Influences Of Atorvastatin And Selenium On Vascular Endothelial Cell Function Affected By SAA

Inflammation plays a vital role in the process of atherosclerosis. Endothelium is the important protection barrier of blood vessels, and the function and structural integrity of vascular endothelial is closely related to the development of atherosclerosis. Vascular endothelium can prevent the infiltration of macrophages and the formation of foam cell, regulate coagulation, impede thrombus formation, thereby preventing the occurrence of atherosclerosis.Serum amyloid A (SAA) is not only a kind of biological markers of acute reactive protein, but a possible biological risk for cardiovascular disease. SAA can promote monocyte chemotaxis, adhesion and sedimentary in atherosclerotic plaques. SAA can increase the cell affinity of high density lipoprotein (HDL) on macrophages and endothelial cell, effect high density lipoprotein cholesterol (HDL-C) absorption via scavenger receptor B-I (SRB-I) and promote cellular HDL-C metabolism. However, the effect and mechanism of SAA on endothelial function is not very clear.This paper is divided into three parts to discuss the effect mechanism of SAA on vascular endothelial cells and the regulation effects of atorvastatin and selenium. Part One The comparison of serum SAA, eNOS, HO-1andselenium levels in acute coronary syndrome patientsObjectiveSerum SAA, eNOS, HO-1and selenium levels has been proposed as a protective role of against cardiovascular disease (CVD), whereas epidemiological data remains controversial. We aimed to investigate the relationship serum SAA, eNOS, HO-1, selenium levels in patients presenting with acute coronary syndrome (ACS) and to explore the pathogenesis of ACS and possible protection factor.Methods90patients with ACS were enrolled in this study.42cases with acute myocardial infarction(AMI),48cases with unstable angina pectoris(UAP),41patients with stable angina pectoris (SAP) and44healthy persons (NC, control group) in this cross-sectional-observational study. The degree of coronary artery lesions was measured by means of Gensini score. These cases were divided0score group,＜16scores group,16～32scores group and＞32scores group by Gensini score. Using enzyme-linked immunosorbent assay (ELISA) to measure serum SAA, eNOS, HO-1and selenium levels in these subjects and detect the systolic blood pressure (SBP), diastolic blood pressure (DBP), triglyceride (TG), total cholesterol(TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and glycosylated hemoglobin Alc(HbAlc) of patients and healthy people in the two groups. The SPSS13.0was used for the statistical analysis.Results1. The control group were sex and age matched with the patients. In the ACS group, SBP, DBP, TC, LDL-C or Gensini score was higher than in the control group. The difference was statistically significant (P＜0.01or P＜0.05). In AMI group,UAP group,SAP group and control group, Gensini score were31.77±16.63,23.51±10.39,24.24±10.06and0respectively.2. The level of SAA was highest in AMI group and was inferior in UAP group and was lowest in control group. The eNOS,HO-1in AMI and UAP group were lower than those in the SAP or control group. The level of Se in ACS and SAP group were lower than those in the control group. The level of Se were difference among AMI, UAP, SAP group,but the difference wasn’t statistically significant (P＞0.05). In AMI, UAP, SAP and NC group,the level of SAA were16.27±2.49,14.74±3.10,6.99±4.78,2.01±0.79respectively. In AMI, UAP, SAP and NC group,the level of eNOS were6.14±0.77,7.10±1.32,8.59±1.26,14.50±3.74respectively. In AMI, UAP, SAP and NC group,the level of HO-1were33.98±2.47,39.07±3.30,46.76±4.97,53.41±3.92respectively. In AMI, UAP, SAP and NC group,the level of Se were64.73±6.86,66.92±6.70,64.36±6.15,79.81±7.09respectively.3. The level of SAA was to step-up along with the Gensini score increase and there were difference in four groups(P＜0.05). The level of eNOS, HO-1were to cut down along with the Gensini score increase. However, it did not reach a statistical difference (P＞0.05) in＜16scores group and16～32scores group. The level of selenium was highest in0score group and there was difference in0score group and＜16scores group,0score group and16～32scores group or0score group and＞32scores group(P＜0.01). Nevertheless, it did not reach a statistical difference (P＞0.05)in any of the other two groups.4. SAA was positively correlated with Gensini scores and coefficient correlation was r=0.706. The levels of eNOS, HO-1and selenium were negatively correlated with SAA level and the coefficient correlation were r=-0.715, r=-0.828, r=-0.476respectively. The levels of eNOS was negatively correlated with Gensini scores and coefficient correlation was r=-0.675. The levels of HO-1and selenium were positively correlated with eNOS level and the coefficient correlation were r=0.772, r=0.570respectively. The levels of HO-1was negatively correlated with Gensini scores and the coefficient correlation were r=-0.651. The levels of selenium was positively correlated with HO-1level and the coefficient correlation were r=0.520. The levels of selenium was negatively correlated with Gensini scores and the coefficient correlation was r=-0.482.Conclusions1. Hypertension, high cholesterol and hyperglycemia are the relevant factors of ACS development.2. In ACS group, level of SAA was significantly higher and eNOS, HO-1and selenium levels were lower. This indicates SAA, eNOS, HO-1and selenium are involved in the occurrence and development of ACS.3. The level of SAA was to step-up along with the Gensini score increase. The level of eNOS, HO-1or selenium was to cut down along with the Gensini score increase. This indicates SAA, eNOS, HO-1and selenium are correlated with degree of pathological changes. When the degree of pathological changes is more serious, the level of SAA is higher. When the degree of pathological changes is more serious, the level of eNOS, HO-1or selenium is lower.4. Correlation analysis results showed that SAA level was negatively correlated with eNOS, HO-1and selenium levels, for that SAA at high level may take part in the occurrence and development of AS by decreasing the levels of cell and vascular protection factors, such as eNOS and HO-1. Part Two The approach of effect and mechanism of SAA on human umbilical vein endothelial cellObjectiveTo study the effect of SAA on eNOS, HO-1, SelS molecule expression and nitric oxide (NO), total antioxidant capacity (T-AOC) and reactive oxygen species (ROS) in human umbilical vein endothelial cells (HUVECs).MethodsTo culture HUVECs and carry out the synchronization processing when cell grew to about70%, then divided the HUVECs into different groups. Cells were treated with different concentrations (0mg/L, lmg/L,10mg/L,20mg/L) of SAA for24h and SAA20mg/L for various periods of time (Oh,6h,12h,24h).After incubation for given duration, the cells were harvested.To determine eNOSmRNA, HO-1mRNA and SelSmRNA transcript levels with real-time fluorescence quantitative (RT-PCR). To detect eNOS, HO-1, SelS protein expression with Western blotting in all groups. At the same time, to determine NO, T-AOC, ROS content and cell activity with MTT in cell culture medium and statistically analysis.Results1. The RT-PCR results showed that the concentration of SAA10mg/L,20mg/L could restrain eNOS mRNA, HO-1mRNA, SelS mRNA transcription and present a concentration dependence. In SAA10mg/L, The value of eNOSmRNA, HO-1mRNA, SelS mRNA were (2.43±0.71),(1.87±0.33),(1.65±0.48) respectively. In SAA20mg/L were (1.55±0.51),(1.61±0.40),(1.33±0.45) respectively.20mg/L SAA effecting for6h,12h or24h was able to inhibit the transcriptions of eNOS mRNA, HO-1mRNA, SelS mRNA and the effect displayed as time dependent. Treated with20mg/L SAA for (Oh,6h,12h,24h), the value of eNOS mRNA were (3.88±0.77),(2.97±0.24),(1.87±0.56),(1.54±0.51) respectively.The value of HO-1mRNA were (2.48±0.44),(2.20±0.46),(1.71±0.41),(1.59±0.24) respectively. The value of SelS mRNA were(2.86±0.73),(2.11±0.47),(1.63±0.39),(1.33±0.45) respectively.2. Western blotting results showed that the concentration of SAA10mg/L,20mg/L could restrain eNOS, HO-1, SelS protein expression, and present a concentration dependence. In SAA10mg/L, the density value of bands of eNOS, HO-1, SelS protein were (0.26±0.06),(0.34±0.09),(0.27±0.08) respectively.In SAA20mg/L, the density value of bands of eNOS, HO-1, SelS protein were (0.21±0.05), (0.20±0.06),(0.06±0.01) respectively.20mg/L SAA effecting for6h,12h or24h were able to inhibit eNOS, HO-1, SelS protein expression and present a time dependence.20mg/L function Oh,6h,12h,24h, the density value of bands of eNOS protein were (0.49±0.12),(0.39±0.10),(0.21±0.05),(0.20±0.06) respectively and the density value of bands of HO-1protein were (0.44±0.12),(0.42±0.11),(0.36±0.09),(0.21±0.05) respectively. The density value of bands of SelS protein were (0.36±0.10),(0.28±0.08),(0.09±0.03),(0.06±0.01) respectively.3. MTT results showed that the10mg/L,20mg/L SAA effecting for12h,24h could decrease the activity of endothelial cell.4.10mg/L,20mg/L SAA effecting for24h obviously reduced NO production (P＜0.05)and the content of NO was to decrease along with the concentration of SAA magnify. In SAA10mg/L, the content of NO was (59.25±15.49) μmol/L. In SAA20mg/L, the content of NO was (44.36±12.47) μmol/L. When treated with10mg/L,20mg/L SAA, NO production were decreased by21.7%and41.4%in HUVECs, respectively, compared with Omg/L (P＜0.05).5. ROS levels were significantly increased in10mg/L,20mg/L SAA-treated cells for24h in a concentration-dependent manner. In SAA10mg/L, ROS levels was (22.34±4.42)μmol/L. In SAA20mg/L, ROS levels was (28.54±4.97)μmol/L.6. T-AOC levels were significantly decreased in10mg/L,20mg/L SAA-treated cells for24h in a concentration-dependent manner. In SAA10mg/L, T-AOC levels was (0.72±0.20)μmol/L. In SAA20mg/L, T-AOC levels was (0.58±0.18)μmol/L.Conclusions1. SAA at high concentration can obviously inhibit the genic transcription and protein expression of eNOS, HO-1, SelS and the effects display as concentration and time dependent, which reveale that the concentration of SAA increasing is able to damage cells and the effect time is longer, the damage to cells is more obvious.2. SAA at high concentration can decrease the activity of endothelial cell and T-AOC of cell, reduce NO production, enhance oxidative stress, increase ROS level, thereby lead to the damage of endothelial cell function and the occurrence of atherosclerosis. Part Three The regulation effects of atorvastatin and selenium on the eNOS, HO1, SelS induced by SAAObjectiveTo discuss the influence and mechanism of atorvastatin and selenium on eNOS, HO-1, SelS molecule expression and NO, T-AOC, ROS induced by SAA in HUVECs.MethodsHUVECs were divided into SAA group(SAA20mg/L), selenium group (SAA20mg/L and selenium100nmol/L), atorvastatin group (SAA20mg/L and atorvastatin10μmol/L), atorvastatin combining selenium group (SAA20mg/L,atorvastatin10μmol/L and selenium100nmol/L), PDTC group (prior to end100μmol/L of the concentration of NF-κB blockers PDTC pretreated for1h, then adding SAA20mg/L). To determine eNOSmRNA, HO-1mRNA, SelSmRNA transcript levels with real-time fluorescence quantitative (RT-PCR)and detect eNOS, HO-1, SelS protein expression with Western blotting in all groups after24hours. At the same time, to determine and statistically analyze cell activity, NO, T-AOC and ROS content in cell culture medium.Results1. Atorvastatin group and selenium group showed similar results. Levels of eNOSmRNA, HO-1mRNA, SelSmRNA and protein expression were significantly higher than the SAA group and the results were statistically significant. Treated with atorvastatin, the value of eNOS mRNA HO-1mRNA, SelS mRNA were (2.57±0.05),(2.19±0.57),(2.22±0.58). The eNOS, HO-1, SelS protein were (0.24±0.06),(0.29±0.07),(0.25±0.05). Treated with selenium, eNOSmRNA, HO-1mRNA, SelS mRNA were (2.13±0.40),(1.85±0.64),(2.01±0.55).The eNOS, HO-1, SelS protein were (0.26±0.06),(0.33±0.08),(0.25±0.05).2. The combined application results of atorvastatin and selenium showed that the eNOS, HO-1or SelS molecular level was higher than atorvastatin group or selenium group. The results were statistically significant compared to SAA group.3. PDTC,one of the specific inhibitors for NF-κ was used to pretreat HUVECs for1h before SAA (20mg/L) treatment for24h, effectively blocked the SAA-induced eNOS, HO-1, SelS mRNA and protein expression decrease. The results were statistically significant compared to SAA group.4. Given four kinds of treatment measures, could increase cell activity, increased NO content and T-AOC, decreased ROS levels. PDTC group, selenium group, atorvastatin group and atorvastatin combining selenium group, NO content were (74.97±10.74)μmol/L,(52.58±7.16)μmol/L,(55.95±9.67)μmol/L,(70.94±10.92) μmol/L, respectively. T-AOC were (1.96±0.51)mmol/ml,(1.74±0.49) mmol/ml,(1.87±0.42)mmol/ml,(2.37±0.65)mmol/ml, respectively. ROS levels were (9.67±2.47,18.97±4.69,16.75±4.86,10.64±2.36), respectively.Conclusions1. Atorvastatin and selenium can both improve transcription of eNOS mRNA, HO-1mRNA, SelS mRNA and protein expression and enhance cell activity. They increase NO production, strengthen the cell total antioxidant capacity, and reduce the content of ROS. Atorvastatin combining with selenium has cooperative effect. This indicates atorvastatin and selenium are able to mitigate oxidative stress and protect the cell.2. The NF-κB blocking agent (PDTC) is capable of blocking the action of high concentration SAA. The transcription of eNOS mRNA, HO-1mRNA, SelS mRNA and protein expression can be reinforced. It can not only increase the cell activity and raise NO content, but also strengthen the cell total antioxidant ability and decrease the content of ROS which suggests SAA regulates the function of endothelial cell through the NF-κB pathway.