Kyoung-Tae Lee

SIRT1 attenuates palmitate-induced endoplasmic reticulum stress and insulin resistance in HepG2 cells via induction of oxygen-regulated protein 150

Endoplasmic reticulum (ER) stress has been implicated in the pathology of type 2 diabetes mellitus (T2DM). Although SIRT1 has a therapeutic effect on T2DM, the mechanisms by which SIRT1 ameliorates insulin resistance (IR) remain unclear. In this study, we investigated the impact of SIRT1 on palmitate-induced ER stress in HepG2 cells and its underlying signal pathway. Treatment with resveratrol, a SIRT1 activator significantly inhibited palmitate-induced ER stress, leading to the protection against palmitate-induced ER stress and insulin resistance. Resveratrol and SIRT1 overexpression induced the expression of oxygen-regulated protein (ORP) 150 in HepG2 cells. Forkhead box O1 (FOXO1) was involved in the regulation of ORP150 expression because suppression of FOXO1 inhibited the induction of ORP150 by SIRT1. Our results indicate a novel mechanism by which SIRT1 regulates ER stress by overexpression of ORP150, and suggest that SIRT1 ameliorates palmitate-induced insulin resistance in HepG2 cells via regulation of ER stress.

Anti-inflammatory effects of β-hydroxyisovalerylshikonin in BV2 microglia are mediated through suppression of the PI3K/Akt/NF-kB pathway and activation of the Nrf2/HO-1 pathway.

In the present study, we investigated whether β-hydroxyisovalerylshikonin (β-HIVS) affects the production of proinflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2) in BV2 microglial cells. Our data showed that β-HIVS inhibited secretion of NO and PGE2 and downregulated expression of their main regulatory genes, inducible NO synthesis (iNOS) and cyclooxygenase-2 (COX-2). β-HIVS also reduced the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) by suppressing nuclear translocation of the NF-κB subunits and inhibiting the degradation and phosphorylation of IκBα. Furthermore, an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), attenuated LPS-stimulated iNOS and COX-2 expression, suggesting that NF-κB inhibition is a main effector in the expression of iNOS and COX-2. We also found that LPS-induced NF-κB activation is regulated through inhibition of PI3K/Akt phosphorylation in response to β-HIVS. Additionally, β-HIVS caused the induction of heme oxygenase-1 (HO-1) via upregulation of nuclear factor-erythroid 2-related factor 2 (Nrf2), both of which are involved in the secretion of proinflammatory mediators such as NO and PGE2. Taken together, our data indicate that β-HIVS diminishes the proinflammatory mediators NO and PGE2 and the expression of their regulatory genes, iNOS and COX-2, in LPS-stimulated BV2 microglial cells by inhibiting PI3K/Akt-dependent NF-κB activation and inducing Nrf2-mediated HO-1 expression.

Anti-inflammatory mechanism of α-viniferin regulates lipopolysaccharide-induced release of proinflammatory mediators in BV2 microglial cells.

α-Viniferin is an oligostilbene of trimeric resveratrol and has anticancer activity; however, the molecular mechanism underlying the anti-inflammatory effects of α-viniferin has not been completely elucidated thus far. Therefore, we determined the mechanism by which α-viniferin regulates lipopolysaccharide (LPS)-induced expression of proinflammatory mediators in BV2 microglial cells. Treatment with α-viniferin isolated from Clematis mandshurica decreased LPS-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2). α-Viniferin also downregulated the LPS-induced expression of proinflammatory genes such as iNOS and COX-2 by suppressing the activity of nuclear factor kappa B (NF-κB) via dephosphorylation of Akt/PI3K. Treatment with a specific NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), indirectly showed that NF-κB is a crucial transcription factor for expression of these genes in the early stage of inflammation. Additionally, our results indicated that α-viniferin suppresses NO and PGE2 production in the late stage of inflammation through induction of heme oxygenase-1 (HO-1) regulated by nuclear factor erythroid 2-related factor (Nrf2). Taken together, our data indicate that α-viniferin suppresses the expression of proinflammatory genes iNOS and COX-2 in the early stage of inflammation by inhibiting the Akt/PI3K-dependent NF-κB activation and inhibits the production of proinflammatory mediators NO and PGE2 in the late stage by stimulating Nrf2-mediated HO-1 signaling pathway in LPS-stimulated BV2 microglial cells. These results suggest that α-viniferin may be a potential candidate to regulate LPS-induced inflammation.

Downregulation of NO and PGE2 in LPS-stimulated BV2 microglial cells by trans-isoferulic acid via suppression of PI3K/Akt-dependent NF-κB and activation of Nrf2-mediated HO-1.

Little is known about whether trans-isoferulic acid (TIA) regulates the production of lipopolysaccharide (LPS)-induced proinflammatory mediators. Therefore, we examined the effect of TIA isolated from Clematis mandshurica on LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in BV2 microglial cells. We found that TIA inhibited the production of LPS-induced NO and PGE2 without accompanying cytotoxicity in BV2 microglial cells. TIA also downregulated the expression levels of specific regulatory genes such as inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) by suppressing LPS-induced NF-κB activity via dephosphorylation of PI3K/Akt. In addition, we demonstrated that a specific NF-κB inhibitor PDTC and a selective PI3K/Akt inhibitor, LY294002 effectively attenuated the expression of LPS-stimulated iNOS and COX-2 mRNA, while LY294002 suppressed LPS-induced NF-κB activity, suggesting that TIA attenuates the expression of these proinflammatory genes by suppressing PI3K/Akt-mediated NF-κB activity. Our results showed that TIA suppressed NO and PGE2 production through the induction of nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent heme oxygenase-1 (HO-1). Taken together, our data indicate that TIA suppresses the production of proinflammatory mediators such as NO and PGE2, as well as their regulatory genes, in LPS-stimulated BV2 microglial cells, by inhibiting PI3K/Akt-dependent NF-κB activity and enhancing Nrf2-mediated HO-1 expression.

Compound K modulates fatty acid-induced lipid droplet formation and expression of proteins involved in lipid metabolism in hepatocytes.

A key factor in the development of type 2 diabetes and non‐alcoholic fatty liver disease (NAFLD) is hepatic steatosis. Incubation of human hepatic cells with free fatty acids (FFAs) causes accumulation of neutral lipids in lipid droplets (LDs) and serves as a model for hepatic steatosis. Ginsenosides, active constituents of ginsengs, have demonstrated beneficial effects in various pharmacological areas, including diabetes, however their effect on lipid accumulation in hepatocytes remains unclear. Here, we examine the effect of compound K (ComK), an active metabolite of ginsenosides, on the regulation of LD formation and on the expression of proteins involved in lipid homeostasis in hepatocytes.

Potential chemoprevention of LPS-stimulated nitric oxide and prostaglandin E2 production by α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranosyl-3-indolecarbonate in BV2 microglial cells through suppression of the ROS/PI3K/Akt/NF-κB pathway

α-l-Rhamnopyranosyl-(1→6)-β-d-glucopyranosyl-3-indolecarbonate (RG3I) is a chemical constituent isolated from the commonly used Asian traditional medicinal plant, Clematis mandshurica; however, no studies have been reported on its anti-inflammatory properties. In the present study, we found that RG3I attenuates the lipopolysaccharide (LPS)-induced DNA-binding activity of nuclear factor-κB (NF-κB) via the dephosphorylation of PI3K/Akt in BV2 microglial cells, leading to a suppression of nitric oxide (NO) and prostaglandin E2 (PGE2) production, along with that of their regulatory genes, inducible NO synthase (iNOS) and cyclooxygenase-2 (Cox-2). Further, the PI3K/Akt inhibitor, LY294002 diminished the expression of LPS-stimulated iNOS and COX-2 genes by suppressing NF-κB activity. Moreover, RG3I significantly inhibited LPS-induced reactive oxygen species (ROS) generation similar to the ROS inhibitors, N-acetylcysteine (NAC) and glutathione (GSH). Notably, NAC and GSH abolished the LPS-induced expression of iNOS and Cox-2 in BV2 microglial cells by inhibiting NF-κB activity. Taken together, our data indicate that RG3I suppresses the production of proinflammatory mediators such as NO and PGE2 as well as their regulatory genes in LPS-stimulated BV2 microglial cells by inhibiting the PI3K/Akt- and ROS-dependent NF-κB signaling pathway, suggesting that RG3I may be a good candidate to regulate LPS-induced inflammatory response.

Isobutyrylshikonin inhibits lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in BV2 microglial cells by suppressing the PI3K/Akt-mediated nuclear transcription factor-κB pathway

Microglia are important macrophages to defend against pathogens in the central nervous system (CNS); however, persistent or acute inflammation of microglia lead to CNS disorders via neuronal cell death. Therefore, we theorized that a good strategy for the treatment of CNS disorders would be to target inflammatory mediators from microglia in disease. Consequently, we investigated whether isobutyrylshikonin (IBS) attenuates the production of proinflammatory mediators, such as nitric oxide (NO) and prostaglandin E2, in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Treatment with IBS inhibited the secretion of NO and prostaglandin E2 (as well as the expression of their key regulatory genes), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2). Isobutyrylshikonin also suppressed LPS-induced DNA-binding activity of nuclear transcription factor-κB (NF-κB), by inhibiting the nuclear translocation of p50 and p65 in addition to blocking the phosphorylation and degradation of IκBα. Pretreatment with pyrrolidine dithiocarbamate, a specific NF-κB inhibitor, showed the down-regulation of LPS-induced iNOS and COX-2 messenger RNA by suppressing NF-κB activity. This indirectly suggests that IBS-mediated NF-κB inhibition is the main signaling pathway involved in the inhibition of iNOS and COX-2 expression. In addition, IBS attenuated LPS-induced phosphorylation of PI3K and Akt, which are upstream molecules of NF-κB, in LPS-stimulated BV2 microglial cells. The functional aspects of the PI3K/Akt signaling pathway were analyzed with LY294002, which is a specific PI3K/Akt inhibitor that attenuated LPS-induced iNOS and COX-2 expression by suppressing NF-κB activity. These data suggest that an IBS-mediated anti-inflammatory effect may be involved in suppressing the PI3K/Akt-mediated NF-κB signaling pathway.

The Quantities of Methyl Orsellinate and Sparassol of Sparassis latifolia by Host Plants

It is known not only that antifungal compounds such as sparassol, methyl orsellinate (ScI) and methyl-dihydroxymethoxy-methylbenzoate (ScII) were produced during submerged culture from Sparassis crispa, but also that ScI and ScII were appeared higher antifungal activity than sparassol. The aim of this study, antifungal compounds of Sparassis latifolia were purified from mycelial culture media and identified by using NMR and ESI-MS. Based on HPLC analysis, methyl orsellinate and sparassol were detected at 15 min and 31 min of retention time, respectively. The compounds derived from S. latifolia were classified into four production patterns according to their strains. The strains originated from host plant Larix kaempferi and Pinus koraiensis showed different patterns of compound production, whereas the strains originated from host plant P. densiflora and Abies holophylla showed almost same patterns. There was no correlation between mycelial biomass and compound production. KFRI 645 strain from L. kaempferi exhibited higher methyl orsellinate production (0.170 mg/ml). Sparassol was produced by KFRI 747 from P. densiflora (0.004 mg/ml). Thus, our result revealed the new fact that methyl orsellinate and sparassol have different patterns according to the strains originated from different host plants.

New compound, 5-O-isoferuloyl-2-deoxy-D-ribono-γ-lacton from Clematis mandshurica: Anti-inflammatory effects in lipopolysaccharide-stimulated BV2 microglial cells

Microglia are main immune cells to exacerbate neural disorders in persistent overactivating. Therefore, it is a good strategy to regulate microglia for the treatment of neural disorders. In the present study, we isolated and characterized a novel compound, 5-O-isoferuloyl-2-deoxy-D-ribono-γ-lacton (5-DRL) from Clematis mandshurica, and evaluated its anti-inflammatory effect in lipopolysaccharide (LPS)-treated BV2 microglial cells. 5-DRL inhibited the expression of LPS-stimulated proinflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2), as well as their regulatory genes inducible NO syntheses (iNOS) and cyclooxygenase-2 (COX-2). 5-DRL also downregulated the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) through suppression of the nuclear translocation of the NF-κB subunits, p65 and p50. Consistent with the inhibition of iNOS and COX-2 via NF-κB activity with 5-DRL, an inhibitor of NF-κB, pyrrolidine dithiocarbamate (PDTC), also led to the suppression of LPS-induced iNOS and COX-2 expression. Additionally, 5-DRL corresponding with antioxidants, N-acetylcysteine (NAC) and glutathione (GSH), remarkably inhibited reactive oxygen species (ROS) generation. Both NAC and GSH, thus attenuated the expression of iNOS and COX-2 by suppressing NF-κB activation, indicating that 5-DRL suppresses LPS-induced iNOS and COX-2 expression through downregulation of the ROS-dependent NF-κB signaling pathway. The present study also indicated that 5-DRL suppresses NO and PGE2 production by inducing heme oxygenase-1 (HO-1) via nuclear factor erythroid 2-related factor 2 (Nrf2). Taken together, the present data indicate that 5-DRL attenuates the production of proinflammatory mediators such as NO and PGE2 as well as their regulatory genes in LPS-stimulated BV2 microglial cells by inhibiting ROS-dependent NF-κB activation and stimulating the Nrf2/HO-1 signal pathway. These data may be implicated in the application of 5-DRL in LPS-stimulated inflammatory disease.

Antagonistic effects of acetylshikonin on LPS-induced NO and PGE2 production in BV2 microglial cells via inhibition of ROS/PI3K/Akt-mediated NF-κB signaling and activation of Nrf2-dependent HO-1

Although acetylshikonin (ACS) is known to have antioxidant and antitumor activities, whether ACS regulates the expression of proinflammatory mediators in lipopolysaccharide (LPS)-stimulated microglial cells remains unclear. In this study, it was found that ACS isolated from Lithospermum erythrorhizon inhibits LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) release by suppressing the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in BV2 microglial cells. Furthermore, ACS reduced the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) and subsequently suppressed iNOS and COX-2 expression. Consistent with these data, ACS attenuated the phosphorylation of PI3K and Akt and suppressed the DNA-binding activity of NF-κB by inducing the generation of reactive oxygen species (ROS) in LPS-stimulated cells. In addition, ACS enhanced heme oxygenase-1 (HO-1) expression via nuclear factor-erythroid 2-related factor 2 (Nrf2) activation. Zinc protoporphyrin, a specific HO-1 inhibitor, partially attenuated the antagonistic effects of ACS on LPS-induced NO and PGE2 production. By contrast, the presence of cobalt protoporphyrin, a specific HO-1 inducer, potently suppressed LPS-induced NO and PGE2 production. These data indicate that ACS downregulates proinflammatory mediators such as NO and PGE2 by suppressing PI3K/Akt-dependent NF-κB activity induced by ROS as well as inducing Nrf2-dependent HO-1 activity. Taken together, ACS might be a good candidate to regulate LPS-mediated inflammatory diseases.