ABSTRACT
AIMS OF STUDY: 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-1ß (IL-1ß) induced proteoglycan (PG) degradation and apoptosis in rabbit articular cartilage and chondrocytes. MATERIALS AND METHODS: Rabbit articular cartilage explants and chondrocytes were cultured with 10 ng/ml IL-1ß 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-1ß 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-1ß 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-1ß-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. CONCLUSION: 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.
Subject(s)
Cartilage, Articular/drug effects , Chondrocytes/drug effects , Drugs, Chinese Herbal/pharmacology , Interleukin-1beta/metabolism , Proteoglycans/metabolism , Pyrazines/pharmacology , Animals , Apoptosis/drug effects , Cartilage, Articular/cytology , Cartilage, Articular/metabolism , Caspase 3/metabolism , Cell Survival/drug effects , Cells, Cultured , Chondrocytes/metabolism , Chondrocytes/physiology , Dose-Response Relationship, Drug , Glycosaminoglycans/metabolism , Matrix Metalloproteinase 3/metabolism , Membrane Potential, Mitochondrial/drug effects , Methylene Blue/analogs & derivatives , Methylene Blue/analysis , Rabbits , Reactive Oxygen Species/metabolism , Tissue Culture Techniques , Tissue Inhibitor of Metalloproteinase-1/metabolismABSTRACT
Sinomenine (SIN), an alkaloid extracted from the stem of the Chinese medicinal plant sinomenium acutum, has been used for treating rheumatoid arthritis. But little is known whether SIN has a protective effect on osteoarthritis (OA). In this study, we investigated the protective effect of SIN on IL-1beta-induced proteoglycan degradation and apoptosis in rabbit articular cartilage and chondrocytes. Treatment with 10 ng/ml IL-1beta increased the level of glycosaminoglycan (GAG) released into the culture media, and up-regulated the activity and mRNA expression of matrix metalloproteinase 13 (MMP-13) and down-regulated the activity and mRNA expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) in cartilage explants, as confirmed by the methods of GAG quantitation, MMP-13/TIMP-1 enzyme-linked immunosorbent assay (ELISA) and real-time quantitative RT-PCR. Treatment with 10 ng/ml IL-1beta resulted in marked apoptosis in chondrocytes, as demonstrated by decreased cell viability, occurrence of DNA laddering and increased caspase-3 activity and annexin V binding of phosphatidylserine. However, simultaneous treatment with SIN (10, 50 or 250 microM) inhibited the GAG release and the activity and mRNA expression of MMP-13, and enhanced the activity and mRNA expression of TIMP-1 in a dose-dependent manner in cartilage explants. Furthermore, DNA fragment, caspase-3 activity and apoptosis rate were down-regulated, and cell viability was up-regulated dose-dependently in chondrocytes. Thus, SIN has the protective capacity to antagonize cartilage degradation and chondrocyte apoptosis, which suggest that SIN may act as an agent for pharmacological intervention in the progress of OA.