Pharmacological Research 99 (2015) 101–115

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Ilexgenin A inhibits endoplasmic reticulum stress and ameliorates endothelial dysfunction via suppression of TXNIP/NLRP3 inflammasome activation in an AMPK dependent manner Yi Li a,b , Jie Yang a,b , Mei-Hong Chen a,b , Qiang Wang a,b , Min-Jian Qin a , Tong Zhang a , Xiao-Qing Chen c , Bao-Lin Liu a , Xiao-Dong Wen a,∗ a

State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China Department of Chinese Medicines Analysis, China Pharmaceutical University, Nanjing 210009, China c School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China b

a r t i c l e

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Article history: Received 1 April 2015 Received in revised form 28 May 2015 Accepted 28 May 2015 Available online 6 June 2015 Chemical compounds studied in this article: Acetylcholine (PubChem CID: 75271) AICA riboside (PubChem CID: 17513) BAPTA-AM (PubChem CID: 2293) Compound C (PubChem CID: 11524144) Dimethyl sulfoxide (PubChem CID: 679) Mitoquinone mesylate (PubChem CID: 11388331) Palmitic acid (PubChem CID: 985) Phenylephrine (PubChem CID: 5284443) Tauroursodeoxycholic acid (PubChem CID: 46782978) Thapsigargin (PubChem CID: 446378) Keywords: Ilexgenin A AMPK Endothelial dysfunction Endoplasmic reticulum stress NLRP3 inflammasome

a b s t r a c t Ilexgenin A is a natural triterpenoid with beneficial effects on lipid disorders. This study aimed to investigate the effects of ilexgenin A on endothelial homeostasis and its mechanisms. Palmitate (PA) stimulation induced endoplasmic reticulum stress (ER stress) and subsequent thioredoxin-interacting protein (TXNIP)/NLRP3 inflammasome activation in endothelial cells, leading to endothelial dysfunction. Ilexgenin A enhanced LKB1-dependent AMPK activity and improved ER stress by suppression of ROS-associated TXNIP induction. However, these effects were blocked by knockdown of AMPK␣, indicating AMPK is essential for its action in suppression of ER stress. Meanwhile, ilexgenin A inhibited NLRP3 inflammasome activation by down-regulation of NLRP3 and cleaved caspase-1 induction, and thereby reduced IL-1␤ secretion. It also inhibited inflammation and apoptosis exposed to PA insult. Consistent with these results in endothelial cells, ilexgenin A attenuated ER stress and restored the loss of eNOS activity in vascular endothelium, and thereby improved endothelium-dependent vasodilation in rat aorta. A further analysis in high-fat fed mice showed that oral administration of ilexgenin A blocked ER stress/NLRP3 activation with reduced ROS generation and increased NO production in vascular endothelium, well confirming the beneficial effect of ilexgenin A on endothelial homeostasis in vivo. Taken together, these results show ER stress-associated TXNIP/NLRP3 inflammasome activation was responsible for endothelial dysfunction and ilexgenin A ameliorated endothelial dysfunction by suppressing ER-stress and TXNIP/NLRP3 inflammasome activation with a regulation of AMPK. This finding suggests that the application of ilexgenin A is useful in the management of cardiovascular diseases in obesity. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Abbreviations: ACC, acetyl-CoA carboxylase; ACh, acetylcholine; AICAR, AICA riboside; AMPK, AMP-activated protein kinase; ATF, activating transcription factor; ER, endoplasmic reticulum; FBS, fetal bovine serum; FFAs, free fatty acids; HFD, high-fat diet; IRE1␣, inositol-requiring enzyme-1␣; Mito Q, mitoquinone mesylate; NAFLD, nonalcoholic fatty liver disease; NLRP3, NOD-like receptor family, pyrin domain containing 3; PA, palmitate; PERK, RNA-dependent protein kinase like ER kinase; ROS, reactive oxygen species; TG, thapsigargin; TRX, thioredoxin; TUDCA, tauroursodeoxycholic acid; TXNIP, thioredoxin-interacting protein; UPR, unfolded protein response; VCAM-1, vascular cell adhesion protein 1; VECs, vascular endothelial cells. ∗ Corresponding author. Tel.: +86 13952095836. E-mail address: [email protected] (X.-D. Wen). 1043-6618/© 2015 Elsevier Ltd. All rights reserved.

The endoplasmic reticulum (ER) is not only a site for lipid synthesis, protein folding and maturation in eukaryotic cells, but also a major signal-transducing organelle that senses cellular stress [1]. Under stress conditions, the accumulation of newly synthesized proteins leads to the unfolded protein response (UPR), which is associated with inductions of transmembrane sensors/transducers, including inositol-requiring enzyme-1␣ (IRE1␣), RNA-dependent protein kinase-like ER kinase (PERK), and activating transcription factor 6 (ATF6) [2,3]. Although UPR is an attempt to restore cellular homeostasis, the altered UPR, namely endoplasmic reticulum


Y. Li et al. / Pharmacological Research 99 (2015) 101–115

stress (ER stress), can trigger a stress cascade with pathological consequences including oxidative stress, inflammation and apoptosis in specialized cells and tissues [4,5]. Recently it has been shown that thioredoxin-interacting protein (TXNIP) is necessary for the inflammasome activation in pancreatic ␤ cells by the protein ER stress-induced NOD-like receptor family, pyrin domain containing 3 (NLRP3) [6]. As suggested as its name, TXNIP is a regulatory protein of thioredoxin (TRX) by binding to TRX and functions as an oxidative stress mediator through inhibiting TRX activity, and thereby promoting oxidative stress as well as perturbing the activities of proteins that rely on the presence of TRX, such as NLRP3 inflammasome [7,8]. NLRP3 inflammasome is critical for the regulation of the innate immune and inflammatory responses. In response to oxidative stress, TXNIP dissociates from TRX and then induces NLRP3 inflammasome activation, which promotes IL-1␤ maturation and secretion in a caspase-1-dependent manner [6,9]. The resulting maturation of IL-1␤ is considered to be responsible for inflammation and cell apoptosis. These findings elucidate the important role of NLRP3 inflammasome activation in ER stress-induced inflammation and cell apoptosis. Lipid disorders are tightly associated with endothelial dysfunction which is the principal cause of the initial lesion in the vessel. Although oxidative stress, inflammation, cell apoptosis and impaired vasodilation are manifested in endothelial dysfunction, new observations suggested that ER stress is a principle cause for the initial lesion in the endothelium. UPR activation is increased in endothelial cells of obese adults with impaired endothelial function [10]; Endothelium in atherosclerosis susceptible regions shows activation of ER stress and the altered expression of proinflammatory markers and oxidant/antioxidant pathways [11]. Recently, it has been proved that TXNIP induction and NLRP3 inflammasome activation are implicated in vascular dysfunction. TXNIP promotes endothelial cell inflammation in response to disturbed flow [12] and induces apoptosis in vascular pericytes exposed to high glucose [13]; TXNIP/NLRP3 activation is also documented to be responsible for inflammation and cell death in endothelial cells [14]. Despite the functional interaction between ER stress and NLRP3 in inflammasome activation in the endothelium remains to be elucidated, these events raise the possibility that ER stress disturbs endothelial homeostasis through TXNIP/NLRP3 inflammasome activation. ShanLv Cha, the leaves of Ilex hainanensis Merr. are commonly used as a folk remedy for the treatment of lipid disorders and cardiovascular diseases in the south of China. Moreover, ShanLv Cha tablets, a preparation in China pharmacopeia 2010 edition, have significant therapeutic effect on hypertension and hyperlipidemia. Chemical investigation on ShanLv Cha revealed ilexgenin A (3␤, 19␣-dihydroxyurs-12-ene-24, 28-dioic acid) is the main triterpenoid in this herb. Pharmacological studies showed the extract from ShanLv Cha reduced the blood lipids and prevents nonalcoholic fatty liver disease (NAFLD) in high-fat diet rats, and our previous work further revealed ilexgenin A is the predominant constituent responsible for these beneficial effects [15–17]. Ilexgenin A prevents lipid disorders, but up to now, little is known about its potential implication in the improvement of vascular function. In view of the involvement of lipid disorders in endothelial dysfunction, we hypothesized that the beneficial action of ilexgenin A in the regulation of lipid metabolism contributes to protecting endothelial function against the challenge of lipid load. To test this hypothesis, we investigated the effects of ilexgenin A on endothelial homeostasis by focusing on the regulation of TXNIP/NLRP3 inflammasome activation in the setting of ER stress condition. We stimulated endothelial cells with palmitate (PA) to induce ER stress and showed that ilexgenin A ameliorated endothelial dysfunction by the suppression of TXNIP/NLRP3 inflammasome activation and required activation of the AMP-activated protein kinase (AMPK).

We expect this new information will contribute to the design of novel strategies for the application of ilexgenin A in the management of cardiovascular diseases in which lipid disorders were involved. 2. Materials and methods 2.1. Materials Ilexgenin A (purity ≥ 98%) was obtained from Chengdu Preferred Biological Technology Co., Ltd., Chengdu, China. Palmitate (PA, Sinopharm Chemical Rwagent Co., Ltd., shanghai, China) was dissolved in absolute ethanol as a stock solution and then was further diluted with medium containing 10% of bovine serum albumin (BSA) before use. AICA riboside (AICAR) and mitoquinone mesylate (Mito Q) were provided by Beyotime Institute of Biotechnology (Shanghai, China). BAPTA-AM was obtained from TCI (Shanghai) Development Co., Ltd., Shanghai, China. Compound C, thapsigargin (TG) and tauroursodeoxycholic acid (TUDCA) were purchased from Sigma (St. Louis, MO, USA). These agents were dissolved in dimethyl sulfoxide (DMSO) as a stock solution and the final working concentration of DMSO was

NLRP3 inflammasome activation in an AMPK dependent manner.

Ilexgenin A is a natural triterpenoid with beneficial effects on lipid disorders. This study aimed to investigate the effects of ilexgenin A on endoth...
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