Ca1210

A standard rat primary liver sinusoidal endothelial cells (LSECs) were used for in vitro study (RA-6017, Cell Biologics, Chicago, USA). The LSECs were cultured in complete rat endothelial cell medium (M1266, Cell Biologics, Chicago, USA). The LSECs at passage 4 to 6 were used for following experiments. Before that, the fecal supernatants from HSOS rats (HSOS-FSN) and normal rats (Norm-FSN) were prepared. In brief, feces were dissolved and homogenized in PBS solution (1 g/5 ml), and then the supernatants were collected after centrifugation (5000 rpm, 4 °C, 10 min) filtered through a 0.22 μm-sized filter, and stored at − 80 °C. The LSECs were seeded in standard 12-well plates and incubated with 200 μl HSOS-FSN or 200 μl Norm-FSN in 2 ml culture medium for 48 h, before which the LSECs were pretreated with 200 μM Ficz (MedChemExpress, China) for 24 h, while PBS was conducted as vehicle control. At the end of intervention, cell viability was measured via a commercial cell counting kit-8 (CA1210, Solarbio, Beijing, China), and was normalized as the percentage of control. Finally, the LSECs were collected and used for mRNA and protein quantification via Real-time PCR and western blot analysis.

PASMC in good growth condition were taken, washed three times with sterile PBS, and the cells were incubated in a culture incubator (P6730, Solarbio) for 2 min after the addition of appropriate amount of trypsin, PASMC were taken out, and the digestion was terminated by the addition of DMEM complete medium, centrifugation was carried out at 1200 rpm for 3 min, the cells were resuspended by adding the appropriate amount of medium, and the cells were cultured at 3000 cells per well. Cells were added to 96-well plates, incubated overnight, and different drugs were added to pretreat the cells for 24 h. Pulmonary arterial hypertension was modeled as previously described, and 10 μL of CCK8 (CA1210, Solarbio) solution was added to each well after 24 h of incubation in an incubator, and the absorbance at 450 nm was measured by an enzyme marker (Flash, ReadMax 1200) after 3 h of incubation.

Measurement of cell growth was done using the CCK8 kit based on the directions provided by the manufacturer (Solarbio, #CA1210). A 5,000-cell density was used in 96-well plates, and loperamide was applied for 8 h to HT22 cells. The cells were then given 10 L of CCK8 to each well, and they were left to react for 4 h. On a plate reader (Heales, #MB-580), the optical densities of the medium in the wells were determined at 450 nm.