Portulaca Oleracea L. (Purslane) Extract Protects Endothelial Function by Reducing Endoplasmic Reticulum Stress and Oxidative Stress through AMPK Activation in Diabetic Obese Mice

Abstract

Portulaca oleracea L. (purslane) is a food and a traditional drug worldwide. It exhibits anti-inflammatory, anti-oxidative, anti-tumor, and anti-diabetic bioactivities; but its activity on diabetic-associated endothelial dysfunction is unknown. This study aimed to investigate the effect of purslane on endothelial function and the underlying mechanisms. Male C57BL/6 mice had 14-week ad libitum access to a high-fat rodent diet containing 60% kcal% fat to induce obesity and diabetes whereas purslane extract (200 mg/kg/day) was administered during the last 4 weeks via intragastric gavage. Primary rat aortic endothelial cells and isolated mouse aortas were cultured with a risk factor, high glucose or tunicamycin, together with purslane extract. By ESI-QTOF-MS/MS, flavonoids and their glycoside products were identified in the purslane extract. Exposure to high glucose or tunicamycin impaired acetylcholine-induced endothelium-dependent relaxations in aortas and induced endoplasmic reticulum (ER) stress and oxidative stress with the downregulation of 5' AMP-activated protein kinase (AMPK)/ endothelial nitric oxide synthase (eNOS) signaling. Co-incubation with purslane significantly ameliorated these impairments. The effects of purslane were abolished by Compound C (AMPK inhibitor). Four-week purslane treatment ameliorated aortic relaxations, ER stress, and oxidative stress in diabetic obese mice. This study supported that purslane protected endothelial function, and inhibited ER stress and oxidative stress in vasculature through AMPK/eNOS activation, revealing its therapeutic potential against vascular complications in diabetes.

Keywords: diabetes mellitus; endoplasmic reticulum stress; endothelial function; nitric oxide; purslane; reactive oxygen species.

Figure 1

Extracted ion chromatogram (EIC) of identified components in the purslane extract. EIC of polyphenols for 1: Cryptochlorogenic acid;2: Luteolin 8-C-glucoside; 3: Quercetin 3-galactoside; 4: Kaempferol-3-O-glucoside; 5: Luteolin-7-O-glucoside; 6: Luteolin; 7: Quercetin; and 8: Kaempferol.

Figure 2

Purslane extract improves endothelial dysfunction and diabetes symptoms in diet-induced obese (DIO) mice. (A) Body weight changes of C57 BL/6J mice with standard chow or high-fat diet for a total of 14 weeks and 4-week oral treatment with vehicle (0.3% CMC-Na solution) or purslane extract (200 mg/kg/day). (B) Oral glucose tolerance test (OGTT) results after fasting for 6 h. (C) Insulin tolerance test (ITT) results after fasting for 2 h. (D,E) Systolic (SBP) and diastolic (DBP) blood pressure changes in all groups of mice detected by the tail-cuff method. (F) Summarized data revealing the effect of 4-week purslane treatment on endothelium-dependent relaxations (EDRs) induced by acetylcholine (ACh), and (G) sodium nitroprusside(SNP)-induced endothelium-independent relaxations in all groups of mouse aortas. (H) Representative traces of ACh-induced EDRs in mouse aortas in vivo. Data are shown as mean ± SEM (n = 5). # p < 0.05 DIO vs. Control; ∗ p < 0.05 DIO + Purslanevs. DIO.

Figure 3

Purslane extract activates AMPK/eNOS signaling pathway and suppresses endoplasmic reticulum (ER) stress in DIO mice. Representative Western blotting images and summarized graphs showing (A) eNOS phosphorylation at Ser1177 and total eNOS (140 kDa), (B) phosphorylation of AMPKα at Thr172 and total AMPKα (62 kDa) expression levels, and ER stress markers including (C) spliced XBP1 (56 kDa), (D) cleaved ATF6 (50 kDa), (E) phosphorylation of eIF2α at Ser52 and total eIF2α expression levels (38 kDa) and (F) CHOP (27 kDa) compared to GAPDH in aortas of lean control, DIO and purslane extract (200 mg/kg/day, 4 weeks) treated DIO mice. Data are shown as mean ± SEM (n= 4–5). # p< 0.05 DIO vs. Control; ∗ p< 0.05 DIO + Purslane vs. DIO.

Figure 4

Purslane extract protects against high glucose- and tunicamycin-triggered endothelial dysfunction on AMPK activation. (A) Summarized data of the effects of purslane extract (100 and 400 μg/mL) on ACh-induced EDRs and (B) SNP-induced relaxations in mouse aortas exposed to high glucose (HG, 30 mM, 48 h). (C,D) Effect of AMPK inhibitor Compound C (Cpd C, 5 μM) on ACh-induced EDRs in mouse aortas co-treated with high dose purslane extract (400 μg/mL) under the high glucose (HG, 30 mM, 48 h) or tunicamycin (Tuni, 2 μg/mL, 24 h) stimulated condition. Data are shown as mean ± SEM (n = 4). # p < 0.05 HG vs. NG (5.55 mM glucose with mannitol added as osmotic control) or Tuni vs. Control; ∗ p < 0.05 vs. HG or Tuni; † p < 0.05 vs. HG+ Purslane or Tuni+ Purslane.

Figure 5

Purslane extract activates AMPK/eNOS signaling and inhibits ER stress in high glucose-induced mouse aortas (mimic hyperglycemia). Representative Western blotting images and analyzed graphical results for (A) phospho (p)-eNOS at Ser1177 (140 kDa) and (B) phospho (p)-AMPKα at Thr172 (62 kDa) in ratio to their respective total proteins in aortic segments stimulated with high glucose (HG, 30 mM, 48 h) with or without the co-treatment of purslane extract (400 μg/mL). Western blotting results showing ER stress markers including (C) spliced XBP1 (sXBP1; 56 kDa), (D) cleaved ATF6 (50 kDa), (E) phospho (p)-eIF2α at Ser52 (38 kDa) and (F) CHOP (27 kDa) normalized to their total proteins or GAPDH. Data are expressed as the mean ± SEM (n = 4). # p < 0.05 HG vs. NG; ∗ p < 0.05 HG + Purslane vs. HG.

Figure 6

Purslane extract activates AMPKα/eNOS pathway and attenuates ER stress in high glucose-stimulated rat aortic endothelial cells (RAECs). (A) NO release from RAECs treated with normal glucose (11 mM present in RPMI-1640 medium), high glucose (44 mM), purslane extract (400 μg/mL), and Compound C (5 μM) for 48 h. Representative blots and summarized data showing (B) p-eNOS at Ser1177 (140 kDa), (C) p-AMPKα at Thr172 (62 kDa), (D) cleaved ATF6 (50 kDa), (E) p-eIF2α at Ser52 (38 kDa), and (F) CHOP (27 kDa) in the ratios to their total proteins or GAPDH in cultured RAECs. Data are presented as mean ± SEM (n = 4). # p < 0.05 HG vs. NG; ∗ p < 0.05 HG + Purslane vs. HG; † p < 0.05 HG + CpdC + Purslane vs. HG + Purslane.

Figure 7

Purslane extract increases eNOS phosphorylation and attenuates ER stress in RAECs exposed to tunicamycin. RAECs were cultured with ER stress inducer tunicamycin (Tuni, 2 μg/mL), purslane extract (400 μg/mL), and Compound C (5 μM) for 24 h. (A) Nitrite concentrations representing NO release of RAECs. (B) Phosphorylation of eNOS at Ser1177 (140 kDa) compared to total eNOS. Expressions of ER stress markers: (C) spliced XBP1 (56 kDa), (D) cleaved ATF6 (50 kDa), (E) phosphorylation of eIF2α at Ser52 (38 kDa), and (F) CHOP (27 kDa) compared to GAPDH or total protein. Data are mean ± SEM of 4–5 experiments. # p < 0.05 vs. Control; ∗ p < 0.05 vs. Tuni; † p < 0.05 vs. Tuni + Purslane.

Figure 8

Purslane extract inhibits oxidative stress in aortas and endothelial cells in vivo and in vitro. (A) Representative fluorescence images and analyzed data of DHE intensity detected in mouse aortas from lean control and DIO mice with 4-week treatment of purslane extract. (B–D) DHE intensity in mice aortas and RAECs treated with normal glucose (5.55 mM for aortas, 11 mM for RAECs), high glucose (30 mM for aortas, 44 mM for RAECs), purslane extract (400 μg/mL) and AMPK inhibitor Compound C (0.5 μM), shown as summarized graphs and representative images. (E,F) Summarized graphs showing the suppressive effects of Purslane extract on tunicamycin (2 μg/mL)-induced oxidative stress in mice aortas and RAECs, which were abolished by Compound C. Data are mean ± SEM of four experiments. # p <0.05 vs. Control/NG; ∗ p <0.05 vs. DIO/HG/Tuni; † p <0.05 vs. HG/Tuni + Purslane extract.