运脾化浊法干预血脂异常大鼠microRNA-33调控脂肪细胞因子信号通路的研究

The multi-target and multi-channel mode of TCM prescription is very similar to the microRNA regulation. The latest researches show that microRNA-33 is one of the most critical regulatory factors for dyslipidemia. Adipocytokine signaling pathway，which regulated by microRNAs-33，is the most closely related to Lipid Metabolism. The Activating Spleen Hua Zhuo prescription （ASHZ）could restore the balance of between the microRNA and target gene, regulate the body's imbalance of yin and yang，maintain the dynamic equilibrium of environment in the body to treat dyslipidemia. The object of this research is rat with dyslipidemia，and many technologies such as qRT-PCR，western blot，bioinformatics analysis are used in this research. ASHZ prescription，which has a clear efficacy in clinical practice，are also used. This research will observe ASHZ prescription's impact on adipocytokine signaling pathway，microRNA-33 and target gene in the inheritance of "Blood Zhuo" theory，which come from Emperor's Canon of Medicine，and many years of clinical experience. This research will elucidate the mechanism of TCM prescription treatment of dyslipidemia on the level of post-transcriptional gene. This research will provides an important basis for further study in clinical treatment of dyslipidemia, it will also provide new targets for the development of effective TCM prescription for dyslipidemia.

中药复方的多靶点和多途径的起效模式和microRNA的调控方式极为相似，最新研究表明，microRNA-33是血脂异常最关键的调控因子之一，microRNA-33调控的脂肪细胞因子信号通路与脂质代谢关系最密切。运脾化浊方药可能是通过恢复相关microRNA与靶基因之间的消长平衡，调节机体阴阳失衡，保持机体内环境动态平衡来治疗血脂异常。本课题在继承《内经》"血浊"学说和结合多年临床经验的基础上，以血脂异常大鼠为对象，利用qRT-PCR、western blot、生物信息学分析等技术，用临床有明确疗效的运脾化浊方药- - 化浊调脂颗粒进行干预，观察其对microRNA-33及靶基因调控的脂肪细胞因子信号通路的影响，从基因转录后水平阐明中药复方治疗血脂异常的机制，为临床治疗血脂异常提供重要的研究基础，也为研制治疗血脂异常有效方药提供新的靶标。

本课题在继承《内经》“血浊学说”和名老中医临床经验的基础上，通过高脂饮食建立血脂异常大鼠模型，运用化浊调脂方干预，同时采用普伐他汀钠片作西药对照及血脂康胶囊作中药对照治疗，采集多时点与大鼠（84只雄性Wistar大鼠）血脂代谢相关信息，运用全自动生化分析、HE染色、RT-PCR、Western-blot、miRNA芯片等技术检测。结果：干预8周后发现①对血脂水平的影响：化浊调脂1号方高剂量组、中剂量组、低剂量组、化浊调脂2号方组4组大鼠血清CHOL、LDL-C均明显低于高脂对照组（P=0.003-0.031，0.000-0.027）；化浊调脂1号方低剂量组大鼠血清CHOL明显低于高脂对照组（P=0.017）。②对脂肪细胞因子（ADPN、LP）的影响：化浊调脂1号方高剂量组、中剂量组、化浊调脂2号方组3组大鼠血浆ADPN明显高于高脂对照组（P=0.010-0.048）；各中药给药组大鼠血浆LP、血管内皮炎症因子（IL-6、TNF-α）、血管内皮功能（NO、ICAM-1）、乙酰辅酶A，与高脂对照组比较，差异无统计学意义（P＞0.05）。③组织切片观察：化浊调脂1号方高剂量组：肝细胞索排列较高脂对照组整齐，肝细胞脂肪样变性程度较高脂对照组明显减轻，汇管区可见炎性细胞浸润，点状坏死灶较高脂对照组明显减少；等。④microRNA芯片技术分析：与高脂对照组比较，化浊调脂1号方使34个microRNA表达上调，19个microRNA表达下调；化浊调脂2号方可使72个microRNA表达上调，47个microRNA表达下调。同时运用RT-PCR定量检测了与脂质代谢密切相关的8个差异microRNA（包括rno-miR-122-5p、rno-miR-30b-5p、rno-miR-148b-3p等），并在mirbase, miranda和 targetscan三个数据库进行靶基因预测，筛选得到了PPARG、LDLR、ABCG1等三个靶基因。采用Western blot技术，检测了靶基因相对应蛋白的表达。结论：①运脾化浊法具有明确的调脂作用（降低血清CHOL、LDL-C）。②运脾化浊法是通过调控rno-miR-122-5p、rno-miR-30b-5p、rno-miR-148b-3p等表达上调PPARG和调控LDLR的转录，进而影响脂连素（ADPN）和LDL水平，达到调脂作用。