Hye Gwang Jeong

HEPATOPROTECTIVE EFFECTS OF 18β-GLYCYRRHETINIC ACID ON CARBON TETRACHLORIDE-INDUCED LIVER INJURY: INHIBITION OF CYTOCHROME P450 2E1 EXPRESSION

The protective effects of 18beta-glycyrrhetinic acid (GA), the aglycone of glycyrrhizin (GL) derived from licorice, on carbon tetrachloride-induced hepatotoxicity and the possible mechanisms involved in this protection were investigated in mice. Pretreatment with GA prior to the administration of carbon tetrachloride significantly prevented an increase in serum alanine, aspartate aminotransferase activity and hepatic lipid peroxidation in a dose-dependent manner. In addition, pretreatment with GA also significantly prevented the depletion of glutathione (GSH) content in the livers of carbon tetrachloride-intoxicated mice. However, reduced hepatic GSH levels and glutathione-S-transferase activities were unaffected by treatment with GA alone. Carbon tetrachloride-induced hepatotoxicity was also prevented, as indicated by a liver histopathologic study. The effects of GA on the cytochrome P450 (P450) 2E1, the major isozyme involved in carbon tetrachloride bioactivation, were also investigated. Treatment of mice with GA resulted in a significant decrease of the P450 2E1-dependent hydroxylation of p-nitrophenol and aniline in a dose-dependent manner. Consistent with these observations, the P450 2E1 expressions were also decreased, as determined by immunoblot analysis. GA also showed antioxidant effects upon FeCl(2)-ascorbate-induced lipid peroxidation in mice liver homogenate and upon superoxide radical scavenging activity. These results show that protective effects of GA against the carbon tetrachloride-induced hepatotoxicity may be due to its ability to block the bioactivation of carbon tetrachloride, primarily by inhibiting the expression and activity of P450 2E1, and its free radical scavenging effects.

Inhibition of cytochrome P450 2E1 expression by oleanolic acid: hepatoprotective effects against carbon tetrachloride-induced hepatic injury.

The protective effects of oleanolic acid on carbon tetrachloride-induced hepatotoxicities and the possible mechanisms involved in this protection were investigated in mice. Pretreatment with oleanolic acid prior to the administration of carbon tetrachloride significantly prevented the increase in serum alanine aminotransferase and lactate dehydrogenase activity and liver lipid peroxidation in a dose-dependent manner. Hepatic glutathione levels and glutathione-S-transferase activities were not affected by treatment with oleanolic acid alone but pretreatment with oleanolic acid protects carbon tetrachloride-induced depletion of hepatic glutathione levels. The effects of oleanolic acid on the cytochrome P450 (P450) 2E1, the major isozyme involved in carbon tetrachloride bioactivation were investigated. Treatment of mice with oleanolic acid resulted in a significant decrease of P450 2E1-dependent p-nitrophenol and aniline hydroxylation in a dose-dependent manner. Consistent with these observations, the P450 2E1 expressions were also decreased, as determined by immunoblot analysis. These results show that the protective effects of oleanolic acid against the carbon tetrachloride-induced hepatotoxicity may, at least in part, be due to its ability to block bioactivation of carbon tetrachloride mainly by the inhibition of expression and activities of P450 2E1.

Suppressive effects of the kahweol and cafestol on cyclooxygenase‐2 expression in macrophages

Inducible cyclooxygenase‐2 (COX‐2) has been suggested to play a role in the processes of inflammation and carcinogenesis. Recent studies have shown the chemoprotective effects of kahweol and cafestol, which are coffee‐specific diterpenes. This study investigated the effects of kahweol and cafestol on the expression of COX‐2 in lipopolysaccharide (LPS)‐activated RAW 264.7 macrophages. Kahweol and cafestol significantly suppressed the LPS‐induced production of prostaglandin E2, COX‐2 protein and mRNA expression, and COX‐2 promoter activity in a dose‐dependent manner. Furthermore, kahweol blocked the LPS‐induced activation of NF‐κB by preventing IκB degradation and inhibiting IκB kinase activity. These results will provide new insights into the anti‐inflammatory and anti‐carcinogenic properties of kahweol and cafestol.

The coffee diterpene kahweol induces heme oxygenase-1 via the PI3K and p38/Nrf2 pathway to protect human dopaminergic neurons from 6-hydroxydopamine-derived oxidative stress.

In this study, we investigated the mechanisms of kahweol protection of neuronal cells from cell death induced by the Parkinsons disease‐related neurotoxin 6‐hydroxydopamine (6‐OHDA). Pretreatment of SH‐SY5Y cells with kahweol significantly reduced 6‐OHDA‐induced generation of ROS, caspase‐3 activation, and subsequent cell death. Kahweol also up‐regulated heme oxygenase‐1 (HO‐1) expression, which conferred neuroprotection against 6‐OHDA‐induced oxidative injury. Moreover, kahweol induced PI3K and p38 activation, which are involved in the induction of Nrf2, HO‐1 expression, and neuroprotection. These results suggest that regulation of the anti‐oxidant enzyme HO‐1 via the PI3K and p38/Nrf2 signaling pathways controls the intracellular levels of ROS.

Protective effect of Platycodi radix on carbon tetrachloride-induced hepatotoxicity

The protective effects of a Platycodi radix (Changkil: CK), the root of Platycodon grandiflorum A. DC (Campanulaceae) on carbon tetrachloride (CC14)-induced hepatotoxicity and the possible mechanisms involved in this protection were investigated in mice. Pretreatment with CK prior to the administration of CC14 significantly prevented the increased serum enzymatic activities of alanine and aspartate aminotransferase in a dose-dependent manner. In addition, pretreatment with CK also significantly prevented the elevation of hepatic malondialdehyde formation and the depletion of reduced glutathione content in the liver of CC14-intoxicated mice. However, hepatic reduced glutathione levels and glutathione S-transferase activities were not affected by treatment with CK alone. CC14-induced hepatotoxicity was also essentially prevented, as indicated by a liver histopathologic study. The effects of CK on the cytochrome P450 (P450) 2E1, the major isozyme involved in CC14 bioactivation were also investigated. Treatment of mice with CK resulted in a significant decrease of P450 2E1-dependent p-nitrophenol and aniline hydroxylation in a dose-dependent manner. CK showed antioxidant effects in FeCl2-ascorbate-induced lipid peroxidation in mice liver homogenate and in superoxide radical scavenging activity. Our results suggest that the protective effects of CK against CC14-induced hepatotoxicity possibly involve mechanisms related to its ability to block P450-mediated CC14 bioactivation and free radical scavenging effects.

Increased expression of Nrf2/ARE-dependent anti-oxidant proteins in tamoxifen-resistant breast cancer cells.

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem in breast cancer patients. In this study, we found that the expressions of anti-oxidant proteins (gamma-glutamylcysteine ligase heavy chain (gamma-GCL h), heme oxygenase-1, thioredoxin and peroxiredoxin1) in TAM-resistant MCF-7 (TAMR-MCF-7) cells were higher than control MCF-7 cells. Molecular analyses using antioxidant response element (ARE)-containing reporters and gel-shift supported the critical role of NF-E2-related factor2 (Nrf2)/ARE in the overexpression of antioxidant proteins in TAMR-MCF-7 cells. Intracellular peroxide production was significantly decreased in TAMR-MCF-7 cells and TAM resistance was partially reversed by Nrf2 siRNA. The basal phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase were increased in the TAMR-MCF-7 cells and the inhibition of ERK significantly decreased the activity of minimal ARE reporter and gamma-GCL h protein expression in TAMR-MCF-7 cells. However, exposure of TAMR-MCF-7 cells to 17-beta-estradiol or ICI-182,780 did not significantly change gamma-GCL h expression. These results suggest that the persistent activation of Nrf2/ARE is critical for the enhanced expression of anti-oxidant proteins in TAM-resistant breast cancer cells and the pathway of ERK, but not of estrogen receptor signaling are involved in the up-regulation of Nrf2/ARE.

A Leu-Lys-rich antimicrobial peptide: activity and mechanism.

To develop novel antibiotic peptides useful as therapeutic drugs, the analogues were designed to increase not only net positive charge by Lys substitution but also hydrophobic helix region by Leu substitution from cecropin A (1-8)-magainin 2 (1-12) hybrid peptide (CA-MA). In particular, CA-MA analogue P5 (P5), designed by flexible region (GIG-->P) substitution, Lys (positions 4, 8, 14, 15) and Leu (positions 5, 6, 12, 13, 16, 17, 20) substitutions, showed an enhanced antimicrobial and antitumor activity without hemolysis. Confocal microscopy showed that P5 was located in the plasma membrane. The antibacterial effects of analogues were further confirmed by using 1,6-diphenyl-1,3,5-hexatriene as a plasma membrane probe. Flow cytometric analysis revealed that P5 acted in an energy-independent manner. This interaction is also independent of the ionic environment. Furthermore, P5 causes significant morphological alterations of the bacterial surfaces as shown by scanning electron microscopy and showed strong membrane disrupting activity when examined using liposomes (phosphatidyl choline/cholesterol; 10:1, w/w). Its potent antibiotic activity suggests that P5 is an excellent candidate as a lead compound for the development of novel antiinfective agents.

Augmentation of macrophage functions by an aqueous extract isolated from Platycodon grandiflorum.

Platycodon grandiflorum has been claimed to have a wide range of health benefits, which include immunostimulation and antitumor activity. The associated biological mechanisms are unclear; however, of the wide diversity of effects, it is believed that their activities may be exerted through several potent effector cells such as macrophages. Therefore, the effects of an aqueous extract from the root of P. grandiflorum (Changkil: CK) on mouse peritoneal macrophage function were investigated. It was found that CK stimulated macrophage proliferation, spreading ability, phagocytosis, cytostatic activity, and nitric oxide production in a dose-dependent manner, and that the production of cytokines such as TNF-alpha, IL-1beta and IL-6 were similarly increased. CK significantly affected secretion at concentrations greater than 10 microg/ml; its maximal effects were at the concentration of 100 microg/ml. Reverse transcription-polymerase chain reaction showed that CK increased the appropriate cytokine mRNAs. These results suggest that CK is a potent enhancer of macrophage function.

Protective effect of metallothionein-III on DNA damage in response to reactive oxygen species

Metallothionein (MT)-III is a member of a brain-specific MT family, in contrast to MT-I and MT-II that are found in most tissues and are implicated in metal ion homeostasis and as an antioxidant. To investigate the defensive role of MT-III in terms of hydroxyl radical-induced DNA damage, we used purified human MT-III. DNA damage was detected by single-strand breaks of plasmid DNA and deoxyribose degradation. In this study, we show that MT-III is able to protect against the DNA damage induced by ferric ion-nitrilotriacetic acid and H(2)O(2), and that this protective effect is inhibited by the alkylation of the sulfhydryl groups of MT-III by treatment with EDTA and N-ethylmaleimide. MT-III was also able to efficiently remove the superoxide anion, which was generated from the xanthine/xanthine oxidase system. These results strongly suggest that MT-III is involved in the protection of reactive oxygen species-induced DNA damage, probably via direct interaction with reactive oxygen species, and that MT-III acts as a neuroprotective agent.

Ursolic acid enhances nitric oxide and tumor necrosis factor-α production via nuclear factor-κB activation in the resting macrophages

Ursolic acid (UA), a pentacyclic triterpene acid, is reported to have anti‐tumor activities; however, the mechanism underlying its anti‐tumorigenic effects is poorly understood. To further determine the mechanism of UA, we investigated the effects of UA on the release of nitric oxide (NO) and tumor necrosis factor‐α (TNF‐α), and on the level of inducible nitric oxide synthase (iNOS) and TNF‐α gene expression in mouse resting macrophages. We found that UA elicited a dose‐dependent increase in NO and TNF‐α production, and the level of iNOS and TNF‐α mRNA. Transient expression and electrophoretic mobility shift assays with nuclear factor‐κB (NF‐κB) binding sites revealed that the increased level of iNOS mRNA and TNF‐α mRNA induced by UA were mediated by the NF‐κB transcription factor complex. These results demonstrate that UA stimulates NO and TNF‐α release and is able to upregulate iNOS and TNF‐α expression through NF‐κB transactivation in the resting macrophages.