TRAIL对活化肝星状细胞的靶向调控作用及肝纤维化干预的研究

Some anti-fibrotic agents include a few cytokines such as interferon (IFN)-α and interferon (IFN)-γ that have been investigated in many animal experimental studies and the reversibility of hepatic fibrosis can be observed. But no approved anti-fibrotic clinical effects have yet been observed. The main reasons are inability to specifically target responsible cells to produce available drug concentration and side-effects on non-target cells. To resolve the serious limitations of IFN-γ in clinic application, we had successfully developed a new targeting drug-carrier (pPB-SSL) utilizing a cyclic peptide (pPB)-affinity for platelet derived growth factor receptor-β to modify sterically stable liposomes (SSL) to deliver interferon (IFN)-γ. IFN-γ encapsulated in pPB-SSL obtained an extended circulation half-life and was selectively delivered to activated hepatic stellate cells (HSC). Additionally, pPB-SSL-IFN-γ further enhanced anti-fibrotic effects and showed a reduction in some contributing to targeted therapies on hepatic fibrosis. Apoptosis is nature's way of eliminating unwanted and damaged cells. There are 3 phases of HSC activation: initiation, perpetuation and resolution. The resolution phase of HSC activation is characterized by apoptosis. Apoptosis can occur by 2 fundamental pathways: the extrinsic pathway (death receptor, DR) and intrinsic pathway (based on intracellular organelle). Many studies had confirmed HSC showed activation-dependent DR expression and the death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) - mediated apoptosis. And DR are not or are only very weakly expressed in whole human liver, normal hepatocytes and quiescent HSC. So TRAIL may potentially induced apoptosis in activated HSC without associated collateral damage to hepatocytes, and been developed as a targeted therapeutic agent in fibrosis. Additional it was found that IFN-γ could induced expression of TRAIL and its DR , and the effects of TRAIL-mediated apoptosis were in IFN-dependent manner. In the present study, we expect to investigate the effects of TRAIL as a targeted agent to activated HSC on hepatic fibrosis in vitro and vivo and the relation between TRAIL/Caspases signaling pathway ,IFN-γ/STAT1 and TGF-β1/Smads signal passway . We also investigate whether the TRAIL liposome by extending the half-time of TRAIL or TRAIL in combination with IFN-γ liposome could increase anti-hepatic fibrotic efficacy. These could be a more significantly novel opportunity for clinical therapy for hepatic fibrosis.

靶向活化肝星状细胞（HSC）抑制其增殖和（或）促其凋亡策略是目前抗肝纤维化研究热点和希望。本课题组已构建了靶向活化HSC上血小板源性生长因子受体-β的环肽pPB连接的包封IFN-γ的长循环脂质体，验证了其增强抗肝纤维化的效能。但构建靶向载药系统工艺复杂。若药物不需要化学连接靶头，本身就是活化HSC的靶向作用分子，必定是更理想药物之选。研究证实肿瘤坏死因子相关凋亡诱导配体（TRAIL）就具有选择性诱导活化HSC的靶向凋亡特性，其杀伤活性是IFN-γ依赖的。因此，本研究拟考察TRAIL促凋亡、抑制胶原和TGF-β1表达，阐明其与IFN-γ/STAT1及TGF-β1/Smads通路关联性；采用小动物活体成像和荧光双标方法，验证其对活化HSC的靶向性；并在动物肝纤维化模型上考察TRAIL的抗肝纤维效能。还为进一步的临床应用，制备能延长半衰期的TRAIL脂质体，提供药代学参数、理论基础和治疗新途径。

我们前期工作提示靶向和长循环策略可作为发挥更满意IFN-γ临床疗效的抗肝纤维治疗方法。本课题在此基础上，继续对肝纤维化和肝癌的靶向治疗药物（蛋白和基因类）深入探索。①已证实肿瘤坏死因子相关凋亡诱导配体（TRAIL）具有选择性诱导活化HSC的靶向凋亡特性，推测TRAIL本身即可作为不需要靶向头基连接的新的抗肝纤维化靶向分子。以活化ＨＳＣ为靶向策略，本课题率先在国内外开展了外源重组TRAIL蛋白相关的治疗肝纤维化的临床前系统研究。证明了TRAIL对HSC上死亡受体4的靶向性。揭示了TRAIL能通过caspase及uPA信号通路获得活化的HSC的凋亡效果，进而达到抗纤维化作用。并率先成功制备了能够靶向PDGFR-β的环肽（pPB）修饰的TRAIL脂质体（pPB-SSL-TRAIL），同时合成PEG修饰的TRAIL（PEG-TRAIL）。通过动物活体成像、免疫荧光法及药代动力学等验证了2种TRAIL新制剂对肝脏、活化HSC的靶向性及长循环性。证明了与TRAIL及无pPB靶的TRAIL脂质体对比，增强的抗肝纤维化效果。筛选出了可能更好的作用于HSC的抗纤维化作用的新靶点-TRAIL。我们研究提供的兼具有靶向性及长循环性pPB-SSL-TRAIL和PEG-TRAIL，也许能更近距离的解决TRAIL这类细胞因子I-II期临床试验中疗效差的问题。因此，除了提供抗肝纤维化治疗的理论基础、药代学参数及治疗的新制剂；研究成果也有极大的临床转化的应用前景。②还率先成功制备了pPB修饰的包封IFN-α的脂质体（pPB-SSL-IFN-α）。证实了pPB-SSL-IFN-α靶向脂质能够表现出增强的抗肝纤维化作用是通过抑制TGF-β1和α-SMA作用。解决了临床上IFN-α非靶向性造成的副作用大和半衰期短进而疗效低的应用限制，提供了原有IFN-α适应症疾病，如肿瘤等，兼具有靶向和长循环性的IFN-α新制剂类型。③还参与构建了靶向肝纤维化的HSP47/gp46siRNA稳定核酸脂质纳米递送系统pPB-SNALP，并分别在体内外进行了靶向性和治疗效果的验证。对于靶向治疗肝纤维化的相关siRNA药物的开发具有指导意义。④首次验证了过表达的miR-128-3p可以使HCC细胞对索拉非尼诱导细胞凋亡增敏，这种效应可通过PTEN/PI3K/Akt信号通路被DJ-1消除。为肝癌的发病机制及靶向治疗提供新思路。