Objectives: To investigate the effect and mechanism of lipid nanoparticle (LNP) delivery of small interfering RNA (siRNA) targeting Cyp2e1 gene on subacute alcoholic liver injury in mice.
Methods: siRNA targeting Cyp2e1 gene was encapsulated in LNP (si-Cyp2e1 LNP) by microfluidic technique and the resulting LNPs were characterized. The optimal dose of si-Cyp2e1 LNP administration was screened. Forty female C57BL/6N mice were randomly divided into blank control group, model control group, si-Cyp2e1 LNP group, LNP control group and metadoxine group. The subacute alcoholic liver injury mouse model was induced by ethanol feeding for 10 d plus ethanol gavage for the last 3 d. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, and the superoxide dismutase (SOD) activity as well as malondialdehyde, reactive oxygen species, glutathione, triacylglycerol, total cholesterol contents in liver tissue were measured in each group, and liver index was calculated. The expression of genes related to oxidative stress, lipid synthesis and inflammation in each group of mice were measured by realtime RT-PCR.
Results: Compared with the model control group, the levels of liver index, serum ALT, AST activities, malondialdehyde, reactive oxygen species, triacylglycerol, total cholesterol contents in liver tissue decreased, but the SOD activity as well as glutathione increased in the si-Cyp2e1 LNP group (all P<0.01). Hematoxylin-eosin staining result showed disorganized hepatocytes with sparse cytoplasm and a large number of fat vacuoles and necrosis in the model control group, while the si-Cyp2e1 LNP group had uniformly sized and arranged hepatocytes with normal liver tissue morphology and structure. Oil red O staining result showed si-Cyp2e1 LNP group had lower fat content of the liver compared to the model control group (P<0.01), and no fat droplets accumulated. Anti-F4/80 monoclonal antibody fluorescence immunohistochemistry showed that the si-Cyp2e1 LNP group had lower cumulative optical density values compared to the model control group (P<0.01) and no significant inflammatory reaction. Compared with the model control group, the expression of catalytic genes P47phox, P67phox and Gp91phox were reduced (all P<0.01), while the expression of the antioxidant enzyme genes Sod1, Gsh-rd and Gsh-px were increased (all P<0.01). The mRNA expression of the lipid metabolism genes Pgc-1α and Cpt1 were increased (all P<0.01) and the lipid synthesis-related genes Srebp1c, Acc and Fasn were decreased (all P<0.01); the expression of liver inflammation-related genes Tgf-β, Tnf-α and Il-6 were decreased (all P<0.01).
Conclusions: The si-Cyp2e1 LNP may attenuate subacute alcoholic liver injury in mice mainly by reducing reactive oxygen levels, increasing antioxidant activity, blocking oxidative stress pathways and reducing ethanol-induced steatosis and inflammation.
目的: 研究脂质纳米粒（LNP）递送靶向Cyp2e1基因的小干扰RNA（siRNA）减轻小鼠亚急性酒精性肝损伤的作用及机制。方法: 通过微流控技术将靶向Cyp2e1基因的siRNA封装在LNP中得si-Cyp2e1 LNP，并进行表征。筛选si-Cyp2e1 LNP给药的最佳剂量，将40只C57BL/6N雌鼠随机分为空白对照组、模型对照组、si-Cyp2e1 LNP组、LNP对照组和美他多辛组，经10 d的乙醇喂养及最后3 d的乙醇灌胃处理，构建亚急性酒精性肝损伤小鼠模型。测定各组血清中丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）活性，肝组织中超氧化物歧化酶（SOD）活性及丙二醛、活性氧、谷胱甘肽、三酰甘油和总胆固醇含量，计算肝脏指数；光学显微镜观察小鼠肝组织病理学变化。实时逆转录聚合酶链反应检测各组小鼠体内氧化应激、脂质合成和炎症相关基因表达的变化情况。结果: 与模型对照组比较，si-Cyp2e1 LNP组肝脏指数降低，血清ALT、AST活性降低，肝组织丙二醛、活性氧、三酰甘油和总胆固醇含量下降，SOD活性升高，谷胱甘肽含量增加（均P<0.01）。苏木精-伊红染色结果显示模型对照组肝细胞排列紊乱，细胞质稀疏，有大量的脂肪空泡和坏死，而si-Cyp2e1 LNP组肝细胞大小均匀，排列整齐，肝组织形态结构正常；油红О染色结果显示si-Cyp2e1 LNP组相比模型对照组肝脏脂肪含量减少，未见脂肪滴积聚；抗F4/80单克隆抗体荧光免疫组织化学结果显示si-Cyp2e1 LNP组相比模型对照组累积光密度比值降低（P<0.01），无明显炎症反应。与模型对照组比较，si-Cyp2e1 LNP组活性氧形成相关催化基因P47phox、P67phox和Gp91phox的表达减少（均P<0.01），而抗氧化酶相关基因Sod1、Gsh-rd和Gsh-px的表达增加（均P<0.01）；脂质代谢相关基因Pgc-1α、Cpt1的mRNA表达增加（均P<0.01），脂质合成相关基因Srebp1c、Acc和Fasn的mRNA表达减少（均P<0.01）；肝脏炎症相关基因Tgf-β、Tnf-α和Il-6的表达减少（均P<0.01）。结论: si-Cyp2e1 LNP主要通过降低活性氧水平，增加抗氧化活性，阻断氧化应激途径，减少乙醇诱发的脂肪变性和炎症，保护肝脏免受乙醇引起的损伤，为亚急性酒精性肝损伤提供了一种新的靶向治疗方法。.
Keywords: Inflammation; Lipid metabolic synthesis; Lipid nanoparticles; Mice; Oxidative stress; Small interfering RNA; Subacute alcoholic liver injury.