Macrophage exosomes mediate palmitic acid-induced metainflammation by transferring miR-3064-5p to target IκBα and activate NF-κB signaling.

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.

Gut Microbiota Plays Essential Roles in Soyasaponin's Preventive Bioactivities against Steatohepatitis in the Methionine and Choline Deficient (MCD) Diet-Induced Non-Alcoholic Steatohepatitis (NASH) Mice.

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-A1 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-A1 , SS-Bb, or not for 16 weeks. Antibiotics-treated pseudo germ-free (PGF) mice are fed with MCD diet containing SS-A1 , 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-A1 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-A1 and SS-Bb decrease Firmicutes, Erysipelotrichaceae, unidentified-Clostridiales, Eggerthellaceae, Atopobiaceae, Aerococcus, Jeotgalicoccus, Gemella, Rikenella, increase Proteobacteria, Verrucomicrobia, Akkermansiaceae, Romboutsia, and Roseburia. SS-A1 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-A1 and SS-Bb against NASH. CONCLUSION: GM plays essential roles in soyasaponin's preventive bioactivities against steatohepatitis in MCD diet-induced NASH mice.

Milk Exosomes from Gestational Diabetes Mellitus (GDM) and Healthy Parturient Exhibit Differential miRNAs Profiles and Distinct Regulatory Bioactivity on Hepatocyte Proliferation.

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.

Sodium Butyrate Inhibits Oxidative Stress and NF-κB/NLRP3 Activation in Dextran Sulfate Sodium Salt-Induced Colitis in Mice with Involvement of the Nrf2 Signaling Pathway and Mitophagy.

BACKGROUND: Sodium butyrate (NaB) is a short-chain fatty acid produced by intestinal microbial fermentation of dietary fiber, and has been shown to be effective in inhibiting ulcerative colitis (UC). However, how NaB regulates inflammation and oxidative stress in the pathogenesis of UC is not clear. AIMS: The purpose of this study was to use a dextran sulfate sodium salt (DSS)-induced murine colitis model, and determine the effects of NaB and the related molecular mechanisms. METHODS: Colitis model was induced in mice by administration of 2.5%(wt/vol) DSS. 0.1 M NaB in drinking water, or intraperitoneal injection of NaB (1 g/kg body weight) was given during the study period. In vivo imaging was performed to detect abdominal reactive oxygen species (ROS). Western blotting and RT-PCR were used to determine the levels of target signals. RESULTS: The results showed that NaB decreases the severity of colitis as determined by an improved survival rate, colon length, spleen weight, disease activity index (DAI), and histopathological changes. NaB reduced oxidative stress as determined by a reduction in abdominal ROS chemiluminescence signaling, inhibition of the accumulation of myeloperoxidase and malondialdehyde, and restoration of glutathione activity. NaB activated the COX-2/Nrf2/HO-1 pathway by increasing the expressions of COX-2, Nrf2, and HO-1 proteins. NaB inhibited the phosphorylation of NF-κB and activation of NLRP3 inflammasomes, and reduced the secretion of corresponding inflammatory factors. Furthermore, NaB promoted the occurrence of mitophagy via activating the expression of Pink1/Parkin. CONCLUSIONS: In conclusion, our results indicate that NaB improves colitis by inhibiting oxidative stress and NF-κB/NLRP3 activation, which may be via COX-2/Nrf2/HO-1 activation and mitophagy.

Advances in the Bioactivities of Phytochemical Saponins in the Prevention and Treatment of Atherosclerosis.

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.

The Accelerated Progression of Atherosclerosis Correlates with Decreased miR-33a and miR-21 and Increased miR-122 and miR-3064-5p in Circulation and the Liver of ApoE-/- Mice with Streptozocin (STZ)-Induced Type 2 Diabetes.

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.

Inflammation Induced by Lipopolysaccharide and Palmitic Acid Increases Cholesterol Accumulation via Enhancing Myeloid Differentiation Factor 88 Expression in HepG2 Cells.

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.

Extracellular vesicles regulate the transmission of insulin resistance and redefine noncommunicable diseases.

Noncommunicable diseases (NCDs), such as diabetes and related neurological disorders, are considered to not be directly transmissible from one person to another. However, NCDs may be transmissible in vivo through extracellular vesicles (EVs). A long-term high-fat diet (HFD) can induce a series of health issues like hyperlipidemia, type 2 diabetes mellitus (T2DM), and diabetic peripheral neuropathy (DPN) due to insulin resistance. Multiple molecular signaling changes can stimulate insulin resistance, especially blocking insulin signaling by increased insulin resistance inducer (phosphorylation of negative regulatory sites of insulin receptor substrate (IRS) proteins) and decreased tyrosine phosphorylation of insulin receptor substrate (phosphorylation of positive regulatory sites of IRS), thus leading to reduced phosphorylation of AKT enzymes. Current efforts to treat T2DM and prevent its complications mainly focus on improving insulin sensitivity, enhancing insulin secretion, or supplementing exogenous insulin based on a common assumption that insulin resistance is noncommunicable. However, insulin resistance is transmissible within multiple tissues or organs throughout the body. Exploring the regulatory roles of EVs in developing insulin resistance may provide novel and effective preventive and therapeutic strategies.

Soyasaponin A2 Alleviates Steatohepatitis Possibly through Regulating Bile Acids and Gut Microbiota in the Methionine and Choline-Deficient (MCD) Diet-induced Nonalcoholic Steatohepatitis (NASH) Mice.

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.

Soyasaponins reduce inflammation by downregulating MyD88 expression and suppressing the recruitments of TLR4 and MyD88 into lipid rafts.

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-1ß, 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.

A dose-response evaluation of purified anthocyanins on inflammatory and oxidative biomarkers and metabolic risk factors in healthy young adults: A randomized controlled trial.

OBJECTIVES: Anthocyanins derived from different plant sources have been found to possess a variety of health-promoting effects, including antiinflammatory properties and protection from oxidative stress. The aim of this study was to investigate the dose-response relationship between anthocyanins and metabolic risk factors as well as inflammatory and oxidative biomarkers in healthy adult volunteers. METHODS: We conducted a randomized, double-blind, placebo-controlled trial, which included an increasing dosing schedule of 20, 40, 80, 160, and 320 mg of purified anthocyanins or placebo. Participants (n = 111) were administered either agent for 14 consecutive days. RESULTS: No significant differences in either baseline characteristics or daily intake of dietary nutrients were detected between the experimental and control groups. After anthocyanin supplementation, there was a significant difference in adjusted fasting plasma glucose levels. The group receiving 80 mg/d of anthocyanin had the lowest baseline-adjusted fasting plasma glucose when compared with placebo (F = 3.556, P = 0.007). Logarithmically adjusted plasma interleukin-10 levels were negatively correlated with increasing anthocyanin dose (F = 2.738, P = 0.025). Similarly, 8-iso-prostaglandin F2α levels decreased with increasing anthocyanins dose (F = 3.513, P = 0.009). CONCLUSIONS: Taken together, our results suggest that anthocyanin supplementation at a dose greater than 80 mg/d is an effective antioxidant and antiinflammatory agent in healthy young adults.

Soyasaponins A1 and A2 exert anti-atherosclerotic functionalities by decreasing hypercholesterolemia and inflammation in high fat diet (HFD)-fed ApoE-/- 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.

Effects of Tart Cherry Juice on Biomarkers of Inflammation and Oxidative Stress in Older Adults.

Inflammation and oxidative stress are important factors in the development of cardiovascular disease and atherosclerosis. The findings of our previous study suggest that 12 weeks consumption of tart cherry juice lowers the levels of systolic blood pressure (BP) and low-density lipoprotein (LDL) cholesterol in older adults. The present study investigated the effects of tart cherry juice on blood biomarkers of inflammation and oxidative stress. In this randomized-controlled clinical trial, a total of 37 men and women between the ages of 65⁻80 were randomly assigned to consume 480 mL of tart cherry juice or control drink daily for 12 weeks. Several blood biomarkers of inflammation and oxidative stress were assessed at baseline and after 12 weeks intervention. After the 12 weeks intervention, tart cherry juice significantly increased the plasma levels of DNA repair activity of 8-oxoguanine glycosylase (p < 0.0001) and lowered (p = 0.03) the mean c-reactive protein (CRP) level compared to the control group. There was a significant group effect observed for plasma CRP (p = 0.03) and malondialdehyde (MDA) (p = 0.03), and a borderline significant group effect observed for plasma oxidized low-density lipoprotein (OxLDL) (p = 0.07). Within group analysis showed that the plasma levels of CRP, MDA, and OxLDL decreased numerically by 25%, 3%, and 11%, respectively after 12 weeks of tart cherry juice consumption compared with corresponding baseline values. The present study suggests that the ability of tart cherry juice to reduce systolic BP and LDL cholesterol, in part, may be due to its anti-oxidative and anti-inflammatory properties. Larger and longer follow-up studies are needed to confirm these findings.

Soyasaponins Reduce Inflammation and Improve Serum Lipid Profiles and Glucose Homeostasis in High Fat Diet-Induced Obese Mice.

SCOPE: Obesity is linked to a chronic low-grade inflammatory state that contributes to the development of obesity-associated metabolic disorders. The anti-inflammatory activities and mechanisms of soyasaponin monomers (A1 , A2 , and I) have been recently demonstrated in cell models. However, their potential in vivo abilities to reduce chronic inflammation and alleviate metabolic disorders in obese status remain unclear. METHODS AND RESULTS: High fat diet (HFD)-fed obese male C57BL/6J mice are intervened by aspirin (0.1 mg kg-1 body weight) or 20 mg kg-1 of soyasaponins A1 , A2 , or I for 8 weeks. Soyasaponins A1 , A2 , and I significantly reduce pro-inflammatory cytokines/mediators in serum, liver, and white adipose tissues (WATs), improve serum lipid profiles, decrease liver cholesterol, triglyceride and steatosis, and promote fecal excretion of cholesterol, triglycerides, and bile acids. Soyasaponins A1 , A2 , and I also decrease IKKα/ß phosphorylation in liver and WATs and reduce NF-κB p65 phosphorylation and CD68 mRNA and protein expression in WATs. Soyasaponins A1 and A2 but not I decrease NF-κB p65 phosphorylation in liver and adipocytes hypertrophy in WATs. In addition, Soyasaponin A2 but not A1 nor I decreases fasting blood glucose and improved insulin resistance. CONCLUSION: Soyasaponins reduce inflammation and improve serum lipid profiles and glucose homeostasis in HFD-induced obese mice.

Soyasaponin Bb inhibits the recruitment of toll-like receptor 4 (TLR4) into lipid rafts and its signaling pathway by suppressing the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent generation of reactive oxygen species.

SCOPE: We and others recently showed that soyasaponin Bb (SSBb ) inhibited lipopolysaccharide (LPS)-induced inflammation in macrophages. Since the recruitment of toll-like receptor 4 (TLR4) into lipid rafts is vital for LPS-initiated signaling, we investigated whether this process would be modulated by SSBb . METHODS AND RESULTS: By using sucrose gradient ultracentrifuge, we found that pretreatment of macrophages with SSBb inhibited LPS-induced recruitments of TLR4, myeloid differentiation primary response protein 88 (MyD88) and Toll/IL-1 receptor domain-containing adaptor inducing interferon-ß (TRIF) into fractions enriched with lipid rafts marker flotillin-1. We also found SSBb decreased co-localization of TLR4 and lipid rafts by utilizing confocal immunofluorescence microscopy. Additionally, we observed that SSBb suppressed LPS-induced formation of TLR4/MyD88 and TLR4/TRIF complexes, production of pro-inflammatory molecules, and activation of nuclear factor kappa B (NF-κB). Furthermore, we found that these inhibitory effects of SSBb were associated with reduced reactive oxygen species (ROS) because pretreating cells with N-acetyl-L-cysteine and NADPH oxidase inhibitor diphenyleneiodonium (DPI) inhibited LPS-induced TLR4 recruitment into lipid rafts and NF-κB activation. SSBb also inhibited NADPH oxidase activation by blocking interaction between gp91(phox) and p47(phox) similarly as DPI. CONCLUSION: SSBb can inhibit TLR4 recruitment into lipid rafts and its signaling by suppressing the NADPH oxidase-dependent ROS generation.

Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis.

PURPOSE: Butyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated autophagy, and whether there was crosstalk between autophagy and the sodium butyrate-induced apoptotic response in human colorectal cancer cells. METHODS: Human colorectal cancer cell lines (HCT-116 and HT-29) were treated with sodium butyrate at concentrations ranging from 0.5-5mM. Cell proliferation was assessed using MTT tetrazolium salt formation. Autophagy induction was confirmed through a combination of Western blotting for associated proteins, acridine orange staining for acidic vesicles, detection of autolysosomes (MDC staining), and electron microscopy. Apoptosis was quantified by flow cytometry using standard annexinV/propidium iodide staining and by assessing PARP-1 cleavage by Western blot. RESULTS: Sodium butyrate suppressed colorectal cancer cell proliferation, induced autophagy, and resulted in apoptotic cell death. The induction of autophagy was supported by the accumulation of acidic vesicular organelles and autolysosomes, and the expression of autophagy-associated proteins, including microtubule-associated protein II light chain 3 (LC3-II), beclin-1, and autophagocytosis-associated protein (Atg)3. The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine inhibited sodium butyrate induced autophagy. Furthermore, sodium butyrate treatment markedly enhanced the expression of endoplasmic reticulum stress-associated proteins, including BIP, CHOP, PDI, and IRE-1a. When endoplasmic reticulum stress was inhibited by pharmacological (cycloheximide and mithramycin) and genetic (siRNA targeting BIP and CHOP) methods, the induction of BIP, PDI, IRE1a, and LC3-II was blocked, but PARP cleavage was markedly enhanced. DISCUSSION: Taken together, these results suggested that sodium butyrate-induced autophagy was mediated by endoplasmic reticulum stress, and that preventing autophagy by blocking the endoplasmic reticulum stress response enhanced sodium butyrate-induced apoptosis. These results provide novel insights into the anti-tumor mechanisms of butyric acid.

The expression profile of aquaporin 1 in rat myocardium after severe burns.

BACKGROUND/AIM: To investigate the myocardial expression profile of aquaporin 1 (AQP1) in rats after severe burns. MATERIALS AND METHODS: Ninety healthy male adult Wistar rats were randomly assigned to the following treatments: sham operation (control group, n = 6), immediate fluid resuscitation treatment post scalding (IF group, n = 42), and delayed fluid resuscitation treatment after scalding (DF group, n = 42). At 3, 6, 12, 24, 48, 72, and 120 h after scalding, myocardial water contents were assayed and AQP1 expressions were detected by real-time polymerase chain reaction and western blot. The AQP levels in the myocardium at 12 h after scalding were assayed by gene microarrays. RESULTS: Scald injuries resulted in significantly synchronized increases in the myocardial water contents and the myocardial mRNA and protein expression of AQP1, with a peak at 12 h after scalding. Rats receiving delayed fluid resuscitation treatment had more severe myocardial edema and significantly higher myocardial AQP1 expressions than the rats receiving immediate fluid resuscitation treatment. The mRNAs of 6 other AQPs (AQP2, 3, 4, 6, 7, and 12b) were found to be changed in the myocardium among rats with different treatments. CONCLUSION: AQP1 may play a functional role in the development of myocardial edema after scalding. Targeting AQP1 may provide opportunities for therapeutic intervention in myocardial edema following severe burns.

Soyasaponins prevent H2O2-induced inhibition of gap junctional intercellular communication by scavenging reactive oxygen species in rat liver cells.

It appears to be more practical and effective to prevent carcinogenesis by targeting the tumor promotion stage. Gap junctional intercellular communication (GJIC) is strongly involved in carcinogenesis, especially the tumor promotion stage. Considerable interest has been focused on the chemoprevention activities of soyasaponin (SS), which are major phytochemicals found in soybeans and soy products. However, less is known about the preventive effects of SS (especially SS with different chemical structures) against tumor promoter-induced inhibition of GJIC. We investigated the protective effects of SS-A1, SS-A2, and SS-I against hydrogen peroxide (H2O2)-induced GJIC inhibition and reactive oxygen species (ROS) production in Buffalo rat liver (BRL) cells. The present results clearly show for the first time that SS-A1, SS-A2, and SS-I prevent the H2O2-induced GJIC inhibition by scavenging ROS in BRL cells in a dose-dependent manner at the concentration range of from 25 to 100 µg/mL. Soyasaponins attenuated the H2O2-induced ROS through potentiating the activities of superoxide dismutase and glutathione peroxidase. This may be an important mechanism by which SS protects against tumor promotion. In addition, various chemical structures of SS appear to exhibit different protective abilities against GJIC inhibition. This may partly attribute to their differences in ROS-scavenging activities.

Soyasaponins can blunt inflammation by inhibiting the reactive oxygen species-mediated activation of PI3K/Akt/NF-kB pathway.

We and others have recently shown that soyasaponins abundant in soybeans can decrease inflammation by suppressing the nuclear factor kappa B (NF-kB)-mediated inflammation. However, the exact molecular mechanisms by which soyasaponins inhibit the NF-kB pathway have not been established. In this study in macrophages, soyasaponins (A1, A2 and I) inhibited the lipopolysaccharide (LPS)-induced release of inflammatory marker prostaglandin E2 (PGE2) to a similar extent as the NF-kB inhibitor (BAY117082). Soyasaponins (A1, A2 and I) also suppressed the LPS-induced expression of cyclooxygenase 2 (COX-2), another inflammatory marker, in a dose-dependent manner by inhibiting NF-kB activation. In defining the associated mechanisms, we found that soyasaponins (A1, A2 and I) blunted the LPS-induced IKKα/ß phosphorylation, IkB phosphorylation and degradation, and NF-kB p65 phosphorylation and nuclear translocation. In studying the upstream targets of soyasaponins on the NF-kB pathway, we found that soyasaponins (A1, A2 and I) suppressed the LPS-induced activation of PI3K/Akt similarly as the PI3K inhibitor LY294002, which alone blocked the LPS-induced activation of NF-kB. Additionally, soyasaponins (A1, A2 and I) reduced the LPS-induced production of reactive oxygen species (ROS) to the same extent as the anti-oxidant N-acetyl-L-cysteine, which alone inhibited the LPS-induced phosphorylation of Akt, IKKα/ß, IkBα, and p65, transactivity of NF-kB, PGE2 production, and malondialdehyde production. Finally, our results show that soyasaponins (A1, A2 and I) elevated SOD activity and the GSH/GSSG ratio. Together, these results show that soyasaponins (A1, A2 and I) can blunt inflammation by inhibiting the ROS-mediated activation of the PI3K/Akt/NF-kB pathway.

A small amount of dietary carbohydrate can promote the HFD-induced insulin resistance to a maximal level.

Both dietary fat and carbohydrates (Carbs) may play important roles in the development of insulin resistance. The main goal of this study was to further define the roles for fat and dietary carbs in insulin resistance. C57BL/6 mice were fed normal chow diet (CD) or HFD containing 0.1-25.5% carbs for 5 weeks, followed by evaluations of calorie consumption, body weight and fat gains, insulin sensitivity, intratissue insulin signaling, ectopic fat, and oxidative stress in liver and skeletal muscle. The role of hepatic gluconeogenesis in the HFD-induced insulin resistance was determined in mice. The role of fat in insulin resistance was also examined in cultured cells. HFD with little carbs (0.1%) induced severe insulin resistance. Addition of 5% carbs to HFD dramatically elevated insulin resistance and 10% carbs in HFD was sufficient to induce a maximal level of insulin resistance. HFD with little carbs induced ectopic fat accumulation and oxidative stress in liver and skeletal muscle and addition of carbs to HFD dramatically enhanced ectopic fat and oxidative stress. HFD increased hepatic expression of key gluconeogenic genes and the increase was most dramatic by HFD with little carbs, and inhibition of hepatic gluconeogenesis prevented the HFD-induced insulin resistance. In cultured cells, development of insulin resistance induced by a pathological level of insulin was prevented in the absence of fat. Together, fat is essential for development of insulin resistance and dietary carb is not necessary for HFD-induced insulin resistance due to the presence of hepatic gluconeogenesis but a very small amount of it can promote HFD-induced insulin resistance to a maximal level.