Materials and animals
SB203580 (p38 MAPK inhibitor, 22,898), SP600125 (JNK1 inhibitor, 13,701), and U0126 (ERK1/2 inhibitor, 19,826) were bought from MedChem Express (NJ, USA). P38 MAPK Rabbit monoclonal antibody(1:1000, 8690), Phospho-p38 MAPK (p-p38 MAPK) Rabbit monoclonal antibody(1:1000, 4511), ERK1/2 MAPK Rabbit monoclonal antibody(1:1000, 4695), Phospho-ERK1/2 (p-ERK1/2) MAPK Rabbit monoclonal antibody(1:2000, 4370), JNK1 Rabbit monoclonal antibody(1:1000, 9252), and Phospho-JNK1 (p-JNK1) Rabbit monoclonal antibody(1:1000, 4668) were bought from Cell Signaling Technology. Rabbit IL-6 Polyclonal Antibody was bought from Bioss (1:500, bs-0782R, Beijing, China), Rabbit TNF-α antibody was bought from Abcam (1:1000, ab205587, USA). Peroxidase-conjugated goat anti-rabbit IgG(H + L) was purchased from Bioss (1:5000, bs-40295G, Beijing, China). Sixty male Sprague-Dawley (SD) rats (3–4 weeks, 80-100 g) were bought from Shanghai SLAC Laboratory Animal Co. Ltd. (License No. SCXK (HU)2017–0005). All rats were raised in a barrier environment of light/darkness (12/12 h alternations) at 22 °C ~ 24 °C, and were provided with adequate food and water. All experimental protocols are consistent with Institute of Laboratory Animal Resources, National Academy Press, Washington, DC1996.
Experimental group
Sixty male SD rats were randomly divided into the sham group (including S1-S3 groups, n = 6) and the PH-LHD group (including M1-M7 group, n = 6) according to different inhibitors and mechanical tension. S1), S3): Mechanical tension: 0 g, 60 min. S2): Mechanical tension: 2.0 g, 60 min. M1), M7): Mechanical tension: 0 g, 60 min.: M2): Mechanical tension: 2.0 g, 60 min. M3): Mechanical tension: 2.0 g, 60 min, SB203580 (p38 MAPK inhibitor) pretreatment for 60 min. M4): Mechanical tension: 2.0 g, 60 min, SP600125 (JNK1 inhibitor) pretreatment for 60 min. M5): Mechanical tension: 2.0 g, 60 min, U0126 (ERK1/2 inhibitor) pretreatment for 60 min. M6): Mechanical tension: 2.0 g, 60 min, Streptomycin (SAC inhibitor) pretreatment for 60 min. SB203580, SP600125, U0126 and Streptomycin were dissolved in dimethyl sulfoxide (DMSO, HY-10999 MedChem Express, USA), and diluted with K-H solution to 200 μmol/L, 200 μmol/L, 200 μmol/L, and 1000 μmol/L at 37 °C, respectivel y[8].
PH-LHD model establishment and echocardiography
PH-LHD model was established by banding procedure described by Siegfried B[9]. Three to four weeks old rat’s anesthesia was performed by intraperitoneal injection of 1.5% pentobarbital sodium (50 ml/kg) and placed on a ventilator. The thorax was opened at the second intercostal space on the left side of the sternum to expose the ascending aorta. A metal clip with an inner diameter of 0.8 mm was placed on the ascending aorta to cause the constriction of the ascending aorta. The sham group received a similar operation to expose the ascending aorta without clamped.
On day 25, the ultrasonic diagnostic instrument (GE VIVID-7 DIMENSION) equipped with a 10S probe (11.5 MHz, 200 cm/s, depth 2.5 cm) was used. After anesthesia, the probe was placed on the left sternum. The long axis section of the left ventricle point of the probe was placed at about 11 o ‘clock. Interventrieular septum dimension (IVSd), left ventricular posterior wall thickness in diastole (LVPWd), and left ventricular internal end diastolic dimension (LVIDd), ejection fraction (EF), heart rate (HR) and other data were measured. All data were averaged over 3 consecutive cardiac cycles.
Vascular mechanical stretching
On day 25, after rats were sacrificed, the heart was removed, then exposed the pulmonary vein by puncture trocar as Fig. 1 showed. The pulmonary veins, pulmonary artery and lung tissues of the rats in S1-S2, M1-M6 groups were rapidly separated and placed in a K-H solution at 4 °C. Peripheral tissues of pulmonary veins were removed under microscope, and pulmonary veins were cut into 4 mm vascular rings. Then, pulmonary vein ring was hanged on the stainless-steel hook for mechanical stretching by a biofunction experiment system (620 M, Danish Myo Technology A/S, Denmark), and data were collected by the biological system (BL-420S, Chengdu Techman Software, China). During the experiment, the K-H solution was changed every 15 min, and a continuous mixture of 95% O2 and 5% CO2 was provided. Then a mechanical tension of 2.0 g was applied for 60 min, while in the S1 and M1 groups no mechanical tension was applied and inhibitors were added in the M3-M6 group. Finally, the above treated pulmonary vein rings were collected and stored in a − 80°C freezer. Rats in S3 and M7 were kept alive until 35 days. On day 35, The pulmonary veins, pulmonary artery and lung tissues in S3 and M7 groups were collected as above method without mechanical stretching.
ELISA
On day 25, serum was collected and stored at − 80 °C. The serum adds biotinylated antibodies, horseradish peroxidase-labeled enzyme, then TMB coloration to detect the expression of IL-6 and TNF-α in serum. All experiments were repeated three times.
Hematoxylin eosin (HE) staining
On day 25, the lung tissue was fixed with 4% paraformaldehyde solution, dehydrated with ethanol, transparent with xylene and paraffin-embedded. Slices with a thickness of 4 m were cut, then stained with hematoxylin 5 min, differentiated with hydrochloric acid 30s, and stained with eosin 3 min. Pulmonary artery medium thickening, vascular myogenesis and an increase number of new vessels of lung tissues from model group and sham group were observed under microscope (CKX41, Olympus, Japan).
Immunohistochemical analysis (IHC)
On day 25, lung tissues and pulmonary veins slices were placed in an oven at 60 °C for 2 h. The slices were successively placed in xylene for 15 min, and then put slices in 100% alcohol for 5 min, 85% alcohol for 5 min, and 75% alcohol for 5 min. After 15 min of citric acid antigen repair, the slices were incubated in 3% hydrogen peroxide for 10 min, and in goat serum for 30 min. Subsequently, the slices were incubated in IL-6 or TNF-α antibody for 8 h at 4 °C, then the slices were incubated in the secondary antibody for 50 min. Finally, the slices were stained, dehydrated, transparentized. The expression of IL-6 and TNF-α in pulmonary veins, pulmonary artery and lung tissue were observed under a microscope.
Western blotting
RIPA Lysis Buffer (10 mL/mg) and protease inhibitor was added into the lung tissue and pulmonary vein, and then grinded lung tissue and pulmonary vein by tissue grinder (70 Hz, 1 min, Servivebio, China). The tissue homogenate was placed in a centrifuge for 15 min (4 °C, 15000 Rpm). And the supernatant was taken to detect protein concentration by BCA method. Add the heated denatured protein to SDS-PAGE, after protein electrophoresis (80 V) for about 1 h and electrotransfer (300 mA) for 45 min, PVDF membrane was sealed in 7% skim milk at room temperature for 2 h. After that, the PVDF membrane was incubated in primary antibody at 4 °C for 8 h. Then, the PVDF membrane was incubated in secondary antibody at room temperature for 1 h. Subsequently, ECL luminescence drops were placed on the washed PVDF film, and put it into the chemiluminescence instrument (ChemiDoc™ Touch system, Bio-Rad, USA). The gray value was analyzed by “Quantity One”. GAPDH was used as the internal control.
Statistical analysis
The experimental results were expressed as Mean ± Standard Error of Mean. T-test was used for comparison between two groups with homogeneity of variance, one-way ANOVA was used for comparison between multiple groups. P < 0.05 was considered as statistically significant. SPSS 25.0 was used for data analysis, and Graphpad Prism 8.0 was used for data plotting.
