Phlorizin Exerts Direct Protective Effects on Palmitic Acid (PA)-Induced Endothelial Dysfunction by Activating the PI3K/AKT/eNOS Signaling Pathway and Increasing the Levels of Nitric Oxide (NO)

Abstract

BACKGROUND Sodium glucose transporter-2 inhibitors are the newest antidiabetic drugs that seem to be cardioprotective and can prevent type 2 diabetes in patients with high cardiovascular risks. Previous clinical trials have shown that these inhibitors can alleviate endothelial dysfunction, but the mechanism of action remains unknown. How SGLT inhibitor influences the release of NO in PA-induced HUVECs has never been reported. MATERIAL AND METHODS To explore the potential effects of the endothelial-protective mechanism of phlorizin and its impact on nitric oxide (NO), human umbilical vein endothelial cells (HUVECs) were incubated with palmitic acid (PA) and then treated with phlorizin. Western blotting was performed to assess the phosphorylation of AKT, eNOS, and IRS-1. To further explore potential targets, siRNA transfection was used to demonstrate the role of SGLT1 and SGLT2. RESULTS Phlorizin suppressed the expression of SGLT1 and SGLT2, activated the PI3K/AKT/eNOS signaling pathway, increased the output of NO, and promoted the consumption of glucose in PA-induced HUVECs. Through demonstrating siRNA suppression of the expression of SGLT1 and SGLT2 in PA-induced HUVECs, this study provides a new understanding of the mechanism behind SGLT1 and SGLT2. CONCLUSIONS Our data demonstrate that phlorizin ameliorates the endothelial dysfunction link with the activation of the PI3K/AKT/eNOS signaling pathway and augmentation of the release of NO, partially through suppressing the expression of SGLT1 and SGLT2 in PA-induced HUVECS.

Figure 1

PA stimulates expression of SGLT1 and SGLT2 in HUVECs. Cells were incubated for 18 h in the presence of PA (300 μM). (A, B) SGLT1 and SGLT2 proteins in the samples were analyzed by Western blotting. Bar plots show the summarized data of the relative density after being normalized to GAPDH and are expressed as average percentage of control ±SEM. (C, D) Quantitative PCR analysis of mRNA expression of SGLT1 and SGLT2, normalized to GAPDH mRNA. ** P<0.01.

Figure 2

The protective effects of phlorizin on PA-induced endothelial dysfunction in HUVECs. (A) The cell viability was determined by MTT assay of HUVECs treated with phlorizin in different concentrations for 30 min. Representative immunofluorescence microscopy (magnification 200, scale bar: 100 mm) showed extracellular HS expression. (G) PA reduced the glucose uptake in endothelial cells in a dose-dependent manner. (B, C) The relative amounts of mRNA of SGLT1 and SGLT2 were analyzed by qPCR. (D–F) Proteins of SGLT1 and SGLT2 were analyzed by Western blotting. (H) The course of phlorizin (50 nM)-induced consumption of extracellular glucose. (I) NO production was detected by the nitric oxide assay kit. ^# P<0.01 vs. controls, * P<0.05, ** P<0.01, *** P<0.001 for a chance difference compared with the relevant group.

Figure 3

The effect of phlorizin on phospho-eNOS, phospho-AKT, and NO release in HUVECs treated with PA. (A) The effect of dose-dependent stimulation of phlorizin on AKT phosphorylation after 30-min incubation in PA-induced HUVECs. (B) Time course of effect of phlorizin (50 nM) on AKT phosphorylation. (C, D, H) Phosphorylation of AKT, eNOS, and IRS-1 were analyzed by Western blotting. (H) Phlorizin did not induce phosphorylation of IRS-1. (E, F) Effects of phlorizin (50 nM) on the levels of p-AKT and p-eNOS, co-treated with LY294002 (LY; a PI3K inhibitor; 10 μM, 1 h). (G) The nitric oxide assay kit for detecting the release of NO in the presence or absence of the specific PI3K inhibitor, LY294002 (10 μM, 1 h) or L-NAME (A special eNOS inhibitor; 100 nM, 1h). ^# P<0.01 vs. the control group, * P<0.05, ** P<0.01, *** P<0.001 for a chance difference compared with the relevant group.

Figure 4

AKT and eNOS activity is stimulated in siRNA transfected HUVECs. (A, B) A scrambled sequence for the SGLT siRNA was taken to be the negative control (si-NC). The siRNA-treated HUVECs for 12 h were collected to be analyzed by Western blotting. Band intensities normalized to GAPDH. (C, D) The SGLT1 and SGLT2 mRNA levels determined by real-time RT-PCR and normalized to GAPDH mRNA. (E–G) Then, transfected HUVECs were collected for SDS-PAGE and immunoblotted with phospho-AKT, phospho-eNOS, and phospho-IRS-1 specific antibodies. Values were normalized using GAPDH controls. Representative of three experiments with similar results. ^# P<0.01 vs. the control group, * P<0.05, ** P<0.01 compared with relevant control.