In-Ah Lee

High Fat Diet-Induced Gut Microbiota Exacerbates Inflammation and Obesity in Mice via the TLR4 Signaling Pathway

Background & Aims While it is widely accepted that obesity is associated with low-grade systemic inflammation, the molecular origin of the inflammation remains unknown. Here, we investigated the effect of endotoxin-induced inflammation via TLR4 signaling pathway at both systemic and intestinal levels in response to a high-fat diet. Methods C57BL/6J and TLR4-deficient C57BL/10ScNJ mice were maintained on a low-fat (10 kcal % fat) diet (LFD) or a high–fat (60 kcal % fat) diet (HFD) for 8 weeks. Results HFD induced macrophage infiltration and inflammation in the adipose tissue, as well as an increase in the circulating proinflammatory cytokines. HFD increased both plasma and fecal endotoxin levels and resulted in dysregulation of the gut microbiota by increasing the Firmicutes to Bacteriodetes ratio. HFD induced the growth of Enterobecteriaceae and the production of endotoxin in vitro. Furthermore, HFD induced colonic inflammation, including the increased expression of proinflammatory cytokines, the induction of Toll-like receptor 4 (TLR4), iNOS, COX-2, and the activation of NF-κB in the colon. HFD reduced the expression of tight junction-associated proteins claudin-1 and occludin in the colon. HFD mice demonstrated higher levels of Akt and FOXO3 phosphorylation in the colon compared to the LFD mice. While the body weight of HFD-fed mice was significantly increased in both TLR4-deficient and wild type mice, the epididymal fat weight and plasma endotoxin level of HFD-fed TLR4-deficient mice were 69% and 18% of HFD-fed wild type mice, respectively. Furthermore, HFD did not increase the proinflammatory cytokine levels in TLR4-deficient mice. Conclusions HFD induces inflammation by increasing endotoxin levels in the intestinal lumen as well as in the plasma by altering the gut microbiota composition and increasing its intestinal permeability through the induction of TLR4, thereby accelerating obesity.

Ginsenoside Rb1 and its metabolite compound K inhibit IRAK-1 activation—The key step of inflammation

In the preliminary study, ginsenoside Rb1, a main constituent of the root of Panax ginseng (family Araliaceae), and its metabolite compound K inhibited a key factor of inflammation, nuclear transcription factor κB (NF-κB) activation, in lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages. When ginsenoside Rb1 or compound K were orally administered to 2,4,6-trinitrobenzene sulfuric acid (TNBS)-induced colitic mice, these agents inhibited colon shortening, macroscopic score, and colonic thickening. Furthermore, treatment with ginsenoside Rb1 or compound K at 20mg/kg inhibited colonic myeloperoxidase activity by 84% and 88%, respectively, as compared with TNBS alone (p<0.05), and also potently inhibited the expression of tumor necrosis factor-α, interleukin (IL)-1β and IL-6, but increased the expression of IL-10. Both ginsenoside Rb1 and compound K blocked the TNBS-induced expressions of COX-2 and iNOS and the activation of NF-κB in mice. When ginsenoside Rb1 or compound K was treated in LPS-induced murine peritoneal macrophages, these agents potently inhibited the expression of the proinflammatory cytokines. Ginsenoside Rb1 and compound K also significantly inhibited the activation of interleukin-1 receptor-associated kinase-1 (IRAK-1), IKK-β, NF-κB, and MAP kinases (ERK, JNK, and p-38); however, interaction between LPS and Toll-like receptor-4, IRAK-4 activation and IRAK-2 activation were unaffected. Furthermore, compound K inhibited the production of proinflammatory cytokines more potently than did those of ginsenoside Rb1. On the basis of these findings, ginsenosides, particularly compounds K, could be used to treat inflammatory diseases, such as colitis, by targeting IRAK-1 activation.

Berberine ameliorates TNBS-induced colitis by inhibiting lipid peroxidation, enterobacterial growth and NF-κB activation

Berberine, which is a major constituent of the rhizome of Coptidis japonica (CJ), inhibits IL-8 production in colonic epithelial cells and improves 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. In our preliminary studies, berberine inhibited lipid peroxidation in liposomes prepared from l-α-phosphatidylcholine as well as TLR-4-linked NF-κB activation in HEK cells. Therefore, to clarify its anticolitic mechanism, we examined the inhibitory effects of berberine in TNBS-induced colitic C3H/HeN and C3H/HeJ mice. Its oral administration inhibited macroscopic score, body weight gain, colon shortening, myeloperoxidase activity, and lipid peroxidation in the colons of TNBS-treated C3H/HeN and C3H/HeJ mice. Berberine inhibited colonic expression of iNOS, COX-2, IL-1β, IL-6, and TNF-α, but increased IL-10 expression in the colons of TNBS-treated C3H/HeN and C3H/HeJ mice. Berberine also inhibited NF-κB activation in TNBS-treated C3H/HeN and C3H/HeJ mice, and inhibited TLR-4 expression in C3H/HeN, but not C3H/HeJ, mice. Treating C3H/HeN and C3H/HeJ mice with berberine significantly reduced the number of Enterobacteriaceae induced by TNBS, but restored the number of Bifidobacteria reduced by TNBS. Furthermore, berberine potently inhibited LPS-induced inflammation in peritoneal macrophages mainly via NF-κB and weakly via MAPKs. Based on these findings, berberine may improve colitis by inhibiting lipid peroxidation, enterobacterial growth and NF-κB activation.

Arctigenin ameliorates inflammation in vitro and in vivo by inhibiting the PI3K/AKT pathway and polarizing M1 macrophages to M2-like macrophages.

Seeds of Arctium lappa, containing arctigenin and its glycoside arctiin as main constituents, have been used as a diuretic, anti-inflammatory and detoxifying agent in Chinese traditional medicine. In our preliminary study, arctigenin inhibited IKKβ and NF-κB activation in peptidoglycan (PGN)- or lipopolysaccharide (LPS)-induced peritoneal macrophages. To understand the anti-inflammatory effect of arctigenin, we investigated its anti-inflammatory effect in LPS-stimulated peritoneal macrophages and on LPS-induced systemic inflammation as well as 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. Arctigenin inhibited LPS-increased IL-1β, IL-6 and TNF-α expression in LPS-stimulated peritoneal macrophages, but increased LPS-reduced IL-10 and CD204 expression. Arctigenin inhibited LPS-induced PI3K, AKT and IKKβ phosphorylation, but did not suppress LPS-induced IRAK-1 phosphorylation. However, arctigenin did not inhibit NF-κB activation in LPS-stimulated PI3K siRNA-treated peritoneal macrophages. Arctigenin suppressed the binding of p-PI3K antibody and the nucleus translocation of NF-κB p65 in LPS-stimulated peritoneal macrophages. Arctigenin suppressed blood IL-1β and TNF-α level in mice systemically inflamed by intraperitoneal injection of LPS. Arctigenin also inhibited colon shortening, macroscopic scores and myeloperoxidase activity in TNBS-induced colitic mice. Arctigenin inhibited TNBS-induced IL-1β, TNF-α and IL-6 expression, as well as PI3K, AKT and IKKβ phosphorylation and NF-κB activation in mice, but increased IL-10 and CD204 expression. However, it did not affect IRAK-1 phosphorylation. Based on these findings, arctigenin may ameliorate inflammatory diseases, such as colitis, by inhibiting PI3K and polarizing M1 macrophages to M2-like macrophages.

Dextran sulfate sodium and 2,4,6-trinitrobenzene sulfonic acid induce lipid peroxidation by the proliferation of intestinal gram-negative bacteria in mice.

AbstrectBackgroundTo understand whether TLR-4-linked NF-kB activation negatively correlates with lipid peroxidation in colitic animal models, we caused colitis by the treatment with dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzenesulfonic acid (TNBS) to C3H/HeJ (TLR-4-defective) and C3H/HeN (wild type) mice, investigated inflammatory markers, lipid peroxidation, proinflammatory cytokines and TLR-4-linked NF-κB activation, in colon and intestinal bacterial composition in vivo.MethodsOrally administered DSS and intrarectally injected TNBS all caused severe inflammation, manifested by shortened colons in both mice. These agents increased intestinal myeloperoxidase activity and the expression of the proinflammatory cytokines, IL-1β, TNF-α and IL-6, in the colon.ResultsDSS and TNBS induced the protein expression of TLR-4 and activated transcription factor NF-κB. However, these colitic agents did not express TLR-4 in C3H/HeJ mice. Of proinflammatory cytokines, IL-1β was most potently expressed in C3H/HeN mice. IL-1β potently induced NF-κB activation in CaCo-2 cells, but did not induce TLR-4 expression. DSS and TNBS increased lipid peroxide (malondialdehyde) and 4-hydroxy-2-nonenal content in the colon, but reduced glutathione content and superoxide dismutase and catalase activities. These colitic inducers increased the number of Enterobacteriaceae grown in DHL agar plates in both mice, although the number of anaerobes and bifidobacteria grown in GAM and BL agar plates was reduced. E. coli, K. pneumoniae and Proteus mirabilis isolated in DHL agar plates increased lipid peroxidation in liposomes prepared by L-α-phosphatidylcholine, but B. animalis and B. cholerium isolated from BL agar plates inhibited it.DiscussionThese findings suggest that DSS and TNBS may cause colitis by inducing lipid peroxidation and enterobacterial proliferation, which may deteriorate the colitis by regulating proinflammatory cytokines via TLR-4-linked NF-κB activation pathway.

Soyasaponin I Attenuates TNBS-Induced Colitis in Mice by Inhibiting NF-κB Pathway

Soybean, which contains soyasaponins and isoflavones as representative constituents, exhibits anti-inflammatory and antioxidant effects. To understand the anti-inflammatory effects of soyasaponins, we isolated soyasaponin I, a major constituent of soybean, and investigated the inhibitory effects on inflammatory markers in LPS-stimulated mouse peritoneal macrophages and 3,4,5-trinitrobenzenosulfonic acid (TNBS)-induced colitic mice. Soyasaponin I, which exhibited lipid peroxidation-inhibitory effects in vitro, inhibited the production of proinflammatory cytokines (TNF-α and IL-1β), inflammatory mediators (NO and PGE2), and inflammatory enzymes (COX-2 and iNOS) in LPS-stimulated peritoneal macrophages. Soyasaponin I also suppressed the phosphorylation of IκB-α and the nuclear translocation of NF-κB. However, these soyasaponins barely inhibited mitogen-activated protein kinases. Oral administration of soyasaponin I (10 and 20 mg/kg) to TNBS-treated colitic mice significantly reduced inflammatory markers, colon length, myeloperoxidase, lipid peroxide (malondialdehyde and 4-hydroxy-2-nonenal), proinflammatory cytokines and NF-κB activation in the colon, as well as increased glutathione content, superoxide dismutase, and catalase activity. Based on these findings, soyasaponin I may attenuate colitis by inhibiting the NF-κB pathway.

β-Sitosterol attenuates high-fat diet-induced intestinal inflammation in mice by inhibiting the binding of lipopolysaccharide to toll-like receptor 4 in the NF-κB pathway.

SCOPE β-Sitosterol, a common phytosterol, has been shown to exhibit anti-inflammatory effects. Here, we investigated the effect of β-sitosterol on high-fat diet (HFD) induced colitis in mice and on LPS-stimulated mouse intestinal macrophages. METHODS AND RESULTS C57BL/6J mice were maintained on an LFD (10 kcal% fat), an HFD (60 kcal% fat), or an HFD with β-sitosterol (20 mg/kg) administration for 8 weeks. The increased levels of body weight and epididymal fat pad weight as well as the concentrations of circulating proinflammatory cytokines and LPS in HFD mice compared with LFD mice were decreased by oral administration of β-sitosterol. The HFD-induced colonic inflammation evidenced by the increased expression of proinflammatory cytokines and the activation of nuclear factor kappa B (NF-κB) in the colon was also inhibited by β-sitosterol. In LPS-stimulated intestinal macrophages, β-sitosterol inhibited the production of proinflammatory cytokines and inflammatory enzymes as well as NF-κB activation. In addition, β-sitosterol significantly prevented the binding of LPS to intestinal as well as peritoneal macrophages. Furthermore, β-sitosterol potently inhibited the interaction between LPS and toll-like receptor 4 in intestinal macrophages transfected with control siRNA or MyD88 siRNA. CONCLUSION These findings indicate that β-sitosterol ameliorates HFD-induced colitis by inhibiting the binding of LPS to toll-like receptor 4 in the NF-κB pathway.

Inhibitory Effect of β-Sitosterol on TNBS-Induced Colitis in Mice

β-Sitosterol, a common sterol in herbal medicines, exhibits anti-inflammatory effects beneficial in the treatment of lung inflammation, asthma, and bronchospasm. To evaluate whether β-sitosterol also has anticolitic benefits, we tested the effect of β-sitosterol on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. β-Sitosterol inhibited colon shortening and led to lowered macroscopic scores and myeloperoxidase activity in TNBS-treated colitic mice. β-Sitosterol also inhibited the expression of proinflammatory cytokines TNF-α, IL-1β, and IL-6, and an inflammatory enzyme, cyclooxygenase (COX)-2, in the colons of TNBS-induced colitic mice, as well as the activation of NF-κB. Based on these findings, β-sitosterol may ameliorate colitis by inhibiting the NF-κB pathway.

Artemisia princeps Pamp. Essential Oil and Its Constituents Eucalyptol and α-terpineol Ameliorate Bacterial Vaginosis and Vulvovaginal Candidiasis in Mice by Inhibiting Bacterial Growth and NF-κB Activation

To investigate the inhibitory effects of Artemisia princeps Pamp. (family Asteraceae) essential oil (APEO) and its main constituents against bacterial vaginosis and vulvovaginal candidiasis, their antimicrobial activities against Gardnerella vaginalis and Candida albicans in vitro and their anti-inflammatory effects against G. vaginalis-induced vaginosis and vulvovaginal candidiasis were examined in mice. APEO and its constituents eucalyptol and α-terpineol were found to inhibit microbe growths. α-Terpineol most potently inhibited the growths of G. vaginalis and C. albicans with MIC values of 0.06 and 0.125 % (v/v), respectively. The antimicrobial activity of α-terpineol was found to be comparable to that of clotrimazole. Intravaginal treatment with APEO, eucalyptol, or α-terpineol significantly decreased viable G. vaginalis and C. albicans numbers in the vaginal cavity and myeloperoxidase activity in mouse vaginal tissues compared with controls. These agents also inhibited the expressions of proinflammatory cytokines (IL-1 β, IL-6, TNF- α), COX-2, iNOS, and the activation of NF- κB and increased expression of the anti-inflammatory cytokine IL-10. In addition, they inhibited the expressions of proinflammatory cytokines and the activation of NF- κB in lipopolysaccharide-stimulated peritoneal macrophages, and α-terpineol most potently inhibited the expressions of proinflammatory cytokines and NF- κB activation. Based on these findings, APEO and its constituents, particularly α-terpineol, ameliorate bacterial vaginosis and vulvovaginal candidiasis by inhibiting the growths of vaginal pathogens and the activation of NF- κB.

Soyasaponin Ab Ameliorates Colitis by Inhibiting the Binding of Lipopolysaccharide (LPS) to Toll-like Receptor (TLR)4 on Macrophages

Many clinical studies have shown that daily intake of soybean [ Glycine max (L.) Merr., Fabacease] or its foods may reduce the risk of osteoporosis, heart attack, hyperlipidemia, coronary heart disease, cardiovascular and chronic renal diseases, and cancers, including prostate, colon, and breast cancers. Of the soy constituents, soyasaponins exhibit anti-aging, antioxidant, apoptotic, and anti-inflammatory effects. However, the anti-inflammatory effect of soyasaponin Ab has not been thoroughly studied. Therefore, we investigated its anti-inflammatory effects in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitic mice and lipopolysaccharide (LPS)-stimulated peritoneal macrophages. Soyasaponin Ab inhibited colon shortening, myeloperoxidase activity, the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), and activation of the transcription factor nuclear factor-κB (NF-κB). Soyasaponin Ab (1, 2, 5, and 10 μM) inhibited the production of NO (IC(50) = 1.6 ± 0.1 μM) and prostaglandin E(2) (IC(50) = 2.0 ± 0.1 ng/mL), the expression of tumor necrosis factor (TNF)-α (IC(50) = 1.3 ± 0.1 ng/mL), interleukin (IL)-1β (IC(50) = 1.5 ± 0.1 pg/mL), and toll-like receptor (TLR)4, and the phosphorylation of interleukin-1 receptor-associated kinase (IRAK)-1 in LPS-stimulated peritoneal macrophages. Soyasaponin Ab weakly inhibited the phosphorylation of ERK, JNK, and p38. Soyasaponin Ab significantly reduced the binding of Alexa-Fluor-594-conjugated LPS to peritoneal macrophages. Soyasaponin Ab did not affect TLR4 expression or LPS-induced NF-κB activation in TLR4 siRNA-treated peritoneal macrophages (knockdown efficiency of TLR4 > 94%). On the basis of these findings, soyasaponin Ab may ameliorate colitis by inhibiting the binding of LPS to TLR4 on macrophages.