• Tuberculosis and Respiratory Diseases
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• 제71권2호
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• pp.88-96
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• 2011

Background: It is known that cigarette smoke (CS) causes cell death. Apoptotic cell death is involved in the pathogenesis of CS-related lung diseases. Some members of the protein kinase C (PKC) family have roles in cigarette smoke extract (CSE)-induced apoptosis. This study was conducted to investigate the role of PKC epsilon in CSE-induced apoptosis in human lung fibroblast cell line, MRC-5. Methods: Lactate dehydrogenase release was measured using a cytotoxicity detection kit. The MTT assay was used to measure cell viability. Western immunoblot, Hoechst 33342 staining and flow cytometry were used to demonstrate the effect of $PKC{\varepsilon}$. Caspase-3 and caspase-8 activities were determined using a colorimetric assay. To examine $PKC{\varepsilon}$ activation, Western blotting was performed using both fractions of membrane and cytosol. Results: We showed that CSE activated $PKC{\varepsilon}$ by demonstrating increased expression of $PKC{\varepsilon}$ in the plasma membrane fraction. Pre-treatment of $PKC{\varepsilon}$ peptide inhibitor attenuated CSE-induced apoptotic cell death, as demonstrated by the MTT assay (13.03% of control, 85.66% of CSE-treatment, and 53.73% of $PKC{\varepsilon}$ peptide inhibitor-pre-treatment, respectively), Hoechst 33342 staining, and flow cytometry (85.64% of CSE-treatment, 53.73% of $PKC{\varepsilon}$ peptide inhibitor-pre-treatment). Pre-treatment of $PKC{\varepsilon}$ peptide inhibitor reduced caspase-3 expression and attenuated caspase-3, caspase-8 activity compared with CSE treatment alone. Conclusion: $PKC{\varepsilon}$ seem to have pro-apoptotic function and exerts its function through the extrinsic apoptotic pathway in CSE-exposed MRC-5 cells. This study suggests that $PKC{\varepsilon}$ inhibition may be a therapeutic strategy in CS-related lung disease such as chronic obstructive pulmonary disease.

• Natural Product Sciences
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• 제25권2호
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• pp.103-110
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• 2019

We investigated the anti-inflammatory effect of Pyunkang-tang extract (PGT), a complex herbal extract based on traditional Chinese medicine that is used in Korea for controlling diverse pulmonary diseases, on cigarette smoke-induced pulmonary pathology in a rat model of chronic obstructive pulmonary disease (COPD). The constituents of PGT were Lonicerae japonica, Liriope platyphylla, Adenophora triphilla, Xantium strumarinum, Selaginella tamariscina and Rehmannia glutinosa. Rats were exposed by inhalation to a mixture of cigarette smoke extract (CSE) and sulfur dioxide for three weeks to induce COPD-like pulmonary inflammation. PGT was administered orally to rats and pathological changes to the pulmonary system were examined in each group of animals through measurement of tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) and interleukin-6 (IL-6) levels in bronchoalveolar lavage fluid (BALF) at 21 days post-CSE treatment. The effect of PGT on the hypersecretion of pulmonary mucin in rats was assessed by quantification of the amount of mucus secreted and by examining histopathologic changes in tracheal epithelium. Confluent NCI-H292 cells were pretreated with PGT for 30 min and then stimulated with CSE plus PMA (phorbol 12-myristate 13-acetate), for 24 h. The MUC5AC mucin gene expression was measured by RT-PCR. Production of MUC5AC mucin protein was measured by ELISA. The results were as follows: (1) PGT inhibited CSE-induced pulmonary inflammation as shown by decreased TNF-${\alpha}$ and IL-6 levels in BALF; (2) PGT inhibited the hypersecretion of pulmonary mucin and normalized the increased amount of mucosubstances in goblet cells of the CSE-induced COPD rat model; (3) PGT inhibited CSE-induced MUC5AC mucin production and gene expression in vitro in NCI-H292 cells, a human airway epithelial cell line. These results suggest that PGT might regulate the inflammatory aspects of COPD in a rat model.

• BMB Reports
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• 제54권10호
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• pp.522-527
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• 2021

Lysine-specific demethylase 1 (LSD1) is an epigenetic regulator that modulates the chromatin status, contributing to gene activation or repression. The post-translational modification of LSD1 is critical for the regulation of many of its biological processes. Phosphorylation of serine 112 of LSD1 by protein kinase C alpha (PKCα) is crucial for regulating inflammation, but its physiological significance is not fully understood. This study aimed to investigate the role of Lsd1-S112A, a phosphorylation defective mutant, in the cigarette smoke extract/LPS-induced chronic obstructive pulmonary disease (COPD) model using Lsd1^SA/SA mice and to explore the potential mechanism underpinning the development of COPD. We found that Lsd1^SA/SA mice exhibited increased susceptibility to CSE/LPS-induced COPD, including high inflammatory cell influx into the bronchoalveolar lavage fluid and airspace enlargement. Additionally, the high gene expression associated with the inflammatory response and oxidative stress was observed in cells and mice containing Lsd1-S112A. Similar results were obtained from the mouse embryonic fibroblasts exposed to a PKCα inhibitor, Go6976. Thus, the lack of LSD1 phosphorylation exacerbates CSE/LPS-induced COPD by elevating inflammation and oxidative stress.

• Journal of Microbiology and Biotechnology
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• 제33권5호
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• pp.634-643
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• 2023

Chronic obstructive pulmonary disease (COPD), one of the leading causes of death worldwide, is caused by repeated exposure to harmful matter, such as cigarette smoke. Although Lilium longiflorum Thunb (LLT) has anti-inflammatory effects, there is no report on the fermented LLT bulb extract regulating lung inflammation in COPD. Thus, we investigated the protective effect of LLT bulb extract fermented with Lactobacillus acidophilus 803 in COPD mouse models induced by cigarette smoke extract (CSE) and porcine pancreas elastase (PPE). Oral administration of the fermented product (LS803) suppressed the production of inflammatory mediators and the infiltration of immune cells involving neutrophils and macrophages, resulting in protective effects against lung damage. In addition, LS803 inhibited CSE- and LPS-induced IL-6 and IL-8 production in airway epithelial H292 cells as well as suppressed PMA-induced formation of neutrophil extracellular traps in HL-60 cells. In particular, LS803 significantly repressed the elevated IL-6 and MIP-2 production after CSE and LPS stimulation by suppressing the activity of the nuclear factor kappa-light-chain-enhancer of activated B (NFκB) in mouse peritoneal macrophages. Therefore, our results suggest that the fermented product LS803 is effective in preventing and alleviating lung inflammation.

• Tuberculosis and Respiratory Diseases
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• 제59권5호
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• pp.530-535
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• 2005

연구 배경 : 우리나라에서는 다른 나라에 비해 약초 등을 이용해 만든 흡연대용품으로 금연을 시도하려는 흡연자들도 많고 이에 따라 여러 가지 다양한 제품 등이 판매되고 있는데 이들의 기관지, 폐에 대한 안정성에 관한 연구는 아직 부족한 상황이다. 특히 흡연자중 일부는 이런 금연 보조제를 사용하는 과정에서 이들을 상용하게 되어 시판되는 금연 보조담배의 장기적 사용시 인체의 안정성 여부에 대한 연구는 중요하리라 사료된다. 방 법 : 일반담배 및 금연 보조담배 3종의 담배연기추출물(cigarette smoke extract, CSE)을 기관지 상피세포에 24시간 노출 후 배양액에서 IL-6를 측정하였고 일반담배 및 허브담배, 쑥담배 노출 후 세포에서 IL-6 m RNA를 측정하여 비교하였다. 항산화 효과를 가진 N-acetyl-L-cysteine(NAC)를 일반 담배 및 금연보조담배 추출물에 함께 투여해 IL-6의 증가 억제여부를 보았다. 결 과 : 대조군에 비해 일반담배 및 금연보조담배3종에서 기관지 상피세포에 노출 시에 모두 의미 있는 IL-6증가를 관찰할 수 있었고, IL-6 m RNA도 같은 경향으로 증가 하였다. NAC를 함께 투여 시 일반 담배 및 허브담배 금연초에서는 IL-6의 증가를 의미 있게 억제하였지만 쑥담배에서는 억제하지 못하였다. 결 론 : 현재 우리나라에서 시판되고 있는 금연 보조담배 3종(허브담배, 쑥담배, 금연초)은 일반담배처럼 기관지 상피세포에 염증을 유발할 수 있어 장기적 사용시 인체의 안정성 여부에 대한 연구는 중요하리라 사료된다.

• IMMUNE NETWORK
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• 제24권2호
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• pp.3.1-3.23
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• 2024

Cigarette smoke extract (CSE)-treated mouse airway epithelial cells (MAECs)-derived exosomes accelerate the progression of chronic obstructive pulmonary disease (COPD) by upregulating triggering receptor expressed on myeloid cells 1 (TREM-1); however, the specific mechanism remains unclear. We aimed to explore the potential mechanisms of CSE-treated MAECs-derived exosomes on M1 macrophage polarization and pyroptosis in COPD. In vitro, exosomes were extracted from CSE-treated MAECs, followed by co-culture with macrophages. In vivo, mice exposed to cigarette smoke (CS) to induce COPD, followed by injection or/and intranasal instillation with oe-TREM-1 lentivirus. Lung function and pathological changes were evaluated. CD68⁺ cell number and the levels of iNOS, TNF-α, IL-1β (M1 macrophage marker), and pyroptosis-related proteins (NOD-like receptor family pyrin domain containing 3, apoptosis-associated speck-like protein containing a caspase-1 recruitment domain, caspase-1, cleaved-caspase-1, gasdermin D [GSDMD], and GSDMD-N) were examined. The expression of maternally expressed gene 3 (MEG3), spleen focus forming virus proviral integration oncogene (SPI1), methyltransferase 3 (METTL3), and TREM-1 was detected and the binding relationships among them were verified. MEG3 increased N6-methyladenosine methylation of TREM-1 by recruiting SPI1 to activate METTL3. Overexpression of TREM-1 or METTL3 negated the alleviative effects of MEG3 inhibition on M1 polarization and pyroptosis. In mice exposed to CS, EXO^-CSE further aggravated lung injury, M1 polarization, and pyroptosis, which were reversed by MEG3 inhibition. TREM-1 overexpression negated the palliative effects of MEG3 inhibition on COPD mouse lung injury. Collectively, CSE-treated MAECs-derived exosomal long non-coding RNA MEG3 may expedite M1 macrophage polarization and pyroptosis in COPD via the SPI1/METTL3/TREM-1 axis.

• KSBB Journal
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• 제27권6호
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• pp.367-374
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• 2012

Cigarette smoking (SM) is considered to be well known environmental toxin which contributes to the onset of various diseases. SM cause direct lungs damage, activate lungs inflammatory responses, and in some cases leads to the development of lung cancer. Cytokines in injured starfish (Asterina pectinifera) is the potential changes in its expression during the regeneration process. Especially, expression of TGF-${\beta}1$ has increased in arm cut starfish extract after eight days. Also, starfish including saponin like the ginseng. Saponin is widely used in the world because of some effective pharmacological activities. Therefore, the current study was designed to elucidate the pharmacological activities of starfish extract against cigarette smoking induced damage in cell line and pulmonary tissue. We investigate that the effect of eight days starfish extract after arm cut (8d) and intact starfish extract on cell line and mouse lung injury by SM. In cell proliferation analysis, although cigarette smoking extract (CSE) was co-treated, the higher proliferation ability is shown in 8d treatment than intact starfish extract. 8d and intact starfish extract was directly transported to pulmonary cells through respiratory organ by nebulizer inhalation. In this case of cigarette smoking, the pulmonary structure was damaged and functions become abnormal. However, 8d treated groups showed similar with the control group compared with SM group. Among them, 8d was proved to be more effective than intact starfish extract. These results demonstrate that 8d could more protect pulmonary structure and function than intact starfish extract against cigarette smoking by ginseng like saponin and regulation of inflammatory cytokines.

• Tuberculosis and Respiratory Diseases
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• 제58권1호
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• pp.43-53
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• 2005

배 경 : 폐기종에서 발생하는 폐포 파괴의 원인으로서 전통적으로 protease/anti-protease 불균형과 산화성 스트레스가 주요 가설로 여겨져 왔으나 최근 폐포세포의 아포프토시스가 폐포파괴 및 폐기종의 원인이 된다는 이론이 제기되고 있어서 A549 폐상피세포에서 흡연추출물에 의한 세포사의 특성을 규명하고자 본 연구를 시행하였다. 방 법 : A549 폐상피세포주에서 여러 농도의 흡연추출물 및 억제제를 첨가한 후 MTT assay를 잉용하여 세포생존율을 측정하였다. 세포사 분석은 FACScan을 이용한 DNA 분절확인, 전자현미경 검사, Hoecst/PI 이중염색을 이용한 현광현미경 검사를 이용하였고 cyt-ochrome c 유리는 면역형광법을 이용하였다. Bcl-2 과발현세포주를 이용하여 bcl-2의 역할을 확인하였고 p53 Western blot 및 HPV-E6 과발현 세포주를 이용하여 p53의 역할을 확인하였다. 결 과 : A549 세포주에서 흡연추출물에 의한 세포사는 FACScan에서 DNA 분절에 의한 subG1 분획의 확인 및 Hoecst/PI 이중염색 및 현광현미경 소견 상 아포프토시스임이 확인되었고 전자현미경 소견상 저농도에서는 아포프토시스가 발생하지만 고농도에서는 괴사가 발생함을 확인하였다. Cytochrome c가 세포질로 유리됨을 확인하였으나 caspase 억제제에 의해서 세포사가 차단되지 않았다. 흡연추출물에 의한 세포사는 Bcl-2과발현에 의해 억제되었고 p53활성화를 유도하고 p53이 기능적으로 knock-out 된 세포주에서 억제되었다. 결 론 : 흡연추출물에 의한 폐상피세포의 세포사는 저농도에서는 아포프토시스를 고농도에서는 괴사를 유도하고 bcl-2 및 p53 경로가 중요한 역할을 담당하며 이는 폐기종 발생기전에 있어서 폐세포 세포사 이론을 뒷바침하는 자료로 활용될 수 있다고 생각된다.

• The Korean Journal of Physiology and Pharmacology
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• 제26권2호
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• pp.95-111
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• 2022

Chronic obstructive pulmonary disease (COPD) is an important healthcare problem worldwide. Often, glucocorticoid (GC) resistance develops during COPD treatment. As a classic hypoglycemic drug, metformin (MET) can be used as a treatment strategy for COPD due to its anti-inflammatory and antioxidant effects, but its specific mechanism of action is not known. We aimed to clarify the role of MET on COPD and cigarette smoke extract (CSE)-induced GC resistance. Through establishment of a COPD model in rats, we found that MET could improve lung function, reduce pathological injury, as well as reduce the level of inflammation and oxidative stress in COPD, and upregulate expression of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), multidrug resistance protein 1 (MRP1), and histone deacetylase 2 (HDAC2). By establishing a model of GC resistance in human bronchial epithelial cells stimulated by CSE, we found that MET reduced secretion of interleukin-8, and could upregulate expression of Nrf2, HO-1, MRP1, and HDAC2. MET could also increase the inhibition of MRP1 efflux by MK571 significantly, and increase expression of HDAC2 mRNA and protein. In conclusion, MET may upregulate MRP1 expression by activating the Nrf2/HO-1 signaling pathway, and then regulate expression of HDAC2 protein to reduce GC resistance.

• Tuberculosis and Respiratory Diseases
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• 제58권6호
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• pp.590-599
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• 2005

연구배경 : 만성폐쇄성폐질환에서 나타나는 기도점액의 과다분비는 이 질환의 중요한 병리학적 소견이며 호흡곤란 등 환자의 증상을 악화시키는 요인 중의 하나이다. 기도 점액을 구성하는 여러 성분 중 Muc 유전자에 의해 만들어지는 당 단백이 흡연에 의해 생성이 증가하는데 이에 관여하는 세포 내 신호전달 과정에 대하여 확실히 밝혀진 바가 없다. 저자는 Muc 유전자 중 인체의 기도에 가장 많이 분비되는 Muc5ac 점액 생성을 담당하는 Muc5ac 유전자의 발현이 흡연에 의하여 증가하는데 관여하는 세포 내 신호전달 과정을 알아보고자 하였다. 재료 및 방법 : 사람 폐선암 세포주인 A549 세포를 배양하여 Muc5ac 유전자의 promotor를 luciferase reporter plasmid를 사용하여 세포 내에 transfection시키고 5% 담배연기 추출물로 자극하여 배양하였다. 또 세포 내 신호전달에 관여하는 표피성장인자 수용체 kinase의 억제제인 AG1478, mitogen-activated protein kinase kinase(MAPKK) 억제제인 PD98059, p38 mitogen-activated protein kinase 억제제인 SB203580으로 각각 전 처치 후 역시 5% 담배연기 추출물로 자극 배양하였다. 배양된 세포에서 단백질을 추출하여 luciferase 분석을 통하여 Muc5ac promoter 활성도를 측정하고 Western blot을 이용하여 표피성장인자 수용체와 mitogen-activated protein kinase (MAPK)인 extracellular signalrelated kinase (ERK)1/2, p38 MAPK, c-Jun N-terminal kinase (JNK)의 발현을 확인하였다. 또 세포에서 RNA를 추출한 후 Muc5ac primer를 이용하여 역전사효소 중합연쇄반응을 수행하여 Muc5ac mRNA 발현을 관찰하였다. 결 과 : 1. Muc5ac promoter를 삽입한 A549 세포를 5% 담배연기 추출물로 자극하였을 때 의의 있게 luciferase 활성도가 증가하였고(P<0.001) 자극하는 시간이 3시간이었을 때 luciferase 활성도가 최고치를 보였다(P<0.01). 또 담배연기 추출물 자극은 표피성장인자 수용체를 인산화시켰으며 인산화는 AG1478과 PD98059에 의하여 억제되었다. 2. AG1478 혹은 PD98059로 전 처치 후 5% 담배연기 추출물로 자극한 경우 5% 담배연기 추출물 단독으로 자극한 것에 비하여 유의하게 lucifearse 활성도가 억제되었고 (P<0.01) 세 가지 종류의 MAPK 중 ERK1/2와 p38 MAPK의 인산화는 관찰되었으나 JNK의 인산화는 관찰되지 않았다. 역전사효소 중합연쇄반응을 이용하여 관찰한 Muc5ac mRNA 발현은 담배연기 추출물에 의해 증가되었고 PD98059와 AG1478에 의하여 역시 억제되었다. 3. 담배연기 추출물에 의하여 인산화 된 ERK1/2는 PD98059에 의하여 인산화가 감소하였고 p38 MAPK의 인산화는 PD98059와 SB203580에 의하여 감소하였으며 이 두 가지 억제제는 모두 luciferase 활성도를 유의하게 억제시켰다(P<0.0001). 결 론 : 담배연기 추출물은 Muc5ac 유전자의 발현을 증가시켜 기도 내 점액 분비를 증가시키며 이는 표피성장인자 수용체를 매개로 ERK1/2와 p38 MAPK를 경유하여 세포 내 신호전달이 이루어진다고 생각된다. 따라서 점액 유전자 활성화를 매개하는 신호전달 과정을 차단하는 약제나 방법이 개발된다면 과도한 점액분비를 치료할 수 있을 것으로 생각한다.