糖尿病状态下CTRP13下调介导的肝窦毛细血管化的机制研究

The nonalcoholic fatty liver disease(NAFLD) was closely associated with hyperglycemia-induced hepatic sinusoid capillarization in diabetes. CTRP13 is a new regulated glucolipid metabolic factors, involved in hepatic steatosis. Our previous experiment found that high glucose induceddown-regulation of CTRP13 in hepatic sinusoidal endothelial cells. However, it remained unclear whether CTRP13 was involved in hepatic sinusoid capillarization. In this study, db/db mice model and cell experiment in vitro were established, genetic recombination, siRNA, electron microscope, RT-PCR, WB, immunohistochemical and fat quantitative techniques with ultrasound were used,respectively.In vivo, the relationship between CTRP13 expression and hepatic sinus blood flow, the characteristics of fenestrae, basement membrane formation and inflammatory factor were investigated. In vitro, the influence of high glucose on CTRP13, PLVAP, AMPK, Rho expression and glucolipid-taken function were determined, and whether high glucose/CTRP13 mediated hepatic sinusoid capillarization is related to CaMKK, AMPK and Rho. In clinical studies, the correlation of CTRP13 and glucolipid metabolism and fatty liver were explored in type 2 diabetes mellitus(T2DM). This study aimed to clarify the molecular mechanisms of CTRP13 mediated hepatic sinusoid capillarization in daibetes, and to provide theoretical basis for the prevention and treatment of NAFLD in T2DM.

糖尿病合并脂肪肝的形成与高糖诱导的肝窦毛细血管化有关。CTRP13是一个新的调节糖脂代谢的因子，参与了肝脏的脂肪变性。我们前期研究发现高糖可诱导肝窦内皮细胞CTRP13下调，然而，它在肝窦毛细血管化中的作用机制不清，国内外未见报道。本研究利用db/db小鼠模型和体外细胞实验，运用基因重组、siRNA、电镜、RT-PCR、WB、免疫组化、脂肪超声定量等技术，通过CTRP13过表达从动物水平研究CTRP13与肝脏肝窦血流、窗孔调节、基底膜形成及炎症因子变化的关系；在细胞水平研究高糖对肝窦内皮细胞CTRP13、PLVAP、AMPK、Rho 表达及摄糖摄脂功能的影响以及高糖/CTRP13介导的肝窦毛细血管化是否与CaMKK、AMPK、Rho途径有关；临床研究糖尿病患者CTRP13与糖脂代谢及脂肪肝的相关性。旨在阐明CTRP13介导的肝窦毛细血管化的分子机制，为糖尿病合并脂肪肝的防治提供理论依据。

CTRP13是新发现的脂质代谢因子，参与糖尿病所致的肝窦功能障碍，但具体机制不清。本研究利用Wistar大鼠和db/db小鼠，运用基因重组、电镜、RT-PCR、WB、免疫组化等技术，动物水平研究CTRP13与肝窦血流、窗孔调节、基底膜形成及炎症因子变化的关系；细胞水平研究高糖对LSECs CTRP13表达的影响及高糖/CTRP13介导的肝窦功能障碍的分子机制；临床水平研究糖尿病患者CTRP13与糖脂代谢及脂肪肝的相关性。结果：1.糖尿病性脂肪肝鼠肝组织中CTRP13表达降低、肝窦基底膜增厚、肝细胞脂肪变性增强，自噬小体数目减少。2.高糖状态下CTRP13、Beclin-1、LC3II/I表达下降, PLVAP、LN、CAV-1表达增加。3.CTRP13过表达减少了高糖诱导的LSECs PLVAP和LN的表达，增加了p-CaMKKβ和p-AMPK的表达；STO-609和Compound C干预后转染组细胞PLVAP、p-mTOR、LN和CAV-1表达增加，p-AMPK、Beclin-1和LC3II/I表达下降。Torin1干预后Beclin-1、LC3II/I表达升高, PLVAP表达降低；自噬抑制剂3MA使Beclin-1、LC3II/I表达降低，PLVAP表达升高。4.T2DM+NAFLD组血清CTRP13含量低于T2DM组和NAFLD组，CTRP13与BMI、FPG、HbA1c、HOMA-IR呈负相关，是CTRP13的独立危险因素。结论：1.高糖通过下调肝脏CTRP13的表达诱发肝窦功能障碍；2.CTRP13过表达激活CaMKKβ/AMPK途径减轻高糖诱导的肝窦毛细血管化，加速肝脏微循环障碍和脂肪肝形成。3.CTRP13过表达激活AMPK/mTOR-自噬信号通路下调PLVAP对肝窦功能发挥保护作用，延缓肝窦毛细血管化；4.CTRP13可能是糖尿病患者脂肪肝发生发展的重要催化因子，是监测脂肪肝发生、疗效评估的有效指标和治疗的潜在靶点。

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