High glucose-induced oxidative stress impairs proliferation and migration of human gingival fibroblasts.

Prima Buranasin,Kohei Takeda,Yuichi Izumi,Koji Mizutani,Kengo Iwasaki,Chantida Pawaputanon Na Mahasarakham,Daisuke Kido,Aamir Ahmad

doi:10.1371/journal.pone.0201855

Abstract

Delayed gingival wound healing is widely observed in periodontal patients with diabetes. However, the molecular mechanisms of the impaired function of gingival fibroblasts in diabetes remain unclear. The purpose of this study was to investigate changes in the properties of human gingival fibroblasts (HGFs) under high-glucose conditions. Primary HGFs were isolated from healthy gingiva and cultured with 5.5, 25, 50, and 75 mM glucose for 72 h. In vitro wound healing, 5-ethynyl-2′-deoxyuridine (EdU), and water-soluble tetrazolium salt (WST-8) assays were performed to examine cell migration and proliferation. Lactase dehydrogenase (LDH) levels were measured to determine cytotoxicity. The mRNA expression levels of oxidative stress markers were quantified by real-time PCR. Intracellular reactive oxygen species (ROS) were also measured in live cells. The antioxidant N-acetyl-l-cysteine (NAC, 1 mM) was added to evaluate the involvement of ROS in the glucose effect on HGFs. As a result, the in vitro wound healing assay showed that high glucose levels significantly reduced fibroblast migration and proliferation at 6, 12, 24, 36, and 48 h. The numbers of cells positive for EdU staining were decreased, as was cell viability, at 50 and 75 mM glucose. A significant increase in LDH was proportional to the glucose concentration. The mRNA levels of heme oxygenase-1 and superoxide dismutase-1 and ROS levels were significantly increased in HGFs after 72 h of exposure to 50 mM glucose concentration. The addition of NAC diminished the inhibitory effect of high glucose in the in vitro wound healing assay. The results of the present study show that high glucose impairs the proliferation and migration of HGFs. Fibroblast dysfunction may therefore be caused by high glucose-induced oxidative stress and may explain the delayed gingival wound healing in diabetic patients.

Highlights

Diabetes is a metabolic disease characterized by increased blood glucose levels
A growing body of evidence suggests that high glucose concentrations have deleterious effects on cell function that are dependent on the organ, tissue, and type of cell
It has been reported that glucose levels of 30 and 35 mM cause significant increases in apoptosis in endothelial cells [10] and keratinocytes [11], respectively, but not in fibroblasts

Summary

Introduction

Diabetes is a metabolic disease characterized by increased blood glucose levels. The impaired metabolism of glucose, lipids, and proteins produces alterations in macro- and microvascular circulation, giving rise to the risk of several complications in patients with diabetes, including retinopathy, neuropathy, nephropathy, cardiovascular complications [1], and delayed wound healing [2]. Many studies in various populations have demonstrated that diabetic patients tend to have a higher prevalence of and more severe periodontitis than nondiabetics [4]. It has been demonstrated that high blood sugar promotes inflammation and inhibits wound healing by altering angiogenesis [5]. Hyperglycemia has been shown to reduce migration [6, 7], proliferation [8], and collagen synthesis [9] and increase apoptosis [10, 11] in various cell types

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