The protective effect of tetramethylpyrazine on cartilage explants and chondrocytes.
OBJECTIVE
Ligusticum wallichi Franchat (chuanxiong) is a very common traditional Chinese herbal medicine in China. Tetramethylpyrazine (TMP) is a major active ingredient extracted from Ligusticum wallichi Franchat. We investigated the protective effect of TMP on interleukin-1β (IL-1β) induced proteoglycan (PG) degradation and apoptosis in rabbit articular cartilage and chondrocytes.


METHODS
Rabbit articular cartilage explants and chondrocytes were cultured with 10 ng/ml IL-1β for 72 h in the absence or presence of various concentrations of TMP (50, 100 or 200 μM). Cartilage and chondroprotective effects of TMP were determined by evaluating (1) the degree of PG degradation by measuring the amount of glycosaminoglycan (GAG) released into the culture media with 1,9-dimethylmethylene blue (DMMB) assay in cartilage explants; (2) gene expression of MMP-3 and TIMP-1 by real-time quantitative reverse transcription-polymerase chain reaction analysis in cartilage explants; (3) chondrocytes viability with MTT assay; (4) the production of intracellular reactive oxygen species (ROS) with laser scanning confocal microscopy (LSCM). Anti-apoptotic effects of TMP were determined by measuring (1) apoptosis with flow cytometric analysis; (2) mitochondrial membrane potential assay with LSCM; (3) caspase-3 activity with special assay kit.


RESULTS
IL-1β treatment increased the level of GAG released into the culture media, and induced the gene expression of MMP-3 and inhibited the gene expression of TIMP-1 in cartilage explants. Moreover, IL-1β treatment decreased the cell viability and mitochondrial membrane potential, and enhanced the level of intracellular ROS, apoptosis rate, and caspase-3 activity in chondrocytes. However, simultaneous treatment with TMP attenuated the IL-1β-induced cartilage and chondrocyte destruction in a dose-dependent manner. TMP showed the decrease of GAG degradation and MMP-3 mRNA production, and the enhancement of TIMP-1 mRNA production in cartilage explants. TMP also increased the cell viability in chondrocytes. Furthermore, TMP inhibited the chondrocytes apoptosis through suppression of ROS production, maintaining of mitochondrial membrane potential and downregulation of caspase-3 activity.


CONCLUSIONS
These results demonstrate that TMP has the cartilage and chondroprotective effect, which suggest that TMP could act as an agent for pharmacological intervention in the progress of OA.
Ju
Xiao-dong
XD
Institute of Sports Medicine, Peking University Third Hospital, No. 49, North Garden Road, Haidian District, Beijing 100191, China.
Deng
Min
M
Ao
Ying-fang
YF
Yu
Chang-long
CL
Wang
Jian-quan
JQ
Yu
Jia-kuo
JK
Cui
Guo-qing
GQ
Hu
Yue-lin
YL
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004365:Drugs, Chinese Herbal / Q000494:pharmacology*; D006025:Glycosaminoglycans / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism*; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008751:Methylene Blue / Q000031:analogs & derivatives / Q000032:analysis; D011509:Proteoglycans / Q000378:metabolism*; D011719:Pyrazines / Q000494:pharmacology*; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism; D046509:Tissue Culture Techniques; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism
D004365:Drugs, Chinese Herbal; D006025:Glycosaminoglycans; D053583:Interleukin-1beta; D011509:Proteoglycans; D011719:Pyrazines; D017382:Reactive Oxygen Species; D019715:Tissue Inhibitor of Metalloproteinase-1; D053148:Caspase 3; D019278:Matrix Metalloproteinase 3; D008751:Methylene Blue; C016401:1,9-dimethylmethylene blue; C017953:tetramethylpyrazine