Investigation on the protective mechanism of retinal pigment epithelial cells of photodamaged human retina by Danqi Huayu formula containing serums
Objective:To investigate the preventive effect and optimal dose of serum containing Danqi Huayu-quan 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 for-mula 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 select-ed for the subsequent experiments.The control group,blank serum group,low-dose,medium-dose and high-dose ser-um 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 ex-pression was decreased(P＜0.001).As the concentration of drug-containing serum increased the iNOS,ET-1 mR-NA expression and NOS,NO,VEGF content in the cells was gradually decreased,and eNOS mRNA expression in-creased(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 ser-um 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 neovascular-isation and improving the function of the blood-retinal barrier.
万方数据
维普
