基于TLR4通道的白花丹醌抗肝纤维化及活血化瘀作用机制研究

In both studies the results of preliminary research group of the Natural Science Foundation of China show that featured Guangxi folk medicine Plumbagin have exact hepatic fibrosis and blood circulation effect. But it is the role of specific mechanisms and targets remain unclear. So now put forward the hypothesis that "Plumbagin of liver fibrosis and the role of blood circulation, by affecting TLR4/MyD88/NF-κB, MAPKs signaling pathways generated." This topic will be established liver fibrosis model of blood stasis (TCM model), improved CCl4 hepatic fibrosis(Western medicine model) and activated HSC-LX2(cell model) three research model; Selection TLR4/MyD88/NF-κB,MAPKs this inflammation of the liver, hepatic stellate cell proliferation and apoptosis, the level of pro-fibrotic cytokine secretion pathway has a very important influence; observe the path of the most critical level of transcription 6 signaling molecules, and the expression levels of the organization and locating the specific cell. Contents: ① observation of two animal models and cell models that channel interference situation Plumbagin. Deep into the tissue cells, in combination with previous results, accurately describes Plumbagin hepatic fibrosis, targets and molecular mechanisms of blood circulation effect; may promote the development of research work in Guangxi specialty medicine. ②The similarities and differences between the activation of the channel for Chinese medicine and Western medicine treatment of liver fibrosis and provide a new target for ideas when comparing the modeling of traditional Chinese medicine and Western medicine in animal models of liver fibrosis in both groups did not give drug intervention.

前期两项国基金等研究显示，广西特色民族药白花丹醌具有确切的抗肝纤维化及活血化瘀作用。可作用的机制和靶点尚不明了。提出假说“白花丹醌的抗肝纤维化及活血化瘀作用，通过影响TLR4/MyD88/ NF-κB、MAPKs信号通路产生”。本课题拟建立血瘀证肝纤模型、改良CCl4肝纤模型和活化HSC-LX2细胞三组研究模型；研究TLR4/MyD88/NF-κB、MAPKs这两条对肝脏炎症反应、肝星状细胞增殖和凋亡、促纤维化因子分泌有很重要影响的通路；观察通路中最关键的6个信号分子的转录水平、表达水平及在组织和细胞中具体定位。研究内容：①观察白花丹醌对两组动物模型及细胞模型该通道的干预情况。由组织深入到细胞研究，结合前期结果能准确阐述白花丹醌抗肝纤维化、活血化瘀作用的靶点及分子机制；可推进广西特色药开发研究。②比较中西造模法两组肝纤动物未予药物干预时该通道激活状况，为肝纤维化中西医治疗提供新靶点及思路

课题组前期研究结果的总结，已经明确了民族药白花丹醌具有确切的抑制肝星状细胞增殖及功能的抗肝纤维化作用；还进一步观察到其通过抑制肝窦毛细血管化现象，改善肝脏微循环的活血化瘀作用。故提出假说“白花丹醌所表现的抗肝纤维化、活血化瘀作用是通过影响TLR4/MyD88/ NF-κB、MAPKs信号通路而产生的”。课题通过建立血瘀证肝纤维化大鼠模型（中医模型）、改良CCl4动物模型（西医模型）和活化型HSC-LX2（细胞模型）共三组研究模型；选择研究TLR4/MyD88/NF-κB和TLR4/MyD88/MAPKs这两条对肝脏炎症反应、肝星状细胞增殖和凋亡、促纤维化因子分泌水平有非常重要影响的通路；观察通路中最关键的6个信号分子TLR4、MyD88、NF-κB、ERK、JUK和P38-MARK的转录水平、表达水平及在组织中、细胞中表达的具体定位。本项目历时3年，完成了肝纤维化动物模型的制备、检测及鉴定；完成了药物干预后各组动物标本的采集、处理及检测；完成了细胞实验：HSC的分离鉴定及培养，各组细胞与药物孵育，及细胞标本的采集、处理及检测；完成了动物模型和细胞模型的各组数据的结果处理、资料汇总及数据分析；撰写论文14篇（其中SCI文章1篇，中文核心期刊论文5篇）。项目取得成果如下：运用改良的复合因素造模法成功构建出血瘀证肝纤维化大鼠（中医模型）和CCl4联合乙醇肝纤维化大鼠（西医模型），这为肝纤维化药理毒理研究提供了新模型；中西医两组造模动物的TLR4/MyD88/NF-κB、MAPKs信号通路激活状况差异的比较，为肝纤维化的中西医结合临床治疗提供出了有价值的新靶点和新思路（治疗中医血瘀证，西医门脉高压症是否也可以从TLR4/MyD88/NF-κB、MAPKs信号通路入手）；而动物模型、细胞模型两层面研究所得的结果，更深入的、更准确地揭示出白花丹醌的抗肝纤维化作用、活血化瘀作用的具体机制及靶位点；这为开发利用广西特色抗肝纤维化、活血化瘀中药成分白花丹醌，提供出了很有价值的实验依据和理论依据；本项目的研究还为少数民族地区的人才培养起到了较显著的推进作用，为少数民族地区培养和扶植了一批青年科研骨干。