Exendin-4 regulates endoplasmic reticulum stress to protect endothelial progenitor cells from high-glucose damage

Journal Pre-proof Exendin-4 regulates endoplasmic reticulum stress to protect endothelial progenitor cells from high-glucose damage Yong Yang, Yong Zhou, Yiyong Wang, Xianglong Wei, Tingzhong Wang, Aiqun Ma PII:

S0890-8508(19)30427-X

DOI:

https://doi.org/10.1016/j.mcp.2020.101527

Reference:

YMCPR 101527

To appear in:

Molecular and Cellular Probes

Received Date: 31 October 2019 Revised Date:

13 January 2020

Accepted Date: 23 January 2020

Please cite this article as: Yang Y, Zhou Y, Wang Y, Wei X, Wang T, Ma A, Exendin-4 regulates endoplasmic reticulum stress to protect endothelial progenitor cells from high-glucose damage, Molecular and Cellular Probes (2020), doi: https://doi.org/10.1016/j.mcp.2020.101527. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Ltd.

Yong Yang and Yong Zhou: Conceptualization, Methodology, Software. Yiyong Wang and Xianglong Wei: Data curation, Writing-Original draft preparation. Tingzhong Wang: Visualization, Investigation. Aiqun Ma: Supervision. Yong Yang: Writing-Reviewing and Editing.

Exendin-4 Regulates Endoplasmic Reticulum Stress to Protect Endothelial Progenitor Cells from High-Glucose Damage Yong Yang1&2*, Yong Zhou3*, Yiyong Wang4, Xianglong Wei2, Tingzhong Wang1&5&6, Aiqun Ma1&5&6 1

Department of Cardiovascular Internal Medicine, The First Affiliated Hospital of

Xi’an Jiaotong University, Xi’an, Shaanxi, China 2

Department of Cardiovascular Internal Medicine, Shenzhen Hospital of Southern

Medical University, Shenzhen, Guangdong, China 3

Department of Oncology, The University of Hong Kong-Shenzhen Hospital,

Shenzhen, Guangdong, China. 4

Department of Cardiovascular Medicine, General Hospital of Ningxia Medical

University, Yinchuan, Ningxia, China 5

Key Laboratory of Molecular Cardiology, Xi’an Jiaotong University, Xi’an, Shaanxi,

China 6

Key Laboratory of Environment and Genes Related to Diseases, Xi’an Jiaotong

University, Xi’an, Shaanxi, China *These authors contributed equally to this work. Corresponding author: Aiqun Ma, Department of Cardiovascular Internal Medicine, The First Affiliated Hospital of Xi’an Jiaotong University, No. 277, West Yanta Road, Xi’an, Shaanxi, 710061, China; Key Laboratory of Molecular Cardiology; Key Laboratory of Environment and Genes Related to Diseases, Xi’an Jiaotong University, E-mail: [email protected], Tel: 86-029-85323568 Running title: Role of exendin-4 in diabetes mellitus

Abstract Background: High glucose affects the function of endothelial cells by increasing oxidative stress. Studies have found that exendin-4 can improve wound healing in diabetic mice and mice with normal blood glucose. However, the mechanism of exendin-4 in endothelial progenitor cells under high-glucose condition has not been fully elucidated. Methods: Diabetic mouse models were established to investigate the effects of exendin-4 on endothelial progenitor cells in diabetic mice. Serum superoxide dismutase (SOD) and malondialdehyde (MDA) were determined by WST-8 and thiobarbituric acid (TBA) colorimetry, respectively. Cell viability, apoptosis and oxygen

species

(ROS)

were

detected

by

3-(4,

5-Dimethylthiazol-2-yl)-2,

5-diphenyltetrazolium bromide (MTT) and flow cytometry. Gene and protein expressions were determined by Quantitative reverse transcription PCR (qRT-PCR) assay and Western blot (WB). Results: The results showed that in diabetic mice, exendin-4 did not affect blood glucose or body weight, moreover, it improved aortic diastolic function, increased SOD activity and down-regulated malondialdehyde (MDA) level in the mice. In addition, exendin-4 also increased endothelial progenitor cell (EPCs) viability and reduced cell apoptosis through inhibiting p38 MAPK pathway and reducing endoplasmic reticulum stress and ROS. Conclusion: Exndin-4 can alleviate diabetes-caused damage to mice, moreover, it reduced endoplasmic reticulum stress and ROS through inhibiting p38 MAPK pathway in MPCs cells under high-glucose condition, thus increasing cell viability and reducing cell apoptosis.

Keywords: Exendin-4, Endoplasmic reticulum stress, Endothelial progenitor cells, Diabetes mellitus

1 Introduction Diabetes mellitus (DM) is a global health problem, and one of the rapidly growing chronic diseases in the world [1, 2]. According to the data released by the American Diabetes Association in 2004 [3], the prevalence of diabetes in all age groups was 2.8% in 2000 worldwide and is estimated to increase to 4.4% by 2030, moreover, the total number of diabetic patients is expected to rise from 171 million in 2000 to 366 million in 2030. Studies have found that cardiovascular disease is a major complication of type 2 diabetes, but in fact, diabetes and cardiovascular disease shares a common pathophysiological basis, that is, endothelial dysfunction [4-6]. Previous results have found that endothelial dysfunction is closely related to oxidative stress, under which, functional and structural damages will occur to the endothelium so that oxidative stress response will cause type 2 diabetes or cardiovascular diseases in the body [4-6]. Thus, these findings suggested that oxidative stress is associated with cell damage in DM. First discovered in 1997 [7], endothelial progenitor cells (EPCs) derived from local endothelial cells and bone marrow play important roles in promoting vascular repair after vascular injury. EPCs can aggregate in vascular injury to proliferate and promote the repair of endothelial cells, thus maintaining the integrity of endothelial cells. Therefore, after vascular injury, promoting the proliferation of EPCs may be an effective measure to repair vascular injury. However, EPCs can secrete a variety of vasoactive substances and regulate the vasodilatory function through the balance of these substances [7]. Therefore, the activity of EPCs can be evaluated by detecting the

vasodilatory function. Exendin-4, which is a glucagon-like peptide-1 (GLP-1) analogue, is a peptide agonist that promotes insulin secretion. Exendin-4 has been used as a therapeutic agent for treating type 2 DM, as it exerts protective effects on ischemia-induced

organ

and

tissue

injury

through

its

antioxidant

and

anti-inflammatory properties [8-10]. According to Eunhui Seo et al. [11], local application of exendin-4 can rapidly heal wounds in diabetic mice and those with normal blood glucose. Previous research [12] found that administration of exendin-4 before or after injury in mice improved their neurodegeneration after 72 h. In addition, study [13] also observed that exndin-4 promotes motor function in rats with spinal cord injury through promoting autophagy and inhibiting neuronal apoptosis. The endoplasmic reticulum is the site for synthesizing proteins and lipid steroids, and when the endoplasmic reticulum homeostasis is imbalanced, it will activate the corresponding signal pathways, thus triggering a series of endoplasmic reticulum stress (ERS) responses in the cells [14, 15]. P-p38 and PERK signals can initiate the survival pathway of ERS in the presence of endoplasmic reticulum stress response [16]. GRP78 and CHOP play very important roles in ERS-mediated apoptosis, and activation of CHOP signals will promote apoptosis [17]. In addition, MDA is formed as an end product of lipid peroxidation, and could reflect lipid peroxides after reacting itself with thiobarbituric acid and the degree of cell injury [18]. SOD can scavenge O2 anion to form H2O2, thus reducing the toxicities of such free radicals or of other free radicals generated by secondary reactions [19]. Therefore, in this study, we investigated the effects of exendin-4 on oxidative stress of certain tissues and cell

damage by detecting the expression of CHOP protein, levels of serum superoxide dismutase (SOD) and malondialdehyde (MDA). Taking the protective effects of exendin-4 on certain tissues and cell damage into consideration, we were interested in investigating the effects of exendin-4 on EPCs damage induced by DM. As the mouse genome project is basically completed and its genetic modification technology is mature, DM model in vivo was established in mice to explore the effects and mechanism of exendin-4 in endothelial progenitor cells, aiming to provide new insights into the treatment of DM. 2 Material and methods 2.1 Animals Thirty six C57BL/6 male mice (weighing 18-20 g, aged 5-7 weeks old) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (China). The mice were provided with free access to food and drinking water. The animals were housed under 12 h day/night cycle at between 22 and 25 ℃ in 70% humidity. The animal experiments were operated strictly in accordance with the Regulations of the Care and Use of Laboratory Animals. The study was approved by the Ethics Review Board for Animal Studies of the First Affiliated Hospital of Xi'an Jiaotong University (SH20184152). 2.2 Diabetic mice model establishment The mice were divided into six groups, namely, Control group, EX-4 2 group, Model group, Model+EX-4 1 group, Model+EX-4 2 group and Model+EX-4 4 group. Six mice in the control group were given the same dose of buffer, while another six in

the exendin-4 group was given only 2µg exendin-4. The mice in the Model group were treated as the diabetic mice model. Briefly, 60 mg/kg/d Streptozotocin (Amresco, Solon, Ohio) was intraperitoneally injected into the mice for 5 days and established as DM1 model group. On the fifth day, blood glucose value were detected via tail vein, and mice with whose blood glucose stably higher than 245 mg/dl were defined as diabetic mice. After the DM1 model group was established, the mice in the Model+EX-4 1 group, Model+EX-4 2 group and Model+EX-4 4 group were respectively treated by 1, 2 and 4 µg exendin-4 (Sigma-Aldrich, St Louis, MO, USA) once every 2 days for 3 weeks on the basis of the DM1 model group to observe the effects of exendin-4. The blood glucose and body weight of the mice from all groups were detect at 1, 2, and 3 week. The diastolic function, EPCs in peripheral blood and oxidative stress in vivo were detected from the mice. 2.3 Measurement of diastolic function in mice The aortas of mice were collected, cut into 3 mm and subjected to a water bath (at 37℃) containing 10 ml Krebs solution in 95% O2 and 5% CO2 for 90 min. Acetylcholine (Ach) was added from low to high concentration (from 1×10-8, 1×10-7, 1×10-6, to 1×10-5 mol/L). The maximum stable tension of each concentration was recorded. 2.4 Detection of EPCs in peripheral blood by flow cytometry Blood (1 mL) was collected via the hearts of the mice. Monocyte layer was separated and added with 500 µL erythrocyte lysate, and mononuclear cells were adjusted to 2×106/ cells after mixing. 10 µg/mL FITC anti-mouse CD34 and 10µg/mL

PE anti-mouse FIK-1 antibodies (BD Pharmingen, San Diego, CA) were then added into the cells and incubated on ice for 1 h. Next, the cells were washed three times by PBS and fixed by 2% paraformaldehyde for 10 min. CD34 and FIK-1 doubl e-stained positive cells were determined as EPCs by flow cytometry (BD Biosciences, USA). 2.5 Detection of serum superoxide dismutase (SOD) by WST-8 Mouse blood was extracted and centrifuged at 2500 rpm for 10min to collect upper serum. SOD detection buffer, WST-8 solution and enzyme solution were proportionally mixed into 160 µL WST-8/enzyme working solution and added into each hole of a 96-well plate. Blank control hole 1 was added with 20µL SOD detection buffer, while the blank control hole 2 was added with 40µL SOD detection buffer. Next, the solution were incubated at 37 ℃ for 30 min, and absorbance (value A) at 450 nm was measured using SOD activity determination kit (Beyotime Biotechnology, Shanghai, China). Inhibitory percentage = (A blank control 1-A sample) / (A blank control 1-A blank control 2) ×100%, and SOD enzyme activity of the samples = inhibition percentage / (1-inhibition percentage) units. 2.6 Detection of serum MDA by thiobarbituric acid (TBA) colorimetry Mouse blood was extracted and centrifuged at 2500 rpm for 10min to collect upper serum. The diluent of TBA, storage of TBA and antioxidant were proportionally mixed to 0.2 mL working fluid of MDA detection, added into each pore and mixed. After the centrifugation, 200µL supernatant was added into the 96-well plate. The absorbance was measured at 532 nm, and the contents of MDA were calculated.

2.7 Isolation, culture and identification of EPCs Bone marrow of femur and tibia of mice were collected, and centrifuged at 1500 rpm, 4℃ for 5 min and the supernatant was then discarded. Each tube was added with 500µL erythrocyte lysate and held for 1-2 min, the cells maintained in EGM-2 were washed by phosphate buffer once, gently blown and then planted (3 mL/hole) in the 6-well plate coated with vitamin. After incubating the cells at 37℃ with 5% CO2 for four days, half of the supernatant was discarded, and the same dose of fresh medium (EGM-2 with 2 mL 10% FBS per hole) was added to continue the culture for 3 days. The growth status of EPCs on the 1st, 3rd and 7th day was observed, and the cells were then identified by cytometry. The cell concentration was adjusted to (1×106 cells/mL). 100µL cell suspensions of each sample were added with 2µL Sca-1 antibody (ab25031, Abcam, USA) and 2µL FIK-1 antibody (ab262844, Abcam, USA) and incubated at 37℃ for 30 min. Next, the cells were washed by PBS once, and centrifuged at 1500rpm, 4℃ for 5 min. Finally, flow cytometry was performed to detect double-stained positive cells. 2.8 Cell treatment For establishment of high-glucose (HG) model at cell level, the cells were digested by 0.5 % trypsin to the density of 1 x105 /ml, and inoculated at 2 ml/ well into a 6-well plate. After the cells attached to the wall, the FBS free 1640 medium was was added to the 6-well plate , and after starving the cells for 12 h, the existing medium was replaced by high-glucose M199 medium.

In order to explore the effects of high glucose and exendin-4 on EPCs, the cells were divided into control group (untreated EPCs), EX-4 100 group (EPCs treated by 100 nmol/L exendin-4), HG group (EPCs treated by 33 nmol/L D-glucose), HG+EX-4 25 group (EPCs treated by 25 nmol/L exendin-4 and 33 nmol/L D-glucose), HG+EX-4 50 group (EPCs treated by 50 nmol/L exendin-4 and 33 nmol/L D-glucose), HG+EX-4 100 group (EPCs treated by 100 nmol/L exendin-4 and 33 nmol/L D-glucose (Sigma-Aldrich, St Louis, MO)). To detect whether exendin-4 regulated endoplasmic reticulum activation through p38 under high-glucose condition, EPCs were divided into control group, HG group, HG+EX-4 100 group, HG+EX-4 100+SB203580 group (EPCs treated by 100 nmol/L exendin-4 and 33 nmol/L D-glucose and 10µmol/L SB203580), HG+SB203580 group (EPCs treated by 33 nmol/L D-glucose and 10µmol/L SB203580), SB203580 group (EPCs treated by 10µmol/L SB203580, purchased from Beyotime Biotechnology (Shanghai, China)). The cells in each group were used to detect activity, apoptosis and oxidative stress in vitro. 2.9 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay EPCs were cultured in medium supplemented with different concentrations of d-glucose, exendin-4, and SB203580. The EPCs at 1×104 cells/pore were inoculated into 96-well plate, and cell viability was detected by MTT. Control group were set at the same time. Briefly, after incubation for 48 h, MTT was added into the control cells and incubated for 4 h, and then added with DMSO. When crystallization was dissolved completely, the optical density at wavelength of 490nm was detected using

commercial enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN). 2.10 Apoptosis assay Apoptosis of EPCs was detected by flow cytometric in FACScan flow cytometer (BD Biosciences, USA). The EPCs were stained by Annexin V-fluorescein isothiocyanate (Annexin V, 10 µL) and propidium iodide (PI, 5 µL) (Thermo Fisher Scientific Life Sciences, Waltham, MA) for the determination of apoptosis. Then Annexin V/PI cells were quantified using FACScan flow cytometer (BD Biosciences, USA). 2.11 Detection of oxygen species (ROS) by flow cytometry EPCs of different treatment groups were added with DCFH-DA (Sigma-Aldrich, St Louis, MO, USA) to the final concentration of 10 micromol/ L, incubated at 37℃ for 20 min, and then centrifuged for 5 min at 1 000 r/min. The supernatant was discarded, added with PBS to disperse cells, and the cells were then washed twice to remove unreacted DCFH-DA. 300 mL PBS was added into each tube to disperse cells in the dark, and the average fluorescence of 1×104 cells was measured by flow cytometry (BD Biosciences, USA). The excitation wavelength was 488 nm, while the emission wavelength was 530 nm. 2.12 Quantitative reverse transcription PCR (qRT-PCR) assay Total RNAs were extracted from EPCs using Trizol reagent (In., Carlsbad, USA), and the cDNAs were synthesized using SuperScriptTM III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). Then qRT-PCR reaction was carried out on DNA Engine with Chromo 4 Detector (MJ Research, Waltham, MA) under the

following conditions: at 50 ℃ for 2 min; at 95 ℃ for 5 min, and followed by 50 cycles at 95 ℃ for 15 s and at 60 ℃ for 30 s. CHOP primers were as follows: forward,

5′-CTGCCTTTCACCTTGGAGAC-3′

5′-CGTTTCCTGGGGATGAGATA-3′. 5′-GGTGCAGCAGGACATCAAGTT-3′

GRP78

and primer

was:

and

reverse, forward, reverse

5′-CCCACCTCCAATATCAACTTGA-3′ and the mRNAs were normalized toβ-actin. β-actin primer wasforward, 5′-CCACCATGTACCCAGGCATT-3′ and reverse, 5′-AGGGTGTAAAACGCAGCTCA-3′. The data were measured by 2−∆∆Ct method [20]. 2.13 Western blot (WB) analysis The total proteins were extracted from EPCs, and the concentration of protein was determined by a BCA Protein Assay Reagent kit (Thermo Fisher Scientific, Inc.). The proteins (30 µg) were separated on 12% SDS-PAGE and transferred to a PVDF membrane, which was blocked by 5% non-fat milk for 2 h at 37℃ and incubated overnight at 4℃ with primary antibodies (CHOP (1:1000, ab11419, 31kD, Abcam); GRP78 (1:1000, ab21685,75kD, Abcam); P-p38 (1:1000, ab4822, 38kD, Abcam); P-ERK1/2 (#9101, 1:1000, 44 kD, Cell Signaling Technology, Inc.)). Next, the membrane was incubated with Goat anti-mouse IgG H&L (HRP) (1:2000, ab205719, Abcam), Goat anti-rabbit IgG H&L (HRP) (1:2000, ab205718, Abcam), and Goat anti-rabbit IgG, HRP-linked antibody (#7074, 1:1000, Cell Signaling Technology, Inc.). The results were determined by Image J software (v.1.46, National Institute of Health), and shown as relative to β-actin (1:1000, ab8226, 42kD, Abcam). 2.14 Statistical Analysis The dada were analyzed using SPSS 20.0 software (SPSS Inc., Chicago, IL), and expressed as mean ± standard deviation. Student’s t-test was performed to analyze the

differences between two groups, while one-way ANOVA analysis followed Bonferroni test were used to analyze the differences among three or more groups. Each experiment was independently conducted in triplicate. P value <0.05 was considered to be statistically significant. 3 Results 3.1 Effects of exendin-4 on diabetic model in mice The blood glucose was measured 1 week after the injection of streptozotocin, and the results showed that the blood glucose increased significantly in streptozotocin-induced type I diabetic mice model, but remained stable 2 weeks after the establishment of DM model in the mice, moreover, exendin-4 at different concentrations did not affect blood glucose in the model mice (Fig1A). After the diabetic mice model was established for 2 weeks, body weight of the mice was reduced remarkably, but exendin-4 at different concentrations did not affect the body weight of the mice (Fig1B). In addition, we measured diastolic function of aorta of mice, and the results revealed that at the third week, the diastolic function of aorta in diabetic mice declined, but was significantly improved by exendin-4 in a concentration-dependent manner in the diabetic mice (Fig1C). Moreover, the number of EPCs in peripheral blood of mice was detected, and we found that EPCs in peripheral blood of the diabetic mice were significantly reduced, while exendin-4 significantly could reverse such a result (Fig1D). In this study, the SOD activity in serum was determined by WST-8, and the results revealed that in the diabetic mice, SOD activity was significantly reduced, but greatly increased by exendin-4 (Fig1 F).

In addition, TBA colorimetry demonstrated that MDA level was increased significantly in diabetic mice, but noticeably reduced by exendin-4. 3.2 Effects of high glucose and exendin-4 on EPCs in mice EPCs of mice showed typical spindle-like morphology 7 days after culture (Fig 2A). The EPCs were identified by flow cytometric analysis, and Sca-1 (stem) FLK-1 (endothelial) doble positive cells were considered as EPCs (Fig 2B). Then, we explored the effects of high glucose and exendin-4 on EPCs, and observed that Exendin-4 remarkably increased cell viability and high glucose produced the opposite effect, however, Exendin-4 reversed the effects of high glucose on EPCs in a concentration-dependent manner (Fig 3A). In addition, HG reduced EPCs, which, however, was revered by exendin-4 reversed in a concentration-dependent manner. We further detected the effect of high glucose and exendin-4 on apoptosis of EPCs, and the data found that high glucose significantly increased apoptosis of EPCs, while exendin-4 reversed the results in a concentration-dependent manner (Fig 3B). In addition, we found that high glucose increased ROS content in EPCs, and exendin-4 reversed such an effect of high glucose in a concentration-dependent manner (Fig 3C). Furthermore, WB analysis showed that the expressions of CHOP and GRP78 proteins were greatly increased by high glucose, but reversed by exendin-4 in a concentration-dependent manner (Fig 4AB). In addition, we also found that high glucose remarkably increased the expressions of CHOP and GRP78 mRNAs, but exendin-4

could

reverse

such

effects

induced

by

high

glucose

in

a

concentration-dependent manner (Fig 4C). Moreover, high glucose also noticeably increased the expressions of p-p38 and p-ERK1/2 proteins and the mRNA expressions of

p-p38

and

p-ERK1/2,

which

were

concentration-dependent manner (Fig 4D-F).

reversed

by

exendin-4

in

a

3.3 Effects of exendin-4 on EPCs in mice through p38 under high-glucose condition Cell viability was significantly inhibited by high glucose but reversed by exendin-4, and such an effect of exendin-4 was abolished by SB203580, which further promoted the role of high glucose (Fig 5A). In addition, apoptosis of EPCs was promoted by high glucose, however, exendin-4 reversed the effect of high glucose and SB203580 reversed the role of exendin-4 in EPCs apoptosis (Fig 5B). High glucose significantly increased ROS content in EPCs, which was reduced by exendin-4, moreover, SB203580 showed the opposite effect to exendin-4 (Fig 5C). T The endoplasmic reticulum is the site for synthesizing proteins and lipid steroids, and when the endoplasmic reticulum homeostasis is imbalanced, it will activate the corresponding signal pathways, thus triggering a series of endoplasmic reticulum stress (ERS) responses in the cells [14, 15]. P-p38 and PERK signals can initiate the survival pathway of ERS in the presence of endoplasmic reticulum stress response [16]. GRP78 and CHOP plays a very important role in ERS-mediated apoptosis, and activation of CHOP signals will promote apoptosis [17]. In this study, WB analysis showed that high glucose increased the expressions of CHOP and GRP78 proteins in EPCs, and exendin-4 reversed the effect induced by high glucose, moreover, SB203580 reversed the effect of exendin-4 (Fig 6A-B). The qRT-PCR analysis showed that exendin-4 reversed the effect of high glucose of increasing the expressions of CHOP and GRP78 mRNAs, while SB203580 reversed the effect of exendin-4 (Fig 6C). In addition, we also found that high glucose increased the expression of p-p38 and p-ERK1/2 proteins in EPCs, while exendin-4 reversed such results, moreover, SB203580 showed the opposite effect to exendin-4 and further promoted the effect of high glucose (Fig 6D-E).

4 Discussion The incidence of DM is increasing worldwide [21]. Type 2 diabetes, which is a long-term metabolic disorder, is characterized by hyperglycemia, insulin resistance and insufficiency of insulin [22, 23]. Endothelial dysfunction is considered to be one of the factors contributing to microvascular complications caused by diabetes mellitus and hyperglycemia [24]. The current results revealed that after mice had been treated by streptozotocin, the blood glucose was increased significantly and body weight was reduced greatly, indicating that the diabetic mice model was successful established. We also found that exendin-4 had no significant effect on blood glucose or body weight of the diabetic model mice, suggesting that exendin-4 did not regulate blood glucose level. By examining the aortic diastolic function of the mice, we observed that exendin-4 could significantly improve the aortic diastolic function in the diabetic mice. Poor glycemic control of patients with type 2 diabetes is associated with impaired left ventricular diastolic function, and left ventricular damage will accelerate aortic sclerosis, which may lead to deterioration of diastolic function [26]. Our study is consistent with the findings of a previous study [27], in which exendin-4 has been found to regulate paracrine communication between infiltrating macrophages and fibroblasts in a glucose-dependent manner and preferentially protect extracellular matrix remodeling and diastolic dysfunction in the experimental diabetic mice. Serum SOD and MDA levels were also measured, and the results showed that exendin-4 significantly increased SOD activity and decreased MDA level in the diabetic mice. Lipid peroxidation is an important biomarker of oxidative stress, and MDA, which is an end product of lipid peroxidation, could reflect lipid peroxides after reacting with thiobarbituric acid and the degree of cell injury [18]. SOD can

scavenge O2 anion to form H2O2 to reduce the toxicities of such free radicals or of other free radicals generated by secondary reactions [19]. Thus, the results in our study suggested that exendin-4 can greatly regulated oxidative stress in diabetic mice. Study showed that decreased number and declined function of EPCs not only increases the risk of developing cardiovascular, but also is detrimental to the repair of damaged heart and blood vessels to DM patients [25]. EPCs are precursor cells with the ability of angiogenesis and endothelial repair, and some studies have shown that the number of circulating EPCs in patients with coronary heart disease is significantly reduced [28, 29], and that changes in circulating EPCs are associated with vascular endothelial dysfunction [30]. Thus, we established a diabetic model in EPCs, and analyzed the effects of exendin-4 on the number of endothelial cells induced by high glucose. The results demonstrated that HG reduced EPCs, but exendin-4 reversed such an effect of high glucose in a concentration-dependent manner. In order to further determine the potential mechanism of exendin-4 in alleviating oxidative stress in the diabetic model in EPCs, and we found that exendin-4 regulated ROS and endoplasmic reticulum stress to protect EPCs. Moreover, high glucose up-regulated the expressions of chop-c/ebp homologous protein (CHOP) and glucose regulatory protein 78 (GRP78), however, Exendin-4 reversed the effect of high glucose. In addition, exendin-4 significantly increased the viability and reduced apoptosis of EPCs cells under high-glucose condition. Oxidative stress is the result of excessive production of oxidative free radicals, and could induce endothelial cell dysfunction [31] and ROS, moreover, oxidative stress could reflect the imbalance between the oxidation and antioxidant systems of cells and tissues [32]. Endoplasmic reticulum is an organelle with many functions, and can exert pressures including hypoxia, starvation, and infection to cells, challenging folding ability of cells and

stimulating endoplasmic reticulum stress, thus inducing the expression of death receptor [33]. GRP78 and CHOP are new biomarkers indicating endoplasmic reticulum stress [34], and are predictive of the diagnosis and prognosis of endoplasmic reticulum stress disorders [35]. According to Yeh Siang Lau et al., endoplasmic reticulum stress can induce apoptosis and endothelial dysfunction in mice [36], and the study also found that endoplasmic reticulum stress may be an important factor for initiating endothelial dysfunction through inducing oxidative stress and reducing NO synthesis. In addition, by conducting in vitro experiments, we found that exendin-4 regulated oxidative stress through inhibiting p38 MAPK pathway, thus realizing the protective effect of EPCs. Previous studies have shown that exendin-4 can improve TNF-induced HUVEC-12 cell damage through inhibiting the expression of p38 MAPK protein [37], thus, we speculated that exendin-4 could also inhibit the damage of HG to EPCs through inhibiting the expressions of proteins related to the p38 MAPK pathway. The current study found that exendin-4 regulated endoplasmic reticulum stress through p-38 to protect EPCs under high-glucose condition. We also found that high glucose up-regulated the expressions of p-p38 and pERK1/2, while exendin-4 reversed such an effect of high glucose. Previous study [38] demonstrated that the response of cells to stimuli such as oxidative stress may be co-induced by simultaneous activation of transcription factor networks, including p38 MAPK and ERK-1/2. In addition, activation of p38 MAPK signal plays a critical role in oxidative/nitrification stress-induced reactions. As expected, we found that the expressions of p-p38 and pERK1/2 in EPCs treated by high glucose were up-regulated, and we confirmed that exendin-4 could inhibit endoplasmic reticulum stress and ROS through inhibiting p38 MAPK pathway.

To conclude, the current study found that exendin-4 has protective effects on diabetic mice and EPCs under high-glucose condition, and such effects are related to p38 MAPK signal. In addition, exendin-4 could promote the production of EPCs, and therefore, exendin-4 might be further explored to improve the cardiovascular complications of diabetes. Our research still has some limitations and we lack clinical data for further analysis, which will be addressed in our future research. 5 Conclusion In conclusion, the current study found that exendin-4 has protective effects on diabetic mice. Exendin-4 can inhibit p38 MAPK pathway, endoplasmic reticulum stress and ROS, thereby increasing cell viability and reducing apoptosis. List of abbreviations Serum superoxide dismutase (SOD) malondialdehyde (MDA) thiobarbituric acid (TBA) 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) oxygen species (ROS) Quantitative reverse transcription PCR (qRT-PCR) western blot (WB) endothelial progenitor cell (EPCs) Cardiovascular disease (CVD) glucagon-like peptide-1 (GLP-1) Diabetic (DM1) Propidium iodide (PI)

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Figures legends Figure 1 Effects of exendin-4 on diabetic model in mice. (A) The blood glucose was detected in streptozotocin-induced diabetic mice model. (B) The body weight of diabetic mice model was measured. (C) At the third week, the diastolic function of aorta in the diabetic mice was measured（D）The number of endothelial progenitor cells (EPCs) in peripheral blood of diabetic mice was detected by flow cytometry. (E) In diabetic mice, superoxide dismutase (SOD) activity was detected by WST-8 in diabetic mice. (F) In diabetic mice, malondialdehyde (MDA) level was detected in diabetic mice by thiobarbituric acid (TBA) colorimetry. N=3, *p<0.05,

**

p<0.01,

***

p<0.001, vs. Control; &&p<0.01, &&&p<0.001 vs. Model.

Figure 2. Isolation, culture and identification of EPCs in mice. (A) Endothelial progenitor cells were isolated, cultured and identified. (B) Sca-1 (stem) FLK-1 (endothelial) doble positive cells were characterized by Flow cytometry. (N=3) Figure 3. Effects of high glucose and exendin-4 on EPCs in mice. (A) Cell viability was detected by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). (B) Cell apoptosis was detected by flow cytometry. (C) Reactive oxygen species (ROS) content was detected by Flow cytometry. N=3, *p<0.05,

**

p<0.01,

***

p<0.001, vs. Control; &p<0.05, &&p<0.01, &&&p<0.001 vs. Model.

Figure 4. Effects of high glucose and exendin-4 on relative proteins in EPCs in mice. (A-B) The WB analysis was used to detect the expression of CHOP and GRP78 proteins. (C) The expressions of CHOP and GRP78 mRNAs were detected by qRT-PCR. (D-E) The WB analysis was used to detect the expressions of p-p38 and p-ERK1/2 proteins. (F) QRT-PCR was used to detect the mRNA expressions of p-p38 and p-ERK1/2. N=3, Model.

***

p<0.001, vs. Control; &p<0.05,

&&

p<0.01,

&&&

p<0.001 vs.

Figure 5. Effects of exendin-4 on EPCs through p38 under high-glucose condition. (A) Cell viability was detected by MTT. (B) Cell apoptosis was detected by Flow cytometry. (C) ROS content in EPCs was detected by Flow cytometry. N=3, **p<0.01, ***

p<0.001, vs. Control;

##

&

p<0.05,

&&

p<0.01,

&&&

p<0.001 vs. Model;

#

p<0.05,

p<0.01, ###p<0.001 vs. HG+EX-4 100; ^p<0.05, ^^p<0.01, ^^^p<0.001, vs. HG+EX-4

100+SB203580; △p<0.05, △△p<0.01, △△△p<0.001

vs. HG+SB203580

Figure 6. Effects of exendin-4 on relative proteins in EPCs in mice through p38 under high-glucose condition. (A-B) The WB analysis was used to detect the expressions of chop-c/ebp homologous protein (CHOP) and glucose regulatory protein 78 (GRP78) proteins in EPCs. (C) The qRT-PCR analysis was used to detect the expressions of CHOP and GRP78 mRNAs. (D-E) The WB analysis was used to measure the expressions of p-p38 and p-ERK1/2 proteins. N=3, *p<0.05, **p<0.01, Control; &&p<0.01,

&&&

^^^

HG+EX-4

p<0.001,

vs.

HG+SB203580

p<0.001 vs. Model;

###

***

p<0.001, vs.

p<0.001 vs. HG+EX-4 100; ^^p<0.01,

100+SB203580;

△△p<0.01,

△△△p<0.001

vs.

1.

High glucose destroys cell function

2.

ER stress causes cell damage

3.

Exndin-4 inhibited p38 MAPK pathway