Female BALB/c mice 8 to 10 weeks old were used in this experiment. All mice were purchased from OrientBio (Sungnam, Korea). They were fed standard mouse chow (Ralston Purina, St. Louis, MO, USA) and water ad libitum. All experimental procedures were reviewed and approved by the Animal Research Ethics Committee of the Catholic University of Korea (Seoul, Korea).
BLM and metformin treatment
The mice were anesthetized with isoflurane, and then their backs were shaved. They were given daily subcutaneous injections of BLM for 4 weeks (Dong-A Pharm, Seoul, Korea). BLM was sterilized by filtration before injection under the shaved back skin at a dose of 1 mg/mL in PBS. Metformin treatment commenced 2 weeks after the first injection of BLM. Mice (n = 5 or 6 per group) were administered 100 mg/kg metformin orally every day for 2 weeks and then sacrificed the day after the final treatment. Back skin and lung tissue were acquired and fixed in 10% formalin solution for histological analysis. Spleen tissue was preserved for cryosection.
Harvested tissue was fixed in 10% formalin and embedded in paraffin. Sections (6 µm thick) were stained with H&E and Masson’s trichrome (MT). MT staining was conducted using ready–to-use kit (Trichrome Stain (Masson) Kit, HT15, Sigma-Aldrich). Briefly, the tissue was sliced and placed on standard microscopy slides. After deparaffinisation and rehydration, the slides were immersed in Bouin’s solution (HT 10132, Sigma-Aldrich) at 56°C for 15 minutes. Subsequently, the slides were washed with tap water for 5 minutes. Next, the tissues were stained in Weigert’s hematoxylin for 5 minutes, and then washed again with tap water for 5 minutes. Then, the slides were stained in Biebrich scarlet-acid fuchsin for 5 minutes, rinsed in distilled water, incubated in phosphotungstic-phosphomolybdic acid for 5 minutes, dyed with aniline blue for 5 minutes, and fixed in 1% acetic acid for 2 minutes. Finally, the slides were washed in distilled water, dehydrated and mounted. Dermal thickness was measured as described previously . Lung sections were analyzed, and the severity of fibrosis was scored as described previously . For the quantification of collagen, mouse skin and lung tissue were hydrolyzed and analyzed with hydroxyproline assays.
Immunohistochemistry was performed with VECTASTAIN ABC kits (Vector Laboratories, Burlingame, CA, USA). Tissue was incubated with primary antibodies against IL-6, IL-17, TGF-β, STAT3, phosphorylated mTOR (p-mTOR), phosphorylated AMPK (p-AMPK), and α-smooth muscle actin (α-SMA) overnight at 4°C. The primary antibody was detected with a biotinylated secondary linking antibody, followed by incubation with a streptavidin-peroxidase complex for 1 h. The final color was produced with DAB chromogen (Dako, Carpinteria, CA, USA). Positive cells, which were identified by a dark brown deposit in the nucleus, were enumerated in 10 randomly selected high-power fields (HPFs; 400×; 2.37 mm2). The sections were counterstained with hematoxylin and photographed with an Olympus photomicroscope (Tokyo, Japan).
Spleen tissue sections 7 µm in thickness were used for immunostaining. To analyze populations of Th17 cells, we used fluorescein isothiocyanate (FITC)-conjugated anti-CD4 and PE-conjugated anti-IL-17 antibodies (eBioscience, San Diego, CA, USA). Stained sections were observed with a Zeiss microscope (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany) at ×400 magnification. Positive cells were counted, and values were expressed as means ± standard deviations.
mRNA was extracted with TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer’s instructions. Complementary DNA was synthesized with a SuperScript Reverse Transcription System (Takara Bio, Kyoto, Japan). A Light-Cycler 2.0 (software version 4.0; Roche Diagnostics, Mannheim, Germany) was used for PCR amplification. All reactions were performed with LightCycler FastStart DNA Master SYBR Green I Mix (Takara) following the manufacturer’s instructions. The following primers were used to amplify mouse genes: for IL-1β, 5′-GGA TGA GGA CAT GAG CAC ATT C-3′ (sense) and 5′-GGA AGA CAG GCT TGT GCT CTG A-3′ (antisense); for IL-6, 5′-ATG CTC CCT GAA TGA TCA CC-3′ (sense) and 5′-TTC TTT GCA AAC AGC ACA GC-3′ (antisense); for IL-17, 5′-CCT-CAA-AGC-TCA-GCG-TGT-CC-3′ (sense) and 5′-GAG-CTC-ACT-TTT-GCG-CCA-AG-3′ (antisense); for TNF-α, 5′-ATG AGC ACA GAA AGC ATG ATC-3′ (sense) and 5′-TAC AGG CTT GTC ACT CGA ATT-3′ (antisense); for TGF-β, 5′-GCC TGA GTG GCT GTC TTT TGA-3′ (sense) and 5′-CAC AAG AGC AGT GAG CGC TGA A-3′ (antisense); for Col1a, 5′-ATG GGA GGA GAG CGT GTG-3′ (sense) and 5′-GAG GTC GGA GAG CAG AGG-3′ (antisense); for β-actin, 5′-GTA CGA CCA GAG GCA TAC AGG-3′ (sense) and 5′-GAT GAC GAT ATC GCT GCG CTG-3′ (antisense). All mRNA levels were normalized to that of β-actin.
Murine splenocytes isolation and stimulation
Splenocytes were acquired from spleen tissue of BALB/c mice and sieved through a mesh screen. Then red blood cells were lysed in hypotonic ACK buffer. The remaining splenocytes were maintained in RPMI 1640 medium containing 5% fetal bovine serum. Splenocytes were stimulated with plate-bound anti-CD3 (0.5 µg/mL; BD Pharmingen) for 3 days. Metformin was obtained from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in RPMI 1640 medium including 5% fetal bovine serum. Metformin was administered during the same 3-day period as the anti-CD3 stimulation.
Skin fibroblasts from a healthy donor were cultured with 10ng/ml of IL-6 and metformin for 1 h. Proteins were isolated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Western blotting was performed with a SNAP i.d. Protein Detection System (Millipore, Billerica, MA, USA). Membranes were stained with primary antibodies against p-mTOR, phosphorylated STAT3 (p-STAT3; 727), and β-actin (all from Cell Signaling Technology, Danvers, MA, USA).
Data are presented as means ± SDs of at least three independent experiments or at least three independent samples, with five mice in each group. In vitro experiments were independently repeated three or more times, and each experiment had at least three samples. One-way analysis of variance followed by Bonferroni’s post hoc test was used to compare differences among three or more groups. The Mann–Whitney U test was used to compare numerical data between groups. To assess the Gaussian distribution and equality of variance, we used the Shapiro–Wilk test and Levene’s test, respectively. P < 0.05 was considered statistically significant. Statistical analyses were performed with IBM SPSS Statistics 20 for Windows (IBM, Armonk, NY, USA).