二至丸不同时相调控mTOR信号改善损伤后肝细胞再生失常的作用机制研究

Abnormal liver cell regeneration is a common character of pathological change in anaphase of liver disease. It is very important significance to protect liver by improving status of liver cell regeneration. As a foundational prescriptions treated anaphase of liver disease with liver kidney yin insufficiency, the study of Er Zhi Pill (EZP) is fewer in mechanism and temporal superiority. The hypothesis of study was based on a theory that mTOR signal is a point of signal accumulation regulated cell cycle and cell growth. Mechanism of EZP treated ameliorate abnormal and postinjury liver cell regeneration in rats with prophylactic and remedial administration will been evaluated by observing evaluated index of liver cell regeneration and liver function. While relevant index in priming, proliferative and terminational phases will been tested by flow cytometry, RT-PCR, ELISA, immunohistochemistry to indentified effect of EZP and active priciple interfere in liver cell regenerative process. Factors in upriver, privotal and downstream phase of mTOR signal pathway will been analyzed to explore potential target point and material base of EZP used to ameliorate abnormal and postinjury liver cell regeneration. All results are help to provide scientific experimental evidences for EZP regulated subhealth and treated liver disease.

肝细胞再生失常是肝脏疾患后期的共同病理变化特征，改善肝细胞再生状况，对保肝护肝具有十分的重要意义。二至丸是治疗肝脏疾患后期之肝肾阴虚证的常用方，其作用机制和时间优势相关研究甚少。本课题立足于mTOR信号是调节细胞周期进程和细胞生长的一个信号汇聚点，选用损伤后肝细胞再生抑制复合模型，经二至丸治疗性给药后，在不同时相下，观察肝细胞再生评价指标及肝功能变化，从大体上评价二至丸对损伤后肝细胞再生障碍的预防改善作用及时间优势，同时采用BradU掺入、流式细胞术、RT-PCR、ELISA、免疫组织化学等方法，观察肝细胞再生启动、增殖、终止阶段相关指标变化，以明确二至丸及其有效组分对肝细胞再生过程的干预作用，并观察mTOR信号通路中上游物质，核心物质及下游物质的变化，以初步明确二至丸改善损伤后肝细胞再生失常的可能作用靶点及可能物质基础，为二至丸调节亚健康及肝脏疾患的临床治疗提供科学的实验依据。

肝细胞再生失常是肝脏疾患后期的共同病理变化特征，改善肝细胞再生状况，对保肝护肝具有十分的重要意义。二至丸是治疗肝脏疾患后期之肝肾阴虚证的基础方，其作用机制和时间优势相关研究甚少。本课题选用损伤后肝细胞再生抑制复合模型，经二至丸治疗性给药后，发现二至丸不同时相给药具有有效改善损伤后肝细胞再生障碍，促进肝功能恢复的作用，同时二至丸预防性给药和治疗性给药对大鼠损伤后肝细胞再生障碍均具有良好的疗效，且均可显著抑制损伤后肝细胞再生障碍大鼠肝细胞早期及晚期凋亡，并促进肝细胞增殖及降低Caspase-3表达，肝脏组织中TNF-α、IL-1β、IL-6、TGF-β1、Rheb、Bax、P70s6k、p-mTOR、Fas及FasL的表达显著下降，而p-TSC2、p-TSC2/TSC2、Bcl-2、CyclinD的表达则显著升高，提示二至丸有效防治损伤后肝细胞障碍大鼠可能是通过抑制mTOR信号活化，降低细胞凋亡基因的表达水平，促进肝细胞周期转化和肝细胞再生，恢复肝功能，进而修复肝损伤。