抑制PKC-α/NADPH氧化酶信号通路激活：补骨脂改善幼龄小鼠脂肪肝的分子机制？

Psoralea corylifolia L. (PC), a Chinese herb, has been widely used to treat yang deficiency of both the spleen and kidney pediatric disease. Previous evidence has demonstrated the hypoglycemia, hypolipidemia and antioxidative stress via PKC-α/NADPH oxidase signaling pathway efficacy of PC. Recent researches have shown that the development of pediatric nonalcoholic fatty liver disease (NAFLD) is related to oxidative stress. Given that oxidative stress is crucial for the contribution to NAFLD, we hypothesize that PC triply improve oxidative stress, inflammation and insulin resistant (IR) through inhibiting PKC-α/NADPH oxidase signaling pathway. To test our hypothesis, we will establish animal models of pediatric NAFLD with PKC-α gene expression up-regulated. Experiments have been designed to examine the levels of blood glucose and lipid profile, the concentration of reactive oxygen species, inflammatory biomarkers and insulin sensitivity. For mechanistic study, we will examine PKC-α/NADPH oxidase expression in liver tissue, as well as subsequent changes in PKC-α/NADPH oxidase downstream nuclear transcription factor kappa-B (NF-κB) and the expression of molecules involved in insulin signaling pathway. Different experimental techniques including real time PCR, immunohistochemistry, Western blot will be used. The successful completion of the proposed research at integrative and molecular levels will illustrate the mechanisms of the triply actions of PC, based on which we will be able to develop a new antioxidative-based treatment for pediatric NAFLD. Additionally, the proposed research will provide foundations for the therapeutic value of PC, a warming the spleen and the kidney yang herb, on pediatric NAFLD and promote its wide use in clinical practice.

补骨脂是温脾补肾壮阳的儿科常用中药。前期研究证实：补骨脂能调节糖脂代谢紊乱、改善氧化应激、抑制PKC-α/NADPH氧化酶信号通路。新近发现，儿童非酒精性脂肪肝（NAFLD）与氧化应激密切相关。本课题以“氧化应激是NAFLD疾病发生发展的核心”为切入点，提出“抑制PKC-α/NADPH氧化酶信号通路激活是补骨脂改善儿童脂肪肝氧化应激、炎症和IR三重功效的共同分子机制”的假说，建立幼龄小鼠脂肪肝动物模型，在检测脂质代谢、ROS含量、炎症标志物和胰岛素敏感性的同时，采用RT-PCR、免疫组化、Western-blot等方法，检测肝脏PKC-α、NADPH氧化酶基因的表达，及核转录因子NF-κB炎症信号和胰岛素PI-3K信号通路分子的活性和表达，探讨补骨脂作为PKC-α抑制剂的可能性，促生一种基于抗氧化应激治疗儿童脂肪肝的新方法；并初步揭示温脾补肾中药在治疗儿童脂肪肝中的重要性，促进其临床应用。

项目背景：非酒精性脂肪性肝病（NAFLD）呈全球化、低龄化流行趋势，是儿童就诊消化专科或肝病专科的首要病因。随着我国儿童肥胖症的流行、城市化进程、中产家庭崛起及“二胎”政策的全面开放，NAFLD已取代慢性病毒性肝炎，成为威胁我国儿童健康的重大问题。然而，目前尚缺乏针对儿童NAFLD的有效治疗药物。补骨脂是温脾补肾壮阳的儿科常用中药。近期研究标明补骨脂具有肝脏保护和抗氧化应激功效。但是，补骨脂对儿童脂肪肝的治疗作用及其机制未见报道。主要研究内容：本项目研究目的是通过建立幼龄小鼠脂肪肝动物模型，探讨补骨脂对儿童脂肪肝的防治作用及机制。研究内容分两部分，即①建立幼龄小鼠脂肪肝动物模型，②检测脂质代谢、ROS 含量、炎性标志物和胰岛素敏感性变化的同时，检测肝脏组织 PKC-α /NADPH 氧化酶信号通路、核转录因子 NF-κB 炎症信号通路和胰岛素 PI-3K 信号通路分子的活性和表达的变化，从抑制PKC-α/NADPH氧化酶信号通路激活探讨补骨脂治疗幼龄小鼠脂肪肝的分子机制。重要结果：①采用高脂饮食单纯子代喂养的方法可成功建立幼龄小鼠NAFLD模型，采用高脂饮食母代子代连续喂养的方法可成功建立幼龄小鼠非酒精性脂肪性肝炎（NASH）模型；②母代高脂暴露联合高卡路里脂肪饮食可加重子代病程进展，且母代高脂暴露是更为关键的因素；③补骨脂可有效改善单纯子代或母代子代连续高脂饮食建立的幼龄小鼠NAFLD/NASH进程；④补骨脂具有抗慢性炎症的功效，作用机制可能涉及抑制NF-κB 炎症信号通路减少炎症因子释放；⑤补骨脂具有减轻肝纤维化的功效，作用机制可能涉及下调PI3K-Akt信号通路抑制细胞增殖；⑥补骨脂具有抗氧化应激的功效，作用机制可能涉及抑制PKC-α /NADPH氧化酶信号通路减少ROS释放；⑦补骨脂的上述功效存在剂量依赖性。科学意义：成功建立拟合儿童脂肪肝的动物模型，为后续研究提供强有力的技术支持。首次将补骨脂用于儿童脂肪肝的研究，阐明其具有抗氧化应激、慢性炎症和胰岛素三重功效，探讨了补骨脂作为PKC-α抑制剂的可能性，促生了一种基于抗氧化应激治疗儿童脂肪肝的新方法，后续还可以其活性成分或以其为组方的儿科常用小复方进行深入研究，也为进一步开发以补骨脂为母核的中药 PKC-α 抑制剂奠定实验基础。初步揭示了温脾补肾中药在治疗儿童脂肪肝中的重要性，促进了补骨脂的临床应用。

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