Cell culture and treatment
The study was permitted by the Law of the People’s Republic of China on
the Protection of Wildlife, and the protocol was approved by the
Institutional Animal Care Committee of Shanghai University of medicine
& Health Sciences, China (Permit Number: JCLW01-08). Cardiac
fibroblasts were isolated from the hearts of 3-day-old neonatal
Sprague-Dawley rats and cultured as described previously. Briefly, the
hearts were removed and washed with cold PBS. The atria and aorta were
discarded. The ventricles were cut open and soaked with 75% ethanol for
30s to inactivate epicardial and endocardial endothelial cells. Then
ventricles was finely minced into small pieces and digested with 0.125%
trypsin and and 0.5 g/L collagenase II (Invitrogen, Carlsbad, CA) for 6
min in a 37 °C in six consecutive steps. Cells were centrifuged and
resuspended in DMEM with 10 % FBS. The cells were seeded at a density
of 1×105 cells/ml and incubated for 60 min. the
Unattached cells were discarded by washing with PBS, and the attached
cells were cultured in DMEM supplemented with 10 % FBS. CFs were
identified by their morphology, positive staining for vimentin, and
negative staining for α-smooth muscle actin (α-SMA) and von Willebrand
factor (vWF). The purity of CFs in this study was more than 97 %. The
cells were divided into control, LPS, serelaxin + LPS, serelaxin +
LPS+GW9962 and LPS+ GW9962 groups. In LPS group, the cells were induced
with LPS (1 μg/ml). In serelaxin and LPS groups, the cells were treated
with LPS(1 μg/ml) and serelaxin (100 ng/ml). GW9662 with 100 nM in each
groups.