The effects of high glucose condition on rat tenocytes in vitro and rat Achilles tendon in vivo

Abstract

Objectives: The aim of this study was to investigate the effect of hyperglycaemia on oxidative stress markers and inflammatory and matrix gene expression within tendons of normal and diabetic rats and to give insights into the processes involved in tendinopathy.

Methods: Using tenocytes from normal Sprague-Dawley rats, cultured both in control and high glucose conditions, reactive oxygen species (ROS) production, cell proliferation, messenger RNA (mRNA) expression of NADPH oxidase (NOX) 1 and 4, interleukin-6 (IL-6), matrix metalloproteinase (MMP)-2, tissue inhibitors of matrix metalloproteinase (TIMP)-1 and -2 and type I and III collagens were determined after 48 and 72 hours in vitro. In an in vivo study, using diabetic rats and controls, NOX1 and 4 expressions in Achilles tendon were also determined.

Results: In tenocyte cultures grown under high glucose conditions, gene expressions of NOX1, MMP-2, TIMP-1 and -2 after 48 and 72 hours, NOX4 after 48 hours and IL-6, type III collagen and TIMP-2 after 72 hours were significantly higher than those in control cultures grown under control glucose conditions. Type I collagen expression was significantly lower after 72 hours. ROS accumulation was significantly higher after 48 hours, and cell proliferation after 48 and 72 hours was significantly lower in high glucose than in control glucose conditions. In the diabetic rat model, NOX1 expression within the Achilles tendon was also significantly increased.

Conclusion: This study suggests that high glucose conditions upregulate the expression of mRNA for NOX1 and IL-6 and the production of ROS. Moreover, high glucose conditions induce an abnormal tendon matrix expression pattern of type I collagen and a decrease in the proliferation of rat tenocytes.Cite this article: Y. Ueda, A. Inui, Y. Mifune, R. Sakata, T. Muto, Y. Harada, F. Takase, T. Kataoka, T. Kokubu, R. Kuroda. The effects of high glucose condition on rat tenocytes in vitro and rat Achilles tendon in vivo. Bone Joint Res 2018;7:362-372. DOI: 10.1302/2046-3758.75.BJR-2017-0126.R2.

Keywords: High glucose; Oxidative stress; Tendinitis.

Fig. 1

Fluorescence staining showing reactive oxygen species (ROS) accumulation (green) in tenocytes and nuclei (4’,6-diamidino-2-phenylindole) (blue). Increased ROS accumulation was observed in high glucose conditions compared with those in the control glucose conditions.

Fig. 2

Quantification of the accumulated of reactive oxygen species (ROS). The ROS accumulation was analyzed by fluorescence intensity normalized to cell number. The intensity was higher in the high glucose than in the control glucose conditions and significant difference was seen at 48 hours (*p < 0.05).

Fig. 3

The messenger RNA (mRNA) expressions of NOX1 and NOX4 in the high glucose conditions were significantly higher than those in control glucose conditions at 48 hours. At 72 hours, the expression of NOX1 was significantly higher in the glucose conditions, while there was no significant difference in the expression of NOX4 between the groups. (*p < 0.05). There was no significant difference in the mRNA expression of type I and type III collagen between the two conditions at 48 hours. At 72 hours, in high glucose conditions, the expression of type I collagen was lower while the expression of type III collagen was higher than that in control glucose conditions (*p < 0.05); b) mRNA expressions of MMP-2 and TIMP-1 were significantly higher in high glucose conditions than those in control glucose conditions at 48 hours and 72 hours. There was a significant increase in TIMP-2 expression in high glucose conditions at 72 hours compared with that in the control glucose conditions (*p < 0.05). The mRNA expression of IL-6 was higher in high glucose than that in control glucose conditions at 48 hours and 72 hours, and the significant difference was observed at 72 hours (*p < 0.05).

Fig. 4

Graphs showing relative fold changes in the proliferation of tenocytes in control and high glucose concentrations at 48 and 72 hours, along with similar concentrations of mannitol (*p < 0.05) (N.S., not significant).

Fig 5

Achilles tendon histology. Haematoxylin and eosin staining of control and diabetic Achilles tendons harvested at six weeks following streptozotocin treatment. No obvious pathological difference between control and diabetic tendons was observed.

Fig. 6

Immunohistochemical staining for NADPH oxidase (NOX)1 and NOX4 expression in the Achilles tendon. The brown-stained cells were NOX-positive. Increased expression of NOX1 in Achilles tendon was observed in diabetic rats compared with control rats. There was no difference in the expression of NOX4 between diabetic and control rats.

Fig. 7

Graphs showing the semiquantitative analysis of cells positive for NADPH oxidase (NOX)1 (left) and NOX4 (right). The ratio of NOX1-positive cells in Achilles tendon was significantly higher in the diabetic rat than in the control rat. No significant difference was observed between control and diabetic rats in the ratio of NOX4-positive cells (*p < 0.05).

Fig. 8

Graphs showing relative fold changes in the messenger RNA (mRNA) levels in tendon: a) mRNA expression of NADPH oxidase (NOX)1 in Achilles tendon was significantly higher in diabetic rats than in control rats. No significant difference between control and diabetic rats was seen in the expression of NOX4 (*p < 0.05). The expression of type I collagen was significantly higher in diabetic rats than in control rats. No significant difference was observed between diabetic and control rats in the expression of type III collagen (*p < 0.05); b) mRNA expression of matrix metalloproteinase (MMP)-2, tissue inhibitors of matrix metalloproteinase (TIMP)-2 and interleukin (IL)-6 in diabetic rat Achilles tendon was significantly higher than in the control.