Re: '스퍼미딘' 자가포식 유도.. 폐섬유화 방지.. 2020 네이처!

[문형철]

블레오마이신은 항암제로 쓰이는 약물인데, 

폐섬유화를 일으킬 수 있는 부작용이 있어요. 

폐섬유화는 폐 조직이 딱딱하게 굳는 병인데, 

블레오마이신이 폐에 염증을 일으키고, 이 염증이 섬유화를 유발하는 거죠.

spermidine은 폐섬유화를 방지하는 생체 아민(폴리아민, 트리아민)

1. nature  
2. experimental & molecular medicine  
3. articles  
4. article

Spermidine attenuates bleomycin-induced lung fibrosis by inducing autophagy and inhibiting endoplasmic reticulum stress (ERS)-induced cell death in mice

Download PDF

• Article
• Open access
• Published: 14 December 2020

Spermidine attenuates bleomycin-induced lung fibrosis by inducing autophagy and inhibiting endoplasmic reticulum stress (ERS)-induced cell death in mice

• Ae Rin Baek, 
• Jisu Hong, 
• Ki Sung Song, 
• An Soo Jang, 
• Do Jin Kim, 
• Su Sie Chin & 
• Sung Woo Park 

Experimental & Molecular Medicine volume 52, pages2034–2045 (2020)Cite this article

• 10k Accesses

• 73 Citations

• 15 Altmetric

• Metricsdetails

Abstract

Spermidine is an endogenous biological polyamine that plays various longevity-extending roles and exerts antioxidative, antiaging, and cell growth-promoting effects. We previously reported that spermidine levels were significantly reduced in idiopathic pulmonary fibrosis (IPF) of the lung. The present study assessed the potential beneficial effects of spermidine on lung fibrosis and investigated the possible mechanism. Lung fibrosis was established in mice using bleomycin (BLM), and exogenous spermidine was administered daily by intraperitoneal injection (50 mg/kg in phosphate-buffered saline). BLM-induced alveolar epithelial cells showed significant increases in apoptosis and endoplasmic reticulum stress (ERS)-related mediators, and spermidine attenuated BLM-induced apoptosis and activation of the ERS-related pathway. Senescence-associated β-gal staining and decreased expression‎ of p16 and p21 showed that spermidine ameliorated BLM-induced premature cellular senescence. In addition, spermidine enhanced beclin-1-dependent autophagy and autophagy modulators in IPF fibroblasts and BLM-induced mouse lungs, in which inflammation and collagen deposition were significantly decreased. This beneficial effect was related to the antiapoptotic downregulation of the ERS pathway, antisenescence effects, and autophagy activation. Our findings suggest that spermidine could be a therapeutic agent for IPF treatment.

스페르미딘은

다양한 장수 연장 역할을 수행하며

항산화, 항노화, 세포 성장 촉진 효과를 발휘하는

내인성 생물학적 폴리아민입니다.

우리는 이전 연구에서

폐 특발성 폐섬유증(IPF) 환자의 폐에서

스페르미딘 수치가 유의미하게 감소했음을 보고했습니다.

본 연구에서는

스페르미딘이 폐 섬유화에 미치는 잠재적 유익 효과를 평가하고

그 가능성 있는 기전을 조사했습니다.

폐 섬유화는 블레오마이신(BLM)을 사용하여 마우스에서 유도되었으며,

외인성 스페르미딘은 매일 복강 내 주사(인산염 완충 용액에 용해된 50 mg/kg)로 투여되었습니다.

BLM에 의해 유도된 폐포 상피 세포에서는

세포 사멸과 내소체 스트레스(ERS) 관련 매개체의 유의미한 증가가 관찰되었으며,

스페르미딘은 BLM에 의한 세포 사멸과 ERS 관련 경로의 활성화를 억제했습니다.

노화 관련 β-갈 염색과 p16 및 p21의 발현 감소는

스퍼미딘이 BLM에 의해 유발된 조기 세포 노화를 개선한 것을 나타냅니다.

또한,

스퍼미딘은 IPF 섬유모세포와 BLM에 의해 유발된 쥐의 폐에서

베클린-1 의존성 자가포식 및 자가포식 조절 인자를 강화하여

염증과 콜라겐 침착을 현저히 감소시켰습니다.

이러한 유익한 효과는

ERS 경로의 항세포사멸 조절, 노화 방지 효과 및 자가포식 활성화와 관련이 있습니다.

우리의 연구 결과는

스페르미딘이 IPF 치료에 유용한 치료제가 될 수 있음을 시사합니다.

Similar content being viewed by others

Inhibitory effects of Schisandrin C on collagen behavior in pulmonary fibrosis

Article Open access18 August 2023

Roxithromycin attenuates bleomycin-induced pulmonary fibrosis by targeting senescent cells

Article 02 March 2021

Caveolin-1 scaffolding domain peptide abrogates autophagy dysregulation in pulmonary fibrosis

Article Open access30 June 2022

Introduction

Idiopathic pulmonary fibrosis (IPF) is a progressive, devastating lung disease characterized by alveolar epithelial cell (AEC) injury and the subsequent proliferation of activated fibroblasts known as myofibroblasts. The accumulation of myofibroblasts is responsible for the excessive deposition of extracellular matrix (ECM), resulting in irreversible distortion of the lung parenchymal structure1,2. Although the exact mechanisms of the development of lung fibrosis remain unclear, repeated injury and shortened survival of AECs have been recognized as initiating events3,4.

The precise mechanism of AEC damage remains unclear, but increased ERS or the accumulation of unfolded proteins in AECs have recently been implicated in IPF pathogenesis5,6. In addition, because the incidence of IPF increases with age, age-related mechanisms such as telomerase attrition and increased AEC senescence are also important drivers of the abnormal repair process during the initiation of fibrosis7,8.

Spermidine is a naturally occurring polyamine and is ubiquitous in living organisms as a polycation9,10. Polyamines are involved in various biological processes, including replication, transcription, translation, posttranslational modification, and membrane stability11, and they regulate cellular proliferation, differentiation, and apoptosis12,13. Previous studies have suggested that polyamines have multiple effects, including antioxidant and anti-inflammatory effects, in various animal disease models14. Eisenberg et al.15 reported that the exogenous administration of spermidine prolongs the life span of several organism models and significantly reduces age-related oxidative protein damage in mice. In addition, natural endogenous spermidine has prominent cardioprotective and neuroprotective effects and stimulates anticancer immunosurveillance in animal models14.

We recently reported that spermidine levels were decreased in the IPF lung compared to healthy controls16. Although the beneficial effects of spermidine have been reported in various chronic inflammatory diseases, its role in and effects on lung fibrosis have not yet been elucidated. Given the antiaging and antioxidative activities of spermidine, we hypothesized that spermidine may have beneficial effects on lung fibrosis, particularly in regulating cellular senescence and ERS-induced cell death.

Hence, in this study, we examined whether spermidine exerted beneficial effects on experimental lung fibrosis. The mechanism of the beneficial effects of spermidine was determined using AEC culture models. The therapeutic implication of spermidine in lung fibrosis development was eval‎uated in a mouse model of bleomycin (BLM)-induced lung fibrosis.

소개

특발성 폐 섬유증(IPF)은

폐포 상피 세포(AEC) 손상과 그로 인한 근섬유모세포로 알려진

활성화된 섬유모세포의 증식을 특징으로 하는 진행성, 치명적인 폐 질환입니다.

근섬유모세포의 축적은

세포외 기질(ECM)의 과도한 침착을 유발하여

폐 실질 구조의 불가역적인 변형을 초래합니다1,2.

폐 섬유증의 정확한 발병 메커니즘은 아직 명확하지 않지만,

AEC의 반복적인 손상과 생존 기간 단축이 발병의 초기 단계로 인정되고 있습니다3,4.

AEC 손상의 정확한 메커니즘은 아직 명확하지 않지만,

최근 IPF 병리학에서 AEC 내 ERS 증가 또는 미접힘 단백질 축적이 관련되어 있다는 보고가 있습니다5,6.

또한 IPF 발생률이 연령과 함께 증가함에 따라,

텔로머라제 감소 및 AEC 노화 증가와 같은 연령 관련 메커니즘도

섬유화 초기 단계에서 비정상적인 수리 과정의 중요한 촉진 요인으로 작용합니다7,8.

스퍼미딘은

자연적으로 발생하는 폴리아민으로,

생물체에 널리 분포하는 양전하를 띤 고분자 물질입니다9,10.

Spermidine is a naturally occurring polyamine and is ubiquitous in living organisms as a polycation.

폴리케이션은 양전하를 띤 고분자 물질.

폴리아민은

복제, 전사, 번역, 번역 후 변형, 세포막 안정화 등

다양한 생물학적 과정에 관여하며11,

세포 증식, 분화, 아포토시스 등을 조절합니다12,13.

이전 연구에서는 폴리아민이 다양한 동물 질환 모델에서

항산화 및 항염증 효과를 포함한 다중 효과를 갖는 것으로 제안되었습니다14.

Eisenberg 등15은

스페르미딘의 외인성 투여가 여러 생물 모델의 수명을 연장하고

쥐에서 연령 관련 산화 단백질 손상을 유의미하게 감소시킨다고 보고했습니다.

또한 자연적으로 생성되는 내인성 스페르미딘은

동물 모델에서 심장 보호 및 신경 보호 효과가 두드러지며 항암 면역 감시를 자극합니다14.

우리는 최근

IPF 폐에서 건강한 대조군에 비해

스페르미딘 수치가 감소했다는 사실을 보고했습니다16.

스페르미딘의 유익한 효과는

다양한 만성 염증성 질환에서 보고되었지만,

폐 섬유화에서의 역할과 효과는 아직 명확히 규명되지 않았습니다.

스페르미딘의 항노화 및 항산화 활성을 고려할 때, 우

리는 스페르미딘이 특히

세포 노화 조절과 ERS에 의한 세포 사멸을 통해

폐 섬유화에 유익한 효과를 가질 수 있다고 가설을 세웠습니다.

따라서 본 연구에서는 스페르미딘이 실험적 폐 섬유화에 유익한 효과를 미치는지 조사했습니다. 스페르미딘의 유익한 효과 메커니즘은 AEC 배양 모델을 사용하여 확인했으며, 블레오마이신(BLM) 유도 폐 섬유화 마우스 모델에서 스페르미딘의 치료적 가능성을 평가했습니다.

Materials and methods

Reagents and antibodies

The antibodies used were rabbit anti-p16 (#ab51243; Abcam, Cambridge, UK), rabbit anti-p-Rb (#8516; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-CHOP (#5554; Cell Signaling Technology), rabbit anti-GRP78/Bip (#3177; Cell Signaling Technology), rabbit anti-ATF-6a (#sc22799; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-IRE-1 (#ab37073; Abcam), rabbit anti-light chain (LC) 3B (#3868S; Cell Signaling Technology), rabbit anti-autophagy related gene 7 (ATG7) (#8558S; Cell Signaling Technology), rabbit anti-beclin-1 (#3738S; Cell Signaling Technology), rabbit anti-mTOR (#2972; Cell Signaling Technology), rabbit anti-mTOR (phospho) (#2971; Cell Signaling Technology), and mouse anti-β-actin (#A5316; Sigma-Aldrich, St. Louis, MO, USA). Spermidine (#S2626) and 4-PBA (#P21005) were purchased from Sigma-Aldrich. An LDH assay kit (#mk401; Takara) was used. The in situ cell death detection kit (#11684795910; Roche Diagnostics, Basel, Switzerland), cellular senescence (SA-β-gal staining) assay kit (#CBA-230; Cell Biolabs, San Diego, CA, USA), mouse interleukin-1 beta (IL-1β) enzyme-linked immunosorbent assay (ELISA) kit (#88701322; eBioscience, Inc., San Diego, CA, USA), mouse tumor necrosis factor-alpha (TNF-α) ELISA kit (#88732422; eBioscience, Inc.), and a mouse transforming growth factor-beta 1 (TGF-β1) ELISA kit (#DY1679; R&D Systems, Minneapolis, MN, USA) were also purchased.

Mouse AEC isolation and culture

Primary mouse AECs were isolated from wild-type mice using a previously described protocol17 with minor modifications.

Preparation of lung cell suspensions

Crude cell suspensions were prepared from C57BL/6 mice. The lungs were perfused with 0.9% NaCl using a 10-mL syringe fitted with a 21-gauge needle (BD Pharmingen, San Diego CA, USA) through the right ventricle of the heart until they were visually free of blood. A 21-gauge intravenous catheter was inserted into the trachea and secured tightly with a suture. The lungs were filled with 1–2 mL of dispase via the tracheal catheter and then allowed to collapse naturally, expelling part of the dispase. Low-melting-point agarose (l%, 0.45 mL, stored in a 45 °C water bath; Invitrogen, Paisley, UK) was slowly infused via the catheter. The lungs were immediately covered with crushed ice and incubated for 2 min. Then, they were removed and placed into 12 mL polypropylene culture tubes with 2 mL of dispase (Sigma-Aldrich), incubated for 45 min at room temperature, and kept on ice until the next step. The lungs were transferred into 7 mL of Dulbecco’s modified Eagle’s medium (DMEM) with 0.01% DNase I in 60 mm Petri dishes. The digested tissue was carefully separated from the airways with the curved edge of fine-tipped forceps and gently swirled for 5–10 min. The resulting suspension was successively filtered through 100 and 40 µm Falcon cell strainers and then through 25 µm nylon mesh. The filtered suspension was centrifuged at 130 × g for 8 min at 4 °C and resuspended in 10 mL of 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered DMEM.

Magnetic purification of ATII cells from crude cell suspensions

The cells were incubated with biotinylated anti-CD32 (0.5 µg/million cells; BD Pharmingen, San Diego CA, USA) and biotinylated anti-CD45 (1.5 µg/million cells; BD Pharmingen) for 30 min at 37 °C. Meanwhile, streptavidin-coated magnetic particles (Thermo Fisher Scientific, Waltham, MA, USA) were washed twice in phosphate-buffered saline (PBS) (10 min each wash) in polypropylene culture tubes using a magnetic tube separator (Sigma-Aldrich). After incubation, the cells were centrifuged (130 × g for 8 min at 4 °C), resuspended in 7 mL of DMEM, added to the magnetic particles, and incubated with gentle rocking for 30 min at room temperature. At the end of the incubation, the tubes were attached to the magnetic tube separator with adhesive tape for 15 min. The cell suspension was aspirated from the bottom of the tube using a narrow-stemmed transfer pipette, centrifuged, and resuspended in a culture medium. Cell viability was determined by trypan blue staining. The purity of alveolar type II cells was assessed via pro-SPC immunofluorescence staining. As in previous studies, 4–8 × 105 cells were isolated from a single mouse. In these samples, the purity of type II pneumocytes was 90–93%. The isolated cells were maintained in 10% FBS and 1% penicillin–streptomycin in 25 mM HEPES-buffered DMEM.

Fibroblast isolation and culture

Primary human lung fibroblasts were obtained from SCH Biobank (Soonchunhyang Biobank, Bucheon, Korea) as previously described18. Briefly, the fibroblasts were derived from lung tissue obtained from IPF patients by video-assisted thoracoscopic biopsy. Lung samples were obtained by lung biopsy, usually 1 week after hospital admission. None of the patients had been treated with corticosteroids or immunosuppressive drugs at the time of the biopsy. Control fibroblasts were obtained from individuals who underwent a lobectomy to remove a primary lung tumor. No morphological evidence of disease was found in the tissue samples used for the isolation of control cells. Lung fibroblasts from IPF or control specimens were isolated by mechanical dispersal, and then trypsin digestion of tissues was used to mince them into 1 mm2 fragment. Fibroblast cultures were established in DMEM supplemented with 10% fetal calf serum, 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.25 μg/mL amphotericin B. All cells were cultured at 37 °C in 95% air/5% CO2 until just prior to reaching confluence, which generally occurred in 1–2 weeks. After three passages, immunoblotting was performed using anti-vimentin antibodies on adherent cells harvested from the same culture vessels. All cells showed the morphological characteristics of fibroblasts. All experiments with IPF and control fibroblasts were performed on cells before the sixth passage.

Apoptosis assay

Apoptotic cells in paraffin-embedded lung tissues were labeled using a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay kit (Roche Diagnostics, Basel, Switzerland). The numbers of TUNEL-positive (apoptotic) cells in three sections per sample were counted under a fluorescence microscope at ×400 magnification (Carl Zeiss Microsystems, Thornwood, NY, USA) as described previously19. Mouse AECs were exposed to vehicle or 10 µg/mL BLM with spermidine for 24 h after overnight serum starvation. The dose of spermidine administered was 100 µM. An annexin V-FITC/propidium iodide detection kit (BD Biosciences Pharmingen, San Diego, CA, USA) was used to determine the proportions of apoptotic and necrotic cells. Approximately, 1 × 106 cells/mL were washed in PBS, surface stained, resuspended in binding buffer, incubated with FITC-conjugated annexin V and propidium iodide (PI) for 15 min in the dark at room temperature, washed, and resuspended in binding buffer.

Immunoblotting

Protein were extracted from cells with lysis buffer (#78510; Thermo Fisher Scientific, Waltham, MA, USA) containing proteinase and phosphatase inhibitor cocktails (#05892970001 and #04906837001; Roche Diagnostics, Basel, Switzerland), and the samples were then centrifuged. Immunoblotting was performed as described previously. For each experiment, equal amounts of total protein were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred to a polyvinylidene difluoride membrane (#ISEQ00010; Millipore, Billerica, MA, USA) and incubated with a specific primary antibody for 2 h at 37 °C or for 24 h at 4 °C. After being washed several times with PBS containing Tween, the membrane was incubated with anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)-linked secondary antibody (#7074; Cell Signaling Technology) or anti-mouse IgG HRP-linked secondary antibody (#7076; Cell Signaling Technology), followed by chemiluminescence detection (#34080; Thermo Fisher Scientific and #1705061; BIO-RAD, Berkeley, CA, USA) with a ChemiDocTM Touch Imaging System (BIO-RAD).

SA-β-galactosidase staining

Senescent cells were analyzed using senescence-associated β-galactosidase (SA-β-gal) staining. Cells were grown in six-well plates, washed, fixed, and stained with the SA-β-gal cellular senescence assay kit (Cell Biolabs) according to the manufacturer’s protocol and as previously described20. The sections were examined under a microscope.

Immunofluorescence staining of fibroblasts

Standard protocols for immunofluorescence microscopy were used as described previously21. Human primary fibroblasts were plated on cover-glass-bottom dishes and treated with or without with the indicated agents. The cells on the dishes were washed twice, fixed in 4% paraformaldehyde for 1 h, and washed three times after fixation. The cells were prepared and stained with the indicated primary antibodies (1:50) overnight at 4 °C. The slides were washed twice, incubated with chromogen-labeled secondary antibody (1:100) for 30 min, and washed three times after being stained. Images were obtained with a confocal microscope (Zeiss LSM 510 META). Autophagosomes were identified as LC3B-positive puncta, and autophagolysosomes were identified by the coexpression‎ of LC3B and LAMP-1.

Animal model of BLM-induced lung fibrosis and treatment with spermidine

Specific-pathogen-free C57BL/6 (Orient Bio Inc., Sungnam-Si, Gyeonggi-Do, Korea) mice were maintained under pathogen-free conditions. All animal procedures followed a protocol approved by the Institutional Animal Care and Use Committee of Soonchunhyang University Bucheon Hospital (SCHBC-animal-2016-011). On day 0, the mice were administered 3 U/kg BLM (Sigma-Aldrich) dissolved in a total volume of 200 µL endotoxin-free water by intratracheal instillation. To determine the proper therapeutic dose of spermidine, we performed a pilot study with a small number of mice. We treated bleomycin-induced mice with 10, 30, 50, and 100 mg/kg spermidine from day 10 to day 21, and then the analyzed inflammatory cells in BAL fluid. In addition, to eval‎uate the cellular toxicity of spermidine, LDH levels in BAL fluid were measured. We found that 50 mg/kg spermidine significantly diminished neutrophils and reduced LDH levels in the BAL fluid, indicating that 50 mg/kg spermidine was both effective and safe in mice (Supplementary Fig. 1). On days 10–21, the mice were administered 50 mg/kg spermidine (Sigma-Aldrich) dissolved in endotoxin-free water containing 0.3% dimethyl sulfoxide by intraperitoneal instillation. The sham control mice were treated with PBS only. On day 21, the mice were sacrificed with an overdose of a ketamine/xylazine mixture, and bronchoalveolar lavage (BAL) was performed by instilling 1 mL of PBS, which was gently retrieved, four times as described previously22. Cell numbers were measured using a hemocytometer, and differential cell counts were performed on slides prepared by cytocentrifugation and Diff-Quik staining (E. Merck KG, Darmstadt, Germany). The BAL fluid was centrifuged at 500 × g for 10 min, and the supernatant was stored at −70 °C. Some of the cell-free supernatants were used for biochemical analyses, such as lactate dehydrogenase (LDH) assays. The Institutional Animal Care and Use Committee of Soonchunhyang University Bucheon Hospital approved this study (SCHBC-2016-012).

BLM에 의한 폐 섬유화 동물 모델 및 스페르미딘 치료특정 병원체 무균 C57BL/6 마우스(Orient Bio Inc., Sungnam-Si, Gyeonggi-Do, Korea)는 병원체 무균 조건 하에서 사육되었습니다. 모든 동물 실험 절차는 순천향대학교 부천병원 동물실험윤리위원회(SCHBC-animal-2016-011)의 승인을 받은 프로토콜에 따라 수행되었습니다. 실험 시작일(날짜 0)에 마우스에게 BLM (Sigma-Aldrich) 3 U/kg을 엔도톡신 무함유 물 200 μL에 용해하여 기관지 내 투여했습니다. 스페르미딘의 적절한 치료 용량을 결정하기 위해 소수의 쥐를 대상으로 예비 연구를 수행했습니다. 블레오마이신으로 유도된 쥐에게 10, 30, 50, 및 100 mg/kg의 스페르미딘을 10일부터 21일까지 투여한 후, BAL 액체 내 염증 세포를 분석했습니다. 또한 스페르미딘의 세포 독성을 평가하기 위해 BAL 액체 내 LDH 수준을 측정했습니다. 

50 mg/kg 스페르미딘은 

BAL 액체 내 중성구 수를 유의미하게 감소시키고 LDH 수치를 낮췄으며, 

이는 50 mg/kg 스페르미딘이 쥐에서 효과적이고 안전함을 나타냈습니다(보조 그림 1). 

10일부터 21일까지 마우스에게 엔도톡신 무함유 물에 0.3% 디메틸 설폭사이드를 용해한 50 mg/kg 스페르미딘(Sigma-Aldrich)을 복강 내 주입으로 투여했습니다. 가짜 대조군 마우스는 PBS만 투여했습니다. 21일차에 마우스는 케타민/시클라진 혼합물 과량 투여로 안락사시킨 후, 이전 연구22에서 설명된 방법과 동일하게 PBS 1mL를 4회 투여하여 BAL을 수행했습니다. 세포 수는 헤모시토미터를 사용하여 측정했으며, 세포 분별 계수는 세포 원심분리 및 Diff-Quik 염색(E. Merck KG, Darmstadt, Germany)을 통해 준비된 슬라이드에서 수행되었습니다. BAL 액체는 500×g에서 10분간 원심분리 후 상층액을 −70°C에 보관했습니다. 세포가 없는 상층액 일부는 젖산 탈수소효소(LDH) 분석 등 생화학적 분석에 사용되었습니다. 이 연구는 순천향대학교 부천병원 동물실험윤리위원회(SCHBC-2016-012)의 승인을 받았습니다.

Histological assays

A portion of each left lung was fixed in 4% (v/v) buffered paraformaldehyde and embedded in paraffin. The tissue was cut into 5 µm-thick slices and stained with hematoxylin and eosin (H&E) or Masson’s trichrome. The right lung was snap-frozen by immersion in liquid nitrogen and stored at −80 °C prior to RNA and protein extraction. Lung sections were stained with H&E for histopathological analyses or with Masson’s trichrome to eval‎uate collagen content and distribution. The Ashcroft score for the eval‎uation of lung fibrosis has previously been described23.

Immunohistochemical staining

The lung tissues were dehydrated and embedded in paraffin. For histological examination, 4 μm-thick sections on slides were treated with 1.4% H2O2-methanol for 30 min to block endogenous peroxidase. Then, nonspecific binding was blocked with 1.5% normal saline, and the slides were incubated with rabbit anti-p21 (1:200; #ab188224; Abcam, Cambridge, UK) and rabbit anti-p16 (1:100; #ab51243; Abcam) antibodies. The next day, the sections were incubated with ABC kit reagents (Vector Laboratories, Burlingame, CA, USA). The color reaction was developed by staining with a liquid DAB + substrate kit (Golden Bridge International Inc., Mukilteo, WA, USA). After immunohistochemical staining, the slides were counterstained with Harris’s hematoxylin for 1 min.

Masson’s trichrome staining

The mouse lung sections were placed in Bouin’s solution at 56 °C for 1 h and then stained successively with Mayer’s hematoxylin solution for 5 min, Biebrich scarlet-acid fuchsin solution for 10 min, phosphomolybdic acid-phosphotungstic acid for 15 min, and aniline blue for 2 h (staining reagents from Sigma-Aldrich). The sections were examined under a microscope.

Measurement of hydroxyproline in the mouse lung

To estimate the amount of collagen in the lung, the right lungs were used in a hydroxyproline assay (#MAK008; Sigma-Aldrich) according to the manufacturer’s protocol. Briefly, the lungs were weighed, homogenized in sterile water, and hydrolyzed in 12 N HCl at 120 °C for 3 h. The hydrolyzed samples were incubated with 4-(dimethylamino) benzaldehyde for 90 min at 60 °C, and the absorbance of oxidized hydroxyproline was measured at 560 nm. The amount of collagen is expressed in micrograms per milligram of lung tissue.

Measurement of proinflammatory and profibrotic cytokine levels

ELISA kits were used to measure the concentrations of IL-1β, TNF-α, and the active form of TGF-β1 in lung tissue (IL-1β: #88701322; eBioscience, Inc., TNF-α: #88732422; eBioscience, Inc., TGF-β1: #DY1679; R&D Systems.) according to the manufacturers’ instructions.

Statistical analyses

All data are expressed as the means ± standard error. The data were analyzed using the Kruskal–Wallis test, followed by the Mann–Whitney U test (with Bonferroni correction for intergroup comparisons), and p values < 0.05 were considered significant.

Results

Spermidine attenuates BLM-induced cell death in AECs

BLM generates reactive oxygen species, which are responsible for the induction of cell death, and BLM is generally administered to establish lung fibrosis models for in vivo studies24. We examined the effects of spermidine on cell death in primary mouse AECs induced by BLM in an in vitro system. The selected concentrations of spermidine used in this experiment did not show any significant cytotoxicity at a concentration of 100 µM for up to 48 h of incubation, and cell viability remained >95% as measured using the MTT assay (data not shown). Twenty-four hours after BLM (10 µg/mL) administration in the presence of spermidine (100 µM) or vehicle, the proportions of apoptotic and necrotic cells were assessed by flow cytometry (Fig. 1). Spermidine significantly diminished BLM-induced increases in the fractions of apoptotic and necrotic cells (Fig. 1). These data indicated that spermidine significantly attenuated BLM-induced AEC apoptosis and necrosis.