Ras相关的C3肉毒毒素底物1的小发夹RNA干扰活性氧-核因子κB通路抑制小鼠视网膜新生血管生成

Objective To investigate the effects of knocking down Racl gene (ras-related C3 botulinum toxin substrate 1) by small hairpin RNA (shRNA) on retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Methods One hundred and eight 7-day-old C57BL/6J mice were divided into three groups randomly. The OIR was induced by Smith protocol in 2 groups. OIR mice received an intravitreal injection of Racl-shRNA plasmid or the nonsense plasmid in the gene-intervention group and control group respectively at the age of postnatal day 11 (P11). Non-OIR mice also received an intravitreal injection of Racl-shRNA plasmid at P11 as the blank-intervention group which lived in the normoxic environment. Retinal neovascularization was investigated on flat-mounts after fluorescence angiography at P15 and P17. Endothelial cell nuclei breaking through the internal limiting membrane were counted on pathological section at P17. The expression of Racl and NF-κB p65 subunit was measured by immuohistochemistry, Western blot, real-time polymerase chain reaction (RT-PCR) and in situ hybridization. Results Compared with the blank-control group, the level of Racl mRNA in the geneintervention group decreased obviously(t=4.5, P = 0. 001 ); the retinal non-perfusion areas, fluorescence leakage, neovascularization and the number of endothelial cell nuclei breaking through the internal limiting membrane were reduced significantly(t = 6. 521, P< 0. 001) ; the level of NF-κB p65 nuclear translocation decreased(t= 16. 008, P<0. 001)while the expression of NF-κB p65 mRNA was reduced obviously(t=3. 354, P=0. 006), which was positively correlated with the expression of Ratl mRNA (P=0. 012).Conclusion Intravitreal injection of Racl-shRNA with liposome in mice can effectively inhibit the expression of Racl, and inhibit the retinal neovascularization under relative hypoxia via blocking the ROS-NF-κB pathway.

Ras-related C23 botulinum toxin substrate 1 small hairphin RNA suppress mouse retinal neovascularization in mice