Ethics statement. This study involves the analysis of human IPF patient specimens. Primary fibroblast lines were obtained from unused, existing pathological human tissue samples, and therefore is exempt. Tissue samples were stripped of all identifiers and designated as waste. All patients underwent procedures for diagnostic or therapeutic procedures. Written informed consent was obtained on all patients prior to the procedure being performed. Use of human tissues was approved by the Xiamen University and Wuxi Lung Transplant Center. All methods were performed in accordance with the relevant guidelines and regulations laid down by the Committee.
Human subjects. Cell lines were derived from lungs removed at the time of transplantation. The diagnosis of IPF was supported by history, physical examination, pulmonary function tests, and typical high resolution chest computed tomography findings of IPF. In all cases, the diagnosis of IPF was confirmed by microscopic analysis of lung tissue and demonstrated the characteristic morphological findings of usual interstitial pneumonia. All patients fulfilled the criteria for the diagnosis of IPF as established by the American Thoracic Society (ATS) and the European Respiratory Society (ERS)43. For this study, new primary IPF fibroblast lines were generated as tissue became available. To address concerns of biological variability, we studied 2 control cell lines and 2 IPF cells lines. Primary control and IPF lung fibroblast lines were generated by explant culture and cultured in high glucose DMEM containing 10% FCS. Fibroblasts were used between passages three and five (Supplementary Fig1) by The Xiamen University School of Medicine. Cells were characterized as fibroblasts as previously described44. The levels of SIRT6 in IPF were derived from Meltzer in R2 Genomics Analysis and Visualization Platform.
Mouse Lung Fibrosis Model. The animal procedures were approved by the Institutional Animal Care and Use Committee at Xiamen University (XMVLAC 20190107). All the experiments were performed in accordance with the ARRIVE ethical guidelines. SPF C57BL/6 male mice (6-8 weeks old) were adopted and divided into two groups: normal saline group and bleomycin injection group (2.5 U/kg). Under anesthesia, the neck skin was incised, and the trachea was exposed. Saline or 2.5 U/kg bleomycin (Sigma, USA) was injected into the mouse trachea from the interval of the trachea cartilaginous rings with a 25-guageneedle. The tracheal puncture points were sutured by silk thread. Mouse lungs were collected at 21 days for experiments in central laboratory of The Xiamen University School of Medicine.
Cell Culture and Transfection. Human fetal lung fibroblasts (MRC5) was gained from the American Type Culture Collection (ATCC, Manassas, VA), cultured in minimal essential medium (MEM) (Gibco, USA) with 10% (v/v) FBS and 1% penicillin/streptomycin. For cell transfection, SIRT6 siRNA was synthesized by GenePharma (Shanghai, China). Before the transfection, cells were cultured to 60%–80% confluence and transfected using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s instructions.
Western Blotting. The cell lysates were centrifuged at 12000 rpm at 4°C. Cells were placed for 30 min on ice in RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) in the presence of a cocktail proteinase inhibitor (Sigma-Sigma-Aldrich, St. Louis, MO, USA). Sample proteins were quantitated using pierce BCA protein assay (Thermo Fisher Scientific, Rockford, USA). The protein lysates were separated using SDS-PAGE and transferred to a PVDF (Millipore, Billerica, MA, USA) membrane. The membrane was blotted with specific primary and horseradish peroxidase (HRP) secondary antibodies. The immunoreactive bands were visualized using SuperSignal West Pico Chemiluminescent Substrates (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Antibody binding was detected by enhanced chemiluminescence detection kit (ECL) (Amersham International Plc., Buckinghamshire, UK). The following antibodies were used: anti-SIRT6 (#12486S), anti-Akt (#2920), phospho-Akt at Ser473 (#9271) and phospho-Akt at Thr308 (#4056S), β-actin (#3700), anti-S6 (#2317) and phospho-S6 at Ser240/244 (#5364) (diluted with 5% BSA to 1: 1000) were obtained from Cell Signaling Technology (Beverly, MA, USA). LC3 (L8918) and β-actin (#A5316) from Sigma-Aldrich (St. Louis, MO, USA).
Histological and Masson’s trichrome analysis. Left lungs were fixed in 4% neutral buffered paraformaldehyde, pH 7.4, for 24 hours at room temperature, dehydrated in a series of ethanol and subsequently xylol and embedded in paraffin. Sections of the left lungs were cut at a thickness of 4 μm, rehydrated and stained with H&E (hematoxylin and eosin) and Masson’s Trichrome. Each successive field was individually assessed for the severity of interstitial fibrosis in a blinded method by two pathologists using the Ashcroft scoring system. All slides were evaluated histopathologically applying a semiquantitative grading: 1=minimal, 2=slight, 3= marked, 4=severe, 5= massive.
Immunohistochemistry. Immunostaining was done on formalin-fixed, paraffin-embedded mice lung tissue specimens, and 4 mm thick paraffin sections were cut. These sections were incubated overnight with anti-SIRT6 (1:200, PA5-13225, Sigma, MO, USA) and anti-p-S6 antibody (1:1000, #5364, CST, MA, USA) at a 1: 200 dilution in 5% FBS overnight and then incubated with the horseradish peroxidase-conjugated secondary antibody for 30 min at room temperature. The results were visualized by reaction with diaminobenzidine (DAB, 3,3′-diaminobenzidine tetrahydrochloride) and counterstaining with hematoxylin.
Cell Proliferation Assay. MRC5 was plated at a density of 1.0 ×103 cells/well in 96-well plates. The cells were cultured in the corresponding serum-free medium for 24 h. Cell viability was examined using10 mL/well CCK-8 solution (Dojindo, Kumamoto, Japan). After incubating for 2 h, the absorbance was measured at 450 nm.
Quantitative Real-Time PCR (qRT-PCR). Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. Then, reverse transcription was performed to get the first strand cDNA by using the PrimeScript® RT reagent kit (TaKaRa, Dalian, China). The expression level of a-SMA, CTGF were determined by qPCR reactions and were performed by using the ABI 7500 Fast system (Applied Biosystems, CA) with SYBR green (TaKaRa). The 2−ΔΔCt method was used for quantification. All reactions were triplicated. The relative expression of Collagen1, 3, Fibronectin, MMP3 were respectively normalized to GAPDH.
Statistical Analysis. Results are presented as means ± SEM. Significance of the differences between means was assessed using one-way analysis of variance or a two-tailed Student’s t-test. Values of P less than 0.05 were considered significant.