[Effects of asiatic acid on angiogenesis and Slit-Robo signaling in retinal microvascular endothelial cells induced by high glucose].

Abstract

Objective: To explore the effects and mechanism of asiatic acid (AA) on high glucose (HG)-induced angiogenesis of human retinal microvascular endothelial cells (hRMECs). Methods: In this experimental study, the MTT assay was used to screen the optimal concentration of AA. The HG-induced hRMEC injury models were established and divided into the control group, HG group, Mannitol group, HG+AA group, HG+AA+740Y-P group, HG+LY294002 group and HG+dimethyl sulfoxide group. Cell viability, migration and tube formation were detected by the MTT assay, scratch test and Matrigel assay, respectively. Real-time PCR and ELISA were used to detect the relative mRNA expression of Slit guidance ligand 2 (Slit2) and roundabout guidance receptor 1 (Robo1), as well as Slit2 secretion. Western blot was used to detect the protein expression of phosphatidylinositol 3-kinase p85 subunit (PI3K p85), phosphorylated PI3K p85 (p-PI3K p85), protein kinase B (AKT), phosphorylated AKT (p-AKT), Slit2 and Robo1. Results: The treatment with 10 μmol/L AA did not affect the viability of normal hRMECs (0.803±0.05) (P=0.662), but significantly inhibited cell viability under HG conditions (1.049±0.12) (P<0.001). Compared with the control group, the HG group showed significant increases in cell viability (1.609±0.14), migration rate (70.90%±5.39%), tube number (61.89±2.17), number of master junctions (96.22±3.86), total tube length [(28 043.78±1 472.75) μm], mRNA and protein expression of Slit2 (4.07±0.31 and 4.76±0.72), Slit2 secretion [(5.889±0.746) ng/ml], mRNA and protein expression of Robo1 (4.46±0.17 and 6.00±0.35), as well as protein expression of p-PI3K p85 (6.61±0.46) and p-AKT (6.02±0.68) (all P<0.001). Compared with the HG+dimethyl sulfoxide group, the HG+AA group exhibited significant decreases in cell viability (1.052±0.14) (P=0.002), migration rate (30.70%±3.97%), tube number (42.22±4.17) (P=0.004), number of master junctions (69.89±1.34) (P<0.001), total tube length [(19 853.11±1 141.85) μm] (P<0.001), mRNA and protein expression of Slit2 (2.02±0.04 and 2.12±0.17), Slit2 secretion [(2.991±0.479) ng/ml] (P<0.001), mRNA and protein expression of Robo1 (1.99±0.13 and 2.86±0.28) (P<0.001), and protein expression of p-PI3K p85 (1.84±0.22) and p-AKT (2.30±0.15) (P<0.001). Compared with the HG+AA group, the HG+AA+740Y-P group showed significant increases in cell viability (1.517±0.14) (P=0.012), migration rate (68.12%±3.70%) (P<0.001), tube number (56.44±4.07) (P=0.009), number of master junctions (93.67±3.85) (P<0.001), total tube length [(27 751.22±1 148.29) μm] (P<0.001), mRNA and protein expression of Slit2 (3.47±0.15 and 4.05±0.25) (P<0.001), Slit2 secretion [(4.763±0.565) ng/ml] (P=0.023), mRNA and protein expression of Robo1 (3.95±0.11 and 5.39±0.46) (P<0.001), and protein expression of p-PI3K p85 (9.66±1.79) and p-AKT (10.42±1.93) (P<0.001). No significant differences were observed among groups in total PI3K and AKT protein levels (P>0.05). Conclusions: AA suppresses the HG-induced Slit-Robo signaling activation, proliferation, migration and angiogenesis in hRMECs, at least in part by inhibiting the PI3K pathway, suggesting its potential as a lead compound for diabetic retinopathy therapy.