The Protective Effect And Mechanism Of Small Activating RNA-modified Tetrahedral Framework Nucleic Acids On Oxidative Stress Damage Of Retinal Cells Via Promoting DJ-1 Gene Expression

Purpose:Retinal vascular endothelial cells and retinal pigment epithelial(RPE)cells,as the key components of the inner and outer blood-retinal barriers,play a vital role in maintaining retinal homeostasis.The damage of oxidative stress on vascular endothelial cells and RPE cells is an important risk factor for the occurrence and development of various retinal diseases.Numerous studies have confirmed that DJ-1 has an endogenous anti-oxidative stress effect through various mechanisms.Meanwhile,DJ-1 can protect a variety of retinal cells from oxidative stress damage and inhibit oxidative stress-induced apoptosis.Therefore,specifically promoting DJ-1 gene expression in vascular endothelial cells and RPE cells may be an effective treatment for oxidative stress-related retinal diseases,which has important clinical value.Small activating RNA(saRNA)can promote the expression of target genes at the transcriptional level by binding to the promoter sequence in the nucleus.However,current saRNA delivery systems still have limitations such as limited application range,long-term application safety,and short half-life.On the other hand,tetrahedral framework nucleic acids(tFNAs),a novel DNA nanomaterial,has gradually become a research hotspot in biomedicine based on excellent membrane penetration,low cytotoxicity,stability,and programmability.tFNAs can be linked to functional oligonucleotides or small molecule drugs through simple modifications to function as a high-efficiency carrier.Therefore,the purpose of this study was to investigate the promotion effect of tFNAs-saRNA on DJ-1 gene expression in vascular endothelial cells and RPE cells,and to further explore the protective effect of tFNAs-saRNA on oxidative damage of vascular endothelial cells and RPE cells.Methods:The resuscitated human umbilical vein endothelial cells(HUVECs)and ARPE-19 cells were sub cultured,and the cells were transfected with different saRNA sequences to detect the expression of DJ-1 gene by RT-qPCR and ELISA.After using H2O2 to establish the oxidative damage models,the protective effects of different saRNA sequences on HUVECs and ARPE-19 cells were detected by CCK-8.The bestperforming saRNA was modified onto tFNAs by sequence expansion.Cellular uptake of Cy5-labeled saRNA,tFNAs,and tFNAs-saRNA was analyzed by flow cytometry and confocal microscopy.After recovery,HUVECs and ARPE-19 cells were sub cultured,H2O2 was used to establish an oxidative damage model,and CCK-8 was used to detect cell viability.After collecting HUVECs and ARPE-19 cells,RT-qPCR,ELISA,and WB were used to detect the expression of DJ-1,Bcl-2,Bax,Caspase-3,Erk1/2,pErk 1/2,Elk-1,p-Elk-1,Akt,p-Akt,Nrf2,and HO-1.The expression of reactive oxygen species(ROS)labeled with DCFH-DA probe in cells was detected by flow cytometry.Results:Among different saRNA sequences,saRNA-3 had the best promotion effect on DJ-1 gene expression in HUVECs and ARPE-19 cells,and could alleviate the oxidative damage on HUVECs and ARPE-19 cells.saRNA-3 was modified to tFNAs to successfully construct tFNAs-saRNA.The results of cellular uptake showed that tFNAs could significantly improve the uptake efficiency of saRNA in HUVECs and ARPE-19 cells.Since the proportion of surviving and apoptotic cells in HUVECs and ARPE-19 cells was about 50%after treatment with 200μM and 400μM H2O2 for 6 hours,200μM and 400μM H2O2 were selected as the treatment conditions for establishing oxidative damage models in HUVECs and ARPE-19 cells,respectively.Different concentrations of tFNAs-saRNA can effectively increase the expression of DJ-1 gene in HUVECs and ARPE-19 cells.250nM and 125nM tFNAs-saRNA has the best protective effect on HUVECs and ARPE-19 cells,respectively.Compared with the oxidative damage group,250nM and 125nM tFNAs-saRNA treatment can significantly improve the ratio of Bcl-2/Bax and inhibit the expression of Caspase-3 and ROS in HUVECs and ARPE-19 cells,respectively.In addition,tFNAs-saRNA can significantly increase the ratio of p-Erk/Erk and p-Elk/Elk in HUVECs,and the ratio of p-Akt/Akt and the expression of Nrf2 and HO-1 in ARPE-19 cells,respectively.Conclusions:After the successful modification of saRNA,tFNAs could effectively increase the cellular uptake of saRNA,thereby promoting the expression of DJ-1 gene in HUVECs and ARPE-19 cells.250nM and 125nM tFNAs-saRNA treatment could activate Erkl/2/Elk-1 and Akt/Nrf2HO-1 pathways in HUVECs and ARPE-19 cells,respectively,thereby regulating the expression of apoptosis-related proteins and reducing the level of intracellular ROS,and finally protecting vascular endothelial cells and RPE cells from oxidative stress damage.In the future,tFNAs-saRNA is expected to become an effective treatment for oxidative stress-related retinal diseases.