Zerumbone suppresses high glucose and LPS-induced inflammation in THP-1-derived macrophages by inhibiting the NF-κB/TLR signaling pathway.

Diabetes mellitus is characterized by hyperglycemia. Low-grade bacterial infection with hyperglycemia in patients with diabetes is associated with atherosclerosis development. Therefore, this study hypothesized that macrophages lead to more severe diabetic complications under combined conditions of high glucose and lipopolysaccharide (LPS)-induced inflammation than under normoglycemic conditions. Zerumbone is the main component of Zingiber zerumbet Smith essential oil, a type of wild ginger. It possesses various biomedical activities, including antibacterial, antioxidant, anti-inflammatory, and anticancer activities; however, the precise mechanism of its anti-inflammatory and epigenetic effects is not fully understood. In this study, the effects of zerumbone on the secretion of proinflammatory cytokines and its underlying regulatory mechanism were investigated in THP-1-derived macrophages exposed to high glucose and LPS. THP-1-derived macrophages were cultured under normoglycemic (5.5 mmol/L glucose) or hyperglycemic (25 mmol/L glucose) conditions in the absence or presence of zerumbone (5-50 µM) for 48 hours and then treated with 100 ng/mL LPS for 6 hours. Zerumbone (25 and 50 µM) suppressed the production of tumor necrosis factor-α and interleukin-6 and the activation of cyclooxygenase-2, nuclear factor-κB, histone deacetylases 3 proteins, and Toll-like receptor messenger RNA (mRNA) and increased the transcription of the sirtuin 1 (SIRT1), SIRT3, and SIRT6 mRNAs. Taken together, our results suggest that zerumbone may exert beneficial effects on diabetes and its complications.

Dietary glucosinolates inhibit splenic inflammation in high fat/cholesterol diet-fed C57BL/6 mice.

BACKGROUND/OBJECTIVES: Obesity is associated with chronic inflammation. The spleen is the largest organ of the lymphatic system and has an important role in immunity. Obesity-induced inflammatory responses are triggered by Toll-like receptor (TLR)-myeloid differentiation primary response 88 (MyD88) pathway signaling. Phenethyl isothiocyanate (PEITC) and 3,3'-diindolylmethane (DIM), major dietary glucosinolates present in cruciferous vegetables, have been reported to produce anti-inflammatory effects on various diseases. However, the effects of PEITC and DIM on the obesity-induced inflammatory response in the spleen are unclear. The purpose of this study was to examine the anti-inflammatory effects of PEITC and DIM on the spleen and their mechanism in high fat/cholesterol diet (HFCD)-fed C57BL/6 mice. MATERIALS/METHODS: We established an animal model of HFCD-induced obesity using C57BL/6 mice. The mice were divided into six groups: normal diet with AIN-93G diet (CON), high fat diet (60% calories from fat) with 1% cholesterol (HFCD), HFCD with PEITC 30 mg/kg/day or 75 mg/kg/day (HFCD+P30, HFCD+P75), and HFCD with DIM 1.5 mg/kg/day or 7.5 mg/kg/day (HFCD+D1.5, HFCD+D7.5). Enzyme-linked immunosorbent assay was used to evaluate pro-inflammatory cytokine secretion. Western blot and quantitative polymerase chain reaction were used to analyze protein and mRNA levels of nuclear factor kappa B (NF-κB) p65, interleukin 6 (IL-6), cyclooxygenase 2 (COX-2), TLR2, TLR4, and MyD88 in spleen tissue. RESULTS: Serum IL-6 levels were significantly higher in the HFCD group than in groups fed a HFCD with PEITC or DIM. Levels of NF-κB p65 protein and TLR2/4, MyD88, NF-κB p65, IL-6, and COX-2 mRNA were significantly higher in the HFCD group than in the CON group and were reduced by the PEITC and DIM supplements. CONCLUSIONS: PEITC- and DIM-supplemented diets improved splenic inflammation by modulating the TLR2/4-MyD88 pathway in HFCD-fed mice. We suggest that dietary glucosinolates may at least partially improve obesity-induced inflammation of the spleen.

Hesperetin suppresses LPS/high glucose-induced inflammatory responses via TLR/MyD88/NF-κB signaling pathways in THP-1 cells.

BACKGROUND/OBJECTIVES: Unregulated inflammatory responses caused by hyperglycemia may induce diabetes complications. Hesperetin, a bioflavonoid, is a glycoside in citrus fruits and is known to have antioxidant and anticarcinogenic properties. However, the effect of inflammation on the diabetic environment has not been reported to date. In this study, we investigated the effect of hesperetin on proinflammatory cytokine secretion and its underlying mechanistic regulation in THP-1 macrophages with co-treatment LPS and hyperglycemic conditions. MATERIALS/METHODS: THP-1 cells differentiated by PMA (1 µM) were cultured for 48 h in the presence or absence of hesperetin under normoglycemic (5.5 mM/L glucose) or hyperglycemic (25 mM/L glucose) conditions and then treated with LPS (100 ng/mL) for 6 h before harvesting. Inflammation-related proteins and mRNA levels were evaluated by enzyme-linked immunosorbent assay, western blot, and quantitative polymerase chain reaction analyses. RESULTS: Hesperetin (0-100 µM, 48 h) treatment did not affect cell viability. The tumor necrosis factor-α and interleukin-6 levels increased in cells co-treated with LPS under hyperglycemic conditions compared to normoglycemic conditions, and these increases were decreased by hesperetin treatment. The TLR2/4 and MyD88 activity levels increased in cells co-treated with LPS under hyperglycemic conditions compared to normoglycemic conditions; however, hesperetin treatment inhibited the TLR2/4 and MyD88 activity increases. In addition, nuclear factor-κB (NF-κB) and Acetyl-NF-κB levels increased in response to treatment with LPS under hyperglycemic conditions compared to normoglycemic conditions, but those levels were decreased when treated with hesperetin. SIRT3 and SIRT6 expressions were increased by hesperetin treatment. CONCLUSIONS: Our results suggest that hesperetin may be a potential agent for suppressing inflammation in diabetes.

Phenethyl Isothiocyanate Improves Lipid Metabolism and Inflammation via mTOR/PPARγ/AMPK Signaling in the Adipose Tissue of Obese Mice.

Obesity is defined as excess adipose mass that causes serious health problems. Phenethyl isothiocyanate (PEITC) is a major and relatively nontoxic compound of the isothiocyanates. Although many studies have demonstrated that PEITC is a potent substance with physiological activities, such as anticancer activity, the precise mechanism for the effects of PEITC on inflammation and lipid metabolism in adipose tissue is not clear. Our study aimed to clarify the effects of PEITC supplements on the adipose tissue in obesity induced with a high-fat/cholesterol diet, and the underlying mechanisms. We induced obesity by feeding the mice with high fat with 1% cholesterol diet (HFCD) for 13 weeks. Mice were divided into five groups: normal diet (CON), HFCD, HFCD with 3 mg/(kg·d) gallic acid (HFCD+G), and HFCD with 30 and 75 mg/(kg·d) PEITC (HFCD+P30 and HFCD+P75, respectively). Using western blotting and quantitative polymerase chain reaction (qPCR) analysis of the adipose tissue, we determined the expression of lipid metabolism-related genes and inflammation-related genes. In the HFCD, the expression level of nuclear factor-κB (NF-κB), lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), and cyclooxygenase-2 (COX-2), was higher compared with that in the CON. Moreover, in the HFCD, the expression of p-mechanical targets of the rapamycin (mTOR) was increased, whereas that of p-AMP-activated protein kinase (AMPK) was decreased compared with that in the CON. Nevertheless, these decreased expression levels of p-AMPK and increased levels of LOX-1, p-mTOR, peroxisome proliferator-activated receptor gamma (PPARγ), NF-κB, and COX-2, were alleviated by PEITC supplementation. Therefore, we suggest that PEITC might be a potential preventive agent for ameliorating obesity-induced inflammation and adipogenesis by modulating the mTOR/AMPK/PPARγ pathway.

Protective effects of phenethyl isothiocyanate on foam cell formation by combined treatment of oxidized low-density lipoprotein and lipopolysaccharide in THP-1 macrophage.

Accumulation of cholesterol-laden macrophage foam cells characteristic of early stage atherosclerotic lesions. Phenethyl isothiocyanate (PEITC) is a naturally occurring isothiocyanate found in cruciferous vegetables that has reported a variety of activities including antioxidant and anti-inflammatory properties. However, the protective effect of PEITC on foam cell formation and its precise mechanism is not yet clear. Therefore, we investigated whether PEITC suppresses foam cell formation and regulates the expression of genes related to lipid accumulation, cholesterol efflux, and inflammation in THP-1 derived-macrophages. We exposed THP-1 derived-macrophages to oxidized low-density lipoprotein (ox-LDL) (20 µg/mL) and lipopolysaccharide (LPS) (500 ng/ml) to mimic foam cell formation. Here, PEITC downregulated the expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), cluster of differentiation 36 (CD36), scavenger receptor A1 (SR-A1), and nuclear factor-κB (NF-κB), while upregulated ATP binding cassette subfamily A member 1 (ABCA1)/liver-X-receptor α (LXR-α)/peroxisome proliferator-activated receptor gamma (PPARγ) and sirtuin 1 (SIRT1) expression compared to co-treated with ox-LDL and LPS. Taken together, PEITC, at least in part, inhibits foam cell formation and reduces lipid accumulation in foam cells. Therefore, we suggest that PEITC may be a potential candidate for the treatment and prevention of vascular inflammation and atherosclerosis.

Phenethyl Isothiocyanate Protects against High Fat/Cholesterol Diet-Induced Obesity and Atherosclerosis in C57BL/6 Mice.

This study concerns obesity-related atherosclerosis, hyperlipidemia, and chronic inflammation. We studied the anti-obesity and anti-atherosclerosis effects of phenethyl isothiocyanate (PEITC) and explored their underlying mechanisms. We established an animal model of high fat/cholesterol-induced obesity in C57BL/6 mice fed for 13 weeks. We divided the mice into five groups: control (CON), high fat/cholesterol (HFCD), HFCD with 3 mg/kg/day gallic acid (HFCD + G), and HFCD with PEITC (30 and 75 mg/kg/day; HFCD + P30 and P75). The body weight, total cholesterol, and triglyceride were significantly lower in the HFCD + P75 group than in the HFCD group. Hepatic lipid accumulation and atherosclerotic plaque formation in the aorta were significantly lower in both HFCD + PEITC groups than in the HFCD group, as revealed by hematoxylin and eosin (H&E) staining. To elucidate the mechanism, we identified the expression of genes related to inflammation, reverse cholesterol transport, and lipid accumulation pathway in the liver. The expression levels of peroxisome proliferator activated receptor gamma (PPARγ), liver-X-receptor α (LXR-α), and ATP binding cassette subfamily A member 1 (ABCA1) were increased, while those of scavenger receptor A (SR-A1), cluster of differentiation 36 (CD36), and nuclear factor-kappa B (NF-κB) were decreased in the HFCD + P75 group compared with those in the HFCD group. Moreover, PEITC modulated H3K9 and H3K27 acetylation, H3K4 dimethylation, and H3K27 di-/trimethylation in the HFCD + P75 group. We, therefore, suggest that supplementation with PEITC may be a potential candidate for the treatment and prevention of atherosclerosis and obesity.