| Purpose:With more and more adverse drug reactions linked with Danshen injection,its safety comes under suspicion. The ADRs of Danshen injection include hematuria,hemolytic uremic syndrome, respiratory tract hemorrhage, facial angioneurotic edema, phlebitis and so on,which are related to peripheral vascular injury. The effects of Danshen Injection on mice and human umbilical vein endothelial cells(HUVECs)were observed in vivo and in vitro.To investigate the safety range of the clinical use of Danshen injection,and to provide theoretical basis for clinical mesication.Material and method:1.Material:According to the research needs, weighing 22.9±1.8g of Kunming mice strains 120,male and female sexes.The mice provided by Liao-ning University of Traditional Chinese Medicine. HUVECs were obtained from School of Basic Medical Sciences of China Medical University. Danshen injection was obtained from Chiatai Qingchunbao Pharmaceutical Co.Ltd.2.Methods:The 120 mice before the official start of the experimental animal laboratory medicine center(temperature 18-23 ?,humidity 40%-60%)adaptation of conventional breeding one week.The mice were randomly assigned according to body weight,gender stratification. Mice were divided into four groups( =30 in each group):vehicle, low-, middle-, and high-Danshen group, and each group was intravenously administered with Danshen injection at a dose of 0,0.64, 1.55, and 5.76g/kg/day for 5 days, respectively.Then, Blood and organ of mice were collected after killing the mice to observe the level of vascular injury, detect vascular injury factor(NO,ET-1) and do histopathological examination.HUVCEs were grown in Dulbecco's modified Eagle's medium supplemented with 10%fetal bovine serum, 100mg/ml heparin and penicillin/streptomycin(100U/m L and 100mg/m L)at37 ?, in a humidified atmosphere containing 95% air and 5% carbon dioxide.Experiments were performed with cells grown to a confluency of 80%.The HUVCEs were treated by PBS and Danshen injection(2 ? 10?50?g/ml), respectively. ELISA, flow cytometry, Western blotting and fluorescence staining method were used to observe the effect of different concentrations of Danshen injection on nitric oxide and endothelin,growth activity and apoptosis of HUVECs.Results:1.In vivo:High-dosage Danshen injection induced peripheral vascular function damage,vascular leakage was increased.2. Serum NO and ET-1 of high-dosage Danshen injection group were significantly increased.Only serum ET-1 increased was observed in Middle-dosage Danshen injection group.Detecting HUVECs culture medium was found the same results.3. High-dosage Danshen injection induced peripheral vascular pathology damage. The EC of high-dosage of Danshen injection group showed significant damage with cell membrane break(short white arrowhead) and cytoplasm damage(short black arrowhead), and didn't reveal integrated tight junction either.4. In vitro:High-dosage Danshen injection(10 and 50 mg/ml) increased the apoptosis and total cell death rate, special in the Danshen 50 group.5. ROS involved in high-concentration Danshen injection induced HUVECs' apoptosis.HUVECs were also pretreated with ROS scavenger NAC, followed by the high-dosage Danshen(50 mg/ml) exposure,whether the ROS production was related to the increased cell apoptosis.Conclusion:1.High-Danshen injection can damage peripheral vascular and lead to apoptosis of human umbilical vein endothelial cells. The mechanism is that high-Danshen injection can activate of caspase family of proteins of endothelial cells.2.Low-Danshen injection may be can protect human umbilical vein endothelial cells.All in all,these results suggest that Danshen injection is nontoxic in its recommended clinical dosage, and the 2.4-fold as the recommended clinical dosage might be the highest safety dosage in clinic treatment.However, high concentration dose Danshen injection can cause vascular endothelial cell apoptosis,which also can induce drug-induced vascular injury. |
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