The Study Of The Protective Effects And Mechanism Of CGA On Oxidative-stress-induced Vascular Endothelial Cells Injury

Background and Objective:Ischemic stroke is a common disease in clinical, which mortality and morbidity are high.But the remaining nerve dysfunction is still a major problem plagued neurologist in neuroscience. After ischemia stroke, ischemic neurons can be lead to ischemia, hypoxia and death. Timely restoration the blood circulation of the ischemia is the key treatment of cerebral ischemia. The present study showed that angiogenesis happened in ischemic region after cerebral ischemia. And angiogenesis can not only restore the blood supply of ischemic region, but also can secrete neurotrophic factors, which create a microenvironment around the ischemia, and promote functional recovery of injured neurons. Angiogenesis and the microenvironment created mainly by endothelial cells surrounding the ischemic region.But oxidative stress occurs after cerebral ischemia, which can damage endothelial cells, inhibit angiogenesis and the release of neurotrophic factors. In recent years, studies have shown that chlorogenic acid can effectively remove free radicals, protecting cells from oxidative damage. Whether chlorogenic acid on endothelial cells could against oxidative stress injury and its possible mechanism are still to be proved. The topic is designed through observe the protection of chlorogenic acid on endothelial cells and changes of mitochondrial apoptosis pathway molecular expression, discuss the anti-apoptotic role of chlorogenic acid on endothelial cells and its possible mechanism.Then we may provide a theoretical basis for better use of chlorogenic acid for ischemic stroke in clinical..Methods:1.Hydrogen peroxide (H2O2) effect on HUVECs in a concentration gradient and time gradient, then measure the apoptosis rate by flow cytometry to establish the best model of oxidative stress.2. There were four groups:normal control group, chlorogenic acid group, H2O2 injured group, chlorogenic acid protected group; cells in each experimental group were given the appropriate treatment to be detected after 12h. 3. Apoptosis in all experimental groups were detected by Flow cytometry.4. Apoptosis of experimental groups were detected by mitochondrial membrane potential.5. Apoptosis of experimental groups were detected by Hoechst33258 fluorescence staining.6. The expression of Bcl-2, Caspase-3 mRNA levels were detected by RT-PCR in each experimental group.7. The expression of Bcl-2, Caspase-3 protein levels were detected by Western blot in each experimental group.Results:1. HUVECs was induced apoptosis by H2O2 in a concentration and time-dependent., the best model of oxidative stress condition was 400μM H2O2 treatment for 12h.2. Apoptosis rate (%) of experimental groups cells by flow cytometry:normal control group:5.18±2.13, chlorogenic acid group:9.55±2.82, H2O2 injured group: 66.78±9.71, chlorogenic acid protected group:46.75±6.79. The results can be seen that apoptosis rate of H2O2 injury group was significantly higher than the control group (P<0.05), cell apoptosis of chlorogenic acid protected group was significantly lower than H2O2 injury group (P<0.05). The results proved that H2O2 can induce apoptosis in HUVECs, and chlorogenic acid has a significant function on anti-apoptosis.3. The result of mitochondrial membrane potential of cells showed HUVECs could induce apoptosis by H2O2, and chlorogenic acid has a significant function on anti-apoptosis.4. The result of Hoechst33258 fluorescence staining showed that HUVECs could induce apoptosis by H2O2, chlorogenic acid has a significant function on anti-apoptosis.5. The result of RT-PCR showed the levels of Bcl-2 mRNA expressed of chlorogenic acid protection group were significantly higher than that of H2O2 injured group (P<0.05), the expression of Caspase-3 mRNA was significantly lower than H2O2 injured group (P<0.05). The results showed that when chlorogenic acid protected HUVECs, the expression of Bcl-2 mRNA was increased, the expression of Caspase-3 mRNA was reduced.6. Western blot showed that the expression of Bcl-2 protein levels of chlorogenic acid protection group were significantly higher than that of H2O2 injury group (P <0.05), Caspase-3 protein expression was significantly lower than H2O2 injury group (P<0.05). The results showed that when chlorogenic acid protect HUVECs, the expression of Bcl-2 protein was increased, Caspase-3 protein expression was reduced.Conclusion:1.HUVECs was induced to apoptosis by H2O2 in a concentration and time-dependent.2.Chlorogenic acid has a significant protection on HUVECs to against oxidative stress.3. The protection mechanism of Chlorogenic acid on HUVECs may be related to inhibiting the apoptosis pathway of mitochondrial.