Tongxinluo Reduces Oxidative Stress Induced By Hypoxia In Vascular Endothelial Cells

Objective:Hypoxia is a common pathological factor of vascularendothelial injury. It can damage the structure and function of endothelial cellsand lead to a variety of cardiovascular diseases. Thus, it is important toimprove endothelial dysfunction induced by hypoxia for prevention andtreatment of cardiovascular diseases.Tongxinluo(TXL), a Chinese traditional medicine, is widely used in theclinical treatment of cardiovascular diseases by improving hypoxia injury ofheart and brain, reducing oxidative stress damage. Recent studies have foundthat TXL could inhibit caspase-3activity induced by hypoxia in endothelialcells and reduce hypoxia-induced apoptosis of endothelial cells. TXL couldupregulate the expression of HIF in vascular endothelial cells by thePI-3K/Akt/HIF signaling pathway to improve the tolerance to hypoxia.However, the protection mechanism of TXL oxidative stress induced byhypoxia in vascular endothelial cells is largely unknown. In this study, weprepared animal models of acute and chronic hypoxia to observe the effect ofTXL on hypoxia-induced vascular endothelial injury and studied the effect ofTXL on CoCl2-induced endothelial cell damage.Methods:1C57BL/6J mice were randomly divided into three groups forchronic hypoxia testing: control group, hypoxia group and TXL treatmentgroup. So were KM mice for acute hypoxia testing.We performedimmunohistochemistry assay to investigate the effect of TXL on chronic andacute hypoxia-induced HO-1,p47-phox, p22-phox expression in the lungtissue.2Cultured human cardiac microvascular endothelial cells werepreincubated with TXL and then treated with CoCl2. Cells were collected and total protein and RNA were extracted. Western blot and Real-time PCR assayswere performed to investigate the effect of TXL on CoCl2-induced HO-1,p47-phox and p22-phox expression.Results:1CoCl2induced HO-1, p47-phox and p22-phox expression in atime-dependent mannerEndothelial cells were incubated with200μM CoCl2for the differenttimes, and expression of HO-1, p47-phox and p22-phox was determined byWestern blotting and Real-time PCR. The results indicated that CoCl2inducedHO-1, p47-phox and p22-phox expression in a time-dependent manner.2CoCl2induced HO-1, p47-phox and p22-phox expression in adose-dependent mannerEndothelial cells were stimulated with CoCl2at different doses for12h,and expression of HO-1, p47-phox and p22-phox was determined by Westernblotting and Real-time PCR. The results indicated that CoCl2induced HO-1,p47-phox and p22-phox expression in a dose-dependent manner.3TXL inhibited the expression of HO-1, p47-phox and p22-phox inducedby CoCl2Cultured human cardiac microvascular endothelial cells werepreincubated with TXL and then stimulated by CoCl2. Cells were collectedand total protein and RNA were extracted. Western blot and real-time PCRassays were performed to investigate the effect of TXL on CoCl2-inducedHO-1, p47-phox and p22-phox expression. As a result, TXL largely inhibitedthe expression of HO-1, p47-phox and p22-phox induced by CoCl2.4TXL inhibited the expression of HO-1, p47-phox and p22-phox inducedby chronic hypoxia in animal modelMice were randomly divided into three groups: control group, hypoxiagroup and TXL treatment group. We performed Immunohistochemistry assayto investigate the effect of on acute and chronic hypoxia-induced HO-1,p47-phox and p22-phox expression. The results indicated that TXL couldinhibit the expression of p47-phox and p22-phox induced by chronic hypoxia but not acute hypoxia.Conclusions:1CoCl2induces HO-1,p47-phox and p22-phox expression in a time anddose-dependent manner.2TXL inhibites the expression of HO-1, p47-phox and p22-phox inducedby CoCl2.3TXL inhibites the up regulation of HO-1, p47-phox and p22-phoxinduced by chronic hypoxia in animal model.4TXL improve the endothelial damage induced by hypoxia throughinhibiting the expression of p47-phox and p22-phox.