Bunsoon Choi

Immobilization stress induces cell death through production of reactive oxygen species in the mouse cerebral cortex

Prolonged stress has been shown to impair brain function and increase vulnerability to neuronal injury. To elucidate the in vivo response of neuronal cells to induced stress, we immobilized mice by binding their legs. Levels of reactive oxygen species (ROS) in the cerebral cortex were increased after stress induction. NADPH oxidase, interleukin-1beta (IL-1beta) and cyclooxygenase 2 mRNA (COX-2) expression levels were upregulated, and Fas levels were also increased. The increased expression of these factors was associated with neuronal death, which was confirmed by TUNEL and NeuN staining. OX42 staining was also evident around the TUNEL-stained lesions. From these findings, it appears that immobilization stress induces neuronal death in the mouse cerebral cortex, a process mediated by NADPH oxidase, IL-1beta, COX-2, ROS and Fas. However, this could be inhibited by pretreating the animals with antioxidants such as ebselen or pyrrolidine dithiocarbamate.

Tumor necrosis factor alpha small interfering RNA decreases herpes simplex virus-induced inflammation in a mouse model

BACKGROUND Anti-TNFalpha antibodies have been used for treating inflammation in patients. But, more effective and safer drugs need to be developed for improved future therapeutic use. OBJECTIVES To inhibit the expression of TNFalpha, we used small interfering RNAs (siRNAs) to reduce over expression of TNFalpha in vitro in cell cultures and in an in vivo Behcets disease-like (BD) mouse model for amelioration of chronic inflammation. METHODS TNFalpha siRNA was injected intraperitoneally twice with a 1-week interval. To compare the efficacy of TNFalpha siRNA versus an anti-TNFalpha antibody, Infliximab and Etanercept were administered to symptomatic mice with inflamed tissue. RESULTS Intraperitoneal delivery of TNFalpha siRNA effectively decreased BD symptoms in 18 of 32 cases (56.3%). Scrambled siRNA treatment decreased BD symptoms in 2 of 19 cases (10.5%). Infliximab was effective in 11 of 27 cases (40.7%) and Etanercept was also effective in 9 of 25 cases (36.0%) at the end of the second week after treatment. TNFalpha siRNA reduced serum levels of TNFalpha (1.57 +/- 0.43pg/ml), compared to levels in mice not injected (84.02 +/- 24.59pg/ml) (p<0.01) or scramble injected (118.89 +/- 20.08pg/ml) (p<0.01). After single injection of TNFalpha siRNA, improvement of BD symptoms showed at 9 +/- 7th day on an average, contrary, in Infliximab injected group, improvement was apparent at 15 +/- 4th day after injection (p<0.05). CONCLUSION We show that siRNAs can be employed to inhibit cytokine gene expression in an in vivo disease mouse model. This inhibition may, therefore, be attributed to the improvement of inflammatory symptoms.

Pretreatment with interleukin-6 small interfering RNA can improve the survival rate of polymicrobial cecal ligation and puncture mice by down regulating interleukin-6 production

Interleukin-6 (IL-6) is considered to be an early marker of severe sepsis that is associated with increased morbidity and mortality. Therefore, we pretreated male ICR mice with IL-6 small interfering RNA (siRNA) before cecal ligation and puncture (CLP) and observed the changes in their survival in response to down regulation of IL-6, as well as the role of Th subsets during sepsis. In addition, sham and CLP operated mice were sacrificed at different time points to determine the serum IL-6 levels during early and late sepsis. IL-6 siRNA pretreated septic mice showed markedly extended survival (23.3%) up to 10 days and significantly reduced serum IL-6 levels at day 5 (209.90 ± 0.50 pg/ml; P<0.0001) when compared to CLP mice at day 1. Furthermore, IL-6 mRNA and protein were highly expressed during early sepsis in CLP mice at day 1 and mRNA was not detected in IL-6 siRNA treated CLP mice at days 1 or 5 and serum level of IL-6 was also decreased significantly (P<0.01). In addition, the mRNA expression of C5aR, ROR-γt, PU.1 and protein expression of IL-17 were high at day 5 in IL-6 siRNA treated mice. Taken together, the results of the present study demonstrate that pretreatment with IL-6 siRNA improved CLP induced septic mice survival. Furthermore, the IL-6 level was down-regulated and the transcription factors ROR-γt and PU.1 were up-regulated by IL-6 siRNA at late sepsis. The results presented herein also suggest that IL-6 siRNA could be a potential molecular therapeutic strategy for the treatment of sepsis.

Rebamipide affects the efficiency of colchicine for the herpes simplex virus-induced inflammation in a Behcet's disease mouse model.

Rebamipide inhibits free radicals derived from activated neutrophils and decreases the inhibiting inflammatory cytokine. Behcets disease (BD) is a chronic, multi-systemic inflammatory disorder with arthritic, gastrointestinal, mucocutaneous, ocular, vascular, and central nervous system involvement. This disease has a chronic course with periodic exacerbations and progressive deterioration. To study the effect of rebamipide treatment to BD-like mice, combination treatment with rebamipide and colchicine was compared to colchicine treatment. Colchicine is one of the most frequently prescribed medicine to the patients with BD. For each BD mouse, 200 microl gastric fluid or 2 microg colchicine or 150 microg rebamipide or 2 microg colchicine plus 150 microg rebamipide was treated orally once per day. Treatment was done for 5 consecutive days. Two hour or 20 days after last administration, spleens were isolated for RT-PCR and real time PCR, and serum was collected for ELISA. In the combination treated group, TNF alpha, MIP-1 alpha, p22 phox, p47 phox, and gp91 phox mRNA expressions were lower than rebamipide treated or colchicine treated groups by reverse transcriptase PCR. NADPH oxidase subunits mRNA were markedly downregulated compared to the colchicine treated group by real time PCR. At 20 days after administration, combination treatment decreased 23.5% of the severity score compared to before administration. In contrast, colchicine treatment decreased 14.3% of the severity score compared to before administration. Rebamipide helped the function of colchicine to improve the HSV induced BD-like symptoms by inhibiting the expression of NADPH oxidase in vivo mouse model.

The Relevance of miRNA-21 in HSV-Induced Inflammation in a Mouse Model

The purpose of this study was to clarify the correlation between microRNA-21 (miR-21) expression and inflammation in a herpes simplex virus (HSV)-induced Behçet’s Disease (BD) mouse model. miR-21 was compared between BD patients and healthy controls in peripheral blood mononuclear cells (PBMC). For miR-21 inhibition, miR-21 antagomir was applied to BD mice. The change of symptoms was monitored. The levels of cytokines and related molecules were determined by ELISA and real time qPCR. Treatment with colchicine or pentoxifylline down-regulated the level of miR-21 with improved symptoms in mice. miR-21 inhibition was accompanied by down-regulated serum levels of IL-17 and IL-6. The expression levels of PDCD4, RhoB, PD-1, IL-12p35, and toll-like receptor-4 were also regulated by miR-21 inhibition. miR-21 was correlated with HSV-induced BD-like inflammation in mice and BD patients. The expression of miR-21 was regulated by antagomir in mice.

CCL21 attenuates HSV-induced inflammation through up-regulation of CD8+ memory cells

CCR7 and its ligand, CCL21, are known to establish microenvironments for the initiation of immune responses in secondary lymphoid tissue. It has also been reported that CCR7 ligand gene-deleted mice have defects in lymphocyte homing. In addition, the injection of the CCR7 ligand was shown to induce the expression of memory T cells. In this study, we analyzed the expression of CCR7 and its ligand in HSV-induced Behçets disease (BD)-like inflammation of mice. Additionally, plasmids containing the CCR7 ligand CCL19 or CCL21, pcDNA3.1-CCL19 or pcDNA3.1-CCL21, respectively, were injected into symptomatic mice, and changes in the population of memory T cells were determined. After administration of pcDNA3.1-CCL21, the frequencies of CD8+CD44+, CD8+CD62L- memory T cells were significantly up-regulated and the symptoms were not deteriorated when compared to the control vector injected group. Specifically, the difference in frequencies of CCR7+ peripheral blood mononuclear cells between active BD patients and inactive BD patients was similar to that of HSV-induced BD-like mice. These results suggest that CCR7, its ligand, and CD8+ memory cells are correlated with the regulation of BD symptoms.

The role of classical and alternative macrophages in the immunopathogenesis of herpes simplex virus-induced inflammation in a mouse model

BACKGROUND The exact mechanism of the inflammatory changes occurring during the development of Behçets disease (BD) remains unclear. OBJECTIVE We investigated the role of classical (M1) and alternative (M2) activation of macrophages in a herpes simplex virus (HSV)-induced BD mouse model. METHODS The classical vs. alternative activated macrophage ratio (M1/M2 ratio) was calculated by analyzing the surface markers CD16/32 and CD23 as M1 and M2 markers, respectively, by flow cytometry. mRNA expression of interferon (IFN)-γ and interleukin (IL)-6 as M1 and arginase-1, FIZZ-1, and MHC-II as M2 markers were analyzed by reverse transcription-polymerase chain reaction. Cytokine levels were assessed by enzyme-linked immunosorbent assay. RESULTS The M1 phenotype was upregulated in BD mice, and an increased M1/M2 ratio was observed compared to that in asymptomatic BD normal and normal healthy mice. Recombinant (r)IFN-γ significantly increased the M1/M2 ratio (1.74±0.42) compared with that of rIL-4 (0.83±0.20). BD mice treated with rIL-4 showed a decreased M1/M2 ratio (1.2±0.3) compared to that of the rIFN-γ- (2.1±2.3) treated group and also showed ameliorated BD symptoms accompanied by downregulation of IL-17 and IL-6 and up-regulation of IL-4. CONCLUSION Therefore, modulation of macrophage phenotypes could be an effective therapeutic approach for treating BD in the future.

Synthesized pyridine compound derivatives decreased TNF alpha and adhesion molecules and ameliorated HSV-induced inflammation in a mouse model

Synthesized pyridine compound derivatives (SK94, SK126) from a natural lead source were administered to mice to test for possible anti-TNF alpha and anti-inflammatory activities. Lipopolysaccharide (LPS)-induced TNF alpha production was analyzed in the endothelial cells, Raw 264.7 cells, and serum of normal mice after treatment with SK compounds. These compounds were also orally administered to a herpes simplex virus (HSV)-induced Behcets disease mouse model to investigate their anti-inflammatory therapeutic effect. TNF alpha production was inhibited in a dose-dependent manner in the SK94 treated cells. E-selectin, VCAM-1, and ICAM-1 mRNA levels were also down-regulated. Treatment with 30mg/kg SK94 inhibited 55% of the TNF alpha production in LPS challenged Balb/c mice (n=8). SK94 and SK126 were administered to the Behcets disease-like mice for five consecutive days and SK94 improved in five out of six mice (83%), while it only improved in one out of nine mice (11%) in the pH 1.2 saline (artificial gastric juice) group (P<0.005), four out of ten mice (40%) in the thalidomide group (P<0.05), and six out of seven (86%) in the SK126 group (P<0.005). Soluble ICAM-1 was inhibited by 23.8% in the sera of SK94 treated mice and by 34.6% in SK126 treated mice when compared to artificial gastric juice. Based on these findings, SK compounds could be candidates for clinical trials.

Chitosan as an Immunomodulating Adjuvant on T-Cells and Antigen-Presenting Cells in Herpes Simplex Virus Type 1 Infection

Herpes disease caused by herpes simplex virus type 1 (HSV-1) is an intractable condition. It is a major concern in public health. Our purpose of this study was to verify the function of chitosan as an adjuvant for immune regulation specifically under herpes simplex virus type 1 (HSV-1) infection. Ahead of HSV infection, chitosan, heat inactivated green fluorescent protein expressing HSV (G-HSV), and a combination of chitosan and G-HSV were used to pretreat ICR mice followed by HSV-1 infection. Using flow cytometric analysis, the frequencies of T-cells, monocytes, dendritic cells (DCs), and natural killer (NK) cells were analyzed by surface expression of CD4+, CD8+, CD14+, CD11c+, NK1.1+, and DX5+ cells. In HSV infected mice, chitosan treatment significantly increased the frequencies of CD4+ T-cells (33.6 ± 5.78%) compared to those in the control group (24.02 ± 12.47%, p = 0.05). The frequencies of DC and NK cells were also significantly different between chitosan treated mice and control mice. In addition, anti-HSV IgG antibody was downregulated in chitosan treated mice. These results suggest that chitosan is a potential modulator or immune stimulator as an adjuvant in HSV-1 infected mice.

Tocotrienols enhance melanosome degradation through endosome docking/fusion proteins in B16F10 melanoma cells

Vitamin E inhibits tyrosinase activity and acts as a melanogenesis inhibitor in epidermal melanocytes in vitro. However, there is no direct evidence indicating that melanosomes are degraded in lysosomes in the presence of vitamin E. To determine whether vitamin E-induced melanosome disintegration is related to the expression of endosome docking/fusion proteins in B16F10 melanoma cells, electron microscopy, reverse transcription-polymerase chain reaction (RT-PCR), and real-time PCR were used to observe the effects of tocomin (α-tocopherols and α,γ,δ-tocotrienols in palm oil) on B16F10 melanoma cells. Melanosomal integrity was lost in lysosomes of B16F10 melanoma cells when treated with tocomin, indicating that tocomin caused the degradation of melanosomes in the lysosomal compartment. RT-PCR and real-time PCR analysis demonstrated mRNA expression of tyrosinase and the endosome docking/fusion proteins (syntaxin7, Rab7, Vps11, Vps16, Vps33, Vps39, and Vps41). Expression of syntaxin7, Vps16, Vps33, and Vps41 mRNA increased significantly in cells treated with tocomin compared with that in controls. These results indicate that the tocomin-induced degradation of melanosomes in the lysosomal compartment occurs with an increase in endosome docking/fusion proteins (syntaxin7, Vps16, Vps33, and Vps41) in cultured B16F10 melanoma cells.