RGD-SSL-鳖甲活性肽构建及其在Endoglin/TGFβ1/Smad信号平衡中的抗肝纤维化作用

Hepatic fibrosis is the general pathological character of chronic liver diseases, which is the key stage of liver cirrhosis. Carapax trionycis, a kind of animal medicine of Chinese materia medica, has the effect on softening hardness and dissipating stagnation, which contributes to the anti-liver fibrosis effect and associates with TGFβ1/Smad pathway. Endoglin, considered as a regulatory factor, also named as CD105, is one of the members of TGFβ superfamily. However, because there is no enough content of the active ingredient of ‘Oligopeptides’ in Carapax trionycis, it is difficult to targeted deliver the active oligopeptides to the liver tissues. Hence, there is a limitation of the further researches on the mechanism of anti-liver fibrosis effect. Based on the biological characteristics of the integrin arginine-glycine-asparagic acid ligands(RGD) on hepatic stellate cells and Sterically Stabilized Liposomes(SSL), the project will build a liver targeted delivery system which is constructed by the active peptide H-G-R-F-G of Carapax trionycis and RGD modified SSL. The system has dual function of ‘targeted delivery’ and ‘durable active peptide effect’ on liver tissues. This project will discuss whether RGD-SSL-active peptide plays a role on anti-liver fibrosis effect through Endoglin/TGFβ1/Smad signal balance from three aspects of cell, animal and molecular levels, respectively. The project will provide novel ideas and methods in the application of RGD-SSL-characterized liver delivery system on the fibrosis researches, and elucidate the possible anti-liver fibrosis mechanism of the active peptide of Carapax trionycis.

肝纤维化是慢性肝病的共性病理改变，是肝硬化的关键环节。中药鳖甲具有软坚散结的功效，其抗肝纤维化机制与TGFβ1/Smad通路相关, Endoglin作为TGFβ超家族成员，是其调节因子之一。由于鳖甲的活性成分“寡肽”含量较少、难以直达肝组织，限制了对其进一步研究。本项目拟利用HSC表面整合素RGD配基及SSL的特性，构建具有肝靶向、SSL包封能够延长活性肽在局部作用时间的肝纤维化递药系统，合成经RGD修饰、SSL包封的RGD-SSL-鳖甲H-G-R-F-G活性肽。该体系能够实现鳖甲活性肽在肝组织的富集，发挥持久药理作用。在此基础上，从细胞、动物、分子三个层面探讨RGD-SSL-鳖甲活性肽是否通Endoglin/TGFβ1/Smad通路的信号平衡而起抗肝纤维化作用。本项目的完成将为RGD-SSL递药系统在肝纤维化研究的应用提供新思路和新方法，并阐明鳖甲活性肽抗肝纤维化的可能作用机制。

本项目构建并研究RGD-SSL-鳖甲H-G-R-F-G活性肽递药系统的抗肝纤维化作用。采用薄膜分散法制备RGD-SSL-HGRFG，检测其粒径及多分散系数（PDI），并观察其电镜下的形态，结果为：SSL-HGRFG平均粒径在100 nm左右，PDI为0.104；通过活体成像的方法，提示该体系能够实现鳖甲活性肽在肝组织的富集，从而发挥药理作用。体外研究中，采用激光共聚焦以及流式方法考察细胞对递药系统的摄取情况；采用MTT的方法筛选出HGRG作用于大鼠肝星状细胞（HSCs）细胞的药理毒性作用以及HGRFG的最佳给药浓度，获得其最佳给药浓度为1mM和2mM，同时采用实时荧光定量PCR和Western Blot试验方法表明HGRFG对HSC的增殖具有一定的抑制作用，主要抑制TGF-β、α-SAM、Smad2/3、p-smad2/3等相关基因与蛋白的表达。体内实验中，腹腔注射四氯化碳橄榄油溶液（CCl4：Oil=4:6）建立SD大鼠肝纤维化模型，随机分为不同组别，各组大鼠采用尾静脉或肌肉注射方式给药，连续八周，结束后测定血清中AST、ALT含量，提示，该递药系统能够降低AST、ALT含量，采用试剂盒分析检测肝损伤程度，结果表明。该递药系统可以提高肝组织SOD活力、降低MDA以及Hyp含量；肝脏进行HE染色观察药物干预组的肝组织病理变化比模型组有一定的改善；采用实时荧光定量PCR和Western Blot试验方法表明HGRFG对大鼠肝脏中的TGF-β、α-SAM、Endoglin、CTGF、Smad2/3、P-Smad1/5、PDGF等相关基因与蛋白具有一定的抑制作用。结论：本项目成功的构建了RGD-SSL-鳖甲H-G-R-F-G活性肽递药系统，同时检测该系统的稳定性，包封率、分散系数等相关特性，并从体内外两个方面说明该系统具有一定的抗肝纤维化作用。为RGD-SSL递药系统在肝纤维化研究的应用提供新思路和新方法。