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. 2023 Apr 14;45(4):3434-3445.
doi: 10.3390/cimb45040225.

Elicitation of Inhibitory Effects for AGE-Induced Oxidative Stress in Rotator Cuff-Derived Cells by Apocynin

Takahiro Furukawa  1 , Takashi Kurosawa  1 , Yutaka Mifune  1 , Atsuyuki Inui  1 , Hanako Nishimoto  1 , Yasuhiro Ueda  1 , Takeshi Kataoka  1 , Kohei Yamaura  1 , Shintaro Mukohara  1 , Tomoya Yoshikawa  1 , Issei Shinohara  1 , Tatsuo Kato  1 , Shuya Tanaka  1 , Masaya Kusunose  1 , Yuichi Hoshino  1 , Takehiko Matsushita  1 , Ryosuke Kuroda  1
Affiliations

Elicitation of Inhibitory Effects for AGE-Induced Oxidative Stress in Rotator Cuff-Derived Cells by Apocynin

Takahiro Furukawa et al. Curr Issues Mol Biol. .

Abstract

Advanced glycation end-products (AGEs) play a critical supportive role during musculoskeletal disorders via glycosylation and oxidative stress. Though apocynin, identified as a potent and selective inhibitor of NADPH oxidase, has been reported to be involved in pathogen-induced reactive oxygen species (ROS), its role in age-related rotator cuff degeneration has not been well clarified. Therefore, this study aims to evaluate the in vitro effects of apocynin on human rotator cuff-derived cells. Twelve patients with rotator cuff tears (RCTs) participated in the study. Supraspinatus tendons from patients with RCTs were collected and cultured. After the preparation of RC-derived cells, they were divided into four groups (control group, control + apocynin group, AGEs group, AGEs + apocynin group), and gene marker expression, cell viability, and intracellular ROS production were evaluated. The gene expression of NOX, IL-6, and the receptor for AGEs (RAGE) was significantly decreased by apocynin. We also examined the effect of apocynin in vitro. The results showed that ROS induction and increasing apoptotic cells after treatment of AGEs were significantly decreased, and cell viability increased considerably. These results suggest that apocynin can effectively reduce AGE-induced oxidative stress by inhibiting NOX activation. Thus, apocynin is a potential prodrug in preventing degenerative changes of the rotor cuff.

Keywords: advanced glycation end-products; apocynin; oxidative stress; reactive oxygen species; rotator cuff.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(a) Expression of NOX-1 mRNA in the AGEs group was significantly higher than in the other groups at 48 h. (b) Expression of NOX-4 mRNA; (c) expression of IL-6 mRNA; and (d) expression of RAGE mRNA showed similar findings to that of NOX-1. * p < 0.05.
Figure 2
Figure 2
At 48 h, cell viability in the AGEs group was significantly lower than in the control + apo groups. That is, cell viability in the AGEs + apo group was significantly higher than in the AGEs group. However, there was no difference between the control, control + apo, and AGEs + apo groups. * p < 0.05.
Figure 3
Figure 3
Immunofluorescence labeling depicting apoptotic (green) cells in each group. (a,b) There were few apoptotic cells in the control and control + apo groups. (c,d) There was apoptosis induction in AGEs and AGEs + apo groups. The amount of apoptotic cells in the AGEs + apo group was lower than that in the AGEs group.
Figure 4
Figure 4
Quantification of the number of apoptotic cells. The number of apoptotic cells was analyzed by fluorescence intensity normalized to cell number. At 48 h, the number of apoptotic cells in the AGEs and AGEs + apo groups was considerably higher than in the control + apo group. In the AGEs + apo group, there were fewer apoptotic cells than in the AGEs group, though there was no significant difference between them. * p < 0.05. NS, no significance, DAPI, 4′,6-diamidino-2-phenylindole.
Figure 5
Figure 5
Fluorescence staining showing ROS. ROS production in rotator cuff-derived cells and nuclei (DAPI) (blue). (a,b) Low ROS production was observed in control + apo groups; (c,d) increased ROS production was observed in the AGEs group and the AGEs + apo group compared to the control and control + apo groups. ROS production was decreased in the AGEs + apo group compared to the AGEs group.
Figure 6
Figure 6
The quantification of ROS generation. The generation of reactive oxygen species was evaluated by fluorescence intensity normalized to cell number. ROS production in the AGEs group and the AGE + apo group was significantly higher than in control + apo groups at 48 h. ROS production in the AGEs + apo group was significantly lower than in the AGEs group. * p < 0.05. DAPI, 4′,6-diamidino-2-phenylindole.
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