阿托伐他汀改善胰岛素抵抗内皮功能的作用及机制研究

发布时间：2018-04-10 03:31

本文选题：胰岛素抵抗　切入点：内皮功能障碍　出处：《吉林大学》2015年博士论文

【摘要】：研究背景及目的：内皮功能障碍是多种心血管疾病,尤其是动脉粥样硬化病理过程发生的始动环节。近年来，胰岛素抵抗被公认为是多种代谢性疾病及心血管疾病的共同基础。同时胰岛素抵抗被证实动脉粥样硬化的发病因素之一。内皮细胞作为胰岛素的靶细胞，多项临床和流行病学研究强烈支持内皮功能障碍与胰岛素抵抗之间存在密不可分的关系。然而，二者之间相互作用的病理生理过程尚不十分明确。阿托伐他汀，作为心血管保护药物，是一个已应用临床多年的，经典的他汀类降脂药物。近年来有报道证实阿托伐他汀存在调脂外的血管内皮保护作用，但有关阿托伐他汀对胰岛素抵抗的内皮细胞的保护作用及机制方面的研究较少。体内实验较复杂，研究个体差异大，不确定因素较多，然而体外实验方面，胰岛素抵抗的内皮细胞模型尚鲜见。目前应用脂肪细胞、肝脏细胞制备胰岛素抵抗模型的报道很多，主要关注点在于研究胰岛素抵抗与糖脂代谢之间的关系。胰岛素抵抗的内皮细胞模型尚鲜见。本实验的第一部分，通过高葡萄糖联合高胰岛素刺激内皮细胞，制备胰岛素抵抗的内皮细胞模型，破坏内皮细胞的胰岛素受体信号通路继而导致血管内皮功能障碍。本实验的第二部分，，应用阿托伐他汀作用胰岛素抵抗的内皮细胞，旨在探讨阿托伐他汀对胰岛素抵抗的血管内皮细胞的保护作用及可能作用机制。方法： HUVEC培养在分别含有5，15或30mM葡萄糖或联合10-5M胰岛素的培养基里培养24小时后，弃去原培养基，各组重新加入含100nM胰岛素的正常培养基刺激细胞30min后，由倒置显微镜观察细胞形态和MTT检测细胞活力，再通过Westernblot对胰岛素受体及其底物的磷酸化、Akt磷酸化、eNOS的磷酸化进行检测；eNOS试剂盒检测eNOS活性；硝酸还原法检测NO浓度；RT-PCR检测内皮细胞分泌ET-1mRNA的表达。本实验中，将HUVEC在含有30mM的葡萄糖和10-5M胰岛素的培养基中培养24小时被定义为胰岛素抵抗的血管内皮细胞即IR VEC。正常培养的HUVEC定义为Nor VEC.接下来将加入不同浓度的阿托伐他汀（0，10-6M，10-5M,10-4M）分别加入到Nor VEC和IR VEC中，刺激24小时或10-4M阿托伐他汀作用细胞24小时期间，检测不同时间点（3h、6h、9h、12h、15h、18h、24h）上述指标，观察阿托伐他汀对胰岛素信号通路的干预作用及对内皮功能的影响。最后，应用PI3k抑制剂LY294002封闭其信号通路，探讨阿托伐他汀保护血管内皮的作用机制。结果： 1细胞分别培养在含有5、15、30mM的葡萄糖和（或）10-5M胰岛素的培养基中培养24h。细胞形态及活细胞数量在各组间没有明显差异（p0.05）。 2与对照组相比，单独30mM葡萄糖刺激血管内皮细胞显著降低了胰岛素受体（IR）及其底物(IRS1)的酪氨酸磷酸化表达，分别降低达60%和40%（p0.01）。与单独培养在30mM葡萄糖培养基中的细胞相比，联合了10-5M胰岛素共同刺激细胞时，胰岛素受体及其底物的酪氨酸磷酸化表达进一步下降(p0.05)。 3与对照组相比，单独30mM葡萄糖或者单独10-5M胰岛素刺激细胞，均使Akt的磷酸化表达降低，分别下降了近50%和30%。当联合应用30mM葡萄糖和10-5M胰岛素共同刺激细胞时，Akt磷酸化的表达进一步下降（p0.01）。 4与对照组相比，单独30mM葡萄糖刺激细胞，使内皮细胞分泌的一氧化氮（NO）明显下降（p0.05），联合10-5M胰岛素共同刺激后，NO浓度下降更为明显（p0.01）。与此相反，相比于对照组，联合30mM葡萄糖和10-5M胰岛素刺激细胞，使ET-1mRNA的表达增加近13倍（p0.01）。 530mM葡萄糖刺激细胞，明显降低eNOS的ser1177位点磷酸化表达(p0.05)，联合10-5M胰岛素刺激后表达水平进一步下降(p0.01)。细胞在联合30mM的葡萄糖和10-5M胰岛素共同刺激下，eNOS的ser1177位点磷酸化水平降低至在5mM葡萄糖中培养的22％。与此同时，eNOS的活性变化与eNOS的磷酸化水平变化几乎一致。 6阿托伐他汀使IR vec的胰岛素受体及其底物的酪氨酸磷酸化的表达明显增加（p0.01），不同剂量的阿托伐他汀组之间无明显差异（P0.05）。 7阿托伐他汀使IR vec内Akt的磷酸化表达明显增高（p0.01）。 8阿托伐他汀显著增加IR vec的NO产量呈正向剂量依赖性。与此相反，与未经处理的IR vec相比，阿托伐他汀显著降低ET-1的mRNA表达，对ET-1mRNA表达的最大抑制发生在10-4M组（抑制率为76％）。 9阿托伐他汀显著增加eNOS的ser1177位点磷酸化及活性的表达呈剂量依赖性。 10在IR vec中，阿托伐他汀诱导NO的产生，在用药后的第3小时NO的浓度即开始升高，其最高水平在9小时后出现，并能持续此表达水平24小时。阿托伐他汀无论在Nor vec或IR vec中均降低ET-1mRNA水平。 11阿托伐他汀增加IR vec中eNOS的ser1177位点磷酸化及活性表达呈时间依赖性。 12LY294002部分抑制了阿托伐他汀诱导VEC和IR VEC细胞NO产生增加（P0.05）。同样地，LY294002降低阿托伐他汀诱导的AKt的磷酸化表达、eNOS的ser1177位点磷酸化和eNOS的活性。然而，在ET-1mRNA表达的并未受到LY294002的影响（P0.05）。结论： 1、在胰岛素抵抗早期，胰岛素抵抗并未引起细胞形态的改变，仅影响细胞功能发生障碍。 2、胰岛素信号通路受损后血管内皮细胞功能发生障碍，eNOS的ser1177位点的磷酸化表达减少及活性降低，内皮细胞分泌NO的产生降低，ET-1mRNA的表达增多。 3、当血管内皮细胞在胰岛素抵抗的状态下，胰岛素信号通路受损导致胰岛素受体（IR）、受体底物(IRS1)、蛋白激酶B（Akt）磷酸化表达减少。 4、阿托伐他汀可以改善胰岛素抵抗状态下的血管内皮功能障碍，并呈量效及时效性。 5、阿托伐他汀可以明显恢复受损的胰岛素信号通路传导，部分上调胰岛素受体（IR）、受体底物(IRS1)、蛋白激酶B（Akt）磷酸化表达，改善胰岛素抵抗。 6、阿托伐他汀具有对胰岛素抵抗状态下的血管内皮细胞保护作用，可能主要作用于PI3k/Akt/eNOS通路。
[Abstract]:Background and purpose of the study :

Insulin resistance is recognized as a common base for many metabolic diseases and cardiovascular diseases .

Method :

HUVEC were cultured for 24 hours in medium containing 5 , 15 or 30 mM glucose or 10 - 5M insulin respectively . After 30 min of stimulation of cells with normal medium containing 100 nM insulin , the cell morphology and MTT test cell viability were observed by an inverted microscope , then the phosphorylation of insulin receptor and its substrate was observed by Western blot , and phosphorylation of eNOS and phosphorylation of eNOS were detected by Western blot ;
eNOS assay was used to detect eNOS activity .
NO concentration was detected by nitrate reduction method .
The expression of ET - 1 mRNA was detected by RT - PCR . In this experiment , HUVEC were cultured in medium containing 30 mM glucose and 10 - 5M insulin for 24 hours .

Results :

Cells were cultured for 24 h in medium containing 5 , 15 , 30 mM glucose and / or 10 - 5M insulin , and no significant difference was found between the cell morphology and the number of viable cells ( p < 0.05 ) .

Compared with control group , 30 mM glucose stimulated vascular endothelial cells significantly reduced tyrosine phosphorylation of insulin receptor ( IR ) and its substrate ( IRS1 ) , decreased by 60 % and 40 % ( p0.01 ) , respectively . Compared with the cells cultured in 30 mM glucose medium alone , the expression of tyrosine phosphorylation of insulin receptor and its substrate decreased further ( p < 0.05 ) .

3 Compared with the control group , 30 mM glucose alone or 10 - 5M insulin stimulated the cells , which decreased the phosphorylation and decreased by nearly 50 % and 30 % , respectively . When combined with 30 mM glucose and 10 - 5M insulin to stimulate the cells , the expression of phosphorylation decreased further ( p . 01 ) .

In contrast to the control group , 30 mM glucose and 10 - 5M insulin stimulated the cells in combination with 30 mM glucose and 10 - 5M insulin to increase the expression of ET - 1 mRNA by nearly 13 times ( p0.01 ) .

At the same time , the phosphorylation level of ser1177 in eNOS decreased to 22 % in 5 mM glucose . At the same time , the change of eNOS activity was almost identical to that of eNOS .

The expression of tyrosine phosphorylation of IR vec insulin receptor and its substrate increased significantly ( P0.01 ) . There was no significant difference between atorvastatin group at different doses ( P0.05 ) .

7 - Atorvastatin increased the expression of phosphorylation in IR vec ( p0.01 ) .

In contrast , atorvastatin significantly reduced the mRNA expression of ET - 1 compared to untreated IR vec , and the maximum inhibition of ET - 1 mRNA expression was in the 10 - 4M group ( 76 % inhibition rate ) .

9 atorvastatin significantly increased the phosphorylation and activity of ser1177 sites of eNOS in a dose - dependent manner .

10 In IR vec , atorvastatin induced NO production , the concentration of NO at the 3rd hour after administration began to rise , the highest level appeared after 9 hours , and the expression level was sustained for 24 hours . Atorvastatin lowered the ET - 1 mRNA level in either Nor vec or IR vec .

11 Atorvastatin increased the phosphorylation of ser1177 and the time - dependent expression of eNOS in IR vec .

However , the expression of ET - 1 mRNA was not affected by the expression of ET - 1 mRNA ( P0.05 ) .

Conclusion :

1 . During the early period of insulin resistance , insulin resistance did not induce changes of cell morphology , and only affected the cellular function .

2 . After the insulin signal pathway was damaged , the endothelial function of endothelial cells was impaired , the expression of ser1177 of eNOS decreased and the activity was decreased , the secretion of NO in endothelial cells decreased , and the expression of ET - 1 mRNA increased .

3 . When vascular endothelial cells are in insulin resistance , insulin receptor ( IR ) , receptor substrate ( IRS1 ) and protein kinase B ( phosphorylation ) decrease in insulin signaling pathway .

4 . Atorvastatin can improve vascular endothelial dysfunction in insulin resistance , and can be dose - effective and time - effective .

5 . Atorvastatin can obviously recover the damaged insulin signaling pathway , partially up - regulate the expression of insulin receptor ( IR ) , receptor substrate ( IRS1 ) , protein kinase B , and improve insulin resistance .

6 . Atorvastatin has the protective effect on vascular endothelial cells under the condition of insulin resistance .

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R54

【共引文献】