To investigate the effect of Notch signaling pathway on the intercellular adhesion molecule (ICAM)-1 of pulmonary vascular endothelial cell (PMVEC) induced by serum in burned rats.

Among the 42 SD rats (6-8 weeks), 30 were randomly selected and divided into sham injury group(n=15)and burn group(n=15) according to the random number table method. Rats in burn group were immeresed in hot water at 95 ℃ for 18 s, inflicted with 30% total body surface area full-thickness burn on back, and the rats in the sham injury group were immersed in a 37 ℃ water bath for 18 s to simulate injury. At 6, 12, 24, 48, 72 h after injury, 3 rats were randomly selected from the burn group, and blood was collected from the abdominal aorta. Enzyme-linked immunoadsordent assay(ELISA) were used to detected the content of ICAM-1 in rats’ serum. Rats in the sham injury group were subjected to the same test. Six rats of the remaining 12 rats (6-8 weeks) were taken, and 30% total body surface area full-thickness burn was made according to the aforementioned method, and the serum of the burned rats was prepared at 24 h after the injury; the remaining 6 rats were not treated, and the serum of healthy rats was prepared. Marginal pulmonary tissue was harvested from 3 days old 10 SD rats, and the rat PMVEC were culturned with tissue block method. The morphological characteristics of primary cells cultured for 2 and 6 d were observed under an inverted phase contrast microscope, and the cells in primary cultured for 7 d were identified by flow cytometry. The PMVEC cells of the 4th generation at logarithmic growth stage were taken for experiment. The cells were inoculated on 6-well plates, when the cells grew to 80% confluence, they were divided into 3 groups according to the random number table method: the control group, the dimethyl sulfoxide (DMSO)+ burn serum group and the gamma-secretase inhibitor (GSI)+ burn serum group, with 3 wells in each group. Cells in control group were cultured with 10% healthy rat serum, the latter 2 groups added with 3 μL/mL DMSO and 75 μmol/L GSI respectively, and after 24 h of culture, 10% burn rat serum were added to stimulate the culture for 24 h. The content of ICAM-1 in PMVEC supernatant was determined by ELISA, the content of ICAM-1 in PMVEC of each group was detected by flow cytometry, and the protein expression of ICAM-1 was detected by Western blotting, and the relative protein expression was calculated. The cell adhesion ability of PMVEC was detected. Data were processed with one-way ANOVA and LSD-t test.

(1) The serum contents of ICAM-1 in rats of burn group were (19.77±3.03), (22.09±3.65), (22.44±4.04), (25.40±2.51), (26.37±3.07) pg/mL at 6, 12, 24, 48, and 72 h after injury respectively, which were significantly higher than those in the sham injury group [(10.60±1.51), (11.03±1.95), (10.87±0.89), (9.30±0.89), (10.93±1.22)] pg/mL, the differences were statistically significant between the two groups (t=4.699, 4.466, 3.181, 10.490, 8.097; P<0.05). (2) Primary cells grew slowly after 2 d of culture, cells were seen to migrate from the edge of the tissue mass, and cell fusion was observed at 6 d after culture that them showed a cobblestone appearance. Cells in primary cultured for 7 d were identified by flow cytometry, and the results showed that CD31-positive cells accounted for 98.6%, which was determined to be PMVEC. (3) At 24 h after serum stimulation culture, the contents of ICAM-1 in the supernatant of cells in the control group, the DMSO+ burn serum group, and the GSI+ burn serum group were 1.21±0.25, 2.16±0.12 and 3.07 ±0.30, respectively, and the difference was statistically significant among the three groups(F=46.72, P<0.05). Compared with the control group, the content of ICAM-1 in the supernatant of cells in the DMSO+ burn serum group and GSI+ burn serum group was significantly increased, and the differences were statistically significant (t=5.977, 8.274; P<0.05); the content of ICAM-1 in cell supernatant of GSI+ burn serum group was significantly higher than that in the DMSO+ burn serum group, and the difference between the two groups was statistically significant (t=4.847, P<0.05). (4) At 24 h after serum stimulation culture, the mean fluorescence intensity of ICAM-1 in PMVEC of control group, DMSO+ burn serum group and GSI+ burn serum group were 2 582±143, 3 453±204, 4 559±414, respectively, and the difference was statistically significant among the three groups(F=37.84, P<0.05). Compared with the control group, the content of ICAM-1 in cells of DMSO+ burn serum group and GSI+ burn serum group was significantly increased, and the differences were statistically significant (t=6.056, 7.817; P<0.05); the content of ICAM-1 in the GSI+ burn serum group was significantly higher than that in the DMSO+ burn serum group, and the difference was statistically significant between the two groups (t=4.149, P<0.05). (5) At 24 h after serum stimulation culture, the ratios of ICAM-1/GAPDH in the control group, DMSO+ burn serum group, and GSI+ burn serum group were 0.74±0.08, 1.07±0.08, 1.38±0.28, respectively, and the difference was statistically significant among the three groups(F=30.76, P<0.05). The ratios of ICAM-1/GAPDH in the DMSO+ burn serum group and GSI+ burn serum group were significantly higher than that in the control group, and the differences were statistically significant (t=5.182, 6.990; P<0.05). The ICAM-1/GAPDH ratio in the GSI+ burn serum group was significantly higher than that in the DMSO+ burn serum group, and the difference was statistically significant (t=3.770, P<0.05). (6) After PMVEC was incubated with human mononuclear cell line THP-1 cells for 2 h, the number of human mononuclear cell line THP-1 cells adhered to the cells in the control group, DMSO+ burn serum group and GSI+ burn serum group observed under inverted phase contrast fluorescence microscope showed an increasing trend. The number of cells in the three groups were 152.00±21.07, 265.33±36.83, 345.67±30.66, and the difference was statistically significant among the three groups (F=31.09, P<0.05). The number of adherent cells in the DMSO+ burn serum group and GSI+ burn serum group were significantly higher than that in the control group, and the differences were statistically significant (t=4.626, 9.016; P<0.05); the number of adherent cells in the GSI+ burn serum group was significantly higher than that in DMSO+ burn serum group, the difference was statistically significant (t=2.903, P<0.05).

The content of ICAM-1 in the serum of rats after burn is significantly increased. The stimulation of PMVEC in burn rat serum can lead to an increase in the intracellular ICAM-1 content and the secretion of ICAM-1 into the supernatant. The application of GSI to block Notch signaling pathway can increase the production and expression of ICAM-1 and increase the adhesion of PMVEC to human mononuclear cell line THP-1 cells.