Ji Yun Jung

Regulation of Toll-like Receptor-Mediated Sestrin2 Induction by AP-1, Nrf2, and the Ubiquitin-Proteasome System in Macrophages

The Sestrin2 (Sesn2) is an evolutionary conserved enzyme that scavenges reactive oxygen species and regulates autophagy through the AMPK-mTOR pathway. The present study was aimed at determining whether Toll-like receptor (TLR) signaling regulates Sesn2 expression and identifying the underlying molecular mechanism. Lipopolysaccharide (LPS), a representative TLR4 ligand, significantly increased the levels of Sesn2 protein in macrophages. LPS also increased Sesn2 mRNA levels and luciferase reporter activity; however, the mRNA levels of Sesn1 were not affected by LPS. Moreover, treatment of macrophages with other TLR ligands (eg, polyI:C or peptidoglycan) also induced Sesn2 expression. We found that LPS-mediated Sesn2 induction was transcriptionally regulated by AP-1 and Nrf2, and that overexpression of c-Jun or Nrf2 increased Sesn2 protein levels and Sesn2 promoter-driven luciferase reporter activity. Moreover, deletion of the antioxidant response element (ARE) in the Sesn2 promoter or Nrf2 knockout abolished LPS-mediated induction of Sesn2. LPS induced Sesn2 gene expression through p38 and PI3K activation. Surprisingly, treatment with the proteasome inhibitor MG132, but not the lysosomal inhibitor chloroquine, caused Sesn2 to accumulate in the cells. In the presence of MG132, we observed that Sesn2 was ubiquitinated. However, LPS treatment attenuated Sesn2 ubiquitination induced by MG132, which resulted in Sesn2 accumulation. Mice treated with D-galactosamine (Gal)/LPS exhibited enhanced Sesn2 expression in the liver. Moreover, infection with a recombinant adenovirus encoding Sens2 markedly reduced the number of Gal/LPS-induced TUNEL-positive cells. Our results suggest that TLR-mediated Sesn2 induction is dependent on AP-1, Nrf2, and the inhibition of ubiquitin-mediated degradation of Sesn2 and might protect cells against endotoxin toxicity.

Inhibitory effects of traditional herbal formula pyungwi-san on inflammatory response in vitro and in vivo.

Pyungwi-san (PWS) is a traditional basic herbal formula. We investigated the effects of PWS on induction of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), pro-inflammatory cytokines (interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)) and nuclear factor-kappa B (NF-κB) as well as mitogen-activated protein kinases (MAPKs) in lipopolysaccharide-(LPS-) induced Raw 264.7 cells and on paw edema in rats. Treatment with PWS (0.5, 0.75, and 1 mg/mL) resulted in inhibited levels of expression of LPS-induced COX-2, iNOS, NF-κB, and MAPKs as well as production of prostaglandin E2 (PGE2), nitric oxide (NO), IL-6, and TNF-α induced by LPS. Our results demonstrate that PWS possesses anti-inflammatory activities via decreasing production of pro-inflammatory mediators through suppression of the signaling pathways of NF-κB and MAPKs in LPS-induced macrophage cells. More importantly, results of the carrageenan-(CA-) induced paw edema demonstrate an anti-edema effect of PWS. In addition, it is considered that PWS also inhibits the acute edematous inflammations through suppression of mast cell degranulations and inflammatory mediators, including COX-2, iNOS and TNF-α. Thus, our findings may provide scientific evidence to explain the anti-inflammatory properties of PWS in vitro and in vivo.

Combination of honokiol and magnolol inhibits hepatic steatosis through AMPK-SREBP-1 c pathway

Honokiol and magnolol, as pharmacological biphenolic compounds of Magnolia officinalis, have been reported to have antioxidant and anti-inflammatory properties. Sterol regulatory element binding protein-1 c (SREBP-1 c) plays an important role in the development and processing of steatosis in the liver. In the present study, we investigated the effects of a combination of honokiol and magnolol on SREBP-1 c-dependent lipogenesis in hepatocytes as well as in mice with fatty liver due to consumption of high-fat diet (HFD). Liver X receptor α (LXRα) agonists induced activation of SREBP-1 c and expression of lipogenic genes, which were blocked by co-treatment of honokiol and magnolol (HM). Moreover, a combination of HM potently increased mRNA of fatty acid oxidation genes. HM induced AMP-activated protein kinase (AMPK), an inhibitory kinase of the LXRα-SREBP-1 c pathway. The role of AMPK activation induced by HM was confirmed using an inhibitor of AMPK, Compound C, which reversed the ability of HM to both inhibit SREBP-1 c induction as well as induce genes for fatty acid oxidation. In mice, HM administration for four weeks ameliorated HFD-induced hepatic steatosis and liver dysfunction, as indicated by plasma parameters and Oil Red O staining. Taken together, our results demonstrated that a combination of HM has beneficial effects on inhibition of fatty liver and SREBP-1 c-mediated hepatic lipogenesis, and these events may be mediated by AMPK activation.

Hederagenin, a major component of Clematis mandshurica Ruprecht root, attenuates inflammatory responses in RAW 264.7 cells and in mice

Clematis mandshurica Ruprecht root has been used in Asia as a traditional anti-inflammatory, analgesic, and antitumor agent. Its main active component is hederagenin, a naturally occurring triterpene, and in this study, we examined the anti-inflammatory effects of hederagenin in lipopolysaccharide-stimulated RAW 264.7 cells using an enzyme-linked immunosorbent assay, Western blot, and RT-PCR. In addition, its effects on acute inflammation in vivo were observed using a carrageenan-induced mouse hind paw edema assay. Furthermore, the changes on the histopathology and histomorphometry of hind paw skins were examined using carrageenan-treated mice. Treatment with hederagenin (10, 30 and 100μM) resulted in inhibited levels of protein expression of lipopolysaccharide-stimulated iNOS, COX-2, and NF-κB as well as production of NO, PGE2, TNF-α, IL-1β, and IL-6 induced by lipopolysaccharide. Consistent with these results, hederagenin also dose-dependently reduced the lipopolysaccharide-induced mRNA levels of iNOS and COX-2, and of the above-mentioned cytokines. Interestingly, results of the carrageenan-induced mouse hind paw edema assay showed an anti-edema effect of hederagenin. Furthermore, hederagenin (30mg/kg) inhibited the carrageenan-induced increases in skin thicknesses, infiltrated inflammatory cells, and mast cell degranulation. These results suggest that hederagenin may possess anti-inflammatory activities.

U-Bang-Haequi Tang: A Herbal Prescription that Prevents Acute Inflammation through Inhibition of NF-κB-Mediated Inducible Nitric Oxide Synthase.

Since antiquity, medical herbs have been prescribed for both treatment and preventative purposes. Herbal formulas are used to reduce toxicity as well as increase efficacy in traditional Korean medicine. U-bang-haequi tang (UBT) is a herbal prescription containing Arctii fructus and Forsythia suspensa as its main components and has treated many human diseases in traditional Korean medicine. This research investigated the effects of UBT against an acute phase of inflammation. For this, we measured induction of nitric oxide (NO) and related proteins in macrophage cell line stimulated by lipopolysaccharide (LPS). Further, paw swelling was measured in carrageenan-treated rats. Carrageenan significantly induced activation of inflammatory cells and increases in paw volume, whereas oral administration of 0.3 or 1 g/kg/day of UBT inhibited the acute inflammatory response. In RAW264.7 cells, UBT inhibited mRNA and protein expression levels of iNOS. UBT treatment also blocked elevation of NO production, nuclear translocation of NF-κB, phosphorylation of Iκ-Bα induced by LPS. Moreover, UBT treatment significantly blocked the phosphorylation of p38 and c-Jun NH2-terminal kinases by LPS. In conclusion, UBT prevented both acute inflammation in rats as well as LPS-induced NO and iNOS gene expression through inhibition of NF-κB in RAW264.7 cells.

Epimedium koreanum Ameliorates Oxidative Stress-Mediated Liver Injury by Activating Nuclear Factor Erythroid 2-Related Factor 2

Oxidative stress induced by reactive oxygen species is the main cause of various liver diseases. This study investigated the hepatoprotective effect of Epimedium koreanum Nakai water extract (EKE) against arachidonic acid (AA)[Formula: see text][Formula: see text][Formula: see text]iron-mediated cytotoxicity in HepG2 cells and carbon tetrachloride (CCl4-)-mediated acute liver injury in mice. Pretreatment with EKE (30 and 100[Formula: see text][Formula: see text]g/mL) significantly inhibited AA[Formula: see text][Formula: see text][Formula: see text]iron-mediated cytotoxicity in HepG2 cells by preventing changes in the expression of cleaved caspase-3 and poly(ADP-ribose) polymerase. EKE attenuated hydrogen peroxide production, glutathione depletion, and mitochondrial membrane dysfunction. EKE also increased the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), transactivated anti-oxidant response element harboring luciferase activity, and induced the expression of anti-oxidant genes. Furthermore, the cytoprotective effect of EKE against AA[Formula: see text][Formula: see text][Formula: see text]iron was blocked in Nrf2 knockout cells. Ultra-performance liquid chromatography analysis showed that EKE contained icariin, icaritin, and quercetin; icaritin and quercetin were both found to protect HepG2 cells from AA[Formula: see text][Formula: see text][Formula: see text]iron via Nrf2 activation. In a CCl4-induced mouse model of liver injury, pretreatment with EKE (300[Formula: see text]mg/kg) for four consecutive days ameliorated CCl4-mediated increases in serum aspartate aminotransferase activity, histological activity index, hepatic parenchyma degeneration, and inflammatory cell infiltration. EKE also decreased the number of nitrotyrosine-, 4-hydroxynonenal-, cleaved caspase-3-, and cleaved poly(ADP-ribose) polymerase-positive cells in hepatic tissues. These results suggest EKE is a promising candidate for the prevention or treatment of oxidative stress-related liver diseases via Nrf2 activation.

Activation of AMPK by Buddleja officinalis Maxim. Flower Extract Contributes to Protecting Hepatocytes from Oxidative Stress

The Buddleja officinalis Maxim. flower is used in traditional Chinese and Korean medicine to treat inflammation, vascular diseases, headache, and stroke, as well as enhance liver function. This research investigated the effects of B. officinalis Maxim. flower extract (BFE) on hepatotoxicity. The cytoprotective effects and mechanism of BFE against severe mitochondrial dysfunction and H2O2 production in hepatotoxicity induced by coadministration of arachidonic acid (AA) and iron were observed in the HepG2 cell line. In addition, we performed blood biochemical, histopathological, and histomorphometric analyses of mice with carbon tetrachloride- (CCl4-) induced acute liver damage. BFE inhibited the AA + iron-mediated hepatotoxicity of HepG2 cells. Moreover, it inhibited mitochondrial dysfunction, H2O2 production, and glutathione depletion mediated by AA + iron in the same cells. Meanwhile, the cytoprotective effects of BFE against oxidative stress were associated with the activation of AMP-activated protein kinase (AMPK). In particular, based on the histopathological observations, BFE (30 and 100 mg/kg) showed clear hepatoprotective effects against CCl4-induced acute hepatic damage. Furthermore, it inhibited 4-hydroxynonenal and nitrotyrosine immunoreactivity in hepatocytes. These results provide evidence that BFE has beneficial hepatoprotective effects against hepatic damage via the activation of AMPK pathway. Accordingly, BFE may have therapeutic potential for diverse liver disorders.

Glycyrrhizae Radix Methanol Extract Attenuates Methamphetamine-Induced Locomotor Sensitization and Conditioned Place Preference

Glycyrrhizae Radix modulates the neurochemical and locomotor alterations induced by acute psychostimulants in rodents via GABAb receptors. This study investigated the influence of methanol extract from Glycyrrhizae Radix (MEGR) on repeated methamphetamine- (METH-) induced locomotor sensitization and conditioned place preference (CPP). A cohort of rats was treated with METH (1 mg/kg/day) for 6 consecutive days, subjected to 6 days of withdrawal, and then challenged with the same dose of METH to induce locomotor sensitization; during the withdrawal period, the rats were administered MEGR (60 or 180 mg/kg/day). A separate cohort of rats was treated with either METH or saline every other day for 6 days in METH-paired or saline-paired chambers, respectively, to induce CPP. These rats were also administered MEGR (180 mg/kg) prior to every METH or CPP expression test. Pretreatment with MEGR (60 and 180 mg/kg/day) attenuated the expression of METH-induced locomotor sensitization dose-dependently, and 180 mg/kg MEGR significantly inhibited the development and expression of METH-induced CPP. Furthermore, administration of a selective GABAb receptor antagonist (SCH50911) prior to MEGR treatment effectively blocked the inhibitory effects of MEGR on locomotor sensitization, but not CPP. These results suggest that Glycyrrhizae Radix blocked repeated METH-induced behavioral changes via GABAb receptors.

Amelioration of inflammatory responses by Socheongryong-Tang, a traditional herbal medicine, in RAW 264.7 cells and rats

Socheongryong-Tang (SCRT) is a natural medicine prescription that has been mainly used in East Asia for the treatment of inflammatory disorders, including asthma and allergic rhinitis. The present study evaluated the anti-inflammatory effects of SCRT on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and in a rat model of carrageenan (CA)-induced paw edema. Levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and prostaglandin E2 (PGE2) in the culture supernatant were quantified and nitric oxide (NO) production was monitored. In addition, the effect of SCRT on the protein expression of nuclear factor-κB (NF-κB), mitogen-activated protein kinases (MAPKs), inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) was assessed by western blot analysis. Furthermore, the effects of SCRT on acute inflammation in vivo and changes in the histomorphometry and histopathology of paw skin were observed using CA-treated rats. SCRT (1 mg/ml) inhibited the LPS-induced changes in the protein expression of NF-κB, JNK, ERK1/2, iNOS and COX-2, as well as the production of NO, PGE2 and cytokines. In the rat paw edema assay, administration of 1 g/kg of lyophilized powder obtained from the aqueous extracts of SCRT for 3 consecutive days inhibited the CA-induced increases in skin thickness, mast cell degranulation, and infiltration of inflammatory cells in the ventral and dorsal pedis skin within 4 h. These results demonstrated that SCRT exerts its anti-inflammatory activities in LPS-stimulated RAW 264.7 cells through decreasing the production of inflammatory mediators, including PGE2, NO and cytokines, via suppression of the NF-κB and JNK and ERK1/2 signaling pathways. In addition, the data of the CA-induced paw edema indicated an anti-edema effect of SCRT. SCRT (1 g/kg) reduced acute edematous inflammation through inhibition of mast cell degranulation and infiltration of inflammatory cells. Therefore, the present study provided scientific evidence for the anti-inflammatory activities of SCRT as well as the underlying mechanisms.

Combination of Pelargonium sidoides and Coptis chinensis root inhibits nuclear factor kappa B-mediated inflammatory response in vitro and in vivo

BackgroundPelargonium sidoides (PS) and Coptis chinensis root (CR) have traditionally been used to treat various diseases, including respiratory and gastrointestinal infections, dysmenorrhea, and hepatic disorders. The present study was conducted to evaluate the anti-inflammatory effects of a combination of PS and CR in vitro and in vivo.MethodsThe in vitro effects of PS + CR on the induction of inflammation-related proteins were evaluated in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The levels of nitric oxide (NO) and of inflammatory cytokines and prostaglandin E2 (PGE2) were measured using the Griess reagent and enzyme-linked immunosorbent assay (ELISA) methods, respectively. The expression of inflammation-related proteins was confirmed by Western blot. Additionally, the effects of PS + CR on paw edema volume, skin thickness, and numbers of infiltrated inflammatory cells, mast cells, COX-2-, iNOS-, and TNF-α-immunoreactive cells in dorsum and ventrum pedis skin were evaluated in a rat model of carrageenan (CA)-induced paw edema.ResultsPS + CR significantly reduced production of NO, PGE2 and three pro-inflammatory cytokines (tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6) and also decreased levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Treatment with PS + CR significantly reduced the protein expression levels of LPS-stimulated nuclear factor kappa B (NF-κB) and phosphorylated inhibitor of NF-κB (p-I-κBα). Additionally, PS + CR significantly inhibited the increases in paw swelling, skin thickness, infiltrated inflammatory cells, mast cell degranulation, COX-2-, iNOS-, and TNF-α-immunoreactive cells in the rat model of CA-induced acute edematous paw.ConclusionsThese results demonstrate that PS + CR exhibits anti-inflammatory properties through decreasing the production of pro-inflammatory mediators (NO, PGE2, TNF-α, IL-1β, and IL-6), suppressing NF-κB signaling in LPS-induced RAW 264.7 cells. Additionally, the results of the CA-induced rat paw edema assay revealed an anti-edema effect of PS + CR. Furthermore, it is suggested that PS + CR also inhibits acute edematous inflammation by suppressing mast cell degranulation and inflammatory mediators (COX-2, iNOS, and TNF-α). Thus, PS + CR may be a potential candidate for the treatment of various inflammatory diseases, and it may also contribute to a better understanding of the molecular mechanisms underlying inflammatory response regulation.