Hy 12031

BEAS-2B cells were seeded at a density of 2 × 10⁴ cells/cm² in a chamber precoated with 5 μg/mL rat tail type I collagen (Hangzhou Shengyou Biotechnology Co., Ltd., Hangzhou, China); they were cultured for about 1 day and installed on the cell tensile loading device (Chongqing Deyansheng Technology Co., Ltd., Chongqing, China) (Figure 1A). The desired stretching frequency was selected on the human–computer interaction display screen, and the rotation of the servo motor was controlled by the controller and power box to achieve stretching changes (Figure 1B). In this study, we exposed the cells in a silicone resin chamber to a tensile treatment at 2 Hz and 20% strain. For the stretching experiments with the actin stabilizer Jasplakinolide (Jasp, 20 nM, Abcam, ab141409), the c-Jun N-terminal kinase (JNK) inhibitor SP600125 (20 μM, Sellcek, S1460), and the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor U0126 (10 μM, HY-12031, MedChemExpress), stretching was induced for 12 h in the medium with reagents added. As a control, static cells were cultured in a small chamber under the same conditions without any stretching.

Dental follicle tissue was microdissected from the peripheral region of the tooth germ of an E60‐miniature pig embryo with a dissecting microscope. The tissue was digested with dispase II (4 mg/ml) and collagenase type I (3 mg/ml) at 37°C for 1 h. The cell slurry was filtered and centrifuged at 300 g for 5 min to obtain the cells. Single‐cell suspensions were seeded onto culture dishes and cultured with MEM‐α supplemented with 15% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. The cells were incubated at 37°C and 5% CO₂.
To inhibit ERK1/2, the chemical U0126 (70 nM; HY‐12031; MedChem Express, Monmouth Junction, NJ) was added to the medium; the same concentration of DMSO was added to the control group medium. To block integrin β1, a mouse monoclonal antibody against integrin β1 (5 μg/ml; ab30388; Abcam, Cambridge, UK) was added to the medium; the same amount of PBS (10 μl) was added to the control group medium. To inhibit Wnt signaling, IWR‐1‐endo (5 μM; 10161; Sigma‐Aldrich) was added to the medium; the same amount of PBS was added to the control group medium. For all groups, the treatment lasted 2 h before cells were harvested.

A total of 48 SD rats were randomly divided into the sham group (n = 12) and the PH-LHD group (n = 36). Corresponding to different inhibitors and mechanical strength used to treat pulmonary venous rings, the sham group was further divided into two subgroups: Sham 1 (S1, mechanical strength: 0 g) and Sham 2 (S2, mechanical strength: 2.0 g); and the PH-LHD group was also further divided into 2 subgroups: Model 1 (M1, banding+0 g), Model 2 (M2, banding+2.0 g). In addition, a broad spectrum inhibitor of MEK1 and MEK2 (U0126, HY-12031 MedChem Express, USA), p38 MAPK inhibitor (SB203580, HY-10256 MedChem Express, USA), SAC inhibitor (Streptomycin, HY-B0472 MedChem Express, USA) and broad-spectrum inhibitor of JNK1, JNK2, and JNK3 (SP600125, HY-12041 MedChem Express, USA) were dissolved in dimethyl sulfoxide (DMSO, HY-10999 MedChem Express, USA), and diluted with K-H solution to 250 μmol/L, 200 μmol/L, 1000 μmol/L, and 250 μmol/L, respectively [12 (link)]. Specifically, the pulmonary veins were immersed in the K-H solution containing the corresponding inhibitors (SB203580, SP600125, U0126 or Streptomycin) at 37°C under the condition of continuous oxygen infusion for 60 mins, and then was stretched with 2.0 g mechanical strength for 60 mins.