Animals and cells

Specific-pathogen free -grade male DBA/1 mice (30 mice, 6–8 weeks old, body weight 24 ± 2 g) were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. (Quality Certificate No. 202243188, Laboratory Animal License No. SCXK (Su) 2021–0013) and were adaptively fed for 1 week. Human embryonic lung fibroblasts (HLFs,#CL-0106) were kindly provided by Procell Life Science & Technology Co., Ltd.

Drugs and reagents

Bovine type II collagen was purchased from Chondrex (#20,022). Freund's complete adjuvant (#F5881) and Freund's incomplete adjuvant (#F5506) were purchased from Sigma. Baricitinib (LY3009104) was purchased from Adooq Bioscience. The following antibodies were used: mouse antibodies: β-actin (HuaBio,#EM21002); TGF-βR2 (Proteintech,#66,636–1-Ig); rabbit antibodies were purchased from affinity: SMA(#14,395–1-AP),Jak2, collagen 4 (Col4, #AF0510),fibronectin(FN,#AF5335),p-Jak2(#AF3002),Stat3(#10,253–2-AP), p-Stat3(#AF3293), Smad3(#AF6362), and p-Smad3(#AF8315); goat anti-mouse IgG antibody (EarthOx, #E030110-01) and goat anti-rabbit IgG antibody (EarthOx, #E030120-01); and transforming growth factor-β1 (Peprotech). si-Jak2 (#stB0004834B, refseq GCCGAGTTGTTACTATCCA) was purchased from Ribo. A Jak2 overexpression lentiviral vector was purchased from Obio Technology (H27516,pcSLenti-EF1-EGFP-P2A-Puro-CMV-JAK2-3Xflag-WPRE). Upstream and downstream primers were purchased from Sangon Biotech (Table 1 for the primer sequences). Reverse transcription kits were purchased from Takara(#RR036A). Wound healing plug-ins were purchased from Ibidi(#80,209).

Main equipment

The following instruments were used: carbon dioxide incubator (ESCO Singapore CCL-0508–8-IVF), microplate reader (Shenzhen Huakerui HR801), real-time fluorescence quantitative PCR instrument (Bioer LineGene 9600Plus), chemiluminescence imaging system (Clinx, Shanghai, Chemi Scope e6000EXP), high-speed refrigerated centrifuge (Zonkia, Anhui, HC-3016R), nanospectrophotometer (Kairo, Beijing, K5500PLUS), and integrated ultrasonic cell disruptor (Xiao Mei Chao Sheng, Kunshan).

Experimental groupsCell experiments

HLFs at passages 3–5 were obtained and divided into the following groups for the cell scratch experiment: (1) control group, normal HLFs; (2) model group, HLFs + TGFβ1 (10 ng/ml, best concentration determined in previous experiment); and (3) intervention group, HLFs + TGFβ1 (10 ng/ml) + baricitinib (2000 nmol/L). Photographs were taken after 48 h of culture.

For downregulation of the Jak2 gene, HLFs were divided into the following groups and transfected with Jak2 siRNA: (1) the control group, in which HLFs were cultured normally; (2) the siNC group, in which HLFs were transfected with the siRNA non-specific control (siNC) and treated with TGFβ1 (10 ng/ml); and (3) the si-Jak2 group, in which HLFs were transfected with siRNA and treated with TGFβ1 (10 ng/ml).

For overexpression of the Jak2 gene, HLFs were divided into the following groups and transfected with lentiviral vectors: (1) the empty virus group, in which HLFs were transfected with empty virus (multiplicity of infection (MOI) = 80) and treated with TGFβ1 (10 ng/ml) and (2) the lentiviral vector group, in which HLFs were transfected with Jak2-expressing lentiviral vector (MOI = 100) and treated with TGFβ1 (10 ng/ml).

Animal experiments

Nine mice in the control group were received normal saline by gavage. There were 21 mice in the model group. These mice received injections of 0.1 ml Freund's adjuvant complete (FAC) emulsified bovine collagen type II (bCII) in the base of the tail on day 0 and were boosted with 0.1 ml Freund's incomplete adjuvant (FIA) emulsified bCII on day 21. The severity of arthritis was assessed three days after the second immunization using a scoring system ranging from 0 to 4 points [16], with a maximum total score of 4 points per mouse, and the assessment was performed once every 3 days. The mice in the model group were equally divided into two groups with similar arthritis scores, one of which served as the intervention group; the mice in this group received baricitinib suspended in 0.5% methylcellulose (3 mg/kg 0.1 ml per mouse) by gavage. Mice in the control group and the model group received 0.1 ml of normal saline every other day by gavage. One mouse was killed to confirm successful modeling.

Histological examination

Lung tissue was fixed in 4% paraformaldehyde for 24 h, dehydrated with gradient alcohol, embedded in paraffin, and sliced into 4-µm sections; the sections were subjected to conventional H&E staining, and alveolar and inflammatory cell infiltration were observed under a light microscope. Lung tissue inflammation was scored using the Szapiel score [17]. Masson staining was used to observe alveolar morphology and collagen deposition under a microscope. Pulmonary fibrosis was scored using the Modified scale [18].

Immunohistofluorescence staining

Lung tissue sections were obtained and blocked with serum, incubated with anti-p-Jak2 antibody (1:50) at 4 °C overnight in the dark, and incubated with fluorescent secondary antibody (1:100, FITC, labeled, green) at 37 °C for 1 h in a humidified box. DAPI was added dropwise, and the sections were incubated in the dark for 5 min. The specimens were stained and mounted with anti-fluorescence quenching reagent. Images were obtained under a fluorescence microscope.

Real-time quantitative PCR (RT‒qPCR)

RNA extraction RNA was extracted from lung tissue following the kit instructions, air dried for 5–10 min, dissolved in 20 µl of enzyme-free water, and stored in a − 80 ℃ freezer for future use.

Reverse transcription of RNA to cDNA RT reaction solution was prepared following the reverse transcription kit instructions. The total volume of the reaction system was 10 μl, and the reaction conditions were 37 °C for 15 min, 85 °C for 5 s, and 4 °C for 59 min. RT‒qPCR:The total volume of the PCR system was 20 μl: upstream and downstream primers, 0.4 μl each; cDNA, 2 μl; enzyme-free water, 8.2 μl; and SYBR Green, 10 µl. A dissolution curve was created, and the data were analyzed using the 2-ΔΔCt method.

Western blotting analysis

Total protein was extracted from adherent cell monolayers and lung tissue. The protein concentration was determined using the BCA method. After equivalent amounts of protein were denatured, the protein was electrophoresed and then transferred to a PVDF membrane. The PVDF membrane was soaked in blocking solution and placed on a shaking table at room temperature for 30 min. The membrane was incubated with primary antibodies (β-actin, TGF-βR2, SMA, Jak2, p-Jak2, Col4, FN, Stat3, p-Stat3, Smad3, p-Smad3) overnight at 4 °C, after which excess primary antibodies were washed off. Then, the membrane was incubated with goat anti-rabbit secondary antibody or goat anti-mouse secondary antibody at 37 °C for 1 h on a shaker. The secondary antibodies were washed off, and the protein bands were visualized. The gray values of the protein bands were analyzed using ImageJ. β-Actin was used as an internal reference to calculate the relative protein expression levels of SMA, TGF-βR2, Jak2, p-Jak2, Col4, FN, Stat3, p-Stat3, Smad3, and p-Smad3.

Statistical analysis

GraphPad Prism 9.0 software was used for the data analysis and to create graphs. The data are expressed as the mean ± standard error. One-way analysis of variance (one-way ANOVA) was used to compare multiple groups, and the t test was used for pairwise comparisons. P < 0.05 was regarded as a statistically significant difference.