Microalgae Oil Improves Hepatic Lipid Metabolism in A High-Fat Diet–Induced Mouse Model

Summary

Using a high-fat diet–induced mouse model of metabolic dysfunction–associated steatotic liver disease, this study evaluates the effects of docosahexaenoic acid (DHA)-rich microalgae oil supplementation and shows that the intervention improves hepatic lipid profiles and is associated with changes in gut microbiota composition.

Abstract

Metabolically, dysfunctional steatotic liver disease is a prevalent metabolic disorder associated with gut microbiota dysbiosis and hepatic lipid imbalance. In this study, a high-fat diet–induced mouse model was established to evaluate the effects of supplementation with DHA-rich microalgae oil. Mice (n = 4 per group) were fed a high-fat diet for 8 weeks and received daily oral administration of microalgae oil, probiotics, or the combination of DHA-rich microalgae oil and probiotics. Metabolic parameters, gut microbiota composition (16S rRNA sequencing), microbial functional pathways, and hepatic metabolomic profiles were assessed. The results showed that DHA-rich microalgae oil improved lipid homeostasis, as indicated by reduced serum LDL-c and hepatic triglyceride levels and increased high-density lipoprotein (HDL-c), and was associated with alleviation of liver injury and oxidative stress. Microbiome analysis revealed selective changes in gut microbial composition, including enrichment of Lactobacillus and Bifidobacterium and reduction of high-fat diet–associated taxa such as Clostridium and Ruminococcus. Functional profiling indicated alterations in microbial metabolic pathways, including the L-methionine salvage cycle and phenylethylamine degradation. Integrated microbiome–metabolome analysis further identified associations between microbial taxa and hepatic metabolites involved in fatty acid metabolism, bile acid turnover, and amino acid pathways. These findings indicate that DHA-rich microalgae oil supplementation is associated with improvements in hepatic lipid metabolism and gut microbiota composition in this model, without implying a direct causal mechanism.

Introduction

Metabolically-dysfunction-associated steatotic liver disease (MASLD) is a highly prevalent global condition, impacting roughly 32.4% of the world's population¹. MASLD is defined by excessive fat in the liver, which contributes to various associated health problems. The human digestive tract contains a sophisticated and balanced microbial community, including bacteria, viruses, fungi, and parasites². Due to the interaction between this microbial community and the liver, probiotics are seen as a promising method to prevent and treat MASLD by modulating this critical connection³. Emerging researc....

Protocol

Details of all animals, diets, reagents, kits, instruments, software, and service providers used in this protocol are provided in the Table of Materials. All animal studies were performed following the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals and received approval from the Experimental Animals Committee of Shandong Provincial Hospital (ethical approval no. 2022–007, approved on January 24, 2022).

CAUTION: Animal blood, fecal samples, and tissues should be handled as potentially biohazardous materials. All procedures involving live animals, biological samples, sharps, or....

Results

DHA-rich microalgae oil improves lipid metabolism, liver injury, and hepatic oxidative stress in HFD-induced MASLD mice

To evaluate the effect of dietary supplements on glucose and lipid homeostasis, biochemical parameters were assessed across five groups: regular chow (RC), high-fat diet (HFD), DHA-rich microalgae oil-supplemented, probiotic-supplemented, and combined DHA-rich microalgae oil + probiotic-supplemented mice. Because the primary objective of this study was to eva.......

Discussion

In recent years, several studies have investigated changes in the microbiome linked to MASLD. However, most of these studies have focused on comparing healthy individuals with MASLD patients or examining different steatosis grades²⁴. Consequently, there is still limited evidence detailing the specific gut microbiota changes associated with liver fibrosis in MASLD, even though fibrosis is the most critical factor in predicting patient outcomes. In this study, we show that DHA-rich microalgae oil pr.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2x PCR Master Mix	Takara Bio Inc., Japan	RR902A	Used for 16S rRNA PCR amplification
Acetonitrile	Merck, Germany	100030	Solvent used for UHPLC mobile phase
Anesthesia vaporizer	RWD Life Science	R580	Used to deliver isoflurane anesthesia to mice
Automatic biochemical analyser	Jian Cheng Biological Engineering Institute, Nanjing, China	A110-1 & A111-1	Measures serum parameters (glucose, TC, TG, LDL-c, HDL-c, AST, ALT) and liver lipid levels
Bacillus coagulans (GIM 1.645)	Guangdong Microbial Culture Collection Center, China	GIM 1.645	Component strain of probiotic formulation
Carboxymethylcellulose sodium (CMC-Na)	Sigma-Aldrich, USA	C4888	Used as emulsifier component for DHA-rich microalgae oil preparation
Catalase (CAT) assay kit	Nanjing Construction Ltd.	A007-1-1	Detects CAT activity (an indicator of antioxidant capacity) in liver tissue
DADA2 software package	DADA2 Development Team	Included in QIIME 2 v2019.4	Used for denoising and chimera filtering of sequencing reads
DHA-rich microalgae oil (Schizochytrium sp.-derived)	-	-	Rich in docosahexaenoic acid (DHA); administered daily by gavage at 10µL/g as the experimental intervention
Experimental animals (Male C57BL/6J mice)	Beijing Vital River Laboratory Animal Technology Co.	-	6 weeks old, n = 4 per group; used for establishing the MASLD model via HFD feeding
Formic acid	Sigma-Aldrich, USA	F0507	Mobile phase additive for metabolomics analysis
Greengenes database	Greengenes	Version 13_8	Used for microbial taxonomic assignment
High-Fat Diet (HFD)	Research Diets Inc., USA	D12492	Composed of 20% carbohydrates, 20% protein, 60% fat (kcal); fed for 8 weeks to induce MASLD
High-resolution mass spectrometer	Thermo Fisher Scientific, USA	Q Exactive Orbitrap	Used for metabolite detection in positive and negative ion modes
Human Metabolome Database (HMDB)	University of Alberta, Canada	2024 release	Public metabolite annotation database
Illumina MiSeq platform	Illumina, Inc., USA	MiSeq System	Used for paired-end 2 × 250 bp sequencing
Isoflurane	Merck, Germany	PHR2874	Used for inhalation anesthesia in mice
Kyoto Encyclopedia of Genes and Genomes (KEGG)	Kanehisa Laboratories, Japan	Version 2023	Database used for pathway enrichment and metabolite annotation
Lactobacillus acidophilus (KDB-03)	Shandong Academy of Agricultural Sciences, China	-	Component strain of probiotic formulation
Lactobacillus casei (KDB-LC)	Shandong Academy of Agricultural Sciences, China	-	Component strain of probiotic formulation
Lactobacillus plantarum (DY-1)	Shandong Academy of Agricultural Sciences, China	-	Component strain of probiotic formulation
Liquid nitrogen	Air Liquide	-	Used for rapid freezing and preservation of fecal and liver samples
Malondialdehyde (MDA) assay kit	Nanjing Construction Ltd.	A003-1	Detects MDA (a marker of lipid peroxidation/oxidative damage) in liver tissue
MassLynx software	Waters Corporation, USA	Version 4.1	Used for metabolomics raw data processing, feature extraction, and normalization
Methanol	Merck, Germany	106035	Solvent used for metabolite extraction
Microbiome data analysis platform	Genescloud	V1	Online platform: https://www.genescloud.cn/home; used for PCoA, NMDS, LEfSe and other microbiome visualization analyses
OMEGA Soil DNA Kit	Omega Bio-Tek, Norcross, GA, USA)	(M5635-02)	DNA Isolation
OTUs reference sequences	Genescloud		Referenced from McDonald et al. 2012; used for taxonomy assignment of amplicon sequence variants (ASVs)
Phosphate-buffered saline (PBS)	Gibco, Thermo Fisher Scientific, USA	10010023	Used for bacterial pellet resuspension
Primer 341F/806R	Tsingke Biotechnology Co., Ltd., China	-	Used for amplification of the 16S rRNA V3–V4 region. 341F: 5′-CCTACGGGNGGCWGCAG-3′; 806R: 5′-GGACTACHVGGGTATCTAAT-3′
Probiotics	-	-	Specific strains not explicitly mentioned; used as a comparative intervention (mainly improves glucose homeostasis)
QIIME 2 software	-	2019.4	Used for microbiome bioinformatics analysis (ASV processing, alpha/beta diversity calculation)
R software	-	4.4.1	Used for liver metabolite enrichment analysis and Spearman correlation analysis of microbial-metabolite associations
Refrigerated centrifuge	Eppendorf, Germany	5810R	Used for serum and metabolomics sample centrifugation
Reverse-phase C18 column	Waters Corporation, USA	186002350	Used for chromatographic separation of metabolites
Saline	Shijiazhuang No.4 Pharmaceutical	2211112304	Administered daily by gavage to the control group (vs. microalgae oil treatment)
Standard chow diet	Keao Xieli Feed Ltd., China	1016706714625204224	Composed of 70% carbohydrates, 20% protein, 10% fat (kcal); fed to the control (RC) group
Sterile feeding needle	Instech Laboratories, USA	FTP-20-38	Used for oral gavage administration
Superoxide dismutase (SOD) assay kit	Nanjing Construction Ltd.	A001-3	Detects SOD activity (an indicator of antioxidant capacity) in liver tissue
Tween-80	Sigma-Aldrich, USA	P4780	Used as emulsifier component for DHA-rich microalgae oil preparation
Ultrasonic homogenizer	Scientz Biotechnology, China	JY92-IIN	Used for liver tissue sonication during metabolite extraction
Untargeted Hepatic Metabolomics	Metabo-Profile Biotechnology (Shanghai) Co., Ltd.	Project Code: P035Y23A13_SFMU_MJWang_SH
Vortex mixer	Thermo Fisher Scientific, USA	LP Vortex Mixer	Used for sample homogenization and emulsification