SUMO1/UBC9‑decreased Nox1 activity inhibits reactive oxygen species generation and apoptosis in diabetic retinopathy

Abstract

Diabetic retinopathy (DR) is an increasing global health concern that causes vision loss and blindness. Reactive oxygen species (ROS) are considered to be a principal cause of DR. An important source of ROS is the oxidization of NADPH. In the present study, NADPH oxidase 1 (Nox1)‑expressing human retinal epithelial cell (HREC) lines were generated and infected with small ubiquitin‑like modifier 1 (SUMO1) and/or ubiquitin conjugating enzyme E2 I (UBC9) lentiviral pGMLV constructs. The viabilities, apoptotic capacities and ROS production levels of the HREC lines were quantified using Hoechst 33258, annexin V/propidium iodide and dichlorodihydrofluorescein diacetate assays, respectively. Additionally, rat DR models were established. From these models, the apoptotic capacities of retinal tissues were visualized using terminal deoxynucleotidyl transferase dUTP nick end labeling assays, and the pathologies were evaluated. The mRNA and protein expression levels of SUMO1, UBC9 and Nox1 were analyzed using reverse transcription‑quantitative polymerase chain reaction and western blot analyses, respectively. Compared with controls, the relative mRNA levels of SUMO1 and UBC9 were significantly upregulated, and the Nox1 levels significantly downregulated, in cells infected with SUMO1 or UBC9 alone or in combination. The ROS production and apoptosis rates of cells and retinal tissues were decreased. In addition, pathological symptoms in DR tissues improved when they were simultaneously transfected with SUMO1 and UBC9 via intraocular injection. In conclusion, the SUMO1/UBC9 axis may regulate Nox1‑mediated DR by inhibiting ROS generation and apoptosis in rat and cellular model systems.

Figure 1.

Constructions of SUMO1 and UBC9 lentiviral transfections and their effects on the expressions of SUMO1, UBC9 and Nox1 in Nox-1-expressing HRECs with high-glucose treatment. (A) Relative mRNA expression of SUMO1 and UBC9. (B) Representative images of western blot analysis with the relative protein expression of SUMO1 and UBC9 in Nox1-expressing HRECs, post-lentiviral transfection with SUMO1 and/or UBC9. (C) Relative mRNA expression of SUMO1, UBC9 and Nox1 in Nox-1-expressing HRECs following treatment with glucose (30 mM) and lentiviral transfection with SUMO1, UBC9 or SUMO1 + UBC9. *P<0.05, **P<0.01 vs. control. SUMO1, small ubiquitin-like modifier 1; UBC9, ubiquitin conjugating enzyme E2 I; Nox1, NADPH oxidase 1; HREC, human retinal microvascular endothelial cell; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 2.

Evaluation of HREC cells by Hoechst 33258. (A) Morphology of apoptotic HRECs using Hoechst 33258 staining (×200) and (B) cell death rates of HRECs were assessed. HRECs were divided in five groups: Nox1-expressing controls (control), Nox1-expressing cells treated with 30 mM glucose (high sugar), Nox1-expressing cells treated with 30 mM glucose and transfected with SUMO1 (high-sugar + SUMO1), Nox1-expressing cells treated with 30 mM glucose and transfected with UBC9 (high-sugar + UBC9), and Nox1-expressing cells treated with 30 mM glucose and transfected with SUMO1 and UBC9 (high-sugar + SUMO1 + UBC9). ***P<0.001 vs. control. HREC, human retinal microvascular endothelial cell; SUMO1, small ubiquitin-like modifier 1; UBC9, ubiquitin conjugating enzyme E2 I; Nox1, NADPH oxidase 1.

Figure 3.

Evaluation of HREC cells by flow cytometry. Apoptosis (A) analysis and (B) quantification of HRECs across the five treatment groups. **P<0.01 vs. controls. PI, propidium iodide; FITC, fluorescein isothiocyanate; SUMO1, small ubiquitin-like modifier 1; UBC9, ubiquitin conjugating enzyme E2 I.

Figure 4.

Intracellular reactive oxygen species quantities were assessed using DCFH-DA staining by flow cytometry at 488 nm excitation and 525 nm emission. DCFH-DA, dichlorodihydrofluorescein diacetate; SUMO1, small ubiquitin-like modifier 1; UBC9, ubiquitin conjugating enzyme E2 I.

Figure 5.

Relative mRNA expression of SUMO1 and UBC9, analyzed by reverse transcription-quantitative polymerase chain reaction in rat retinal tissues following infections with lentiviruses expressing SUMO1 and/or UBC9. β-actin was used as the reference/housekeeping gene. **P<0.01 vs. controls. SUMO1, small ubiquitin-like modifier 1; UBC9, ubiquitin conjugating enzyme E2 I.

Figure 6.

Effect of lentiviral transfection of SUMO1 and UBC9 on retinal tissue pathology and mRNA and protein levels of SUMO1, UBC9 and Nox1 in an established DR model. (A) Pathological evaluation of retinal tissues. Relative (B) mRNA and (C) protein expressions of SUMO1, UBC9 and Nox1 in an established rat DR model across five treatment groups: Healthy controls (control), DR affected animals (DR), DR animals infected with SUMO1 expressing lentiviruses (DR + SUMO1), DR animals infected with UBC9 lentiviruses (DR + UBC9), and DR animals infected with both SUMO1 and UBC9 lentiviruses (DR + SUMO1 + UBC9). *P<0.05, **P<0.01 vs. control. SUMO1, small ubiquitin-like modifier 1; UBC9, ubiquitin conjugating enzyme E2 I; Nox1, NADPH oxidase 1; DR, diabetic retinopathy.

Figure 7.

Representative images of apoptotic events were visualized following TUNEL assays in retinal tissues from the DR models. DR, diabetic retinopathy; SUMO1, small ubiquitin-like modifier 1; UBC9, ubiquitin conjugating enzyme E2 I.