Hepatic steatosis is a notable feature of metabolic-associated fatty liver disease (MAFLD), which is associated with exosomal miR-122-5p. However, the mechanisms by which exosomal miR-122-5p is involved in the transport of free fatty acid (FFA) remain uncharacterized. The aim of the present study was to investigate the functional role of exosomal miR-122-5p derived from steatotic hepatocytes in hepatic CD36 translocation. Exosomes from PBS- or FFA-treated AML12 cells were extracted via ultracentrifugation (Ctrl-exo or steatotic hepatocyte-derived exosome (SH-exo)), and recipient AML12 cells were treated with them for 24 h. Additionally, AML12 cells were transfected with miR-122-5p mimic or inhibitor for 24 h. A dual-luciferase reporter assay was applied to identify miR-122-5p targeting 3' UTR of ATP6V1H mRNA. RT-qPCR and western blot assessed the expressions of miR-122-5p, CD36, and ATP6V1H in exosomes and recipient AML12 cells. The miR-122-5p down-regulates ATP6V1H expression by directly targeting the 3' UTR of ATP6V1H mRNA. SH-exo were absorbed by recipient AML12 cells and exhibited elevated levels of miR-122-5p. The SH-exo increased FFA uptake, promoted CD36 translocation, and disrupted v-ATPase assembly in recipient AML12 cells by down-regulating the ATP6V1H expression in an insulin-independent manner. MiR-122-5p mimic similarly increased FFA uptake and CD36 translocation by inhibiting ATP6V1H expression in AML12 cells. Furthermore, miR-122-5p inhibitor reduced the FFA content and increased ATP6V1H expression in SH-exo-treated AML12 cells. Our findings indicated that exosomal miR-122-5p, derived from steatotic hepatocytes, can increase FFA uptake and promote CD36 translocation in recipient hepatocytes by directly targeting ATP6V1H expression, suggesting that hepatic exosomal miR-122-5p may serve as a potential therapeutic target for MAFLD.

In this study, Seriola dumerili was used as the raw material and subjected to ultrasonic-assisted wet curing with NaCl solutions of varying concentrations. Physicochemical parameters, such as water-holding capacity and moisture content, were measured. Using a combination of e-nose, e-tongue, GC-IMS, and GC-MS technologies, the study systematically analyzed the effects of ultrasonic-assisted low-salt wet curing on fish meat quality and flavor. The results showed that as the NaCl concentration increased, the firmness, chewiness, and water-holding capacity of the fish meat gradually increased, while the TVB-N value and TCA-soluble peptide content decreased significantly. E-nose analysis revealed that the response values for nitrogen oxides and sulfides gradually increased; e-tongue results indicated that saltiness and richness showed an upward trend, while bitterness gradually decreased. GC-IMS and GC-MS analyses jointly indicated that the total content of volatile compounds increased with rising brine concentration, with ultrasonic treatment significantly promoting the accumulation of aldehydes, alcohols, and ketones. GC-MS identified a total of 84 volatile compounds, and OAV analysis screened out 10 key odor-active compounds. Among these, the US4% group exhibited the highest total OAV, indicating that this condition was most conducive to the development of characteristic flavors in the salted fish. In summary, US4% represents the optimal processing condition for low-salt salted fish products, providing scientific evidence and technical support for optimizing low-salt wet curing processes and producing high-quality, reduced-salt flavored fish products.

Food-derived phytochemicals represent promising strategies for preventing and treating nonalcoholic steatohepatitis (NASH). Existing evidences support the preventive bioactivities of soyasaponins, a family of phytochemicals from soybeans and its products, against NASH. Nevertheless, the underlying mechanisms remain incompletely understood. In this study, soyasaponins attenuated steatohepatitis in high-fat diet (HFD)-induced NASH ApoE-/- mice. Meanwhile, soyasaponins increased DEAD-box protein 5 (DDX5), inhibited mTOR phosphorylation and augmented autophagy (LC3, Beclin, p62) in liver. Further, the molecular docking results revealed that soyasaponins interacted with DDX5. Moreover, in vitro (HepG2 and L02 cells) models of NASH, soyasaponins reduced total cholesterol and triglycerides, upregulated DDX5, inhibited mTOR phosphorylation and augmented autophagy (LC3, Beclin, p62). Furthermore, DDX5 silencing abolished the effects of soyasaponins on TSC1/2-mTOR signaling, autophagy and lipids accumulation. In summary, soyasaponins alleviate NASH by augmenting the DDX5-TSC1/2-mTOR signaling-mediated autophagy, contributing to a deeper understanding of the mechanisms underlying the ameliorative bioactivities of soyasaponins against NASH.

Soyasaponin Bb has various health-promoting bioactivities. However, the bioavailability of soyasaponin Bb is not fully understood. This study aimed to explore the absorption and metabolism of soyasaponin Bb by using both in vivo and in vitro methods. Soyasaponin Bb (100 mg/kg) was orally administrated in Sprague–Dawley (SD) rats, and the content of soyasaponin Bb and soyasapogenol B in plasma, urine and feces were determined by HPLC–MS/MS. The Caco-2 intestinal epithelial cell model was established by culturing on Transwell plates and assessing through cell morphology, transepithelial electrical resistance, alkaline phosphatase activity, and phenol red flux. Then, the apical (AP) to basolateral (BL) transport or uptake of soyasaponin Bb in the model were determined. In SD rats, soyasaponin Bb reached a maximum of 19.8 ng/mL in plasma and showed two material peaks. The cumulative excretion of soyasaponin Bb at 168 h was (0.0022 ± 0.0006)

INTRODUCTION:High palmitic acid (PA) levels trigger metainflammation, facilitating the onset and progression of chronic metabolic diseases. Recently, exosomes were identified as new inflammation mediators. However, the mechanism by which macrophage exosomes mediate PA-induced inflammation remains unclear. OBJECTIVES:To explore how PA induces metainflammation through macrophage exosomes. METHODS:Exosomes secreted by RAW264.7 mouse macrophages stimulated with PA (ExosPA) or not (Exos) were prepared by ultracentrifugation. The differential miRNAs between ExosPA and Exos were identified by high-throughput sequencing, and their targeted mRNAs and proteins were bioinformatically analyzed and verified by qPCR and western blot. Mouse macrophages and metabolic cells (AML-12 hepatocytes, C2C12 myocytes or 3T3-L1 adipocytes) were treated with ExosPA or Exos. The verified miRNAs and its targeted molecules related to inflammation were analyzed in recipient cells. Furthers, exosomes were prepared from primary peritoneal macrophages isolated from AIN93G diet-fed (Control PM-Exos) or HPD-fed (PA PM-Exos) mice. Control or PA PM-Exos were then tail vein injected (30 μg) into mice (n = 10), once a week for 2 weeks. The verified miRNA and its targets in blood, blood exosomes, and metabolic tissues were detected. Finally, measured the levels of miRNA, inflammatory factors, and fatty acids in the blood of 20 obese/overweight individuals and 20 healthy individuals. RESULTS:ExoPA activate NF-κB signaling and enhance inflammatory enzyme/cytokine production in macrophages and metabolic cells. ExoPA enrich miR-3064-5p and target to inhibit IκBα as verified by exosome inhibitors and miR-3064-5p mimics and inhibitors. HPD elevates exosomal miR-3064-5p, macrophage exosomal miR-3064-5p, and inflammatory cytokine levels in mice circulation. PA PM-Exos from HPD-fed mice triggered inflammation in the circulation and metabolic tissues/organs of chow diet-fed mice. Overweight/obese individuals exhibit increased levels of circulating palmitoleic acid, exosomal miR-3064-5p, and high-sensitivity C-reactive proteins. CONCLUSIONS:Macrophage exosomes transferring miR-3064-5p to target IκBα and activate NF-κB signaling in metabolic cells is a mechanism of PA-induced metainflammation.

This study aims to investigate whether human milk exosomes from gestational diabetes mellitus (GDM-EXO) and healthy (HEA-EXO) parturients differ in regulating intestinal development in offspring. The differential miRNAs associated with intestinal development in GDM-EXO and HEA-EXO were verified by using qPCR and their relationships with gut microbiota (GM) in infants were analyzed. C57BL/6J mice were gavaged with 50 mg/kg·BW HEA-EXO or GDM-EXO. The intestinal morphology, gut barriers, ZO-1 and Occludin, and GM were determined by histological staining, Western blotting, and 16S rDNA amplicon sequencing, respectively. Hsa-miR-19b-3p, hsa-miR-148a-3p, and hsa-miR-320a-3p were upregulated, and hsa-miR-429 was decreased in GDM-EXO compared to HEA-EXO. The GDM parturients' infants had increased intestinal Coriobacteriaceae, Clostridiaceae, Erysipelotrichaceae, Erysipelatoclostridiaceae, and fewer Lactobacillaceae than the healthy parturient's infants. The four differential miRNAs in GDM-EXO all correlated with the infants' GM. GDM-EXO- and HEA-EXO-fed mice had greater villus lengths, villus length-to-crypt depth ratios, goblet cell numbers, elevated ZO-1 and Occludin, and lower crypt depths than control mice. HEA-EXO-fed mice had better intestinal morphology and gut barrier integrity than GDM-EXO-fed mice. GDM-EXO-fed mice had significantly decreased Lachnospiraceae and Oscillospiraceae than HEA-EXO-fed mice. GDM-EXO demonstrate weaker ability to promote intestinal development in offspring than HEA-EXO.

Scope Gut microbiota (GM) is involved in nonalcoholic steatohepatitis (NASH) development. Phytochemicals soyasaponins can prevent NASH possibly by modulating GM. This study aims to investigate the preventive bioactivities of soyasaponin monomers (SS‐A 1 and SS‐Bb) against NASH and explores the mechanisms by targeting GM. Methods and results Male C57BL/6 mice are fed with methionine and choline deficient (MCD) diet containing SS‐A 1 , SS‐Bb, or not for 16 weeks. Antibiotics‐treated pseudo germ‐free (PGF) mice are fed with MCD diet containing SS‐A 1 , SS‐Bb, or not for 8 weeks. GM is determined by 16S rRNA amplicon sequencing. Bile acids (BAs) are measured by UPLC‐MS/MS. In NASH mice, SS‐A 1 and SS‐Bb alleviate steatohepatitis and fibrosis, reduce ALT, AST, and LPS in serum, decrease TNF‐α, IL‐6, α‐SMA, triglycerides, and cholesterol in liver. SS‐A 1 and SS‐Bb decrease Firmicutes , Erysipelotrichaceae , unidentified‐Clostridiales , Eggerthellaceae , Atopobiaceae , Aerococcus , Jeotgalicoccus , Gemella , Rikenella , increase Proteobacteria , Verrucomicrobia , Akkermansiaceae , Romboutsia , and Roseburia . SS‐A 1 and SS‐Bb alter BAs composition in liver, serum, and feces, activate farnesoid X receptor (FXR) in liver and ileum, increase occludin and ZO‐1 in intestine. However, GM clearance abrogates the preventive bioactivities of SS‐A 1 and SS‐Bb against NASH. Conclusion GM plays essential roles in soyasaponin's preventive bioactivities against steatohepatitis in MCD diet‐induced NASH mice.

Statins inhibit mevalonate synthesis and successfully lower plasma cholesterol levels and decrease the risk of cardiovascular diseases in humans, but also lead to myalgia in some patients. We hypothesize that statins may modulate glucose metabolism and insulin signaling in the skeletal muscle cells during and after differentiation, and in turn lead to side effects. Here, differentiating and differentiated L6 muscle cells were treated with 1 μM of different class of statins (compactin, pravastatin, atorvastatin, lovastatin and simvastatin) with or without insulin or mevalonate for extended periods of time. The glucose consumption and expression levels of proteins for glucose metabolism and insulin receptor (IR)/Akt signaling were determined. The prolonged statin treatments (except pravastatin) decreased glucose consumption in L6 skeletal muscle cells. In differentiating L6 cells, compactin, lovastatin or simvastatin decreased the expression levels of proteins involved in glucose metabolism and insulin signaling, including glucose transporter 4 (GLUT4), pyruvate dehydrogenase (PDH), glycogen synthase (GS), glycogen synthase kinase 3β (GSK3β) and insulin receptor β subunit (IRβ). In differentiated L6 cells, long-term treatment of compactin or simvastatin also decreased levels of proteins in glucose metabolism and IR/Akt signaling, including GLUT4, GSK3β, IRβ and PI3K p110α. Insulin treatment restored statin-mediated impairments in L6 cells. The insulin-mediated phosphorylation of Akt Ser473 was attenuated in differentiating and differentiated L6 cells in the presence of atorvastatin (differentiated only), compactin, lovastatin or simvastatin. In addition, mevalonate supplementation reversed the statin-mediated impairments in differentiated and differentiating L6 cells. Statin affected glucose usage and insulin signaling by inhibiting mevalonate synthesis in L6 cells. Our results provides a possible mechanism of adverse effects of statins in skeletal muscle and calls for cautious use of the medication in patients with impaired insulin sensitivity and glucose metabolism.

SCOPE:Exosomes, a novel type of bioactive component in human milk (HM), affect infant development, growth, and health. Recent studies indicate that HM exosomes and miRNAs relate to gestational diabetes mellitus (GDM). However, the miRNAs profiles and functionalities of HM exosomes from GDM parturient remain unclear. This study aims to compare the differential miRNAs in HM exosomes from GDM and healthy parturient, and investigate the HM exosomes bioactivities in regulating hepatocyte proliferation and insulin sensitivity.METHODS AND RESULTS:This study extracted HM exosomes from GDM (GDM-EXO) and healthy (NOR-EXO) parturient by ultracentrifugation, high-throughput sequenced and compared the exosomal miRNAs profiles, and explored the regulatory bioactivities on hepatocyte proliferation in HepG2 cells and Balb/c mice. As compared to NOR-EXO, GDM-EXO has similar morphology, size, concentration, and exosome-specific markers (CD9 and TSG101) expression. GDM-EXO and NOR-EXO specifically harbor 1299 and 8 miRNAs, respectively. Moreover, GDM-EXO had 176 upregulated and 47 downregulated miRNAs compared with NOR-EXO. Both GDM-EXO and NOR-EXO were absorbed in cultured HepG2 hepatocytes and mice liver. GDM-EXO inhibited hepatocytes proliferation by downregulating mammalian target of rapamycin (mTOR) possibly via exosomal miR-101-3p delivery.CONCLUSION:HM exosomes from GDM and healthy parturient exhibit differential miRNAs profiles and distinct regulatory bioactivity on hepatocyte proliferation.

Atherosclerosis is a major risk factor for type 2 diabetes (T2D) mortality. We aim to investigate the changes in miR-21, miR-122, miR-33a and miR-3064-5p in circulation and the liver of ApoE-/- mice with streptozocin (STZ)-induced T2D. Twenty 5-week-old male ApoE-/- mice were randomly assigned to the control (n = 10) and T2D group (n = 10) and intraperitoneally injected with a citrate buffer and streptozotocin (STZ) (40 mg/kg BW) once a day for three consecutive days. The successfully STZ-induced T2D mice (n = 5) and control mice (n = 5) were then fed with a high-fat diet (HFD) for 34 weeks. Compared to the control mice, ApoE-/- mice with STZ-induced T2D had slower (p < 0.05) growth, increased (p < 0.05) total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), decreased (p < 0.05) high-density lipoprotein cholesterol (HDL-C) in serum, reduced (p < 0.05) TC and sterol regulatory element-binding protein-2 (Srebp-2), elevated (p < 0.05) ATP-binding-cassette-transporter-A1 (Abca1) in the liver, aggravated (p < 0.05) atherosclerotic lesions in the aorta, downregulated (p < 0.05) miR-21 and miR-33a, and upregulated (p < 0.05) miR-122 and miR-3064-5p in serum and the liver. In addition, the aortic lesions showed a positive correlation with miR-122 (r = 1.000, p = 0.001) and a negative correlation with miR-21 (r = −1.000, p = 0.001) in ApoE-/- mice with T2D. In conclusion, T2D-accelerated atherosclerosis correlates with a reduction in miR-21 and miR-33a and an elevation in miR-122 and miR-3064-5p in circulation and the liver of ApoE-/- mice.

Atherosclerosis (AS) is a chronic inflammatory disease characterized by hardening and narrowing of arteries. AS leads to a number of arteriosclerotic vascular diseases including cardiovascular diseases, cerebrovascular disease and peripheral artery disease, which pose a big threat to human health. Phytochemicals are a variety of intermediate or terminal low molecular weight secondary metabolites produced during plant energy metabolism. Phytochemicals from plant foods (vegetables, fruits, whole grains) and traditional herb plants have been shown to exhibit multiple bioactivities which are beneficial for prevention and treatment against AS. Many types of phytochemicals including polyphenols, saponins, carotenoids, terpenoids, organic sulfur compounds, phytoestrogens, phytic acids and plant sterols have already been identified, among which saponins are a family of glycosidic compounds consisting of a hydrophobic aglycone (sapogenin) linked to hydrophilic sugar moieties. In recent years, studies have shown that saponins exhibit a number of biological activities such as anti-inflammation, anti-oxidation, cholesterol-lowering, immunomodulation, anti-platelet aggregation, etc., which are helpful in the prevention and treatment of AS. This review aims to summarize the recent advances in the anti-atherosclerotic bioactivities of saponins such as ginsenoside, soyasaponin, astra-galoside, glycyrrhizin, gypenoside, dioscin, saikosaponin, etc.

Recently, multiple studies have shown that chronic inflammation disturbs cholesterol homeostasis and promotes its accumulation in the liver. The underlying molecular mechanism remains to be revealed. The relationship between the toll-like receptor 4 (TLR4) inflammatory signaling pathway and cholesterol accumulation was investigated in HepG2 cells treated with lipopolysaccharide (LPS) or palmitic acid (PA) for different lengths of time. In addition, the effects of pretreatment with 20μmol/L ST2825 (MyD88 inhibitor) were also studied in LPS- or PA-treated HepG2 cells and myeloid differentiation factor 88 (MyD88)-overexpressing HEK293T cells. The intracellular total and free cholesterol levels were measured using a commercial kit and filipin staining, respectively. The expression levels of sterol regulatory element-binding protein-2 (SREBP-2) and components in the TLR4 signaling pathway were determined using Western blotting. The treatments with LPS for 12 h and with PA for 24 h significantly increased the contents of intracellular total and free cholesterol, as well as the expression levels of SREBP-2 and components in the TLR4 signaling pathway. The inhibition of MyD88 by ST2825 significantly decreased the cholesterol content and the expression levels of SREBP-2 and components of the TLR4/MyD88/NF-κB pathway in HepG2 cells, as well as MyD88-overexpressing HEK293T cells. These results indicated that LPS and PA treatments increase SREBP-2-mediated cholesterol accumulation via the activation of the TLR4/MyD88/NF-κB signaling pathway in HepG2 cells.

β-carotene, a member of the carotenoid family, is a provitamin A, and can be converted into vitamin A (retinol), which plays essential roles in the regulation of physiological functions in animal bodies. Microalgae synthesize a variety of carotenoids including β-carotene and are a rich source of natural β-carotene. This has attracted the attention of researchers in academia and the biotech industry. Methods to enrich or purify β-carotene from microalgae have been investigated, and experiments to understand the biological functions of microalgae products containing β-carotene have been conducted. To better understand the use of microalgae to produce β-carotene and other carotenoids, we have searched PubMed in August 2021 for the recent studies that are focused on microalgae carotenoid content, the extraction methods to produce β-carotene from microalgae, and the bioactivities of β-carotene from microalgae. Articles published in peer-reviewed scientific journals were identified, screened, and summarized here. So far, various types and amounts of carotenoids have been identified and extracted in different types of microalgae. Diverse methods have been developed overtime to extract β-carotene efficiently and practically from microalgae for mass production. It appears that methods have been developed to simplify the steps and extract β-carotene directly and efficiently. Multiple studies have shown that extracts or whole organism of microalgae containing β-carotene have activities to promote lifespan in lab animals and reduce oxidative stress in culture cells, etc. Nevertheless, more studies are warranted to study the health benefits and functional mechanisms of β-carotene in these microalgae extracts, which may benefit human and animal health in the future.

The mechanisms of soyasaponin A1 (SS-A1) on antagonizing inflammation via regulating the lipid raft-mediated toll-like receptor 4 (TLR4) signaling are not completely understood. In this study, SS-A1 inhibited palmitic acid (PA)-induced recruitments of TLR4 and its adaptor molecules into lipid rafts in Raw264.7 macrophage cell line by using ultracentrifugation and confocal microscopy. SS-A1 also suppressed the PA-caused dimerization of TLR4 with and its adaptor molecules by applying immunoprecipitation and fluorescence resonance energy transfer. Meanwhile, SS-A1 modified the formation, clustering, and size of lipid rafts and decreased cholesterol in lipid rafts. Cholesterol replenishment abrogated SS-A1’s inhibition on the lipid rafts recruitment and dimerization of TLR4 and its adaptor molecules and its anti-inflammatory activity. Additionally, SS-A1 inhibited the mature sterol regulatory element-binding protein 2 and increases ATP-binding cassette transporter A1. Collectively, SS-A1 inhibits the lipid raft recruitment and dimerization of TLR4 and its adaptor molecules by maintaining cholesterol homeostasis in PA-stimulated inflammatory Raw264.7 macrophage cell line.

SCOPE:Nonalcoholic steatohepatitis (NASH) is a chronic progressive disease with complex pathogenesis of which the bile acids (BAs) and gut microbiota are involved. Soyasaponins (SS) exhibits many health-promoting effects including hepatoprotection, but its prevention against NASH is unclear. This study aims to investigate the preventive bioactivities of SS monomer (SS-A2 ) against NASH and further clarify its mechanism by targeting the BAs and gut microbiota.METHODS AND RESULTS:The methionine and choline deficient (MCD) diet-fed male C57BL/6 mice were intervened with obeticholic acid or SS-A2 for 16 weeks. Hepatic pathology is assessed by hematoxylin-eosin and Masson's trichrome staining. BAs in serum, liver, and colon are measured by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-TQMS). Gut microbiota in caecum are determined by 16S rDNA amplicon sequencing. In the MCD diet-induced NASH mice, SS-A2 significantly reduces hepatic steatosis, lobular inflammation, ballooning, nonalcoholic fatty liver disease activity score (NAS) scores, and fibrosis, decreases Erysipelotrichaceae (Faecalibaculum) and Lactobacillaceae (Lactobacillus) and increases Desulfovibrionaceae (Desulfovibrio). Moreover, SS-A2 reduces serum BAs accumulation and promotes fecal BAs excretion. SS-A2 changes the BAs profiles in both liver and serum and specifically increases the taurohyodeoxycholic acid (THDCA) level. Faecalibaculum is negatively correlated with serum THDCA.CONCLUSION:SS-A2 alleviates steatohepatitis possibly through regulating BAs and gut microbiota in the MCD diet-induced NASH mice.

Atherosclerosis is a chronic inflammatory disease causing coronary heart attacks and strokes. Soyasaponins (SS), the phytochemicals naturally existing in soybeans and their products, have been shown to reduce hypercholesterolemia and inflammation, which are intimately related to the genesis and development of atherosclerosis. However, the anti-atherosclerotic functionality of soyasaponins remains unknown. The aim of this study was to investigate the effects of the supplementation of two types of soyasaponin monomers (A1 and A2) on atherosclerotic plaque formation, serum lipid profiles, and inflammation in ApoE gene knockout (ApoE-/-) mice. Sixty 5-week-old ApoE-/- male mice were fed with a high-fat diet (HFD) and intervened by SSA1 and SSA2 (10 and 20 μmol per kg BW, respectively) or simvastatin (10 μmol per kg BW) for 24 weeks. The atherosclerotic lesions in the aorta, aortic root, and innominate artery, lipid profile and inflammatory markers in serum, and TLR4/MyD88/NF-κB signaling in arterial tissues were determined. SSA1 and SSA2 decreased the plaque ratio in the aortic root and innominate artery but not in the entire aorta. In serum, SSA1 reduced TG, TC, and LDL-C but increased HDL-C; SSA2 decreased TC, TG, and LDL-C but did not affect HDL-C. Meanwhile, SSA1 increased TG, SSA2 increased TC, and both of them increased bile acids in the feces. SSA1 and SSA2 lowered TNF-α, MCP-1, and hs-crp in serum. Furthermore, SSA1 and SSA2 reduced the TLR4 and MyD88 expressions in the aorta and innominate artery and inhibited NF-κB p65 and IκBα phosphorylation in the aorta. These results suggest that SSA1 and SSA2 exert anti-atherosclerotic functionalities by decreasing hypercholesterolemia and inflammation in HFD-fed ApoE-/- mice.

Abstract Background Previous studies indicate that soyasaponins may reduce inflammation via modulating toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) signaling. However, its underlying mechanisms are still not fully understood. Methods Lipopolysaccharide (LPS)-challenged inflamed male ICR mice were intervened by intragastrical administration with 10 and 20 μmol/kg·BW of soyasaponin A1, A2 or I for 8 weeks. The serum inflammatory markers were determined by commercial kits and the expression of molecules in TLR4/MyD88 signaling pathway in liver by real-time PCR and western blotting. The recruitments of TLR4 and MyD88 into lipid rafts of live tissue lysates were detected by sucrose gradient ultracentrifugation and western blotting. LPS-stimulated RAW264.7 macrophages were treated with 10, 20 and 40 μmol/L of soyasaponin A1, A2 or I for 2 h. MyD88-overexpressed HEK293T cells were treated with 20 and 40 μmol/L of soyasaponins (A1, A2 or I) or 20 μmol/L of ST2825 (a MyD88 inhibitor) for 6 h. The expression of molecules in TLR4/MyD88 signaling pathway were determined by western blotting. Data were analyzed by using one way analysis of variance or t-test by SPSS 20.0 statistical software. Results Soyasaponins A1, A2 or I significantly reduced the levels of tumor necrosis factor alpha (TNFα), interleukin (IL)-6 and nitric oxide (NO) in serum (p < 0.05), and decreased the mRNA levels of TNFα, IL-6, IL-1β, cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) (p < 0.05), the protein levels of myeloid differentiation protein 2 (MD-2), TLR4, MyD88, toll-interleukin1 receptor domain containing adaptor protein (TIRAP), phosphorylated interleukin-1 receptor-associated kinase 4 (p-IRAK-4), phosphorylated interleukin-1 receptor-associated kinase 1 (p-IRAK-1) and TNF receptor associated factor 6 (TRAF6) (p < 0.05), and the recruitments of TLR4 and MyD88 into lipid rafts in liver (p < 0.05). In LPS-stimulated macrophages, soyasaponins A2 or I significantly decreased MyD88 (p < 0.05), soyasaponins A1, A2 or I reduced p-IRAK-4 and p-IRAK-1 (p < 0.05), and soyasaponin I decreased TRAF6 (p < 0.05). In MyD88-overexpressed HEK293T cells, soyasaponins (A1, A2 or I) and ST2825 significantly decreased MyD88 and TRAF6 (p < 0.05). Conclusion Soyasaponins can reduce inflammation by downregulating MyD88 expression and suppressing the recruitments of TLR4 and MyD88 into lipid rafts. This study provides novel understanding about the anti-inflammatory mechanism of soyasaponins.

Soyasaponin A1 (SSA1) was previously shown to inhibit the palmitate (PA)-induced inflammation via regulating the toll-like receptor 4 (TLR4) signaling in macrophages. Since the lipid raft recruitment and dimerization of TLR4 and its downstream adaptor molecules are vital for PA-initiated TLR4 signaling, we explored whether this process would be modulated by SSA1. Murine macrophage RAW264.7 were stimulated with PA (200 μmol/L) in the presence or absence of SSA1 (40 μmol/L). The lipid raft fractions were separated by sucrose density gradient ultracentrifugation and immunoblotted with anti-flotillin-1, anti-TLR4, anti-myeloid differentiation primary response protein 88 (MyD88), or anti-Toll/IL-1 receptor domain-containing adaptor inducing interferon-β (TRIF) antibody. Lipid rafts, TLR4, MyD88 and TRIF were fluorescently labeled and analyzed by confocal microscopy to visualize the recruitment of molecules into lipid rafts and investigate the clustering and size of lipid rafts. The complexes of TLR4/MyD88 and TLR4/TRIF were immunoprecipitated by anti-TLR4 antibody first and then immunoblotted by anti-MyD88 or anti-TRIF antibody. PA-induced recruitment of TLR4, MyD88 and TRIF into fractions enriched with lipid rafts marker flotillin-1 was inhibited (P < 0.05) by SSA1. Meanwhile, the PA-induced co-localization of TLR4, MyD88, and TRIF with lipid rafts was also decreased (P < 0.05) by SSA1 as visualized by confocal immunofluorescence microscopy. Furthermore, the PA-induced clustering of lipid rafts was diminished (P < 0.05) by SSA1. However, the PA-decreased size of lipid rafts was increased (P < 0.05) by SSA1. The formation of TLR4/MyD88 and TLR4/TRIF complexes was suppressed (P < 0.05) by SSA1, whereas the protein expressions of TLR4, MyD88 and TRIF were not changed (P > 0.05) by SSA1 in PA-stimulated macrophages. SSA1 inhibits the recruitment of TLR4 and its adaptor molecules (MyD88 and TRIF) into lipid raft as well as their dimerization (TLR4/MyD88 and TLR4/TRIF) in PA-stimulated inflammatory macrophages. This work was supported by grants from National Natural Science Foundation of China (NSFC).