Study Of The Protective Effect And Mechanism Of Scutellaria Baicalensis Stem-leaf Total Flavonoid Against Human Umbilical Vascular Endothelial Cells Oxidative Injury Induced By Oxidized Low Density Lipoprotein

Objectives:To investigate the protective effects and mechanisms of Scutellaria Baicalensis Stem-leaf Total Flavonoid(SSTF) in oxidative injury of human umbilical vein endothelial cells (HUVECs) induced by oxidized low density lipoprotein (ox-LDL).Methods:1. Fresh human serum was collected, low density lipoprotein (LDL) was prepared by heparin precipitation, and then ox-LDL was oxidized by CuSO4for24hours. And the degree of oxidation was tested by thiobarbituric acid (TBA), the concentration was tested by coomassie brilliant blue G-250.2. HUVEC was cultured in vitro together with different concentrations of ox-LDL (12.5,25,50,100,200mg/L) for24hours. The cell morphological changes was observed by light microscope, the cell vitality (OD value) was measured by tetrazolo colorimetric method (MTT), malondialdehyde (MDA) content was measured by thibabituric acid (TBA), superoxide dismutase (SOD) activity was measured by spectrophotometric assay, and intracellular ROS level was detected by flow cytometry (FCM); at last we selected an optimum concentration for endothelial cells oxidative indury induced by ox-LDL.3. To observe the changes of HUVEC NOX4expression after induced by ox-LDL with different concentrations through detecting NOX4mRNA expression by RT-PCR and protein by Western blot. 4. HUVEC was cultured in vitro together with different concentrations of SSTF group (25,50,100,200,400mg/L), the cell viability (OD value) was measured by MTT after24h,48h and72h. HUVEC oxidative injury induced by ox-LDL was cultured in vitro together with different concentrations of SSTF (low, medium, high) for24hours. We could investigate its protective effects and mechanisms through detecting morphological changes, cell vitality, MDA content, SOD activity, intracellular ROS level, NOX3mRNA and protein expression.Results:1. LDL was extracted and separated by heparin precipitation, then definited by single band in the0.45%agarose gel electrophoresis. LDL was successfully oxidized to generate ox-LDL by CuSO4. The MDA value of ox-LDL was higher than the native LDL. The ox-LDL consentration was480μg/mL.2. Results showed that compared with the control group, the cell vitality (OD value) was significantly lower (P<0.05), the content of MDA was increased (P<0.01), the activity of SOD was decreased (P<0.05) in ox-LDL groups (12.5、25、50、100、200mg/L), the ROS level was significantly increased (P<0.05) in a concentration-dependent manner. Especially in100,200mg/L concentrations of ox-LDL effect was most obvious.3. Compared with control group, the cell shape of ox-LDL group (100mg/L) by the light microscope was distorted while the cell boundary was still clear. The ox-LDL group (200mg/L) was damaged seriously, the cell became round and swell, and the boundary was not clear. Some dead cells fall off the bottom of wells.4. Results from the RT-PCR and Western blot showed that compared with the control group, NOX4mRNA and protein expression were increased significantly (P<0.01, P<0.01) in ox-LDL groups (50、100、200mg/L).5. Different concentrations of SSTF group (25,50,100,200,400mg/L) compared with control group after24、48and72h, the cell vitality (OD value) was significantly increased (P<0.05, P<0.01, P<0.05). Our experiment data showed that SSTF had no adverse reaction and had an facilitative effection on the HUVEC. 6. HUVEC oxidative injury induced by ox-LDL was in vitro cultured together with different concentrations of SSTF (low, medium, high), results showed that compared with ox-LDL group, the cell vitality (OD value) was significantly increased (P<0.05), the content of MDA was decreased (P<0.01), the activity of SOD was increased (P<0.01), the ROS level was increased (P<0.05). NOX4mRNA and protein expression were decreased significantly (P<0.01, P<0.01) in SSTF groups.Conclusion:1. Oxidized low-density lipoprotein could induce vascular endothelial cells oxidative injury, the degree of the injury in a concentration-dependent manner.2. The vascular endothelial cells oxidative injury induced by oxidized low-density lipoprotein was through the pathway of NADPH oxidase, NOX4oxidase played an important role in this process.3. SSTF had obvious protective effects against the vascular endothelial cells oxidative injury induced by oxidized low-density lipoprotein in cellular level, its mechanism may be associated with inhibition of NADPH oxidase (NOX4).