背景与目的：

代谢综合征是心血管疾病(cardiovascular disease，CVD)的主要危险因素之一，是指一组包括肥胖、高血糖、血脂异常等症状的代谢紊乱。代谢综合征的发病率在过去的几十年里迅速增长，并与近年来心血管疾病的发病率增加两倍有关。CVD是全球范围内死亡的主要原因。血管内皮功能障碍是CVD发展的早期事件，常伴随代谢综合征的进展。

血管内皮细胞是位于血液和血管内膜下组织之间的单层细胞，在维持血管的完整性和功能方面起着至关重要的作用。高脂饮食引起的持续高脂血症，血浆游离脂肪酸含量(free fatty acids，FFA )升高，可引起血管内皮功能障碍，增加血管通透性，从而导致一系列的病理改变，最终导致CVD的发生。

葛根素（puerarin，PUE）是从传统中药葛根中提取的一种异黄酮，具有抗炎，抗氧化，降血糖，降血脂及调节血栓形成等多种药理活性，广泛应用于CVD的预防和治疗。然而PUE对血管内皮损伤的影响尚未得到重视，本研究拟通过体外棕榈酸（Palmitic acid，PA）诱导损伤的人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVEC）模型和体内高脂饮食引起血管损伤大鼠模型，探讨PUE对血管内皮损伤的作用及其机制。

方法：

体外实验：将HUVEC细胞用于体外培养，给予不同浓度PA分别刺激HUVEC细胞24h后，利用油红染色检测细胞脂毒性， CCK8法检测细胞活力，分析PA作用的浓度依赖性，筛选出建立细胞损伤模型的最佳PA作用浓度；以PA（400μmol·L^-1）刺激HUVEC细胞不同时间（0,12,24,48h），通过CCK8法检测细胞活性，分析PA作用的时间依赖性，筛选出最佳的PA作用时间；以不同浓度的PUE（0，0.01，0.1，1，10 μmol·L^-1）预处理HUVEC细胞4h后，PA（400μmol·L^-1）刺激24h，CCK8法和油红染色检测PUE的作用，选择最佳的PUE作用浓度进行后续的机制研究；实验细胞分为四组，正常对照组（control，CON）、棕榈酸组（PA400μmol·L^-1，PA）组、葛根素组（PA 400μmol·L^-1+PUE 1μmol·L^-1，PUE）及辛伐他汀对照组（PA 400μmol·L^-1+simvastatin（SIM）1μmol·L^-1，SIM）组，不同药物预处理4h后再加入PA刺激HUVEC细胞24 h，采用流式细胞术检测细胞凋亡，NO探针法检测细胞内NO的水平，PCR检测炎症因子TNF-α、IL-6及IL-1β mRNA的表达，Western blot检测细胞内p-AKT、p-eNOS、SIRT1及NF-kB等蛋白的表达。

体内实验：30只健康雄性SD大鼠高脂饲料连续喂养6周建立高脂血症大鼠模型。将高脂饮食大鼠随机分为模型组 （HFD）、模型+PUE 100 mg·kg^-1• day^-1(PUE)组和模型+SIM 10mg·kg^-1• day^-1（SIM）阳性对照组,同时设正常对照组（CON）10只大鼠。每周称重，连续每日灌胃给药6周后，麻醉取血测定血浆生化指标：血浆总胆固醇（total plasma cholesterol，TC）、高密度脂蛋白（high density lipoprotein，HDL）、C反应蛋白（C-reactive protein，CRP）、低密度脂蛋白（low density lipoprotein，LDL）及甘油三酯（triglyceride，TG）等的水平并计算HDL/LDL比值；测定与血管内皮损伤及血栓形成相关的指标血栓素B2（thromboxane B2，TXB2）、一氧化氮（nitric oxide，NO）含量及内皮素1（endothelin-1，ET-1）的含量；半自动凝血分析仪检测大鼠血浆凝血四项既纤维蛋白原含量（fibrinogen，FIB）、凝血酶时间（thrombin time，TT）、活化部分凝血活酶时间（activated partial thromboplastin time，APTT）及凝血酶原时间（prothrombin time，PT）并检测ADP诱导的血小板最大聚集率（maximum aggregation rate，MAR）；Western blot检测胸主动脉P-AKT、P-eNOS、SIRT1及NF-kB等蛋白的表达；取胸主动脉固定切片油红0及茜素红染色观察病理变化。

结果：

体外实验：PA引起HUVEC细胞脂毒性，减少细胞活力，且具有浓度依赖性和时间依赖性；低浓度PUE（0.01-1μmol·L^-1）不影响HUVEC细胞的存活率，高浓度PUE（大于等于10μmol·L^-1）降低HUVEC细胞的存活率；PUE在一定浓度范围内（0.01-1μmol·L^-1）预处理可呈浓度依赖性的改善PA诱导的HUVEC细胞活力降低及PA诱导的脂质堆积；PA引起HUVEC细胞凋亡，PUE预处理减少HUVEC细胞凋亡并增加凋亡相关蛋白Bcl-2及Bax两者的比值；PA减少HUVEC细胞内NO水平，PUE预处理可明显增加HUVEC细胞内NO水平并增加p-eNOS及p-AKT的表达；此外，PUE可降低PA诱导的炎症相关因子TNF-α、IL-6及IL-1β mRNA的表达，并降低NF-kB的表达提高SIRT1的表达；PUE和对照药SIM结果类似。

体内实验：相较对照组高脂喂养加快模型组大鼠的体重增长，升高血浆TG、CHO、LDL水平并降低HDL水平，增加大鼠体内炎症水平，加快PT、APTT、TT时间并升高FIB、 MAR及TXB2水平；与模型组相比，PUE治疗改善高脂饮食引起的脂代谢紊乱、炎症反应、高凝状态及钙质沉积，增加主动脉P-AKT、P-eNOS、SIRT1蛋白的表达并降低NF-kB蛋白的表达。

结论：

体外实验表明低浓度PUE对PA诱导的HUVEC细胞的脂毒性具有保护作用，可减少细胞凋亡增加胞内NO可用量；体内实验表明，口服PUE调节血脂、抑制血浆炎症水平、抑制凝血。体内外实验均表明，PUE可能通过调控AKT/eNOS通路增加细胞内NO的生成，通过SIRT1/NF-kB通路抑制血管炎症反应，以及调节脂质代谢抑制脂肪沉积，从而发挥血管内皮保护作用。

Background and purpose:

Metabolic syndrome, a group of metabolic disorders including obesity, hyperglycemia, dyslipidemia and hypertension, is one of the main risk factors of cardiovascular disease (CVD). The incidence rate of metabolic syndrome has increased rapidly in the past decades, and the morbidity of cardiovascular disease has doubled in recent years. CVD is the leading cause of death worldwide. Vascular endothelial dysfunction, which is often accompanied by the progression of metabolic syndrome, is an early event in the development of CVD.

Vascular endothelial cells are a layer of monolayer cells located between blood and subintimal tissue. They can synthesize and secrete a variety of cytokines and active substance which participate in many physiological processes such as regulating the balance between cholesterol and lipid, blood coagulation and fibrinolysis, signal transduction, immune inflammation and so on. Endothelium plays an important role in maintaining the integrity and function of blood vessels. Continuous hyperlipidemia caused by high-fat diet increases of plasma free fatty acid (FFA), which can cause vascular endothelial dysfunction and increase vascular permeability, that result in a series of pathological changes and eventually lead to CVD.

Puerarin (PUE) is an isoflavone extracted from Pueraria lobata, a traditional Chinese medicine. It has many pharmacological activities, such as anti-inflammatory, antioxidant, hypoglycemic, hypolipidemic and reducing thrombosis. It is widely used in the prevention and treatment of CVD. However, the effect of PUE on vascular endothelium injury has not been known. This study intends to explore the effect and mechanism of PUE on vascular endothelial injury through the model of human umbilical vein endothelial cells (HUVECs) injury induced by palmitic acid (PA) in vitro and the hyperlipidemia rat model induced by high-fat diet in vivo.

Method:

In vitro experiment: HUVECs were cultured in vitro and given different concentrations of PA  (0， 25，50，100，200，400，800，1600 μmol·L^-1)  24 hours, the lipotoxicity was detected by oil red staining, the cellular activity was detected by CCK8 , the concentration dependence effect of PA was analyzed, and the optimum concentration of PA was selected to establish the cell injury model; HUVECs were stimulated with PA  (400μmol·L^-1) for different times (0,12,24,48 h). The cellular activity was detected by CCK8 , the time-dependent effect of PA was analyzed, and the optimum time of PA was selected; Pretreatment with different concentrations of PUE  (0，0.01，0.1，1，10 μmol·L^-1) for 4h, HUVECs were stimulated with PA  (400μmol·L^-1)  for 24 hours, CCK8 and oil red staining were used to detect the effect of PUE, and the optimum concentration of PUE was selected for subsequent mechanism studies; The experimental cells were divided into four groups: control group (CON) and palmitic acid group  (PA400μmol·L^-1，PA), puerarin group  (PA 400μmol·L^-1+PUE 1μmol·L^-1，PUE)  and positive control group (PA 400μmol·L-1+simvastatin (SIM) 1μmol·L-1, SIM). Pretreatment with different drugs for 4h, HUVECs were stimulated for 24 hours, apoptosis was detected by flow cytometry, intracellular NO level was detected by NO probe, and mRNA expression of inflammatory related factors TNF-α，IL-6 and IL-1β was detected by PCR, The protein expression of p-AKT, p-eNOS, SIRT1 and NF-kB were detected by Western blot.

In vivo experiment: The hyperlipidemia model was established in 30 healthy male SD rats with high-fat diet for 6 weeks. These hyperlipidemia rats were randomly divided into hyperlipidemia group (HFD), hyperlipidemia + PUE 100 mg·kg^-1•day^-1(PUE) group and hyperlipidemia + SIM 10 mg·kg^-1•day^-1(SIM) group as positive control group. 10 rats were set up as the normal control group (CON). These rats were administered daily by gavage for 6 weeks and weighed weekly. Blood samples were taken from anesthetized rats, and plasma parameters (C-reactive protein (CRP), high density lipoprotein (HDL), total plasma cholesterol (TC), low density lipoprotein (LDL) and triglyceride (TG), and the ratio of HDL / LDL) were measured by automatic blood biochemical detection; Thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 (6-keto-pgf1) were determined by ---; The content of nitric oxide (NO) and endothelin-1 (ET-1) were measured by ---. The parameters of plasma coagulation (fibrinogen (FIB), thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (APTT)) of rats were detected by semi-automatic coagulation analyzer, and the maximum platelet aggregation rate(MAR) induced by ADP. The thoracic aorta was fixed and stained with HE and oil red 0 to observe the pathological changes of the aorta; The expressions of p-AKT, p-eNOS, SIRT1 and NF-kB in thoracic aorta were detected by Western blot.

Results:

The results of vitro experimental: PA caused HUVECs lipotoxicity and decreased cell viability in a concentration dependent and time-dependent manner; Low concentration PUE (0.01-1μmol·L^-1) did not affect the survival rate of HUVECs. High concentration PUE (greater than or equal to 10μmol·L^-1) decreased the cell survival rate of HUVECs; Pretreatment with PUE (0.01-1μmol·L^-1) could increase cellular viability and decrease lipotoxicity in a concentration dependent manner in HUVECs stimulated with PA; PA induced HUVECs apoptosis, pretreatment with PUE decreased HUVECs apoptosis and increased the ratio of apoptosis related protein Bcl-2/Bax. PA decreased the level of NO in HUVECs, and pretreatment with PUE could significantly increase the level of NO in HUVECs stimulated with PA; In addition, compared with PA group, PUE could also increase the expression of p-AKT, p-eNOS and SIRT1 protein and significantly reduce the expression of NF-kB protein; There is similar effects between PUE and SIM.

The results of vivo experimental: high fat diet increased the body weight of rats, increased the levels of plasma TG, CHO and LDL-C, decreased the level of HDL-C, increased the level of inflammation in rats, accelerated the time of PT, APTT and TT, and increased the levels of FIB, MAR and TXB2; Compared with the HFD group, PUE treatment improved the disorder of lipid metabolism, inflammatory reaction and hypercoagulability caused by high-fat diet, increased the expression of p-AKT, p-eNOS and SIRT1 protein in aorta, and decreased the expression of NF-kB protein.

Conclusion:

The vitro experiments showed that low concentration PUE could protect HUVECs from lipotoxicity induced by PA, reduce apoptosis and increase the level of intracellular NO; The vivo experiments showed that oral PUE can regulate blood lipid, inhibit inflammation, inhibit coagulation and exert a protective role on vascular endothelium injury. In vivo and in vitro experiments, PUE may increase the production of intracellular NO by regulating AKT/eNOS pathway, alleviate the injury of endothelial cells, and inhibit vascular inflammatory response through SIRT1/NF-kB pathway.