几丁寡糖对脂肪酸代谢紊乱的抑制作用及分子机制研究

发布时间：2018-02-01 11:39

  本文关键词： 几丁寡糖 高脂饮食 脂肪酸 脂代谢紊乱 炎症　出处：《西南医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:探讨几丁寡糖(Chitin oligosaccharide,NACOS)对机体脂代谢紊乱的抑制作用及其潜在的分子机制。方法:体外细胞学实验:(1)MTT法筛选用药浓度:分别采用浓度梯度的PA(0、25、50、100和200μM)预先作用24 h,MTT法用酶标仪检测HepG2细胞增殖的OD值,计算细胞存活率,判断最适PA浓度。将HepG2细胞经NACOS(0、50、100μg/ml)预处理12 h后,加入PA处理24 h,MTT法检测细胞存活率,判断最适NACOS浓度。(2)油红O染色法:HepG2细胞经NACOS(0、50、100μg/ml)预处理12 h后,再用PA刺激24 h。倒置显微镜观察HepG2细胞形态学,以此评价NACOS对PA诱导所致的HepG2细胞中脂质沉淀的影响以及NACOSA抑制HepG2细胞活化的最适浓度。(3)实时荧光定量PCR(Quantitative Real-time PCR)检测HepG2细胞经PA刺激不同时间后过氧化物酶体增殖体受体共激活因子-1α(PGC1α)、炎症因子IL-1β、乙酰辅酶A1(ACC1)、细胞色素氧化酶亚单位-5b(Cox5b)、中链酰基辅酶A脱氧酶(Mcad)的转录表达水平,以评价PA对HepG2细胞的最适处理时间;将HepG2细胞经NACOS(100μg/m L)预处理12 h后,加入PA处理24 h,检测过PGC1α、Il-1β、ACC1、Cox5b、Mcad的转录表达水平,以评价NACOS对PA诱导HepG2细胞脂代谢紊乱的抑制作用。(4)免疫印迹法(Western b Lot)检测HepG2细胞经PA不同时间刺激后MAPKs及PI3K/Akt通路中相关蛋白激酶p-p38、ERK1/2、Akt的蛋白磷酸化水平的变化,以评价PA对HepG2细胞的最适处理时间;将HepG2细胞经NACOS(0、50、100μg/ml)预处理12 h后,再用PA刺激0.5 h,检测MAPKs及PI3K/Akt通路中相关蛋白激酶p-p38、p-ERK1/2、p-Akt的蛋白水平的变化,以评价NACOS对PA诱导HepG2细胞脂代谢紊乱的抑制作用。体内实验雄性C57BL/6小鼠随机分为4组(n=5),即正常对照(normal control group,NCD)组、高脂饮食(high fat diet,HFD)组、NACOS组、NACOS+HFD组,造模20周,乙醚麻醉杀死小鼠,提取肝脏组织进行检查,用于制备组织匀浆。主要检测方法如下:(1)考察高脂模型C57BL/6小鼠在给予NACOS后各项生理指标的的变化,如体重、饮水、饮食上的变化。(2)RT-PCR方法检测肝脏组织中过氧化物酶体增殖体受体共激活因子-1α、白介素因子1-β、乙酰辅酶A1、细胞色素氧化酶亚单位-5b、中链酰基辅酶A脱氧酶的转录表达水平的变化。(3)Western blotting方法检测MAPKs及PI3K/Akt通路中相关蛋白激酶p-p38、p-ERK1/2、p-Akt的蛋白水平的变化。结果:体外细胞学实验:(1)MTT细胞活性实验表明,PA在25-200μM范围内对HepG2细胞的活力没有明显的抑制作用,表明PA在该浓度下没有细胞毒性,可在此浓度以下进行体外药效学实验;NACOS 100μg/m L可略微增加HepG2的活力,但没有统计学差异,且NACOS与PA联合进行药物处理时亦没有表现出细胞毒作用。(2)油红O染色实验显示,PA处理后则细胞浆中的红色脂肪颗粒呈显著聚集趋势。与PA单相比,NACOS预处理可明显抑制PA诱导所致的HepG2细胞中脂滴的形成。(3)RT-PCR实验结果表明,HepG2细胞经PA 100μM处理3-24 h后,PGC1α、IL-1β、ACC1、Cox5b、Mcad均表现出不同程度的升高(P0.05或0.01),并在作用6 h后达峰值。(4)Western blotting结果显示,HepG2细胞经PA 100μM刺激后,丝裂原活化蛋白激酶(Mitogen activated protein kinases,MAPKs)及磷脂酰肌醇3-激酶/蛋白激酶B(Phosphoinositide 3-kinase,PI3K;Proteinkinase B,Akt)通路中的p38、ERK1/2、Akt激酶迅速激活,其磷酸化水平在0-1 h内呈时间依赖性增加。其中,p-ERK1/2及p-Akt在0.5 h时达峰值(P0.05,vs对照组),而p-p38则在1 h时达最高水平(P0.01,vs对照组)。体内实验结果表明:(1)与给予基础饲料的正常对照组(NCD)小鼠比较,高脂模型组(HFD)小鼠的平均体重显著增加(P0.01)。相反,当高脂小鼠同时给予NACOS(1 mg/ml)处理时,可显著抑制其体重的增加(P0.05或0.01)。高脂饲料和或NACOS均对小鼠的采食量及饮水量没有明显影响。(2)RT-PCR实验结果表明,HFD组小鼠较NCD组小鼠肝脏组织中PGC1α、ACC1及Mcad显著升高(P0.05或0.01),炎症因子IL-1β亦增加明显(P0.05),但HFD处理对Cox5b的转录表达无显著影响。与HFD组比较,小鼠经NACOS处理后,其肝脏组织中脂代谢调控因子PGC1α、Mcad及ACC1的转录水平均不同程度地得到抑制(P0.05或0.01),炎症因子IL-1β亦显著降低(P0.05或0.01),Cox5b转录表达水平变化不明显。(3)Western blotting结果显示,与NCD组小鼠相比,HFD组小鼠肝脏的p-p38、p-Akt蛋白的表达水平显著增高(P0.05),而p-ERK1/2则无明显差别。与HFD组相比,MACOS可显著抑制高脂饮食喂养所致p-p38及p-Akt的激活(P0.01或0.05)。此外,NACOS亦可降低p-ERK1/2的蛋白表达水平(P0.05)。结论:(1)体外细胞学实验结果表明:NACOS与PA联合进行药物处理时没有表现出细胞毒作用NACOS预处理明显抑制PA诱导所致的HepG2细胞中脂滴的形成;NACOS抑制PA刺激引起的HepG2细胞脂代谢的紊乱及相关炎症因子的过表达;NACOS预处理显著抑制p-p38、p-ERK1/2及p-Akt的表达水平。(2)体内实验结果表明:高脂小鼠同时给予NACOS(1 mg/ml)处理时,可显著抑制小鼠体重的增加,高脂饲料和或NACOS对小鼠的采食量及饮水量没有明显影响;NACOS处理可有效逆转高脂饮食喂养所致的小鼠肝脏脂代谢紊乱;NACOS抑制高脂饮食造成的脂代谢紊乱可能是通过抑制MAPKs通路的激活,下调炎症反应的发生,从而预防脂代谢紊乱的发生。
[Abstract]:Objective: To investigate the effect of chitooligosaccharide (Chitin oligosaccharide, NACOS) inhibitory effects on lipid metabolism and its underlying molecular mechanisms. Methods: in vitro experiments: (1) MTT method for screening drug concentration: the concentration gradient of PA (0,25,50100 and 200 M) pre 24 h OD value by MTT method ELISA detection of HepG2 cell proliferation, cell survival rate was calculated, the optimum PA concentration of HepG2 judgment. Cells were treated with NACOS (0,50100 g/ml) pretreatment of 12 h after treated with PA 24 h, the survival rate of cells was detected by MTT, the optimal concentration of NACOS. (2) by oil red O staining: NACOS HepG2 cells (0,50100 g/ml) pretreatment 12 h after stimulation with PA 24 h. inverted microscope was used to observe the morphology of HepG2 cells, in order to evaluate the optimal concentration of lipid precipitation of NACOS on PA induced HepG2 in NACOSA cells and inhibit the activation of HepG2 cells. (3) real time fluorescence quantitative PCR (Qu Antitative Real-time PCR) detection of HepG2 cells stimulated by PA in different time after peroxisome proliferator receptor coactivator -1 alpha (PGC1 alpha), inflammatory factor beta IL-1, acetyl coenzyme A1 (ACC1), cytochrome oxidase subunit -5b (Cox5b), medium chain acyl coenzyme A oxygenase (Mcad) expression level of transcription, to evaluate PA on HepG2 cells and the optimal treatment time; HepG2 cells were treated with NACOS (100 g/m L) pretreatment of 12 h after treated with PA 24 h, detection of PGC1 alpha, Il-1 beta, ACC1, Cox5b, the expression level of Mcad transcription, to evaluate the inhibitory effect of NACOS on PA induced HepG2 cells lipid metabolism disorder. (4) immunoblotting (Western B Lot) related protein kinase p-p38, HepG2 cells were detected by PA in different time after stimulation of MAPKs and PI3K/Akt in ERK1/2 pathway, changes in protein phosphorylation levels of Akt, PA on HepG2 cells to evaluate the optimal treatment time; HepG2 cells by NACOS ( 0,50100 g/ml) pretreatment 12 h after stimulation with PA 0.5 h, detection of protein kinase MAPKs and PI3K/Akt pathway in p-p38, p-ERK1/2, changes of p-Akt protein level, to evaluate the inhibitory effect of NACOS on PA induced HepG2 cell lipid metabolism in vivo. Male C57BL/6 mice were randomly divided into 4 groups (n=5) normal control (normal, control, group, NCD) group, high fat diet (high fat, diet, HFD) group, NACOS group, NACOS+HFD group, at 20 weeks, the ether killed mice, extracted from liver tissue were examined for tissue homogenate was prepared. The following main detection methods: (1) effects of high fat the model of C57BL/6 mice treated with NACOS changes in various physiological indexes such as body weight, drinking, diet change. (2) factor -1 alpha CO activation of peroxisome proliferation in liver tissue was determined in RT-PCR receptor, interleukin beta factor 1-, acetyl coenzyme A1, cytochrome oxidase Subunit -5b, the expression of transcription chain acyl coenzyme A dehydrogenase. (3) associated protein kinase p-p38, Western MAPKs and PI3K/Akt blotting method to detect the pathway of p-ERK1/2, changes of p-Akt protein level. Results: in vitro experiments: (1) MTT cell activity experiment showed that PA in 25-200 the range of M on the viability of HepG2 cells had no obvious inhibitory effect, PA showed no cytotoxicity in the concentration, the concentration of the following in vitro pharmacodynamic experiment; NACOS 100 g/m L can slightly increase the activity of HepG2, but the difference was not statistically significant, also showed no cytotoxicity and NACOS were combined with PA drug treatment (2). Oil red O staining showed red fat particles in cytoplasm after PA treatment showed a significant accumulation trend. Compared with PA, NACOS pretreatment could significantly inhibit the formation of PA induced by HepG2 cells in lipid droplets. .(3)RT-PCR瀹為獙缁撴灉琛ㄦ槑,HepG2缁嗚優缁廝A 100渭M澶勭悊3-24 h鍚，

本文编号：1481770