Liu Y, Cheng F, Luo Y X, Hu P, Ren H, Peng M L
Key Laboratory of Molecular Biology for Infectious Diseases, Institute for Viral Hepatitis, Department of Infectious Diseases, Chinese Ministry of Education, the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China.
Zhonghua Gan Zang Bing Za Zhi. 2017 Apr 20;25(4):285-290. doi: 10.3760/cma.j.issn.1007-3418.2017.04.010.
To clarify the role of cytochrome P450 in nonalcoholic fatty liver disease (NAFLD) by RNA-Seq and bioinformatics analysis. A total of 20 male C57BL/6 mice were used. Ten mice were fed with high-fat diet (D12492, 60% kcal fat) for 16 weeks to establish a mouse model of NAFLD, and the other 10 mice were fed with low-fat diet (D12450B, 10% kcal fat) as control group. At the end of the experiment, the body weight, liver weight, and hepatic triglyceride (TG) content were measured. Meanwhile, HE staining and RNA-Seq analysis were performed for the liver tissues. The differentially expressed genes were screened out and subjected to bioinformatics analysis, including KEGG and GO BP enrichment analyses and interaction network analysis. Comparison of means between the two groups was made using t-test. Compared with the control group, the mice in the model group were obviously obese, with significantly increased body weight (41.41 ± 6.01 g vs 28.78 ± 1.79 g, = 6.04, < 0.01) and liver weight (1.38 ± 0.30 g vs 1.08 ± 0.10 g, = 2.89, < 0.01). The mice in the model group showed obvious steatosis, accompanied by a small amount of inflammatory cell infiltration, but with no obvious fibrosis, according to the results of HE staining. In addition, the hepatic TG content in the model group was significantly increased compared with that in the control group (0.64 ± 0.01 mg/mg vs 0.29 ± 0.06 mg/mg, = 10.11, = 0.04). Compared with the control group, a total of 367 differentially expressed genes, including 211 down-regulated and 156 up-regulated ones, were identified in the model group according to the RNA-seq results. Meanwhile, 19 CYP450 subtypes, accounting for 5% of the differentially expressed genes, were identified, and CYP2E1, CYP2C70, CYP3A11, CYP3A25, CYP2D26, CYP4A10, CYP17A1, CYP2B10, and CYP2C38 were involved in oxidative stress, steroid hormone metabolism, fatty acid metabolism, arachidonic acid metabolism, and the PPAR signaling pathway. An interaction network was constructed with 30 nodes, and CYP2E1 and CYP2C70 were identified as key nodes. RT-PCR validation results showed that the expression changes of CYP450 subtypes and lipid metabolism-related genes were consistent with the findings of sequencing. The CYP450 family plays a vital role in the pathogenesis of fatty liver by regulating lipid metabolism-related pathways, including oxidative stress, arachidonic acid metabolism, steroid hormone metabolism , and fatty acid metabolism.
通过RNA测序和生物信息学分析阐明细胞色素P450在非酒精性脂肪性肝病（NAFLD）中的作用。总共使用了20只雄性C57BL/6小鼠。10只小鼠喂食高脂饮食（D12492，60%千卡脂肪）16周以建立NAFLD小鼠模型，另外10只小鼠喂食低脂饮食（D12450B，10%千卡脂肪）作为对照组。实验结束时，测量体重、肝脏重量和肝甘油三酯（TG）含量。同时，对肝脏组织进行苏木精-伊红（HE）染色和RNA测序分析。筛选出差异表达基因并进行生物信息学分析，包括京都基因与基因组百科全书（KEGG）和基因本体生物学过程（GO BP）富集分析以及相互作用网络分析。两组间均值比较采用t检验。与对照组相比，模型组小鼠明显肥胖，体重显著增加（41.41±6.01克对28.78±1.79克，t = 6.04，P < 0.01），肝脏重量增加（1.38±0.30克对1.08±0.10克，t = 2.89，P < 0.01）。根据HE染色结果显示，模型组小鼠出现明显脂肪变性，伴有少量炎性细胞浸润，但无明显纤维化。此外，模型组肝TG含量与对照组相比显著增加（0.64±0.01毫克/毫克对0.29±0.06毫克/毫克，t = 10.11，P = 0.04）。根据RNA测序结果，与对照组相比，模型组共鉴定出367个差异表达基因，包括211个下调基因和156个上调基因。同时，鉴定出19种细胞色素P450（CYP450）亚型，占差异表达基因的5%，其中CYP2E1、CYP2C70、CYP3A11、CYP3A25、CYP2D26、CYP4A10、CYP17A1、CYP2B10和CYP2C38参与氧化应激、类固醇激素代谢、脂肪酸代谢、花生四烯酸代谢和过氧化物酶体增殖物激活受体（PPAR）信号通路。构建了一个包含30个节点的相互作用网络，CYP2E1和CYP2C70被确定为关键节点。逆转录-聚合酶链反应（RT-PCR）验证结果表明，CYP450亚型和脂质代谢相关基因的表达变化与测序结果一致。CYP450家族通过调节脂质代谢相关途径，包括氧化应激、花生四烯酸代谢、类固醇激素代谢和脂肪酸代谢，在脂肪肝发病机制中起重要作用。
