Jae-Myung Yoo

Effect of endocannabinoids on IgE-mediated allergic response in RBL-2H3 cells.

Recently, some endocannabinoids were reported to show anti-inflammatory and anti-allergic activities. In this respect, various arachidonoyl endocannabinoids were screened for the inhibition of allergic response in IgE-activated RBL-2H3 cells. Among arachidonoyl endocannabinoids with a low cytotoxicity, only NA-5HT remarkably inhibited the release of β-hexosaminidase (IC(50), 13.58 μM), a marker of degranulation, and tumor necrosis factor-α (IC(50), 12.52 μM), a pro-inflammatory cytokine, in IgE-activated RBL-2H3 cells. Additionally, NA-5HT markedly suppressed the formation of prostaglandin D(2) (PGD(2)) with IC(50) value of 1.27 μM and leukotriene B(4) (LTB(4)) with IC(50) value of 1.20 μM, and slightly LTC4. When effect of NA-5HT on early stage of FcεRI cascade was investigated, it significantly inhibited phosphorylation of Syk, but not Lyn. Furthermore, NA-5HT suppressed phosphorylation of PLCγ1/2 and PKCδ, related to degranulation process, as well as phosphorylation of LAT, ERK1/2, p38, JNK, Gab2, PI3K and Akt, implicated in the expression of pro-inflammatory cytokines. Relative to its effect on the late stage, NA-5HT slightly reduced phosphorylation of 5-lipoxygenase (5-LO) and cyclooxygenase-2 (COX-2). Additionally, NA-5HT significantly reduced the level of p40(phox), and partially inhibited the expression of p47(phox) and p67(phox). From these results, it is suggested that NA-5HT expresses anti-allergic action by suppressing the activation of Syk, LAT, p38, JNK, PI3K and Akt, as well as the expression of ERK1/2 and NADPH oxidase subunits. Further, a strong inhibition of PGD(2) or LTB(4) biosynthesis by NA-5HT may be an additional mechanism for its anti-allergic action. Such anti-allergic actions of NA-5HT may contribute to further information about its biological functions.

Anti-Allergic Action of Aged Black Garlic Extract in RBL-2H3 Cells and Passive Cutaneous Anaphylaxis Reaction in Mice

Garlic (Allium sativum) has been used as a food as well as a component of traditional medicine. Aged black garlic (ABG) is known to have various bioactivities. However, the effect of ABG on allergic response is almost unknown. In the present study, we investigated whether ABG can inhibit immunoglobulin E-mediated allergic response in RBL-2H3 cells as well as in vivo passive cutaneous anaphylaxis (PCA). In in vitro tests, ethyl acetate extract (EBG) of ABG significantly inhibited the release of β-hexosaminidase (IC₅₀, 1.53 mg/mL) and TNF-α (IC₅₀, 0.98 mg/mL). Moreover, BG10, an active fraction of EBG, dramatically suppressed the release of β-hexosaminidase (IC₅₀, 53.60 μg/mL) and TNF-α (IC₅₀, 27.80 μg/mL). In addition, BG10 completely blocked the formation of prostaglandin E₂ and leukotriene B₄ at ≥25 μg/mL. When the effect of BG10 on FcɛRI receptor cascade was investigated, BG10 significantly inhibited the phosphorylation of Syk, but not Lyn. Furthermore, BG10 dose dependently decreased the phosphorylation of cytosolic phospholipase A₂ (cPLA₂) and 5-lipoxygenase (5-LO) as well as the expression of cyclooxygenase-2 (COX-2). Consistent with what has been mentioned earlier, BG10 also significantly inhibited the PCA reaction in mice. In conclusion, these results indicate that ABG suppresses the allergic response, and the mechanism for its anti-allergic action may involve suppressions of Syk, cPLA₂, 5-LO, and COX-2. The anti-allergic actions of ABG, EBG, or BG10 suggest that they may be useful as functional foods for allergic diseases.

Neuroprotective action of N-acetyl serotonin in oxidative stress-induced apoptosis through the activation of both TrkB/CREB/BDNF pathway and Akt/Nrf2/Antioxidant enzyme in neuronal cells

N-acetyl serotonin (NAS) as a melatonin precursor has neuroprotective actions. Nonetheless, it is not clarified how NAS protects neuronal cells against oxidative stress. Recently, we have reported that N-palmitoyl serotonins possessed properties of antioxidants and neuroprotection. Based on those, we hypothesized that NAS, a N-acyl serotonin, may have similar actions in oxidative stress-induced neuronal cells, and examined the effects of NAS based on in vitro and in vivo tests. NAS dose-dependently inhibited oxidative stress-induced cell death in HT-22 cells. Moreover, NAS suppressed glutamate-induced apoptosis by suppressing expression of AIF, Bax, calpain, cytochrome c and cleaved caspase-3, whereas it enhanced expression of Bcl-2. Additionally, NAS improved phosphorylation of tropomyosin-related kinase receptor B (TrkB) and cAMP response element-binding protein (CREB) as well as expression of brain-derived neurotrophic factor (BDNF), whereas the inclusion of each inhibitor of JNK, p38 or Akt neutralized the neuroprotective effect of NAS, but not that of ERK. Meanwhile, NAS dose-dependently reduced the level of reactive oxygen species, and enhanced the level of glutathione in glutamate-treated HT-22 cells. Moreover, NAS significantly increased expression of heme oxygenase-1, NAD(P)H quinine oxidoreductase-1 and glutamate-cysteine ligase catalytic subunit as well as nuclear translocation of NF-E2-related factor-2. Separately, NAS at 30 mg/kg suppressed scopolamine-induced memory impairment and cell death in CA1 and CA3 regions in mice. In conclusion, NAS shows actions of antioxidant and anti-apoptosis by activating TrkB/CREB/BDNF pathway and expression of antioxidant enzymes in oxidative stress-induced neurotoxicity. Therefore, such effects of NAS may provide the information for the application of NAS against neurodegenerative diseases.

Inhibitory effect of fermented Arctium lappa fruit extract on the IgE-mediated allergic response in RBL‑2H3 cells

Arctium lappa fruit has been used in traditional medicine, and it is known to exert beneficial effects, such as antioxidant, anti-inflammatory and anticancer effects. However, the effects of the Arctium lappa fruit on the allergic response remain unknown. In this study, we evaluated the anti-allergic effects of Arctium lappa fruit extract (AFE) and its fermented form (F-AFE) using immunoglobulin E (IgE)-activated RBL‑2H3 cells. To investigate the anti-allergic effects of AFE or F-AFE, we examined the release of β-hexosaminidase, a key biomarker of degranulation during an allergic reaction, and the production of pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) in the cells treated with or without the above-mentioned extracts. AFE weakly inhibited the release of β-hexosaminidase, whereas F-AFE significantly suppressed the release of β-hexosaminidase in a dose-dependent manner. Consistently, F-AFE suppressed the production of TNF-α and PGE2 in a dose-dependent manner. F-AFE exerted an inhibitory effect on the production of β-hexosaminidase, TNF-α and PGE2 with an IC50 value of 30.73, 46.96 and 36.27 µg/ml, respectively. Furthermore, F-AFE inhibited the phosphorylation of Lyn, Fyn and Syk, which are involved in the FcεRI signaling pathway, that of phosphoinositide phospholipase C (PLC)γ1/2 and protein kinase C (PKC)δ, which are associated with the degranulation process, as well as that of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK), p38 and Akt, which are associated with cytokine expression. In the late phase, F-AFE partially suppressed the phosphorylation of cytosolic phospholipase A2 (cPLA2), but not the expression of cyclooxygenase (COX)-2. To compare and identify the major components of the two extracts, we used high-performance liquid chromatography. The levels of arctigenin, one of the major compounds, were elevated 6-fold in F-AFE compared with AFE, whereas the levels of arctiin, an arctigenin glycoside, were decreased in F-AFE by approximately 57.40%. These results suggest that arctigenin plays an important role in the anti-allergic effects of F-AFE. Taken together, F-AFE containing anti-allergic phytochemicals, including arctigenin, inhibited the activation of the FcεRI receptor induced by the antigen‑IgE complex. Such effects may provide further information for the development of a phytomedicine for allergic diseases.

IL-32γ enhances TNF-α-induced cell death in colon cancer

Interleukin (IL)‐32 is a recently discovered cytokine that appears to play an important role in human colon cancer growth. We investigated that IL‐32γ in combination with TNF‐α remarkably inhibited cell growth of human colon cancer cells (HCT116 and SW620) and tumor growth in xenograft‐bearing nude mice. The transient enforced overexpression of IL‐32γ potentiated the inhibitory effect of TNF‐α on DNA synthesis, cell number and protein content, and enhanced apoptosis in colon cancer cells. We also found that knockdown of IL‐32γ by siRNA showed the abolishment of cell growth inhibitory effect of TNF‐α. The IL‐32γ‐overexpressing colon cancer cells further increased TNF‐α‐mediated expression of p38 MAPK as well as that of Bax, cleaved caspase‐3 and ‐9, but decreased that of antiapoptotic proteins such as Bcl‐2, cellular inhibitor of apoptosis protein (IAP) and X chromosome IAP. In xenograft model, the lipopolysaccharide (LPS)‐injected (1.25 mg/kg) mice inoculated with IL‐32γ‐transfected HCT116 colon cancer cells were more decrease tumor volume and weight than inoculated with vector. Tumor tissues isolated from LPS‐injected mice inoculated with IL‐32γ‐overexpressing colon cancer cells potentiated the expression levels of pro‐apoptotic proteins such as cleaved caspase‐3, 9 and Bax, but decreased that of Bcl‐2. Furthermore, the mice increased IL‐10 production, but decreased IL‐6 levels in serum. In conclusion, our results suggest that IL‐32γ may potentiate TNF‐α‐induced cell growth inhibition through activation of p38 MAPK pathways.

Inhibitory effects of mulberry fruit extract in combination with naringinase on the allergic response in IgE-activated RBL-2H3 cells.

In this study, we investigated the anti-allergic action of mulberry fruit extract (MFE) or MFE in combination with naringinase (MFEN) in IgE-activated RBL-2H3 cells, and investigated the mechanisms responsible for the anti-allergic effects of MFEN. β-hexosaminidase release assay was used to measure the amount of β-hexosaminidase released from the cells, and ELISA was used to measure the levels of tumor necrosis factor-α (TNF-α). We found that MFE significantly reduced the release of β-hexosaminidase (IC(50), 10.59 mg/ml) and TNF-α (IC(50), 4.87 mg/ml). Moreover, MFEN enhanced the inhibitory effects on the release of β-hexosaminidase (IC(50), 123.10 µg/ml) and TNF-α (IC(50), 65.01 µg/ml). Furthermore, MFEN had no cytotoxicity at the concentration range used to exert the anti-allergic effects. In addition, we evaluated the effects of MFEN on the formation of pro-inflammatory lipid mediators, such as prostaglandin D(2) (PGD(2)), leukotriene C(4) (LTC(4)) and leukotriene B(4) (LTB(4)) using enzyme immunoassay (EIA) kits. MFEN markedly reduced the formation of PGD(2) (IC(50), 6.47 µg/ml) and LTC(4) (IC(50), 0.31 µg/ml), but not LTB(4) (IC(50), 25.75 µg/ml). In mechanistic analyses, we measured the phosphorylation of Syk, Lyn and Fyn by immunoblot analysis. MFEN significantly inhibited the phosphorylation of Syk, but not that of Lyn or Fyn. MFEN also suppressed the phosphorylation of phospholipase C (PLC)γ1/2, protein kinase C (PKC)δ, linker for activation of T cells (LAT), extracellular signal-regulated protein kinase (ERK)1/2, JNK, GRB2-associated binding protein 2 (Gab2), phosphoinositide-3-kinase (PI3K), Akt, cytosolic phospholipase A2 and 5-lipoxygenase, as well as the expression of cyclooxygenase-2. In conclusion, these results suggest that MFEN exerts potent inhibitory effects on allergic response through the suppression of the activation of the FcεRI signaling cascade. Our data demonstrating the anti-allergic effects of MFEN may provide further insight into the therapeutic application of MFEN or its use as a functional food.

Effect of endocannabinoids on soybean lipoxygenase-1 activity

Endocannabinoids appear to be involved in a variety of physiological processes. Lipoxygenase activity has been known to be affected by unsaturated fatty acids or phenolic compounds. In this study, we examined whether endocannabinoids containing both N-acyl group and phenolic group can affect the activity of soybean lipoxygenase (LOX)-1, similar to mammalian 15-lipoxygenase in physicochemical properties. First, N-arachidonoyl dopamine and N-oleoyl dopamine were found to inhibit soybean LOX-1-catalyzed oxygenation of linoleic acid in a non-competitive manner with a Ki value of 3.7 μM and 6.2 μM, respectively. Meanwhile, other endocannabinoids failed to show a remarkable inhibition of soybean LOX-1. Separately, N-arachidonoyl dopamine and N-arachidonoyl serotonin were observed to inactivate soybean LOX-1 with Kin value of 27 μM and 24 μM, respectively, and k3 value of 0.12 min(-1) and 0.35 min(-1), respectively. Furthermore, such an inactivation was enhanced by ascorbic acid, but suppressed by 13(S)-hydroperoxy-9,11-octadecadienoic acid. Taken together, it is proposed that endocannabinoids containing polyunsaturated acyl moiety and phenolic group may be efficient for the inhibition as well as inactivation of 15-lipoxygenase.

Ethanol Extract of Sanguisorbae Radix Inhibits Mast Cell Degranulation and Suppresses 2,4-Dinitrochlorobenzene-Induced Atopic Dermatitis-Like Skin Lesions

Sanguisorbae Radix (SR) is well known as herbal medicine named “Zi-Yu” in Korea, which is the dried roots of Sanguisorba officinalis L. (Rosacease). We investigated the underlying mechanism on the inhibition of atopic dermatitis (AD) of an ethanol extract of SR (ESR) using 2,4-dinitrochlorobenzene- (DNCB-) induced AD mice model. Oral administration of ESR significantly suppressed DNCB-induced AD-like symptoms such as scratching behavior, ear thickness, epidermal thickness, and IgE levels. To investigate the effects of ESR treatment on degranulation of IgE/Ag-activated mouse bone marrow-derived mast cells (BMMCs), we measured the release of β-hexosaminidase (β-HEX, degranulation marker). ESR decreased the infiltration of eosinophils and mast cells into the AD skin lesions. Furthermore, ESR significantly inhibited degranulation of IgE/Ag-activated BMMCs. We have demonstrated that ESR decreased AD symptoms in mice and inhibits degranulation of IgE/Ag-activated mast cells. Our study suggests that ESR may serve as a potential therapeutic candidate for the treatment of AD symptoms.

Anti-inflammatory effects of Perillae Herba ethanolic extract against TNF-α/IFN-γ-stimulated human keratinocyte HaCaT cells

ETHNOPHARMACOLOGICAL RELEVANCE Perillae Herba is a perennial plant that is widely distributed throughout Asia. The leaves of Perillae Herba have been widely used to treat various diseases, such as cold due to wind-cold, headache, cough, abdominal fullness, distention, and fish and crab poisoning. MATERIALS AND METHODS To assess the anti-inflammatory activity of Perillae Herba leaf ethanolic extract (PHE) in human keratinocytes, we measured the tumor necrosis factor (TNF)-α/interferon (IFN)-γ-induced mRNA expression and production of proinflammatory chemokines such as thymus and activation-regulated chemokines; regulated on activation, normal T cell expressed and secreted; interleukin (IL)-6; and IL-8 in HaCaT cells. We evaluated the ability of PHE to decrease the expression of proinflammatory marker proteins, such as mitogen-activated protein kinase (MAPK), STAT-1, and NK-κB, using western blot analysis and immunocytochemistry. RESULTS PHE inhibited activation of p38, ERK, and JNK and suppressed the phosphorylation of STAT-1 and NK-κB in TNF-α/IFN-γ-stimulated HaCaT cells. PHE also suppressed chemokine mRNA and protein levels in TNF-α/IFN-γ-stimulated HaCaT cells. PHE appears to regulate chemokine formation by inhibiting activation of MAPK, as well as the STAT-1 and NK-κB pathways. CONCLUSIONS PHE suppresses the expression and production of TNF-α/IFN-γ-stimulated proinflammatory chemokines by blocking NF-κB, STAT-1, and MAPK activation.

Corrigendum to “Anti-inflammatory effects of Perillae Herba ethanolic extract against TNF-α/IFN-γ-stimulated human keratinocyte HaCaT cells” [J. Ethnopharmacol. 211 (2018) 217–223]

Corrigendum Corrigendum to “Anti-inflammatory effects of Perillae Herba ethanolic extract against TNF-α/IFN-γ-stimulated human keratinocyte HaCaT cells” [J. Ethnopharmacol. 211 (2018) 217–223] Ju-Hye Yang, Jae-Myung Yoo, Esther Lee, BoHyoung Lee, Won-Kyung Cho, Kwang-Il Park, Jin Yeul Ma Korean Medicine (KM) Application Center, Korea Institute of Oriental Medicine (KIOM), 70 Cheomdan-ro, Dong-gu, Daegu 701-300, Republic of Korea