Tetramethylpyrazine Inhibits The Proliferation Induced By PDGF On The Rat Airway Smooth Muscle Cell

Objective: The abnormal proliferation of airway smooth muscle cells is a well-documented pathologic character of the asthmatic airway. Measures which try to control the proliferations of airway smooth muscle cells are very important to retard or reverse asthmatic airway remodeling. Tetramethylpyrazine(TMP), a drug originally extracted from the rhizome of Ligusticum walliichi, because of so many pharmacologic action, has been widely treated respiratory diseases besides asthma. We established a proliferative model of airway smooth muscle cells to investigate inhibited effects of TMP on hyperplasia of airway smooth muscle cells, and study its mechanism by both cytology and molecular biology, and investigate the signal transduction pathways and medicinal targets involved in this process. This may give experimental supports for the application of TMP to the treatment of asthma. Methods: 1. ASMC were cultured by improved tissue-piece digestion inoculation. 2. A model of proliferative airway smooth muscle cells was established inducted by PDGF-BB. The absorbance ( A490 ) value of ASMC with treatment with PDGF in the presence and absence TMP were detected by MTT colorimetric assay to observe the proliferation induced by PDGF-BB and the antiproliferation induced by TMP. 3. Using Flow Cytometry analyzed DNA content of ASMC, of which DNA is stained with PI, after treatment with TMP to detect cell cycle phases. 4. Both of the levels of ERK1/2 and p-ERK1/2 protein were determined by western blotting. 5. Cell apoptosis after treatment with TMP assessed by staining with Hoechst 33342. Results: 1. We established a model of rat airway smooth muscle cells by modified primary culture. 2. A490 value of ASMC by MTT assay increased with the elevation of PDGF-BB concentration (2~80 ng/ml). Compared with control group, A490 value of 20 ng/ml PDGF group and 80 ng/ml PDGF group both were increased significantly after treatment with PDGF (P <0.01). 3. After 12 hours incubation with TMP, compared with control group, the average cell inhibition rates of 50μmol/L, 100μmol/L, 200μmol/L group were increased significantly (P<0.05). After 48 hours incubation with TMP, compared with control group, each group average cell inhibition rates were increased significantly (12.5μmol/L and 25μmol/L group, P<0.05, 50μmol/L and 100μmol/L and 200μmol/L group P<0.01) .4. The average G0/G1 phase rates of 50μmol/l group, 100μmol/l group and 200μmol/l group were increased significantly, the average S phase rates were decreased significantly (CV<8), compared with control group after 24 hours treatment with TMP. 5. The levels of p- ERK1/2 protein was significantly decreased at 30minutes and 60 minutes after treatment 200μmol/l TMP with 20 ng/ml PDGF. 6. Hyperfluorescence(50μmol/L,200μmol/L and 400μmol/L group)can be observed compared with control group after 24 hours treatment with TMP. Conclusions: 1. We established a model of rat airway smooth muscle cell by modified primary culture. 2. The proliferative model of rat airway smooth muscle cell were induced by PDGF. 3. The proliferation of airway smooth muscle cell was inhibited by TMP was related to cell cycle arresting in G0/G1 phase. 4. The possible inhibitory mechanism of TMP is related to phosphorylation of ERK1/2 in MAPK signal pathway. 5. The inhibitory effect after treatment with TMP in high concentration was related to cell apoptosis.