Eun-Ha Joh

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.

Echinocystic acid ameliorates lung inflammation in mice and alveolar macrophages by inhibiting the binding of LPS to TLR4 in NF-κB and MAPK pathways

Orally administered lancemaside A, which is isolated from Codonopsis lanceolata (family Campanulaceae), showed anti-colitic effect in mice. However, its metabolite echinocystic acid was absorbed into the blood. Therefore, its anti-inflammatory effects were investigated in lipopolysaccharide (LPS)-stimulated alveolar macrophages in vitro and acute lung injury in vivo. Alveolar macrophages from mice were stimulated with LPS and were treated with echinocystic acid. Acute lung injury was induced by intratracheal administration of LPS in mice. Mice were treated with echinocystic acid or dexamethasone. Echinocystic acid potently suppressed the production of the pro-inflammatory cytokines, TNF-α and IL-1β, as well as of the activations of NF-κB and MAPKS, in LPS-stimulated alveolar macrophages. Echinocystic acid also down-regulated the production of inflammatory markers, which included inducible nitric oxide synthase and cyclooxygenase-2, as well as the inflammatory mediators, nitric oxide and prostaglandin E(2), in LPS-stimulated alveolar macrophages. Echinocystic acid also inhibited the activation of IL-1 receptor-associated kinases, and the activation of mitogen-activated protein kinases in LPS-stimulated alveolar macrophages. Furthermore, echinocystic acid potently inhibited the interaction between LPS and TLR4 in alveolar macrophages transfected with or without MyD88 siRNA, although it did not inhibit the binding in the macrophages transfected with TLR4 siRNA. Echinocystic acid suppressed LPS-induced acute lung inflammation in mice, as well as the expression of pro-inflammatory cytokines, such as IL-1β and TNF-α, and their transcription factor, NF-κB. On the basis of these findings, echinocystic acid, a metabolite of lancemaside A, may express anti-inflammatory effects by inhibiting the binding of LPS to TLR4 on macrophages.

Ginsenoside Rg5 ameliorates lung inflammation in mice by inhibiting the binding of LPS to toll-like receptor-4 on macrophages.

Heating and steaming processes have been applied to various natural medicines for either enhancing or altering their pharmacological activities, and the chemical compositions of the active components. While ginsenoside Rb1, which is the major constituent of raw ginseng, has been studied extensively for its anti-inflammatory effect, the biological activity of ginsenoside Rg5, a major constituent of steamed ginseng, remains to be explored. Here, we isolated Rg5 and examined anti-inflammatory effect in lipopolysaccharide (LPS)-stimulated macrophages and on LPS-induced lung inflammation. Rg5 inhibited the expression of proinflammatory cytokines, IL-1β and TNF-α, as well as inflammatory enzymes, COX-2 and iNOS in LPS-stimulated alveolar macrophages. Rg5 also reduced LPS-induced phosphorylation of IL-1 receptor-associated kinases (IRAK)-1 and IKK-β, as well as the degradation of IRAK-1 and IRAK-4. Rg5 inhibited the phosphorylation of NF-κB as well as the translocation of p65 into the nucleus. When macrophages were treated with Alexa Fluor 594-conjugated LPS in the presence of Rg5, the fluorescence intensity of LPS observed outside the cell membrane was lower than that in LPS-stimulated alveolar macrophages alone. Rg5, inhibited the levels of protein and neutrophils in bronchoalveolar lavage fluid of LPS-stimulated mice, as well as pro-inflammatory cytokines, TNF-α and IL-1β. Rg5 also inhibited iNOS and COX expressions, and NF-κB activation in LPS-stimulated lung inflammation of mice. The inhibitory effect of Rg5 (10 mg/kg) was comparable to that of dexamethasone (5 mg/kg). Based on these findings, Rg5 can ameliorate lung inflammation possibly by inhibiting the binding of LPS to toll-like receptor (TLR)-4 on macrophages.

Kalopanaxsaponin A ameliorates experimental colitis in mice by inhibiting IRAK-1 activation in the NF-κB and MAPK pathways

BACKGROUND AND PURPOSE Kalopanaxsaponin A, a triterpenoid saponin isolated from Kalopanax pictus (family Araliaceae), potently inhibited nuclear factor‐kappa B (NF‐κB) activation in lipopolysaccharide (LPS)‐stimulated peritoneal macrophages during a screening programme for anti‐colitis agents from natural products. Its anti‐inflammatory mechanism remains unknown. Therefore, we investigated its anti‐inflammatory effects in lipopolysaccharide (LPS)‐ or peptidoglycan‐stimulated murine peritoneal macrophages and trinitrobenzene sulphonic acid (TNBS)‐induced colitic mice.

Anti-pruritic effect of baicalin and its metabolites, baicalein and oroxylin A, in mice

AbstractAim:To explore whether intestinal microflora plays a role in anti-pruritic activity of baicalin, a main constituent of the rhizome of Scutellaria baicalensis (SB).Methods:Baicalin was anaerobically incubated with human fecal microflora, and its metabolites, baicalein and oroxylin A, were isolated. The inhibitory effect of baicalin and its metabolites was accessed in histamine- or compound 48/80-induced scratching behavior in mice.Results:Baicalin was metabolized to baicalein and oroxylin A, with metabolic activities of 40.2±26.2 and 1.2±1.1 nmol·h−1·mg−1 wet weight of human fecal microflora, respectively. Baicalin (20, 50 mg/kg) showed more potent inhibitory effect on histamine-induced scratching behavior when orally administered than intraperitoneally. In contrast, baicalein and oroxylin A had more potent inhibitory effect when the intraperitoneally administered. The anti-scratching behavior activity of oral baicalin and its metabolites was in proportion to their inhibition on histamine-induced increase of vascular permeability with oroxylin A more potent than baicalein and baicalin. In Magnus test using guinea pig ileum, oroxylin A is more potent than baicalein and baicalin in inhibition of histamine-induced contraction. The anti-scratching behavioral effect of oral baicalin was significantly reduced when oral antibiotics were simultaneously administered, whereas the effect of baicalein and oroxylin A were not affected.Conclusion:Oral baicalin may be metabolized by intestinal microflora into baicalein and oroxylin A, which ameliorate pruritic reactions through anti-histamine action.

Lancemaside A isolated from Codonopsis lanceolata and its metabolite echinocystic acid ameliorate scopolamine-induced memory and learning deficits in mice.

The rhizome of Codonopsis lanceolata (family Campanulaceae), which contains lancemaside A as a main constituent, has been used as herbal medicine to treat inflammation, insomnia, and hypomnesia. Lancemaside A and echinocystic acid, which is its metabolite by intestinal microflora, potently inhibited acetylcholinesterase activity in a dose-dependent manner, with IC₅₀ value 13.6 μM and 12.2 μM, respectively. Its inhibitory potency is comparable with that of donepezil (IC₅₀=10.9 μM). Lancemaside A and echinocystic acid significantly reversed scopolamine-induced memory and learning deficits on passive avoidance task. Lancemaside A orally administered 5h before treatment with scopolamine reversed scopolamine-induced memory and learning deficits more potently than one orally administered 1h before. Echinocystic acid more potently reversed it than lancemaside A. Lancemaside A and echinocystic acid significantly reversed scopolamine-induced memory and learning deficits on the Y-maze and Morris water maze tasks. Lancemaside A and echinocystic acid also increased the expression of brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP response element binding protein (p-CREB). Based on these findings, orally administered lancemaside A may be metabolized to echinocystic acid, which may be absorbed into the blood and ameliorate memory and learning deficits by inhibiting AChE activity and inducing BDNF and p-CREB expressions.

Lancemaside A inhibits lipopolysaccharide‐induced inflammation by targeting LPS/TLR4 complex

In our previous study, lancemaside A isolated from Codonopsis lanceolata (family Campanulaceae) ameliorated colitis in mice. In this study, the anti‐inflammatory effects of lancemaside A was investigated in lipopolysaccharide (LPS)‐stimulated mice and their peritoneal macrophage cells. Lancemaside A suppressed the production of pro‐inflammatory cytokines, TNF‐α and IL‐1β, in vitro and in vivo. Lancemaside A also down‐regulated inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2), as well as the inflammatory mediators, nitric oxide (NO), and PGE2. Lancemaside A also inhibited the expression of IL‐1 receptor‐associated kinase‐4 (IRAK‐4), the phosphorylation of IKK‐β and IκB‐α, the nuclear translocation of NF‐κB and the activation of mitogen‐activated protein kinases in LPS‐stimulated peritoneal macrophages. Furthermore, lancemaisde A inhibited the interaction between LPS and TLR4, as well as IRAK‐4 expression in peritoneal macrophages. Based on these findings, lancemaside A expressed anti‐inflammatory effects by regulating both the binding of LPS to TLR4 on macrophages. J. Cell. Biochem. 111: 865–871, 2010.

Metabolism of Cyanidin‐3‐O‐β‐D‐Glucoside Isolated from Black Colored Rice and Its Antiscratching Behavioral Effect in Mice

Black-colored rice (BCR), the main constituent of which is cyanidin-3-O-beta-D-glucoside (C3G), exhibits an anti-allergic effect, and orally administered C3G is mainly metabolized to protocatechuic acid in rats. Therefore, to understand the relationship between the metabolism of C3G and its pharmacological effect, we isolated C3G from BCR, anaerobically incubated it with fecal microflora, investigated its metabolite(s) by LC-MS/MS, and measured the antiscratching behavioral effects of C3G and its metabolites. C3G was metabolized to protocatechuic acid via cyanidin. Protocatechuic acid and cyanidin were identified as the metabolites. The activities transforming C3G to protocatechuic acid and cyanidin were 28.2 +/- 11.7 and 21.8 +/- 5.2 nmol/h/mg fecal microflora, respectively. C3G and its metabolites showed inhibitory effects against histamine- or compound 48/80-induced scratching behaviors in mice. C3G more potently inhibited scratching behaviors following oral administration than following intraperitoneal administration. However, protocatechuic acid and cyanidin showed more potent inhibition when administered intraperitoneally than when administered orally. These metabolites also inhibited the expression of allergic cytokines, IL-4 and TNF-alpha, and the activation of their transcription factor, NF-kappaB, in RBL-2H3 cells stimulated with IgE-antigen. These findings suggest that C3G-rich BCR may be a beneficial food for diseases involving scratching behaviors, such as chronic dermatitis, rhinitis, and psoriasis.

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.