孔石莼多糖磺化衍生物的降血脂作用及机制研究

The studies have demonstrated that the strong causal relations between the levels of lipid parameters and hyperlipidemic. A decrease of serum high density lipoprotein cholesterol (HDL-cholesterol) and increments of low density lipoprotein cholesterol (LDL-cholesterol), and triglyceride (TG) were considered to be significant risk factors. In our previous study, we investigated antihyperlipidemic activity of polysaccharide (U) extracted from Ulva pertusa, however, the activity was not strong. On this basis, we obtained high sulfate content ulvan derivtive (HU) prepared with sulfur trioxide/N, N-dimethylformamide (SO3-DMF) in formamide. The results proved that the antihyperlipidemic activity of HU was stronger than that of U, especially on decreasing the levels of LDL-c. The elevation of serum cholesterol especially LDL-c levels is by far one of the most relative risk factors that cause atherosclerosis and CHD. The majority of LDL-c are taken up and cleared by hepatic LDLR. LDL-R is a transmembrane receptor located on cell surface, the expression level of which is tightly regulated by sterols or non-sterols both on the transcription and post-transcription level. Upregulation of hepatic LDLR is the most effective method to lower serum LDL-c levels. To study if HU can enhance the transcriptional activity of LDLR promoter, we would determine the expression of LDLR on post-transcriptional level by stabilizing LDLR mRNA in vitro and in vivo. In addition, the activities of HMG-CoA reductase, (Squalene Synthase)SQS and CYP7A1 would be determined. Altogether, evidence from molecular mechanism study of HU to cellular level research in vivo and in vitro would be interesting in researching of new marine drug.

高血脂疾病已经成为威胁人类健康的常见病，降血脂产品的开发和应用是解决这一问题的重要途径。申请人前期研究已明确孔石莼多糖（U）具有降血脂活性，但作用不显著，进一步研究发现U经磺化衍生化、分离纯化得到的磺化衍生物（HU）的降血脂作用明显优于U。本课题在此基础上，基于LDL受体（LDLR）介导的LDL内吞途径是机体清除血液中LDL的主要途径，通过检测动物血液中LDL的变化和肝脏组织中LDL受体mRNA的变化，以及与之密切相关的胆固醇合成关键酶：羟甲基戊二酰辅酶A（HMG-CoA）还原酶，鲨烯合成酶（SQS）和7α-羟化酶（CYP7A1）的mRNA表达状况，从分子水平阐明HU的作用机制，深入理解胆固醇合成关键酶与LDLR之间的关系。本项目的研究对阐明孔石莼多糖磺化衍生物HU的降血脂机制有重要的理论意义，并为降血脂药物的研发及我国孔石莼资源高值化利用提供科学依据。

本课题基于孔石莼硫酸杂聚糖HU能够降低大鼠血液中的血脂水平，且基于活性多糖大多能干扰胆固醇-胆汁酸代谢途径的学说，通过检测大鼠肝脏中LXR、FXR、CYP7A1和PPARγ的mRNA的变化；基于LDL受体介导的LDL内吞途径是机体清除血液中LDL的主要途径，通过检测HepG2细胞中LDL受体的mRNA及蛋白的变化以及通过检测动物血液中LDL的变化和肝脏组织中LDL受体 mRNA的变化，从分子水平阐明HU降血脂的作用机制。.材料与方法：1．HepG2细胞的培养。体外给药，检测U和HU对HepG2细胞中LDL受体的mRNA及蛋白的影响； 2．通过喂食高脂饲料建立高血脂Wistar大鼠模型，检测大鼠肝脏组织中LDL受体mRNA的变化；检测LXR、FXR、CYP7A1和PPARγ的mRNA的变化；以及肝脏中抗氧化活性的变化。.结果发现：1. 在Wistar大鼠动物实验中，实验结束时，高血脂组的大鼠体重明显增加，与空白对照组相比，存在显著性差异(P< 0.05)；其食物摄取量各组别之间不存在显著性差异；降血脂动物实验表明，U和HU都具有良好的降血脂活性，而且，HU表现出更好的降血脂活性。总体来说，HU对TC的影响更显著，尤其是雌性大鼠HU中低剂量组与U组相比，存在显著性差异（P< 0.05），均降低22.7%；2. 在体内抗氧化实验中， 与高血脂组相比，HU中剂量组（250mg/kg）能够显著提高大鼠肝脏中SOD的活力（提高57.3%）；HU低剂量组（125mg/kg）能够显著增加GSH-Px的活力，与高血脂组对比均存在显著性差异（P< 0.01）；3. 在体外细胞培养实验中，与空白对照组相比U和HU明显促进了HepG2 LDLR蛋白表达量。而且在相同剂量下，HU对HepG2 LDLR蛋白表达的促进作用明显高于U；HepG2细胞经不同浓度的U和HU作用后，其LDLR mRNA的含量与空白对照组相比均明显升高。在剂量是0.1 mg/mL时，U对HepG2 LDLR mRNA表达的促进作用明显强于HU。且我们还发现随着剂量的增大，HU对HepG2 LDLR mRNA的促进作用逐渐减弱；4. HU对大鼠肝脏中各靶基因和受体的激活程度不一，对LDL-R HU中剂量组（250mg/kg）表现出最强的激活能力，与高血脂模型组比较，存在显著性差异（P<0.01），HU组对FXR，LXR