Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Oct:37:101728.
doi: 10.1016/j.redox.2020.101728. Epub 2020 Sep 15.

Procyanidin B2 mitigates endothelial endoplasmic reticulum stress through a PPARδ-Dependent mechanism

Xin Nie  1 , Weiqi Tang  2 , Zihui Zhang  3 , Chunmiao Yang  3 , Lei Qian  1 , Xinya Xie  3 , Erjiao Qiang  1 , Jingyang Zhao  2 , Wenfei Zhao  3 , Lei Xiao  4 , Nanping Wang  5
Affiliations

Procyanidin B2 mitigates endothelial endoplasmic reticulum stress through a PPARδ-Dependent mechanism

Xin Nie et al. Redox Biol. 2020 Oct.

Abstract

Hyperglycemia-induced endothelial endoplasmic reticulum (ER) stress is implicated in the pathophysiology of diabetes and its vascular complications. Procyanidins are enriched in many plant foods and have been demonstrated to exert several beneficial effects on diabetes, cardiovascular and other metabolic diseases. In the present study, we investigated the effect of procyanidin B2 (PCB2), the most widely distributed natural procyanidin, on ER stress evoked by high glucose in endothelial cells (ECs) and the underlying mechanisms. We showed that PCB2 mitigated the high glucose-activated ER stress pathways (PERK, IRE1α and ATF6) in human vascular ECs. In addition, we found that PCB2 attenuated endothelial ER stress via the activation of peroxisome proliferator-activated receptor δ (PPARδ). We demonstrated that PCB2 directly bound to and activated PPARδ. Conversely, GSK0660, a selective PPARδ antagonist, attenuated the suppressive effect of PCB2 on the ER stress signal pathway. Functionally, PCB2 ameliorated the high glucose-impaired endothelium-dependent relaxation in mouse aortas. The protective effect of PCB2 on vasodilation was abolished in the aortas pretreated with GSK0660 or those from the EC-specific PPARδ knockout mice. Moreover, the protective effects of PCB2 on ER stress and endothelial dysfunction required the inter-dependent actions of PPARδ and AMPK. Collectively, we demonstrated that PCB2 mitigated ER stress and ameliorated vasodilation via a PPARδ-mediated mechanism beyond its classic action as a scavenger of free radicals. These findings further highlighted the novel roles of procyanidins in intervening the ER stress and metabolic disorders related to endothelial dysfunction.

Keywords: Endoplasmic reticulum stress; Endothelium-dependent relaxation; Peroxisome proliferator-activated receptor δ; Procyanidin B2.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
PCB2 attenuated high glucose-triggered ER stress in ECs. HUVECs were pretreated with or without PCB2 (10 μM, 12 h) before the exposure to high glucose (HG, 30 mM) or mannitol for 24 h. (A) Protein levels of p-PERK, PERK, ATF4, GRP78 and PDI were detected by using western blotting. (B) Quantification of p-PERK/PERK, ATF4, GRP78 and PDI levels as in (A) (n = 3). (C) HUVECs were pretreated with or without PCB2 (10 μM, 12 h) before the exposure to tunicamycin (TM, 2 μg/ml) for 16 h. Protein levels of p-PERK, PERK, ATF4, GRP78 and PDI were detected. (D) Quantification of p-PERK/PERK, ATF4, GRP78 and PDI levels as in (C) (n = 3). (E, G) Protein levels of p-IRE1α, IRE1α and ATF6 were assessed, *, deglycosylated ATF6. (F, H) Quantification of p-IRE1α/IRE1α and ATF6 levels as in (E) and (G) (n = 3). (I, J) The mRNA levels of CHOP, GRP78, ATF4 and ATF3 were assessed by using qRT-PCR (n = 3). All data were expressed as mean ± SEM. *P < 0.05, **P < 0.01 vs. Vehicle; #P < 0.05, ##P < 0.01 vs. HG or TM.
Fig. 2
Fig. 2
PCB2 activated PPARδ in ECs. (A) Model structures showing the complex formed by the PPARδ ligand-binding pocket and PCB2 based on molecular docking. PCB2 is shown in green. (B) Binding affinity of PCB2 to PPARδ. (C) BAECs were co-transfected with pPPRE-TK-luc with either pcDNA-PPARδ or pcDNA3.1 and treated with DMSO, PCB2 (10 μM) or GW501516 (1 μM) for 24 h. The luciferase activities were shown as fold changes in relation to the control (n = 4). (D) BAECs were transfected with pPPRE-TK-luc and PPARδ plasmids and then pretreated with or without GSK0660 (1 μM, 1 h) before the exposure to PCB2 (10 μM, 24 h) (n = 4). (E) HUVECs were pretreated with or without GSK0660 (1 μM, 1 h) before the exposure to PCB2 (10 μM) for 24 h. The mRNA levels of PDK4, ADRP and ANGPTL4 were assessed (n = 6). All data were expressed as mean ± SEM. *P < 0.05, **P < 0.01 vs. Vehicle; #P < 0.05, ##P < 0.01 vs. PCB2. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3
Fig. 3
Inhibition of PPARδ abrogated the suppressive effects of PCB2 on ER stress. (A) HUVECs were pretreated with or without GSK0660 (1 μM) for 1 h, then incubated with PCB2 (10 μM, 12 h) before the exposure to HG (30 mM, 24 h). Protein levels of p-PERK, PERK, ATF4, GRP78 and PDI were assessed. (B) Quantification of p-PERK/PERK, ATF4, GRP78 and PDI levels as in (A) (n = 3). (C) Protein levels of p-IRE1α, IRE1α and ATF6 were assessed. (D) Quantification of p-IRE1α/IRE1α and ATF6 levels as in (C) (n = 3). (E) The mRNA levels of CHOP, GRP78, ATF4 and ATF3 were assessed (n = 3). All data were expressed as mean ± SEM. *P < 0.05, **P < 0.01 vs. Vehicle; #P < 0.05, ##P < 0.01 vs. HG; †P < 0.05, ††P < 0.01 vs. PCB2+HG.
Fig. 4
Fig. 4
PCB2 improved vascular relaxation impaired by high glucose. C57BL/6J mouse thoracic aortic rings were pretreated with PCB2 (10 μM, 12 h) or vehicle before the exposure to HG (30 mM, 36 h) or mannitol. (A) Representative traces of the ACh-induced relaxation of the Phe-precontracted rings. The dots represented cumulative addition of increasing doses of ACh (5 × 10-10 to 10-5 M). (B) PCB2 ameliorated the ACh-induced relaxations which were impaired by HG. (C) SNP-induced relaxations were not affected by HG. (D) Alternatively, the aortic rings were treated with indomethacin (Indo, 1 μM), ODQ (3 μM) or (E)l-NAME (100 μM) or 1400 W (100 nM) for 30 min before the Phe-contraction and ACh-induced relaxation. All data were expressed as mean ± SEM. n = 5, *P < 0.05 vs. Vehicle; #P < 0.05 vs. HG; †P < 0.05 vs. PCB2+HG; NS, not significant vs. PCB2+HG.
Fig. 5
Fig. 5
PCB2 enhanced endothelium-dependent vasodilatation via PPARδ activation. Thoracic aortic rings were isolated from PPARδ WT littermates (A) or PPARδEC-/- mice (B) and pretreated with PCB2 (10 μM, 12 h) before the exposure to HG (30 mM, 36 h). ACh-induced vasodilatory responses were measured (n = 5). (C) C57BL/6J mouse aortic rings were pretreated with GSK0660 (1 μM) for 1 h, then incubated with PCB2 (10 μM, 12 h) before the exposure to HG (30 mM, 36 h) (n = 5). All data were expressed as mean ± SEM. *P < 0.05 vs. Vehicle; #P < 0.05 vs. HG; †P < 0.05 vs. PCB2+HG.
Fig. 6
Fig. 6
Inhibition of PPARδ abrogated the effects of PCB2 on NO and ROS production. ECs were pretreated with GSK0660 (1 μM) for 1 h and then treated with PCB2 (10 μM) for 12 h before exposure to HG (30 mM) for 24 h. (A) Confocal microscopic detection of superoxide with DHE. (B) The mean fluorescence intensity was evaluated. Scale bar: 25 μm (n = 3). (C) Production of ROS was measured with L-012 chemiluminescence (n = 5). (D) Summerized levels of intracellular NO production in ECs detected as DAF FM-DA signals before (F0) and after (F1) the addition of A23187 (n = 5). (E) Nitrite levels in EC supernatants was measured by using the Griess reagent (n = 4). All data were expressed as mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle; #P < 0.05, ##P < 0.01 vs. HG; †P < 0.05, ††P < 0.01 vs. PCB2+HG.
Fig. 7
Fig. 7
PCB2 increased AMPK phosphorylation via PPARδ activation. (A) HUVECs were exposed to PCB2 (10 μM) for indicated time periods. Phosphorylated-AMPK and AMPK levels were detected. (B) Quantification of p-AMPK/AMPK level as in (A) (n = 3). All data were expressed as mean ± SEM. **P < 0.01 vs. vehicle. (C) HUVECs were treated with GSK0660 (1 μM) for 1 h before the exposure to PCB2 (10 μM) for 6 h, the levels of p-AMPK and AMPK were detected. (D) Quantification of p-AMPK/AMPK level as in (C) (n = 3). All data were expressed as mean ± SEM. *P < 0.05 vs. vehicle; ##P < 0.01 vs. PCB2. (E) HUVECs were pretreated with GSK0660 (1 μM, 1 h) and, then, incubated with PCB2 (10 μM, 12 h) before the exposure to HG (30 mM, 24 h). Protein levels of p-AMPK and AMPK were measured. (F) Quantification of p-AMPK/AMPK level as in (E) (n = 3). All data were expressed as mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle; ##P < 0.01 vs. HG; †P < 0.05 vs. PCB2+HG.
Fig. 8
Fig. 8
AMPK activation mediated the PCB2/PPARδ improved endothelial function. (A) HUVECs were pretreated with compound C (CC, 20 μM, 1 h) and then incubated with PCB2 (10 μM, 12 h) before the exposure to HG (30 mM, 24 h). Protein levels of p-PERK, PERK, ATF4, GRP78 and PDI were measured. (B) Quantification of p-PERK/PERK, ATF4, GRP78 and PDI levels as in (A) (n = 3). (C) Protein levels of p-IRE1α, IRE1α and ATF6 were assessed. (D) Quantification of p-IRE1α/IRE1α and ATF6 levels as in (C) (n = 3). C57BL/6J mouse thoracic aortic rings were pretreated with CC (20 μM, 1 h) and, then, incubated with PCB2 (10 μM, 12 h) before the exposure to HG (30 mM, 36 h). (E) ACh-induced endothelium-dependent and (F) SNP-induced endothelium-independent relaxations were measured (n = 5). All data were expressed as mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle; #P < 0.05, ##P < 0.01 vs. HG; †P < 0.05, ††P < 0.01 vs. PCB2+HG. (G) The proposed mechanisms: PCB2 activated PPARδ-AMPK to prevent ER stress and ameliorated endothelial dysfunction against high glucose.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Walter P., Ron D. The unfolded protein response: from stress pathway to homeostatic regulation. Science. 2011;334:1081–1086. - PubMed
    1. Hetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat. Rev. Mol. Cell Biol. 2012;13:89–102. - PubMed
    1. Battson M.L., Lee D.M., Gentile C.L. Endoplasmic reticulum stress and the development of endothelial dysfunction. Am. J. Physiol. Heart Circ. Physiol. 2017;312:H355–H367. - PubMed
    1. Hotamisligil G.S. Endoplasmic reticulum stress and the inflammatory basis of metabolic disease. Cell. 2010;140:900–917. - PMC - PubMed
    1. Civelek M., Manduchi E., Riley R.J., Stoeckert C.J., Davies P.F. Chronic endoplasmic reticulum stress activates unfolded protein response in arterial endothelium in regions of susceptibility to atherosclerosis. Circ. Res. 2009;105 453-U127. - PMC - PubMed

Publication types

LinkOut - more resources