内质网应激介导高脂饮食和棕榈酸诱导的血管损伤及非诺贝特的干预

发布时间：2018-06-08 23:12

本文选题：内质网应激 + 非诺贝特　；参考：《安徽医科大学》2015年硕士论文

【摘要】：目的：1.观察高脂饮食(HFD)能否诱导SD大鼠胸主动脉血管环舒缩功能变化及其病理学改变,以及非诺贝特(FF)是否能够保护血管。2.研究棕榈酸(PA)是否减弱正常雌性大鼠内皮依赖性血管舒张反应及诱导小鼠胸主动脉内皮细胞(MAEC)产生内质网应激(ERS),以及非诺贝特酸(FA)的保护作用是否与缓解内质网应激有关。方法：1.体内实验部分：依据随机分配原则,将雌性大鼠随机分为正常饮食组(SCD)、高脂饮食组(HFD)和HFD加非诺贝特治疗组(HFD+FF),以5个月高脂饮食建立SD大鼠高脂血症模型,非诺贝特灌胃治疗2个月。每周称量大鼠体重,测定血清生化指标TG、TC、HDL-C、LDL-C,气相色谱法(GC)检测FFA、PA含量,硝酸还原酶法检测内皮功能紊乱指示剂NO生成变化；HE染色研究胸主动脉病理学改变,Weigert染色检测胸主动脉弹力纤维变化,油红O染色检测脂质沉积；RT-PCR分析胸主动脉中CHOP及GRP78基因表达的改变；Western blot法检测内质网应激标志物CHOP、GRP78的蛋白表达以及ERK、ERK磷酸化,JNK、JNK磷酸化和eNOS和eNOS的磷酸化水平变化；血管张力实验观察梯度浓度乙酰胆碱(ACh)对血管环的作用变化。2.体外实验部分：以正常雌性SD大鼠血管环为研究对象,不同浓度棕榈酸和非诺贝特孵育,观察棕榈酸和非诺贝特对乙酰胆碱(ACh)诱导的血管舒张反应的影响。将体外培养的MAEC分为正常对照组(CON),4-PBA组(PBA),棕榈酸组(PA),4-PBA+棕榈酸组(PA),棕榈酸+非诺贝特酸干预组(FA+PA),衣霉素阳性对照组(TM), MTT法研究棕榈酸组和非诺贝特酸组细胞活力变化；硝酸还原酶法检测非诺贝特对棕榈酸作用后培养液NO生成的变化；RT-PCR分析各组CHOP, GRP78, IRE1α和XBP1-s基因水平变化；Western blot法检测内质网应激标志物CHOP、GRP78的蛋白表达以及ERK、ERK磷酸化,JNK、JNK磷酸化和eNOS和eNOS的磷酸化变化。结果：1.体内实验部分：5个月HFD饮食后,与对照组比较①HFD组大鼠体重明显高于SCD组；②HFD组大鼠血清TC、TG、LDL-C显著增高,HDL-C显著降低；③HFD组大鼠血清FFA、PA水平显著高于正常组；④HFD组血清NO水平显著高于对照组；⑤HE及Weigert染色显示：HFD组胸主动脉管壁增厚,弹力纤维排列紊乱,局部有断裂现象；⑥油红O染色提示HFD组血管脂质生成显著增加；⑦HFD组胸主动脉组织CHOP和GRP78基因表达显著增加；⑧CHOP、 GRP78、ERK磷酸化及JNK磷酸化表达明显高于SCD组,eNOS磷酸化表达显著降低；⑨HFD组胸主动脉内皮依赖性血管舒张反应显著降低；⑩非诺贝特灌胃治疗2个月,与HFD组相比,显著降低大鼠体重,逆转血脂水平改变,明显改善胸主动脉病理学变化,CHOP和GRP78基因表达显著降低,各蛋白表达CHOP、GRP78、p-ERK、 p-JNK明显降低,p-eNOS显著增加；舒张反应明显改善。2.体外实验部分：与CON组相比,①不同浓度PA作用正常雌性SD大鼠血管环3 h后,内皮依赖性血管舒张变化明显减弱,以0.2 mmol/L PA减弱最为明显；内质网应激抑制剂4-PBA或FA预处理后,血管环舒张反应明显改善；TM孵育血管环90 min后,舒张反应显著降低②不同浓度PA对MAEC活力明显减弱,以0.5mmol/L PA作用24 h最明显；③PA处理显著减少细胞上清液NO的水平,FA孵育后逆转PA作用下NO水平变化；④PA显著诱导CHOP、GRP78、IRE1α、XBP1-s基因表达;⑤CHOP、GRP78、p-ERK、p-JNK表达显著增加,p-eNOS表达显著降低；⑥内质网应激抑制剂4-PBA或FA预处理后,各基因表达显著降低,CHOP、GRP78、p-ERK、p-JNK蛋白表达明显降低,p-eNOS显著增加；⑦内质网应激阳性对照物TM组CHOP、GRP78基因和蛋白水平均明显升高。结论：1.5个月的高脂饮食可以成功构建雌性大鼠高脂血症模型且减弱胸主动脉内皮依赖的血管舒张反应,非诺贝特可改善其变化,其机制可能与缓解内质网应激,增加eNOS的表达和磷酸化有关。2.棕榈酸可抑制MAEC增殖,引起内质网应激,非诺贝特(酸)可能是通过促进细胞增殖、抑制内质网应激、改善内皮依赖性血管舒张反应来保护内皮细胞的。
[Abstract]:Objective: 1. to observe whether the high fat diet (HFD) can induce the changes in the vasoconstrictor function of the thoracic aorta of SD rats and its pathological changes, and whether FF can protect the blood vessel.2. to study whether palmitic acid (PA) attenuated the endothelium dependent vasodilatation of normal female rats and induced the production of MAEC in the thoracic aorta of mice. Endoplasmic reticulum stress (ERS) and the protective effect of fenofibrate (FA) were associated with endoplasmic reticulum stress. Methods: 1. in vivo, the female rats were randomly divided into normal diet group (SCD), high fat diet group (HFD) and HFD plus fenofibrate (HFD+FF), and SD rats were established by high fat diet for 5 months. Hyperlipidemia model, fenofibrate gavage for 2 months. Weighing rats weekly, serum biochemical indexes TG, TC, HDL-C, LDL-C, gas chromatography (GC) were used to detect FFA, PA content, and nitric acid reductase assay for endothelial dysfunction indicator NO formation change; HE staining for pathological changes in thoracic aorta, Weigert staining for thoracic aorta Changes in elastic fiber, oil red O staining and lipid deposition; RT-PCR analysis of CHOP and GRP78 gene expression in thoracic aorta; Western blot method for detecting endoplasmic reticulum stress markers CHOP, GRP78 protein expression, ERK, ERK phosphorylation, JNK, JNK phosphorylation and phosphorylation level; vascular tension test observed gradient concentration. The effect of degree acetylcholine (ACh) on vascular ring changes in vitro.2. experimental part: the effects of palmitic acid and fenofibrate on the vasodilatation induced by acetylcholine (ACh) were observed in normal female SD rats. The effects of palmitic acid and fenofibrate on the vasodilatation induced by acetylcholine (ACh) were observed. The MAEC in vitro was divided into normal control group (CON), 4 Group -PBA (PBA), palmitic acid group (PA), 4-PBA+ palmitic acid group (PA), palmitic acid + fenofibric acid intervention group (FA+PA), mycophencin positive control group (TM), MTT method to study the change of cell vitality in palmitic acid group and fenofibrate group; nitrate reductase method was used to detect the change of NO generation after the action of palmitic acid on fenofibric acid; RT-PCR analysis of CHOP in each group. GRP78, IRE1 alpha and XBP1-s gene level changes; Western blot assay was used to detect endoplasmic reticulum stress markers CHOP, GRP78 protein expression, ERK phosphorylation, JNK, JNK phosphorylation, eNOS and phosphorylation. Results: 1. after 5 months of diet, the body weight of the rats was significantly higher than that of the control group. (2) the serum TC, TG, LDL-C and HDL-C significantly decreased in the HFD group, and the level of FFA and PA in the HFD group was significantly higher than that in the normal group; (4) the serum NO level of the group HFD was significantly higher than that of the control group. The expression of CHOP and GRP78 in thoracic aorta was significantly increased in group HFD, and the expression of CHOP, GRP78, ERK phosphorylation and JNK phosphorylation was significantly higher than that of group SCD, and the expression of phosphorylation of eNOS was significantly lower in HFD, and the vascular endothelial dependent blood Guan Shuzhang reaction in thoracic aorta decreased significantly in HFD group; After 2 months of treatment, compared with group HFD, the body weight, the change of blood lipid level, the pathological changes of the thoracic aorta, the expression of CHOP and GRP78 genes were significantly reduced, the expressions of CHOP, GRP78, p-ERK, p-JNK were significantly decreased and p-eNOS increased significantly, and the diastolic reaction obviously improved the experimental part of.2. in vitro: compared with the CON group, (1) no After 3 h of normal female SD rats with the same concentration of PA, endothelium dependent vasodilatation decreased obviously, and the most obvious decrease was 0.2 mmol/L PA. After endoplasmic reticulum stress inhibitor 4-PBA or FA preconditioning, the vasodilatation reaction of vascular ring was obviously improved; after TM incubated with 90 min, the diastolic reaction was significantly reduced by PA to MAEC alive at different concentrations. The strength was obviously weakened and the 0.5mmol/L PA was the most obvious effect of 24 h; (3) PA treatment significantly reduced the level of NO in the cell supernatant, and FA was incubated to reverse the change of NO level under the action of PA; (4) PA significantly induced CHOP, GRP78, IRE1 alpha, gene expression; After pretreatment with 4-PBA or FA, the expression of each gene was significantly reduced. The expression of CHOP, GRP78, p-ERK, p-JNK protein was significantly decreased, and p-eNOS was significantly increased; and the level of CHOP, GRP78 gene and protein in TM group of endoplasmic reticulum stress positive control was significantly increased. Conclusion: high fat diet in 1.5 months could successfully construct hyperlipidemia model and weaken in female rats. The endothelium-dependent vasodilatation response of the thoracic aorta, fenofibrate can improve its changes, its mechanism may be associated with alleviating endoplasmic reticulum stress, increasing the expression of eNOS and phosphorylation of.2. palmitic acid to inhibit MAEC proliferation, causing endoplasmic reticulum stress, fenofibrate (acid) may be by promoting cell proliferation, inhibiting endoplasmic reticulum stress and improving endothelium dependence. The vasodilatation response is used to protect endothelial cells.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R589

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