Animals

A total of 18 female SPF-grade C57BL/6 mice (8 weeks old) were provided by Weitonglihua Animal (Beijing, China). The mice received housing in a room with adjustable temperature (22–26 °C) and acclimating for seven days, providing full access to food and water. The animal research was approved by the animal ethics committee of the Second Affiliated Hospital of Chongqing Medical University and performed according to our institute’s guidelines. Besides, the manuscript is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).

Establishment of a mouse myocardial fibrosis model and dosing scheme

The mouse myocardial fibrosis model was established by injection of isoproterenol (ISO) [26]. Briefly, mice were randomly allocated to the Control group, the ISO group, and the ISO + Baricitinib group (n = 6 per group). The ISO group and the ISO + Baricitinib group were subcutaneously injected with ISO (Solarbio, China) at 10 mg/kg/d, whereas the Control group received physiological saline via subcutaneous injection. Continuous dosing for seven days induced the mice myocardial fibrosis model, and starting from the 8th day of the experiment, the ISO + Baricitinib group began oral administration of baricitinib (AdooQ, USA) at 5 mg/kg/d. The control group and the ISO group were subjected to 0.5% sodium carboxymethyl cellulose (CMC-Na; MCE, China) via oral administration for 14 consecutive days.

Following the final dosing, the body weight of the mice was detected. The mice were anesthetized by avertin (200 mg/kg) via intraperitoneal injection and euthanatized by cervical dislocation, the protocol was approved by the animal ethics committee of the Second Affiliated Hospital of Chongqing Medical University. The hearts were swiftly removed and rinsed with phosphate-buffered saline (PBS), excessive tissues were eliminated, surface moisture was blotted with filter paper, and the heart weight was determined. The heart of each mouse was bisected horizontally, with the apex section fixed in 10% formaldehyde for subsequent histological analysis, and the remaining portion rapidly frozen and stored at -80 °C for heart homogenization. The heart index was computed, and the disparities among groups were assessed. The formula for calculating the heart index is: Heart index = Heart weight (mg) / Body weight (g).

Histological measurement

The fixed cardiac tissue was dehydrated, embedded, and sliced. The slices were stained by hematoxylin-eosin (H&E; Beyotime, China) and Masson’s trichrome (Beyotime, China). The cell apoptosis was detected using a DAB TUNEL Cell Apoptosis Detection Kit (Servicebio, China). The slices were examined using an optical microscope, and images were recorded. Image J 6.0 software (NIH, USA) was employed to evaluate the cardiomyocyte size and myocardium’s collagen volume fraction (CVF), computed as the collagen fiber area divided by the total area multiplied by 100%. Measurements were taken from 5 fields in each sample.

Immunohistochemistry and immunofluorescence

After being deparaffinized, rehydrated, and subjected to antigen retrieval in a citrate-sodium hydrochloric acid buffer, the slices were incubated with 5% bovine serum albumin (Takara, Japan) to block non-specific protein binding sites. Next, the slices were incubated with primary antibodies against Col1 (1:100; Proteintech, China) and α-SMA (1:100; Proteintech, China), CD4 (1:100; Servicebio, China), CD8 (1:100; Servicebio, China), and CD68 (1:100; BIOSS, China) overnight at 4 °C. The slices were then incubated with a secondary antibody against rabbit IgG (1:500; Proteintech, China). Staining was then carried out using a diaminobenzidine (DAB) reagent kit (Beyotime, China). The expression of Col1 and α-SMA was observed, and images were captured using an optical microscope (Olympus, China). For immunofluorescence, the slices were then incubated with fluorescently labeled secondary antibodies (1:500; Servicebio, China). The expression of Col1 and α-SMA and the number of CD4, CD8, and CD68 positive cells were observed, and images were captured using a microscope (Olympus, China).

Culture and treatment of cardiac fibroblasts

Mouse cardiac fibroblasts (MCFs; Procell, China) were cultured for 24 h in F12 medium (Fuheng, China) supplemented with 10% fetal bovine serum (FBS; Biochannel, China) and 1% penicillin-streptomycin. The culture was maintained in an incubator at 37 °C with 5% CO2. The cells were seeded, and the following treatment groups were established: TGF-β1 + Baricitinib (10 nM), TGF-β1 + Baricitinib (100 nM), TGF-β1 + Baricitinib (1000 nM), and TGF-β1 + Baricitinib (2000 nM). These groups were exposed to baricitinib at 10, 100, 1000, and 2000 nM concentrations and treated with TGF-β1 (10 ng/mL) (R&D Systems, USA) for 48 h. The TGF-β1 group was exposed to equal PBS and treated with TGF-β1 (10 ng/mL) for 48 h. The control group remained untreated. Cells were harvested for western blot assays.

RNA interference

Small interfering RNA (siRNA) was used to knock down JAK2 in MCFs at a concentration of 50 nM (RiboBio, China). For transfection, cells were cultured in the F12 medium without penicillin and streptomycin. Transfection with LipoGene 2000 Star (Sbjbio, China) was performed, followed by a 6-hour incubation in Opti-MEM (Gibco, China). Subsequently, the MCFs were treated with TGF-β1 (10 ng/mL) for 48 h, and cell harvest was conducted for western blot assays.

Quantitative polymerase chain reaction (qPCR)

Total RNA was isolated from frozen cardiac tissue with the Trizol reagent (Takara, Japan) and reverse-transcribed to cDNA via PrimeScriptTM RT Master Mix (Takara, Japan). The qPCR was conducted via SYBR Green Master Mix (Yeason, China) per the supplier’s directions. The 2−ΔΔCT method was used to determine the relative expression, and each sample was normalized to the GAPDH level. Primer sequences are listed in Table 1.

Table 1 The primer sequences of qPCRWestern blot

Total proteins were extracted from frozen cardiac tissue and cultured cells with RIPA lysis buffer and homogenized thoroughly at 4 °C using a low-temperature tissue grinder. Proteins were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride membrane (Sunbio, China). Following a 1-hour blocking step with 5% skim milk (Yili, China), the membranes were incubated with primary antibodies, including Col1 (1:1000), Col3 (1:2000), α-SMA (1:1000), Fn (1:2000), MMP9 (1:500), TIMP1 (1:1000), TGF-βRII (1:2000), GAPDH (1:10000) (Proteintech, China), p-STAT3 (1:1000), STAT3 (1:1000), TGF-β1 (1:1000), p-JAK2 (1:1000), and JAK2 (1:1000) (Affinity, China). The membrane was then incubated for 1 h with corresponding secondary antibodies (1:5000; Proteintech, China). Enhanced chemiluminescence reagent (4 A BIOTECH, China) was used to detect the blots. Band intensities were analyzed using Image J 6.0 software.

Statistical analysis

All experimental data were analyzed with SPSS 25.0 statistical software (IBM, USA). Continuous data were presented as mean ± standard deviation. Graphs were created using Prism 9 GraphPad software. One-way analysis of variance (ANOVA) followed by Tukey’s test was adopted for comparisons among multiple groups. P < 0.05 was regarded as statistically significant.