Blue mussel (Mytilus edulis) lipid extract prevents metabolic dysfunction‐associated steatotic liver disease

Abstract

BACKGROUND

The prevalence of metabolic dysfunction‐associated steatotic liver disease (MASLD) is increasing year by year and it has become an important factor that endangers health. Blue mussel lipid extract (BMLE) is rich in n‐3 polyunsaturated fatty acids (PUFAs) and astaxanthin, which has strong anti‐inflammatory activity and has been shown to have a good effect on type 2 diabetes, atherosclerosis, and so forth. The aim of the present study was to investigate the effect of BMLE on MASLD and its possible mechanism.

RESULTS

The results showed that BMLE could significantly reduce liver lipid accumulation. In BMLE group the serum levels of total cholesterol (TC), triglyceride (TG), low‐density lipoprotein cholesterol (LDL‐C), aspartate aminotransferase (AST) and liver levels of lipid synthesis proteins fatty acid synthase (FAS), acetyl‐CoA carboxylase (ACC), sterol regulatory element binding protein‐1 (SREBP‐1) and peroxisome proliferator‐activated receptor γ (PPARγ) were significantly lower than the corn oil (CO) group. And BMLE down‐regulated the NLRP3/Caspase‐1/GSDMD pyroptosis pathways, reduced the serum levels of tumor necrosis factor α (TNF‐α), interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6) inflammatory factors. In addition, BMLE increased gut microbiota unclassified_Muribaculaceae, and reduced uncultured_Bacteroidales_bacterium and Dubosiella in genus level. Unclassified_Muribaculaceae was negatively correlated with IL‐1β, TNF‐α, IL‐6, FAS and PPARγ. Uncultured_Bacteroidales_bacterium was positively correlated with TG, IL‐1β, TNF‐α, IL‐6, SREBP1, ACC and Caspase‐1. Dubosiella and TC are also significantly positively correlated.

CONCLUSION

BMLE may improve MASLD by regulating fat synthesis and reducing inflammatory factors, as well as regulating gut microbiota. © 2026 Society of Chemical Industry.

INTRODUCTION

Metabolic dysfunction‐associated steatotic liver disease (MASLD) is a clinicopathologic syndrome characterized by the excessive accumulation of fat in hepatocytes. It is an acquired metabolic stress‐induced liver injury closely associated with obesity, insulin resistance, type 2 diabetes mellitus (T2DM), hypertension, hyperlipidemia, and metabolic syndrome. ¹ The disease spectrum of MASLD include simple fatty liver disease (SFL), non‐alcoholic steatohepatitis (NASH), and cirrhosis. ² The global prevalence of MASLD has increased from 25.5% in 2005 to 32.4% in 2022. ³ There is no specific therapeutic agent for MASLD up to now. Therefore, increasing attention has been paid to the prevention of MASLD through nutritional strategies. ⁴

Mussel are bivalve mollusks widely distributed along the coastal area of China. ⁵ Blue mussel lipid extract (BMLE) is rich in n‐3 polyunsaturated fatty acids (PUFAs), mainly eicosapentaenoic acid (EPA, C20:5n‐3) and docosahexaenoic acid (DHA, C22:6n‐3). Previous studies have demonstrated that supplementation with n‐3 PUFA can reduce glycemic parameters and liver fat content.^{6, 7} In addition, BMLE also contains astaxanthin, a carotenoid present in various microorganisms and marine organisms that possesses potent antioxidant activity – 500 times stronger than that of vitamin E.^{8, 9} Astaxanthin has also been shown to alleviate MASLD and reduce liver and adipose tissue inflammation in mouse experiments.^{10, 11} Our previous studies has indicated that BMLE can effectively improve glycemic traits, inflammatory cytokines and lipid profiles in patients with T2DM, and it is also superior to fish oil in ameliorating atherosclerosis in APOE^−/− mice.^{5, 12} Moreover, the beneficial effect of BMLE on T2DM is strongly correlated with gut microbiota. ¹³ In conclusion, BMLE has beneficial effects on glucose and lipid metabolism, as well as anti‐inflammatory properties. Moreover, MASLD is also closely related to glucose and lipid metabolism disorders and inflammation. Therefore, we believe that BMLE has a certain preventive effect on MASLD. However, the preventive effect of BMLE on MASLD remains unclear.

Given the beneficial effects of BMLE on glucose and lipid metabolism as well as its anti‐inflammatory properties, we hypothesized that BMLE also exerts a preventive effect on MASLD. Therefore, the present study aimed to explore the effect of BMLE on MASLD and its underlying mechanism in high‐fat diet (HFD)‐induced MASLD mice.

MATERIALS AND METHODS

Ethical approval

The study was approved by the Ethics Committee of Medical College of Qingdao University (QDU‐AEC‐2024748). All animal experimental procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Qingdao University, China.

Treatment oils

Mussel meat was freeze‐dried by vacuum, homogenized into powder, and mussel oil (MO) was separated by supercritical fluid extraction (Essen Biotechnology Co. Ltd, Harbin, China). ⁵ Fish oil (FO) was produced by Prospect Biotechnology Co. Ltd, Shanghai, China. Corn oil (CO) was purchased from a local supermarket (Brand: LONGEVITY FLOWER). BMLE and FO contained 199.25 and 233.96 mg/g of EPA, 257.37 and 175.13 mg/g of DHA, respectively. The total n‐3 PUFA content in BMLE and FO was 54.59% and 52.33%, respectively. BMLE contained 191 mg/kg astaxanthin, which was under the detection limit in FO and CO.

Study design

The study design is shown in Fig. 1. Healthy male C57BL/6J mice (6‐week‐old, 18 ± 2 g), purchased from Vital River Laboratory Animal Technology Co., Ltd, Beijing, China, were raised in a standardized environment (12 h:12 h light/dark cycle, temperature 21 ± 1 °C, relative humidity 60%). After 1‐week of adaptive feeding, 46 mice were randomly allocated into four groups: (1) control group (n = 7): normal‐diet (ND, 30% fat) + CO (0.1 mL per 20 g of body weight); (2) CO group (n = 10): HFD (60% fat) + CO (0.1 mL per 20 g of body weight); (3) BMLE group (n = 10): HFD + BMLE (0.1 mL per 20 g of body weight); (4) FO group (n = 9): HFD + FO (0.1 mL per 20 g of body weight). The detailed composition and fatty acid composition of the feed are shown in Supporting Information Tables S1 and S2. After 12 weeks of treatment, mice were sacrificed to collect tissues and blood samples for subsequent detection.

Figure 1.

Diagram of study design.

Fatty acid determination

The fatty acids compositions (percent in total fatty acids) in three treatment oils and the liver of mice were detected by a gas chromatography (GC) system equipped with an Agilent DB‐23 column (60 m, 0.25 mm × 0.25 μm; Agilent Technologies, Santa Clara, CA, USA) according to our previous study. ¹² The lipids in the samples were extracted with chloroform/methanol (1:1, v/v). The extracted lipids were mixed with 3 mL methanol/sulfuric acid (20: 1, v/v) and 1 mL toluene and stored at 70 °C for 2 h to produce fatty acid methyl esters. The fatty acid methyl esters were filtered by Sep‐Pak silica gel column, the solvent was removed by a nitrogen dryer, and then dissolved in n‐hexane for GC detection. The sample inlet temperature was 260 °C, while the pressures of the gases nitrogen and hydrogen were 50 and 75 kPa, respectively. The temperature program was as follows: 0–2 min, 140 °C; 2–3 min, 140–160 °C; 3–8 min, 160 °C; 8–9 min, 160–180 °C; 9–21 min, 180 °C; 21–22 min, 180–200 °C; 22–30 min, 200 °C; 30–30.25 min, 200–205 °C; 30.25–41.25 min, 205 °C.

Determination of serum lipids and inflammatory factors

Biochemical kits (Jiancheng Biological Engineering Institute, Nanjing, China) were used to detect blood lipids, aspartate aminotransferase (AST) and alanine aminotransferase (ALT): triglyceride (TG; A110‐1‐1), total cholesterol (TC; A111‐1‐1), high‐density lipoprotein cholesterol (HDL‐C; A112‐1‐1), low‐density lipoprotein cholesterol (LDL‐C; A113‐1‐1), AST (C0109‐2‐1), ALT (C009‐2‐1). Enzyme‐linked immunosorbent assay (ELISA) kits (Jining Industrial Co., Ltd, Shanghai, China) were used to detect serum inflammatory factors: interleukin‐6 (IL‐6; JN16894), interleukin‐10 (IL‐10, JN17305), interleukin‐1β (IL‐1β, JN16939), tumor necrosis factor‐α (TNF‐α; JN17113).

Detection of the protein levels

Liver protein lysates were obtained by adding radioimmunoprecipitation assay (RIPA) buffer to liver tissue and homogenizing. The liver protein lysates were isolated by 10% or 6% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to 0.45 μm polyvinylidene difluoride (PVDF) membrane (IPVH00010; Merck Millipore, Burlington, MA, USA). After blocking in 5% skim milk (GC310001; Service bio, Donghu new technology zone, Wuhan, Hubei, China) for 2 h, the membranes were incubated overnight at 4 °C with primary antibodies: acetyl‐CoA carboxylase (ACC, 1:1000, ET1609; HuaAn Bio, Laiwu, Jinan, Shandong, China); sterol regulatory element binding protein‐1 (SREBP‐1, 1:1000, 14088‐1‐AP; Proteintech, Donghu new technology zone, Wuhan, Hubei, China); peroxisome proliferator‐activated receptor γ (PPARγ, 1:1000, 340844; Zen bio, Wuhou, Chengdu, Sichuan, China); fatty acid synthase (FAS, 1:5000, ab128856; abcam, Cambridge, UK); NLR family pyrin domain‐containing protein 3 (NLRP3, 1:1000, 68102‐1‐Ig; Proteintech); Caspase‐1 (1:1000, A0964; ABclonal, Donghu new technology zone, Wuhan, Hubei, China), Gasdermin D (GSDMD, 1:1000, ab215203; abcam); Gasdermin D‐N (GSDMD‐N, 1:1000, ab215203; abcam); and 𝛽‐actin (1:1000, GB15003; Service bio). The membranes were then incubated with horseradish peroxidase (HRP)‐conjugated secondary antibody for 90 min at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL) solution on Ultra Sensitive Multifunctional Imager and analyzed using the ImageJ software.

Liver tissue H&E and Oil Red‐O staining

Liver tissue was fixed with 4% paraformaldehyde solution. Subsequently, the liver tissue sections were stained with hematoxylin and eosin (H&E) and Oil Red‐O to observe the histological changes and lipid accumulation in the liver. Image analysis using ImageJ software.^{14, 15}

16S rRNA sequencing

Total DNA was extracted using the TGuide S96 magnetic bead soil/fecal genomic DNA extraction kit (DP812; Tiangen Biochemical Technology Co. Ltd, Beijing, China). The V3–V4 region of the bacterial 16S rRNA gene was amplified with primers as follow (338F: 5′‐ ACTCCTACGGGAGGCAGCA′ and 806R: 5′‐ GGACTACHVGGGTWTCTAAT‐3′). Sequencing was performed using Illumina novaseq6000 sequencer (Illumina, San Diego, CA, USA). The platform BMKCloud (www.biocloud.net) was used for subsequent bioinformatics analysis.

Statistical analysis

Data were expressed as mean ± standard deviation unless otherwise specified. One‐way analysis of variance (ANOVA) was used for significance test, and least significant difference (LSD) post hoc test was used for multi‐group comparison. Spearman correlation analysis was used to evaluate the linear relationship between continuous variables (P value was adjusted for false discovery rate (FDR)). In the correlation analysis, a q value less than 0.2 is considered to be statistically significant. ¹⁶ In other analyses, a P value less than 0.05 is regarded as statistically significant. All statistical analyses were conducted using SPSS 27.0. Figures were generated using GraphPad Prism 8.02.

RESULTS

Effect of treatment oil on body weight, serum biochemical indices and inflammatory factors

Successful induction of the MASLD model by HFD was confirmed by significant increases in body weight, serum AST and evident hepatic lipid accumulation (hepatic Oil Red‐O staining) in the CO group compared to the control group.^{17, 18} At 12 weeks, all three groups of mice fed the HFD had a higher body weight than the ND‐fed control group (Fig. 2(A)) (P < 0.05). There was no difference in average food intake among the four groups (Fig. 2(B)) (P > 0.05). However, the body weight of BMLE was lower than that of the CO group (P < 0.05), and there was no significant difference between the FO group and the CO group or the BMLE group. Compared with the control group, the HFD induced an increase in TC, TG, LDL‐C (P < 0.05), but BMLE could significantly alleviate the increase (Fig. 2(C)–(E)) (P < 0.05). TC and LDL‐C were also reduced in the FO group compared with the CO group (P < 0.05), but LDL‐C was still higher than that in the control group (P < 0.05). There were no significant differences in HDL‐C among the four groups (Fig. 2(F)). Serum level of IL‐1β, IL‐6, IL‐10 and TNF‐α were shown in Fig. 2(G)–(J)). The levels of IL‐1β, IL‐6 and TNF‐α in the BMLE group were significantly lower than those in the CO and FO groups (P < 0.05).

Figure 2.

Effect of BMLE on body weight, serum biochemical indices and inflammatory factors. (A) Body weight, (B) food intake, (C) TC, (D) TG, (E) LDL‐C, (F) HDL‐C, (G) IL‐1β, (H) IL‐6, (I) IL‐10 and (J) TNF‐α levels. Data are expressed as mean ± standard deviation. Groups sharing the same superscript have no significant difference from each other in the post hoc analysis (P > 0.05).

BMLE reduced liver lipid accumulation and liver damage

AST in BMLE and control groups was significantly lower than that in the CO group (P < 0.05), and there was no difference between FO and the other three groups (Fig. 3(A)). There was no significant difference in ALT among the four groups (Fig. 3(B)). Oil Red‐O staining of liver tissue sections showed obvious lipid deposition in the CO group (Fig. 3(C),(D)). Compared with the CO group, the positive area of Oil Red‐O staining was obviously decreased in the BMLE and FO groups (P < 0.05), and the improving effect of BMLE was better than that of FO (Fig. 3(C),(D)) (P < 0.05). H&E staining showed that in the normal control group, the structure of the hepatic cords was normal, which was arranged radially with normal hepatocyte morphology (Fig. 3(E)). Lipid droplets of varying size and quantity appeared in most hepatocytes of the CO and FO groups. Compared with the CO group, the number of lipid droplets in hepatocytes was reduced in the BMLE group.

Figure 3.

Effects of treatment oil on the liver. (A) AST, (B) ALT, (C) positive Oil Red‐O staining area, (D) Oil Red‐O staining results, (E) H&E staining results (n = 6). Data are expressed as mean ± standard deviation. Groups sharing the same superscript have no significant difference from each other in the post hoc analysis (P > 0.05).

Correlation between fatty acid content in liver and blood lipids and inflammatory factors

The fatty acid composition of mice liver in the four groups is shown in Table 1. We analyzed correlations between liver fatty acids and blood lipids and inflammatory factors (Fig. 4 and Table S3). No significant results were observed in this correlation analysis (q value > 0.2).

Table 1.

Fatty acids composition (percent of total fatty acids) of mice liver in four groups

	Control	CO	MO	FO
C14:0	0.72 ± 0.34	0.43 ± 0.22	0.66 ± 0.21	0.48 ± 0.21
C15:0	0.66 ± 1.42	0.37 ± 0.54	1.08 ± 0.85	0.91 ± 1.32
C16:0	19.05 ± 1.63	21.19 ± 3.21	26.27 ± 3.06	26.30 ± 2.10
C16:1n‐7	1.55 ± 0.79	1.65 ± 0.89	1.93 ± 0.41	1.64 ± 0.60
C17:0	2.61 ± 2.67	1.44 ± 1.82	1.36 ± 1.53	0.79 ± 0.42
C18:0	9.41 ± 1.95	7.33 ± 2.49	9.37 ± 1.33	9.26 ± 1.07
C18:1n‐9	20.60 ± 5.54	22.24 ± 4.35	19.42 ± 4.29	18.65 ± 2.93
C18:2n‐6	21.30 ± 2.60	24.09 ± 3.85	12.66 ± 2.82	13.35 ± 1.67
C18:3n‐3	1.27 ± 1.16	0.87 ± 0.63	1.77 ± 0.54	1.41 ± 1.33
C20:0	0.37 ± 0.30	0.20 ± 0.15	0.20 ± 0.07	0.18 ± 0.10
C20:1n‐9	0.64 ± 0.44	0.28 ± 0.12	0.27 ± 0.16	0.45 ± 0.28
C20:2n‐6	0.46 ± 0.15	0.28 ± 0.10	0.24 ± 0.19	0.16 ± 0.04
C20:3n‐6	0.61 ± 0.24	0.59 ± 0.14	0.32 ± 0.06	0.30 ± 0.09
C20:4n‐6	9.26 ± 3.05	9.79 ± 1.86	4.23 ± 0.65	5.11 ± 1.34
C20:4n‐3	1.48 ± 2.81	0.67 ± 0.85	0.27 ± 0.26	0.49 ± 0.75
C20:5n‐3	1.21 ± 1.50	0.76 ± 0.70	4.06 ± 1.25	4.49 ± 0.91
C22:1n‐9	3.78 ± 1.44	2.47 ± 0.87	1.78 ± 0.74	0.48 ± 0.48
C22:6n‐3	5.03 ± 1.77	5.34 ± 1.21	14.11 ± 2.28	15.56 ± 2.76
Total n‐3 PUFA	9.37 ± 4.17	7.64 ± 2.01	20.20 ± 2.89	21.96 ± 1.74
Total n‐6 PUFA	31.63 ± 5.06	34.75 ± 5.06	17.45 ± 2.93	18.91 ± 2.62
Total n‐3 PUFA/Total n‐6 PUFA	0.31 ± 0.16	0.22 ± 0.07	1.19 ± 0.24	1.18 ± 0.20

Note: CO, corn oil; FO, fish oil; MO, mussel oil; PUFA, polyunsaturated fatty acid.

Figure 4.

Correlation between fatty acids content in liver and blood lipids and inflammatory factors. (A) Spearman correlation analysis, *, P < 0.05, (B) FDR correction performed based on (A).

Effect of treatment oil on hepatic lipid synthesis proteins

The protein levels of FAS, SREBP‐1, ACC and PPARγ were significantly higher in the CO group than the control group (P < 0.05) (Fig. 5(A)–(D)). BMLE but not FO significantly inhibited the expression of FAS, SREBP‐1, ACC and PPARγ (P < 0.05).

Figure 5.

Effect of treatment oil on hepatic lipid synthesis proteins (n = 4). (A) FAS, (B) SREBP‐1, (C) ACC, (D) PPARγ. Data are expressed as mean ± standard deviation. Groups sharing the same superscript have no significant difference from each other in the post hoc analysis (P > 0.05).

Effect of treatment oils on pyroptosis pathway in the liver

The protein levels of NLRP3, Caspase‐1 and GSDMD‐N in the control and BMLE groups were significantly lower than those in the CO and FO groups (P < 0.05) (Fig. 6(A),(B),(D)). Furthermore, the BMLE group showed a significantly lower protein level of GSDMD than the CO group (P < 0.05), but did not differ significantly with the FO group (P>0.05) (Fig. 6(C)).

Figure 6.

Effect of treatment oils on inflammatory factors in the liver (n = 4). (A) NLRP3, (B) Caspase‐1, (C) GSDMD, (D) GSDMD‐N. Data are expressed as mean ± standard deviation. Groups sharing the same superscript have no significant difference from each other in the post hoc analysis (P > 0.05).

Effects of BMLE on gut microbiota

There was no significant difference in alpha‐diversity between groups (Supporting Information Fig. S1). We analyzed the distribution histogram at the genus level (Fig. 7(A)). The relative abundance of unclassified_Muribaculaceae in the CO group was lower than that in the control group and BMLE group (P < 0.05) (Fig. 7(B)). The relative abundance of uncultured_Bacteroidales_bacterium in the CO and FO groups was higher than that in the control and BMLE groups (P < 0.05) (Fig. 7(C)). And the relative abundance of Dubosiella in the CO group was higher than that in the other three groups (P < 0.05) (Fig. 7(D)). Further correlation analysis was performed between genus level of gut microbiota and serum indices, lipid synthesis proteins and pyroptosis pathway proteins (Fig. 7(E) and Table S4). The results showed that unclassified_Muribaculaceae was negatively correlated with IL‐1β, TNF‐α, IL‐6, FAS and PPARγ. Uncultured_Bacteroidales_bacterium was positively correlated with IL‐1β, TNF‐α, IL‐6, SEREBP1, ACC and Caspase‐1. Dubosiella and TC, FAS are also significantly positively correlated (q value < 0.2).

Figure 7.

Effect of treatment oils on gut microbiota (n = 6). (A) Genus level distribution histogram, (B) unclassified_Muribaculaceae, (C) uncultured_Bacteroidales_bacterium, (D) Dubosiella, (E) correlation between gut microbiota at genus level and blood lipids and inflammatory factors, (F) FDR correction performed based on (E). Groups sharing the same superscript have no significant difference from each other in the post hoc analysis (P > 0.05). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ^, after FDR correction; q value < 0.2.

DISCUSSION

The main cause of MASLD is excessive accumulation of fat in the liver. The major fatty acids in both MO and FO are long‐chain n‐3 PUFAs (mainly C20:5n‐3 and C22:6n‐3). Previous results from animal and human studies have shown that n‐3 PUFA can reduce lipid accumulation in the liver and improve lipid degeneration and insulin resistance.^{19, 20} Furthermore, n‐3 PUFA is able to ameliorate TC, TG and LDL‐C elevations induced by HFD.^{6, 21} However, in the present study, correlation analyses between hepatic fatty acid content and blood lipids or inflammatory factors did not yield significant results. This finding suggests that the observed reductions in blood lipids and inflammatory factors in the MO group may be independent of n‐3 PUFAs. Notably, BMLE also contains astaxanthin. In our previous study, the content of astaxanthin in BMLE was higher than that in FO. ¹² Therefore, we hypothesize that the lipid‐lowering and anti‐inflammatory effects may be attributable to astaxanthin. Astaxanthin, as a carotenoid with strong antioxidant activity, has also become a research hotspot in the prevention of MASLD. A clinical study showed astaxanthin can reduce liver steatosis in NASH patients, ²² prevent weight gain in mice induced by HFD, and reduce fat droplet formation. ²³ Our previous studies also revealed that the anti‐inflammatory effect of BMLE is superior to that of FO.^{5, 12} BMLE has high concentrations of astaxanthin. The presence of astaxanthin may be the reason why BMLE improves blood lipids slightly better than FO. The liver is the central hub of lipid transport between different organs and plays a crucial role in regulating lipid synthesis and metabolism. ²⁴ MASLD includes a range of liver diseases, the first stage of which is characterized by simple steatosis, where liver fat accumulates in liver cells. ²⁵ In the present study, through the intake of BMLE and FO, the stained area of fat droplets in Oil Red‐O staining was significantly reduced, and the effect of BMLE was still significantly better than that of FO.

PPARγ is a hormone receptor that is critical for both lipid metabolism and adipocyte differentiation.^{26, 27, 28} SREBP‐1 is a transcription factor that regulates lipid metabolism and participates in fatty acid synthesis mainly by regulating the expression of downstream target genes FAS and ACC.^{29, 30, 31} In the process of lipid synthesis, ACC and FAS cooperate with each other to regulate fatty acid synthesis. ACC catalyzes the carboxylation of acetyl‐CoA to malonyl‐CoA, which provides the substrate for FAS to synthesize fatty acids. FAS catalyzes acetyl‐CoA and malonyl‐CoA to produce long chain fatty acids.^{32, 33} In the present study, BMLE significantly decreased the expression of SREBP‐1, ACC, FAS, and PPARγ in the liver of mice on HFD. These results indicated that BMLE could improve MASLD by down‐regulating the expression of proteins related to fat synthesis.

The occurrence and development of MASLD is also closely related to the NLRP3 inflammasome. ³⁴ The pro‐inflammatory cytokine IL‐1β plays a key role in the development and progression of non‐alcoholic fatty liver disease, and IL‐1β is activated via the classic NLRP3 inflammasome activation pathway. ³⁵ Caspase‐1 is a pro‐inflammatory protease that cleaves pro‐IL‐1β into IL‐1β, and the NLRP3 inflammasome is involved in Caspase‐1 activation.^{36, 37} At the same time, activated Caspase‐1 can also lyse GSDMD to form GSDMD‐N, which can induce the formation of cell membrane pores and lead to the release of IL‐1β into the extracellular. ³⁸ In the present study, BMLE significantly reduced the expression of NLRP3, Caspase‐1, GSDMD, and GSDMD‐N in liver, and the level of IL‐1β in serum. TNF‐α is also a key cytokine in the pathogenesis of MASLD. TNF‐α is produced in the early stages of the MASLD process and plays an important role in the pathogenesis of NASH and MASLD. ³⁹ In the present study, serum TNF‐α level was significantly lower in the BMLE group than in the CO and FO groups. Our previous study also found that in patients with type 2 diabetes, serum TNF‐α and IL‐1β were significantly lower in patients consuming BMLE than those taking FO. ⁵ These results suggest that BMLE has good anti‐inflammatory activity and can improve MASLD by inhibiting the NLRP3/Caspase‐1/GSDMD pyroptosis pathway.

The occurrence and development of MASLD is also closely related to gut microbiota. Previous clinical and animal studies have shown that the gut microbiota and its metabolites have a profound impact on the onset and progression of MASLD by regulating gut function, liver metabolism, and inflammatory responses.^{40, 41, 42} In the present study, we also found that BMLE can regulate some bacteria that are significantly related to blood lipids or inflammatory factors at the genus level, such as unclassified_Muribaculaceae, uncultured_Bacteroidales_bacterium and Dubosiella. The BMLE intervention significantly reversed the decrease in unclassified_Muribaculaceae, and Muribaculaceae could inhibit obesity by participating in mitochondrial energy metabolism and inflammatory processes. ⁴³ Protective pretreatment with Bifidobacterium pseudolongum CCFM1253 also significantly reduced the relative abundance of uncultured_Bacteroidales_bacterium in mice with acute liver injury. ⁴⁴ Monascus red pigment, which can ameliorate MASLD, also reduced the relative abundance of Dubosiella in HFD‐induced mice. ⁴⁵ This suggests that BMLE may improve MASLD by improving the abundance of gut microbiota.

There is a close relationship between pyroptosis pathway, fat synthesis and gut microbiota. The previous studies have shown that liver lipid accumulation, accompanied by the proliferation of inflammatory cytokines and dysbiosis of the intestinal flora, is considered to be the main mechanism underlying its occurrence and development. ⁴⁶ The occurrence and development of MASLD is also related to the intestinal‐liver axis.^{47, 48} Previous studies have shown that NLRP3 inflammasome inhibitor can reverse obesity, systemic inflammation and astrogliosis induced by HFD in mice. ⁴⁹ And changes in the diversity and variety of the gut microbiota affect neuroinflammation by activating the NLRP3 inflammasome and releasing inflammatory cytokines, and in turn, gut microbiota composition is influenced by NLRP3 inflammasome activation. ⁵⁰ Another previous study has shown that unclassified_Muribaculaceae is negatively correlated with the levels of IL‐1β, TNF‐α, and IL‐6. ⁵¹ In the present study, BMLE could inhibit the NLRP3/Caspase‐1/GSDMD pyroptosis pathway, reduce fat synthesis protein, and regulate gut microbiota, from these three aspects the prevention of MASLD can be achieved. Our results indicate that BMLE exerts its protective effects against MASLD via a multi‐targeted network. Based on the results of this study, we have proposed a comprehensive mechanism model (Fig. 8). The process appears to be initiated by shifts in the gut microbiota, notably an increase in unclassified_Muribaculaceae alongside decreases in uncultured_Bacteroidales_bacterium and Dubosiella. These microbial alterations may, in turn, reduce the delivery of pro‐inflammatory mediators to the liver, leading to the inhibition of the NLRP3 inflammasome and the downstream Caspase‐1/GSDMD‐mediated pyroptotic cascade. Consequently, the dampened intrahepatic inflammatory environment likely suppresses SREBP‐1 and other key transcription factors, thereby decreasing the expression of lipogenic enzymes such as FAS and ACC. Although a direct causal relationship between the microbiota, inflammation, and lipid metabolism remains to be fully elucidated, this proposed model provides a holistic mechanistic basis for BMLE's efficacy and highlights critical avenues for future research.

Figure 8.

The mechanism by which BMLE prevents MASLD. The changes in the gut microbiota regulated by BMLE, including an increase in unclassified_Muribaculaceae and a decrease in uncultured_Bacteroidales_bacterium and Dubosiella, might be the starting point of its effect. The alteration of the microbiota may inhibit the activation of the NLRP3 inflammasome in the liver and the subsequent Caspase‐1/GSDMD‐mediated cell pyroptosis pathway, reducing IL‐1β, IL‐6 and THF‐α. This may further lead to the down‐regulation of core transcription factors such as SREBP‐1, ultimately resulting in a reduction in the expression of key enzymes for fat synthesis such as FAS and ACC and an improvement in lipid accumulation.

The study also has limitations. Due to the short half‐life of astaxanthin and its low content in tissues, the amount of astaxanthin in the liver has not been measured. ⁵² Therefore, the correlation between astaxanthin and blood lipids and inflammatory factors was not explored. In addition, we did not verify the results in humans, and thus caution should be exercised when extending the results to humans.

CONCLUSION

Our findings demonstrated that BMLE can prevent MASLD in mice by down‐regulating fat synthesis proteins, inhibiting the NLRP3/Caspase‐1/GSDMD pyroptosis pathway, and improving gut microbiota. These findings provide new insights into the development of BMLE as a functional food for the prevention of MASLD.

FUNDING INFORMATION

This work was supported by the Key Scientific Research Projects in Shandong Province China (grant number 2017YYSP007).

CONFLICT OF INTEREST

The authors have no conflicts of interest to declare.

AUTHOR CONTRIBUTIONS

KL and SL conceived and designed the study. KL and SL developed the protocol and conducted the study with all other authors. SL, XS and CH performed the experiments. SL and BL analyzed the data. SL and BL wrote the manuscript. KL revised the manuscript. All authors interpreted the data and revised the manuscript for important intellectual content and approved the final draft.

Supporting information

Table S1. Fatty acids composition (% of total fatty acids) of the experimental diets.

Table S2. Detailed composition of the experimental diets.

Table S3. Correlation of liver fatty acid composition and biological indicators in mice.

Table S4. Correlation of gut microbiota and serum indexes, lipid synthesis proteins and pyroptosis pathway proteins.

Figure S1. Alpha diversity of gut microbiota.

DATA AVAILABILITY STATEMENT

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