【作者】 李伟；

【导师】 孙伟；

【作者基本信息】 南京中医药大学 ， 中西医结合， 2013， 博士

【摘要】 目的观察毛蕊异黄酮、丹参酮ⅡA、大黄素对LPS诱导损伤的人脐静脉血管内皮细胞(HUVECs)的干预作用,探讨三种中药单体通过调控Rho/ROCK通路、AKT通路对内皮细胞细胞骨架及通透性的影响,为其对内皮细胞功能的保护作用提供实验支持,同时为中医采用补气活血清热泄浊法治疗内皮损伤相关性疾病提供理论依据。方法首先以0.2μg/ml LPS作用于人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)24h,造成血管内皮损伤模型。Y27632(ROCK特异性抑制剂)和缬沙坦(西药对照,AngⅡ受体1拮抗剂,Rho/ROCK信号通路的间接抑制剂)作为阳性对照药物。我们分别依次观察了毛蕊异黄酮、丹参酮ⅡA、大黄素对LPS诱导的内皮损伤后的HUVECs的变化：MTT法观察细胞存活率、流式细胞仪Annexin V-FITC/PI染色观察凋亡率,transwell观察细胞迁移能力,10mg/mL波连蛋白预包被/无包被96孔板观察细胞黏附能力,硝酸还原酶法观察细胞培养上清中的cNOS、iNOS和NO浓度,免疫荧光观察细胞骨架和黏着斑蛋白的结构和分布。研究中,三种中药单体都表现出卓越的内皮细胞保护能力。然后我们试图对三种中药单体的内皮细胞保护机制进行探讨。首先,以内皮细胞生物学功能PCR基因芯片技术筛选可能的基因谱,发现细胞骨架重构相关基因,如IRho/ROCK、AKT和FAK-PI3K信号通路相关基因在其中起重要作用,尤以Rho/ROCK通路为主。结合基因芯片结果和文献中各基因功能,拟定观察Rho/ROCK通路中的关键基因如纤连蛋白(fibronectin、FN),整合素A5(integrin A5, ITGA5), Ras同源基因家族成员(Ras homolog gene family member A, RhoA),肌球蛋白轻链磷酸酶(myosin light chain phosphatase, MLCP),磷脂酰肌醇-4,5-二磷酸肌醇3-激酶(phosphatidylinositol-4,5-bisphosphate3-kinase, PI3K),黏着斑激酶(focal adhesion kinase, FAK),血管内皮生长因子(vascular endothelial growth factor, VEGF),血管内皮生长因子受体2(vascular endothelial growth factor receptor2, VEGFR2)的mRNA表达水平,进行实时定量PCR验证,同时以Western blotting对ITGA5, RhoA,4,5-二磷酸磷脂酰肌醇(Phosphatidylinositol4,5-bisphosphate, PIP2)和磷酸化的肌球蛋白轻链(Phosphorylated myosin light chain, PMLC)进行蛋白水平的研究。结果(1)LPS成功诱导构建了HUVECs细胞骨架损伤模型。HUVECs细胞增殖减弱、凋亡增加,细胞骨架肌动蛋白皱缩、黏着斑蛋白聚集,LPS活化内皮、诱导肌球蛋白收缩,导致细胞间隙开放、内皮通透性增加、细胞迁移能力和黏附能力下降。LPS作用24h后,cNOS减低,tNOS、iNOS、NO升高,呈现内皮损伤和炎症状态。同时,LPS激活Rho/ROCK信号通路,提高Rho/ROCK通路相关基因、蛋白的表达(FN, ITG A5, RhoA, MLCP, PI3K FAK, VEGF, VEGFR2)。Y27632和缬沙坦特异性阻断了Rho/ROCK通路,显现出对细胞骨架损伤的较强的保护作用。(2)毛蕊异黄酮诱导HUVECs细胞凋亡、提高细胞迁移能力、降低黏附能力、cNOS升高、iNOS减低、细胞骨架和黏着斑蛋白形态和分布状况改善,即减少细胞数量、提高细胞质量。毛蕊异黄酮的内皮保护作用涉及多条信号通路。毛蕊异黄酮通过增加NO的生成促进MLCP的激活,MLCP活化后降低PMLC,直接对抗Rho/ROCK通路的活化,同时通过降低、VEGF、 VEGF.R2和PI3K阻断AKT信号通路,发挥其内皮保护作用。(3)丹参酮ⅡA抑制HUVECs细胞凋亡、改善了细胞骨架肌动蛋白和黏着斑蛋白的形态和分布状况、提高细胞迁移和黏附能力,提高cNOS浓度。丹参酮ⅡA是内皮细胞强保护剂,其机制主要是通过下调ITG A5的表达而阻断Rho/ROCK通路的激活。(4)大黄素诱导HUVECs细胞凋亡、提高细胞迁移能力和黏附能力、cNOS升高、iNOS减低,但对细胞骨架和黏着斑蛋白基本没有改善作用。大黄素的内皮保护作用复杂,涉及多条信号通路,通过轻度抑制Rho/ROCK通路、调节AKT和/或FAK-PI3K信号通路发挥调节保护作用。结论(1)LPS诱导了HUVECs细胞骨架损伤,毛蕊异黄酮、丹参酮ⅡA、大黄素通过稳定细胞骨架结构、调节凋亡率、调节细胞合成和利用NO的能力、改变细胞迁移和黏附能力,而调节血管内皮通透性、改善内皮屏障功能。(2)丹参酮ⅡA抑制HUVECs凋亡,毛蕊异黄酮和大黄素促进HUVECs凋亡,维持调节内皮细胞数量的稳定。(3)三种中药单体对细胞骨架的保护机制分别涉及多个信号通路和信号通路间的串话,以Rho/ROCK信号通路为最主要的调节通路,还涉及AKT和/或FAK-PI3K通路,及其彼此间的串话。毛蕊异黄酮、丹参酮ⅡA、大黄素均参与了Rho/ROCK通路的调节,其中,毛蕊异黄酮同时还通过降低VEGF、VEGF.R2和PI3K阻断AKT信号通路,发挥作用；丹参酮ⅡA以下调ITGA5的表达而阻断Rho/ROCK通路的激活为主,同时也经PI3K和NO与其他通路互相协调：大黄素的调节机制较为复杂,通过轻度抑制Rho/ROCK通路、调节AKT和/或FAK-PI3K信号通路发挥调节保护内皮细胞的作用。(4)虽然机制和过程不同,但三个中药单体均显示了对细胞骨架损伤后修复的调节功效和对内皮细胞的保护作用。研究意义本研究首次将中药单体的内皮保护机制与基因芯片技术联系起来,从对细胞骨架损伤后修复、内皮细胞通透性的调节和机制的角度进行了研究。从细胞和分子水平观察了炎症损伤时,血管内皮细胞的变化；选用补气、活血、清热解毒类中药单体对炎症损伤进行干预治疗时血管内皮细胞的改善状况,并从分子水平初步探讨其药效机制(以Rho/ROCK信号通路为主、涉及可能的AKT和FAK-PI3K信号通路关键的基因和蛋白的表达)。从客观的角度为补气活血清热解毒类药物治疗内皮损伤相关疾病提供了有力的实验依据和理论基础。更多还原

【Abstract】 ObjectiveTo observe the protective effect of Calycosin, tanshinone Ⅱ A and emodin on LPS-induced endothelial cell injury, and to discuss the effect of three monomers on cytoskeleton and endothelial permeability by adjusting RhO/ROCK, AKT and FAK-PI3K pathway. The study was to provide experimental and theoretical basis for the protective effect on vascular endothelium, and the Chinese "Buqi Huoxue and Qingre Xiezhuo" method in the treatment of CKD and other endothelial injury-related diseases.MethodsEndothelial cell injury was induced by treatment of human umbilical vein endothelial cells (HUVECs) with0.2μg/ml LPS for24h. Y27632and valsartan were taken as positive controls. We studied the effect of calycosin, Tan ⅡA and emodin on LPS-induced cell viability of HUVECs by MTT assay; apoptosis rate with Annexin V-FITC/PI by flow cytometry; cell migration assay with transwell insert system; adhesion assay with96-well plate with or without the pre-coat with10mg/mL vitronectin; cNOS, iNOS and NO in culture supernatant with nitrate reductase assay; and cytoskeleton reorganization and vinculin distribution with immunofluorescence assay, separately. The three monomers of TCM all demonstrated superiorly protective effects on LPS-induced endothelial cell injury in the study.Then we dipped into the vascular endothelium protective mechanism of the three monomers. First, gene chip technology was employed to screening the genes which might involved in vascular endothelium function, and genes associated with cytoskeleton remolding, such as Rho/ROCK pathway, AKT pathway and FAK-PI3K pathway, performed to be predominant. Key genes, including fibronectin(FN), integrin A5(ITG A5), Ras homolog gene family member A (RhoA), myosin light chain phosphatase(MLCP), phosphatidylinositol-4,5-bisphosphate3-kinase (PI3K), focal adhesion kinase (FAK), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor2(VEGFR2) were selected to confirm the changes obtained from microarray with quantitive real-time PCR. And then and Western blotting was used for further study. And three monomers demonstrated different influence on gene and protein express related to skeleton remolding and vascular permeability barrier function.Results1. LPS succeeded in inducing cytoskeleton injury of HUVECs. The proliferation of HUVECs decreased, apoptosis rate increased, actin cytoskeleton polymerizated and vinculin contracted. LPS activated the endothelium and induce actomyosin contraction, and resulting in the opening of intercellular gaps, making the endothelium permeable to fluid and molecules, and cell migratory and adhensive capability was decreased significantly. LPS induced24h, cNOS decreased, tNOS, iNOS, NO increased, showing vascular endothelial injury and inflammation. LPS indeed activated Rho/ROCK pathway and increased the mRNA and protein expression associated with Rho/ROCK pathway (FN, ITG A5, RhoA, MLCP, PI3K FAK, VEGF and VEGFR2). Y27632and valsartan blocked Rho/ROCK pathway specifically, and demonstrated strong protective effect on cytoskeleton injury of HUVECs.2. Calycosin induced HUVECs apoptosis, migration capability increased, adhesion capacity decreased, beneficial cNOS levels increased, the concentration of harmful iNOS decreased and cytoskeleton and vinculin distribution improved. That is to reduce the number and improve the quality. Multiple pathways involved in the regulation of calycosin on HUVECs. It was considered that calycosin activated MLCP through the promotion of generation of NO, decreased PMLC, and directly suppressed the cytoskeleton remodeling caused by activation of Rho/ROCK pathway; at the same time, blocked AKT pathway through decrease of VEGF, VEGF.R2and PI3K.3. Tan IIA suppressed HUVECs apoptosis, improved actin cytoskeleton, migration and adhesion capacity increased and cNOS concentration increased. Tan ⅡA exerted a strong protective effect on HUVEC, and the mechanism was, at least in part, due to the blockade of Rho/ROCK pathway, presumably through downregulation of ITG A5.4. Emodin induced HUVECs apoptosis, migration and adhesion capability increased, capacity decreased, beneficial cNOS levels increased and harmful iNOS decreased. But emodin performed poor in improving cytoskeleton remolding. Emodin restrained slightly Rho/ROCK pathway, and exerted protection on HUVECs by adjusting AKT and/or FAK-PI3K pathway.Conclusions1. LPS induced cytoskeleton damage in HUVECs. Calycosin, tanshinone ⅡA and emodin regulated the endothelial permeability and improved endothelial barrier function by stabilizing cell skeleton structure, regulating apoptosis, regulate NO synthesis and utilization, change migration and adhesion capability.2. Tanshinone ⅡA suppressed apoptosis, while calycosin and emodin induced apoptosis of HUVECs, thus to maintain the stability of the number of endothelial cells.3. The mechanism of three monomers on cytoskeleton protection was associated with multiple signaling and crosstalk among signaling pathways. Rho/ROCK signaling pathway is the main pathway, AKT and FAK-PI3K pathways are also involved. Calycosin also suppressed the AKT signaling pathway by down-regulating VEGF, VEGF.R2and PI3K. Tanshinone ⅡA blocked Rho/ROCK pathway by down-regulating the expression of ITG A5, and coordinated with other pathway by PI3K and NO. The mechanism of emodin was complex, which is by inhibiting the Rho/ROCK pathway lightly, and regulating of AKT and/or FAK-PI3K pathway at the same time.4. Although the mechanism and procession was different, three monomers demonstrated the beneficial modulatory capability on cytoskeleton repairment and protective effect on endothelial cells.SignificanceThis is the first study on effect of TCM monomers on recovery after HUVECs cytoskeleton injury, and regulation and mechanism of permeability by microarray analysis technology. Changes of vascular endothelial cells during inflammation and after treatment with "Buqi Huoxue and Qingre Xiezhuo" monomers were observed at the cellular and molecular levels in vitro. And the effect mechanisms were explored and discussed at molecular level (key gene and protein expression in Rho/ROCK signaling pathway, and AKT and FAK-PI3K pathways also involved). The present study provided experimental and theoretical basis from an objective point of view on "Buqi Huoxue and Qingre Xiezhuo" method treatment in endothelial injury related diseases.更多还原