二十二碳六烯酸抑制炎症及内质网应激改善肺血管重塑
发布时间：2018-08-23 13:38
【摘要】：目的:肺高压(PH)是肺血管阻力持续升高,右心室负荷不断增加,进而导致进行性右心衰竭甚至死亡的常见严重并发症。近年来研究认为肺血管炎症及血管细胞内质网应激反应在PH的发生发展中起重要作用。二十二碳六烯酸(DHA)是n-3多不饱和脂肪酸(n-3PUFA)的主要成分,具有调节内质网应激及抗炎等效应。我们前期研究发现DHA能改善低氧性肺动脉高压。本研究拟采用野百合碱(MCT)诱导的大鼠肺高压模型,进一步探讨DHA的防治肺高压作用及其机制。方法:1.实验动物分组:SD (200-250 g)大鼠40只,每组10只,随机分为4组:对照组(生理盐水注射),MCT造模组(腹腔单次注射MCT,4周),MCT造模组+DHA灌胃预防组(腹腔注射MCT当天即给予DHA灌胃,持续4周),MCT造模+DHA治疗组(腹腔注射MCT两周后给予DHA每天灌胃,持续2周)。(1)右心导管法检测各组大鼠肺动脉平均压;沿室间隔分离右心室(RV),左室+室间隔(LV+S)分别称取质量,计算右心室肥厚指数:RV/ (LV+S) ; (2)实时定量PCR检测肺组织细胞因子IL6,TNF-α,MCP-1及IL1β基因表达;(3)免疫组化法检测①肺动脉中膜厚度②肺组织及阻力性肺动脉周围CD3+T细胞及CD68+巨噬细胞计数;(4)免疫印迹法检测肺组织内质网应激相关蛋白表达水平;(5)免疫荧光双染检测内质网应激相关蛋白在肺组织中膜的表达差异。2.组织块贴壁法培养大鼠肺动脉平滑肌细胞(PASMC)并进行免疫荧光细胞鉴定。(1)免疫印迹及细胞免疫荧光检测PASMC内质网应激蛋白表达水平;(2)实时定量PCR检测PASMC的NFATcl-4基因表达水平;(3)免疫印迹检测NFATcl的总蛋白水平及核转移情况;(4)CCK8及BrdU摄入检测PASMC增殖;(5)流式细胞术检测PASMC细胞周期;(6)免疫印迹检测细胞周期相关蛋白表达水平。结果:(1)与对照组大鼠相比,MCT组大鼠4周后平均肺动脉压显著升高(MCT: 48.2±3.1 mmHg vs control: 20.5±1.3 mmHg;p0.05) ; DHA预防及治疗可显著降低MCT诱导的PH大鼠平均肺动脉压(MCT+DHA预防组:25.3±2.1mmHg; MCT+DHA 治疗组:31.5±3.4mmHg;p0.05 vs MCT组)及右心指数(MCT+DHA预防组:0.37±0.02;]MCT+DHA治疗组:0.42±0.01; p0.05vsMCT组);并显著抑制MCT诱导的大鼠肺动脉中膜增厚。(2) DHA预防及治疗可显著减少MCT诱导的大鼠肺组织IL1β,TNFα,IL-6和MCP-1基因表达(p值均0.05);并抑制MCT诱导的大鼠肺组织及肺血管周围CD3+T细胞及CD68+巨噬细胞的聚集。(3) DHA预防及治疗可显著减少MCT诱导的大鼠肺组织内质网应激相关蛋白p-IRE1,PDI和GRP78蛋白表达(p值均0.05);免疫荧光双染表明DHA主要减少肺动脉平滑肌层PDI的表达。(4)在培养的PASMC,PDGF-BB刺激能显著增加内质网应激相关蛋白p-IRE1,PDI和GRP78蛋白表达(p值均0.05)而DHA预孵能抑制PDGF-BB诱导的内质网应激相关蛋白的表达;免疫荧光表明PDGF-BB刺激PASMC后PDI荧光信号增强,而预孵DHA能减弱荧光信号。(5)PDGF-BB 刺激能诱导 PASMC 的 NFATc1,NFATc2 及 NFATc3基因表达增加(p0.05 vs对照组)而对NFATc4无显著影响;预孵DHA能逆转PDGF-BB诱导的NFATc1及NFATc2的基因表达,而对NFATc3无显著影响。(6) PDGF-BB刺激能诱导PASMC的NFATc1总蛋白表达增加,蛋白核转移增多,而预孵DHA及内质网应激抑制剂4-PBA能产生类似的抑制PDGF-BB诱导性NFATc1总蛋白表达增加及活性增强。(7) PDGF-BB刺激PASMC能显著促进细胞周期蛋白cyclin D1表达,抑制G1期相关负性蛋白p21和p27表达,处于细胞周期S期百分比增多,细胞增殖增加;而DHA预孵或NFATc1 siRNA干扰能产生类似的抑制PDGF-BB诱导的cyclinD1表达增加,恢复p21和p27的蛋白水平,将细胞阻滞于G1期,抑制PDGF-BB诱导的PASMC的增殖。结论:(1) DHA抑制MCT诱导的PH的肺组织及肺血管周围炎症反应。(2) DHA抑制PDGF-BB诱导的PASMC内质网应激,减少PASMC增殖,改善肺血管重塑,降低MCT诱导的大鼠PH平均肺动脉压。
[Abstract]:AIM: Pulmonary hypertension (PH) is a common and serious complication of progressive right heart failure (RHF) and death caused by increased pulmonary vascular resistance (PVR) and right ventricular load. Recent studies have shown that pulmonary vascular inflammation and vascular endoplasmic reticulum stress play an important role in the development of PH. Saturated fatty acids (n-3PUFA) are the main components of endoplasmic reticulum (ER) stress and anti-inflammatory effects. Our previous study found that DHA can improve hypoxic pulmonary hypertension. In this study, monocrotaline (MCT)-induced pulmonary hypertension in rats was used to further explore the preventive and therapeutic effects of DHA on pulmonary hypertension and its mechanism. (200-250 g) rats were randomly divided into four groups: control group (saline injection), MCT modeling group (single injection of MCT, 4 weeks), MCT modeling group + DHA gastric lavage prevention group (intraperitoneal injection of MCT on the day of DHA gastric lavage for 4 weeks, continuous 4 weeks), MCT modeling + DHA treatment group (intraperitoneal injection of MCT for two weeks after DHA daily gastric lavage for 2 weeks). The mean pulmonary artery pressure (PAP) was measured by catheterization, the right ventricle (RV) was separated along the interventricular septum, and the left ventricular + septum (LV + S) was weighed and the right ventricular hypertrophy index (RV / LV + S) was calculated. CD3 + T cells and CD68 + macrophages were counted around tissues and pulmonary artery resistance; (4) The expression of ER stress related protein in lung tissues was detected by Western blotting; (5) The expression of ER stress related protein in lung tissues was detected by immunofluorescence double staining. 2. PASMC was cultured by tissue block adherence method and progressed. Immunofluorescent cell identification was performed. (1) Immunoblotting and cytoimmunofluorescence were used to detect the expression of ER stress protein in PASMC; (2) Real-time quantitative PCR was used to detect the expression of NFATcl-4 gene in PASMC; (3) Immunoblotting was used to detect the total protein level and nuclear transfer of NFATcl; (4) CCK8 and BrdU uptake was used to detect the proliferation of PASMC; (5) Flow cytometry was used to detect the expression of PASMC. Results: (1) Compared with the control group, the mean pulmonary artery pressure in MCT group increased significantly after 4 weeks (MCT: 48.2 (+ 3.1) mmHg vs control: 20.5 (+ 1.3) mmHg; p0.05); DHA prevention and treatment could significantly reduce the mean pulmonary artery pressure (MCT + DHA) in MCT-induced PH rats. Group B: 25.3 (+ 2.1 mmHg); MCT + DHA group: 31.5 (+ 3.4 mmHg); P0.05 vs MCT group) and right heart index (MCT + DHA prevention group: 0.37 (+ 0.02); MCT + DHA treatment group: 0.42 (+ 0.01); P0.05 vs MCT group); and significantly inhibited MCT-induced pulmonary artery media thickening in rats. (2) DHA prevention and treatment can significantly reduce MCT-induced pulmonary tissue IL-1 beta, IL-6 and IL-1 and MMC-1. Gene expression (p value was 0.05), and inhibited the accumulation of CD3 + T cells and CD68 + macrophages in lung tissue and perivascular tissues of rats induced by MCT. (3) DHA could significantly reduce the expression of stress-related proteins p-IRE1, PDI and GRP78 (p value was 0.05). Immunofluorescence double staining showed that DHA mainly reduced the expression of lung stress-related proteins p-IRE1, PDI and GRP78 in lung tissue of rats induced by MCT. PDI expression in arterial smooth muscle layer. (4) PDGF-BB stimulation significantly increased the expression of ER stress-related proteins p-IRE1, PDI and GRP78 in cultured PASMC (p 0.05), while DHA pre-incubation inhibited the expression of ER stress-related proteins induced by PDGF-BB. Immunofluorescence showed that PDI fluorescence signal was enhanced after PDGF-BB stimulation of PASMC, while DHA pre-incubation could inhibit the expression of ER stress-related proteins induced by PDGF-BB. (5) PDGF-BB stimulation could induce the expression of NFATc1, NFATc2 and NFATc3 genes in PASMC, but had no significant effect on the expression of NFATc4. Preincubation DHA could reverse the expression of NFATc1 and NFATc2 genes induced by PDGF-BB, but had no significant effect on the expression of NFATc1 protein in PASMC. (6) PDGF-BB stimulation could induce the expression of NFATc1 protein in PASMC. Plus, nuclear transfer increased, while pre-incubation DHA and ER stress inhibitor 4-PBA produced similar inhibition of PDGF-BB-induced total NFATc1 protein expression and activity increase. (7) PDGF-BB stimulated PASMC could significantly promote cyclin D1 expression, inhibit G1-related negative protein p21 and p27 expression, in the S-phase percentage of cell cycle. DHA pre-incubation or NFATc1 siRNA interference could similarly inhibit the increase of cyclin D1 expression induced by PDGF-BB, restore the protein levels of p21 and p27, block the cells in G1 phase and inhibit the proliferation of PASMC induced by PDGF-BB. Inhibiting PDGF-BB-induced endoplasmic reticulum stress, reducing PASMC proliferation, improving pulmonary vascular remodeling, and reducing MCT-induced PH mean pulmonary artery pressure in rats.
【学位授予单位】：江苏大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R544.1
本文编号：2199235
[Abstract]:AIM: Pulmonary hypertension (PH) is a common and serious complication of progressive right heart failure (RHF) and death caused by increased pulmonary vascular resistance (PVR) and right ventricular load. Recent studies have shown that pulmonary vascular inflammation and vascular endoplasmic reticulum stress play an important role in the development of PH. Saturated fatty acids (n-3PUFA) are the main components of endoplasmic reticulum (ER) stress and anti-inflammatory effects. Our previous study found that DHA can improve hypoxic pulmonary hypertension. In this study, monocrotaline (MCT)-induced pulmonary hypertension in rats was used to further explore the preventive and therapeutic effects of DHA on pulmonary hypertension and its mechanism. (200-250 g) rats were randomly divided into four groups: control group (saline injection), MCT modeling group (single injection of MCT, 4 weeks), MCT modeling group + DHA gastric lavage prevention group (intraperitoneal injection of MCT on the day of DHA gastric lavage for 4 weeks, continuous 4 weeks), MCT modeling + DHA treatment group (intraperitoneal injection of MCT for two weeks after DHA daily gastric lavage for 2 weeks). The mean pulmonary artery pressure (PAP) was measured by catheterization, the right ventricle (RV) was separated along the interventricular septum, and the left ventricular + septum (LV + S) was weighed and the right ventricular hypertrophy index (RV / LV + S) was calculated. CD3 + T cells and CD68 + macrophages were counted around tissues and pulmonary artery resistance; (4) The expression of ER stress related protein in lung tissues was detected by Western blotting; (5) The expression of ER stress related protein in lung tissues was detected by immunofluorescence double staining. 2. PASMC was cultured by tissue block adherence method and progressed. Immunofluorescent cell identification was performed. (1) Immunoblotting and cytoimmunofluorescence were used to detect the expression of ER stress protein in PASMC; (2) Real-time quantitative PCR was used to detect the expression of NFATcl-4 gene in PASMC; (3) Immunoblotting was used to detect the total protein level and nuclear transfer of NFATcl; (4) CCK8 and BrdU uptake was used to detect the proliferation of PASMC; (5) Flow cytometry was used to detect the expression of PASMC. Results: (1) Compared with the control group, the mean pulmonary artery pressure in MCT group increased significantly after 4 weeks (MCT: 48.2 (+ 3.1) mmHg vs control: 20.5 (+ 1.3) mmHg; p0.05); DHA prevention and treatment could significantly reduce the mean pulmonary artery pressure (MCT + DHA) in MCT-induced PH rats. Group B: 25.3 (+ 2.1 mmHg); MCT + DHA group: 31.5 (+ 3.4 mmHg); P0.05 vs MCT group) and right heart index (MCT + DHA prevention group: 0.37 (+ 0.02); MCT + DHA treatment group: 0.42 (+ 0.01); P0.05 vs MCT group); and significantly inhibited MCT-induced pulmonary artery media thickening in rats. (2) DHA prevention and treatment can significantly reduce MCT-induced pulmonary tissue IL-1 beta, IL-6 and IL-1 and MMC-1. Gene expression (p value was 0.05), and inhibited the accumulation of CD3 + T cells and CD68 + macrophages in lung tissue and perivascular tissues of rats induced by MCT. (3) DHA could significantly reduce the expression of stress-related proteins p-IRE1, PDI and GRP78 (p value was 0.05). Immunofluorescence double staining showed that DHA mainly reduced the expression of lung stress-related proteins p-IRE1, PDI and GRP78 in lung tissue of rats induced by MCT. PDI expression in arterial smooth muscle layer. (4) PDGF-BB stimulation significantly increased the expression of ER stress-related proteins p-IRE1, PDI and GRP78 in cultured PASMC (p 0.05), while DHA pre-incubation inhibited the expression of ER stress-related proteins induced by PDGF-BB. Immunofluorescence showed that PDI fluorescence signal was enhanced after PDGF-BB stimulation of PASMC, while DHA pre-incubation could inhibit the expression of ER stress-related proteins induced by PDGF-BB. (5) PDGF-BB stimulation could induce the expression of NFATc1, NFATc2 and NFATc3 genes in PASMC, but had no significant effect on the expression of NFATc4. Preincubation DHA could reverse the expression of NFATc1 and NFATc2 genes induced by PDGF-BB, but had no significant effect on the expression of NFATc1 protein in PASMC. (6) PDGF-BB stimulation could induce the expression of NFATc1 protein in PASMC. Plus, nuclear transfer increased, while pre-incubation DHA and ER stress inhibitor 4-PBA produced similar inhibition of PDGF-BB-induced total NFATc1 protein expression and activity increase. (7) PDGF-BB stimulated PASMC could significantly promote cyclin D1 expression, inhibit G1-related negative protein p21 and p27 expression, in the S-phase percentage of cell cycle. DHA pre-incubation or NFATc1 siRNA interference could similarly inhibit the increase of cyclin D1 expression induced by PDGF-BB, restore the protein levels of p21 and p27, block the cells in G1 phase and inhibit the proliferation of PASMC induced by PDGF-BB. Inhibiting PDGF-BB-induced endoplasmic reticulum stress, reducing PASMC proliferation, improving pulmonary vascular remodeling, and reducing MCT-induced PH mean pulmonary artery pressure in rats.
【学位授予单位】：江苏大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R544.1
【参考文献】
相关期刊论文 前2条
1 卢志强;张艳军;庄朋伟;张金保;徐利满;孙凤娇;;肺动脉压的检测方法优化研究[J];中国药理学通报;2015年07期
2 王静;戴爱国;;原代大鼠肺动脉平滑肌细胞的提取和鉴定以及缺氧对其增殖的影响[J];中国呼吸与危重监护杂志;2012年02期
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