Study Guanxinkang Granules On The Oxidative Injury Of Endothelial Cells Induced By Homocysteine

Objective: HHcy is one of independent risk factors for AS. But the mechanism in atherogenesis of Hcy is not clear till to now. Currently, most research results show that it is the initial stage for AS that endothelial dysfunction leads to HHcy-induced vascular injury. So it is of great significance to investigate the mechanism about atherogensis of Hcy. As we all know, EC can induce both platelet antiaggregation and vasodilation by producing NO and ET-1. In normal situation, they keep the balance and stability. When the endothelium is injured, NO is produced, the lacuna is released and the equilibrium tends to ET. Then, vascular contract will be predominant and sketch function decreases. The impaired endothelial function is the earliest phenomenon of AS. Some studies show that, in physical conditions, NO is generated by eNOS and ET-1 is the major member of ET systems. Other experiments reveal that Hcy can reduce NO level, but it can increase ROS, ET-1 level. We suppose that HHcy can affect ROS, NO and ET-1 expression. In this study, we test ROS, NO and ET-1 in HUVEC cultured with different Hcy concentrations to investigate the possible molecular mechanisms of Hcy on atherogenesis. Furthermore, the protective effect of Guanxinkang granules on the injury of HUVECs induced by HCY is discussed.Method:prepare rabbit serum. HUVECs cultured in vitro are randomly divided into 4 groups: (1)Contral group; (2)Model group: HCY group (with the final concentrations being 0.25,0.5,1.0,2.0 mmol /L); (3)Chinese herbs group: after being incubated for 2 hours by HCY, it will be added by the final concentrations 1.0mmol/L+10% rabbit serum with Chinese herbs; (4)western medicine(fluvastatin) group: after being incubated for 2 hours by HCY with the final concentrations being 1.0mmol/L+10% rabbit serum with fluvastatin. Each group has 6 samples. After being coincubated for 24 hours by HCY and drugs , intracellular ROS levels were detected by flow cytometry(FCM),The level of NO and ET-1 in the supernatant was assessed by nitrate reduction test and ELLSA(enzyme linked immunosorbent assay) respectively.Result:Exposure of HUVECs to HCY for 24h significantly increased HUVECs ROS production and ET-1 release(7.80±1.19, 15.37±1.27, 21.00±2.65, 35.56±2.96 vs 2.96±0.53; 87.67±10.95, 122.29±8.13, 139.03±5.39, 166.48±13.12 vs 40.61±0.64; P<0.05, respectively), while NO production significantly decreased (185.97±15.36, 147.75±15.86, 115.22±10.98, 80.54±9.93 vs 214.09±18.38; P<0.05); comparing to the control group. HCY caused a significant increase the production of ROS and the release of ET-1, while significantly decreased HUVECs for the production of NO(P<0.05) induced by HCY; and these effects were dose-dependent. but compared to the model group(HCY1.00), Guanxinkang granules and fluvastatin significantly decreased HUVECs ROS production and ET-1 release (3.77±0.60,10.89±0.72 vs 21.00±2.65; 46.18±5.96,74.35±4.26 vs 139.03±5.39 ; P<0.05) , while NO production significantly increases (203.95±12.85,186.68±18.35 vs 115.22±10.98;P<0.05).Conclusion: 1.Establishing a HCY-induced endothelial impairment model in cultured human umbilical vein endothelial cells is one of new reasonable methods in vitro. 2. HCY can obviously stimulate the production of ROS and ET-1 and decrease NO secretion in HUVECs. 3. Guanxinkang granules and fluvastatin can protect HUVECs against injury induced by HCY as a result of the increased production of NO and inhibition of ROS and ET-1 secretion, these effects are dose- dependent. 4. The results suggest that Guanxinkang granules can delay, protect HUVECs induced by HCY and resist atherosclerosis.