枇杷叶倍半萜调控CYP2E1/JNK通路对高脂膳食诱导NAFLD的干预作用及机制研究

Nonalcoholic fatty liver disease (NAFLD) induced by high fat diet is a serious metabolic disease, but its pathogenesis is still not clear. The present study suggests that insulin resistance (IR) and oxidative stress are important elements in its pathogenesis. Cytochrome P450 2E1 (CYP2E1) is a member of the oxido-reductase cytochrome family, whose high expression can activate downstream JNK signaling to induce insulin resistance and oxidative stress. Our previous study found that unique sesquiterpenes of loquat leaf has therapeutic effect on NAFLD. This study will further isolate 4 sesquiterpene compounds from the total sesquiterpene with biological activity, and apply cell experiment and animal experiment. Combined with methods like immunohistochemistry, quantitative PCR, Western blot and other means, we will screen sesquiterpene with active effect on NAFLD. The key of this study is to investigate the regulation of CYP2E1 and JNK signaling pathway and the mechanism of sesquiterpenes from loquat leaf in treating NAFLD by improving IR and reducing oxidative stress. This study provides a theoretical basis for understanding the pathogenesis of NAFLD and exploring new therapeutic targets, and provides the experimental basis for the application of sesquiterpenes from loquat leaf in treat NAFLD. The study also provides experimental basis for expanding the application range of loquat leaf.

高脂膳食引起的非酒精性脂肪肝病（Non-alcoholic fatty liver disease, NAFLD）是一种危害严重的代谢性疾病，但发病机制尚不明确，研究认为胰岛素抵抗和氧化应激是其发病的重要环节。细胞色素P450 2E1（Cytochrome P450 2E1, CYP2E1），是一种细胞色素氧化还原酶，它的高表达可激活JNK信号引起胰岛素抵抗和氧化应激。课题组前期研究发现枇杷叶独特倍半萜具有治疗NAFLD的作用，本研究将进一步从中分离得到4种倍半萜单体，通过细胞和动物实验，结合免疫组化、定量PCR、免疫印迹等手段，筛选出活性倍半萜，并重点研究此类倍半萜调控CYP2E1及下游JNK通路，改善IR，减轻氧化应激，治疗NAFLD的作用机制。本研究为深入了解NAFLD发病机制、探索新的治疗靶点提供理论支撑，为枇杷叶独特倍半萜应用于NAFLD的治疗、拓展枇杷叶的应用范围提供实验依据。

高脂膳食引起的非酒精性脂肪肝病（Non-alcoholic fatty liver disease, NAFLD）是一种危害严重的代谢性疾病，研究认为胰岛素抵抗和氧化应激是其发病的重要环节。前期研究发现枇杷叶倍半萜具有治疗NAFLD的作用，本项目在前期研究的基础上，利用植物化学的研究方法，对枇杷叶治疗NAFLD的倍半萜类成分进一步进行分离纯化与结构鉴定，富集得到4个倍半萜单体，包括3个新化合物，1个新天然产物。.细胞色素P450 2E1（Cytochrome P450 2E1, CYP2E1），是一种细胞色素氧化还原酶，它的高表达可激活JNK信号引起胰岛素抵抗和氧化应激。本研究采用高脂饮食诱导NAFLD小鼠，研究枇杷叶倍半萜治疗NAFLD是否由CYP2E1介导。在细胞水平，我们通过油酸诱导HEPG2细胞构NAFLD模型，考察枇杷叶倍半萜对NAFLD细胞模型的干预作用，进一步确证CYP2E1的作用。.动物研究发现，枇杷叶总倍半萜可以降低NAFLD模型小鼠血中TC、TG、HDL、LDL含量，降低ALT水平，升高SOD水平，减少MDA含量，达到减少脂质堆积、改善氧化应激及肝损伤的作用；蛋白水平检测表明，枇杷叶总倍半萜通过抑制CYP2E1的高表达，并抑制下游JNK信号分子的磷酸化水平，从而达到改善氧化应激、调节脂质代谢治疗NAFLD的作用。进一步的细胞学实验表明，枇杷叶倍半萜A（化学式：Nerolidol-3-O-α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnpyranosyl-(1→6)]-β-Dglucopyranoside）可以显著降低油酸诱导的NAFLD细胞模型中TC、TG、FFA的含量，增强SOD活性，减少MDA、4-hydroxynonenal的含量，具有良好的调节脂质代谢、抗氧化应激的活性，作用机制是通过抑制CYP2E1的表达，并降低其下游信号分子JNK、c-Jun磷酸化水平，从而改善高脂诱导的细胞模型中脂质堆积和氧化应激，达到治疗NAFLD的作用。.本研究的科学意义在于证实枇杷叶倍半萜具有治疗NAFLD的作用，CYP2E1可能是其一个重要靶点，显示了枇杷叶倍半萜类化学成分具有良好的治疗NAFLD，并作为创新药物研究开发应用前景。