| Part one:Protective effect of Rhodiola on hypoxia induced oxidative stress in apoE-/-mice atherosclerosisObjective:Utilized the intermittent hypoxia to induce oxidative stress in apoE-/-mice, and combined with high fat diet to establish the animal model of atherosclerosis, to observe the effects of Rhodiola on mice body weight, blood lipids and aortic plaque formation. Detection of mouse aortic internal oxidation and antioxidant indexes of MDA, SOD and oxLDL. At the same time, detection of aortic tissue of NF-κB and HIF-1a, VEGF expression changes, so as to explore the effect of Rhodiola on prevention of atherosclerosis and the possible mechanis.Methods:1, Animal model:Hypoxia induced atherosclerosis model was established by feeding high fat diet and exposed to hypoxia feeding box to12hours daily intermittent hypoxia (10%O2) stimulation for12weeks in8weeks old male apoE-/-mice.2, Grouping and administration:The mice were randomly divided into the hypoxia and normoxia group, two group were subdivided into6subgroups.(1) normal diet group (normal group diet, ND);(2) high fat diet group (high fat diet group, HFD);(3) high fat diet plus low dose Rhodiola group (Low dose group Rhodiola, Rd-L,2.5g/kg.d);(4) high-fat diet+moderate dose group of Rhodiola (Moderate dose group Rhodiola, Rd-M,5g/kg.d);(5) high fat diet+high dose Rhodiola group (High dose Rhodiola group, Rd-H, lOg/kg.d);(6) high fat diet+positive drug control vitamin E group (group VitE, VE,500mg/kg.d). The intervention groups were given different dose drug daily morning, and the mice of control group were given the same volume of distilled water during12weeks.3, Specimen collection and detection:Collection the serum and aortic specimens from the mice for the detection of the following indicators:(1) ELISA for the detection of serum oxidized low density lipoprotein (oxLDL).(2) Oil red O and HE staining to observe the pathological changes of aortic plaque size and pathological change.(3) The expression of NF-κB in aortic plaque was detected by immumohistochemical technique.(4) The ROS level of aortic was detected by DHE anionic fluorescent probe.(5)The superoxide dismutase (SOD) activity or malondialdehyde (MDA) content was detected by spectrophotometry.(6) Real-time fluorescent quantitative PCR and Western blot were used to analysis the HIF-1a and VEGF mRNA expression and protein conten of mice aortaResults:1, Gross observation of animal model:4mice were died of gastric perfusion. After12weeks of intervention, the visible yellow lipid streaks and atheromatous plaque adhered to aorta vessel wall, distributed in the arch and abdominal aorta. Under hypoxic conditions, plaque area and the thickness increased.2, Aortic overall oil red O staining The plaque area in hypoxia group was significantly higher than that in normoxia group (hypoxia ND:11.10%±4.56%vs normoxia ND:2.1%±1.3%, P<0.01; hypoxia HFD:35.33%±4.51%vs normoxia HFD:25%±3.61%, P<0.01). Under hypoxic conditions, compared with that of high fat diet group, the plaque area in vitamin E or each dose of Rhodiola subgroup was significantly reduced (P<0.05); and the high dose Rhodiola subgroup was the most significant in the whole group,(hypoxia Rd-H:4.34%±2.06%vs hypoxia VE:12.03%±3.6%, P<0.05). While the normoxic each dose Rhodiola subgroup, plaque area compared with the high fat diet subgroup, has no significant change (P<0.05).3, DHE staining The fluorescence intensity of DHE of hypoxia mice aorta in situ compared with normoxia group was significantly increased (hypoxia ND:45.1±5.78vs. normoxia ND:8.0±1.15, P<0.05; hypoxia HFD:73.2±4.16vs. normoxic HFD:34.2±7.93, P<0.05).Compared with the high fat diet subgroup, DHE fluorescence intensity of each dose Rhodiola subgroup in hypoxia was significantly decreased (P<0.01); the high dose salidroside group was decreased most significantly (hypoxia Rd-H:8.3±3.06vs. hypoxia VE:23.67±6.17, P<0.05). While the normoxic each dose Rhodiola subgroup, the fluorescence intensity of DHE compared with the high fat diet subgroup, has no significant change (P<0.05).4, Serum content of oxLDL The content of serum oxLDL in hypoxia with high fat diet subgroup were significantly increased (hypoxia HFD:20.98±2.61ng/ml vs. normxia, HFD:11.27±1.55ng/ml, P<0.05). The content of oxLDL of low, moderate and high dose subgroup in hypoxia group (10.25±2.07,6.13±1.15,5.12±1.02ng/ml) were significantly lower than those in high fat diet subgroup P<0.01. The oxLDL content of hypoxia high dose Rhodiola subgroup, was lower than vitamin E subgroup (hypoxia Rd-H:5.12±1.02ng/ml vs. hypoxia VE:10.52±2.15, P<0.05). Compared with the high fat diet group, the content of oxLDL in each dose Rhodiola subgroup in normoxic group has no significant difference (P>0.05).5, The content of oxidant and antioxidant index The content of MDA in hypoxia mice aorta was significantly increased than that of normoxic mice (hypoxia ND:3.53±0.54nmol/mg.pro vs. normoxic ND:1.28±0.18nmol/mg.pro; hypoxia HFD:8.09±0.67nmol/mg.pro vs. normoxic HFD:4.82±1.11nmol/mg.pro, P<0.05). The MDA content of hypoxia each dose Rhodiola subgroup was significantly lower than that of high fat diet subgroup, the difference was statistically significant (P<0.05). While the MDA content of normoxia Rhodiola subgroup, compared with the conternt of the high fat diet subgroup, there was no significant difference (P>0.05). Hypoxia mice aortic SOD activity compared with normoxia mice decreased significantly (hypoxia ND:17.21±4.26Umg/mg.pro vs. normoxia ND:31.43±0.77Umg/mg.pro; hypoxia HFD:7.01±1.12Umg/mg.pro vs. normoxic HFD:13.92±2.97, P<0.05). The SOD activity of moderate and high dose Rhodiola subgroup in hypoxia group increased significantly, compared with the high fat diet group, the difference was statistically significant (P<0.01), and the activity of SOD in the high dose subgroup, compared with the vitamin E group, there was no significant difference (P>0.05).6, Immunohistochemical result The NF-κB p65positive cell number of normal diet group mice aortic tissue sections in hypoxia was significantly higher than that of the normoxic control group, optical density relative value has significant difference (hypoxia ND:39.67±3.93vs. normoxia ND:20.33±2.91, P<0.05). Hypoxia with high fat diet intervention significantly increased positive staining nuclear number of foam cell (hypoxia HFD:70.1±10.39vs. hypoxia ND:39.67±3.93, P<0.01). Normoxic high dose of Rhodiola group, compared with the high fat diet group, there were significant differences (P<0.05).The decreasion of NF-κB p65expression in moderate and high dose Rhodiola subgroup in hypoxia group, compared with the high fat diet subgroup, the relative value of optical density had significant difference (P<0.05); the effect was most obvious in high dose subgroup, the plaque had only a small amount of nuclear positive cells, compared with the vitamin E group, there was statistically significant difference (hypoxia Rd-H:8.67±3.48vs. hypoxia VE:23.2±7.31, P<0.05).7, Real-time fluorescent quantitative PCR The expression of HIF-1α and VEGF mRNA in each intervention group (including high fat diets and Rhodiola subgroups), compared with the normal diet control group, there was no significant difference (P>0.05).8, Western blot detection Compared with the normal diet control group, hypoxia or high fat diet can induce HIF-1α protein in aorta P<0.05. Compared with the high fat diet group, HIF-1α protein level of each dose Rhodiola subgroup in normoxia group, has no significant difference (P>0.05). The expression of HIF-1α protein significantly increased in each dose Rhodiola subgroup in hypoxia group, and compare with the high fat diet group, there are significant differences (P<0.01). The expression of HIF-1α protein in Vitamin E group had no significant difference with that of the normal diet group (P>0.05).Conclusion:1. Intermittent hypoxia induced oxidative stress in apoE-/-mice, resulting in increased levels of ROS in the aorta, lipid peroxidation, enhanced the expression of NF-κB p65, resulting in the formation of atherosclerotic plaque. Hypoxia with high fat diet intervention aggravated the level of oxidative stress and the atherosclerosis plaque formation.2. Hypoxia or high fat diet could increase the content of HIF-1α in apoE-/-mice aortic.3. Only under the hypoxic condition, Rhodiola can effectively inhibit aortic ROS overproduction, reducing lipid peroxidation, reducing NF-κB p65aggregation in plaque, inhibit atherosclerotic plaque formation.4. Under the hypoxic condition, the effection of antioxidant stress and inhibition of plaque formation of Rhodiola was more effective than vitamin E. In hypoxic conditions, Rhodiola significantly increased the content of HIF-1α protein in aorta. Part two:Investigate the mechanism of salidroside inhibit oxidative stress from the interaction between ROS and HIF-1αObjective:HUVECs were treated with Hydrogen peroxide to produce oxidative stress damage cell model, to investiagte the relation between the ROS and HIF-1α, and to observe the effects of salidroside on HIF-1α and ROS level, and finally to elucidate the possible mechanism of the Rhodiola inhibiting atherosclerosis.Method:Treatment of oxidative stress HUVEC cell model with Salidroside,then using (1) WST-1method to observe the cell viability;(2) the DCFH method to observe the changes of intracellular ROS levels;(3) the expression of HIF-1a mRNA cells were detected by Realtime PCR;(4) Western blot to detect the content of HIF-la protein and degradation speed,and PI3K/AKT/mT0R signal pathway;(5) Dual luciferase reporter system to detect the HIF-1a transcription activity;(6) Western Blot detection;(7) Utilizing the RNA interference technology and the over-expression vector of the HIF-1α gene to downward or upward expression of HIF-1α in cells,then used DCFH to detected effects of salidroside on ROS level. Result:(1) Exogenous H2O2induced HUVEC death and intracellular ROS level in a concentration-dependent manner. The salidroside could protect the HUVEC from damage induced by H2O2in a dose-dependent manner, decreased ROS production and improved cell survival rate. Compared with the H2O2group, cell survival rate increased to79%±4.5%in100p.g/ml salidroside group, there was significant differences (p<0.05).(2) Compared with the control group, H2O2significantly increased the level of HIF-1α mRNA and protein expression in HUVECs (P<0.05).(3) Both in normoxic or under conditions of oxidative stress, salidroside had no significant effect on intracellular HIF-1α mRNA (P>0.05).(4) under normoxic conditions, salidroside had no significant effect on HIF-1α protein content in the cell, but in oxidative stress condition, salidroside significantly promoted expression of HIF-1α protein in cells, compared with the H2O2group, there was significant statistical difference (P<0.05).(5) Addition of cycloheximide (CHX) treatment of the cells, we observed the salidroside significantly inhibited the degradation rate of HIF-1α.(6) H2O2could increase the HIF-1α transcription activity in HUVECs. In the oxidative stress condition, the effect of salidroside on HIF-1α transcription activity was stronger than H2O2(p<0.01).(7) Salidroside could promote related protein in the PI3K/AKT/mTOR signaling pathway, and promotes the expression of HIF-1α. When PI3K inhibitor LY294002was treated cells, the expression of HIF-1α, which was mediated by PI3K/AKT/mTOR signal transduction pathway was also lower.(8) By Western blot identification,3siRNA sequences showed silencing of HIF-1α role, the silence effect of HF1sequence was the strongest (p<0.05), and pCDH-HIF-1α over-expression plasmids were successfully transfected into HUVEC cells to upregulate the expression of HIF-1α.(9)When the endogenous HIF-1α level were inhibited, the antioxidant ability of cells significantly decreased. After H2O2stimulation, intracellular ROS level increased sharply. Compared with the random primer sequence of negative control group (SCR group), the level of ROS in salidroside+HIF-1α siRNA group, showed no statistically significant difference (P>0.05). And Compared with the the cells transfected with the empty vector group, after H2O2stimulation induced oxidative stress, the intracellular ROS level of cells transfected with pCDH-HIF-1α plasmid was significantly decreased (p<0.05), suggest that up-regulation of HIF-1α expression could inhibit the production of ROS. Compared with the pCDH-HIF-1α group, the intracellular ROS level in salidroside group, showed no statistically significant difference (P>0.05), so that the inhibition of HIF-1α expression can inhibit the antioxidant capacity of salidroside.Conclusion:1. ROS has a positive effect on HUVEC cells of HIF-1α, and make its transcriptional activity enhancement. HIF-1α has a feedback effect on ROS, can inhibit the intracellular ROS overproduction.2. Salidroside could inhibit H2O2induced intracellular oxidative stress injury, promote the proliferation of HUVEC.3. Salidroside had no significant effect on HIF-1αgene transcription in HUVEC cells. But in conditions of oxidative stress, Salidroside could promote the level and transcriptional activity of HIF-1α protein in HUVECs through the PI3K/AKT/mTOR pathway. In the state of oxidative stress, the antioxidant effects of salidroside significantly dependent on the HIF-1α. |
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