基于自身免疫调控的二至丸对毒性肝损伤模型促进肝细胞再生的机制研究

Toxic liver injury is one of the public health problems that need to be solved urgently at present, Immunological stress - complement activation - Hepatocyte regeneration is one of the hotspots of current research. The Chinese Traditional Medicine for the treatment of toxic liver injury curative effect. A representative formula Erzhi Pills has integrity immune regulation function, promote liver tissue repair, reduce liver damage, but can the complement activation and hepatocyte regeneration is unclear. On the basis of previous studies, Our research team propose innovative the hypothesis of liver regeneration on multiple target mechanism effect of Erzhi Pills through Immunological stress - complement activation - Hepatocyte regeneration. Our research Liver injury model induced by CCl4, to study the regulation of inflammatory cytokines and oxidative stress, and the expression of complement activity and its products. The establishment of immune regulation and complement activation related research; through the liver tissue microcirculation assessment and liver cell proliferation, differentiation, apoptosis and regeneration initiation factor, transcription factor, regeneration effect factor, cyclin dependent kinase, PCNA, liver regeneration signal regulated protein, two to explore the damage effect of multi pill target effect molecular mechanism to promote regeneration of liver cells of liver toxicity, and provide a theoretical basis and experimental support for the clinical application of science, for the interpretation of TCM to provide new ideas and methods for the prevention and treatment of toxic liver injury.

毒性肝损伤是当前亟待解决的公共卫生问题之一，“免疫应激-补体活化-肝再生”途经是当前研究的热点。中医药防治毒性肝损伤疗效确切，二至丸具有整体性的免疫调控作用，可促进肝组织修复，减少肝损伤，但能否使补体活化以及促肝细胞再生目前还不清楚。我们在前期研究的基础上，创新性提出二至丸通过“免疫应激-补体活化-肝再生”途经促肝再生的多靶点作用机制假说，采用CCl4诱导肝损伤模型，研究其对炎症因子和氧化应激反应的调控，以及补体活性及其产物的表达，建立免疫调控与补体活化关联性；通过研究肝组织微循环评估和肝细胞的增殖、分化、凋亡及其相关的再生起始因子、转录激活因子、再生效应因子、细胞周期蛋白依赖性激酶、细胞核增殖抗原、肝再生信号调节蛋白等，探索二至丸对毒性肝损伤促肝细胞再生的多靶点效应分子作用机制，为临床应用提供科学的理论依据和实验支撑，为阐释中医药防治毒性肝损伤的作用机理提供新的思路和方法。

目的：研究二至丸对调控自身免疫应激-补体活化-肝再生的影响，探索二至丸促肝细胞再生的多途径、多靶点分子效应机制。方法：基于CCl4诱导肝损伤体内体外模型和肝缺血再灌注损伤体内体外模型，采用ELISA、免疫荧光、Western blot等实验方法验证其对免疫应激、补体活化、肝再生的影响及作用机制。结果：（1）二至丸可改善CCl4所致肝组织病理损伤、免疫应激和氧化应激，抑制炎性因子的表达，促进Kupffer细胞向M2型极化;（2）二至丸可促肝细胞再生和肝脏补体活化，其肝增殖情况与补体表达水平相一致，尤以造摸36h后效果最佳;（3）网络药理学筛选预测二至丸保肝作用可能涉及的通路包括PI3K-Akt信号通路，MAPK信号通路，RAS信号通路，RAP1信号通路，FOXO信号通路等，并通过实验对MAPK通路进行验证，发现二至丸可促进Erk1/2磷酸化，进一步调控HGF、EGF表达，从而促进肝细胞增殖;（4）二至丸可缓解肝脏缺血再灌注损伤，改善肝脏线粒体功能，促进肝细胞再生。实验验证表明二至丸可促进PI3K和Akt蛋白的磷酸化水平，采用抑制剂LY294002干扰PI3K/Akt的表达后，p-PI3K、p-AKT、Sirt1蛋白表达明显减弱，ac-PGC1蛋白表达明显增加，提示二至丸可能通过影响PI3K/Akt/Sirt1/PGC1α通路发挥肝保护作用。结论：本课题验证了二至丸促进肝细胞增殖的作用可能与“免疫应激-补体活化-肝再生”途经有关，并应用网络药理学和分子生物学技术阐明了二至丸促肝细胞增殖作用的分子机制可能与促进补体活化和调控MAPK、PI3K/Akt通路有关。本研究对揭示中药及其复方调控肝再生的作用机制和防治毒性肝损伤具有重要的现实意义和实用价值，也为二至丸后续开发提供了理论依据和实验支撑，为阐释中医药防治毒性肝损伤的作用机理提供新的思路和方法。