Inhibitory effect of rosmarinic acid on high glucose-induced angiogenesis of human retinal microvascular endothelial cells and the activation of NLRP3 inflammasome pathway

Objective

To investigate the inhibitory effects of rosmarinic acid (RA) on high glucose-induced angiogenesis of human retinal microvascular endothelial cells (HRMEC) and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome pathway-related proteins.

Methods

The HRMEC were divided into control group, high glucose group, high glucose+ low concentration RA group, high glucose+ medium concentration RA group, and high glucose+ high concentration RA group, and were cultured in vitro with conventional medium, 30 mmol/L D-glucose medium, 30 mmol/L D-glucose+ 25 μmol/L RA medium, 30 mmol/L D-glucose+ 50 μmol/L RA medium and 30 mmol/L D-glucose+ 100 μmol/L RA medium accordingly.The 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to detect the cell proliferation.Transwell assay was performed to detect the cell migration.Matrigel assay was employed to determine the tube formation ability of cells.Western blot was utilized to detect the expression levels of NLRP3, apoptosis-associated speck like protein (ASC) and cysteinyl aspartate-specific protease-1 (Caspase-1). Enzyme-linked immunosorbent assay (ELISA) kit was used to detect the concentrations of interleukin (IL)-1β and IL-18 in supernatant of cell culture.

Results

The cell proliferation rate, the number of migrated cells and the number of formed tubes were (100.00±0.92)%, 37.67±9.02 and 45.00±4.58 in the control group, (163.56±1.46)%, 117.33±7.23 and 95.00±9.54 in the high glucose group, (152.29±2.90)%, 78.67±4.04 and 84.67±1.53 in the high glucose+ low concentration RA group, (147.72±2.22)%, 65.33±4.16 and 71.00±3.61 in the high glucose+ medium concentration RA group, (132.47±0.74)%, 52.67±6.81 and 60.00±1.00 in the high glucose+ high concentration RA group, respectively.There were statistically significant differences in cell proliferation rate, the number of migrated cells and formed tubes among all groups (F=537.07, 64.63, 45.58; all at P<0.001). Compared with the control group, the cell proliferation rate, the number of migrated cells and formed tubes were significantly increased in the high glucose group, high glucose+ low concentration RA group, high glucose+ medium concentration RA group and high glucose+ high concentration RA group, showing statistical significances (all at P<0.05). Compared with the high glucose group, the cell proliferation rate, the number of migrated cells and formed tubes were significantly decreased in the different concentrations RA groups (all at P<0.05). With the increase of RA concentration, the cell proliferation rate, the number of migrated cells and formed tubes were decreased, and there were statistical differences among high glucose+ low/medium/high concentrations RA groups (all at P<0.05). There were significantly differences in the relative expression levels of NLRP3, ASC and Caspase-1 proteins in cells and the concentrations of IL-1β and IL-18 in cell culture supernatant among all the five groups (F=145.12, 422.82, 463.79, 2 019.96, 33 406.97; all at P<0.001). Compared with the control group, the relative expression levels of NLRP3, ASC and Caspase-1 proteins as well as the concentrations of IL-1β and IL-18 in cell culture supernatant were significantly increased in the high glucose group, high glucose+ low concentration RA group, high glucose+ medium concentration RA group and high glucose+ high concentration RA group (all at P<0.05). Compared with the high glucose group, the expression levels of NLRP3, ASC and Caspase-1 proteins as well as the concentrations of IL-1β and IL-18 were decreased in the different concentrations RA groups, and the differences were statistically significant (all at P<0.05). With the increase of RA concentration, the expression levels of NLRP3, ASC and Caspase-1 proteins as well as the concentrations of IL-1β and IL-18 were decreased, and there were statistically significant differences among high glucose+ low/medium/high concentrations RA groups (all at P<0.05).

Conclusions

RA can inhibit proliferation, migration and tube formation of HRMEC induced by high glucose, and inhibit high glucose-induced activation of NLRP3 inflammasome signaling pathway.

NLRP3 inflammasome; Rosmarinic acid; High glucose; Human retinal microvascular endothelial cells; Angiogenesis