Protective effects of aucubin on osteoarthritic chondrocyte model induced by hydrogen peroxide and mechanical stimulus.
BACKGROUND
During the onset of osteoarthritis (OA), certain biochemical events have been shown to accelerate cartilage degradation, including the dysregulation of cartilage ECM anabolism, abnormal generation of reactive oxygen species (ROS) and overproduction of proteolytic enzymes and inflammatory cytokines. The potency of aucubin in protecting cellular components against oxidative stress, inflammation and apoptosis effects are well documented, which makes it a potential candidate for OA treatment. In this study, we aimed to evaluate the protective benefits of aucubin against OA using H2O2and compression induced OA-like chondrocyte models.


METHODS
The effects of aucubin were studied in porcine chondrocytes after 1 mM H2O2stimulation for 30 min or sustained compression for 24 h. Effects of aucubin on cell proliferation and cytotoxicity of chondrocytes were measured with WST-1 and LDH assays. ROS production was evaluated by the Total ROS/Superoxide Detection Kit. Caspase-3 activity was evaluated by the CaspACE assay system. The levels of apoptosis were evaluated by the Annexin V-FITC apoptosis detection kit. OA-related gene expression was measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Total DNA quantification was evaluated by the DNeasy Blood and Tissue kit. Sulfated-glycosaminoglycans (sGAGs) production and content were evaluated by DMMB assay and Alcian blue staining.


RESULTS
The results showed that the ROS scavenge effects of aucubin appeared after 1 h of pretreatment. Aucubin could reduce the caspase-3 activity induced by H2O2, and reduced the apoptosis cell population in flowcytometry. In RT-qPCR results, aucubin could maintain ACAN and COL2A1 gene expressions, and prevent IL6 and MMP13 gene up-regulation induced by H2O2and compression stimulations. In the DMMB assay and Alcian blue staining, aucubin could maintain the sGAG content and protect chondrocytes against compressive stress, but not oxidative stress from H2O2.


CONCLUSIONS
These results indicated that aucubin has protective effects in an osteoarthritic chondrocyte model induced by H2O2and mechanical stimulus.
Institute of Biomedical Engineering, National Taiwan University, No. 49, Fanglan Rd, Taipei, 10672, Taiwan.
In-Chi
IC
Young
Institute of Pharmacology, College of Medicine, National Taiwan University, No. 1, Sec. 1, Ren-Ai Rd, Taipei, 10051, Taiwan.
Sung-Ting
ST
Chuang
Institute of Biomedical Engineering and Nanomedicine, National Health Research Institute, No. 35, Keyan Rd, Miaoli, 35053, Taiwan.
Chia-Hsien
CH
Hsu
Institute of Biomedical Engineering, National Taiwan University, No. 49, Fanglan Rd, Taipei, 10672, Taiwan.
Yu-Jun
YJ
Sun
Department of Orthopaedic Surgery, Taiwan Adventist Hospital, No. 424, Sec. 2, Bade Rd, Taipei, 10556, Taiwan.|Department of Orthopaedic Surgery, National Taiwan University Hospital, No.7, Chung Shan S. Rd, Taipei, 10002, Taiwan.
Hwa-Chang
HC
Liu
Institute of Biomedical Engineering, National Taiwan University, No. 49, Fanglan Rd, Taipei, 10672, Taiwan.
Yo-Shen
YS
Chen
Institute of Biomedical Engineering, National Taiwan University, No. 49, Fanglan Rd, Taipei, 10672, Taiwan. double@ntu.edu.tw.
Feng-Huei
FH
Lin
D053674:Aggrecans / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D019902:Chondrocytes / Q000187:drug effects*; D024043:Collagen Type II / Q000235:genetics; D004195:Disease Models, Animal; D015870:Gene Expression / Q000187:drug effects; D006861:Hydrogen Peroxide; D066298:In Vitro Techniques; D015850:Interleukin-6 / Q000235:genetics; D057888:Iridoid Glucosides / Q000627:therapeutic use* / Q000633:toxicity; D053509:Matrix Metalloproteinase 13 / Q000235:genetics; D010003:Osteoarthritis / Q000188:drug therapy* / Q000235:genetics; D010812:Physical Stimulation; D017382:Reactive Oxygen Species / Q000378:metabolism; D013552:Swine
D053674:Aggrecans; D024043:Collagen Type II; D015850:Interleukin-6; D057888:Iridoid Glucosides; D017382:Reactive Oxygen Species; C006650:aucubin; D006861:Hydrogen Peroxide; D053148:Caspase 3; D053509:Matrix Metalloproteinase 13
Aucubin; Inflammation; Mechanical stress; Osteoarthritis; ROS