芍药内酯苷对泡沫细胞及动脉粥样硬化形成的影响

发布时间：2018-05-11 14:11

本文选题：芍药内酯苷 + 泡沫细胞　；参考：《吉林大学》2017年博士论文

【摘要】：目的:近年来,随着人们生活水平的提高及饮食习惯的改变,动脉粥样硬化(AS)的发生率逐年上升,成为导致人类死亡主要原因之一。单核细胞源性巨噬细胞在AS的发生发展中发挥着极其重要的作用,血液中单核细胞在各种外界因素的刺激下穿过血管内膜并分化为巨噬细胞,当机体脂质代谢发生紊乱,血液中脂质浓度过高时,巨噬细胞通过其表面A型清道夫受体吞噬大量氧化低密度脂蛋白(ox-LDL),造成其胞质内的脂质沉积最终形成泡沫细胞,泡沫细胞为AS斑块中具有特征性的病理细胞,在AS斑块中大量存在。大量研究证实,AS的发生为机体脂质代谢紊乱及慢性炎症反应共同作用的结果,并且另有研究表明LPS可通过激活TLR4/NF-κB通路加剧AS的发生。芍药内酯苷为一种中药成分,临床研究显示其具有良好的抗炎作用,并对一些炎症疾病如肠炎、类风湿性关节炎等具有很好的治疗效果。本研究通过建立体外泡沫细胞模型及小鼠动脉粥样硬化模型并给予芍药内酯苷处理,观察芍药内酯苷对泡沫细胞的形成及AS的发生的影响,并探究其具体作用机制。方法:1.芍药内酯苷对THP-1源性泡沫细胞形成的影响1)为研究芍药内酯苷对泡沫细胞形成的影响,需建立稳定的THP-1源性泡沫细胞模型,将THP-1细胞分为两组分别为THP-1组及巨噬细胞组,巨噬细胞组细胞给予160nmol/L的PMA并孵育24小时,通过镜下观察细胞形态和对细胞进行CD14免疫荧光染色来对诱导成的细胞进行鉴定。随后将诱导后的细胞分为两组分别为巨噬细胞组及泡沫细胞组,泡沫细胞组细胞给予ox-LDL处理48小时,并分别对细胞进行油红O染色,观察细胞胞质内脂质沉积情况。2)为研究芍药内酯苷对泡沫细胞形成的影响,将经PMA诱导成功的巨噬细胞分为三组:巨噬细胞组(对照组);泡沫细胞组:将巨噬细胞以50mg/L ox-LDL,静置培养48小时,诱导为泡沫细胞;芍药内酯苷组:将巨噬细胞以芍药内酯苷预处理1小时后,加入50mg/L ox-LDL共同孵育48小时,通过油红O染色分别观察各组细胞胞质脂质沉积情况,并对细胞裂解液中胆固醇(TC)及甘油三脂(TG)含量进行测定。3)为研究泡沫细胞形成过程中LOX-1/NF-κB通路的变化,将THP-1经PMA诱导后形成的巨噬细胞分别以不同浓度的ox-LDL(25/50/100mg/L)处理48小时,以未经处理的巨噬细胞作为对照组。收集各处理组细胞总RNA及胞质蛋白,分别以定量PCR及western blot方法测定细胞表达的LOX-1及NF-κB在转录及蛋白水平的表达量。并测定NF-κB信号通路下游IL-6及TNF-a的变化情况。4)为进一步确定LOX-1/NF-κB在泡沫细胞形成过程中的作用,将细胞分为四组:巨噬细胞组(对照组);ox-LDL组:细胞给予50mg/L ox-LDL处理48小时;LOX-1中和抗体组:细胞给予LOX-1中和抗体预处理1小时,再加入50mg/L ox-LDL处理48小时,抗体终浓度为10ug/ml;NF-κB抑制剂(PDTC)组:给予细胞NF-κB抑制剂预处理24小时,再加入50mg/L ox-LDL处理48小时,抑制剂终浓度为40umol/L。油红O染色观察各组细胞胞质脂质沉积情况,并分别以定量PCR及western blot方法测定细胞表达的LOX-1及NF-κB在转录及蛋白水平的表达量。5)为观察芍药内酯苷在泡沫细胞形成过程中对LOX-1/NF-κB通路的影响,将THP-1细胞经PMA诱导成的巨噬细胞分为三组,分别为巨噬细胞组:对照组;泡沫细胞组:将巨噬细胞以50mg/L ox-LDL,静置培养48小时,诱导为泡沫细胞;芍药内酯苷组:将巨噬细胞以芍药内酯苷预处理1小时后,加入50mg/L ox-LDL共同孵育48小时。分别提取细胞总RNA及细胞蛋白,定量PCR及western blot检测细胞LOX-1和NF-κB在转录及蛋白水平的表达量,并测定NF-κB信号通路下游IL-6及TNF-a的变化情况。2.芍药内酯苷对小鼠动脉粥样硬化模型的影响1)为研究芍药内酯苷对小鼠动脉粥样硬化形成的影响,将ApoE-/-小鼠随机分成三组,分别为:对照组(NC)、高脂组+生理盐水组(HFD)、高脂组+芍药内酯苷组(HFD+A)。检测试剂盒测定其血清中甘油三酯(TG)、胆固醇(TC)、低密度脂蛋白(LDL)及高密度脂蛋白(HDL)的含量变化,并通过对胸主动脉组织进行HE及油红O染色,观察主动脉病理变化及脂质沉积情况。2)为研究芍药内酯苷对小鼠动脉粥样硬化模型LOX-1/NF-κB通路的影响,将ApoE-/-小鼠随机分成三组,分别为:对照组(NC)、高脂组+生理盐水组(HFD)、高脂组+芍药内酯苷组(HFD+A)。分离胸主动脉组织蛋白及总RNA,定量PCR及western blot测定其LOX-1和NF-κB表达水平的变化,并测定NF-κB信号通路下游IL-6及TNF-a的变化情况。结果:1.芍药内酯苷对THP-1源性泡沫细胞形成的影响1)通过PMA对THP-1细胞处理后,镜下观察细胞呈贴壁生长,大量细胞生出伪足,成巨噬细胞样改变,免疫荧光染色细胞大量表达CD14。给予PMA诱导后的巨噬细胞ox-LDL处理后,油红O染色细胞胞质大量脂质沉积。THP-1源性泡沫细胞模型成功建立。2)ox-LDL诱导巨噬细胞形成泡沫细胞的过程中给予芍药内酯苷预处理结果发现,其胞质中脂质沉积与未芍药内酯苷处理的细胞相比明显减少,并且细胞裂解液中胆固醇(TC)及甘油三酯(TG)含量也显著降低。3)以不同浓度ox-LDL处理巨噬细胞后,定量PCR及western blot结果发现LOX-1及NF-κB的表达水平随着ox-LDL的浓度的增加也具有上升趋势,并且下游IL-6及TNF-a的表达量也随之上调。4)在以ox-LDL诱导泡沫细胞形成的过程中给予LOX-1中和抗体及NF-κB抑制剂处理后,油红O染色发现胞质脂质沉积情况明显改善,定量PCR及western blot结果发现LOX-1及NF-κB的表达水平明显下降,并且下游IL-6及TNF-a的表达量也显著下调。5)在以ox-LDL诱导泡沫细胞形成的过程中给予芍药内酯苷处理后,定量PCR及western blot结果发现LOX-1及NF-κB的表达水平明显下降,并且下游IL-6及TNF-a的表达量也显著下调。2.芍药内酯苷对小鼠动脉粥样硬化模型的影响1)给予小鼠动脉粥样硬化模型芍药内酯苷处理后,小鼠血清甘油三酯(TG)、胆固醇(TC)、低密度脂蛋白(LDL)及高密度脂蛋白(HDL)的含量下降,胸主动脉组织进行HE及油红O染色结果发现,病理变化及组织脂质沉积明显改善。2)给予小鼠动脉粥样硬化模型芍药内酯苷处理后,胸主动脉组织LOX-1及NF-κB的表达水平明显下降,并且下游IL-6及TNF-a的表达量也显著下调。结论:1.芍药内酯苷通过调节LOX-1/NF-κB信号通路在巨噬细胞吞噬大量ox-LDL后减少其胞质内脂质堆积,阻断其泡沫化进程。2.ox-LDL通过上调LOX-1的表达进而激活NF-κB的表达促进泡沫细胞的形成,芍药内酯苷在此过程中通过调节LOX-1/NF-κB通路及炎症分子的表达抑制动脉粥样硬化斑块的形成。
[Abstract]:Objective: in recent years, with the improvement of people's living standard and the change of dietary habits, the incidence of atherosclerosis (AS) is increasing year by year, which has become one of the main causes of human death. Monocyte derived macrophages play an extremely important role in the development of AS, and the mononuclear cells in the blood are in various external factors. When the lipid metabolism of the body is disorganized and the macrophage is differentiated, when the body's lipid metabolism is disturbed and the lipid concentration in the blood is too high, the macrophage phagocytosis a large amount of oxidized low density lipoprotein (ox-LDL) through its surface A type scavenger receptor, resulting in the formation of the lipid deposition in the cytoplasm at the end of the foam cells, and the foam cells are special in the AS plaques. A large number of studies have proved that the occurrence of AS is the result of the common effect of lipid metabolism disorder and chronic inflammatory reaction in the body, and other studies show that LPS can aggravate the occurrence of AS by activating TLR4/NF- kappa B pathway. Paeoniflorin is a kind of traditional Chinese medicine, and the clinical study shows that LPS has good effect. The effect of anti-inflammatory effects on some inflammatory diseases such as enteritis and rheumatoid arthritis is very good. In this study, the effects of paeoniflorin on the formation of foam cells and the occurrence of AS were observed through the establishment of an in vitro foam cell model and a mouse atherosclerosis model, and the effects of paeoniflorin on the formation of foam cells and the specific effects were explored. Mechanism. Method: 1. the effect of paeoniflorin on the formation of THP-1 derived foam cells (1). To study the effect of paeoniflorin on the formation of foam cells, a stable THP-1 derived foam cell model should be established, and the THP-1 cells were divided into two groups, THP-1 and macrophage groups, and the macrophage cells were given 160nmol/L PMA and incubated for 24 small groups. The cell morphology and CD14 immunofluorescence staining were used to identify the cells. Then the induced cells were divided into two groups, macrophage group and foam cell group. The cells of the foam cell group were treated with ox-LDL for 48 hours, and the cells were stained with oil red O, and the cytoplasm was observed. Internal lipid deposition.2) in order to study the effect of paeoniflorin on the formation of foam cells, the macrophages were divided into three groups: macrophage group (control group); foam cell group: macrophages were cultured with 50mg/L ox-LDL, cultured for 48 hours, and induced as foam cells; paeoniflorin group: macrophage with paeoniflorin After 1 hours of pretreatment, 50mg/L ox-LDL was added to incubate for 48 hours. The cytoplasmic lipid deposition of each cell was observed by oil red O staining, and the contents of cholesterol (TC) and glycerol three fat (TG) in the cell lysate were measured.3) to study the changes of LOX-1/NF- kappa B pathway during the formation of the foam cells, and the THP-1 was induced by PMA to form. The macrophages were treated with different concentrations of ox-LDL (25/50/100mg/L) for 48 hours, and the untreated macrophages were used as the control group. The total RNA and cytoplasmic proteins were collected and the expressions of LOX-1 and NF- kappa B expressed in the cells were measured by quantitative PCR and Western blot, and the NF- kappa B signal was measured. The change of IL-6 and TNF-a in the downstream of the channel.4) to further determine the role of LOX-1/NF- kappa B in the formation of foam cells, the cells were divided into four groups: macrophage group (control group); ox-LDL group: the cells were treated with 50mg/L ox-LDL for 48 hours; LOX-1 neutralization antibody group: fine cell given LOX-1 neutralization antibody for 1 hours, then 50mg/L ox-LD was added. L was treated for 48 hours, the final antibody concentration was 10ug/ml, and the NF- kappa B inhibitor (PDTC) group was treated with NF- kappa B inhibitor for 24 hours and then 50mg/L ox-LDL treatment for 48 hours. The final concentration of the inhibitor was 40umol/L. oil red O staining, and the cell cytoplasmic lipid deposition was observed by 40umol/L. oil red O staining. The expression of OX-1 and NF- kappa B at the transcriptional and protein level.5) was to observe the effect of paeoniflorin on the LOX-1/NF- kappa B pathway during the formation of foam cells. The macrophages induced by PMA in THP-1 cells were divided into three groups: macrophage group, control group, and foam cell group: macrophages were incubated with 50mg/L ox-LDL and cultured for 48 hours, Induced as foam cells; paeoniflorin group: after pretreating macrophages with paeoniflorin for 1 hours, the cells were incubated with 50mg/L ox-LDL for 48 hours. The total RNA and cell protein were extracted respectively. Quantitative PCR and Western blot were used to detect the expression of LOX-1 and NF- kappa B in the transcriptional and protein level, and the downstream IL-6 of NF- kappa B signaling pathway was determined. And the change of TNF-a.2. the effect of paeoniflorin on the atherosclerotic model of mice 1) to study the effect of paeoniflorin on the formation of atherosclerosis in mice. The ApoE-/- mice were randomly divided into three groups: the control group (NC), the high fat group + physiological saline group (HFD), the high fat group + paeoniflorin group (HFD+A) and the detection kit The serum levels of triglyceride (TG), cholesterol (TC), low density lipoprotein (LDL) and high density lipoprotein (HDL) were changed, and the pathological changes of aorta and lipid deposition.2 were observed by HE and oil red O staining on the thoracic aorta tissue. The effect of paeoniflorin on the LOX-1/NF- kappa B pathway in the atherosclerotic model of mice was studied. ApoE-/- mice were randomly divided into three groups: control group (NC), high fat group + physiological saline group (HFD), high fat group + paeoniflorin group (HFD+A), separation of thoracic aorta tissue protein and total RNA, quantitative PCR and Western blot to determine the expression of LOX-1 and NF- kappa B expression, and determine the changes in the downstream of NF- kappa signaling pathway. Fruit: 1. the effect of paeoniflorin on the formation of THP-1 derived foam cells 1. After the treatment of THP-1 cells by PMA, the cells were observed under the microscope, and a large number of cells produced pseudo foot and became macrophage like changes. The immunofluorescent staining cells expressed a large number of CD14. to PMA induced macrophage ox-LDL treatment, and the cytoplasm of the oil red O staining cells was large. The lipid deposition.THP-1 derived foam cell model successfully established.2) in the process of ox-LDL induced macrophage formation of foam cells, the results showed that the lipid deposition in the cytoplasm decreased significantly compared with the cells treated with paeoniflorin, and the content of cholesterol (TC) and triglyceride (TG) content in the cell lysate. After treating macrophages with different concentrations of ox-LDL, the results of quantitative PCR and Western blot showed that the expression level of LOX-1 and NF- kappa B also increased with the increase of ox-LDL concentration, and the expression of IL-6 and TNF-a downstream of the downstream was also up regulation of.4) in the process of inducing the formation of foam cells. After the treatment of the antibody and NF- kappa B inhibitor, the oil red O staining showed that the cytoplasmic lipid deposition was obviously improved. The quantitative PCR and Western blot results showed that the expression level of LOX-1 and NF- kappa B decreased obviously, and the downstream IL-6 and TNF-a expressed the.5) in the process of inducing the formation of foam cells. The results of quantitative PCR and Western blot showed that the expression level of LOX-1 and NF- kappa B decreased significantly, and the expression of IL-6 and TNF-a in the lower reaches significantly reduced the effect of.2. paeoniflorin on the atherosclerotic model in mice. 1) after the mice were treated with paeoniflorin, the serum triglyceride (TG) and cholesterol (TC) in mice were given. The levels of low density lipoprotein (LDL) and high density lipoprotein (HDL) were decreased. The results of HE and oil red O staining in thoracic aorta tissue showed that the pathological changes and tissue lipid deposition significantly improved.2). The expression level of LOX-1 and NF- kappa B in the thoracic active vein tissue decreased significantly after the treatment of paeoniflorin in the atherosclerotic model of the mice. The expression of IL-6 and TNF-a decreased significantly. Conclusion: 1. paeoniflorin can reduce the accumulation of lipid in the cytoplasm of macrophages by regulating the LOX-1/NF- kappa B signaling pathway and blocking its foaming process.2.ox-LDL by up regulation of the expression of LOX-1 and activating the expression of NF- kappa B to promote the formation of foamy cells, paeoniflorin During this process, the formation of atherosclerotic plaques was inhibited by regulating the expression of LOX-1/NF- - B pathway and inflammatory molecules.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R543.5

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