miR-322调节血管新生参与肝纤维化的作用研究及中药复方的干预

Our previous work supported by national natural science foundation of China discovered that: (1) Some miRNAs are differentially expression both in liver and renal fibrosis model; (2) Fuzheng Huayu recipe (FZHY) could regulate miRNA expression both in liver and renal fibrosis model; (3) Regulating TGF-β1 signal transduction pathway is the same mechanism of action of FZHY acting against liver and renal fibrosis; (4) Salvianolic acid B and rosmarinic acid may be the common active compounds in FZHY acting against liver fibrosis and renal fibrosis; (5) miR-322 was up-regulated in different liver fibrosis model and FZHY could decrease its expression. Based on the above progress, we have published 4 papers, 3 are included in SCI, and obtained Shanghai Science and Technology Progress Award. Based on the results relating to miR-322, which is involved in angiogenesis, in this study: (1) Rat liver fibrosis model will be established and intervened by miR-322 Agomir or miR-322 Antagomir, the dynamic changes of miR-322, angiogenesis, liver fibrosis will be observed; in vitro, liver sinusoid endothelial cell (LSEC) hypoxia injury model will be established and intervened by miR-322 mimics or inhibitor, the expression of miR-322, hypoxia, tube formation will be investigated; (2) Rat liver fibrosis model and LSEC hypoxia injury model will be established and intervened by FZHY, miR-322 inhibitor will be used as positive control, the expression of miR-322, hypoxia and angiogenesis will be analyzed. This study will verify the hypothesis: (1) miR-322 plays an important role in the development of liver fibrosis through regulating angiogenesis; (2) Regulating miR-322 expression and inhibiting angiogenesis are the mechanism of action of FZHY against liver fibrosis. This study will expand the pathogenesis of liver fibrosis, and provide a new target for the treatment of liver fibrosis.

申请人主持的青年基金发现：1、肝与肾纤维化共同的差异miRNA；2、扶正化瘀方（FZHY）调控的肝与肾纤维化差异miRNA；3、FZHY抗肝与肾纤维化的共同作用机制：调节TGF-β1信号转导；4、丹酚酸B与迷迭香酸可能是FZHY抗肝与肾纤维化的共同物质基础；5、miR-322在多种肝纤维化模型中表达均上调，FZHY可下调其表达。以上结果已发表论文4篇，SCI收录3篇，获上海市科技进步奖1项；基于miR-322前期结果结合其与血管新生的研究进展，本课题拟：1、复制肝纤维化与肝窦内皮细胞缺氧模型，以miR-322模拟物与抑制剂分别使其过表达或低表达，观察血管新生、肝纤维化等变化，明确miR-322与纤维化、血管新生的关系；2、采用上述模型，FZHY干预，以miR-322抑制剂为对照，观察miR-322、血管新生、肝纤维化等变化，明确FZHY抗纤维化的作用机制。本项目为青年项目的拓展与延续。

肝血管新生贯穿肝纤维化病理发生发展之始终，是抗肝纤维化的重要治疗措施。本课题基于血管新生在肝纤维化中的作用及miR-322与血管新生的关系等研究进展，探讨miR-322调节血管新生参与肝纤维化发生发展的作用及其作用机制，以及抗肝纤维化有效中药复方扶正化瘀方及复肝丸调节miR-322表达、抑制肝血管新生的抗肝纤维化作用机制。首先体内复制CCl4染毒小鼠肝纤维化动物模型，以miR-322拮抗剂或激动剂干预，体外采用人肝窦内皮细胞系，以miR-424（小鼠miR-322与人miR-424具有同源性）模拟物或抑制剂转染细胞，分别使其过表达或低表达，观察miR-322对肝纤维化及血管新生的影响，结果表明miR-322参与了肝纤维化的发生发展过程，其作用机制与下调其靶基因Cullin-2表达，使VCBCR复合体稳定性降低， HIF-1α不能被降解，导致HIF-1α表达升高，促进肝脏血管新生相关；继之，体内复制CCl4染毒小鼠肝纤维化模型，扶正化瘀方及复肝丸干预，体外诱导肝窦内皮细胞缺氧模型，扶正化瘀方药物血清干预，观察中药复方抗肝纤维化的作用机制，结果发现扶正化瘀方及复肝丸抗肝纤维化的部分作用机制均与下调miR-322表达，促进HIF-1α降解，抑制肝脏血管新生相关。该工作的完成，为肝纤维化的发病机制增添基础，并为其治疗提供新的有效的靶点；同时本课题研究证实调节miRNAs表达是中药复方防治肝纤维化的重要作用环节，有助于发展中医药防治肝纤维化的新策略与新理论。本课题已发表论文7篇，其中SCI期刊收录4篇，待发表2篇；参加国际国内学术会议交流6次；协助培养博士2名、硕士1名。