Effects And Mechanisms Of Tetramethylpyrazine On The Proliferation Of Glomerular Mesangial Cells Induced By Lipopolysaccharide

The abnormal proliferation of glomerular mesangial cells is the common pathologic character of many glomerular diseases. Measures which try to control the proliferations of glomerular mesangial cells are very important to retard the development of glomerular damages. Tetramethylpyrazine is a kind of active component extracted from the alkaloid in szechwan lovge rhizome. It has many pharmacological effects. It has been widely used for the treatment of glomerulus disease, chronic renal failure, renal syndrome et al. In this subject, effects of tetramethylpyrazine on the proliferation of rat glomerular mesangial cells induced by lipopolysaccharide were studied. The potential mechanisms was probed from several aspects such as cell cycle, cell apoptosis and the expression of intercellular adhesion molecule-1 et al. The aim is to explore the possible mechanism of tetramethylpyrazine in treating renal diseases on cell level.This may give experimental supports for the application of tetramethylpyrazine to the treatment of renal diseases.In this subject rat glomerular mesangial cells were cultured in vitro. After glomerular mesangial cells were induced by lipopolysaccharide, tetramethylpyrazine was used to observe its effects on the proliferations and the growth curve of glomerular mesangial cells by MTT colorimetric assay and trypan blue staining .Cell cycle and apoptosis were analyzed by flow cytometry. The levels of intercellular adhesion molecule-1, Cyclin-dependent kinases inhibitor P27 , Apoptosis protein Bcl-2 , Bak were detected by immunohistochemistry.The result showed that tetramethylpyrazine could inhibit the proliferations of glomerular mesangial cells at the concentration of 400~2500mg/L.The inhibition rate was 25% at the concentration of 400mg/L while 65.9% at the concentration of 2500mg/L, which was in a dose dependent manner. The cell growth curve showed that Tetramethylpyrazine could make glomerular mesangial cells grow slowly at the concentration of 500mg/ L whileinhibit them from growing at the concentration of 2500mg/ L. By cell cycle analysis it was found that tetramethylpyrazine could block GO/G1 phase and make the cell ration of GO/G1 phase increased but S+G2/M phase decreased gradually. Then proliferation index declined. The levels of p27 in tetramethylpyrazine groups were increased 1.35, 1.52, 1.82 times than that of the lipopolysaccharide groups(P<0.05, .PO.01). Tetramethylpyrazine could promote the apoptosis of glomerular mesangial cells. The apoptosis rate in the largest dose group of tetramethylpyrazine was 29.8% . Meanwhile the levels of Bcl-2 in tetramethylpyrazine groups were decreased 15.79%, 29.19%, 46.41% than that of the lipopolysaccharide group (PO.05; PO.01) . But the Bax positive optical density changed slightly. The ratio of Bcl-2/Bax was decreased compared with the lipopolysaccharide groups. Lipopolysaccharide was found to be able to enhance the expression of intercellular adhesion molecule-1 on the surface of glomerular mesangial cells .The level was three times than the control groups (PO.01) .While the dose of tetramethylpyrazine was increased ,the intercellular adhesion molecule-1 expression level was decreased. The intercellular adhesion molecule-1 expression level in the lOOmg/L, 500mg/L, 2500mg/L tetramethylpyrazine groups were decreased by 14.05%, 23.97%, 30.58% respectively than that of the lipopolysaccharide groups (PO.05, PO.01) .The result suggested that the inhibiting effects of tetramethylpyrazine on the proliferation of rat glomerular mesangial cells induced by lipopolysaccharide may lie in that tetramethylpyrazine can modulate cell cycle, promote apoptosis and decrease the expression level of intercellular adhesion molecule-1.The possible mechanisms of its effects on cell cycle and cell apoptosis may have some relationship with its ability to modulate the expressions of p27 and Bcl-2 /Bax.