Ji Youn Lim

The new diterpene isodojaponin D inhibited LPS-induced microglial activation through NF-kappaB and MAPK signaling pathways.

Neuroinflammation with prolonged microglial activation leads to increased levels of pro-inflammatory mediators and subsequently contributes to neuronal dysfunction and neuronal loss. Therefore, pharmacological suppression of neuroinflammation would theoretically slow the progression of neurodegenerative disease. In this study, we investigated the anti-inflammatory effects and possible mechanisms of isodojaponin D (19-hydroxy-1alpha,6-diacetoxy-6,7-seco-ent-kaur-16-en-15-one-7,20-olide), a new diterpene isolated from Isodon japonicus against lipopolysaccharide(LPS)-induced microglial activation in BV2 cells. Results from RT-PCR and Western blot showed that pretreatment with isodojaponin D (5 and 10 microg/ml) prior to treatment with LPS (1 microg/ml) significantly decreased LPS-induced production of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in a dose-dependent manner. In addition, LPS-induced pro-inflammatory cytokines, including IL-1beta, IL-6, and TNF-alpha, were also decreased by pretreatment with isodojaponin D. This effect was accompanied by a decrease in translocations of Nuclear Factor-KappaB (NF-kappaB) p50 and p65 from the cytoplasm to the nucleus and by a decrease in I kappaB (IkappaB) degradation. In addition, pretreatment with isodojaponin D significantly attenuated LPS-induced mitogen-activated protein kinase (MAPK) activation. Taken together, these results suggest that isodojaponin D suppressed LPS-induced microglial activation and production of pro-inflammatory mediators by inhibition of the NF-kappaB signaling pathway and phosphorylation of MAPKs. These results suggest that isodojaponin D could play a beneficial role in treatment of neurodegenerative disease.

Identification of toxicological biomarkers of di(2-ethylhexyl) phthalate in proteins secreted by HepG2 cells using proteomic analysis.

The effects of di(2‐ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells were studied using a proteomic approach. HepG2 cells were exposed to various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 μM) for 24 or 48 h. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) and comet assays were then conducted to determine the cytotoxicity and genotoxicity of DEHP, respectively. The MTT assay showed that 10 μM DEHP was the maximum concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells exposed to DEHP was found to increase in a dose‐ and time‐dependent fashion. Proteomic analysis using two different pI ranges (4–7 and 6–9) and large size 2‐DE revealed the presence of 2776 protein spots. A total of 35 (19 up‐ and 16 down‐regulated) proteins were identified as biomarkers of DEHP by ESI‐MS/MS. Several differentiated protein groups were also found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up‐ or down‐regulated. Among these, the identities of cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, cofilin‐1, and haptoglobin‐related protein were confirmed by Western blot assay. Therefore, these proteins could be used as potential biomarkers of DEHP and human disease associated with DEHP.

The effect of polaprezinc on gastric mucosal protection in rats with ethanol-induced gastric mucosal damage: comparison study with rebamipide.

AIMS Polaprezinc (PZ), which consists of l-carnosine and zinc, is widely used to treat gastric ulcers. We compared the effects of PZ with those of rebamipide (RM) on the expression of inflammatory cytokines, antioxidants, growth factors, and heat shock proteins (HSP) in a rat model. MAIN METHODS Seventy Sprague-Dawley rats were randomly assigned to test groups according to the dose of PZ at 5, 10, or 30 mg/kg or RM at 10, 30, or 100 mg/kg. Next, we obtained ulcer indices from rats with ethanol-induced gastric mucosal damage. Western blot analysis was used to evaluate the expression of various target proteins. KEY FINDINGS Pathological ulcer indices in the PZ and RM groups were significantly lower than those in the control group. The levels of inflammatory cytokines (interleukin 1β [IL-1β], IL-6, IL-8, and tumor necrosis factor α) decreased, whereas the levels of platelet-derived growth factor-B, vascular endothelial growth factor, and nerve growth factor significantly increased after PZ administration. Furthermore, the expression of antioxidants (superoxide dismutase 1 [SOD-1], SOD-2, heme oxygenase-1, glutathione S-transferase, peroxidredoxin-1, and peroxidredoxin-5) was significantly higher in the PZ group, and the levels of HSP 90, 70, 60, 47, 27, and 10 significantly increased with an increase in PZ dose. SIGNIFICANCE In a rat model of ethanol-induced gastric mucosal damage, PZ administration ameliorated ethanol-induced mucosal injury and showed protective effects on the mucosa by reducing the levels of inflammatory cytokines and increasing the expression of antioxidant enzymes and growth factors. Furthermore, PZ showed cytoprotective effects by increasing the HSP levels.

Neuroprotective effects of constituents of Eragrostis ferruginea against Aβ-induced toxicity in PC12 cells

A new flavonoid, 7-demethylageconyflavone A (1), and five known compounds, tricin (2), ageconyflavone A (3), corylin (4), nectandrin B (5), and 4-ketopinoresinol (6) were isolated from the aerial parts of Eragrostis ferruginea. Their structures were determined using spectroscopic techniques, including 1D- and 2D-NMR. All compounds were tested for the neuroprotective effects against amyloid beta peptide (Aβ) using PC12 cells, a major cause of the pathology of Alzheimer’s disease. Tricin (2) was found to have a neuroprotective effect with an ED50 value of 20.3 μM against Aβ-induced toxicity in PC12 cells. Ageconyflavone A (3), nectandrin B (5) and 4-ketopinoresinol (6) demonstrated moderate neuroprotective effects with ED50 values of 58.7, 44.1, and 54.8 μM, respectively.

Delphinidin Ameliorates Beta-Amyloid-Induced Neurotoxicity by Inhibiting Calcium Influx and Tau Hyperphosphorylation

Beta-amyloid (Aβ) has been suggested to induce neurotoxicity in Alzheimer’s disease. We evaluated the neuroprotective effects of delphinidin, an anthocyanidin commonly present in pigmented fruits and vegetables, against Aβ-induced toxicity. Aβ (25–35) significantly decreased the viability of PC12 cells, and this was accompanied by an increase in intracellular calcium levels and tau phosphorylation. However, treatment with delphinidin rescued PC12 cells from Aβ by attenuating the elevation of intracellular calcium levels and tau phosphorylation. Taken together, these results suggest that delphinidin protects PC12 cells against Aβ-induced toxicity by attenuating intracellular calcium influx and tau hyperphosphorylation.

Suppression of LPS-induced inflammatory responses by inflexanin B in BV2 microglial cells.

Microglia are a type of resident macrophage that functions as an inflammation modulator in the central nervous system. Over-activation of microglia by a range of stimuli disrupts the physiological homeostasis of the brain, and induces inflammatory response and degenerative processes, such as those implicated in neurodegenerative diseases, including Alzheimers disease and Parkinsons disease. Therefore, we investigated the possible anti-inflammatory mechanisms of inflexanin B in murine microglial BV2 cells. Lipopolysaccharide (LPS) activated BV2 cells and induced the production of pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), and cytokines (interleukins-1β and -6, and tumour necrosis factor α). The LPS-induced production of pro-inflammatory mediators was associated with the enhancement of nuclear factor-kappaB (NF-κB) nuclear translocation and the activation of mitogen-activated protein kinase (MAPK) including ERK1/2 and JNK. Conversely, pretreatment of cells with inflexanin B (10 and 20 μg/mL) significantly reduced the production of pro-inflammatory mediators. This was accompanied with the reduced nuclear translocation of NF-κB and reduced activation of MAPKs. These results suggest that inflexanin B attenuated the LPS-induced inflammatory process by inhibiting the activation of NF-κB and MAPKs.

The expression of HSPs, anti-oxidants, and cytokines in plasma and bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome.

OBJECTIVES We studied several acute inflammatory materials (AIM) such as various inflammatory cytokines, oxidative stress, and heat shock proteins in ARDS patients by simultaneously measuring from bronchoalveolar lavage fluid (BALF) and plasma. DESIGN AND METHODS AIM were measured by using plasma and BALF sampling obtained from ARDS group (n=12) and non-ARDS group (n=12). RESULTS In the BALF, only HSP 47 was significantly increased in ARDS group than non-ARDS group. In plasma, GRP 94, HSP 90, HSP 60, HSP 47, GPx-3, and IL-8 were increased significantly in ARDS group. In short, most of the AIM in BALF or plasma were not significantly different in ARDS group as compared with non-ARDS group. Ninety-day mortality was significantly related to HSP90, HSP 60 and GPx-3 in plasma but not in BALF. CONCLUSION Alteration of AIM levels in both BALF or plasma of ARDS group was not remarkable compared with the non-ARDS group. Our result suggests the need to reconsider ARDS pathophysiology and therapeutic strategy.

Proteomic analysis of proteins secreted by HepG2 cells treated with butyl benzyl phthalate.

Proteomic changes in proteins secreted by human hepatocellular carcinomas (HepG2) cells exposed to butyl benzyl phthalate (BBP) were evaluated. HepG2 cells were treated with three different concentrations of BBP (0, 10, or 25 μM) for 24 or 48 h. Following incubation, the cells were subjected to proteomic analysis using two different pI ranges (4–7 and 6–9) and large-size two-dimensional gel electrophoresis. Results showed resolution of a total of 2776 protein spots. Of these, 29, including 19 upregulated and 10 downregulated proteins, were identified by electrospray ionization–mass spectrometry–mass spectrometry (ESI-MS/MS). Among these, the identities of cystatin C, Rho guanine nucleotide dissociation inhibitor, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, heptaglobin-related protein, inter-alpha-trypsin inhibitor heavy chain H2, and electron transfer flavoprotein subunit beta were confirmed by Western blot analysis. These proteins were found to be involved in apoptosis, signaling, tumor progression, energy metabolism, and cell structure and motility. Therefore, these proteins have potential to be employed as biomarkers of BBP exposure and may be useful in understanding mechanisms underlying the adverse effects of BBP.

Benexate hydrochloride betadex modulates nitric oxide synthesis and cytokine expression in gastric ulcers.

The present study investigated benexate hydrochloride betadex (BHB)-mediated ulcer healing, and changes to microcirculation modulated through nitric oxide synthase (NOS) and anti-inflammatory activity. A rat model of gastric mucosal injury was established through injection of a 60% acetic acid solution into the stomach. Following ulcer induction, the rats were administered BHB orally for 5 days at doses of 0, 100, 300 or 1,000 mg/kg. The highest dose of BHB was also administered with or without L-NG-nitroarginine methyl ester (L-NAME). The area of gastric ulcers was determined by planimetry, and expression of cyclooxygenases (COX), cytokines and NOS in stomach tissues were measured using western blotting. Compared with the control group, gastric ulcer size was significantly decreased in the 1,000 mg/kg BHB-treated group (P<0.05). Administration of BHB led to a significant increase in endothelial (e)NOS expression (P<0.05). Although acetic acid co-treatment with L-NAME induced more severe mucosal damage, BHB decreased COX expression and tumor necrosis factor-α levels when administered with the nitric oxide inhibitor, L-NAME (P<0.05). BHB exhibited protective effects in a rat model of gastric ulcers, which were associated with a decrease in pro-inflammatory cytokine levels and the activation of eNOS.

Evaluation of toxicological biomarkers in secreted proteins of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and their expressions in the plasma of rats and incineration workers.

Toxicological biomarkers of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) were investigated in proteins secreted by HepG2 cells and their expression levels were determined in the plasma of rats exposed to 2,3,7,8-TCDD and in the plasma of incineration workers exposed to dioxins. HepG2 cells were treated with various concentrations of 2,3,7,8-TCDD (0, 0.25, 0.5, 1, 2.5, 5, 10, 25 nM) for 24 or 48 h. MTT and Comet assays were performed to determine cytotoxicities and genotoxicities to select exposure concentrations for the proteomic analysis of proteins secreted by 2,3,7,8-TCDD-treated cells. In the proteomic analysis, dose- and time-dependent toxicological biomarkers were evaluated using two pI ranges (4-7 and 6-9) using a large gel 2-DE system. Fifteen secreted proteins were identified by a nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS and the identities of eight secreted proteins including glyoxalase 1 (GLO 1), homogentisate dioxygenase (HGD), peroxiredoxin 1 (PRX 1), proteasome subunit beta type (PSMB) 5 and 6, UDP-glucose 6-dehydrogenase (UDP-GlcDH), hydroxyacyl-coenzyme A dehydrogenase (HADH) and serotransferrin (STF) were confirmed by western blotting. Of these, PSMB 5 and PRX 1 were also found in the plasma of rats exposed to 2,3,7,8-TCDD, whereas GLO 1, HGD, PSMB 6 and PRX 1 were found in the plasma of incineration workers exposed to dioxins.