Protective Effect Of Tetramethylpyrazine On Lipopolysaccharide-induced Injury In Human Umbilical Vein Endothelial Cells

Objective: To explore the effects of tetramethylpyrazine(TMP) on expressions of Rho A, Rho associated coiled coil forming protein kinase 2(ROCK2), myosin light chain(MLC) phosphorylation and ezrin-radixin-moesin(ERM) phosphorylation on lipopolysaccharide(LPS)-induced injury in human umbilical vein endothelial cells(HUVECs), and the actin filament(F-actin) cytoskeleton changes of morphology and distribution in endothelial cells.Methods: HUVECs were cultured in vitro and randomized into 5 groups of Control group, LPS group(0.1 μg/ml) and TMP groups(0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml), n=6.Preprocessings: ① Control group and LPS group: adding separately an equal amount of the culture medium, without any other reagent; ② TMP groups: to join separately different concentrations of TMP into medium for 12 h of pretreatment. After the preprocessings: ① Control group: adding an equal amount of the culture medium; ②LPS group and TMP groups: adding the LPS into nutrient solution separately training for 12 h. Using immunofluorescence method to observe the changes of form and distribution on actin cytoskeleton, the expressions of Rho A, ROCK2 and p-Ezr m RNA in endothelial cells were detected by real-time fluorescence quantitative PCR and the protein expressions of Rho A, ROCK2, p-MLC and p-ERM were examined using western blotting.Results: Compared with the Control group, there was an apparent change of the F-actin morphology in LPS group, the stress composed of F-actin showed a trend of increasing and enlarge gradually within cytoplasm, and peripheral dense was obviously fractured,increasing the hollow shape crack among cells. In TMP groups, the F-actin peripheral dense zone and hollow shape crack were decrescent, and cell morphology obviously was better than which of LPS group. Compared with Control group, the protein expressions of Rho A, ROCK2, p-MLC, p-ERM and the m RNA expressions of Rho A,ROCK2, p-Ezr in LPS group were significantly increased(P<0.01); Compared with LPS group, the protein and the m RNA expressions of Rho A were no statistical significance in TMP groups and there was no significant differences among the TMP groups in the Rho A expression, while lowered the protein expressions of ROCK2,p-MLC, p-ERM and the m RNA expressions of ROCK2 and p-Ezr(P<0.01), and there was significant differences among TMP groups in the above experimental results.Conclusion: Tetramethylpyrazine may protect HUVECs against LPS-induced injury by inhibiting the expressions of ROCK2, p-MLC and p-ERM in Rho/ROCK signaling pathways, reducing the injury of lipopolysaccharide on endothelial cell skeleton.