Metformin ameliorates the proinflammatory state in patients with carotid artery atherosclerosis through sirtuin 1 induction

ORIGINAL ARTICLE Metformin ameliorates the proinflammatory state in patients with carotid artery atherosclerosis through sirtuin 1 induction WEI XU, YANG-YANG DENG, LIN YANG, SIJIA ZHAO, JUNHUI LIU, ZHAO ZHAO, LIJUN WANG, PRABINDRA MAHARJAN, SHANSHAN GAO, YULING TIAN, XIAOZHEN ZHUO, YAN ZHAO, JUAN ZHOU, ZUYI YUAN, and YUE WU XI’AN, SHAANXI, CHINA

Metformin is a widely used classic antidiabetic drug. However, its clinical pharmacologic mechanism remains poorly understood. In the present study, we investigated the anti-inflammatory effects of metformin on circulating peripheral blood mononuclear cells (MNCs) of patients with carotid artery atherosclerosis (AS). A total of 42 patients with carotid artery AS were randomly assigned to metformin (500 mg twice a day; Met; n 5 21) or placebo control (Con; n 5 21) groups. After 12 weeks of treatment, plasma concentrations of high-sensitivity C-reactive protein (hs-CRP), interleukin 6 (IL-6), and tumor necrosis factor a (TNF-a) significantly decreased in the Met group compared with the Con group. In addition, treatment with metformin significantly reduced the expression of IL-6 and TNF-a at the messenger RNA level and attenuated nuclear factor kappa B (NF-kB) DNA binding activity in MNCs. Intriguingly, metformin did not alter the expression of NF-kB p65 subunit, but markedly inhibited its acetylation. Furthermore, metformin significantly induced sirtuin 1 (SIRT1) expression in MNCs. Moreover, we found that metformin treatment dramatically induced SIRT1 expression, blocked p65 acetylation, and inhibited NF-kB activity and the expression of inflammatory factors in MNCs in vitro. We conclude that metformin has a novel direct protective role to ameliorate the proinflammatory response through SIRT1 induction, p65 acetylation reduction, NF-kB inactivation, and inflammatory inhibition in peripheral blood MNCs of patients with carotid artery AS. (Translational Research 2015;-:1–8) Abbreviations: AS ¼ atherosclerosis; IL-6 ¼ interleukin 6; IMT ¼ intima-media thickness; Met ¼ metformin group; MNCs ¼ mononuclear cells; SIRT1 ¼ sirtuin 1; TNF-a ¼ tumor necrosis factor a

From the Department of Cardiovascular Medicine, First Affiliated Hospital of the Medical School, Xi’an Jiaotong University, Xi’an, Shaanxi, China; Cardiovascular Department of Key Laboratory of Environment and Genes Related to Diseases, Xi’an Jiaotong University, Ministry of Education, Xi’an, Shaanxi, China; Department of Vascular Surgery, First Affiliated Hospital of the Medical School, Xi’an Jiaotong University, Xi’an, Shaanxi, China.

Submitted for publication January 21, 2015; revision submitted May 30, 2015; accepted for publication June 2, 2015.

Wei Xu and Yang-Yang Deng contributed equally to this study.

http://dx.doi.org/10.1016/j.trsl.2015.06.002

Reprint requests: Yue Wu or Zuyi Yuan; e-mail: [email protected]. edu.cn or [email protected]. 1931-5244/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved.

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AT A GLANCE COMMENTARY Xu W, et al. Background

The clinical pharmacologic mechanism of metformin, a classic antidiabetic drug, is still poorly understood. Here, we investigated the antiinflammatory effects of metformin on circulating peripheral blood mononuclear cells (MNCs) in patients with carotid artery atherosclerosis (AS). Translational Significance

In the present study, we found that metformin has a novel direct protective role to ameliorate the proinflammatory response through sirtuin 1 induction, p65 acetylation reduction, NF-kB inactivation, and inflammatory inhibition in peripheral blood MNCs of patients with carotid artery AS.

INTRODUCTION

Nowadays, atherosclerosis (AS) is one of the leading causes of morbidity and mortality in developing and developed countries. Furthermore, carotid artery AS has become a strong predictor for future stroke. It is known that AS is a chronic low-grade inflammatory disease within the arterial wall.1,2 Previous pathologic data have shown that lymphocytes, macrophages, and foam cells are involved in the pervasive inflammation occurring in atherosclerotic arterial walls.3 Activation of nuclear factor kappa B (NF-kB), a multiprotein complex of transcriptional factors, induces a variety of proinflammatory mediators and cytokines, such as interleukin 6 (IL-6) and tumor necrosis factor a (TNF-a).1,4 Metformin is a safe, inexpensive, and widely used glucose-lowering drug for diabetes treatment in clinical practice as well as a potential anti-inflammatory agent.5 In several randomized clinical studies, metformin has been shown to confer atheroprotective effects by decreasing the progression of carotid intimal medial thickness and coronary AS.6,7 Remarkably, in the UK Prospective Diabetes Study, patients taking metformin demonstrated a lower risk of both microvascular and macrovascular events, including cardiovascular death, nonfatal myocardial infarction, and nonfatal stroke.8,9 In another study, metformin was shown to have antiinflammatory and antiatherogenic effects in vascular cells in vitro and to limit atherosclerotic lesion development in vivo10 Moreover, it has been shown in animal models that metformin can improve endothelium function.11 Our previous data demonstrated that metformin

attenuates cardiac remodeling in failing rat hearts by ameliorating overactivated endoplasmic reticulum stress.12 However, it remains unknown how and to what extent metformin exerts an atheroprotective and anti-inflammatory role in patients. Peripheral blood mononuclear cells (MNCs) provide a representative view of the overall inflammatory status in the body.13,14 Proinflammatory transcription factor NF-kB activated in MNCs induces expression of proinflammatory genes and correlates with circulating systemic markers of inflammation.13-15 Our previous data have also shown that MNCs are involved in the inflammatory process and may potentially reflect the degree of AS and proinflammatory state.15-17 In the present study, we will investigate whether metformin exerts anti-inflammatory effects on circulating peripheral blood MNCs of patients with carotid artery AS. We examined the plasma concentration of proinflammatory mediators and their gene expression in MNCs. In this study, we also evaluated NF-kB binding in the nucleus of MNCs. Moreover, we assessed the expression of NF-kB subunit p65 and its regulator as a measure of NF-kB activity and inflammation. MATERIALS AND METHODS Patients and study design. A double-blind placebocontrolled study was performed at the First Affiliated Hospital of Medical College, Xi’an Jiaotong University. A total of 45 Chinese real-world Han patients (31 males and 14 females) with carotid artery AS were consecutively recruited from a real-world clinical practice. Metformin and placebo were purchased from Beijing Shengyong Pharmaceutical Co, Ltd (Beijing, China). To minimize potential confounding factors, patients with histories of stroke, recent acute coronary syndrome, acute inflammation, and treatment with steroids or immunosuppressive drugs were excluded from the study15,18 Patients contraindicated to metformin treatment or already under metformin administration were also excluded from the study. Three patients met our exclusion criteria. The remaining 42 patients were randomly assigned to the following 2 groups: placebo control group (Con; n 5 21) and metformin group (Met, n 5 21). All patients and physicians were blinded to the patient assignment. Table I summarizes the demographic data of all subjects. Day 2 after randomization, either study medication (500 mg twice a day) or placebo was administered to patients for 12 weeks. Fasting blood samples were collected before administration. Patients were followed twice (4 and 12 weeks after discharge) and fasting blood samples were collected again at the end of the 12-week study

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Table I. Baseline characteristics of the study population Characteristics

Placebo (n 5 21)

Metformin (n 5 21)

P

Sex, male/female, n/n Age, y Systolic BP, mm Hg Diastolic BP, mm Hg Smoking Disease Hypertension Diabetes Stroke Fasting glucose, mmol/L Total cholesterol, mmol/L Triglycerides, mmol/L HDL cholesterol, mmol/L LDL cholesterol, mmol/L hs-CRP, mg/L Treatment Aspirin b-Blocker ACE inhibitors/ARBs Insulin Sulfonylureas CCB Statins

14/7 55.1 6 10.9 128 6 18 79 6 10 7

15/6 55.2 6 10.4 129 6 17 76 6 10 9

1.00 0.88 0.89 0.55 0.56

7 2 5 5.28 6 0.57 3.87 6 0.65 1.75 6 1.03 0.99 6 0.16 2.36 6 0.57 3.1 (0.9, 7.8)

8 3 4 5.32 6 0.37 3.71 6 0.47 1.71 6 1.01 0.95 6 0.18 2.21 6 0.39 3.5 (1.3, 9.9)

1.00 1.00 1.00 0.86 0.48 0.89 0.74 0.34 0.58

12 1 2 1 2 8 5

14 1 3 2 1 11 7

0.75 1.00 1.00 1.00 1.00 0.54 0.74

Abbreviations: ACE, angiotensin-converting enzyme; ARB, angiotensin II type 1 receptor blocker; BP, blood pressure; CCB, calcium antagonists; HDL, high-density lipoprotein; hs-CRP, high-sensitivity C-reactive protein; LDL, low-density lipoprotein. Data are reported as the mean 6 standard deviation, median (interquartile range), or n.

period. The study protocol was explained and all participating patients gave written informed consents. This study was conducted in compliance with the Declaration of Helsinki, and the research protocol was approved by the Ethics Committee of Xi’an Jiaotong University. Carotid AS judgment. The extent of carotid AS was examined by ultrasound based on carotid plaque or intima-media thickness (IMT). Ultrasonographic scanning of the carotid artery was performed by an ultrasonic phase-locked echo-tracking system, which was equipped with a high-resolution real-time 10MHz linear scanner (HP Sonos 5500 Color Doppler ultrasonic system). Measurements were determined by a reader blinded to all clinical information. Briefly, longitudinal images of the bilateral carotid arteries, including the proximal (.10 mm proximal to bulb bifurcation) and distal common carotid artery, bulb as well as internal and external carotid arteries (10 segments), were acquired. Carotid IMT was determined in 3 segments as follows: the distal common carotid artery (1 cm proximal to the carotid bulb); the carotid artery bifurcation (1 cm proximal to the flow divider); and the proximal internal carotid artery (1 cm of length). The maximum IMT was recorded in the longitudinal and transverse projections at the site of the most advanced atherosclerotic lesion. Mean values of all IMT measurements from the right and left sides were calculated for

each patient. Plaque was defined as carotid IMT (cIMT) of 1.5 mm or more, or focal encroachment of 0.5 mm or more into the arterial lumen7,18,19 Isolation of MNCs. Peripheral blood MNCs were isolated by Ficoll standard density gradient centrifugation. The upper layer containing MNCs was harvested and washed with Hanks’ balanced salt solution and then with phosphate-buffered saline. Plasma concentrations of proinflammatory mediators. Concentrations of plasma IL-6, TNF-a, and IL-4

were assayed with enzyme-linked immunosorbent assay kits according to the manufacturers’ instructions. The lower limits of detection (or lowest concentration of standard sample) were 0.2 pg/mL for both IL-6 and TNF-a and 0.4 pg/mL for IL-4. All antibodies were purchased from Excell. High-sensitivity C-reactive protein (hs-CRP) assays were performed in the clinical laboratory in our hospital. Western blot analysis and quantitative polymerase chain reaction. Cytoplasmic protein levels of p65, sub-

units of MNCs were detected by Western blotting as previously described.12 Densitometry was performed using the Bio-Rad molecular analyst software and all values were corrected by loading b-actin. Total RNA isolation and real-time quantitative reverse transcription– polymerase chain reaction were performed as described previously.20

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NF-kB DNA binding activity. Nuclear proteins were extracted according to the manufacturer’s instructions (Pierce). NF-kB DNA binding activity was measured with NF-kB transcription factor assay kit (Abcam) according to the manufacturers’ instructions.20 Short interfering RNA transfection in MNCs. Human short interfering-sirtuin 1 (si-SIRT1) and mock short interfering RNA (siRNA) were obtained from Santz Cruz Biotechnology, and transient siRNA transfection was carried out according to the protocol of the manufacturer as described previously.12 Briefly, siRNAs were dissolved in double distilled water to prepare a 10 mM of stock solution. Isolated MNCs grown in 6-well plates were transfected with siRNA in OptiMEM (Invitrogen) containing RNAiMax (Invitrogen). For each transfection, 250 mL of transfection medium containing 5 mg of siRNA was gently mixed with 250 mL of transfection medium containing 5 mL of transfection reagent. After 20 minutes of incubation at room temperature, the mixture was added to cells in 2 mL of culture medium and cultured for 24 hours before treatment. Statistical analysis. Statistical analyses were performed using Statistical Product and Service Solutions (SPSS) for Windows (version 13.0, SPSS Inc, Chicago, IL). Discrete variables were expressed as numbers and percentages and analyzed using c2 test. Summary values were expressed as the mean 6 standard error. Skewed data were reported as medians (interquartile range). Paired t test was used to analyze changes in baseline concentrations. All multiple comparisons between the Con and Met groups were carried out using the Holm-Sidak 2-way repeated-measures analysis of variance (TWRMANOVA) method. All P values ,0.05 were considered statistically significant.

RESULTS Clinical data. All 42 patients fulfilled the 12-week follow-up without any drug-related adverse effects or cardiovascular events. Table I shows that there were no differences between the Met and Con groups in terms of baseline characteristics. Table II demonstrates that no significant changes in body mass index, body weight, systolic blood pressure, diastolic blood pressure, fasting glucose, and insulin levels were observed after 12 weeks of treatment. Metformin significantly decreased low-density lipoprotein cholesterol (LDL-C) after 12 weeks of treatment, although it did not alter levels of total cholesterol, triglycerides, and highdensity lipoprotein cholesterol (Table II). CRP is a well-established inflammatory marker that has been demonstrated to be increased in patients with clinical AS. We found that placebo did not alter plasma CRP level in the Con group. However, the plasma CRP

level was dramatically decreased after 12 weeks of metformin administration, indicating the antiinflammatory effect of metformin. Metformin reduces plasma proinflammatory mediators. To

concentrations

of

determine whether metformin attenuated the proinflammatory state in AS patients, we assessed plasma levels of IL-6, TNF-a, and IL4 before and after treatment. After 12 weeks of metformin treatment, plasma concentrations of IL-6 and TNF-a were decreased to 71 6 9% (P 5 0.02) and 64 6 9% (P 5 0.03) of the baseline, respectively, whereas in the Con group, plasma concentrations of these factors were increased to 107 6 9% (P 5 0.66) and 109 6 11% (P 5 0.59) of the baseline, respectively. Therefore, placebo did not alter the levels of proinflammatory factors. Furthermore, compared with the Con group, plasma levels of IL-6 and TNF-a were significantly decreased after 12 weeks of metformin administration (TWRMANOVA, P 5 0.001 and 0.015, respectively; Fig 1), suggesting that metformin attenuated proinflammatory mediators in AS patients. In addition, both metformin and placebo did not alter IL-4 level (TWRMANOVA, P 5 0.690; Fig 1, C). Metformin suppresses gene transcription of proinflammatory factors. We performed quantitative

real-time polymerase chain reaction to examine whether the effect of metformin on plasma proinflammatory mediators was attributable to messenger RNA (mRNA) expression of MNCs. In the Met group, the mRNA levels of IL-6 and TNF-a were decreased compared with the baseline (Fig 2), whereas in the Con group, the mRNA levels of these 2 factors were not significantly changed. The mRNA levels of IL-6 and TNF-a were significantly decreased in the Met group compared with the Con group (TWRWANOVA, P 5 0.001 and 0.002, respectively; Fig 2). In addition, the mRNA level of IL-4 remained unchanged in either the Met group or the Con group. Metformin inhibits NF-kB DNA binding activity in MNCs. We examined the NF-kB DNA binding activity

to explore whether metformin could affect the proinflammatory transcription factor NF-kB in MNCs. We found that the NF-kB DNA binding activity was significantly decreased to 46 6 10% of the baseline after metformin administration (P 5 0.006; Fig 2, D), suggesting that metformin could inhibit NF-kB DNA binding activity in vivo. Metformin inhibits p65 acetylation in vivo. Furthermore, we investigated the NF-kB signaling pathway in MNCs. The protein levels of NF-kB subunit p65 and acetylated p65 were detected. Interestingly, the p65 acetylation was significantly blocked by metformin administration, whereas the total protein level of p65 showed no significant changes compared with baseline in either

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Table II. Metabolic and other parameters at baseline and after 12 wk Placebo controls (n 5 21)

Metformin (n 5 21)

Marker

0 wk

12 wk

0 wk

12 wk

Weight, kg Systolic BP, mm Hg Diastolic BP, mm Hg Fasting glucose, mmol/L Fasting insulin, U/L Total cholesterol, mmol/L Triglycerides, mmol/L HDL cholesterol, mmol/L LDL cholesterol, mmol/L hs-CRP, mg/L

65.4 6 12.7 128 6 18 79 6 10 5.28 6 0.57 6.13 6 2.98 3.87 6 0.65 1.75 6 1.03 0.99 6 0.16 2.36 6 0.57 3.1 (0.9, 7.8)

65.6 6 11.3 127 6 17 78 6 8 5.30 6 0.49 6.10 6 3.02 3.85 6 0.71 1.68 6 0.91 1.01 6 0.41 2.33 6 0.43 2.9 (0.8, 7.2)

66.9 6 9.1 129 6 17 76 6 10 5.32 6 0.37 6.09 6 2.44 3.71 6 0.47 1.71 6 0.61 0.95 6 0.18 2.21 6 0.39 3.5 (1.3, 9.9)

66.3 6 8.9 126 6 17 74 6 9 5.31 6 0.45 5.89 6 2.62 3.59 6 0.43 1.73 6 0.51 0.92 6 0.41 1.99 6 0.30*‡ 1.3 (0.5, 4.8)†‡

Abbreviations: BP, blood pressure; HDL, high-density lipoprotein; hs-CRP, high-sensitivity C-reactive protein; LDL, low-density lipoprotein. n 5 21 for each group, values are reported as the mean 6 standard deviation or median (interquartile range). *P , 0.05 compared with baseline. † P , 0.01 compared with baseline. ‡ P , 0.01 compared with the placebo group.

B

P=0.001

* 10

5

0

0wk 12wk 0wk 12wk Con Met

6

C

P=0.015

* 4

2

0

0wk 12wk 0wk 12wk Met Con

Plasma IL-4 (pg/ml)

15

Plasma TNF-α (pg/ml)

Plasma IL-6 (pg/ml)

A

6

P=0.690

4

2

0

0wk 12wk 0wk 12wk Con Met

Fig 1. Metformin significantly reduced plasma concentrations of IL-6 and TNF-a in patients with carotid artery AS. Changes in plasma concentrations of proinflammatory mediators are presented as raw data. Plasma IL-6 and TNF-a levels were significantly decreased in the metformin group (Met) compared with the control group (Con) after 12 weeks (12 wk) of treatment (2-way repeated-measures analysis of variance, P 5 0.001 and 0.015, respectively). *P , 0.05, compared with baseline (0 wk). Both metformin and placebo did not alter IL-4 concentrations (2-way repeated-measures analysis of variance, P 5 0.690). AS, atherosclerosis; IL-6, interleukin 6; TNF-a, tumor necrosis factor alpha.

the Con or Met groups after 12 weeks of treatment (Fig 2, E). Metformin induces SIRT1 expression in vivo. As a novel and most important deacetylase in cell, SIRT1 has been known to play an important role in p65 acetylation. Therefore, we further detected the level of SIRT1 in MNCs. Fig 2, F shows that metformin, but not placebo, significantly increased the SIRT1 expression in MNCs, suggesting a novel mechanism of NF-kB regulation in vivo. in

Metformin inhibits NF-kB activity through SIRT1 induction vitro. We further verified whether the anti-

inflammatory effect of metformin was mediated through acetyl-p65–NF-kB inactivation at the cellular level. MNCs isolated from AS patients were transfected with either si-SIRT1 or mock siRNA, and

then treated with metformin or vehicle. Fig 3, A shows that metformin markedly induced SIRT1 and blocked p65 acetylation in isolated MNCs. SIRT1 knockdown, but not the mock siRNA, significantly abolished metformin-inhibited p65 acetylation, NF-kB activation, and IL-6 and TNF-a induction, revealing that metformin-inhibited NF-kB activity through SIRT1 induction in vitro. DISCUSSION

In the present study, we provided solid evidence that metformin could lessen the circulating inflammatory responses through the suppression of NF-kB activity and inhibition of proinflammatory mediators in MNCs. Furthermore, inhibition of p65 acetylation by SIRT1 in MNCs plays an important role in metformin treatment, which could serve a novel mechanism in clinical treatment for AS patients. Vascular inflammation is recognized as the fundamental mechanism for AS, and proinflammatory mediators play pivotal roles in AS.21 IL-6 and TNF-a are classic proinflammatory cytokines to promote AS, whereas IL-4 is an anti-inflammatory cytokine.22 It appears to be a promising strategy for AS treatment by reducing circulating IL-6 and TNF-a. Our study demonstrates that metformin administration significantly reduced plasma concentrations and the expression of IL-6 and TNF-a at mRNA level, indicating that metformin could directly attenuate the proinflammatory state of circulating MNCs. Inhibition of proinflammatory factors can further result in the reduction of neointima volume in metformin treatment.23 However, neither IL-4 protein nor mRNA level was altered by metformin. The main reason might be that

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B **

6 4 2 0

D Veh Met

P=0.736

4 3

6

2

4

1

2

0

0wk 12wk 0wk 12wk Con Met

E

150

0wk 12wk 0wk 12wk Met Con

F

Ac-p65

Sirt1

p65

Gapdh

100

** 50

0

0wk 12wk 0wk 12wk

6 100

**

50

0

0wk 12wk 0wk 12wk

Sirt1 (fold)

NF-κB activity (%)

**

8

0

0wk 12wk 0wk 12wk Met Con

C

P=0.002

IL-4 mRNA (au)

P=0.001

TNF-α mRNA (au)

IL-6 mRNA (au)

A8

Ac-p65 (%)

6

**

4 2 0

0wk 12wk 0wk 12wk

Fig 2. Metformin markedly attenuated NF-kB–related proinflammation in vivo. (A–C) Changes in mRNA expression of MNCs in baseline (0 wk) and after 12 weeks (12 wk) of treatment, including IL-6, TNF-a, and IL-4. (D) Changes in NF-kB binding activity of MNCs in baseline (0 wk) and after 12 weeks (12 wk) of treatment. (E and F) The protein level of p65, acetyl-p65, and SIRT1 in MNCs of baseline (0 wk) after treatment for 12 weeks (12 wk). **P , 0.01, compared with baseline. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-6, interleukin 6; MNCs, mononuclear cells; mRNA, messenger RNA; NF-kB, nuclear factor kappa B; SIRT1, sirtuin 1; TNF-a, tumor necrosis factor alpha.

Fig 3. SIRT1 induction is required for metformin-blocked p65 acetylation and NF-kB activation in vitro. MNCs isolated from patients with carotid artery AS were transfected with siRNA (mock or si-SIRT1) and then treated with metformin (Met). (A) Western blots were performed for SIRT1, acetyl-p65, and loading control (GAPDH). (B) NF-kB binding activity in MNCs. (C) The mRNA levels of proinflammatory genes, including IL-6 and TNF-a. *P , 0.05 compared with the Met group, #P , 0.05 compared with mock group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-6, interleukin 6; MNCs, mononuclear cells; mRNA, messenger RNA; NF-kB, nuclear factor kappa B; SIRT1, sirtuin 1; TNF-a, tumor necrosis factor alpha.

different cytokines may have different characteristics. First, IL-6 and IL-4 have different roles in the inflammatory pathway. IL-6 mainly promotes inflammation, whereas IL-4 mainly plays anti-inflammatory role. Second, the promoter of IL-4 does not contain NF-kB sequence, so IL-4 is not regulated by NF-kB. More importantly, IL-4 and IL-6 are derived from different

cell types. The cell source of IL-4 is T cell, which might react differently to metformin from MNCs where IL-6 and TNF-a are derived from mcarophage.24,25 Several randomized clinical studies have confirmed the benefits of metformin treatment,6,8 including cardiovascular protection, lipid control, nonalcoholic simple fatty liver alleviation, and tumor inhibition.

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Metformin has exhibited several antiatherosclerotic effects, including modulation of blood pressure and lipid concentrations, matrix remodeling activation of matrix proteases, and inhibition of inflammation.10,26 In the present study, we investigated the effect of metformin on each of these mechanisms. We did not observe a significant reduction in blood pressure, which was in accordance with other reports and might be attributable to the fact that the blood pressure of all subjects was already under control by optimal treatment. Consistent with other reports, metformin treatment did not alter total cholesterol, high-density lipoprotein, and triglyceride (TG), but it markedly lowered LDL. We also did not observe any changes in levels of blood glucose and insulin. The possible explanation could be that not all patients included in this study were diabetics, and metformin did not exert significant influence on the blood glucose of patients without diabetes. NF-kB, a key nuclear factor in inflammatory signaling, initiates the transcription of various cytokines for AS pathogenesis, including TNF-a and IL-6.27 Previous study28 has demonstrated that metformin inhibits NF-kB activity; however, its exact underlying mechanism remains unclear. In the present study, we found that oral administration of metformin significantly suppressed NF-kB binding activity in MNCs, thus resulting in a decrease in proinflammatory factors. As one major component of NF-kB, p65 has been known for some modifications.28 When it is acetylated, the DNA binding function is enhanced, which may aggregate AS progression. Interestingly, we found that acetyl-p65, instead of the total level of p65, played an important role in regulating NF-kB binding activity in vivo. SIRT1 is an nicotinamide adenine dinucleotide (NAD1)-dependent protein deacetylase. It has been shown to suppress NF-kB signaling through deacetylation of the p65 subunit of NF-kB, leading to the reduction of the inflammatory responses mediated by this transcription factor.27,29,30 Herein, we describe, for the first time, metformin-mediated SIRT1 induction in MNCs in vivo, which might be an important and novel finding of the mechanism, by which metformin alleviates inflammatory state. The major limitation of this study is that we could not fully exclude the potential effect of comedication, such as statin, a well-established anti-inflammatory drug. As shown in Table I, statins were also administrated to patients in both groups, which may be a potential confounding factor in the present study. Furthermore, metformin, but not placebo, significantly decreased LDL-C after 12 weeks of treatment as depicted in Table II. Therefore, we could not exclude the potential contribu-

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tion of the LDL-lowering effect of metformin, which requires further investigation. CONCLUSIONS

Our study demonstrated that 3 months of metformin treatment attenuated proinflammation state in MNCs through induction of SIRT1, blockade of NF-kB activation, and inhibition of inflammatory mediators. These findings suggest that metformin possessed a novel, direct atheroprotective role by modulating the circulating inflammatory responses in AS patients. ACKNOWLEDGMENTS

Conflicts of Interest: All authors have read the journal’s policy on conflicts of interest and have none to declare. This study was supported by the National Natural Science Foundation of China (grant no. 81300226, 81470550, 81400302, 81400660, 91339116, and 81100209), the National Basic Research Program of China (‘‘973 Project’’, no. 2012CB517804), and the Clinical Innovation Funds of the First Affiliated Hospital of XJTU (grant no. 14ZD20). Dr Yuan is a recipient of the National Natural Fund for Distinguished Young Scholars of China (81025002). All authors have read the journal’s authorship agreement. REFERENCES

1. Lind L. Circulating markers of inflammation and atherosclerosis. Atherosclerosis 2003;169:203–14. 2. Del Rincon I, Polak JF, O’Leary DH, et al. Systemic inflammation and cardiovascular risk factors predict rapid progression of atherosclerosis in rheumatoid arthritis. Ann Rheum Dis 2014; 74:1118–23. 3. van Diepen JA, Berbee JF, Havekes LM, Rensen PC. Interactions between inflammation and lipid metabolism: relevance for efficacy of anti-inflammatory drugs in the treatment of atherosclerosis. Atherosclerosis 2013;228:306–15. 4. Schreck R, Albermann K, Baeuerle PA. Nuclear factor kappa B: an oxidative stress-responsive transcription factor of eukaryotic cells (a review). Free Radic Res Commun 1992;17:221–37. 5. Woo SL, Xu H, Li H, et al. Metformin ameliorates hepatic steatosis and inflammation without altering adipose phenotype in dietinduced obesity. PLoS One 2014;9:e91111. 6. Riksen NP, el Messaoudi S, Rongen GA. It takes more than one CAMERA to study cardiovascular protection by metformin. Lancet Diabetes Endocrinol 2014;2:105–6. 7. Preiss D, Lloyd SM, Ford I, et al. Metformin for non-diabetic patients with coronary heart disease (the CAMERA study): a randomised controlled trial. Lancet Diabetes Endocrinol 2014;2: 116–24. 8. Holman RR, Paul SK, Bethel MA, Matthews DR, Neil HA. 10year follow-up of intensive glucose control in type 2 diabetes. N Engl J Med 2008;359:1577–89. 9. Effect of intensive blood-glucose control with metformin on complications in overweight patients with type 2 diabetes (UKPDS

8

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

Xu et al

34). UK Prospective Diabetes Study (UKPDS) Group. Lancet 1998;352:854–65. Oliveira PW, de Sousa GJ, Caliman IF, et al. Metformin ameliorates ovariectomy-induced vascular dysfunction in non-diabetic Wistar rats. Clin Sci 2014;127:265–75. Arunachalam G, Samuel SM, Marei I, Ding H, Triggle CR. Metformin modulates hyperglycaemia-induced endothelial senescence and apoptosis through SIRT1. Br J Pharmacol 2014;171: 523–35. Zhuo XZ, Wu Y, Ni YJ, et al. Isoproterenol instigates cardiomyocyte apoptosis and heart failure via AMPK inactivation-mediated endoplasmic reticulum stress. Apoptosis 2013;18:800–10. Ghanim H, Aljada A, Hofmeyer D, Syed T, Mohanty P, Dandona P. Circulating mononuclear cells in the obese are in a proinflammatory state. Circulation 2004;110:1564–71. Satoh M, Tabuchi T, Itoh T, Nakamura M. NLRP3 inflammasome activation in coronary artery disease: results from prospective and randomized study of treatment with atorvastatin or rosuvastatin. Clin Sci 2014;126:233–41. Wu Y, Zhang W, Liu W, Zhuo X, Zhao Z, Yuan Z. The doublefaced metabolic and inflammatory effects of standard drug therapy in patients after percutaneous treatment with drugeluting stent. Atherosclerosis 2011;215:170–5. Zhao Z, Wu Y, Cheng M, et al. Activation of Th17/Th1 and Th1, but not Th17, is associated with the acute cardiac event in patients with acute coronary syndrome. Atherosclerosis 2011;217: 518–24. Gao S, Liu W, Zhuo X, et al. The activation of mTOR is required for monocyte proinflammatory response in patients with coronary artery disease. Clin Sci 2014;128:517–26. Tan C, Liu Y, Li W, et al. Associations of matrix metalloproteinase-9 and monocyte chemoattractant protein-1 concentrations with carotid atherosclerosis, based on measurements of plaque and intima-media thickness. Atherosclerosis 2014;232:199–203. Koyama H, Maeno T, Fukumoto S, et al. Platelet P-selectin expression is associated with atherosclerotic wall thickness in carotid artery in humans. Circulation 2003;108:524–9.

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20. Liu J, Zhuo XZ, Liu W, et al. Drug-eluting stent, but not bare metal stent, accentuates the systematic inflammatory response in patients. Cardiology 2014;128:259–65. 21. Lang JK, Cimato TR. Cholesterol and hematopoietic stem cells: inflammatory mediators of atherosclerosis. Stem Cells Transl Med 2014;3:549–52. 22. Popko K, Gorska E, Stelmaszczyk-Emmel A, et al. Proinflammatory cytokines Il-6 and TNF-alpha and the development of inflammation in obese subjects. Eur J Med Res 2010; 15(suppl 2):120–2. 23. Lu J, Ji J, Meng H, et al. The protective effect and underlying mechanism of metformin on neointima formation in fructose-induced insulin resistant rats. Cardiovasc Diabetol 2013;12:58. 24. Bruun JM, Lihn AS, Pedersen SB, Richelsen B. Monocyte chemoattractant protein-1 release is higher in visceral than subcutaneous human adipose tissue (AT): implication of macrophages resident in the AT. J Clin Endocrinol Metab 2005;90:2282–9. 25. Hyun B, Shin S, Lee A, et al. Metformin down-regulates TNFalpha secretion via suppression of scavenger receptors in macrophages. Immune Netw 2013;13:123–32. 26. Madiraju AK, Erion DM, Rahimi Y, et al. Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase. Nature 2014;510:542–6. 27. Yang H, Zhang W, Pan H, et al. SIRT1 activators suppress inflammatory responses through promotion of p65 deacetylation and inhibition of NF-kappaB activity. PLoS One 2012;7:e46364. 28. Rothgiesser KM, Fey M, Hottiger MO. Acetylation of p65 at lysine 314 is important for late NF-kappaB-dependent gene expression. BMC Genomics 2010;11:22. 29. Kauppinen A, Suuronen T, Ojala J, Kaarniranta K, Salminen A. Antagonistic crosstalk between NF-kappaB and SIRT1 in the regulation of inflammation and metabolic disorders. Cell Signal 2013;25:1939–48. 30. Clarke NE, Belyaev ND, Lambert DW, Turner AJ. Epigenetic regulation of angiotensin-converting enzyme 2 (ACE2) by SIRT1 under conditions of cell energy stress. Clin Sci 2014; 126:507–16.