Triptolide10Prevents Hypoxia-induced Angiogenic Events

Objective To investigate the mechanism of triptolide(T10) in hypoxia-inducedretinal neovascularization.Method1.normal and hypoxic models of human umbilical vein endothelialcells(HUVEC) were built. It was to determine the best Inhibitory concentration of T10byMTT. Endothelial cell with the effects of triptolide (triptolid, T10) proliferation wasassayed by MTT, and expression of t-PAmRNA, VEGFmRNA and ANXA2mRNA inHUVEC cell lines were observed by RT-PCR and western blotting.2.Ninety-six mice ofC57BL/6J after birth were randomly divided into three groups: control group (32mice/64eyes), treatment group (32mice/64eyes) and model group (32mice/64eyes). At day5(P5),treatment group and model group are placed in a concentration of75%oxygen, return tonormal air at day12(P12). Treatment group received intraperitoneal injection of T10.Control group was always placed in the normal air. At day17(P17), the retinalnovascularization was examined by fluorescence angiography with FITC-Dextran afterperfusion and pathological section. Expressive levels of VEGFmRNA、ANXA2mRNA andt-PAmRNA were detected by real-time PCR in all groups.Results1. The optimum inhibitory concentration of T10was100nmol/L.Compared with the model group, after added Triptolide10, migration and proliferation ofthe endothelial cells were significantly inhibited (P<0.05). At the same time, T10decreased the gene and protein expression of VEGF and ANXA2in HUVEC cell lines byinhibiting t-PA (P>0.05).2. Fluorescein angiography showed a pattern of pathologicalretinal neovascularization such as central non-perfused areas, tortuous and dilated bloodvessels and leaky neovascular tufts in model group, at day17but showed normal retinalblood vessels, no fluorescein leakage and non-perfusion area in control group. A largearea of non-perfusion and a small area of the vascular network in peripheral retina wereunobserved at day17in treatment group. And the serial HE staining sections confirmed theresults. It showed81.95±1.93retinal vascular cell nuclei anterior to the internal limitingmembrane in model group compared with1.05±0.28in control group and there wasstatistical significance between two group(P<0.05). But there was no statisticalsignificance between treatment group and control group (P>0.05), because treatment grouponly has42.25±1.16.3.The t-PAmRNA, VEGF mRNA, AnxA2mRNA expressive levels were significantly between the Hypoxia-induced group and treatment group atdifferent, there was significant difference between two group (P<0.05), there was statisticaldifference in the expression of t-PA mRNA VEGFmRNA and AnxA2mRNA. But therewas no statistical significance between treatment group and control group (P>0.05).Conclusions1. T10can inhibit HUVEC cell migration and proliferation, itsmechanism maybe inhibit the expression of t-PA, which reduce the expression of VEGFand ANXA2.2. The mice of model group with hypoxia-induced retinal neovascularizationwas stable, reliable, reproducible, it can be used as the study of the pathogenesis of retinalvascular diseases and prevention methods in animal models.3. T10can inhibit theexpression of VEGF, ANXA2, t-PA and PAI-1in retinal neovascularization.