The protective effects of orexin a against high glucose-induced activation of NLRP3 inflammasome in human vascular endothelial cells

Accepted Manuscript The protective effects of orexin a against high glucose-induced activation of NLRP3 inflammasome in human vascular endothelial cells Chengxi Zhang, Mamately Abdukerim, Mulati Abilailieti, Leile Tang, Yesheng Ling, Sinian Pan PII:

S0003-9861(19)30383-2

DOI:

https://doi.org/10.1016/j.abb.2019.07.017

Reference:

YABBI 8052

To appear in:

Archives of Biochemistry and Biophysics

Received Date: 21 May 2019 Revised Date:

3 July 2019

Accepted Date: 23 July 2019

Please cite this article as: C. Zhang, M. Abdukerim, M. Abilailieti, L. Tang, Y. Ling, S. Pan, The protective effects of orexin a against high glucose-induced activation of NLRP3 inflammasome in human vascular endothelial cells, Archives of Biochemistry and Biophysics (2019), doi: https://doi.org/10.1016/ j.abb.2019.07.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Title: The protective effects of orexin A against high glucose-induced activation of NLRP3 inflammasome in human vascular endothelial cells Authors: Chengxi Zhang1, Mamately Abdukerim2, Mulati Abilailieti2, Leile Tang1,

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Yesheng Ling1, Sinian Pan3 Affiliations: 1.

Department of Cardiology, the Third Affiliated Hospital, Sun Yat-Sen University,

2.

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Guangzhou, 510630, China

Department of Cardiology, the First People’s Hospital of Xinjiang Kashi Area,

3.

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Kashi, 844000, Xinjiang, China

Department of Pediatrics, the Third Affiliated Hospital, Sun Yat-Sen University,

Guangzhou 510630, China

Sinian Pan

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Corresponding to:

Department of Pediatrics,

the Third Affiliated Hospital, Sun Yat-Sen University,

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Guangzhou 510630, China

Address: No.600 Tianhe Road, Tianhe District, Guangzhou city, Guangdong

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Province, 510630, China

Tel/Fax: +086-020-82179710 Email: [email protected]

ACCEPTED MANUSCRIPT Abstract Vascular disease is one of the most significant threats to the lives of patients suffering from diabetes, and chronic exposure of vascular endothelial cells to high glucose has been shown to significantly contribute to the process of endothelial cell dysfunction,

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one of the earliest events in diabetes-associated vascular disease. Nucleotide oligomerization domain (NOD)-like receptor pyrin domain-containing 3 (NLRP3) inflammasome plays a key role in initiating the inflammatory process by facilitating the production of interleukin-1β (IL-1β) and IL-18. ASC and caspase 1 are also

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implicated in NLRP3 inflammasome-mediated chronic inflammation. While under normal conditions, a balance exists between oxidants and antioxidants, exposure to

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high glucose significantly increases the production of ROS, which is enhanced by NOX4 expression. In the present study, we explored the role of orexin A, an endogenous peptide produced in the hypothalamus, in high glucose-induced activation of the NLRP3 inflammasome, oxidative stress, and expression of several key cytokines. Our findings demonstrate that orexin A exerts potent antioxidant effects in

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human aortic endothelial cells exposed to high glucose by inhibiting mitochondrial ROS and expression of NOX4 at both the mRNA and protein levels as revealed by MitoSOX staining, real-time PCR, and western blot analysis. We also show that

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orexin A inhibits high glucose-induced expression of TxNIP, which is crucial to the activation of the NLRP3 inflammasome, as well as that of HMGB1. We confirmed via

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real-time PCR and western blot analysis that orexin A suppressed the production of the inflammatory cytokines IL-1β and IL-18. Additionally, through SIRT1 knockdown siRNA experimentation, we confirmed that SIRT1 knockdown abolishes the effects of orexin A described above, thereby indicating a critical role of SIRT in the capacity of orexin A to ameliorate high glucose-induced oxidative stress and activation of NLRP3 inflammasome. Keywords: NLRP3 inflammasome; orexin A; endothelial dysfunction; high glucose; SIRT1; IL-1β; IL-18; HMGB1; TxNIP

ACCEPTED MANUSCRIPT 1. Introduction Increased levels of glucose are a major contributor to the development of type II diabetes as well as various associated pathologies, including diabetes-related vascular disease. Vascular disease resulting from exposure to high glucose is the leading cause

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of death among type II diabetes patients [1; 2]. The early stage of diabetes-related vascular disease is characterized by endothelial dysfunction, chronic inflammation, and oxidative stress, leading to loss of vasoconstrictive function of the endothelium. In part, this is due to impaired signal transduction and an imbalance in the expression

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of endothelial constricting factors and endothelium-derived relaxing factor [3]. Numerous studies have shown that high glucose induces increased production of

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reactive oxygen species (ROS), as well as NADPH oxidase-4 (NOX-4). [4; 5; 6]. NOX-4 further increases the production of ROS and inhibition of NOX-4 expression has been suggested as a potential treatment strategy against endothelial dysfunction [7; 8].

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Activation of the nucleotide oligomerization domain (NOD)-like receptor pyrin domain-containing 3 (NLRP3) inflammasome is regarded as a critical step in the initiation of endothelial inflammation. The NLRP3 inflammasome regulates and

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drives the production of interleukin-1β (IL-1β) and IL-18, two important proinflammatory cytokines involved in the initiation and prolongment of endothelial

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damage and dysfunction. Specifically, the NLRP3 inflammasome manufactures pro-IL-1β/pro-IL-18 and induces the activation of caspase-1. Caspase-1 triggers the production of mature IL-1β and IL-18 by cleaving pro-IL-1β and pro-IL-18, which then initiate the inflammatory response [9;10]. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) plays a key role in NLRP3 activation by recruiting caspase 1 to the NLR protein complex [11]. Inhibition of activation of the NLRP3 inflammasome has been proposed as a potential treatment strategy against IL-1β- and IL-18-induced diseases including diabetes-associated vascular disease [12;13;14]. Thioredoxin-interacting protein (TxNIP) is intimately involved in activating the NLRP3 inflammasome and contributes to oxidative stress

ACCEPTED MANUSCRIPT by downregulating the expression of the antioxidant thioredoxin [15]. The class III histone deacetylase sirtuin 1 (SIRT1) plays an inhibitory role against activation of the NLRP3 inflammasome in endothelial cells [16]. Exposure to high glucose has been shown to downregulate SIRT1, thereby inhibiting its protective effect against NLRP3

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inflammasome-induced endothelial dysfunction [17]. Therefore, preventing inhibition of SIRT1 is also considered a promising strategy against high glucose-induced endothelial dysfunction [18].

Orexin A, also known as hypocretin 1, is a naturally occurring neuropeptide that has

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been demonstrated to regulate key physiological functions including wakefulness, cognitive function, diet, and mood. Animal models have demonstrated the

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involvement of orexin A in obesity [19]. Small molecules targeting orexin receptors are viewed as a promising treatment for several diseases including diabetes [20]. Importantly, treatment with orexin A has also been shown to be involved in regulating cardiovascular function [21]. However, there is little to no existing research regarding the role of orexin A in the activation process of the NLRP3 inflammasome. In the

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present study, we examined the potential of orexin A as an inhibitor of high glucose-induced NLRP3 inflammasome activation in human vascular endothelial cells. Our findings demonstrate that orexin A may indeed serve as a potential therapeutic

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strategy against diabetes-associated vascular disease by mitigating endothelial dysfunction caused by increased expression of proinflammatory cytokines and

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oxidative stress. We also provide evidence of the involvement of SIRT1 in mediating these beneficial effects.

2. Materials and methods 2.1 Cell culture and treatment The primary human coronary endothelial cells (HAECs) used in this study were purchased from Lonza, Switzerland. was Endothelial Cell Medium (Lonza,

ACCEPTED MANUSCRIPT Switzerland) supplemented with endothelial cell growth factor and 10% fetal bovine serum (Gbico, USA). 1×106 Cells were plated onto 25 cm2 flasks and 6 well-plates (TPP, Trasadigen, Switzerland) and cultured at 37°C in a humidified atmosphere at 5% CO2. When the cells were subcultured, they were splitted by treatment with trypsin.

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HAECs were stimulated with 25 mM high glucose in the presence or absence of 2.5 and 5 µM orexin A (Sigma-Adrich, USA) for 24 h. 2.2 MitoSOX Red staining

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After the necessary treatment, the level of mitochondrial ROS was determined by staining HAECs with MitoSOX Red (Life technology, USA) dye. Briefly, cells were

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exposed to 25 mM high glucose in the presence or absence of 2.5 and 5 µM orexin A for 24 h, then washed 3 times with PBS buffer. HAECs were then loaded with 5 µM MitoSOX Red in darkness at a temperature of 37 °C for 30 min. A fluorescence microscope (Zeiss, Germany) was used to detect fluorescent signals with an excitation wavelength at 510 nM and emission wavelength at 580 nM.

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2.3 Real-time polymerase chain reaction (PCR) analysis For the extraction of total RNA, HAECs were stimulated with 25 mM high glucose in

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the presence or absence of 2.5 and 5 µM orexin A for 24 h. The cells were harvested, and total RNA was extracted using Qiazol Reagent (Life Technology, USA) and A NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific,

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stored at -80 ℃.

USA) was used to assess the extracted RNA. Then, a total of 1 µg of isolated RNA was used to synthesize cDNA via reverse transcription PCR (RT-PCR) analysis. The SYBR Green Master Mix method was then used to detect target gene expression via real-time

PCR.

The

results

are

presented

as

normalized

to

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the 2-ΔΔCt method. The following

primers

were

used

in

this

study:

NOX-4

(for:

5’-CTTTTGGAAGTCCATTTGAG-3’; rev: 5’ -GTCTGTTCTCTTGCCAAAAC-3’); TXNIP

(for:

5’-

CAGCCTACAGCAGGTGAGAAC-3’;

rev:

5’-

ACCEPTED MANUSCRIPT CTCATCTCAGAGCTCGTCCG-3’);

SIRT1

(for:

5’-TCACCACCAGATTCTTCAGTG-3’; 5’-CCTCTTGATCATCTCCATCAGTC-3’);

rev: human

GAPDH,

(for:

5’-CCACATCGCTCAGACACCAT-3’; rev: 5’-CCAGGCGCCCAATACG-3’).

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2.4 Western blot analysis

Protein expression of target genes was assessed using western blot analysis. After the indicated treatment, HAECs were lysed using RIPA buffer (50 mM Tris-HCl at pH 7.4,

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150 mM NaCl, 0.1% SDS,1 mM Na2EDTA, 1 mM EGTA,1% NP-40, 1% sodium deoxycholate) containing protease inhibitor cocktail. Perotein lysates were stored at

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-80℃. Then, equal amounts of cell lysates from each group were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, USA). Cells were then washed 3 times with TBST and blocked with 5% nonfat dry milk. Membranes were probed with antibodies on a shaker overnight at 4 °C and

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HRP-conjugated antibody for 1 h at RT. An electrochemiluminescence (ECL) kit (Millipore, USA) was used to detect the immunoblot bands on the PVDF membranes. The following primary and secondary antibodies were used in this study: NOX4

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(1:2000, # ab154244, Abcam, USA); TXNIP (1:1000, # ab231966, Abcam, USA); NLRP3 (1:1000, # ab214185, Abcam, USA); ASC (1:1000, #67824, Cell Signaling

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Technology, USA); Cleaved caspase-1 p10 (1:500, #89332, Cell Signaling Technology, USA); SIRT1 (1:2000, # AF7714, R&D Systems, USA); β-actin (1:10000, #3700, Cell Signaling Technology, USA); anti-rabbit IgG, HRP-linked secondary antibody (1:3000, #7074, Cell Signaling Technology, USA); anti-mouse IgG, HRP-linked antibody (1:3000, #7076, Cell Signaling Technology, USA). 2.5 Enzyme-linked immunosorbent assay (ELISA) For measurement of IL-1β and IL-18, HAECs culture media was collected for analysis. ELISA kits were purchased from R&D Systems. Data were collected by

ACCEPTED MANUSCRIPT 96-plate reader spectrometry, and the results were normalized to total protein extracts, represented by fold change. 2.6 SIRT1 knockdown

SIRT1-specific

DNA

sequence.

5’-CCACCUGAGUUGGAUGAUA-3’)

SIRT1 was

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For our SIRT1 knockdown experiments, we used a SIRT1 siRNA to target the siRNA

transfected

into

(siSIRT1:

cells

using

Lipofectamine RNAi Max reagent (Life technology, USA). The transfected cells were

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cultured for 48-72 h, and successful knockdown was confirmed by blotting SIRT1

2.7 Statistical analysis

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protein with its specific antibody.

Experimental data are expressed as means ± standard deviation (S.D.). All the experiments have been repeated for 3 times. Resutls were statistically analyzed using SPSS 17.0 software (SPSS, Inc., US). Analysis of variance (ANOVA) and the

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Mann-Whitney U-test were used to compare differences between groups. A P value of

3. Results

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less than 0.05 was considered to represent statistical significance.

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3.1 Orexin A ameliorates high glucose-induced oxidative stress First, we investigated the effects of treatment with orexin A on HAECs exposed to high glucose conditions for 24 h. As shown in Figure 1, high glucose induced a significant increase in the production of ROS, raising the level of ROS to 3.5-fold baseline. However, treatment with 2.5 and 5 µM orexin A reduced the level of high glucose-induced ROS production to only 2.3- and 1.7-fold, respectively. We also determined the effects of orexin A treatment on the production of NOX4 induced by high glucose. As demonstrated by the results of real-time PCR and western blot

ACCEPTED MANUSCRIPT analyses in Figure 2, high glucose increased the expression of NOX4 to 4.3-fold at the mRNA level and 3.6-fold at the protein level. However, treatment with the two doses of orexin A reduced the mRNA expression of NOX4 to only 2.9- and 1.8-fold, respectively, while the protein expression of NOX4 was reduced to 2.3- and 1.7-fold,

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respectively. 3.2 Orexin A suppresses high glucose-induced expression of TxNIP and HMGB1

Next, we measured the effects of orexin A on high glucose-induced expression of

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TxNIP. Briefly, cells were exposed to high glucose for 24 h in the presence or absence of 2.5 and 5 µM orexin A. As demonstrated by the results of real-time PCR in Figure

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3 exposure to 25 mM high glucose resulted in 4.2-fold expression of TxNIP at the mRNA level, which was reduced to 2.7- and 1.87-fold by orexin A in a dose-dependent manner. The results of western blot analysis in Figure 3 show that high glucose increased the protein expression of TxNIP to 3.4-fold, which was reduced to 2.3- and 1.6-fold by orexin A in a dose-dependent manner. To investigate

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the effects of orexin A on high glucose-induced expression of high mobility group box 1 protein (HMGB1), cells were treated as described above and secretion of HMGB1 was assessed via western blot analysis. As shown by the results in Figure 4, high

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glucose increased the secretion of HMGB1 o 3.4-fold, which was reduced by orexin A in a dose-dependent manner to only 2.3- and 1.5-fold, respectively.

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3.3 Orexin A inhibits activation of NLRP3 inflammasome Western blot analysis was used to determine the effects of orexin A on high glucose-induced activation of the NLRP3 inflammasome as well as the release of ASC and cleaved caspase 1. As shown in Figure 5, high glucose increased the protein level of NLRP3 to 3.8-fold, which was reduced by orexin A in a dose-dependent manner to 2.4- and 1.6-fold. The release of ASC was increased to 2.9-fold by exposure to high glucose, which was reduced to only 2.1- and 1.5-fold by orexin A in a dose-dependent manner. High glucose increased the expression of cleaved caspase 1

ACCEPTED MANUSCRIPT (P10) to 3.5-fold, which was reduced by orexin A in a dose-dependent manner to only 2.3- and 1.7-fold, respectively. 3.4 Orexin A inhibits the production of IL-1β and IL-18

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Next, we confirmed the inhibition of NLRP3 inflammasome activation by determining the levels of IL-1β and IL-18, the production of which requires the NLRP3 inflammasome. As shown in Figure 6, the results of ELISA show that exposure to high glucose significantly increased the expression of these two cytokines

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to 5.3- and 4.6-fold, respectively. However, treatment with orexin A reduced the protein expression of IL-1β to only 3.1- and 1.9-fold and the protein expression of

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IL-18 to only 2.5- and 1.6-fold, indicating a significant decrease in the NLRP3 inflammasome-driven production of these inflammatory cytokines and confirming our finding that orexin A mitigates high glucose-induced NLRP3 inflammasome activation.

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3.5 The effects of orexin A are mediated through SIRT1

Since SIRT1 is an important inhibitor of NLRP3 inflammasome activation, we next determined the involvement of SIRT1 in mediating the effects of orexin A against

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high glucose-induced NLRP3 inflammasome activation. As shown in Figure 7, the results of real-time PCR indicate that high glucose suppressed the mRNA expression

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of SIRT1 to only roughly 36% baseline, which was remarkably rescued by treatment with the two doses of orexin A to 57% and 93%, respectively. Concordantly, the results of western blot analysis show that high glucose reduced the protein expression of SIRT1 to only 43% baseline, which was rescued to 69% and 91% baseline by the two respective doses of orexin A. Finally, to confirm that the beneficial effects of orexin A against high glucose-induced activation of the NLRP3 inflammasome are mediated through SIRT1, we performed a SIRT1 knockdown experiment. HAECs were transfected with SIRT1 siRNA and successful SIRT1 knockdown was confirmed via western blot analysis (Figure 8A). As shown in Figure 8B, blockage of SIRT1

ACCEPTED MANUSCRIPT completely abolished the inhibitory effects of orexin A on NLRP3 inflammasome activation, expression of ASC, and the level of cleaved caspase 1 (P10). Additionally, the results in Figure 8C demonstrate that knockdown of SIRT1 also abolished the inhibitory effect of orexin A on IL-1β and IL-18 expression. Thus, SIRT1 is essential

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for mediating the effects of orexin A against high glucose-induced NLRP3 inflammasome activation. 4. Discussion

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Oxidative stress is known to be a crucial factor in the pathogenesis of various diseases, including diabetes and diabetes-associated vascular disease. Recent research has

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demonstrated that ROS contributes to NLRP3 inflammasome-mediated pyroptosis, a particularly inflammatory form of apoptosis, by acting as a “kindling” effector that triggers NLRP3 inflammasome activation in aortic endothelial cells. Additionally, once the NLRP3 inflammasome is activated, chemokines recruit immune cells into the endothelial wall, which then greatly exacerbates the production of ROS and

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induces a state of severe oxidative stress. These ROS are referred to as “bonfire” ROS [22]. A recent study showed that NOX4 is involved in high glucose-induced generation of ROS, while NOX2 is not. This study also showed that treatment with

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rutin and a NOX4 inhibitor significantly ameliorated high glucose-induced generation of TxNIP, NLRP3, caspase-1, and IL-1β [23]. In line with these findings, our

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experimental results demonstrate that orexin A significantly reduced NOX4 and ROS, which resulted in a similar inhibition of TxNIP expression, NLRP3 inflammasome activation, and activation of caspase-1, IL-1β, and IL-18, thereby exerting a powerful anti-inflammatory and antioxidative stress effect in HAECs exposed to high glucose. HMGB1 is closely associated with NLRP3 inflammasome activation and serves as a facilitator of endothelial permeability [24;25]. Additionally, HMGB1 has been shown to promote vascular expression of IL-1β through NLRP3 inflammasome activation, thereby playing a key role in promoting vascular endothelial cell dysfunction [26]. Concordant with our findings, high glucose has been shown to significantly increase the release of HMGB1 and modulating the expression of HMGB1 has been

ACCEPTED MANUSCRIPT considered

as

a

potential

therapeutic

strategy

against

diabetes-associated

inflammation and oxidative stress in vascular disease [27]. Here, we show that orexin A significantly ameliorated the expression of HMGB1 induced by high glucose conditions.

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Activation of the NLRP3 inflammasome plays a critical role in driving endothelial dysfunction induced by exposure to high glucose by upregulating the production of IL-1β and IL-18, two key proinflammatory cytokines. The role of IL-1β in diabetes-associated vascular disease has been studied extensively, but a reliable

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treatment method against IL-1β-mediated chronic inflammation remains elusive. A recent review demonstrated the importance of counteracting overproduction of IL-1β

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by the NLRP3 inflammasome in diabetes-associated vascular complications and highlighted several treatment approaches focused on IL-1β antagonism [28]. Increased levels of IL-18 are associated with the pathogenesis of cardiovascular disease and atherosclerosis through its enhancement of proinflammatory cytokine production and expression of vascular adhesion molecules [29]. Numerous studies

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have suggested inhibition of IL-1β and IL-18 production as a means to combat endothelial dysfunction, including through inhibition of caspase 1 [30;31;32]. While antagonism of IL-1β and IL-18 may be a promising therapeutic approach against the

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sequelae of high glucose-mediated chronic inflammation, our aim was to inhibit IL-1β and IL-18 production by suppressing activation of the NLRP3 inflammasome. As

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demonstrated by our results, orexin A significantly inhibited activation of the NLRP3 inflammasome and subsequent production of IL-1β and IL-18. Notably, we also demonstrated the involvement of SIRT1 in the NLRP3 inflammasome-mediated anti-inflammatory and antioxidative stress capacity of orexin A. As a key inhibitor of NLRP3 inflammasome activation, SIRT1 is considered a valuable therapeutic target. SIRT1 has also been shown to inhibit the release of HMGB1 and, concordant with our results, is significantly downregulated in response to high glucose [33]. Recent research suggests that modulation of SIRT1 may serve as a promising strategy against NLRP3 inflammasome-mediated pathology in high glucose-induced endothelial

ACCEPTED MANUSCRIPT dysfunction and cardiovascular disease [34]. Here, we confirmed that orexin A rescues high glucose-induced reduced SIRT1 expression, thereby inhibiting activation of the NLRP3 inflammasome and subsequent production of IL-1β, IL-18, ASC, and caspase 1. Mechanistically, the results of our SIRT1 knockdown experiment revealed

effects of orexin A on NLRP3 inflammasome activation.

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that SIRT1 knockdown via transfection with SIRT1 siRNA abolished the inhibitory

In conclusion, the present study revealed the potential of orexin A, an endogenous peptide produced in the hypothalamus, to inhibit high glucose-induced oxidative

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stress via suppression of NOX4 and ROS production in HAECs. Additionally, orexin A can potently inhibit inflammation by downregulating the expression of TxNIP,

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HMGB1, and activation of the NLRP3 inflammasome, thereby reducing the production of IL-1β and IL-18, two key proinflammatory cytokines. Mechanistically, we demonstrate that knockdown of the NLRP3 inhibitor SIRT1 abolished the effects of orexin A, thereby revealing that SIRT1 is a necessary mediator of the beneficial effects of orexin A. Further research is required to fully elucidate the role of orexin A

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in high glucose-induced endothelial dysfunction.

None

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[31] Krishnan SM, Sobey CG, Latz E, Mansell A, Drummond GR. IL‐1β and IL‐18: inflammatory markers or mediators of hypertension?. British journal of pharmacology. 2014;171(24):5589-602.

[32] Chvatchko Y, Tedgui A, Mallat Z, inventors; Merck Serono SA, assignee. Use of

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IL-18BP for treatment of peripheral vascular diseases. United States patent US 9,592,267. 2017 Mar 14.

[33] Liu L, Patel P, Steinle JJ. PKA regulates HMGB1 through activation of IGFBP-3

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and SIRT1 in human retinal endothelial cells cultured in high glucose. Inflammation

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Research. 2018;67(11-12):1013-9. [34] Jiang T, Jiang D, Zhang L, Ding M, Zhou H. Anagliptin ameliorates high glucose-induced endothelial dysfunction via suppression of NLRP3 inflammasome activation mediated by SIRT1. Molecular immunology. 2019;107:54-60. Legends Figure 1. Orexin A suppresses high glucose-induced production of mitochondrial ROS in human aortic endothelial cells (HAECs). Cells were treated with 25 mM high glucose with or without Orexin A (2.5 and 5 µM) for 24 h. Mitochondrial ROS was

ACCEPTED MANUSCRIPT determined by MitoSox Red (*, #, $, P<0.01 vs. previous column group, N=5 for each experiment). Figure 2. Orexin A inhibits high glucose-induced expression of NADPH oxidase 4 (NOX-4) in human aortic endothelial cells (HAECs). Cells were treated with 25 mM

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high glucose with or without Orexin A (2.5 and 5 µM) for 24 h. (A). mRNA of NOX-4 as determined by real-time PCR; (B). Protein of NOX-4 as determined by western blot analysis (*, #, $, P<0.01 vs. previous column group, N=5 for each

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experiment).

Figure 3. Orexin A inhibits high glucose-induced expression of TxNIP in human

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aortic endothelial cells (HAECs). Cells were treated with 25 mM high glucose with or without Orexin A (2.5 and 5 µM) for 24 h. (A). mRNA of TxNIP as determined by real-time PCR; (B). Protein of TxNIP as determined by western blot analysis (*, #, $, P<0.01 vs. previous column group , N=5 for each experiment). Figure 4. Orexin A inhibits high glucose-induced secretion of high mobility group

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box-1 (HMGB-1) in human aortic endothelial cells (HAECs). Cells were treated with 25 mM high glucose with or without orexin A (2.5 and 5 µM) for 24 h. Secretion of

experiment).

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HMGB-1 was assessed (*, #, $, P<0.01 vs. previous column group, N=5 for each

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Figure 5. Orexin A inhibits high glucose-induced NLRP3 inflammasome in human aortic endothelial cells (HAECs). Cells were treated with 25 mM high glucose with or without Orexin A (2.5 and 5 µM) for 24 h. (A). Protein levels of NLRP3; (B). Protein levels of ASC; (C). Protein levels of cleaved caspase 1 (P10) (*, #, $, P<0.01 vs. previous column group, N=5 for each experiment). Figure 6. Orexin A inhibits high glucose-induced maturation of IL-1β and IL-18. Cells were treated with 25 mM high glucose with or without Orexin A (2.5 and 5 µM) for 24 h. (A). Protein of IL-1β as determined by ELISA; (B). Protein of IL-18 as determined by ELISA (*, #, $, P<0.01 vs. previous column group, N=5 for each

ACCEPTED MANUSCRIPT experiment). Figure 7. Orexin A restores high glucose-induced decrease in the expression of SIRT1. Cells were treated with 25 mM high glucose with or without Orexin A (2.5 and 5 µM) for 24 h. (A). mRNA of SIRT1 as determined by real-time PCR; (B). Protein of SIRT1

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as determined by western blot analysis (*, #, $, P<0.01 vs. previous column group, N=5 for each experiment).

Figure 8. Inhibition of SIRT1 abolished the inhibitory effects of Orexin A in NLRP3

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inflammasome activation. Cells were transfected with SIRT1 siRNA. Non-specific negative siRNA (NS) was used as a negative control. At 24 h post transfection, cells

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were stimulated with high glucose (25 mM) with or without Orexin A (5 µM) for 24 h. (A). Western blot analysis revealed the successful knockdown of SIRT1; (B). Blockage of SIRT1 abolished the inhibitory effects of Orexin A in the expressions of NLRP3, ASC, and P10; (C). Knockdown of SIRT1 abolished the inhibitory effects of Orexin A on IL-1β and IL-18 secretion (*, #, $, P<0.01 vs. previous column group,

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