左卡尼汀改善子痫前期样小鼠妊娠结局的实验研究：循环及蜕膜单核巨噬细胞的变化及其潜在机制

发布时间：2018-05-11 23:07

本文选题：子痫前期 + 左卡尼汀　；参考：《天津医科大学》2017年博士论文

【摘要】：研究目的:子痫前期(Preeclampsia,PE)不仅是导致孕产妇死亡和围产儿死亡的主要原因,而且患者与其子代远期心血管疾病(Cardiovascular Disease,CVD)发病率明显增加。近年来研究显示循环单核细胞亚群及蜕膜巨噬细胞(Decidual Macrophage,DMs)M2表型偏移参与妊娠期间免疫抑制和螺旋动脉重塑,是成功妊娠的关键。左卡尼汀(L-Carnitine,LC)抗炎、抗氧化,应用于许多CVD中,同时还可以促进体外胚胎发育并提高体外受精成功率、上调组织PPAR-γ的表达。因此本研究通过观察LC对PE样小鼠妊娠结局及M2型DMs的影响,探索LC在PE中的应用前景及潜在机制,并探讨pStat6/Stat6-PPAR-γ信号通路在LC中介的干预效应中的作用。内容与方法:(1)7-8周龄雌性和雄性C57BL/6J小鼠适应性喂养一周后,无创监测鼠尾血压,随机将血压稳定的雌鼠分为8组,分别为对照组(A组)、PE组[妊娠第7天(Gestation Day 7,GD7)皮下注射L-NAME,B组]、PE+LC高剂量干预组(800mg/kg/d、C至E组)和PE+LC低剂量干预组(400mg/kg/d、F至H组)。C/F组、D/G、E/H组分别于孕前1周、GD0、GD9开始给予LC灌胃直至GD18结束妊娠。监测各组血压、24小时尿蛋白;高频小动物超声评价各组GD18血管功能;观察子代宫内生长发育状况以及胎盘/肾脏结构改变;分析A至E组外周血单核细胞Ly6Chi亚群、蜕膜巨噬细胞表型偏移的变化;检测A至E组血浆氧化三甲胺(Trimethylamine Oxide,TMAO)浓度;监测B组和C组动脉粥样硬化程度;检测A至E组粪便16S rDNA分析菌群分布情况。(2)L-NAME、LC以及GW9662(PPAR-γ阻滞剂)共同干预RAW 264.7细胞,观察iNOS、IL-1β、TNF-α、CD206、ARG1、IL-10和PPAR-γm RNA表达水平以及PPAR-γ和Stat6信号通路相关蛋白pStat6/Stat6表达水平,探索LC对小鼠巨噬细胞表型偏移的影响及其机制。结果:(1)L-NAME可以引起C57BL/6J小鼠PE样表现,如高血压、蛋白尿、宫内生长受限和血管功能受损等,同时还可以诱导外周血Ly6Chi单核细胞亚群增加,蜕膜巨噬细胞M2表型偏移减少。Ly6Chi亚群与血压、蛋白尿呈正相关;与胎鼠重、顶臀长和胎盘直径呈负相关(r=0.6363,r=0.6752,r=-0.6694,r=-0.6623,r=-0.6623,all P0.001);蜕膜组织巨噬细胞M2表型与血压、蛋白尿呈负相关,与胎鼠重、顶臀长和胎盘重呈正相关(r=-0.5214,r=-0.6562,r=0.4882,r=0.5701,r=0.5701,all P0.05)。(2)不同时间点给予不同剂量LC可以不同程度降低PE样小鼠血压、24小时尿蛋白,改善血管内皮功能,改善子代宫内生长受限,降低脐动脉和子宫-胎盘动脉的搏动指数(Plusatility Index,PI)和阻力指数(Resistance Index,RI);LC改善围产期结局的程度随着LC剂量的增加而增加,差异有统计学意义(all P0.05)。(3)流式细胞术分析结果显示与A组相比,B组外周血单核细胞Ly6Chi亚群升高(61.7±8.66%vs.72.2±6.02%),蜕膜组织巨噬细胞M2表型表达减少(65.4±9.66 vs.53.7±4.08),差异有统计学意义(all P0.01)。孕前1周给予高剂量LC后Ly6Chi亚群比例下调(72.2±6.02%vs.61.25±3.21%),蜕膜巨噬细胞M2表型表达增加(53.7±4.08 vs.65.8±6.14),差异有统计学意义(all P0.01)。B组蜕膜组织M1表型标志物(TNF-α、IL-1β、iNOS)基因相对表达量较对照组增加,差异有统计学意义(1.00 vs.1.41±0.15;1.00 vs.5.35±1.14;1.00 vs.81.3±16.3;all P0.001);孕前1周、GD0给予高剂量LC干预后TNF-α、IL-1β、iNOS基因表达量均下降,差异有统计学意义(1.42±0.15 vs.0.34±0.14;5.35±1.14 vs.3.64±1.13;81.3±16.3 vs.8.84±1.41;all P0.001;1.42±0.15 vs.0.41±0.11;5.35±1.14vs.4.39±0.89;81.3±16.3 vs.12.32±1.04;all P0.05);GD9给予高剂量LC干预后iNOS基因表达量下降,差异有统计学意义(81.3±16.3 vs.20.2±3.37;P0.001)。B组蜕膜组织M2表型标志物CD206基因相对表达量较A组下调,差异有统计学意义(1.00 vs.0.60±0.17;P0.001);IL-10、ARG1基因表达量与对照组差异无统计学意义(1.00 vs.1.46±0.31;1.00 vs.1.04±0.15;P0.05);孕前1周给予高剂量LC干预后CD206、IL-10基因表达量上升,差异有统计学意义(0.60±0.17vs.1.00±0.20;1.46±0.31 vs.2.57±0.66;all P0.001);GD0给予高剂量LC干预后,IL-10基因表达量上升,差异有统计学意义(1.46±0.31 vs.1.77±0.48;P0.05)。(4)蜕膜组织M2型巨噬细胞与孕鼠GD18血压、24小时蛋白尿呈负相关(r=-0.5196,P0.001;r=-0.5132,all P0.001);与胎鼠重、顶臀长呈正相关(r=0.5408,r=0.5779,all P0.001)。(5)围产期LC总量与蜕膜M2型巨噬细胞呈正相关(r=0.59,P0.001);与蜕膜组织TNF-α、IL-1β、iNOS基因表达量呈负相关(r=-0.80,r=-0.50,r=-0.96,all P0.001);与蜕膜组织CD206、IL-10基因表达量呈正相关(r=0.47,r=0.53,all P0.001)。(6)L-NAME可刺激RAW264.7细胞系M1巨噬细胞表型标志物(TNF-α、IL-1β、iNOS)基因表达量增加,差异有统计学意义(1.00 vs.1.56±0.19;1.00 vs.1.82±0.42;1.00 vs.3.90±0.80;all P0.05);联合LC干预后TNF-α表达量下降,差异有统计学意义(1.56±0.19 vs.0.81±0.16;P0.01),IL-1β、iNOS表达量有下降趋势,但差异无统计学意义(1.82±0.42 vs.1.58±0.42;2.67±0.80 vs.3.90±0.80;all P0.05),M2巨噬细胞表型标志物CD206、IL-10和ARG1基因表达量以及PPAR-γ基因表达量增加,差异有统计学意义(1.50±0.70 vs.6.43±1.36;3.45±0.65 vs.5.51±1.84;1.11±0.20 vs.2.21±0.53;1.02±0.27 vs.1.47±0.28;all P0.05);同时给予L-NAME、LC和GW9662(PPAR-γ阻滞剂)干预后M1、M2巨噬细胞表型标志物和PPAR-γ基因表达量与单纯L-NAME刺激组相似,差异无统计学意义(all P0.05);L-NAME+LC组PPAR-γ和Stat6信号通路相关蛋白pStat6/Stat6表达水平较对照组增加,差异有统计学意义(all P0.05);加用GW9662后上述蛋白表达量下降,与对照组差异无统计学意义(all P0.05)。(7)A至E组血浆TMAO浓度差异无统计学意义;B组与C组相比主动脉瓣、主动脉及其主要分支动脉粥样硬化差异无统计学意义(all P0.05)。A至E组小鼠粪便16S rDNA高通量测序结果显示各组厚壁菌门和拟杆菌门占主要门类分布,组间差异无统计学意义,与血浆TMAO、围产期服用LC总量无相关性;属水平组间梭状芽胞杆菌、甲型变形菌纲、梭菌属XlVa、厌氧细杆菌属、棍状厌氧菌、欧陆森氏菌属和八叠球菌属差异有统计学意义,其中欧陆森氏菌属与围产期口服LC总量呈负相关(r=-0.7989,P0.01)。结论:本研究结果显示孕鼠GD7皮下注射L-NAME可以引起PE样表现,是比较合理的PE样模型。LC可能通过pStat6/Stat6-PPAR-γ信号通路介导蜕膜巨噬细胞M2表型偏移,改善PE样小鼠围产期妊娠结局;PE样小鼠孕前1周给予口服LC直至GD18结束妊娠(800 mg/kg/d)对动脉粥样硬化的发生发展无明显影响,提示LC可能是防治PE的一个新的方法。
[Abstract]:Objective: Preeclampsia (PE) is not only the main cause of maternal mortality and perinatal death, but also the incidence of Cardiovascular Disease (CVD) in the patients and their progeny is significantly increased. In recent years, the study showed the circulating mononuclear subgroup and the M2 form of the decidual macrophage (Decidual Macrophage, DMs). Type migration involved in immunosuppression and spiral artery remodeling during pregnancy is the key to successful pregnancy. L-Carnitine (LC), anti-inflammatory and antioxidant, is used in many CVD, and can also promote the development of in vitro embryos and improve the success rate of in vitro fertilization and up regulation of the expression of tissue PPAR- gamma. Therefore, this study was conducted by observing LC to PE like mice. The effect of pregnancy outcome and M2 type DMs, explore the potential application and potential mechanism of LC in PE, and explore the role of pStat6/Stat6-PPAR- gamma signaling pathway in the intervention effect of LC mediator. (1) 7-8 weeks old female and male C57BL/6J mice are fed for one week after adaptive feeding, and the blood pressure of the rat tail is not monitored, and the female rats with stable blood pressure are randomly divided. In the 8 groups, the control group (group A), group PE [seventh days of pregnancy (Gestation Day 7, GD7) subcutaneous injection of L-NAME, B group], PE+LC high dose group (800mg/kg/d, C to E group) and PE+LC low dose intervention group, respectively at 1 weeks before pregnancy. Hourly urine protein; high frequency small animals to evaluate GD18 vascular function in each group; observe the development of intrauterine and placental / renal structural changes in the offspring; analyze the Ly6Chi subgroup in the peripheral blood mononuclear cells from A to E group, the change of the phenotypic migration of decidual macrophages; detect the concentration of Trimethylamine Oxide (TMAO) in the plasma of A to E, and monitor B. The degree of atherosclerosis in group and C group and the distribution of 16S rDNA in the stool of A to E. (2) L-NAME, LC and GW9662 (PPAR- gamma blocker) were used to interfere with RAW 264.7 cells. The effect of cable LC on the phenotypic migration of mouse macrophages and its mechanism. Results: (1) L-NAME can cause PE like performance in C57BL/6J mice, such as hypertension, proteinuria, intrauterine growth restriction and vascular dysfunction, and can also induce the increase of Ly6Chi monocyte subgroup in peripheral blood, and the M2 phenotypic migration of decidual macrophages to decrease the.Ly6Chi subgroup and blood. Positive correlation between pressure and proteinuria; negative correlation with fetal weight, top hip length and placental diameter (r=0.6363, r=0.6752, r=-0.6694, r=-0.6623, r=-0.6623, all P0.001). The M2 phenotype of decidual macrophages was negatively correlated with blood pressure and proteinuria, and was positively correlated with fetal weight, top hip length and placental weight (r=-0.5214, r=-0.6562, r=0.4882, r=0.5701, r=0.5701). 0.05) (2) different doses of LC at different time points can reduce blood pressure in PE like mice to varying degrees, 24 hours of urine protein, improve vascular endothelial function, improve intrauterine growth restriction, and reduce the pulsation index (Plusatility Index, PI) and resistance index (Resistance Index, RI) of the umbilical artery and uterine placental artery (Resistance Index, RI); LC improves the perinatal outcome. With the increase of LC dose, the difference was statistically significant (all P0.05). (3) flow cytometry analysis showed that compared with the A group, the Ly6Chi subgroup of peripheral blood mononuclear cells in the B group increased (61.7 + 8.66%vs.72.2 + 6.02%), and the M2 phenotype expression in decidual macrophages decreased (65.4 + 9.66 vs.53.7 + 4.08), and the difference was statistically significant (all P0.. 01) the proportion of Ly6Chi subgroup decreased (72.2 + 6.02%vs.61.25 + 3.21%) and M2 phenotype expression of decidual macrophages increased (53.7 + 4.08 vs.65.8 + 6.14) after 1 weeks of pregnancy. The difference was statistically significant (all P0.01) and the relative expression of M1 phenotypic markers (TNF- a, IL-1 beta, iNOS) was increased in.B group Decidua Tissue (TNF- a, IL-1 beta, iNOS), and the difference was statistically significant Significance (1 vs.1.41 + 0.15; 1 vs.5.35 + 1.14; 1 vs.81.3 + 16.3; all P0.001); TNF- a, IL-1 beta, iNOS gene expression decreased in 1 weeks before pregnancy, and the difference was statistically significant (1.42 + 0.15 vs.0.34 + 0.14, 5.35 + 1.14 vs.3.64 + 1.13, 5.35 + 1.14) 1.14vs.4.39 + 0.89, 81.3 + 16.3 vs.12.32 + 1.04, all P0.05), iNOS gene expression decreased after GD9 was given to high dose LC, and the difference was statistically significant (81.3 + 16.3 vs.20.2 + 3.37; P0.001).B group M2 phenotype marker. 0, there was no significant difference between the ARG1 gene expression and the control group (1 vs.1.46 + 0.31; 1 vs.1.04 + 0.15; P0.05); the prognosis of IL-10 gene expression was higher in the 1 weeks before pregnancy, and the expression of IL-10 gene increased, and the difference was statistically significant (0.60 + 0.17vs.1.00 + 0.20; 1.46 + 0.31 vs.2.57 + 0.66; all P0.001). The difference was statistically significant (1.46 + 0.31 vs.1.77 + 0.48; P0.05). (4) the M2 type macrophages in the decidua tissue were negatively correlated with the blood pressure of GD18 in pregnant rats and 24 hours of proteinuria (r=-0.5196, P0.001; r=-0.5132, all P0.001); and the weight of the fetal rat, the top hip length Cheng Zhengxiang (r=0.5408, r=0.5779, all). (5) the perinatal total and decidua giant macrophages Positive correlation (r=0.59, P0.001), and negative correlation with TNF- alpha, IL-1 beta and iNOS gene expression in decidua (r=-0.80, r=-0.50, r=-0.96, all P0.001), and a positive correlation with the CD206 of decidual tissue. (6) it stimulates the phenotype marker of macrophage The difference was statistically significant (1 vs.1.56 + 0.19; 1 vs.1.82 + 0.42; 1 vs.3.90 + 0.80; all P0.05); TNF- alpha expression decreased after combined LC intervention, and the difference was statistically significant (1.56 + 0.19 vs.0.81 + 0.16; P0.01), IL-1 beta and iNOS expression decreased, but the difference was not statistically significant (1.82 + 0.42 vs.1.58 + 0.42; 2.; 2.) 67 + 0.80 vs.3.90 + 0.80; all P0.05), M2 macrophage phenotypic markers CD206, IL-10 and ARG1 gene expression and PPAR- gamma gene expression increased, the difference was statistically significant (1.50 + 0.70 vs.6.43 + 1.36; 3.45 + 0.65 vs.5.51 + 1.84; 1.11 + 0.20 vs.2.21 + 0.53; 1.02 + 0.27 vs.1.47) PAR- gamma blocker) after M1, the phenotype markers of M2 macrophages and the expression of PPAR- gamma gene were similar to those of the L-NAME stimulus group, and there was no statistical difference (all P0.05). The pStat6/Stat6 expression level of the PPAR- gamma and Stat6 signaling pathway related proteins in L-NAME+LC group was higher than that in the control group, and the difference was statistically significant (all). There was no significant difference between the expression of the protein and the control group (all P0.05). (7) there was no significant difference in plasma TMAO concentration in group A to E; there was no significant difference between the aortic valve and the aorta and the main branches of the aorta and the main branches of the B group (all P0.05), and the high throughput sequencing of the.A to E group of the.A to E group showed that the high throughput sequencing of the fecal 16S There was no significant difference in the distribution of the main phylum and bacteriobacteria. There was no significant difference between the group and the plasma TMAO and the total amount of LC in perinatal period. The difference between the group of Clostridium Clostridium, a Proteus, Clostridium, XlVa, anaerobes, anaerobes, oontococcus and eight The total amount of LC was negatively correlated with the total perinatal oral administration (r=-0.7989, P0.01). Conclusion: the results of this study showed that GD7 subcutaneous injection of L-NAME in pregnant rats can cause PE like performance. It is a more reasonable PE like model.LC may mediate the migration of the M2 phenotype of the decidua macrophage by pStat6/Stat6-PPAR- gamma pathway and improve the perinatal pregnancy induced pregnancy in PE like mice. The outcome of pregnancy; PE like mice given LC 1 weeks before pregnancy until the end of GD18 (800 mg/kg/d) has no significant influence on the development of atherosclerosis, suggesting that LC may be a new method for the prevention and control of PE.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R714.244

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