聚乙二醇姜黄素衍生物对非酒精性脂肪肝的改善作用及机制的研究

发布时间：2018-07-06 13:57

本文选题：姜黄素衍生物 + 非酒精性脂肪肝　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:非酒精性脂肪与二型糖尿病、肥胖症、胰岛素抵抗等代谢综合症患者最常见的肝病,目前尚无有效治疗NAFLD的药物。姜黄素是姜黄中提取的天然多酚化合物,研究证实其对非酒精性脂肪肝具有明显改善,但是姜黄素水溶性差、生物利用度低限制了其临床应用。低分子PEG修饰后形成的聚乙二醇姜黄素衍生物(Curc-mPEG454),显著提高母本姜黄素生物活性,组织分布更为广泛,前期研究证实其抗炎、抗氧化能力显著高于普通姜黄素。本研究采用高脂饮食喂养C57BL/6J小鼠复制NAFLD小鼠模型,用Curc-mPEG454进行干预,以探讨Curc-mPEG454对高脂饮食诱导的小鼠非酒精性脂肪肝脂肪变性的改善作用及可能的机制,为NAFLD的治疗提供新的思路。方法:1.40只6-8周龄雄性C57BL/6J小鼠饲养在SPF级别屏障中心动物房,适应一周后,随机分为4组,对照组、模型组、Curc-mPEG454低剂量组、Curc-mPEG454高剂量组,每组10只。对照组给予低脂饲料,其余各组均给以高脂饲料喂养,自由摄取水和食物,高脂饮食16周构建NAFLD小鼠模型。造模期间每周测小鼠体质量。对照组和模型组给予腹腔注射生理盐水(0.9%NS 50mg/kg体重)、Curc-mPEG454低剂量组给予姜黄素衍生物50mg/kg/d腹腔注射、Curc-mPEG454高剂量组给予姜黄素衍生物100mg/kg/d腹腔注射,造模期间每周测小鼠体质量,根据小鼠体质量调整Curc-mPEG454注射剂量。2.Curc-mPEG454治疗16周后,禁食12小时后称小鼠体质量、心包采血、取肝脏组织标本。称肝质量,计算肝指数。检测血清中肝功能及血脂水平:谷丙转氨酶(ALT)、谷草转氨酶(AST)、甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)、游离脂肪酸(FFA);肝组织HE染色观察脂肪变程度;荧光定量PCR(Real-Time PCR)检测肝脏组织中参与脂肪生成、脂肪酸摄取、脂肪酸β氧化、TG分泌及胆汁酸代谢的细胞因子。Western Blot及免疫组化检测小鼠肝组织中CD36、PPARγ、CREB的蛋白表达水平。3.所有实验数据均以平均数±标准差表示,组间数据比较采用SPSS20.0软件中单因素方差分析程序进行,当P0.05认为组间差异具有统计学意义。结果:1.Curc-mPEG454对小鼠体质量、肝质量及血清脂质的影响实验结束时,各组小鼠体质量均显著增高(P0.05)。对照组、模型组、Curc-mPEG454低剂量治疗组、Curc-mPEG454高剂量治疗组体质量增加值分别为(9.51±2.45)g、(22.95±5.14)g、(16.26±3.42)g、(17.12±4.56)g。与模型组相比,Curc-mPEG454低剂量(50mg/kg/d)与Curc-mPEG454高剂量(100mg/kg/d)治疗后,显著降低体质量及体质量增加值(P0.05),Curc-mPEG454低剂量治疗组与Curc-mPEG454高剂量组间体质量增加值无统计学意义(P0.05);与模型组相比,Curc-mPEG454治疗组血清TG水平显著降低(P0.05),但治疗组间差异无统计学意义(P0.05)。2.Curc-mPEG454对肝脏脂质沉积的影响肝脏HE及油红O染色可见对照组肝细胞形态正常,排列整齐,无脂肪变性;模型组可见弥漫性肝脂肪变性,肝细胞内大量大小不等的脂肪空泡,未见明显的炎症细胞浸润及纤维化,模型组脂肪变分级处于最高级,主要分布于3-4级;Curc-mPEG454治疗后,肝脏脂肪变性程度较模型组明显减轻,脂肪空泡明显减少,脂肪变分级显著降低,主要分布于1-2级,差异具有统计学意义(P0.05)。Curc-mPEG454治疗后显著降低肝脏TG水平,差异具有统计学意义(P0.05)。3.Curc-mPEG454对脂代谢相关因子m RNA及蛋白表达的影响高脂饮食喂养后,与对照组相比,模型组肝脏脂肪酸转运蛋白CD36及其上游PPARγ的m RNA及蛋白表达均显著升高,差异具有统计学意义(P0.05);Curc-mPEG454治疗后,肝脏CD36及PPARγ水平与模型组相比均显著降低,差异具有统计学意义(P0.05);治疗组间肝脏CD36及PPARγ的表达差异无统计学意义(P0.05)。Curc-mPEG454治疗对参与脂肪酸合成SREBP-1C、ACC1、FAS、SCD-1基因,脂肪酸氧化PPARα、CPT1α基因,TG分泌DGAT2、apo B、MPT基因及胆汁酸合成代谢FXR及CYP7a1基因与模型组相比差异均无统计学意义(P0.05)。4.Curc-mPEG454对肝脏CREB及P-CREB m RNA及蛋白表达的影响与对照组相比,模型组肝脏CREB的m RNA及蛋白水平均显著降低(P0.05),Curc-mPEG454治疗后,与模型组相比,显著上调CREB及磷酸化CREB的表达(P0.05)。结论:1.Curc-mPEG454能够减轻小鼠体质量及肝质量,降低血清及肝脏TG含量,减少肝脏脂质积聚,具有减肥降脂,改善非酒精性脂肪肝的作用。2.Curc-mPEG454通过激活CREB的磷酸化,抑制肝脏PPARγ/CD36信号路,从而减少肝脏脂肪酸摄取和甘油三酯的合成改善肝脏脂质沉积。
[Abstract]:Objective: Nonalcoholic Fat is the most common liver disease in patients with type two diabetes, obesity, insulin resistance and other metabolic syndrome. There is no effective treatment for NAFLD. Curcumin is a natural polyphenol compound extracted from turmeric. Research has proved that it has obvious improvement on nonalcoholic fatty liver, but the water solubility of curcumin is poor and biological. Low degree of use limits its clinical application. The polyethylene glycol curcumin derivative (Curc-mPEG454), formed by low molecular PEG modification, significantly improves the biological activity of the mother curcumin, and the tissue distribution is more extensive. Earlier studies have confirmed that its anti-inflammatory and antioxidant capacity is significantly higher than that of ordinary Jiang Huang. This study used high fat diet to feed C57BL/6J mice to copy N The AFLD mouse model, with Curc-mPEG454 intervention, was used to explore the effect and possible mechanism of Curc-mPEG454 on nonalcoholic fatty liver degeneration induced by high fat diet in mice. Methods: 1.40 6-8 weeks male C57BL/6J mice were fed in the SPF level barrier center animal room for a week, Randomly divided into 4 groups, the control group, the model group, the Curc-mPEG454 low dose group, the Curc-mPEG454 high dose group, 10 in each group. The control group was given low fat diet, the other groups were fed with high fat diet, free intake of water and food, and the high fat diet for 16 weeks to construct the NAFLD mice model. Intraperitoneal injection of normal saline (0.9%NS 50mg/kg body weight), Curc-mPEG454 low dose group given curcumin derivative 50mg/kg/d intraperitoneal injection, Curc-mPEG454 high dose group to give curcumin derivative 100mg/kg/d intraperitoneal injection, test the body mass per week of mice, according to the constitution of mice adjusted Curc-mPEG454 injection dose.2.Curc-mPEG454 treatment 16 After 12 hours of fasting, the body mass of the mice was called the body mass, the pericardium was collected and the liver tissue specimens were collected. The liver quality and the liver index were calculated. The serum liver function and blood lipid levels were measured: ALT, AST, triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), and free of high density lipoprotein cholesterol (HDL-C). Fatty acid (FFA); liver tissue HE staining to observe the degree of adipose degeneration; fluorescence quantitative PCR (Real-Time PCR) detection of lipid formation, fatty acid uptake, fatty acid beta oxidation, TG secretion and bile acid metabolism of cytokine.Western Blot and immunohistochemical detection of CD36, PPAR gamma, CREB protein expression level.3. all.3. all The experimental data were expressed with mean standard deviation, and the data were compared with the single factor variance analysis program in SPSS20.0 software. When P0.05 thought the difference between groups was statistically significant. Results: the effect of 1.Curc-mPEG454 on the body mass, liver quality and serum lipid of mice was increased significantly (P0.05 The body mass of the control group, the model group, the Curc-mPEG454 low dose treatment group and the high dose Curc-mPEG454 treatment group were (9.51 + 2.45) g, (22.95 + 5.14) g, (16.26 + 3.42) g and (17.12 + 4.56) g., compared with the model group, and the body mass and body mass were significantly reduced after Curc-mPEG454 low dose (50mg/kg/d) and Curc-mPEG454 high dose (100mg/kg/d) treatment. The value of added value (P0.05), Curc-mPEG454 low dose treatment group and Curc-mPEG454 high dose group had no statistical significance (P0.05). Compared with the model group, the serum TG level of Curc-mPEG454 treatment group was significantly decreased (P0.05), but there was no significant difference between the treatment group (P0.05).2.Curc-mPEG454 on liver lipid deposition in the liver HE and Oil red O staining showed that the liver cells in the control group were normal, orderly and without fatty degeneration. The model group showed diffuse hepatic steatosis, fat vacuoles with large numbers of different sizes in the liver cells, no obvious inflammatory cell infiltration and fibrosis. The model group was in the highest grade, mainly in grade 3-4; after Curc-mPEG454 treatment, the liver was in the liver. The degree of visceral adipose degeneration was significantly lower than that in the model group, the fat vacuoles were significantly reduced and the fat classification was significantly reduced, mainly in the 1-2 level. The difference was statistically significant (P0.05).Curc-mPEG454 treatment significantly reduced the liver TG level, the difference was statistically significant (P0.05).3.Curc-mPEG454 on lipid metabolism related factors m RNA and protein expression After the high fat diet, the liver fatty acid transporter CD36 and its upstream PPAR gamma m RNA and protein expression were significantly increased in the model group, and the difference was statistically significant (P0.05). After Curc-mPEG454 treatment, the liver CD36 and PPAR gamma levels were significantly lower than those in the model group, and the difference was statistically significant (P0.05). There was no significant difference in the expression of CD36 and PPAR gamma (P0.05) between the treatment groups (P0.05), and there was no significant difference in the difference of.Curc-mPEG454 in fatty acid synthesis SREBP-1C, ACC1, FAS, SCD-1 gene, fatty acid oxidation PPAR alpha, CPT1 alpha gene, TG secretory DGAT2, and bile acid synthesis and metabolism. 05) the effect of.4.Curc-mPEG454 on the expression of CREB and P-CREB m RNA and protein in the liver was significantly lower than that of the control group. The level of M RNA and protein in the liver of the model group decreased significantly (P0.05). After Curc-mPEG454 treatment, the expression of CREB and phosphorylated CREB was significantly up to be compared with the model group. Conclusion: the results can reduce the body mass and liver of mice. Quality, reducing TG content in serum and liver, reducing lipid accumulation in liver, reducing fat and reducing fat, improving non-alcoholic fatty liver function,.2.Curc-mPEG454 can inhibit the liver PPAR gamma /CD36 signaling by activating CREB phosphorylation, thus reducing liver fatty acid intake and triglyceride synthesis to improve liver lipid deposition.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575

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