Treatment of hepatic fibrosis in rat treated with DMN by siRNA/HSP47 encapsulated in vitamin A conjugating liposome

迄今为止，还没有一种方法被批准用于人类肝硬化的抗纤维化治疗，主要是由于对特定靶分子或靶细胞的特异性不足。在本研究中，我们首次证实了一种46 kda的胶原结合糖蛋白（gp46）（大鼠热休克蛋白47）的小干扰RNA （siRNA）对正常大鼠肾（NRK）成纤维细胞胶原分泌的实质性抑制。由于已知肝星状细胞（HSC）在纤维形成中起关键作用，通过视黄醇结合蛋白受体（RBPRs）吸收维生素a (VA)，因此我们在不同浓度的FBS存在下，用羧基荧光素（FAM）标记的siRNAgp46包裹在VA偶联脂体（VA-lip- sirnagp46）中，处理原代大鼠HSC和人HSC细胞系（LI90），并证实这些细胞的荧光明显受到抗rbp抗体的抑制。在二甲基亚硝胺（DMN）诱导的肝硬化大鼠体内静脉注射fam标记的VA-lip-siRNAgp46后，也证实了其对HSC的特异性递送，因为荧光主要在肝硬化肝脏的HSC区域观察到，而在肝实质、肺、脾和视网膜中几乎没有观察到。给予^3H-VA-lip-siRNAgp46时，dmn处理大鼠肝脏中主要可见放射性，而正常大鼠肝脏中放射性不明显。VA-lip-siRNAgp46 （iv）治疗DMN大鼠，由于hsc的凋亡，肝硬化几乎完全组织学消退，存活率为100%；相比之下，未经治疗的大鼠无一存活。这种新方法可能不仅适用于肝硬化的治疗，也适用于治疗其他器官的纤维化疾病，如肾硬化和慢性胰腺炎，据报道，星状细胞在这些疾病中起着至关重要的作用。

To date, no modality has been approved for antifibrotic therapy of liver cirrhosis in humans, mainly due to insufficient specificity for particular target molecules or target cells. In the present study, we first confirmed substantial suppression of collagen secretion from normal rat kidney (NRK) fibroblasts by small interfering RNA (siRNA) for a 46-kDa collagen-binding glycoprotein-(gp46)(heat shock protein 47 of rat), a collagen-specific chaperon. Since hepatic stellate cells (HSC), which play a key role in fibrogenesis, are known to take up vitamin A (VA) through retinol-binding protein receptors (RBPRs), we then treated primary rat HSCs and a human HSC cell line (LI90) with carboxy-fluorescein (FAM)-labeled siRNA gp46 encapsulated in VA-coupled liposomes (VA-lip-siRNAgp46) in the presence of various concentrations of FBS, and affirmed that the fluorescence of these cells was appreciably suppressed by anti-RBP antibodies. Specific delivery of FAM-labeled VA-lip-siRNAgp46 to HSCs was also verified following i.v. injection in rats with liver cirrhosis induced by dimethylnitrosamine (DMN), since the fluorescence was mainly observed in HSC areas of the cirrhotic liver but scarcely in the liver parenchyma, lung, spleen or retina. When ^3H-VA-lip-siRNAgp46 was administered, radioactivity was seen mainly in the liver of DMN-treated rats, while in the normal rats, radioactivity in the liver was not prominent. Treatment of DMN rats with VA-lip-siRNAgp46 (i.v.) resulted in nearly complete histological resolution of liver cirrhosis, due to apoptosis of HSCs, and a 100% survival rate; in contrast, no untreated rats survived. This novel approach may be applicable not only to the therapy of liver cirrhosis, but also for treating fibrotic conditions in other organs, such as nephrosclerosis and chronic pancreatitis, in which stellate cells reportedly play an essential role.