Development and Preclinical Evaluation of Nanoformulations in Liver Fibrotic Mice

项目总结 酗酒和肥胖造成的肝脏损伤会导致肝脏发炎、脂肪变性和纤维化。肝细胞 损伤导致肝细胞和枯否细胞(KCs)释放炎性细胞因子，从而导致 肝星状细胞(HSCs)的激活。如果不解决，它可能会导致肝纤维化和进展到 肝硬变。Hedgehog(HH)信号调节肝纤维化的多条途径，包括上皮细胞到肝细胞 间充质转化(EMT)、HSC活化和炎症。此外，慢性肝病与 与miRNAs的失调有关。具体地说，miR-96在肝脏损伤后上调，并促进脂肪肝。 疾病(FLD)。MIR-96导致胰岛素受体(INSR)和胰岛素受体底物(IRS)-1功能障碍 导致胰岛素信号转导和糖原合成受损。此外，miR-96下调了Smad7和 Foxo1-3，并促进转化生长因子-β1(TGFR-β1)介导的肝纤维化。在我们的预赛中 研究表明，包括ptch1、shh和gl2在内的hh信号配体的上调是导致 5%酒精和高脂饮食(HFD)喂养的小鼠，胶原蛋白和脂肪沉积显著增加。我们 合成了一种新型的HH途径抑制剂2-氯-N_1-[4-氯-3-(2-吡啶基)苯基]-N_4，N_4-双(2- 纳米分子中具有GLI1/2抑制活性的1，4-苯二甲酰胺(MDB5) 集中精神。MDB5对HFD和酒精性肝病(ALD)小鼠均有治疗作用 肝损伤标志物天冬氨酸氨基转移酶(AST)和丙氨酸水平显著降低 转氨酶(ALT)，并进一步去除GLI2及其靶基因。我们在ALD和HFD中的miRNA图谱 鉴定出miR-96的小鼠肝脏持续上调。目标扫描分析显示，miR-96目标 几个抗炎和抗纤维化基因。抑制miR-96在肝细胞中的表达 抗miR-96的HSCs修复了受影响的基因Smad7和FOX03。我们合成了 甘草次酸(GA)偶联GA-PEG-P(Asp)-g-DC-g-TEPA共聚物用于肝脏特异性体内给药 分别为MDB5和抗miR-96。肝纤维化患者血清MDB5浓度显著升高。 全身注射MDB5-GA-NPs后1h的肝脏。转化生长因子-β和HH信号串扰 转化生长因子-β1通过RAS信号的下游产物诱导Gli-1的表达。因此，我们假设 GA-NPs联合应用MDB5和抗miR-96可预防酒精性和脂肪性肝损伤， 和纤维化症。我们的具体目标是：1)确定HH抑制剂MDB5对酒精的治疗效果 和高脂饮食诱导的肝损伤；ii)建立miR-96在HFD和ALD小鼠模型中的促纤维化作用， 和iii)确定携带MDB5和抗miR-96的肝靶向NPs的治疗效果 HFD和ALD小鼠模型。我们的长期目标是了解ALD的进展机制和 非酒精性脂肪肝治疗肝纤维化，并建立新的肝脏特异性治疗方法。

PROJECT SUMMARY Liver injury from alcohol abuse, and obesity can lead to liver inflammation, steatosis, and fibrosis. Liver cell damage results in the release of inflammatory cytokines from hepatocytes and Kupffer cells (KCs), which cause the activation of hepatic stellate cells (HSCs). If unresolved, it may result in liver fibrosis and progression to cirrhosis. Hedgehog (Hh) signaling regulates multiple pathways in liver fibrosis, including epithelial-to- mesenchymal transition (EMT), HSC activation, and inflammation. Further, chronic liver diseases are associated with the dysregulation of miRNAs. Specifically, miR-96 is upregulated after liver damage and promotes fatty liver disease (FLD). miR-96 caused malfunctioning of insulin receptor (INSR) and insulin receptor substrate (IRS)-1 results in impaired insulin signaling and glycogen synthesis. Further, miR-96 downregulates SMAD7 and FOXO1-3 and promotes transforming growth factor-beta 1 (TGF-β1) mediated liver fibrosis. In our preliminary studies, we demonstrated the upregulation of Hh signaling ligands including PTCH1, SHH, and GLI2 cause significant increase in collagen and fat deposition in 5% ethanol and high-fat diet (HFD) fed mouse. We synthesized a novel Hh pathway inhibitor 2-chloro-N 1-[4-chloro-3-(2-pyridinyl) phenyl]-N4, N4-bis(2- pyridinylmethyl)-1,4-benzenedicarboxamide (MDB5) with GLI1/2 inhibitory activity in nanomolar (nM) concentration. Treatment of both HFD and alcohol-induced liver disease (ALD) mice with MDB5 resulted in a significant decrease in the levels of liver injury markers aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and further ablate GLI2, and its target genes. Our miRNA profiling in ALD and HFD mouse liver identified miR-96 was consistently upregulated. Target Scan analysis revealed that miR-96 targets several anti-inflammatory and anti-fibrogenic genes. The knockdown of miR-96 expression in hepatocytes and HSCs with anti-miR-96 resulted in the restoration of affected genes SMAD7 and FOXO3. We synthesized glycyrrhetinic acid (GA) conjugated GA-PEG-P(Asp)-g-DC-g-TEPA copolymers for liver-specific in vivo delivery of MDB5 and anti-miR-96, respectively. There was a significant increase in MDB5 concentration in the fibrotic liver at 1h post systemic administration of MDB5 loaded GA-NPs. TGF-β and Hh signaling crosstalk whereby TGF-β1 induces GLI-1 through downstream consequence of RAS signaling. Therefore, we hypothesize that the combination therapy of MDB5 and anti-miR-96 using GA-NPs could prevent alcohol and fat induced liver injury, and fibrosis. Our specific aims are to i) establish the therapeutic efficacy of the Hh inhibitor MDB5 on alcohol and high fat diet induced liver injury; ii) establish the profibrotic role of miR-96 in in HFD and ALD mouse models, and iii) determine the therapeutic efficacy of liver targeted NPs loaded with MDB5 and anti-miR-96 for treating HFD and ALD mouse models. Our long-term goal is to understand the progressive mechanisms of ALD and NAFLD to liver fibrosis, and establish new, liver-specific treatments.