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Abstract. – OBJECTIVE: This study aims to explore whether the inhibitory role of met-formin could inhibit LPS-induced inflammato-ry response in vascular smooth muscle cells (VSMCs) and its underlying mechanism.

MATERIALS AND METHODS: VSMCs were extracted from aorta of Sprague Dawley rats. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was performed to detect VSMCs viability after treatment with dif-ferent concentrations of metformin. Levels of monocyte chemoattractant protein-1 (MCP-1), interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) in VSMCs were detected by ELISA (en-zyme-linked immunosorbent assay) and qRT-PCR (quantitative Real time-polymerase chain reaction). Protein and mRNA levels of toll like receptor 4 (TLR4) and peroxisome prolifera-tors activated receptor γ (PPAR-γ) in VSMCs were detected by Western blot and qRT-PCR, respectively. Finally, VSMCs were treated with the PPAR-γ antagonist GW9662 and inflamma-tory indicators in cells were detected.

RESULTS: No significant difference in VSMCs viability was found after 0-2 mM metformin treat-ment or 500 μg/L LPS induction for 24 h. After 500 μg/L LPS induction in VSMCs for 24 h, levels of MCP-1, TNF-α and IL-6 were remark-ably elevated. Both mRNA and protein levels of TLR4 in VSMCs were upregulated after 500 μg/L LPS induction for 24 h, which were re-markably reversed by the treatment of differ-ent concentrations of metformin. Knockdown of TLR4 remarkably inhibited LPS-induced inflam-matory response in VSMCs, manifesting as de-creased levels of MCP1, TNF-α and IL-6, which were further downregulated after combination treatment of TLR4 knockdown and 20 mM met-formin. Furthermore, both mRNA and protein levels of PPAR-γ in VSMCs were downregulated after 500 μg/L LPS induction for 24 h, which were remarkably reversed by the treatment of different concentrations of metformin. GW9662 treatment resulted in elevated expressions of MCP-1, TNF-α and IL-6, which were reversed by metformin treatment.

CONCLUSIONS: Metformin can effectively in-hibit the mRNA and protein expressions of IL-6, MCP-1, and TNF-α in LPS-induced VSMCs. The anti-inflammatory effects of metformin inhib-it the inflammatory response through downreg-ulating rely on the downregulation of TLR4 ex-pression and upregulation ofng PPAR-γ activity.

Key Words:Metformin, VSMCs, LPS, TLR4, PPAR-γ.

Introduction

Atherosclerosis (AS) is a major cause of car-diovascular and cerebrovascular diseases. It is expected that by 2020, AS will become the lead-ing cause of death throughout the world1. AS is one of the chronic complications of diabetes mellitus. Endothelial dysfunction is the initial step of atherosclerotic vascular complications, which is regulated by a variety of growth factors and cytokines2. Inflammatory response is involved in the occurrence and progression of AS, which is the central link in regulating plaque rupture and thrombosis3,4. Therefore, we considered that AS is an inflammatory disease involving multi-ple inflammatory factors. Vascular smooth muscle cells (VSMCs) are significant components of the arterial wall. VSMCs produce large amounts of inflammatory and adhesion factors under lipopoly-saccharide (LPS) stimulation, thus participating in the inflammatory response in the blood ves-sels5. Inflammation-induced intimal hyperplasia is the common behavior of AS and other vascular proliferative diseases. Multiple pro-inflammatory factors are activated to varying degrees after ar-terial injury6. In recent years, studies have found that metformin not only exerts hypoglycemic ef-

European Review for Medical and Pharmacological Sciences 2019; 23: 4988-4995

R.-N. QU1, W. QU2

1Department of Pharmacy, China-Japan Union Hospital of Jilin University, Changchun, China2Department of Pharmacy, The Affiliated Jiangyin Hospital of Southeast University Medical College, Jiangyin, China

Corresponding Author: Ruoning Qu, MM; e-mail: quruoning@163.com

Metformin inhibits LPS-induced inflammatory response in VSMCs by regulating TLR4 and PPAR-γ

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fect, but also protects cardiovascular diseases via inhibiting the proliferation of VMSCs7. Schol-ars8,9 have shown the diverse pharmacological aspects of metformin, such as anti-inflammatory, anti-proliferative, anti-tumor, and immune regu-lation. Metformin can inhibit the inflammatory response in VSMCs via adenosine monophos-phate activated protein kinase-gene of phosphate and tension homology deleted on chromosome ten (AMPK-PTEN) pathway10. PTEN, as a down-stream factor of AMPK, exerts a crucial role in regulating inflammation11. Meanwhile, metformin can inhibit LPS-induced pulmonary inflammatory response through downregulating toll like receptor 4 (TLR4) expression. Metformin has also been confirmed to activate peroxisome proliferators ac-tivated receptor γ (PPAR-γ)12,13. However, whether metformin has a protective effect on AS has not yet been reported. In this study, we examined whether metformin can inhibit LPS-induced in-flammatory responses in VSMCs.

Materials and Methods

Isolation and Culture of VSMCsThe aorta tissues of Sprague Dawley (SD) rats

aging 8-10 weeks were isolated and adventitia and intima were peeled off. The aorta tissues were cut into 1-2 mm3 for culturing of primary VSMCs. This study was approved by the Animal Ethics Committee of Jilin University Animal Center. Cells were then cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Rockville, MD, USA) containing 10% FBS (fetal bovine serum) (Gibco, Rockville, MD, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (HyClone, South Logan, UT, USA).

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl Tetrazolium Bromide) Assay

Culture medium was replaced with 20 μL of MTT solution (5 g/L) (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37°C for 4 h. The supernatant was discarded and 100 μL of DMSO (dimethyl sulf-oxide) (Sigma-Aldrich, St. Louis, MO, USA) were added into each well. The absorbance value was re-corded at the wavelength of 570 nm with a microplate reader (Bio-Rad, Hercules, CA, USA).

Cell TransfectionCells were seeded in the 6-well plates at a

density of 5×105 per well. Serum-free medium was replaced when the cell confluence was up to

70-80%. 200 μL of medium containing 75 pmol siRNA and 200 μL of medium containing 7.5 μL of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) were mixed together. After 20-min incubation at room temperature, cells were incu-bated with the mixture for 6 h.

ELISA (Enzyme-Linked Immunosorbent Assay)

Cells were seeded in the 96-well plates at a density of 5 × 104 per well. Serum-free medium was replaced when the cell confluence was up to 80-90%. After specific treatment, the supernatant of each group was collected for detecting levels of monocyte che-moattractant protein-1 (MCP-1), interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) using ELISA kits (R&D Systems, Minneapolis, MN, USA). The absorbance value was recorded at the wavelength of 450 nm with a microplate reader.

QRT-PCR (Quantitative Real Time-Polymerase Chain Reaction)

The TRIzol kit (Invitrogen, Carlsbad, CA, USA) was used to extract the total RNA, which was then reversely transcribed into complementary Deoxyri-bose Nucleic Acid (cDNA). After amplification of cDNA, qRT-PCR was performed to detect the ex-pressions of related genes. Primers used in this study were as follows: β-actin Forward: 5’-AATGAGCG-GTTCCGATGC-3’, Reverse: 5’-GGAAGGTGGA-CAGTGAGGC-3’; MCP-1 Forward: 5’-CACGTC-GTAGCAAACCACCAA-3’, Reverse: 5’-GTTG-GTTGTCTTTGAGATCCAT-3’; TNF-α Forward: 5’-CACGTCGTAGCAAACCACCAA-3’, Reverse: 5’-GTTGGTTGTCTTTGAGATCCAT-3’; IL-6 Forward: 5’-TGGTGATAAATCCCGATGAAG-3’, Reverse: 5’-GGCACTGAAACTCCTGGTCT-3’; TLR4 Forward: 5’-CTTTGAAAATGTAAGGC-GGC-3’, Reverse: 5’-ATGTAGGCAGGTGTGT-GGC-3’; PPAR-γ Forward: 5’-GATGACCACTC-CCATTCCTTT-3’, Reverse: 5’-AAACCTGATGG-CATTGTGAGA-3’.

Western BlotCells were lysed for protein extraction. The con-

centration of each protein sample was determined by a BCA (bicinchoninic acid) kit (Abcam, Cam-bridge, MA, USA). Protein sample was separat-ed by gel electrophoresis and transferred to PVDF (polyvinylidene difluoride) membranes (Millipore, Billerica, MA, USA). After incubation with primary and secondary antibody, immunoreactive bands were exposed by enhanced chemiluminescence method (Thermo Fisher Scientific, Waltham, MA, USA).

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Statistical AnalysisStatistical product and service solutions

(SPSS) 17.0 software (IBM, Armonk, NY, USA) was applied for statistical analysis. The quan-titative data were represented as mean ± stan-dard deviation (x̅±s). Differences among groups were analyzed by one-way analysis of variance (ANOVA), followed by Post-Hoc Test (Least Sig-nificant Difference). Multiple averages among groups were compared by SNK analysis. p<0.05 was considered statistically significant.

Results

Metformin Inhibited the Cell Viability of VSMCs

To exclude the possible cytotoxic effect of met-formin on experimental results, cytotoxicity effects of metformin (0-20 mM) and LPS (500 μg/L) on cell viability of VSMCs were determined by MTT assay. No significant difference in VSMCs viabil-ity was found after 0-2 mM metformin treatment or 500 μg/L LPS induction for 24 h (Figure 1A, p>0.05). In the following experiments, 5, 10, and 20 mM metformin were selected as the treatment conditions, which exerted remarkable inhibitory effects on VSMCs viability.

Metformin Reduced Levels of Inflammatory Factors in VSMCs After LPS Induction

After 500 μg/L LPS induction in VSMCs for 24 h, ELISA results showed the upregulated levels of MCP-1, TNF-α and IL-6 in the cell supernatant (Figure 1B-1D). The mRNA levels of MCP-1, TNF-α and IL-6 in VSMCs were also remarkably elevated after LPS induction (Figure 1E-1G). Additionally, levels of inflammatory fac-tors were decreased accompanied by the elevated concentration of metformin.

Metformin Reduced Levels of Inflammatory Factors in VSMCs After LPS Induction via Downregulating TLR4

The binding of LPS and CD14 on the membrane of VSMCs upregulated TLR4 expression, which is the key step in the inflammatory cascades in VSMCs14. Our study showed that both mRNA and protein levels of TLR4 in VSMCs were upregulated after 500 μg/L LPS induction for 24 h, which were remarkably reversed by the treatment of different concentrations of metformin (5, 10, 20 mM) (Figure

2A, p<0.05). To further explore the role of TLR4 in regulating the inflammatory response, TLR4-siRNA was constructed (Figure 2B). Knockdown of TLR4 remarkably inhibited LPS-induced inflammatory re-sponse in VSMCs, manifesting as decreased levels of MCP1, TNF-α and IL-6 (Figure 2C, 2E and 2G). Combination treatments of TLR4 knockdown and 20 mM metformin further decreased their levels in VSMCs (Figure 2D, 2F and 2H).

Metformin Inhibited LPS-Induced Inflammatory Response in VSMCs by Upregulating PPAR-γ Expression

PPAR-γ is an important negative regulator in LPS-induced inflammatory responses15. Our data showed that both mRNA and protein levels of PPAR-γ in VSMCs were downregulated after 500 μg/L LPS induction for 24 h, which were remark-ably reversed by the treatment of different con-centrations of metformin (5, 10, 20 mM) (Figure 3A, p<0.05). After 24 h of treatment with 20 mM metformin in VSMCs, mRNA and protein ex-pressions of PPAR-γ were also upregulated (Fig-ure 3B). To further clarify the anti-inflammatory effect of metformin, VSMCs were treated with the PPAR-γ antagonist GW9662, leading to the elevated expressions of MCP-1, TNF-α and IL-6 (Figure 3C, 3E and 3G). On the contrary, com-bination treatments of GW9662 and metformin remarkably downregulated expressions of these inflammatory factors (Figure 3D, 3F and 3H).

Discussion

In recent years, the role of long-term, low-lev-el inflammation in the formation and destabili-zation of atherosclerotic plaques has been well recognized. Pathological inflammatory response may induce apoptosis of VSMCs, accelerate the formation of atherosclerotic plaques and trans-form stable plaques into vulnerable plaques, thus promoting the occurrence and development of AS16. LPS is a highly potent inflammatory factor inducing the secretion of inflammatory mediators in endothelial cells, VSMCs, and macrophages17. Therefore, the inhibition of the inflammatory response in VSMCs cells has become a vital therapeutic target for alleviating atherosclerotic plaque formation. IL-6 can induce the activation of T cells, increase cytokine synthesis, promote the transformation of B cells into plasma cells with secretory function and induce the produc-tion of large amounts of specific antibodies.

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Figure 1. Metformin reduced levels of inflammatory factors in VSMCs after LPS induction. A, Cell viability was detected by MTT assay after treatment with different concentrations of metformin (0, 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM and 20 mM) and 500 μg/L LPS. B, MCP-1 level in VSMCs treated with metformin and LPS by ELISA. C, TNF-α level in VSMCs treated with metformin and LPS by ELISA. D, IL-6 level in VSMCs treated with metformin and LPS by ELISA. E, The mRNA level of MCP-1 in VSMCs treated with metformin and LPS by qRT-PCR. F, The mRNA level of TNF-α in VSMCs treated with metformin and LPS by qRT-PCR. G, The mRNA level of IL-6 in VSMCs treated with metformin and LPS by qRT-PCR.

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Figure 2. Metformin reduced levels of inflammatory factors in VSMCs after LPS induction via downregulating TLR4. A, The protein level of TLR4 after VSMCs treated with different concentrations of metformin (0, 5 mM, 10 mM and 20 mM) and 500 μg/L LPS. B, Transfection efficacy of TLR4-siRNA. C-D, The mRNA and protein levels of MCP-1 in VMSCs by different treatments. E-F, The mRNA and protein levels of TNF-α in VMSCs by different treatments. G-H, The mRNA and protein levels of IL-6 in VMSCs by different treatments.

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Figure 3. Metformin inhibited LPS-induced inflammatory response in VSMCs by upregulating PPAR-γ expression. A, The protein level of PPAR-γ after VSMCs treated with different concentrations of metformin (0, 5 mM, 10 mM and 20 mM) and 500 μg/L LPS. B, The mRNA level of PPAR-γ after VSMCs treated with different concentrations of metformin (0, 5 mM, 10 mM and 20 mM) and 500 μg/L LPS. C-D, The mRNA and protein levels of MCP-1 in VMSCs by different treatments. E-F, The mRNA and protein levels of TNF-α in VMSCs by different treatments. G-H, The mRNA and protein levels of IL-6 in VMSCs by different treatments.

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Current researches have shown that IL-6 pro-motes the occurrence and development of AS via regulating inflammatory response1. MCP-1 can induce macrophages in the circulatory sys-tem to enter the subendothelial region, thereby accelerating the formation of plaque fatty cores4. TNF-α accelerates atherosclerotic plaque forma-tion and plaque instability by inducing apoptosis of VSMCs in plaque areas, triggering platelet ad-hesion and aggregation, macrophage chemotaxis, and cholesterol deposition. Serum level of TNF-α is positively correlated to the degree of coronary stenosis3,18. Therefore, reversing the pathological upregulations of IL-6, MCP-1, and TNF-α con-tribute to AS alleviation. Toll-like receptor family members are involved in many aspects of inflam-mation-related immune responses, including mo-lecular pattern recognition, antigen presentation, and signal transduction. Among them, TLR4 is a crucial member of the Toll-like receptor family, which is a key regulator in LPS-induced inflammatory response. TLR4 is activated by the binding of LPS and CD14 on the cell membrane, eventually stimulating the release of multiple in-flammatory factors via Myd8819. Therefore, TLR4 inactivation protects the development of AS and cardiovascular diseases. In this study, we focused on the anti-inflammatory effects of metformin on regulating TLR4 activity. The results showed that metformin downregulates TLR4 expression in LPS-induced VSMCs in a concentration-de-pendent manner. TLR4 knockdown achieved the similar anti-inflammatory effect as that of met-formin. Combination treatment of metformin and TLR4 knockdown further enhance the inhibitory effects on releasing inflammatory factors. Hence, we speculated that the inhibitory effect of met-formin on LPS-induced release of inflammatory cytokines from VSMCs may be related to the downregulation of TLR4 expression. PPAR-γ is an important member of the PPAR family and regulates the expressions of downstream tar-get genes after binding to RXR20. PPAR-γ is widely distributed in cardiovascular tissues, and involved in inflammation, cell proliferation and invasion. Functionally, PPAR-γ exerts protective effects on VSMCs and cardiac fibroblasts through inhibiting the release of inflammatory factors21. Previous studies have shown that metformin can inhibit LPS-induced pulmonary inflammatory responses in mice by upregulating PPAR-γ ac-tivity22. In this study, we found that PPAR-γ expression was significantly downregulated in LPS-induced VSMCs. Metformin antagonized

the LPS-induced decrease of PPAR-γ expression in a concentration-dependent manner. Treatment of PPAR-γ antagonist GW9662 in VSMCs could partially counteract the anti-inflammatory effects of metformin by inhibiting PPAR-γ activation.

Conclusions

In this study, we first found that metformin can effectively inhibit the mRNA and protein ex-pressions of IL-6, MCP-1, and TNF-α in LPS-in-duced VSMCs. The anti-inflammatory effects of metformin rely on the downregulation of TLR4 expression and upregulation of PPAR-γ activity.

Conflict of InterestsThe Authors declare that they have no conflict of interests.

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