Title : Inhibitory effects of novel artemisinin derivative on laser-induced choroidal neovascularization via regulating AKT and ERK1/2 pathways
Abstract:
Choroidal neovascularization (CNV) is a major cause of blindness in ocular fundus disease. The major defects of the current first-line anti-VEGF drugs for fundus disease are the high administration frequency and ocular complications. Meanwhile?about 40% of fundus disease patients have anti-VEGF drugs resistance and more than 50% of patients have poor treatment effect. Therefore, there is an urgent need to find other kinds of new drugs for fundus treatment. Artemisinin is isolated from the plant Artemisia annua. Previous evidence showed that artemisinin may inhibit angiogenesis. But because of the physicochemical properties limit its ophthalmology application. We have designed and synthesized an artemisinin derivative P31, which has been granted national invention patents. This compound exhibits excellent solubility and high permeability.
Objective?To investigate the inhibitory effect of P31 on CNV in cell and animal models, and explored the mechanisms.
Methods: ? BN rats (n?18) were randomly divided into 3 groups: control group( treated with normal saline)?model group ( treated with normal saline) and P31 treatment group ( treated with 2.0% P31 eye drops)?with 6 rats in each group. The rats in model group and P31 treatment group were induced by laser (532nm). The eyes of each group were assessed by fundus angiography. The choroidal flat mounts were stained by immunofluorescence. The expression levels of AKT and ERK1/2 pathways proteins were tested by western-blot. ? 10ng/ml VEGF induced Human umbilical vein endothelial cells (HUVEC) were treated with 5, 25 and 50 μM P31 for 24, 48 and 72h, cell proliferation was measured by BrdU assays, cell migration and invasion were measured by wound healing assay and Transwell assay, and cell angiogenesis was determined by tube formation assay. The phosphorylation levels of AKT and ERK pathway were analyzed by western-blot.
Results: ? Compared with model group?the fundus fluorescence leakage and the mean density were significantly reduced in P31 treatment group (P<0.01). The immunofluorescence staining indicated that CNV area were obviously decreased by P31 eye drops (P<0.01). The AKT and ERK1/2 signaling pathway was inhibited by P31, indicated by down regulation of phosphorylation of AKT and ERR1/2 in rat retina tissue (P<0.05). ? VEGF stimulation resulted in increased HUVECs proliferation, whereas P31 attenuated the VEGF-induced cell proliferation in a dose-dependent manner. BrdU staining confirmed that P31 suppressed VEGF-induced HUVECs proliferation (P<0.01). While P31 significantly inhibited the invasion of HUVECs in a dose-dependent manner (p<0.01). The treatment of HUVECs with increasing concentration of P31 caused a concentration-dependent decrease in wound-healing cell migration(p<0.01). In the presence of P31, tube formation was interrupted on VEGF-treated HUVECs (p<0.01). The levels of phosphorylation of ERK1/2 and AKT were examined in comparison with the control (p<0.05), whereas the total expression of ERK1/2 and AKT did not change significantly after P31 treatment.
Conclusion: P31 as a novel artemisinin derivative, attenuates choroidal neovascularization and has a promising application in ocular fundus diseases.
