Tongxinluo Attenuates Endothelial Injury Induced By Sodium Arsenite Through Suppressing Oxidative Stress

Objective:Vascular endothelium is a continuous coating in vascular intima layer. In response to oxygen species, superoxide anion and hydrogen peroxide can cause endothelial cell injury. Oxidative damage of endothelial cells is closely related to atherosclerosis, hypertension, coronary heart disease and other cardiovascular disease. Tongxinluo?TXL?, is a compound traditional Chinese medicine,has been widely used to treat atherosclerosis, coronary heart disease, stroke and other cardiovascular diseases. A recent study found that TXL could improve endothelial function and inhibit oxidative stress. However, its action mechanism is still unclear. In this study we investigated the effect of TXL on endothelial function and its action mechanism.Methods:C57BL/6 mice were randomly assigned to one of the following three groups: control group, model group?NaAsO₂ treatment group? and TXL group.The mice of experimental and treatment group were intraperitoneally injected with sodium arsenite every day?3 mg/kg/d, 20 days?by 7.5 mg/kg/d of TXL was intragastrically administered 3d before sodium arsenite treatment. After 20 days mouse aortas were removed and divided into three parts to make paraffin embedding, extraction of proteins and RNAs. Immunohistochemistry was used to detect the expression of p22, HO-1, CD31, KLF5. Western blot and real-time PCR were used to detect the expression of p22, HO-1 and KLF5.Result: 1 TXL inhibits NaAsO₂ induced vascular permeability increase. Compared with control and TXL group, vascular wall of NaAsO₂ group showed a blue stain when incubated with Evans blue dye. This shows that NaAsO₂ induces vascular permeability increase, and tongxinluo can inhibit the permeability increase induced by NaAsO₂. 2 TXL inhibits the inflammatory response and oxidative stress induced by NaAsO₂. The results showed that TXL suppressed the expression of MCP-1and i NOS which associated with inflammation induced by NaAsO₂ in animal model.Western blot and real time PCR results showed that TXL inhibited the expression of HO-1, p47 and p22 induced by NaAsO₂ in animal model. TXL inhibits the expression of p22 and HO-1 in endothelial cell induced by NaAsO₂. To further investigate the effects of NaAsO₂ on the function of vascular endothelial cell cultured human umbilical vein endothelial cells were treated with different does of NaAsO₂ for 24 hours. Cells were collected and total proteins were extracted, Western blot was used to test the expression of HO-1 and p22. The results indicated that NaAsO₂ significantly induced HO-1 and p22 expression in a dose-dependent manner. When HUVEC was incubated with TXL for 12 hours, and then was treated with NaAsO₂, we found that TXL dose-dependently inhibited the expression of p22 and HO-1 induced by NaAsO₂ in endothelial cells. TXL inhibits the expression of ROS induced by NaAsO₂. Cultured human umbilical vein endothelial cells were treated with different does of NaAsO₂ for 24 hours. Results showed that NaAsO₂ does-dependentiy induced the expression of ROS. HUVEC was incubated with TXL for 12 hours, and then was treated with NaAsO₂ for 24 hours. Fluorescence microscopy was used to observe DCFH-DA fluorescence intensity. Results showed that TXL inhibited the expression of ROS induced by NaAsO₂. 3 TXL improves the endothelial injury induced by NaAsO₂ via promoting HUVEC proliferation. After HUVEC was treated with different does of NaAsO₂ for 24 hours, the expression of PCNA and CD31 was reduced, suggesting that NaAsO₂ inhibits endothelial cell proliferation. After incubation TXL, with the expression of CD31 and PCNA was determined by Western blotting. TXL dose-dependently promoted the expression of CD31 and PCNA. Immunohistochemical staining of CD31 and PCNA also obtained the similar results. 4 TXL reduces the expression of KLF5 induced by NaAsO₂. NaAsO₂ induces the expression of KLF5 in endothelial cells. Cultured human umbilical vein endothelial cells were treated with different does of NaAsO₂ for 24 hours,western blot and real time PCR showed that NaAsO₂ dose-dependently induced the expression of KLF5 in endothelial cells. HUVEC was incubated with TXL for 12 hours, and then was treated with NaAsO₂ for 24 hours. Western blot and real time PCR showed that TXL downregulated KLF5 expression induced by NaAsO₂. Immunohistochemistry assay also suggested that TXL inhibited the expression of KLF5 in animal model. 5 TXL improves NaAsO₂-induced oxidative stress via activating AKT signaling. HUVEC was incubated with TXL for 12 hours, and then was treated with NaAsO₂ for 24 hours.Western blot assay showed that AKT expression was induced, at the same time, TXL also induced AKT phosphorglation.Conclusions: 1 TXL inhibits NaAsO₂ induced vascular permeability increase. 2 TXL inhibits the inflammatory response and oxidative stress induced by NaAsO₂. 3 TXL improves the endothelial injury induced by NaAsO₂ via promoting HUVEC proliferation. 4 TXL reduces the expression of KLF5 induced by NaAsO₂. 5 TXL improves NaAsO₂-induced oxidative stress via activating AKT signaling.