丹芪化瘀方含药血清对光损伤人视网膜色素上皮细胞保护机制研究

Investigation on the protective mechanism of retinal pigment epithelial cells ofphotodamaged human retina by Danqi Huayu formula containing serums

目的:探讨丹芪化瘀方含药血清对LED冷光源致人视网膜色素上皮细胞(ARPE-19)光损伤的防治作用及最佳药物剂量,探寻其可能存在的保护机制。方法:应用健康的SD大鼠制备丹芪化瘀方空白血清和含药血清,将0%(空白血清)及5%、10%、15%、20%含药血清作用于ARPE-19细胞,每组设6个复孔。各组加入相应的药物于细胞恒温培养箱中培养24 h后采用CCK-8检测细胞活力,筛选最佳含药血清浓度进行后续实验。实验设置对照组、空白血清组、低剂量、中剂量、高剂量含药血清组培养,采用实时荧光定量聚合酶链式反应(Real-time PCR)检测细胞中诱导型一氧化氮合酶(iNOS)、内皮型一氧化氮合酶(eNOS)、内皮素-1(ET-1)mRNA表达;酶联免疫吸附测定法(ELISA)检测一氧化氮合成酶(NOS)、一氧化氮(NO)、血管内皮生长因子(VEGF)含量;蛋白免疫印迹法(Western blot)检测对照组、空白血清组、高剂量组iNOS的蛋白表达水平。结果:筛选出16%丹芪化瘀方含药血清作为后续实验干预浓度。与对照组比较,空白血清组iNOS、ET-1 mRNA表达及NOS、NO、VEGF含量均升高(P<0.001),eNOS mRNA表达降低(P<0.001);随着含药血清浓度的增加细胞中iNOS、ET-1 mRNA表达及NOS、NO、VEGF含量逐渐降低,eNOS mRNA表达增加(P<0.001),与低、中剂量组相比,高剂量组的iNOS、ET-1 mRNA表达及NOS、NO、VEGF含量显著降低(P<0.001或P<0.01或P<0.05),eNOS mRNA显著升高(P<0.001或P<0.05);空白血清组iNOS蛋白水平明显高于对照组,而16%含药血清的iNOS蛋白水平明显低于空白血清组(P<0.001)。结论:丹芪化瘀方含药血清可能通过抑制iNOS/NO途径减轻细胞损伤,上调eNOS表达,下调ET-1、VEGF的表达,抑制脉络膜新生血管形成并改善血-视网膜屏障功能,从而发挥保护作用。

Objective:To investigate the preventive effect and optimal dose of serum containing Danqi Huayuquan formula on light damage to human retinal pigment epithelial cells(ARPE-19)induced by LED cold light source,and to explore the possible protective mechanism.Methods:The drug-containing serum of Danqi Huayu formula was prepared,and light-damaged human ARPE-19 cells were cultured in vitro.A control group,a blank serum group,5%,10%,15%and 20%drug-containing serum groups were set up,and six replicate wells were set up in each group.Each group was added with the corresponding drugs and cultured in a constant temperature incubator for 24 hours.The color viability was detected by CCK-8,and the optimal concentration of drug-containing serum was selected for the subsequent experiments.The control group,blank serum group,low-dose,medium-dose and high-dose serum group were set up for the experiments,and real-time PCR(Real-time PCR)was used to detect the inducible monoclonal antibodies in the cells.Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to detect the mRNA expression of inducible nitric oxide synthase(iNOS),endothelial nitric oxide synthase(eNOS),endothelin-1(ET-1).Enzyme-linked immunosorbent assay(ELISA)was used to detect the content of nitric oxide synthase(NOS),nitric oxide(NO),and vascular endothelial growth factor(VEGF).And western blotting was used to detect the content of control group,blank serum group,low dose,medium dose,and high dose serum groups containing the drug.Western blot to detect the protein expression levels of iNOS in the control group,blank serum group and high dose group.Results:The 16%Danqi Huayu formula drug-containing serum was finally screened out as the intervention concentration for subsequent experiments.Compared with the control group,iNOS,ET-1 mRNA expression and NOS,NO,VEGF content in the blank serum group were elevated(P<0.001),and eNOS mRNA expression was decreased(P<0.001).As the concentration of drug-containing serum increased the iNOS,ET-1 mRNA expression and NOS,NO,VEGF content in the cells was gradually decreased,and eNOS mRNA expression increased(P<0.001),and iNOS,ET-1 mRNA expression and NOS,NO,VEGF content were significantly decreased(P<0.001 or P<0.01 or P<0.05),and eNOS mRNA was significantly elevated(P<0.001 or P<0.05)in the high-dose group compared with those in the low-and medium-dose groups.The blank serum group had iNOS protein level was significantly higher than that of the control group,while the iNOS protein level of 16%drug-containing serum was significantly lower than that of the blank serum group(P<0.001).Conclusion:The drug-containing serum of Danqi Huayu formula may play a protective role by inhibiting the iNOS/NO pathway to attenuate cellular injury,upregulating eNOS expression,downregulating the expression of ET-1 and VEGF,inhibiting choroidal neovascularisation and improving the function of the blood-retinal barrier.