Ginsenoside Rb1 Attenuates High Glucose-Induced Oxidative Injury via the NAD-PARP-SIRT Axis in Rat Retinal Capillary Endothelial Cells

Abstract

(1) Aims: The present study aimed to observe the effects of Ginsenoside Rb1 on high glucose-induced endothelial damage in rat retinal capillary endothelial cells (RCECs) and to investigate the underlying mechanism. (2) Methods: Cultured RCECs were treated with normal glucose (5.5 mM), high glucose (30 mM glucose), or high glucose plus Rb1 (20 μM). Cell viability, lactate dehydrogenase (LDH) levels, the mitochondrial DNA copy number, and the intracellular ROS content were measured to evaluate the cytotoxicity. Superoxide dismutase (SOD), catalase (CAT), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), poly(ADP-ribose) polymerase (PARP), and sirtuin (SIRT) activity was studied in cell extracts. Nicotinamide adenine dinucleotide (NAD⁺)/NADH, NADPH/NADP⁺, and glutathione (GSH)/GSSG levels were measured to evaluate the redox state. The expression of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), SIRT1, and SIRT3 was also evaluated after Rb1 treatment. (3) Results: Treatment with Rb1 significantly increased the cell viability and mtDNA copy number, and inhibited ROS generation. Rb1 treatment increased the activity of SOD and CAT and reduced the activity of NOX and PARP. Moreover, Rb1 enhanced both SIRT activity and SIRT1/SIRT3 expression. Additionally, Rb1 was able to re-establish the cellular redox balance in RCECs. However, Rb1 showed no effect on NMNAT1 expression in RCECs exposed to high glucose. (4) Conclusion: Under high glucose conditions, decreases in the reducing power may be linked to DNA oxidative damage and apoptosis via activation of the NMNAT-NAD-PARP-SIRT axis. Rb1 provides an advantage during high glucose-induced cell damage by targeting the NAD-PARP-SIRT signaling pathway and modulating the redox state in RCECs.

Keywords: Ginsenoside Rb1; NAD+; PARP; high glucose; retinal capillary endothelial cells; sirtuin.

Figure 1

Characterization of established rat retinal capillary endothelial cell (RCEC) cultures. (a) Endothelial cells migrated from the retinal capillary fragments after 5 d. (b) Cells acquired a typical contact-inhibited monolayer with a short fusiform or round morphology after 8 to 10 d. The original magnification was 200×. (c) and (d) The RCECs were analyzed for the expression and localization of endothelial cell markers by immunofluorescence staining of CD31 and the von Willebrand factor (vWf). All nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue fluorescence). Scale bar: 40 μm.

Figure 2

Ginsenoside Rb1 (Rb1) increased the cell viability in rat RCECs exposed to high glucose. (a) MTT assays were used to examine the cell viability. (b) Trypan blue exclusion was used to count the live cells. The data are expressed as the means ± SD. ## p < 0.01 versus 5.5 mM glucose; *p < 0.05 and ** p < 0.01 versus 30 mM glucose.

Figure 3

Rb1 ameliorated high glucose-induced cytotoxicity, DNA damage, and apoptosis in rat RCECs. (a) A lactate dehydrogenase (LDH) assay was used to assess the cell cytotoxicity. (b) The relative mtDNA copy number was estimated with real-time quantitative PCR. (c) Apoptosis was measured with an Annexin V-FITC/PI double staining assay and laser confocal scanning microscopy. The data are expressed as the means ± SD. ## p < 0.01 versus 5.5 mM glucose; * p < 0.05 and ** p < 0.01 versus 30 mM glucose. Scale bar: 33 μm.

Figure 4

Rb1 inhibited ROS generation induced by high glucose in RCECs. (a) Intracellular ROS generation was measured with Amplite ROS Red. (b) A MitoTracker Red CMH-XRos probe was used to monitor ROS production in mitochondria. (c) Fluorescence images were analyzed using Image-Pro Plus 6.0 software. The data are expressed as the means ± SD. ## p < 0.01 versus 5.5 mM glucose; ** p < 0.01 versus 30 mM glucose. Scale bar: 40 μm.

Figure 5

Effects of Rb1 on the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) and antioxidant enzymes in RCECs exposed to high glucose. (a) and (b) The activity of superoxide dismutase (SOD) and catalase (CAT) was evaluated with an enzymatic colorimetric method. (c) The activity of NOX was measured with the chemiluminescence method. The data are expressed as the means ± SD. ## p < 0.01 versus 5.5 mM glucose; ** p < 0.01 versus 30 mM glucose.

Figure 6

Rb1 prevented the reductions in sirtuin1 (SIRT1), SIRT3, and the nicotinamide adenine dinucleotide (NAD⁺)/NADH ratio and abrogated the increase in poly(ADP-ribose) polymerase (PARP) in RCECs exposed to high glucose. (a) Cells were collected to examine PARP activity. (b) NAD⁺ levels. (c) NADH levels. (d) Intracellular NAD⁺/NADH ratios. (e) Sirtuin activity. (f) Western blot analysis was used to determine SIRT1 and SIRT3 protein expression in RCECs. (g) The relative densities of SIRT1 and SIRT3 protein expression were determined by image analysis with Image-Pro Plus 6.0 software. (h) RT-qPCR was used to measure SIRT1 and SIRT3 mRNA expression. The data are expressed as the means ± SD. ## p < 0.01 versus 5.5 mM glucose; * p < 0.05 and ** p < 0.01 versus 30 mM glucose.

Figure 6

Rb1 prevented the reductions in sirtuin1 (SIRT1), SIRT3, and the nicotinamide adenine dinucleotide (NAD⁺)/NADH ratio and abrogated the increase in poly(ADP-ribose) polymerase (PARP) in RCECs exposed to high glucose. (a) Cells were collected to examine PARP activity. (b) NAD⁺ levels. (c) NADH levels. (d) Intracellular NAD⁺/NADH ratios. (e) Sirtuin activity. (f) Western blot analysis was used to determine SIRT1 and SIRT3 protein expression in RCECs. (g) The relative densities of SIRT1 and SIRT3 protein expression were determined by image analysis with Image-Pro Plus 6.0 software. (h) RT-qPCR was used to measure SIRT1 and SIRT3 mRNA expression. The data are expressed as the means ± SD. ## p < 0.01 versus 5.5 mM glucose; * p < 0.05 and ** p < 0.01 versus 30 mM glucose.

Figure 7

Rb1 treatment showed no effect on NMNAT1 expression in rat RCECs exposed to high glucose. (a) Western blot analysis was used to determine NMNAT1 protein expression. (b) The relative densities of NMNAT1 protein expression were determined by image analysis with Image-Pro Plus 6.0 software. (c) RT-qPCR was used to determine NMNAT1 mRNA expression. Data are expressed as the means ± SD. ## p < 0.01 versus 5.5 mM glucose; ** p < 0.01 versus 30 mM glucose.

Figure 8

Rb1 modulated the NADPH/NADP⁺ and glutathione (GSH)/GSSG ratios in RCECs exposed to high glucose. (a) NADPH and GSH levels. (b) NADP⁺ and GSSG levels. (c) NADPH/NADP⁺ and GSH/GSSG ratios. Data are expressed as the means ± SD. ## p < 0.01 versus 5.5 mM glucose; * p < 0.05 and ** p < 0.01 versus 30 mM glucose.

Figure 9

Chemical structure of Ginsenoside Rb1.