Metformin Reduces Bleomycin-induced Pulmonary Fibrosis in Mice

최선미,장안희,김효진,이규화,김영환 대한의학회 2016 Journal of Korean medical science Vol.31 No.9

Metformin has anti-inflammatory and anti-fibrotic effects. We investigated whether metformin has an inhibitory effect on bleomycin (BLM)-induced pulmonary fibrosis in a murine model. A total of 62 mice were divided into 5 groups: control, metformin (100 mg/ kg), BLM, and BLM with metformin (50 mg/kg or 100 mg/kg). Metformin was administered to the mice orally once a day from day 1. We sacrificed half of the mice on day 10 and collected the bronchoalveolar lavage fluid (BALF) from their left lungs. The remaining mice were sacrificed and analyzed on day 21. The right lungs were harvested for histological analyses. The messenger RNA (mRNA) levels of epithelial-mesenchymal transition markers were determined via analysis of the harvested lungs on day 21. The mice treated with BLM and metformin (50 mg/kg or 100 mg/kg) showed significantly lower levels of inflammatory cells in the BALF compared with the BLM-only mice on days 10 and 21. The histological examination revealed that the metformin treatment led to a greater reduction in inflammation than the treatment with BLM alone. The mRNA levels of collagen, collagen-1, procollagen, fibronectin, and transforming growth factor-β in the metformin-treated mice were lower than those in the BLM-only mice on day 21, although statistical significance was observed only in the case of procollagen due to the small number of live mice in the BLM-only group. Additionally, treatment with metformin reduced fibrosis to a greater extent than treatment with BLM alone. Metformin suppresses the inflammatory and fibrotic processes of BLM-induced pulmonary fibrosis in a murine model.

The Impact of Autophagy on the Cigarette Smoke Extract-Induced Apoptosis of Bronchial Epithelial Cells

이창훈,이경희,장안희,유철규 대한결핵및호흡기학회 2017 Tuberculosis and Respiratory Diseases Vol.80 No.1

Background: Previous studies report that apoptosis and autophagy are involved in the pathogenesis of emphysema, and macroautophagy is one of the processes regulating the apoptosis pathway. However, few studies have evaluated whether chaperone-mediated autophagy (CMA) contributes to the regulation of apoptosis. In this study, we investigated the impact of autophagy, including both macroautophagy and CMA, on the apoptosis in bronchial epithelial cells. Methods: Cigarette smoke extract (CSE) was injected intratracheally into C57BL/6 mice, and emphysema and apoptosis were evaluated in the lungs. After treatment with CSE, apoptosis, macroautophagy, and CMA were measured in BEAS2-B cells, and the impact of autophagy on the apoptosis was evaluated following knockdown of autophagy-related genes by short interfering RNAs (siRNAs). Results: Intratracheal CSE injection resulted in the development of emphysema and an increase in apoptosis in mice. CSE increased the apoptosis in BEAS2-B cells, and also elevated the expression of proteins related to both macroautophagy and CMA in BEAS2-B cells. The knockdown experiment with siRNAs showed that macroautophagy increases apoptosis in BEAS2-B cells, while CMA suppresses apoptosis. Conclusion: The intratracheal injection of CSE induces pulmonary emphysema and an increase in apoptosis in mice. CSE also induces apoptosis, macroautophagy, and CMA of bronchial epithelial cells. Macroautophagy and CMA regulate apoptosis in opposite directions.

Targeted Base Editing via RNA-Guided Cytidine Deaminases in Xenopus laevis Embryos

박동석,윤미정,권지연,장안희,김용섭,최순철 한국분자세포생물학회 2017 Molecules and cells Vol.40 No.11

Genome editing using programmable nucleases such as CRISPR/Cas9 or Cpf1 has emerged as powerful tools for gene knock-out or knock-in in various organisms. While most genetic diseases are caused by point mutations, these genome-editing approaches are inefficient in inducing single-nucleotide substitutions. Recently, Cas9-linked cytidine deaminases, named base editors (BEs), have been shown to convert cytidine to uridine efficiently, leading to targeted single-base pair substitutions in human cells and organisms. Here, we first report on the generation of Xenopus laevis mutants with targeted single-base pair substitutions using this RNA-guided programmable deaminase. Injection of base editor 3 (BE3) ribonucleoprotein targeting the tyrosinase (tyr) gene in early embryos can induce site-specific base conversions with the rates of up to 20.5%, resulting in oculocutaneous albinism phenotypes without off-target mutations. We further test this base-editing system by targeting the tp53 gene with the result that the expected single-base pair substitutions are observed at the target site. Collectively, these data establish that the programmable deaminases are efficient tools for creating targeted point mutations for human disease modeling in Xenopus.

Cigarette Smoke Extract Enhances IL-17A-Induced IL-8 Production via Up-Regulation of IL-17R in Human Bronchial Epithelial Cells

이경희,이창훈,우지수,정지영,장안희,유철규 한국분자세포생물학회 2018 Molecules and cells Vol.41 No.4

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine mainly derived from T helper 17 cells and is known to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) has been considered as a primary risk factor of COPD. However, the interaction between CS and IL-17A and the underlying molecular mechanisms have not been clarified. In the current study, we investigated the effects of cigarette smoke extract (CSE) on IL-17A-induced IL-8 production in human bronchial epithelial cells, and sought to identify the underlying molecular mechanisms. IL-8 production was significantly enhanced following treatment with both IL-17A and CSE, while treatment with either IL-17A or CSE alone caused only a slight increase in IL-8 production. CSE increased the transcription of IL-17RA/RC and surface membrane expression of IL-17R, which was suppressed by an inhibitor of the phosphoinositide 3-kinase (PI3K)/Akt pathway (LY294002). CSE caused inactivation of glycogen synthase kinase-3β (GSK-3β) via the PI3K/Akt pathway. Blockade of GSK-3β inactivation by overexpression of constitutively active GSK-3β (S9A) completely suppressed the CSE-induced up-regulation of IL-17R expression and the CSE-induced enhancement of IL-8 secretion. In conclusion, inactivation of GSK-3β via the PI3K/Akt pathway mediates CSE-induced up-regulation of IL-17R, which contributes to the enhancement of IL-17A-induced IL-8 production.

T 림파구의 Uropod 형성에 있어 인산화 Ezrin단백질의 역할

김지현,김경민,최우봉,김정환,김광현,이종환,장안희,윤선미,김지현 대한암예방학회 2009 Journal of cancer prevention Vol.14 No.2

Migrating T cell shows typical hand-mirror shape, leading edge and uropod polarity. EL 4 T cells features partitioned morphology like leading edge and uropod. In order to verify this polarity, EL 4 T cells were stained for nucleus, Golgi apparatus, cell membrane with DAPI, BODIPY-FL C5-ceramide and CD44, respectively. EL4 T cells showed typical migrating T cell shape. Ezrin is a cross-linker between cell membrane and cytoskeleton. Especially, phospho-ezrin may play an important role for uropod region. Phospho-ezrin was accumulated in uropod region immune-stained with anti phospho ezrin antibody. T567D ezrin, phospho-mimetic form of ezrin, was transfected into EL4 T cells. T567D transfectants showed enhanced uropod structure and disrupted leading edge. NH2 terminal domain (NT)-ezrin transfectants showed weak uropod structure. In addition, T567D had ability of cytoplasmic accumulation of F-actin in P3X63,653 myeloma cell. To test cell biological function with enhanced uropod, cell migration assay was carried out with T567D ezrin transfectants. Reduced cell migration was detected in T567D transfectants with F2 endothelial cell, however cell migration was increased in T567D transfectants without F2 cells. Collectively, phospho-ezrin is involved in uropod fromation and uropod facilitates the rapid cell migration.