The Effect And Mechanism Of Atorvastatin On The Improvement Of Endothelial Function

Atherosclerosis (AS) is the main disease that affects the health of human being seriously and the research of its mechanism has been last for centuries. Now it is confirmed that inflammatory reaction play the crucial role in formation and development of AS It is gradually believed that AS is a kind of inflammatory disease proposed by Ross in 1999.Issemann and his colleagues found a new steroid hormone receptor which can be activated by fatty acid derived compound peroxisome proliferators(PP) called peroxisome proliferators-activated receptor( PPAR). PPAR is classified into three subtypes: PPARα, PPARδand PPARγ. PPARγwas thought existed in fat cell only and acted on fat cells differentiation before. It is found in basic studies recently that almost all vascular wall cells which participate in AS formation such as vascular endothelial cell(VEC), vascular smooth muscle cell (VSMC), mononuclear macrophage (Mo/Mφ)can express PPARγ. The endothelial function and inflammatory reaction can be improved through activating PPARγ.It is proved that statins (3-hydroxy-3methylglutaryl-coenzyme A reducta- se inhibition) can not only reduce cholesterol, but also protect vascular endothelium. It is also showed that statins have effects on anti-inflammatory and inhibition of AS in clinical and basic studies. But the mechanism beyond anti-hyperlipemia is not clear. The pathways of PPARγand NF-κB are researched more recently in cardiovascular field. It is reported that both PPARγand NF-κB participate in development of AS. Straus and his colleagues presumed that PPARγcould inhibit vascular inflammatory reaction through NF-κB activated by its ligand and contribute to inhibit AS. It has not confirmed that whether statins can improveendothelial function through the PPARγand NF-κB pathways.The purpose of our study are:1) HUVECs are incubated with different concentrations of atorvastatin(ATV)and lipopolysaccharide (LPS). The endothelial function is observed to confirm whether it can be improved through PPARγand NF-κB pathways. 2) Different dosage of ATV was applied to hypertensives without hyperlipemia. Brachial artery FMD and the level of NO, SOD, sICAM-1 in serum are evaluated in order to approach the effects of statins on endothelium protection function besides its lipid reducing function.PartⅠEffects of different concentration atorvastatin on endothelial function of human umbilical vein endothelial cell stimulated by lipopolysaccharideObjective:To investigate whether the endothelial function can be improved by different concentrations ATV and the mechanism of this effect in HUVECs.Materials and Methods:HUVECs were cultured in vitro and divided into two groups in 2-4 generation. Experiment 1:①control group (group 1);②LPS group (group 2, 1.0μg/ml);③LPS+ATV1 group (group 3, LPS 1μg/ml,ATV 1.0μM);④LPS+ATV2 group (group 4,LPS 1μg/ml,ATV 5.0μM). After HUVECs were incubated with different concentrations of ATV and LPS for 24 hours, the expression of PPARγ,NF-κB-p65,eNOS,ICAM-1,E-selectin in HUVECs were evaluated with reverse transcription- polymerase chain raction (RT-PCR) and Western Blot. The level of NO,sICAM-1,sE-selectin,SOD in cell culture fluid were measured with the way of Nitrate reductase,ELISA and so on. Experiment 2:①control group (group 1);②LPS+ATV2 group (group 2,LPS 1μg/ml,ATV 5.0μM);③GW9662+LPS+ATV2 group(group 3,GW9662 0.2μM, LPS 1μg/ml,ATV 5.0μM). After HUVECs were incubated with different concentrations of ATV,LPS and GW9662(the specific inhibitor of PPARγ) for 24 hours, the expression of PPARγ,NF-κB-p65,eNOS,ICAM-1 in HUVECs were evaluated with RT-PCR and Western Blot. The level of NO,sICAM-1,SOD in cell culture fluid were determined with the way of Nitrate reductase,ELISA and so on.Results:1.The cultivation and identification of HUVECsHUVECs were cultured in vitro. It was definited as HUVECs using morphology and immunofluorescence.2.The results of experiment 12. 1 After HUVECs were incubated with LPS for 24 hours, the contents of NO and SOD in cell culture fluid were decreased, and the contents of sICAM-1 and sE-selectin in cell culture fluid were increased.2.2 After HUVECs were incubated with ATV (1.0μM ) for 24 hours, the contents of NO and SOD in cell culture fluid were significantly increased while compared with group 2, and the concentration of sICAM-1 were significantly decreased while compared with group 2, but the contents of sE-selectin had no change. After HUVECs was incubated with ATV (5.0μM ) for 24 hours, the contents of NO and SOD in cell culture fluid were significantly increased while compared with group 2, and the contents of sICAM-1 were significantly decreased while compared with group 2,but the contents of sE-selectin had no change also. There were statistic difference between the level of NO,SOD and sICAM-1 in group 3 and 4. The concentration dependence of ATV was confirmed. There was no change of content of sE-selectin in different concentration ATV groups.2.3 The PPARγand eNOS of HUVECs had not been affected by stimulated with LPS while the expression of NF-κB-p65,ICAM-1 and E-selectin was upregulated by LPS.2.4 The PPARγof HUVECs were upre gulate d by different concentrations ATV, and the expression of NF-κB-p65 and ICAM-1 induced by LPS was downregulated. The concentration dependence of ATV was also confirmed. ATV had no effect on the expression of eNOS and E-selectin.3.The results of experiment 23.1 Compared with group 2, the contents of NO and SOD significantly decreased and sICAM-1 increased in cell culture fluid when HUVECs were incubated with LPS,ATV and GW9662( the specific inhibitor of PPARγ). This results demonstrated that ATV could increase NO,SOD and decrease sICAM-1 through activating PPARγthat indicate vascular endothelium function maybe improved by ATV through the pathways of PPARγand NF-κB.3.2 The expression of PPARγinduced by ATV was inhibited by GW9662. Our study demonstrated that the expression of NF-κB-p65 was inhibited by ATV through activating PPARγ, but the effect was inhibited by GW9662 and the express of ICAM-1 was increased.Conclusions:1. ATV upregulated the expression of PPARγand inhibited the expression of NF-κB-p65 and ICAM-1 in HUVECs induced by LPS. There are dosage dependence of these effects. There is no significant effect on the expression of eNOS and E- selectin.2. In the cell culture fluid with different concentrations ATV, the level of NO and SOD increase, meanwhile the level of sICAM-1 decrease. It also enhances the release of NO but had no effect on the expression of eNOS. Our results demonstrate that ATV can protect the endothelial function.3. The effects of ATV are partially inhibited by GW9662 (the specific inhibitor of PPARγ) . We supposed that the endothelial function improved by ATV maybe through activating PPARγ.PartⅡThe effect of different dosage atorvastatin on endothelial function in hypertensives without hyperlipemiaObjective:To investigate the effect of different dose atorvastatin on endothelium- dependent vasodilatation function and the changes of NO,SOD,sICAM- 1 in serum of hypertensives without hyperlipemia.Materials and Methods:Fifty-five hypertensives without hyperlipemia were randomly dividedinto 2 groups: twenty-five subjects with atorvastatin 10mg, thirty subjects with atorvastatin 20mg. Twenty-five normotensives were enrolled in control group. Before and after 4 weeks atorvastatin taking, blood samples were taken for determining concentrations of serum cholesterol,aminotransferase,NO,SOD,sICAM-1 and blood pressure were evaluated. Flow-mediated dilation (FMD) and endothelium- independent dilatation(EID) were measured with high-resolution ultrosonography.Results:1. Comparing with normotensives, FMD of the hypertensives without hyperlipemia were significantly decreased, but there was no change in EID between two groups. The level of serum NO and SOD was significantly decreased as compared with those in normotensives( p﹤0.01) , and serum sICAM-1 significantly increased( p﹤0.01) .2. The FMD was positively correlated with the level of serum NO and SOD in the hypertensives without hyperlipemia in one factor correlation analysis, and contiguous coefficient were 0.74 and 0.56 respectivly( p﹤0.01). The FMD were negatively correlated with serum sICAM-1 and contiguous coefficient was﹣0.83( p﹤0.01). There was no correlation between FMD and age,gender,blood pressure and body mass index( p﹥0.05). Logistic analysis show that serum NO, SOD and sICAM-1 were the main factors influencing the FMD. Logistic equation: Y=6.593+0.039NO+0.019SOD+0.046 sICAM-1 with statistics significance .3. The FMD of hypertensives was enhanced significantly, but there was no change in EID after ATV taken for 4 weeks. The FMD of ATV 10 mg group is 7.5±2.7 vs 11.5±3.1, p < 0.05; EID 16.9±4.5 vs 17.4±5.3, p > 0.05. The FMD of ATV 20 mg group is 7.3±3.4 vs 14.7±2.5, p <0.01; EID 17.1±5.5 vs 18.2±5.6, p >0.05. FMD was improved significantly greater in 20 mg group than 10 mg group( p < 0.05) . Serum sICAM-1 was significantly decreased without dose dependence.Conclusions:1. Comparing with normotensives, FMD of the hypertensives without hyperlipemia were significantly decreased. ATV can improve FMD withdosage dependent.2. The FMD of the hypertensives without hyperlipemia is independently relative with the levels of serum NO,SOD and sICAM-1.3. The improvement of FMD in the hypertensives without hyperlipemia properly relates with the improvement of serum activity mediators by ATV.