基于Nrf2信号通路研究肝糖异方治疗肝源性糖尿病的作用机制及其实脾法的效应特征

Although hepatogenous diabetes (HD) has high incidence rate and poor prognosis in chronic liver disease, its pathological mechanism is still unclear and the effective prevention and treatment means are absent. Based on the previous work that Chinese herbal compound (Gantangyi recipe) have good curative effects on HD and that the Nrf2 signal pathways and oxidative stress (OS) play important roles in HD pathological change, we hypothesize that Gantangyi recipe can relieve oxidative stress damage and insulin resistance of hepatocyte cell (HC), inhibit the inflammatory damage of activated hepatic stellate cells (HSC) to HC by regulating Nrf2 signaling pathways, thus exerts therapeutic effects by promoting the glycogen synthesis and reducing gluconeogenesis. And the spleen-firming herbal therapy shows its main influence by up-regulating the Nrf2 signal pathways. The cirrhotic rat model in vivo and the HC in vitro which characterized by oxidative stress damage are set up and treated with Gantangyi recipe and its component recipe or their pharmacologic serum. In vitro, the siRNA or agonist of Nrf2 was transfected or added to the culture BRL HC as control or culture condition. The oxidative damage, fibrosis, insulin resistance, glycogen synthesis and gluconeogenesis of hepatic tissue were observed. Meanwhile, the changes of signal pathway of Nrf2 and its downstream signal pathways were observed too. The pharmacologic serum of each group was cultured with the activated HSC caused by lipopolysaccharide, then this conditioned medium was added to damage the HC, and the impacts of oxidative damage, glycogen contents, and apoptosis of HC were observed. Then to clarify the key pharmacological mechanisms of Gantangyi recipe on treating HD, to illustrate the effect characteristics of the spleen-firming herbal therapy, to provide a new way of prevention and treatment for HD by traditional Chinese medicine.

肝源性糖尿病（HD）发病广、危害重,但其病理机制尚不明确,缺乏有效防治手段。基于肝糖异方有效改善HD的前期基础,及Nrf2信号通路和氧化应激在HD病理中的研究进展,我们提出“肝糖异方重在调节Nrf2信号通路、改善肝细胞氧化应激损伤，并抑制活化肝星状细胞对肝细胞的炎性损伤，从而改善肝细胞胰岛素抵抗、促进其糖原合成与减少糖异生，发挥防治肝源性糖尿病的效应；其中，实脾中药在上调Nrf2信号通路中发挥主要作用”的科学假说。复制氧化应激损伤大鼠体内外模型,分别以肝糖异方及其拆方干预，体外并以Nrf2的沉默siRNA或激动剂为对照,观察肝组织损伤、纤维化、胰岛素抵抗、糖原合成与糖异生，尤其是Nrf2及其下游效应通路的变化。再观察各组药物血清与活化的星状细胞共培养后的条件培养液对肝细胞损伤、糖原含量、凋亡的影响。从而阐明肝糖异方防治HD的关键药理机制，并明确实脾治法作用特点，为中医药防治HD提供新思路。

肝源性糖尿病是肝硬化的重要并发症之一。氧化应激肝损伤在肝纤维化发生、胰岛素抵抗中均发挥重要作用，其可通过多条信号途径调控肝脏的胰岛素抵抗、糖原合成与糖异生。Nrf2是体内最重要的抗氧化应激通路。肝糖异方立足于“扶正化瘀”基础上加强“实脾”，前期临床随机对照研究发现，肝糖异方可以有效改善肝源性糖尿病患者证候及糖代谢异常疗效。本项目在此基础上，基于Nrf2信号通路，开展体内外实验研究，从而揭示肝糖异方的效应机制。.研究工作进展顺利，基本达到预期目标。课题组已在国内核心期刊发表论文4篇，收录待发表2篇，在SCI期刊源发表2篇论文，培养研究生3名，会议交流4次。.体内研究结果表明：成功建立肝硬化合并糖代谢异常大鼠模型。肝糖异方及其拆方可减轻肝损害、抗肝纤维化、纠正糖代谢异常。各药物组分别不同程度通过改善胰岛素抵抗、改善脂代谢、促进Nrf2信号通路抗氧化蛋白的表达、抑制氧化应激而发挥作用。.体外研究结果表明：在H2O2诱导大鼠原代肝细胞和BRL-3A大鼠肝细胞氧化应激模型中，各药物组可不同程度改善肝损害、氧化损伤，改变氧化应激关键酶及胰岛素抵抗、糖原合成、糖异生等通路。经siRNA Nrf2干扰后，肝糖异方组的代谢通路均有不同程度改变。在以LPS活化肝星状细胞株HSC-T6诱导BRL-3A大鼠肝细胞损伤模型中，扶正化瘀方可减轻LPS诱导的星状细胞活化，从而保护对肝细胞氧化应激损伤。.本研究得出的结论是：肝糖异方可调节Nrf2信号通路、改善肝细胞氧化应激损伤，并抑制活化肝星状细胞对肝细胞的炎性损伤，从而改善肝细胞胰岛素抵抗、促进其糖原合成与减少糖异生，发挥防治肝源性糖尿病的效应。实脾方具有抑制肝脏炎症，改善胰岛素抵抗作用；扶正化瘀方具有显著抗肝纤维化，降低血糖，抗脂质过氧化作用；肝糖异方结合了其二个拆方的作用机制及功效，综合发挥疗效。