萝卜硫素的部分降糖机制研究及其脂质体的制备

发布时间：2018-08-30 15:19

【摘要】：糖尿病是内分泌疾病,经长期研究证实跟遗传和环境都有关,严重影响人体健康。合成药物临床治疗糖尿病存在不良反应多等问题,天然药物与其相比具有安全性高,在疾病治疗方便可使用时间长的优点。萝卜硫素(sulforaphane,SFN)是一种从植物中提取的天然单体物质,有研究表明萝卜硫素在缓解2型糖尿病患者的胰岛素抵抗症状方面有良好效果,但是其机制仍有待深入探究。我们的实验证实萝卜硫素能改善IR-Hep G2细胞胰岛素抵抗,其机制可能与抑制Lipin1表达有关。肝脏是Lipin1的主要分布部位,提高药物对肝脏的靶向性也能使药物发挥更好的作用。此外,在实验中我们发现萝卜硫素在高剂量情况下具有一定毒性,限制药物使用剂量,影响了药理作用的发挥。脂质体作为药物载体,具有靶向、减毒,减少药物副作用等特殊优点,因此本研究将萝卜硫素与脂质体这种新剂型相结合,研制了萝卜硫素脂质体,以期提高药物在肝脏中的靶向性,解决药物用量限制,达到增效减毒的目的,将来能更好地服务于临床。本课题主要分为两个部分:1.萝卜硫素改善Hep G2胰岛素抵抗及其机制的研究采用棕榈酸(palmitic acid,PA)建立起Hep G2细胞胰岛素抵抗(insulin resistance,IR)模型,通过检测使用药物前后细胞培养基上清液中葡萄糖消耗量(glucose consumption,GC),细胞内甘油二酯(diacylglycerol,DAG)含量的变化,孕烷X受体(Pregnane X Receptor,PXR)底物蛋白细胞色素P450 3A4酶(cytochrome 3A4,CYP3A4),脂素Lipin1蛋白表达的变化来揭示萝卜硫素对IR-Hep G2细胞的降糖作用可能存在的机制,通过分析葡萄糖消耗量,我们发现模型组与对照组相比数值显著降低,说明IR-Hep G2模型成功建立。跟模型组相比,低、中、高剂量萝卜硫素均能增加细胞培养基上清中葡萄糖的消耗含量,其中高剂量组消耗量最显著。经甘油二酯检测试剂盒结果显示,跟模型组相比,低、中、高剂量萝卜硫素能够使IR-Hep G2细胞内甘油二酯的含量降低。从Western blot实验我们得知,萝卜硫素能够通过抑制PXR受体底物CYP3A4蛋白的表达来抑制蛋白Lipin1的表达。提示萝卜硫素能改善IR-Hep G2细胞胰岛素抵抗,其机制可能与抑制Lipin1表达从而导致甘油二酯含量降低有关。2.萝卜硫素脂质体的制备及其质量评价通过正交设计,采用逆向蒸发法制备萝卜硫素脂质体并对其进行质量评价。使用正交实验方法,通过使用包封率作为评价指标选择用于制备萝卜硫烷脂质体的最合适的制剂配方。研究脂质体的形态,大小,电负性,稳定性和其他性质。选取了合适的组织样品处理方法,建立了萝卜硫素体内分析方法并采用方法学验证。根据实验设计将小鼠分成两组。给药后取组织,处理后对药物体内分析,之后对萝卜硫素脂质体的组织分布性进行初步研究。采用正交实验法筛选出制备萝卜硫素脂质体最合适的制备处方有机相/水相比例为1:3,温度为30℃,水合时间为5min,磷脂胆固醇摩尔比6:1,按该处方得到的脂质体包封率最高可达51.11%,所制得的萝卜硫素脂质体透射电镜下为球形或类似球形单层形状的脂质体,粒径均一,流动性较好。平均粒径为(176.733±5.443)nm,PDI为(0.197±0.032),表明脂质体的分布专一,粒度的大小适中。其平均Zeta电位为(21.13±0.17)mv,显示脂质体很稳定。在4℃条件下,脂质体放置10天后,有少量脂质体出现沉淀现象,一个月以后,脂质体有部分沉淀,包封率也显示有降低趋势,总体脂质体体系较稳定。HPLC法测脂质体在小鼠体内分布研究萝卜硫素脂质体在小鼠体内肝、脾组织部位分布较多。细胞增殖实验证实萝卜硫素脂质体在Hep G2细胞中的毒性低于相同剂量游离萝卜硫素,证实萝卜硫素脂质体这种剂型能使药物减量增效。
[Abstract]:Diabetes mellitus is an endocrine disease. It has been proved by long-term research that it is related to both genetics and environment, and seriously affects human health. Natural monomers extracted from plants have been shown to be effective in alleviating insulin resistance in type 2 diabetes mellitus, but the mechanism remains to be explored. Our experiments have shown that sulforaphane can improve insulin resistance in IR-Hep G2 cells, and the mechanism may be related to the inhibition of Lipin 1 expression in liver. The viscera is the main distribution site of Lipin-1, and improving the drug targeting to the liver can also make the drug play a better role. In addition, we found that sulforaphane has certain toxicity at high doses, limiting the dosage of the drug, affecting the pharmacological effect. Liposomes as drug carriers, with targeting, detoxification and reduction. In order to improve the drug targeting in the liver, solve the drug dosage limitation, achieve the purpose of synergism and detoxification, and serve the clinical better in the future. This topic is mainly divided into two parts: 1. Radish. Improving Insulin Resistance and Its Mechanisms in Hep G2 Cells Using Palmitic Acid (PA) to Establish Insulin Resistance (IR) Model of Hep G2 Cells. Glucose Consumption (GC) and Diacyl Glycerol (DAG) in the Supernatant of Cell Medium before and after the Use of the Drug were detected. Changes in the content of pregnane X Receptor (PXR) substrate protein, cytochrome 3A4 (CYP3A4), and lipoprotein Lipin 1 protein expression were used to reveal the possible mechanism of glucose-lowering effect of sulforaphane on IR-Hep G2 cells. By analyzing glucose consumption, we found that the model group compared with the control group values. Compared with the model group, low, medium and high doses of sulforaphane could increase the glucose consumption in the supernatant of the cell culture medium, especially in the high dosage group. Western blot showed that sulforaphane could inhibit the expression of Lipin-1 by inhibiting the expression of CYP3A4 protein, the substrate of PXR receptor. It suggested that sulforaphane could improve insulin resistance in IR-Hep G2 cells, and the mechanism might be related to the inhibition of Lipin-1 expression leading to the decrease of glycerol-2-ester content. 2. The preparation and quality evaluation of sulforaphane liposomes were carried out by orthogonal design. The sulforaphane liposomes were prepared by reverse evaporation method and evaluated by quality evaluation. The optimum formulation for preparing sulforaphane liposomes was selected by orthogonal experiment using entrapment efficiency as evaluation index. The morphology, size, electronegativity, stability and other properties of the mice were studied. The method of in vivo analysis of sulforaphane was established and validated by methodology. The mice were divided into two groups according to the experimental design. Orthogonal experiment was used to select the optimum preparation formula for sulforaphane liposomes. The ratio of organic phase to water was 1:3, the temperature was 30 C, the hydration time was 5 min, the molar ratio of phospholipid to cholesterol was 6:1, and the encapsulation efficiency of liposomes was up to 51.11%. The average particle size was (176.733 (+5.443) nm and the PDI was (0.197 (+0.032), indicating that the liposomes were well distributed and moderate in size. The average Zeta potential was (21.13 (+0.17) mv, indicating that the liposomes were stable. At 4 (?) C, there were few liposomes after 10 days of storage. After one month, the liposome was partially precipitated and the entrapment efficiency showed a decreasing trend. The overall liposome system was stable. The distribution of sulforaphane liposomes in mice liver and spleen was detected by HPLC. The cytotoxicity of Hep G2 cells was lower than that of free radish sulfur at the same dose, which proved that the dosage form of radish sulfur liposome could reduce the dosage and increase the efficacy of the drug.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R283.6;R285

【参考文献】