Evaluation of apocynin in vitro on high glucose-induced oxidative stress on tenocytes

Abstract

Aims The purpose of this study was to evaluate the in vitro effects of apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase (NOX) and a downregulator of intracellular reactive oxygen species (ROS), on high glucose-induced oxidative stress on tenocytes. Methods Tenocytes from normal Sprague-Dawley rats were cultured in both control and high-glucose conditions. Apocynin was added at cell seeding, dividing the tenocytes into four groups: the control group; regular glucose with apocynin (RG apo+); high glucose with apocynin (HG apo+); and high glucose without apocynin (HG apo–). Reactive oxygen species production, cell proliferation, apoptosis and messenger RNA (mRNA) expression of NOX1 and 4, and interleukin-6 (IL-6) were determined in vitro. Results Expression of NOX1, NOX4, and IL-6 mRNA in the HG groups was significantly higher compared with that in the RG groups, and NOX1, NOX4, and IL-6 mRNA expression in the HG apo+ group was significantly lower compared with that in the HG apo– group. Cell proliferation in the RG apo+ group was significantly higher than in the control group and was also significantly higher in the HG apo+ group than in the HG apo– group. Both the ROS accumulation and the amounts of apoptotic cells in the HG groups were greater than those in the RG groups and were significantly less in the HG apo+ group than in the HG apo– group. Conclusion Apocynin reduced ROS production and cell death via NOX inhibition in high-glucose conditions. Apocynin is therefore a potential prodrug in the treatment of diabetic tendinopathy. Cite this article: Bone Joint Res 2020;9(1):23–28.

In Vitro and in Vivo Tenocyte-protective Effectiveness of Dehydroepiandrosterone Against High Glucose-induced Oxidative Stress

“…High glucose conditions upregulate the expression of NOX1 mRNA and ROS production in rat tenocytes and Achilles tendon 16,17 suggesting that overexpression of NOX may be a therapeutic target for diabetic tendinopathy. Here, the in vitro effectiveness of DHEA on hyperglycemia-induced oxidative stress in rat tenocytes was investigated.…”

“…Under pathological conditions, the upregulation of tissue-and diseasespeci c NOX subtypes can cause overproduction of ROS 15 . Previous reports showed NOX1 expression was increased in rat tenocytes and Achilles tendons under hyperglycemic conditions, and the accompanying ROS overproduction and in ammatory reaction contributed to diabetic tendinopathy 16,17 . Dehydroepiandrosterone (DHEA) is the most abundant circulating steroid hormone produced by the mammalian adrenal cortex 18 .…”

“…According to previous studies 16,17 , nuclear fragmentation was detected by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining with an APO-DIRECT Kit (Phoenix Flow Systems, San Diego, CA, USA) according to the manufacturer's protocol, using xed cells (4% paraformaldehyde/PBS) with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI). For quanti cation, the number of apoptosis-positive and DAPI-positive cells in four rectangular areas (0.75 mm × 1.0 mm) in each slide were counted and the mean values were calculated.…”

“…The list of primer sequences are shown in Table 1. Results were normalized to the mRNA levels of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed relative to their levels in the control culture using the 2 -ΔΔCt method 25 as previous studies 16,17 (n=15 per group).…”

“…According to previous reports 16,17 , intracellular ROS levels in tenocytes of each group were detected by the oxidation-sensitive uorescent probe dichloro-dihydro-uorescein diacetate (DCFH-DA) using the Total In vitro experiments: Immuno uorescence staining for analysis of apoptotic cells…”