| Background:As the improvements of people’s living standards, the change of lifestyle, and the rapid arrival of an aging population, heart cerebrovascular disease has become the main diseases which is harmful to human life and health in the world today. Atherosclerosis (AS) is an important pathologic basis of heart cerebrovascular disease and vascular endothelial cell injury is the tumor-initiating link of AS. In recent years, the domestic and foreign researches show that high blood triglyceride (HTG) is an independent and risk factor of AS’s happending, HTG which can start AS by the way of damaging vascular endothelial cells, promoting the proliferation of vascular smooth muscle cell, causing lipid peroxidation, promoting the formation of blood clots and so on. In the process of AS caused by HTG, the injury of vascular endothelial cell which is the result of oxidative stress caused by the increasing of reactive oxygen species(ROS) may played an extremely important role. ROS is an important signal molecule in adjusting the function and state of blood vessel, in physiological and pathological conditions, a variety of enzyme in the body participate in the ROS generation. Nicotinamide phosphate oxidase(NADPH oxidase) is now considered as the main enzyme of the ROS generation endovascular, and endothelial cells mainly express NADPH oxidase4(NOX4) in NOX family. So whether it through NADPH way in the process of vascular endothelial cells oxidative injury induced by HTG? By now, the research about this has not been reported home and abord. This is such one of the research tasks of my project, expecting reveal the mechanism of AS induced by HTG from a new angle and provide theoretical basis for its prevention.Scutellaria baicalensis stem-leaf total flavonoid(SSTF) whose main ingredients are flavonoids compounds is the extract of scutellaria baicalensis stem-leaf. The pharmacology study has already showed that SSTF has extensive functions in cardiovascular system, such as antioxidant, reduce blood pressure, adjust blood-lipid, improve cardiovascular blood etc. So whether SSTF has protection on the vascular endothelial cells oxidative injury induced by HTG and its mechanism concerns inhibiting the expression of NOX? This is another research task of the project, its research results which can provide experiment basis are conducive to finding new targets of anti-AS drug.Objectives:1. To investigate the oxidative injuries caused by human high triglycerides(HTG) blood serum on the human umbilical vein endotheial cells(HUVEC), and to discuss the functions and molecular mechanisms of nicotinamide adenine dinucleotide phosphate oxidase4(NADPH oxidase4, NOX4) on HUVEC oxidative injury induced by HTG.2. To observe the protective effects of scutellaria baicalensis stem-leaf total flavonoid (SSTF) on HUVEC oxidative injury induced by HTG, and to discuss its possible mechanisms at the molecular level.Methods:1. Dose and time effects of HTG on the oxidative injury of HUVEC:HUVEC were cultured in vitro and were treated with different concentrations of HTG(1%,2%, 3%,4%and5%) for different time course(8h,12h,16h,20h and24h). Then cell vitality(OD value) was measured by tetrazolo colorimetric method (MTT), superoxide dismutase(SOD) activity of cell was examined by spectrophotometric assay, malondialdehyde(MDA) content was determined by thibabituric acid(TBA) method and intracellular ROS level was detected by flow cytometry(FCM). And we obtained an optimum concentration and most appropriate time for endothelial cells oxidative indury induced by HTG.2. The role of NOX4in HUVEC oxidative indury induced by HTG:We could observe the changes of HUVEC NOX4expression after induced by HTG through detecting NOX4mRNA expression by RT-PCR and protein by Western blot.3. The protective effects of SSTF on HUVEC oxidative injury induced by HTG: Different concentrations of SSTF(100,200,400mg/L) and positive control drug Vc(100mg/L) were cultured in vitro together with HUVEC oxidative injury induced by HTG for16hours. We could obsere the protective effects of SSTF through measuring cell vitality, SOD activity, MDA content and intracellular ROS level, then compare with which of VitC.4. The protection mechanisms of SSTF on HUVEC oxidative injury induced by HTG:Different concentrations of SSTF(100,200,400mg/L) and positive control drug VitC(100mg/L) were cultured in vitro together with HUVEC oxidative injury induced by HTG for16hours. We could investigate the changes of HUVEC NOX4expression after treated with SSTF and VitC through detecting mRNA expression by RT-PCR and protein by Western blot.Results:1. Dose and time effects of HTG on the oxidative injury of HUVEC:Results from the MTT assays showed that compared with control group, the the cell vitality (OD value) decreased significantly(P<0.01) of HUVEC oxidative injury induced by 4%HTG for24h and5%HTG for over16h, in a concentration-dependent manner. Results from the activity of SOD showed that compared with control group, the activity of SOD decreased significantly(P<0.01) of HUVEC oxidative injury induced by2%HTG for24h,3%HTG and4%HTG for over20h,5%HTG for over16h, in a concentration-dependent manner. Results from the content of MDA showed that compared with control group, the content of MDA increased significantly(P<0.01) of HUVEC oxidative injury induced by2%HTG for over20h,3%HTG for over16h,4%HTG and5%HTG for over8h, in a concentration-dependent manner. Results from the ROS level showed that intracellular ROS had a baseline production in cultured HUVEC and were increased significantly after HTG treatment in a concentration-dependent manner. Compared with control group, the ROS level increased significantly(P<0.01) of HUVEC oxidative injury induced by1%HTG for over16h and2%HTG for over20h, especially by5%HTG for16h. According to these results, we determined that HUVEC oxidative injury induced by5%HTG for16hours was the optimum study condition.2. The role of NOX4in HUVEC oxidative indury induced by HTG:Results from the RT-PCR showed that NOX4mRNA was detected in cultured HUVEC and HTG stimulation caused a significantly increasing of NOX4mRNA expression. Compared with control group, NOX4mRNA expression increased significantly(P<0.01). Results from the Western blot showed that NOX4protein was detected in cultured HUVEC and HTG stimulation caused a significantly increasing of NOX4protein expression. Compared with control group, NOX4protein expression increased significantly(P<0.01).3. The protective effects of SSTF on HUVEC oxidative injury induced by HTG: Results from the MTT assays showed that compared with the HTG group, the cell vitality(OD value) increased in every SSTF group and VitC(100mg/L) group, increased significantly(P<0.01) in SSTF (400mg/L) group and VitC(100mg/L) group. Results from the activity of SOD showed that compared with the HTG group, the activity of SOD increased significantly (P<0.05or P<0.01) in every SSTF group and VitC (100mg/L) group. Results from the content of MDA showed that compared with the HTG group, the content of MDA decreased significantly (P<0.01) of HUVEC in every SSTF group and VitC (100mg/L) group. Results from the ROS level showed that compared with the HTG group, the intracellular ROS level decreased significantly(P<0.01) in every SSTF group and VitC(100mg/L) group.4. The protection mechanisms of SSTF on HUVEC oxidative injury induced by HTG:Results from the RT-PCR showed that compared with the HTG group, NOX4mRNA expression decreased in every SSTF group and VitC(100mg/L) group, decreased significantly(P<0.01) in SSTF (200,400mg/L) group and VitC(100mg/L) group. Results from the Western blot showed that compared with the HTG group, NOX4protein expression decreased significantly (P<0.01) in every SSTF group and VitC (100mg/L) group.Conclusion:1. High triglycerides blood serum can induce vascular endotheial cells oxidative injury, and the oxidative injury degree positively correlated with the concentrations of high triglycerides blood serum.2. The vascular endothelial cells oxidative injury induced by high triglyceride blood serum is through the pathway of NADPH oxidase, and which is regulated and controled by NOX4.3. SSTF showed obvious protective effects against the vascular endothelial cells oxidative injury induced by high triglyceride blood serum, which can promote vascular endothelial cells antioxidant capacity, inhibit lipid peroxidation effect, reduce intracellular ROS generation.4. The probable mechanisms of SSTF protective effects on HUVEC oxidative injury induced by HTG may be associated with its down-regulating NOX4mRNA and protein expression, through which can reduce intracellular ROS generation. |
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