/fmicb.2021.800470
Gupta, Y., Ernst, A. L., Vorobyev, A., et al. Impact of diet and host genetics on the murine intestinal mycobiome. Nature Communications, 2023, 14: 834. https://doi.org/10.1038/s41467-023-36479-z
Ju, T., Kong, J. Y., Stothard, P., et al. Defining the role of Parasutterella, a previously uncharacterized member of the core gut microbiota. The ISME Journal, 2019, 13: 1520–1534. https://doi.org/10.1038/s41396-019-0364-5
Nie, K., Ma, K., Luo, W., et al. Roseburia intestinalis: a beneficial gut organism from the discoveries in genus and species. Frontiers in Cellular and Infection Microbiology, 2021 , 11: 757718. https://doi.org/10.3389/fcimb.2021.757718
Liu Y-F, Ling N, Zhang B, et al. Flavonoid-Rich mulberry leaf extract modulate lipid metabolism, antioxidant capacity, and gut microbiota in high-fat diet-induced obesity: potential roles of FGF21 and SOCS2. Food & Medicine Homology, 2024, 1(2): 9420016. https://doi.org/10.26599/FMH.2024.9420016
Network-based pharmacological study of potential targets of mulberry leaves and stems active ingredient for the treatment of NASH/NAFLD: This figure shows the intersection of the active ingredient targets of mulberry leaves and stems and NASH/NAFLD disease targets, and also shows the GO and KEGG enrichment analysis results of the intersected targets. (A) The "component-key target-pathway" of mulberry leaves and stems, (B) the Venn diagrams of the active ingredient targets and NASH/NAFLD disease targets of mulberry leaves and stems collected by different databases, (C) the KEGG enrichment analysis of the intersected targets, and (D) the GO enrichment analysis of the intersected targets, categorized into biological process (BP), cellular component (CC), and molecular function (MF).
Experimental design and Impact of interventions on high-fat diet-induced obesity in mice. This figure details the experimental design and evaluates the effects of various interventions on obesity induced by a high-fat diet in mice. (A) Experimental flow. (B-F) Effects of different interventions on several parameters. Body weight (B), Body weight gain (C), Liver/Body weight ratio (D), Abdominal fat/Body weight ratio (E), and Epididymal fat/Body weight ratio (F). (G) Stained tissue sections (200× magnification), highlighting fat vacuoles with straight arrows, cellular inflammatory infiltrates (predominantly mononuclear cells) with dashed arrows, and mononuclear cells with solid arrows. The data are expressed as mean ± SD and analyzed using one-way ANOVA, with different symbols indicating significant intergroup differences, *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the HFC group.
Influence of interventions on lipid profiles and antioxidant capacities. This figure explores the impact of various interventions on lipid metabolism and antioxidant defense systems in mice subjected to a high-fat diet. It assesses plasma TC (A), TG (B), HDL-C (C), LDL-C (D), CAT (E), GSH-Px (F), MDA (G), arteriosclerosis index (H), and liver TC (I), TG (J), HDL-C (K), LDL-C (L), CAT (M), GSH-Px (N), MDA (O), and SOD (P). The data expressed as mean ± SD, were analyzed using one-way ANOVA, with different symbols indicating significant differences between groups. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the HFC group.
Gene sequencing data analysis. This figure provides an in-depth analysis of gene sequencing data obtained from the study. (A) KEGG pathway analysis for differentially expressed genes in NC, HFC, and HFM group comparisons, (B) GO enrichment analysis of 348 genes that are differentially expressed among NC, HFC, and HFS groups, categorized into BP, CC, and MF, (C) Venn diagram illustrating the overlap of genes co-expressed in HFC and NC versus HFC and HFM. (D) Heatmap representing the up- and down-regulation of differentially expressed genes in NC compared to HFC, (E) Volcano plot highlighting the top 20 most up- and down-regulated genes in HFC/NC comparison.
Expression of genes and proteins related to lipid and bile acid metabolism in mice livers. (A-D) Gene expression levels for SREBF-1 (A), SCD-1 (B), BSEP (C), and SHP (D). (E and I) Western blot analysis. (F-K) Relative protein levels normalized to GAPDH for CYP7A1 (F), FXR (G), SHP (H), FGF21 (J), and SOCS2 (K). Data were expressed as mean ± SD and analyzed using one-way ANOVA, with different symbols denoting significant differences between groups. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the HFC group.
MLQE supplementation effects on modulating gut microbiota diversity and composition in mice on a high-fat diet. (A) The number of OTUs in different groups. (B, C) Alpha diversity using Chao 1 and Shannon indexes. (D-F) The relative abundance of gut microbiota species composition at various taxonomic levels, phylum (D), genus (E), and species (F). All species with an average abundance of less than 0.5% within the subgroup in which they are found, as well as those not yet annotated at the taxonomic level in question, should be combined into the category “Others”. (G-K) Compare the relative abundance at the genus level between different groups, HFC vs NC (G), HFC vs HFO (H), HFC vs HFS (J), HFC vs HFM (K). (I) Heat map that provides a detailed illustration of the variations in gut microbiota at the genus level.
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