Astragaloside IV protects rat retinal capillary endothelial cells against high glucose-induced oxidative injury

Abstract

Aim: Diabetic retinopathy is a microvascular complication of diabetes that leads to blindness. Hyperglycemia causes oxidative stress, which is an important cause in the pathogenesis of microangiopathy. The aim of this study was to investigate the potential protective effects of astragaloside IV (AS-IV) in retinal capillary endothelial cells (RCECs) incubated with high glucose conditions.

Methods and results: Based on rat RCECs cultured with high glucose (30 mM) in vitro, a significant increase in cell viability in rat RCECs incubated with both AS-IV and high glucose for 48 or 72 h by MTT assay. The increased viability was accompanied by decreased glucose transporter-1 expression using immunofluorescent assay. Meanwhile, AS-IV reduced intracellular hydrogen peroxide and superoxide, decreased mitochondrial reactive oxygen species in rat RCECs with high glucose by the fluorescent probes, and lowered malondialdehyde levels. In addition, AS-IV increased the activities of total superoxide dismutase, MnSOD, catalase, and glutathione peroxidase. The glutathione content also increased after AS-IV treatment. Furthermore, AS-IV reduced NADPH oxidase 4 expression by western blot method.

Conclusion: These results suggest that the main mechanism underlying the protective effects of AS-IV in high glucose-injured RCECs may be related to its antioxidative function.

Keywords: GLUT1; Nox4; astragaloside IV; oxidative stress; retinal capillary endothelial cells.

Figure 1

Chemical structure of AS-IV. Abbreviation: AS-IV, astragaloside IV.

Figure 2

Verification of rat RCECs. (A) Cells were cultured in endothelial cell medium for 10 days, and the characteristic cobble-stone morphology of RCECs was observed, scale bar: 100 μm, magnification: ×100. Immunocytochemical staining showed that the rat RCECs expressed vWf (B, green fluorescent dye) and CD31 (C, green fluorescent dye), scale bar: 40 μm, magnification: ×200. All nuclei were stained with DAPI (blue fluorescent dye). Abbreviation: RCECs, retinal capillary endothelial cells.

Figure 3

AS-IV increased cell viability in rat RCECs exposed to high glucose. Seeded in 96-well plates, the rat RCECs were incubated with varying concentrations of AS-IV (2, 5, 10, and 20 μM) in high glucose (30 mM glucose). The MTT assay was used to examine cell viability after culturing for 24 h (A), 48 h (B), and 72 h (C). The results showed that 5, 10, and 20 μM of AS-IV increased cell viability after 48 and 72 h, respectively. The experiment was repeated three times. Data represent mean ± SD (n=15). ^##P<0.01 and ^#P<0.05 versus 5.5 mM glucose; **P<0.01 versus 30 mM glucose. Abbreviations: AS-IV, astragaloside IV; RCECs, retinal capillary endothelial cells.

Figure 4

AS-IV inhibited the GLUT1 expression induced by high glucose in rat RCECs. Seeded in 24-well plates, the rat RCECs were incubated with varying concentrations of AS-IV (10 μM) in high glucose (30 mM glucose) for 72 h. Immunofluorescence analysis was performed to detect GLUT1 expression. We found that AS-IV could reduce GLUT1 expression. Data are expressed as mean ± SD (n=4). ^##P<0.01 and ^#P<0.05 versus 5.5 mM glucose; **P<0.01 versus 30 mM glucose. Scale bar: 50 μm. Abbreviations: AS-IV, astragaloside IV; RCECs, retinal capillary endothelial cells; GLUT1, glucose transporter-1.

Figure 5

AS-IV inhibited the ROS and MDA generation induced by high glucose in rat RCECs. Seeded in plates or flasks, the rat RCECs were incubated with varying concentrations of AS-IV (10 μM) in high glucose (30 mM glucose), and mitochondrial ROS as well as intracellular O₂⁻ and H₂O₂ production were measured using the MitoTracker Red CM-H₂XRos, DHE, and DCFH-DA, respectively. Representative images of cells from three independent experiments are shown (A). The values are expressed as mean ± SD per treatment group (n=4) (B). After 72 h of AS-IV treatment, the intracellular concentrations of H₂O₂ and O₂⁻ as well as mitochondrial ROS were decreased. A lipid peroxidation assay showed that AS-IV decreased the MDA content (C). ^##P<0.01 and ^#P<0.05 versus 5.5 mM glucose; **P<0.01 versus 30 mM glucose. Scale bar: 33 μm. Abbreviations: AS-IV, astragaloside IV; RCECs, retinal capillary endothelial cells; ROS, reactive oxygen species; MDA, malondialdehyde; DHE, dihydroethidium; DCFH-DA, 2,7′-dichlorofluorescindiacetate.

Figure 6

AS-IV increases the activities of antioxidant enzymes and regulates the glutathione redox system in rat RCECs exposed to high glucose. Seeded in T-25 cm² flasks, the rat RCECs were incubated with varying concentrations of AS-IV (10 μM) in high glucose (30 mM glucose) for 72 h. The cells were collected and subjected to antioxidant enzymes and glutathione determination. (A) The results showed that AS-IV increased the activities of the total SOD, MnSOD, CAT, and GSH-PX in rat RCECs incubated with 30 mM glucose. (B) The results showed that AS-IV increased the GSH content and reduced the GSSG content after incubation with 30 mM of glucose. Data are expressed as mean ± SD (n=4). ^##P<0.01, ^#P<0.05 versus 5.5 mM glucose; **P<0.01 versus 30 mM glucose. Abbreviations: AS-IV, astragaloside IV; RCECs, retinal capillary endothelial cells; SOD, superoxide dismutase; MnSOD, manganese superoxide dismutase; CAT, catalase; GSH-PX, glutathione peroxidase; GSH, glutathione; GSSG, glutathione disulfide.

Figure 6

AS-IV increases the activities of antioxidant enzymes and regulates the glutathione redox system in rat RCECs exposed to high glucose. Seeded in T-25 cm² flasks, the rat RCECs were incubated with varying concentrations of AS-IV (10 μM) in high glucose (30 mM glucose) for 72 h. The cells were collected and subjected to antioxidant enzymes and glutathione determination. (A) The results showed that AS-IV increased the activities of the total SOD, MnSOD, CAT, and GSH-PX in rat RCECs incubated with 30 mM glucose. (B) The results showed that AS-IV increased the GSH content and reduced the GSSG content after incubation with 30 mM of glucose. Data are expressed as mean ± SD (n=4). ^##P<0.01, ^#P<0.05 versus 5.5 mM glucose; **P<0.01 versus 30 mM glucose. Abbreviations: AS-IV, astragaloside IV; RCECs, retinal capillary endothelial cells; SOD, superoxide dismutase; MnSOD, manganese superoxide dismutase; CAT, catalase; GSH-PX, glutathione peroxidase; GSH, glutathione; GSSG, glutathione disulfide.

Figure 7

AS-IV inhibited the NADPH oxidase 4 (Nox4) expression induced by high glucose in rat RCECs. Seeded in plates or flasks, the rat RCECs were incubated with varying concentrations of AS-IV (10 μM) in high glucose (30 mM glucose) for 72 h. Western blot analysis was performed to detect Nox4 expression. We found that AS-IV could reduce Nox4 expression. Data are expressed as mean ± SD (n=4). ^##P<0.01 versus 5.5 mM glucose; *P<0.05 versus 30 mM glucose. Abbreviations: AS-IV, astragaloside IV; RCECs, retinal capillary endothelial cells.