Liraglutide protects high-glucose-stimulated fibroblasts by activating the CD36-JNK-AP1 pathway to downregulate P4HA1

Biomedicine & Pharmacotherapy 118 (2019) 109224

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Liraglutide protects high-glucose-stimulated fibroblasts by activating the CD36-JNK-AP1 pathway to downregulate P4HA1

T

Tong Zhaoa, Huiqiang Chena, Chao Chenga, Juan Zhanga, Zhi Yana, Jiangying Kuangb, ⁎⁎ ⁎ Feng Kongb, Chunyan Lic, , Qinghua Lua, a

Department of Cardiology, The Second Hospital of Shandong University, Jinan 250033, Shandong, China Central Research Laboratory, The Second Hospital of Shandong University, Jinan 250033, Shandong, China c Department of Gynaecology, Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Diabetes Glucagon-like peptide-1 Prolyl 4-hydroxylases Activator protein-1 Fibroblast

Background: Diabetic cardiomyopathy (DCM) is a serious complication of diabetes mellitus. It’s known that glucagon-like peptide-1 (GLP-1) and prolyl 4-hydroxylase subunit alpha-1 (P4HA1) have significant effect on cardiovascular function, but their interaction in cardiac fibroblasts (CFs) is still being unraveled. Methods and results: The present study demonstrated that glucose promotes CFs proliferation and cardiac fibrosis. Using qRT-PCR, Western blot, CCK-8, EdU, flow cytometry, wound healing and Transwell assays to explore the functions of liraglutide and P4HA1 in high-glucose (HG)-induced CFs, we proved that liraglutide as well as silencing of P4HA1 inhibited cell proliferation, migration and invasion, and promoted cell cycle arrest and apoptosis in HG-induced CFs. In addition, liraglutide downregulated P4HA1 expression, upregulated CD36 and P-JNK expression levels, and enhanced the DNA binding activity of AP-1 on P4HA1. Inhibition of CD36 or p– JNK promoted P4HA1 expression. Conclusions: Liraglutide may down-regulate P4HA1 expression at least partly though CD36-JNK-AP1 pathway, thereby reducing myocardial fibrosis. Therefore, our study provides novel insight into the molecular mechanism and function of liraglutide in HG-mediated CFs.

1. Introduction Unhealthy trends in diet and lifestyle have led to the rise of diabetes mellitus as a global health crisis among adults. The management of diabetes as well as its complications incurs huge cost expenditures [1]. Diabetes mellitus is an independent risk factor for diverse cardiovascular diseases [2]. Studies have documented a disturbingly high rate of diabetic patients developing diabetic cardiomyopathy (DCM) and eventually congestive heart failure [1]. Cardiac remodeling is widely known to be a critical and irreversible pathogenetic process that induces diastolic dysfunction and eventually leadsto heart failure [3,4]. In patients with diabetes mellitus, the incidence of cardiac fibrosis is higher than in nondiabetic controls [5]. However, the pathogenic

molecular mechanisms in cardiac fibroblasts (CFs) in diabetes remain poorly understood. Glucagon-like peptide-1 (GLP-1) is a member of the pro-glucagon incretin family [6]. It is mainly secreted by intestinal L-cells in response to food intake and helps to maintain glucose homeostasis. GLP-1 exerts this function by stimulating glucose-dependent insulin release from pancreatic β-cells and by inhibiting glucagon secretion from pancreatic α-cells [7]. Furthermore, GLP-1 also delays gastric empting and suppresses appetite, thus reducing the absorption of nutrients [6–8]. The GLP-1 receptor (GLP-1R), a 463 amino-acid member of the G proteincoupled receptor (GPCR) superfamily [9], is widely distributed in diverse tissues, including brain, lung, stomach, intestine, pancreas and kidney, as well as the heart [1,10]. The currently used armamentarium

Abbreviations: DCM, diabetic cardiomyopathy; GLP-1, glucagon-like peptide-1; P4HA1, 4-hydroxylase subunit alpha-1; CFs, cardiac fibroblasts; DCM, diabetic cardiomyopathy; GPCR, G protein-coupled receptor; MMPs, matrix metalloproteinases; HG, high-glucose; AP-1, Activator protein-1; JNK, Jun N-terminal kinase; NC, negative control; qRT-PCR, quantitative reverse transcription-PCR; COL I, collagen I; COL III, collagen III; CCK8, Cell Counting Kit-8; EdU, 5-ethynyl-2-deoxyuridine; IF, immunofluorescence; EMSA, electrophoretic mobility shift assay ⁎ Corresponding author at: Department of Cardiology, The Second Hospital of Shandong University, No. 247 Beiyuan Street, Jinan 250033, Shandong, China. ⁎⁎ Corresponding author at: Department of Gynaecology, Provincial Hospital Affiliated to Shandong University, No. 324 Jingwu Road, Jinan 250021, Shandong, China. E-mail addresses: [email protected] (C. Li), [email protected] (Q. Lu). https://doi.org/10.1016/j.biopha.2019.109224 Received 15 March 2019; Received in revised form 10 July 2019; Accepted 10 July 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

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2.3. Real time quantitative PCR (qRT-PCR) assay

for the treatment of type 2 diabetes includes long-acting synthetic GLP1R agonists (GLP1RAs), such as liraglutide [11,12]. These agents have beneficial effects on blood glucose homeostasis and weight control [11,13]. In the present study, we focused on the cardiovascular effect of liraglutide and its potential mechanisms. Prolyl 4-hydroxylases (P4Hs) are key enzymes in the biosynthesis of all known types of collagens. P4Hs are regarded as particularly suitable targets for anti-fibrotic therapy [14]. P4H consists of α and β subunits, and P4HA1 is rate-limiting and essential for collagen maturation and secretion [15,16]. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that play critical roles in diverse physiological and pathological processes, including organ generation and regeneration, wound healing, inflammation and tumorigenesis [17]. MMPs have important functions in vasculature, specifically angiogenesis, collateral artery formation and thrombus resolution [17]. Both P4HA1 and MMPs are essential for the synthesis and degradation of collagens [16]. However, the effect of P4HA1 on high-glucose (HG) induced CFs and its relationship with liraglutide remains largely unknown. In this study, we investigated the effect of liraglutide on P4HA1 expression, cell proliferation, cycle, apoptosis, migration and invasion abilities in HG-mediated CFs. We also explored the underlying mechanism of liraglutide regulation of P4HA1 in HG-induced CFs. All of these findings provide a theoretical basis for the clinical usefulness of liraglutide for the treatment of cardiovascular diseases.

Relative gene expression was analyzed by qRT-PCR. Total RNA from cultured CFs was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Next, the quality and quantity of extracted RNAs were measured by 260 to 280 nm absorbance. Then cDNA was obtained by reverse transcription (RT) using First Strand cDNA Synthesis Kit (Thermo, America) following the manufacturer’s directions. Amplification of the specific genes was performed using SYBR GREEN PCR Master Mix (Takala, Dalian, China), and then the relative expression levels were analyzed with an ABI7500 Real-time PCR system (Applied Biosystems). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), an endogenous housekeeping gene, was used for expression normalization. The specific RT primers and PCR primers are as follows：P4HA1: 5′-ACC ACA GCA CAG TAC AGA GTA T-3′ (forward), 5′-GGA AAC ATC CAG TCC TGT GAG-3′ (reverse); GAPDH: 5′-AAC GAC CCC TTC ATT GAC CT-3′ (forward), 5′-ATG TTA GTG GGG TCT CGC TC-3′ (reverse). 2.4. Western blot assays Total protein extraction from CFs was performed using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China), and protein concentrations were detected with the Bradford method (Bradford, 1976). 20 μg total protein extracts were isolated with 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes. Subsequently, membranes were blocked using tris buffered saline tween (TBST) with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with specific primary antibodies at 4 °C overnight. Afterwards, the blots were incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, Cambridge, UK) at room temperature for 1.5 h. Protein detection was performed with enhanced chemiluminescence (Thermo Scientific, Shanghai, China) according to the manufacturer's instructions. Images were visualized by a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories, Hercules, CA, USA). GAPDH (Sigma Aldrich, St. Louis, MO, USA) served as a loading control. The primary antibodies used in this experiment are as follows: anti-GAPDH (1:2000, Santa Cruz Biotechnology, Santa Cruz, CA), anti-P4HA1 (1:1000, Abcam, ab59497), anti-collagen I (COL-I) (1:1000, Abcam, ab34710), anticollagen III (COL-III) (1:1000, Abcam, ab184993), anti-matrix metalloproteinase-1 (MMP-1) (1:1000, Abcam, ab38929), anti-matrix metalloproteinase-9 (MMP-9) (1:1000, Abcam, ab73734), anti-CD36 (1:1000, Abcam, ab64014), anti-p-JNK (1: 1000, Abcam, ab47337).

2. Material and methods 2.1. Cardiomyocyte culture and HG-stimulated CFs creation Human myocardial fibroblasts were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Cells were cultured in Dulbecco’s modified Eagle’s medium (BioWhittaker, Verviers, Belgium) supplemented with 10% fetal bovine serum (BioWhittaker) and 100 U/ml penicillin/streptomycin (both Invitrogen) and were cultured at 37 °C with 5% CO2 atmosphere. CFs were treated with 25 mmol/L glucose for 48 h to obtain HGstimulated CFs. HG-stimulated CFs were treated with 50 nmol/L liraglutide for 1 h to investigate the effect of liraglutide on HG-stimulated CFs progression. To explore the interaction between liraglutide and the CD36-JNK-AP1 pathway, HG-induced CFs were treated with AP-1 inhibitor (curcumin, 20 μmol/L) for 30 min., JNK inhibitor (SP600125, 50 μmol/L) for 1 h, or CD36 inhibitor (sulfo-N-succinimidyl oleate [SSO, 1 μmol /L]) for 15 min..

2.5. Cell proliferation assays 2.2. Transient transfection Cell viability was detected using the Cell Counting Kit-8 assay (Dojindo Co. Ltd., Kumamoto, Japan). The treated cells were seeded in 96-well plates at a density of 5000/well. At indicated time-points of 0, 12, 24, 48, 72 h, 10 μl CCK-8 was added to each well and incubated at 37 °C for another 2 h. The corresponding absorbance was measured at a wavelength of 450 nm using a microplate reader instrument (Bio-Rad, MA, USA).

Negative control (NC), P4HA1-315 siRNAs (siP4HA1-315), P4HA11182 siRNAs (siP4HA1-1182) and P4HA1-523 siRNAs (siP4HA1-523) were obtained from purchased from GenePharma Co., Ltd. (Shanghai, China). The sequences of siRNAs were as follows: siNC: sense, 5′-UUC UCC GAA CGU GUC ACG UTT-3′, antisense, 5′-ACG UGA CAC GUU CGG AGA ATT-3′; siP4HA1-315: sense, 5′-GGU CAG AUG ACU GAU UUG ATT-3′, antisense, 5′-UCA AAU CAG UCA UCU GAC CTT-3′; siP4HA1-1182: sense, 5′-GGA AAC CGU AAU CCU AAA UTT -3′, antisense, 5′-AUU UAG AUU ACG GUU UCC CTT-3′; siP4HA1-523: sense, 5′-GGA GUG AGU UGG AGA AUC UTT-3′, antisense, 5′-AGA UUC UCC AAC UCA CUC CTT-3′. Cultured CFs were separately transfected with three types of siRNAs targeting P4HA1 (siP4HA1-315, siP4HA1-1182 and siP4HA1-523) and NC RNA duplex using Lipofectamine® RNAiMAX (Invitrogen, Carlsbad, CA, USA) according the manufacturer’s protocol. The mRNA and protein levels of specific genes were detected by qRTPCR and Western blot assays. Cell proliferation, migration and invasion abilities were analyzed.

2.6. EdU assay Cell proliferation was determined by Cell-Light 5-ethynyl-2-deoxyuridine (EdU) DNA Cell Proliferation Kit (RiboBio, Guangzhou, China). Briefly, 1 × 105 CFs were seeded in 24-well plates. After treatment with different stimulators, CFs were exposed to 30 μM EdU at 37 °C for 2 h. Cells were then fixed with 4% paraformaldehyde at room temperature for 30 min. and permeabilized with 0.5% Triton X-100 for 10 min. before detection. After washing with phosphate-buffered saline (PBS), cells were incubated in 100 μl reaction buffer at 37 °C for 2

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Fig. 1. The effect of glucose on proliferation and fibrosis in CFs. Different concentrations of glucose (0, 5, 10, 25, 50 mmol/L) were used to treat CFs for 24 h. (A) CCK8 assay was used to detect cell proliferation activity (*P < 0.05, **P < 0.01). (B) qRT-PCR assay was performed to measure the mRNA expression level of P4HA1 (***P < 0.001). (C) Western blot assay was used to analyze the protein expression levels of COL I, COL III, MMP-1, MMP-9 and P4HA1. Subsequently, CFs were treated with 25 mmol/L glucose for 0, 12, 24, 48 and 72 h respectively. (D) CFs growth was detected by CCK8 assay (*P < 0.05, **P < 0.01). (E) P4HA1 expression was measured by qRT-PCR assay (**P < 0.01). (F) The expression levels of COL I, COL III, MMP-1, MMP-9 and P4HA1 were analyzed by Western blot assay.

2.10. Cell invasion assay

30 min., followed by staining with Hoechst dye (Hoechst 33342, Invitrogen).

Cell invasion ability was determined in by Transwell assay (Costar, Corning Incorporated, Cambridge, MA, USA). Cultured CFs were collected and suspended in FBS-free DMEM medium. A total of 1 × 105 cells were seeded into the top Transwell cell culture chambers that were previously coated with matrigel. Complete culture medium (μl) supplemented with 10% FBS was added to the lower chambers, serving as a chemoattractant. After incubation for 16 h, cells were fixed with 4% paraformaldehyde for 30 min. and stained with 0.1% crystal violet solution for 15 min.. Afterwards, cells remaining in the chambers were removed using a cotton swab. The invaded cells were counted from 5 randomly selected fields using an inverted microscope at 20 × magnification.

2.7. Immunofluorescence (IF) assay Specially treated CFs were plated onto sterilized glass coverslips. Next, cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min. on ice. The slides were incubated with rabbit primary antibody (Santa cruz Biotechnology) overnight at 4 °C, followed by antirabbit FITC-linked goat antibody (Sigma). Subsequently, cells were stained with bulk DNA with 4′,6-diamidino-2-phenylindole (DAPI) for detection. Representative photographs were taken at 40× magnification with confocal microscopy (FV1000 Olympus).

2.11. Electrophoretic mobility shift assay (EMSA) 2.8. Cell cycle and apoptosis analysis The P4HA1 promoters were amplified by Genepharma (Shanghai, China). The PCR products were labeled using chemiluminescent nucleic acid detection module (Pierce) according to the manufacturer's instructions. Afterwards, the labeled DNA were incubated at room temperature for 15 min. in 20 μl of binding buffer containing purified vancomycin resistance-associated regulator (VraR) protein. The complex was then separated by electrophoresis in an 8% native polyacrylamide gel. Finally, the band shifts were detected and analyzed using a Universal Hood 2 electrophoresis imager (Bio-Rad), and the quantitative analysis of the bands was performed using NIH ImageJ (version 1.3).

Cell cycle distribution was performed at indicated times with propidium iodide (Sigma, Aldrich) staining. Cells were harvested and fixed in ice-cold 70% ethanol, then precipitated at 4 °C overnight. After resuspension in PBS, cells were treated with propidium iodide (10 μg/mL) supplemented with RNase (100 mg/l) for 30 min. in the dark before detection. Apoptotic cell death was evaluated using Annexin V-FITC/PI apoptosis detection kit (BestBio, Shanghai, China) according to the manufacturer’s instructions. 1 × 106 cells/ml cells were suspended with 100 μl 1× binding buffer and double stained with Annexin VFITC/PI for 15 min. before detection. Cell cycle and apoptosis ability were detected by flow cytometry (BD Biosciences) using a fluorescenceactivated cell sorting (FACS) Calibur flow cytometer (BD Biosciences, San Jose, CA, USA), and analyzed with the ModFit LT 2.0 software.

2.12. Statistical analysis All results were confirmed by at least three separate experiments. Data were displayed as mean ± standard deviation (SD). Statistical analysis was performed using Student's t-test and one-way analysis of variance (ANOVA) with SPSS 18.0 (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered as statistically significant.

2.9. Wound healing assay Cell migration ability was determined by wound healing assay. CFs (5 × 105 cells/well) were seeded into 6-well plates and cultured until 90% confluency. Then wound areas were created using sterile 100 μl pipette tips. Afterwards, cells were incubated at 37 °C in 5% CO2 for another 48 h. The width of the wound area was then measured with an inverted microscope and evaluated using Leica application Suite software (LASV3.8, Germany).

3. Results 3.1. Glucose promotes cell proliferation ability and fibrosis in CFs To evaluate the effect of glucose on CF proliferation ability, CFs 3

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Fig. 2. Liraglutide inhibits cell proliferation and fibrosis and promotes cell cycle arrest and apoptosis in HG-stimulated CFs. Different concentration of liraglutide (0, 10, 50, 100 nmol/L) were used to treat HG-stimulated CFs for 1 h. (A) qRT-PCR assay was performed to measure the mRNA expression level of P4HA1 (**P < 0.01). (B) Western blot assay was performed to analyze the protein expression levels of P4HA1, COL I, COL III, MMP-1 and MMP-9. (C) CCK8 assay was used to detect cell proliferation activity (**P < 0.01). (D) Cell proliferation ability was accessed by EdU assay. Subsequently, HG-stimulated CFs were treated with 50 nmol/L liraglutide for 1 h. (EF) Cell cycle and apoptosis rate were detected by flow cytometry (**P < 0.01, ***P < 0.001).

glucose-treated CFs. Over time, the mRNA expression level of P4HA1 initially increased, whereafter it became a little lower; P4HA1 expression was highest at 48 h (P < 0.01, Fig. 1E). Results from Western blot assay indicated that glucose upregulated the expression levels of fibrosis indices, including COL I, COL III, MMP-1, MMP-9 and P4HA1, especially at 48 h (Fig. 1F). Based on these results, we determined that CFs would be treated with 25 mmol/L glucose for 48 h to obtain HGstimulated CFs.

were exposed to different concentrations of glucose (0, 5, 10, 25, 50 mmol/L) for 24 h. Cell growth was then detected by CCK8 assay. As glucose concentration increased, the proliferation ability of CFs also increased initially, then decreased slightly, with proliferation ability strongest in the 25 mmol/L glucose group (P < 0.01, Fig. 1A). We then performed qRT-PCR to investigate the expression level of P4HA1 in CFs treated with different concentrations of glucose treated CFs. With increasing glucose concentration, P4HA1 mRNA level increased initially, albeit to a little lower level, with the highest mRNA level of P4HA1 reached in the 25 mmol/L glucose group (P < 0.001, Fig. 1B). Afterwards, we analyzed the expression levels of fibrosis indices in glucose treated CFs. As shown in Fig. 1C, glucose upregulated the protein expression levels of COL I, COL III, MMP-1, MMP-9 and P4HA1, and the impact was greatest at the glucose concentration of 25 mmol/L. Next, we further identified the optimal time for glucose treatment in CFs. Therefore, 25 mmol/L glucose was used to treat CFs for 0、12、 24、48 and 72 h, respectively. Then we performed CCK8 to assess the effects of glucose on CFs proliferation at different time points. As shown in Fig. 1D, over time, CFs proliferation increased initially and then decreased slightly; the optimal treatment duration was 48 h (P < 0.01). Next, P4HA1 expression level was determined by qRT-PCR assay in

3.2. Liraglutide prevents cell growth and fibrosis in HG-stimulated CFs To investigate the effect of liraglutide on cell proliferation ability in HG-stimulated CFs, different concentrations of liraglutide (0, 10, 50,100 nmol/L) were used to treat HG-stimulated CFs for 1 h. The results from qRT-PCR assay demonstrated that with increasing liraglutide concentration, P4HA1 expression decreased; P4HA1 expression was lowest at the liraglutide concentration of 50 nmol/L, (P < 0.01, Fig. 2A). Accordingly, the results from Western blot assay showed that liraglutide downregulated the expression levels of fibrosis indices, such as COL I, COL III, MMP-1, MMP-9 and P4HA in HG-stimulated CFs, and the optimal concentration was 50 nmol/L (Fig. 2B). 4

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Fig. 3. Liraglutide inhibits cell migration and invasion ability in HG-stimulated CFs. HG-stimulated CFs were treated with 50 nmol/L liraglutide for 1 h. (A) Wound healing assay was used to detect cell migration ability (**P < 0.01), (B) Transwell assay was performed to determine cell invasion ability (**P < 0.01).

Fig. 4. P4HA1 was knocked down in CFs. CFs were transfected with P4HA1-315 siRNAs (siP4HA1-315), P4HA1-1182 siRNAs (siP4HA1-1182) and P4HA1-523 siRNAs (siP4HA1-523), respectively. (A) P4HA1 expression was accessed by qRT-PCR assay and Western blot assays (B) (***P < 0.001). (C) Flow cytometry was performed to analyze P4HA1 level.

Additionally, 50 nmol/L liraglutide was used to treat HG-mediated CFs for 1 h. EdU assay showed that liraglutide-treated CFs had weaker proliferation ability than the control group (Fig. 2D), suggesting that liraglutide can prevent growth of HG-mediated CFs. Next, we explored the effects of liraglutide on the cell cycle and apoptosis of HG-mediated CFs. As shown in Fig. 2E, the proportions of cells in G0/G1 phases were 63.85% and 45.69% in the HG + liraglutide group and HG group, respectively, while the proportions in S phase

3.3. Liraglutide inhibits cell proliferation, migration and invasion, and induces cell cycle arrest and apoptosis in HG-mediated CFs To further investigate the effects of liraglutide on the proliferation of HG-mediated CFs, CCK8 assay was performed. The results indicated that, with increasing liraglutide concentration, the proliferation ability of CFs initially decreased and then increased slightly; proliferation ability was weakest in the 50 nmol/L group (P < 0.01, Fig. 2C). 5

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Fig. 5. Silence of P4HA1 inhibits cell proliferation induced by liraglutide in HG-stimulated CFs. HG-stimulated CFs were treated with liraglutide for 1 h and/or transfected with siP4HA1, respectively. P4HA1 expression was detected by qRT-PCR (A) and Western blot assays (B), respectively (**P < 0.01, ***P < 0.001). (C) P4HA1 expression was analyzed by flow cytometry. (D) Cell proliferation ability was measured by EdU assay.

expression and revealed that silencing of P4HA1 or treatment with liraglutide downregulated P4HA1 expression; silencing of P4HA1 also decreased P4HA1 expression mediated by liraglutide (P < 0.01, Fig. 5A-B). In addition, flow cytometry results demonstrated cooperation between silencing of P4HA1 and liraglutide in the downregulation of P4HA1 expression in HG-stimulated CFs (Fig. 5C). Next, we performed EdU assay to measure cell proliferation ability and found that silencing of P4HA1 prevented cell proliferation and enhanced the inhibitory effect of liraglutide on cell proliferation ability in HG-mediated CFs (Fig. 5D). Similarly, the results from flow cytometry showed silence of P4HA1 promoted cell cycle arrest and apoptosis ability in HG-mediated CFs, augmenting the effects of liraglutide. As shown in Fig. 6A-B, the proportions of cells in G0/G1 phases were 49.64%, 63.49%, 47.67%, 57.24% and 70.88% in HG-mediated CFs treated with NC, si-P4HA1, control, liraglutide, and si-P4HA1+liraglutide groups, respectively (P < 0.05, P < 0.01, Fig. 6C). The proportions of apoptotic cells were 10.2%, 24.6%, 9.6%, 16.1% and 32.6% respectively (P < 0.01, Fig. 6D). We further evaluated the effects of P4HA1 on cell migration and invasion abilities in HG-mediated CFs. The silencing of P4HA1 inhibited cell migration and invasion and enhanced the inhibitory effect of liraglutide on cell migration and invasion abilities in HG-mediated CFs (P < 0.05, P < 0.01, Fig. 7).

were 20.04% and 38.25% in the HG + liraglutide group and HG group, respectively (P < 0.01, Fig. 2E). Thus, we suggested that liraglutide induced cell cycle arrest in the G0/G1 phase. Moreover, the proportions of apoptotic cells were 28.8% and 9.6% in the HG + liraglutide group and HG group, respectively (P < 0.001, Fig. 2F), suggesting that liraglutide promoted cell apoptosis of HG-mediated CFs. The roles of liraglutide on migration and invasion abilities of HGmedicated CFs were evaluated by wound healing and Transwell assays, respectively. The results revealed that liraglutide can significantly reduce the migration (P < 0.01, Fig. 3A) and invasion (P < 0.01, Fig. 3B) abilities of HG-mediated CFs. 3.4. The effects of P4HA1 knockdown in CFs To investigate the biological roles of P4HA1 on CFs, CFs were transfected with P4HA1-315 siRNAs (siP4HA1-315), P4HA1-1182 siRNAs (siP4HA1-1182) and P4HA1-523 siRNAs (siP4HA1-523), respectively. The results from qRT-PCR and Western blot assays indicated that the interference effect was greatest with siP4HA1-523 (P < 0.001, Fig. 4A-B). Results of flow cytometry also indicated that silencing of P4HA1 downregulated P4HA1 expression in CFs, with siP4HA1-523 having the greatest inhibitory effect (Fig. 4C). 3.5. Silencing of P4HA1 enhanced liraglutide-mediated inhibition of cell proliferation, migration and invasion and promoted liraglutide-mediated induction of cell cycle arrest and apoptosis in HG-stimulated CFs

3.6. Liraglutide inhibits P4HA1 in HG-stimulated CFs by regulating the CD36-JNK-AP1 pathway

The effects of P4HA1 knockdown on the function of liraglutidetreated, HG-stimulated CFs were investigated. HG-stimulated CFs were treated with liraglutide for 1 h and/or transfected with siP4HA1. qRTPCR and Western blot assays were performed to evaluate P4HA1

Recent studies suggested that the promoter region of P4HA1 contained AP-1 binding sites and that JNK participate in the regulation of P4HA1 [18,19]. It was also reported that liraglutide regulates CD36 6

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Fig. 6. Silence of P4HA1 enhances cell cycle arrest and apoptosis induced by liraglutide in HG-stimulated CFs. HG-stimulated CFs were treated with liraglutide for 1 h and/or transfected with siP4HA1, respectively. Flow cytometry was used to determine cell cycle distribution (A) and cell apoptosis (B). (C) The cell cycle distribution was quantitatively analyzed (*P < 0.05, **P < 0.01). (D) The apoptosis rate was recorded (**P < 0.01).

Western blot assay also showed that inhibition of JNK prevented the expression levels of CD36 and p-JNK, while liraglutide alleviated this effect (Fig. 8F). These results proved that inhibition of JNK increased P4HA1 expression and decreased CD36 and p-JNK expression in HGmediated CFs. Liraglutide reversed the effects mediated by JNK inhibitor. These data demonstrated that liraglutide suppressed P4HA1 expression in HG-stimulated CFs, partly though the JNK /AP-1 pathway. Afterwards, we treated HG-induced CFs with liraglutide or CD36 inhibitor (SSO) to explore the relationship between liraglutide and CD36. EMSA assay was used to detect the DNA binding activity of AP-1 and the results indicated that inhibition of CD36 reduced the DNA binding activity of AP-1 on P4HA1, while liraglutide alleviated this effect in HG-stimulated CFs (Fig. 8G). We also found that inhibition of CD36 upregulated P4HA1 expression, while liraglutide reversed the enhancing effect of CD36 inhibitor on P4HA1 expression in HG-stimulated CFs (Fig. 8H). The same results were observed in Western blot assays (Fig. 8I). Furthermore, we found that inhibition of CD36 reduced p-JNK expression, and liraglutide reversed this effect (Fig. 8I). These results indicated that inhibition of CD36 increased P4HA1 expression and reduced the AP-1 DNA binding activity and p-JNK expression in HG-stimulated CFs, while liraglutide reversed these effects. These data suggested that liraglutide inhibited P4HA1 expression in HG-stimulated CFs at least partly though the CD36/JNK/AP-1 pathway.

expression [20]. Therefore, we hypothesized that liraglutide may reduce myocardial fibrosis by regulating P4HA1 via the CD36-JNK-AP1 pathway. HG-induced CFs were treated with liraglutide for 1 h or 20 μmol/L AP-1 inhibitor (curcumin) for 30 min. Then EMSA assay was performed to evaluate the DNA binding activity of AP-1 on P4HA1. The results indicated that liraglutide enhanced the DNA binding activity of AP-1 on P4HA1, and reversed the inhibitory effect of AP-1 inhibitor on P4HA1 expression (Fig. 8A). Next, qRT-PCR was used to determine the expression level of P4HA1 in HG-mediated CFs treated with liraglutide or curcumin. The results revealed that inhibition of AP-1 upregulated P4HA1 expression and that liraglutide reversed the enhancing effect of AP-1 inhibitor on P4HA1 in hyperglycemic-stimulated CFs (Fig. 8B). The same results were seen in the data from Western blot assays (Fig. 8C). Furthermore, we found that liraglutide promotes CD36 and pJNK expression and reverse the inhibitory effect of AP-1 inhibitor on CD36 and p-JNK expression in HG-stimulated CFs (Fig. 8C). Afterwards, we performed cell IF assay to assess the expression level of AP-1 in CFs. As shown in Fig. 8D, liraglutide promoted the expression of AP-1 and reversed the inhibitory effect of HG or AP-1 inhibitor on AP-1 expression in CFs (Fig. 8D). These results provided further support that liraglutide can upregulate AP-1 expression and that the inhibition of AP-1 increases P4HA1 expression. The enhancing effect of AP-1 inhibition on P4HA1 expression is reversed by liraglutide, suggesting that liraglutide suppresses P4HA1 expression in HG-stimulated CFs, partly though AP1. To further investigate the interaction between liraglutide and JNK, HG-mediated CFs were treated with liraglutide or 50 μmol/L JNK inhibitor (SP600125) for 1 h. Then qRT-PCR was used to detect the expression of P4HA1. Inhibition of JNK promoted P4HA1 expression in HG-stimulated CFs, and liraglutide reversed this effect (Fig. 8E). The same results were seen in the data from Western blot assay (Fig. 8F).

4. Discussion DCM is characterized by structural and functional myocardial impairments in diabetic patients without coronary artery disease, hypertension, or other potential etiological conditions [21]. Alterations in metabolism and energetics of cardiac substrates are the leading cause 7

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Fig. 7. Silencing of P4HA1 inhibits cell migration and invasion ability induced by liraglutide in HG-stimulated CFs. HG-stimulated CFs were treated with liraglutide for 1 h and/or transfected with siP4HA1, respectively. (A) The cell migration ability was accessed by wound healing assay. (B) Cell invasion ability was detected by Transwell assay. (C) The relative wound area was calculated (**P < 0.01). (D) The number of invading cells per field were counted (**P < 0.01, ***P < 0.001).

can change the structure and function of the heart, which is one of the most important pathological features of DCM. In the process of cardiac fibrosis, excessive synthesis of COL I and COL III and excretion by CFs, result in collagen deposition [33,34]. Our study found that glucose promoted COL I, COL III, MMP-1 and MMP-9 expression levels in CFs. For the past decade, GLP-1 and its analogs have been used in the pharmacological management of diabetes. GLP-1 has been reported to increase myocardial glucose uptake in isolated heart preparations [35], and protect the pancreatic β-cells as well as the cardiovascular system [10]. In the present study, we confirmed that liraglutide inhibited cell growth, migration and invasion, and promoted cell cycle arrest and apoptosis in HG-mediated CFs. P4Hs play a critical role in the synthesis of collagens and stabilization of their triple helical structure by catalyzing the formation of 4hydroxyproline in collagen and collagen-like proteins [15,36,37]. P4H a1 (encoded by P4HA1) is the main P4H isoform in most cells [36,38]. Overexpression of P4HA1 is associated with various human diseases, including CFs [39], liver fibrosis [40], diabetic kidney [41], and even tumors [42,43]. Our study demonstrated that silencing of P4HA1 inhibits cell proliferation, migration and invasion abilities, and promotes cell cycle arrest and apoptosis in HG-mediated CFs. Our study also proved, perhaps for the first time, that liraglutide could downregulate the expression of P4HA1. Activator protein-1 (AP-1) was identified as a mammalian transcription factors nearly 30 years ago [44]. It is now recognized that AP1 heterodimerizes with members of the c-Fos, c-Jun, ATF and Maf subfamilies [45,46]. AP-1 members are ubiquitously expressed and regulate a wide range of cellular events, such as proliferation,

for DCM [21,22]. Lipid metabolism disorder, glucotoxicity, oxidative stress and insulin resistance may damage myocardial function in diabetics [23,24]. In addition, cardiac fibrosis is often associated with various cardiovascular disorders, such as hypertension, ischemic injury, and myocardial infarction. Exposure to a diversity of pathological irritants triggers proliferation and differentiation of CFs into cardiac myofibroblasts, which progresses to cardiac fibrosis and heart remodeling and eventually leads to cardiac dysfunction [25–27]. In our study, we first evaluated the effect of glucose on the proliferative ability in CFs and found that glucose promoted the proliferation CFs with increasing concentration and time. Matrix metalloproteinases (MMPs) are a family of Zn2+-dependent proteases that are secreted by connective tissue and involved in extracellular matrix (ECM) degradation [28]. MMPs, such as MMP-1, matrix metalloproteinase-2 (MMP-2) and MMP-9, play major roles in the physiological and pathological processes of myocardial reconstruction, embryonic development, cell migration, angiogenesis, atherosclerosis, malignant tumor infiltration and metastasis [29–31]. Tissue inhibitor of metalloproteinases (TIMPs) are a set of low molecular weight glycoprotein that are widely distributed in tissues and humors; TIMPs are produced and secreted by fibroblasts, epithelial cells and endothelial cells. In addition, TIMPs are multifunctional proteins that can inhibit the activity of MMPs [32]. A delicate balance between MMP and TIMP is maintained in normal biological processes. In DCM, if the delicate balance is broken, abnormal deposition of collagen will occur. Studies have also found that MMP stimulates COL I expression in rat CFs, and MMP expression is associated with the severity of myocardial fibrosis. Overexpression of ECM, such as COL I, collagen II (COL II), and COL III 8

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Fig. 8. Liraglutide inhibits P4HA1 in HG-stimulated CFs by regulating the CD36-JNK-AP1 pathway. HG-stimulated CFs were treated with liraglutide for 1 h and/or AP-1 inhibitor (20 mmol /L curcumin) for 30 min., respectively. (A) EMSA assay was used to detect the changes in the DNA binding activity of AP-1 on P4HA1 after AP-1 inhibitor stimulation. (B) qRT-PCR assay was performed to measure the mRNA expression level of P4HA1 (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Western blot assay was used to analyze the protein expression levels of P4HA1, CD36 and p-JNK. (D) The subcellular distribution of AP-1 was evaluated by IF assay. Subsequently, HG-stimulated CFs were treated with liraglutide for 1 h and/or JNK inhibitor (50 μmol /L SP600125) for 1 h, respectively. (E) qRT-PCR assay was performed to measure P4HA1 expression. (F) Western blot assay was used to analyze P4HA1, CD36 and p-JNK expression levels. Afterwards, HG-stimulated CFs were treated with liraglutide for 1 h and/or CD36 inhibitors (1 μmol /L SSO) for 15 min., respectively. (G) EMSA assay was used to detect the changes in the DNA binding activity of AP-1 on P4HA1 after CD36 inhibitor stimulation. (H) P4HA1 expression was analyzed qRT-PCR assay (*P < 0.05, **P < 0.01). (I) P4HA1, CD36 and pJNK expression levels were assessed by Western blot assay.

5. Conclusions

differentiation and cell apoptosis [45,47]. Binding of AP-1 dimers to cognate DNA at an ‘AP-1 site’, which is located in the promoters of target genes [46–48], regulates gene expression. It’s well known that the Jun N-terminal kinase (JNK) pathway is involved in vital cellular and organ activities as well as in multiple diseases through phosphorylation of c-Jun protein, one of its most important downstream proteins, and translocation of p-c-Jun to the nucleus [49–51]. CD36, an 88kda transmembrane glycoprotein, belongs to the class B scavenger receptor family. CD36 is expressed widely in human tissues, including microvascular endothelium, hematopoietic cells, smooth muscle cells, and the cardiovascular system [52,53]. As a multi-ligand scavenger receptor, CD36 participates in various pathophysiological processes, including blood vessel formation, atherosclerosis, phagocytosis, inflammation, lipid metabolism and apoptosis [53]. Prior studies have also suggested that CD36 promotes JNK phosphorylation [54], whereas JNK inhibitor (SP600125) reduces CD36 [55], demonstrating important interactions between CD36 and JNK [54,55]. JNK a member of the mitogen-activated protein kinase (MAPK) family [56]. Phosphorylated JNK leads to phosphorylation of c-Jun at the amino terminus, which in turn induces c-Jun synthesis. In addition, the synthesis and phosphorylation of c-Jun enhances the activity of c-Jun/AP-1 [57].The critical role of CD36/MAPK/JNK pathway in trimethylamine N-oxide (TMAO)induced formation of foam cells [55], which appears to justify the usage of liraglutide in the treatment of cardiovascular diseases. However, the underlying interactions between liraglutide, P4HA1, CD36 and JNK/ AP-1 pathway remain an enigma. Our work also confirmed this fact with JNK inhibitor (SP600125) and CD36 inhibitor (SSO) and found that inhibition of JNK or CD36 increases the expression level of P4HA1. We therefore speculated that liraglutide suppressed P4HA1 expression in HG-stimulated CFs partly though the CD36-JNK-AP1 pathway.

Our study demonstrated that glucose promotes CF proliferation and cardiac fibrosis. However, liraglutide inhibits CF proliferation, migration and invasion, and promotes cell cycle arrest and apoptosis in HGstimulated CFs. Moreover, our study is the first to demonstrate that liraglutide downregulated P4HA1 expression, and that silencing of P4HA1 enhanced the biological effects of liraglutide on HG-mediated CFs. Liraglutide downregulated P4HA1 expression through inhibition of CD36-JNK-AP1 pathway. Therefore, we hypothesize that liraglutide regulates P4HA1 to reduce myocardial fibrosis by CD36-JNK-AP1 pathway, which provides a valuable theoretical basis for the diagnosis and treatment of DCM. In the future, liraglutide, P4HA1 inhibitors and CD36-JNK-AP1 pathway inhibitors may prove useful in the pharmacological treatment of DCM. A limitation of the current study is the high concentration of glucose, 25 mM glucose, which is substantially higher than the typical blood glucose concentration. Since disease progression is a very slow process, future experiments should use a lower glucose concentration and extend the duration of treatment. Further confirmation of the observations in the current study will also require animal experiments with large sample sizes. Additional research is needed to demonstrate the relationships between AP1, JNK, and CD36 and the possible mechanisms in HG-stimulated CFs.

Ethics approval and consent to participate Not applicable.

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Consent for publication [17]

Not applicable.

[18]

Availability of data and materials [19]

All relevant data are within this published paper.

[20]

Authors’ contributions Tong Zhao and Qinghua Lu conceived and supervised the study; Tong Zhao and Huiqiang Chen designed experiments; Tong Zhao and Chao Cheng performed experiments; Juan Zhang and Zhi Yan provided new tools and reagents; Jiangying Kuang developed new software and performed simulation studies; Feng Kong analysed data; Tong Zhao and Chunyan Li wrote the manuscript; Tong Zhao and Qinghua Lu made manuscript revisions.

[21]

[22]

[23]

Funding

[24]

This work was supported by Natural Science Foundation of Shandong (China, 2009ZRB019N0).

[25]

Declaration of Competing Interest

[26]

The authors declare that they have no competing interests.

[27] [28]

Acknowledgment None.

[29] [30]

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