基于Notch信号通路及肝脏超微结构的针灸拮抗顺铂化疗小鼠肝损伤的机制研究

Liver injury is one of the most common adverse reactions caused by chemotherapy.Combined our early acupuncture and moxibustion against liver injury of chemotherapy research foundation and the research progress of Notch signaling pathways, around cisplatin chemotherapy induced liver injury, and using DAPT Notch signaling pathways, put forward the “Notch signaling pathways is the key role of acupuncture and moxibustion antagonism DDP chemotherapy induced liver injury in mice ”hypothesis.This research adopts the biochip screening, RT-qPCR method validation DDP chemotherapy genetic variations associated with Notch signal pathway in the liver tissue in mice, using immunohistochemical method, the western blot method validation gene protein expression and distribution rule of difference, and combining the ultrastructural morphological changes in the liver, from the perspective of the microscopic molecular biology and ultrastructural pathology acupuncture and moxibustion antagonism DDP chemotherapy induced liver injury combined effect mechanism of action, for acupuncture and moxibustion for diseases associated with liver injury to provide new experimental basis.

肝损伤是化疗所致较为常见的毒副反应之一。结合前期针灸抗化疗肝损伤的研究基础以及Notch信号通路的研究进展，围绕顺铂化疗所致肝损伤，并运用DAPT阻断Notch信号通路，提出“Notch信号通路是针灸抗DDP化疗所致小鼠肝损伤的关键作用途径”假说。本研究采用生物芯片筛选、RT-qPCR法验证DDP化疗小鼠肝脏组织中Notch信号通路相关差异基因，应用免疫组化法、western blot法验证差异基因的蛋白表达及分布规律，并结合肝脏超微病理形态变化，从微观分子生物学和超微病理学的角度阐释针灸拮抗DDP化疗所致肝损伤综合效应的作用机制，为针灸治疗肝损伤相关疾病提供新的实验依据。

本研究以顺铂（DDP）所致小鼠肝损伤为模型，运用DEGseq从肝脏组织中筛选出Notch信号通路上的7个热点差异基因：Notch1、Hes1、Lfng、Adam17、Numb、Hdac1、Notch2，并运用免疫组化法筛选出4个显著性差异基因：Notch1、Hes1、Lfng、Adam17，运用RT-PCR、Western blot法对肝脏组织中Notch1、Hes1、Lfng、Adam17 mRNA转录和蛋白表达量进一步检测与分析，并运用透射电镜观察肝细胞受损与修复过程的超微结构形态变化； RT-PCR、ELISA法检测肾脏组织中Keap1、Nrf2、HO-1、NQO1含量及mRNA转录情况，运用Western Blot检测肾脏组织中TLR4、IκBα、NF-κB、Keap1、Nrf2、HO-1、NQO1蛋白表达量。 .得出以下结论：.1.针灸可以改善DDP所致小鼠肝损伤。在治疗效果上，针刺与艾灸相比无明显差异。.2.Notch信号通路上显著性差异基因Notch1、Hes1、Adam17、Lfng与针灸改善DDP所致小鼠肝损伤的分子生物学机制联系密切。.3.针灸可“良性上调”肝脏组织中Notch1、Hes1、Adam17、Lfng的mRNA表达水平与蛋白含量，进而抑制Notch信号通路的过度激活，改善肝损伤，而DAPT可通过阻断Notch信号通路拮抗针灸改善DDP化疗所致肝损伤疗效。.4.针灸可以改善DDP所致小鼠肝损伤，主要通过修复肝细胞细胞壁的缺损、减轻核膜皱缩程度、抑制线粒体肿胀、减轻内质网排列结构紊乱达到治疗效果。.5.针灸可以调节肾脏组织中Keap1、Nrf2、HO-1、NQO1含量、蛋白表达量及mRNA转录情况，激活Keap1/Nrf2-HO-1信号通路，提高肾损伤模型小鼠抗氧化应激能力，并抑制TLR4/IκBα/NF-κB信号通路的过度激活，减轻肾脏炎症反应，这可能是针灸改善DDP所致小鼠肾损伤的机制之一。.