Yonsei Med J.  2016 Mar;57(2):373-381. 10.3349/ymj.2016.57.2.373.

Anti-Proliferative Effects of Rutin on OLETF Rat Vascular Smooth Muscle Cells Stimulated by Glucose Variability

• Yu SH
• Yu JM
• Yoo HJ
• Lee SJ
• Kang DH
• Cho YJ
• Kim DM

• KMID: 2374042
• DOI: http://doi.org/10.3349/ymj.2016.57.2.373

Abstract

PURPOSE
Proliferation of vascular smooth muscle cells (VSMCs) plays a crucial role in atherosclerosis. Rutin is a major representative of the flavonol subclass of flavonoids and has various pharmacological activities. Currently, data are lacking regarding its effects on VSMC proliferation induced by intermittent hyperglycemia. Here, we demonstrate the effects of rutin on VSMC proliferation and migration according to fluctuating glucose levels.
MATERIALS AND METHODS
Primary cultures of male Otsuka Long-Evans Tokushima Fatty (OLETF) rat VSMCs were obtained from enzymatically dissociated rat thoracic aortas. VSMCs were incubated for 72 h with alternating normal (5.5 mmol/L) and high (25.0 mmol/L) glucose media every 12 h. Proliferation and migration of VSMCs, the proliferative molecular pathway [including p44/42 mitogen-activated protein kinases (MAPK), mitogen-activated protein kinase kinase 1/2 (MEK1/2), p38 MAPK, phosphoinositide 3-kinase (PI3K), c-Jun N-terminal protein kinase (JNK), nuclear factor kappa B (NF-kappaB), and Akt], the migratory pathway (big MAPK 1, BMK1), reactive oxygen species (ROS), and apoptotic pathway were analyzed.
RESULTS
We found enhanced proliferation and migration of VSMCs when cells were incubated in intermittent high glucose conditions, compared to normal glucose. These effects were lowered upon rutin treatment. Intermittent treatment with high glucose for 72 h increased the expression of phospho-p44/42 MAPK (extracellular signal regulated kinase 1/2, ERK1/2), phospho-MEK1/2, phospho-PI3K, phospho-NF-kappaB, phospho-BMK1, and ROS, compared to treatment with normal glucose. These effects were suppressed by rutin. Phospho-p38 MAPK, phospho-Akt, JNK, and apoptotic pathways [B-cell lymphoma (Bcl)-xL, Bcl-2, phospho-Bad, and caspase-3] were not affected by fluctuations in glucose levels.
CONCLUSION
Fluctuating glucose levels increased proliferation and migration of OLETF rat VSMCs via MAPK (ERK1/2), BMK1, PI3K, and NF-kappaB pathways. These effects were inhibited by the antioxidant rutin.

Keyword

Rutin; hyperglycemia; smooth muscle cells; mitogen-activated protein kinases; phosphatidylinositol 3-kinase

MeSH Terms

Animals
Caspase 3/metabolism
Cell Movement/*drug effects
Cell Proliferation/*drug effects
Flavonoids/*pharmacology
Glucose/*metabolism/pharmacology
JNK Mitogen-Activated Protein Kinases
MAP Kinase Kinase 1
Male
Mitogen-Activated Protein Kinase 3
Muscle, Smooth, Vascular/cytology/*drug effects/enzymology
Myocytes, Smooth Muscle/metabolism
NF-kappa B/metabolism
Phosphatidylinositol 3-Kinases
Protein Kinase Inhibitors/*pharmacology
Rats
Rats, Inbred OLETF
Rats, Long-Evans
Reactive Oxygen Species/metabolism
Rutin/*pharmacology
p38 Mitogen-Activated Protein Kinases/metabolism
Caspase 3
Glucose
Flavonoids
JNK Mitogen-Activated Protein Kinases
NF-kappa B
MAP Kinase Kinase 1
Mitogen-Activated Protein Kinase 3
Phosphatidylinositol 3-Kinases
Protein Kinase Inhibitors
Reactive Oxygen Species
Rutin
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Fig. 1 Inhibitory effects of rutin on proliferation of vascular smooth muscle cells (VSMCs) from Otsuka Long-Evans Tokushima Fatty (OLETF) rats at fluctuating glucose concentrations. (A) Representative photomicrographs of morphology of OLETF rat VSMCs (magnification ×100). (B) Cell proliferation was evaluated by the methylthiazoletetrazolium assay and expressed as cell viability (%). VSMCs were incubated for 72 h with or without glucose fluctuation (alternating 5.5 and 25 mM every 12 h) and rutin (1, 10, 30, and 100 µM). Data are expressed as mean±SD from 5 separate experiments. *p<0.05 vs. control, †p<0.05 vs. glucose fluctuations. Cont, control; Fluc, glucose fluctuations.

• 

  Fig. 2 Inhibitory effects of rutin on proliferation of vascular smooth muscle cells (VSMCs) at fluctuating glucose concentrations through the mitogen-activated protein kinase (MAPK) signaling pathway determined by western blot analysis. VSMCs were incubated for 72 h with or without glucose fluctuation (alternating 5.5 and 25 mM every 12 h) and rutin (30 µM). Data are expressed as mean±SD from 5 separate experiments. *p<0.05 vs. control, †p<0.05 vs. glucose fluctuations. C, control; ERK1/2, extracellular signal regulated kinase 1/2; F, glucose fluctuation; JNK, c-Jun N-terminal protein kinase; MEK1/2, mitogen-activated protein kinase kinase 1/2; R, rutin; p, phosphorylated; p38 MAPK, p38 mitogen-activated protein kinase.

• 

  Fig. 3 Inhibitory effects of rutin on proliferation of vascular smooth muscle cells (VSMCs) at fluctuating glucose concentrations through nuclear factor kappa B (NF-κB) and phosphoinositide 3-kinase (PI3K)/Akt pathway determined by western blot analysis. VSMCs were incubated for 72 h with or without glucose fluctuation (alternating 5.5 and 25 mM every 12 h) and rutin (30 µM). Data are expressed as mean±SD from 5 separate experiments. *p<0.05 vs. control, †p<0.05 vs. glucose fluctuations. C, control; F, glucose fluctuation; p, phosphorylated; R, rutin.

• 

  Fig. 4 Effect of ERK inhibitor (PD98059) and PI3K inhibitor (wortmannin) on proliferative protein expression of glucose fluctuation-induced vascular smooth muscle cell (VSMC) with or without rutin determined by western blot analysis. VSMCs were incubated for 72 h with or without glucose fluctuation (alternating 5.5 and 25 mM every 12 h) and rutin (30 µM). And PD98059 10 and 20 µM or wortmannin 10 and 20 µM were added in control (5.5 mM) and alternating (5.5 and 25 mM) glucose medium. Data are expressed as mean±SD from 5 separate experiments. *p<0.05. C, control; ERK1/2, extracellular signal regulated kinase 1/2; F, glucose fluctuation; MEK1/2, mitogen-activated protein kinase kinase 1/2; NF-κB, nuclear factor kappa B; R, rutin; p, phosphorylated; PD, PD98059; PI3K, phosphoinositide 3-kinase; W, wortmannin.

• 

  Fig. 5 Inhibitory effect of rutin on proliferation of vascular smooth muscle cells (VSMCs) through apoptotic pathways determined by western blot analysis. VSMCs were incubated for 72 h with or without glucose fluctuation (alternating 5.5 and 25 mM every 12 h) and rutin (30 µM). Data are expressed as mean±SD from 5 separate experiments. Bcl-2, B-cell lymphoma 2; Bcl-xL, B-cell lymphoma-extra-large; C, control; F, glucose fluctuation; p, phosphorylated; R, rutin.

• 

  Fig. 6 Inhibitory effects of rutin on glucose fluctuation-induced vascular smooth muscle cell migration in Otsuka Long-Evans Tokushima Fatty rats. (A) The percentage inhibitory area of migratory cells was calculated using a microscope. Control (normal glucose, 5.5 mM), glucose fluctuations (alternating 5.5 mM and 25 mM every 12 h), glucose fluctuations with 30-µM rutin (rutin treatment with alternating 5.5 mM and 25 mM every 12 h). (B) Inhibitory effect of rutin on big mitogen-activated protein kinase 1 (BMK1; migration pathway) by western blot analysis. Five separate experiments were performed. *p<0.05 vs. control, †p<0.001 vs. glucose fluctuations. C, control; F, glucose fluctuation; p, phosphorylated; R, rutin.

• 

  Fig. 7 Inhibitory effect of rutin on production of reactive oxygen species determined by fluorescence spectrophotometric analysis. VSMCs were incubated for 72 h with or without glucose fluctuation (alternating 5.5 and 25 mM every 12 h) and rutin (30 µM). Data are expressed as mean±SD from 5 separate experiments. *p<0.05 vs. control, †p<0.05 vs. glucose fluctuations. C, control; F, glucose fluctuation; p, phosphorylated; R, rutin; VSMCs, vascular smooth muscle cells.

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