七氟醚诱导肝损伤大鼠模型microRNA-214的表达变化及其损伤机制探讨

Changes of miR-214 in sevoflurane-induced liver injury and its mechanism

摘要:

目的 通过七氟醚诱导肝损伤大鼠模型探讨七氟醚对肝脏凋亡、炎症因子的影响及其可能作用机制。方法 将16只SD大鼠随机分为对照组和七氟醚组，采用RT-PCR检测七氟醚组肝脏组织中microRNA-214（miR-214）的表达水平。应用Olympus自动化学分析仪检测血清丙氨酸氨基转移酶（ALT）和天门冬氨酸氨基转移酶（AST）的水平；应用RT-PCR检测TNF-α、IL-4、IL-6、MCP-1在肝脏组织中的表达。Western blotting检测Bax和Bcl-2的表达。应用生物信息学方法对miR-214进行靶基因预测及信号通路分析。结果 七氟醚组与对照组miR-214表达水平比较，七氟醚组高于对照组（P <0.05）；与对照组比较，七氟醚组的肝脏系数升高（P <0.05）；与对照组比较，七氟醚组血清ALT和AST水平升高（P <0.05）；与对照组比较，七氟醚组TNF-α、IL-6 mRNA相对表达量升高而IL-4相对表达量降低（P <0.05），MCP-1相对表达量差异无统计学意义（P >0.05）；与对照组比较，七氟醚处理组Bcl-2/Bax比值下降（P <0.05）；通过miRanda和TargetScan数据库预测miR-214的靶基因结果显示，两数据库靶基因数量分别是4 652和395个。通过韦恩图对2个数据库靶基因进行交集选取，结果共有319个相同的基因开展后续研究；DAVID平台的KEGG通路数据库分析miR-214的靶基因信号通路预测，利用P值进行排序选择，结果排在首位的为Natural killer cell mediated cytotoxicity通路。结论 七氟醚处理会使肝脏出现一定程度的损伤，而miR-214在这一过程中发挥着重要作用。

Abstract:

Objective To investigate the mechanism of hepatic apoptosis, inflammatory factors, and possible effects of sevoflurane with constructing an animal model of sevoflurane treatment.Methods Sixteen SD rats were randomly divided into control group and sevoflurane treatment group. RT-PCR was used to verify the expression of miR-214 in liver tissue after sevoflurane treatment. The levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by Olympus automatic chemical analyzer. The key transcriptional gene TNF-α of inflammatory factors in liver tissue was detected by RT-PCR. The expression levels of IL-4, IL-6 and MCP-1 in liver tissues. Western blotting was used to detect the expression of apoptotic marker proteins Bax and Bcl-2. Target gene prediction and pathway analysis of miR-214 were performed with using bioinformatics methods.Results The expression of miR-214 in liver tissues after sevoflurane treatment was significantly higher than that in the control group (P < 0.05); compared with the control group, the liver coefficient in the sevoflurane treatment group was significantly increased (P < 0.05), which explained the phenomenon of increased liver compensation. Compared with the control group, the content of ALT and AST in serum of the sevoflurane-treated group increased significantly (P < 0.05), indicating that the liver appeared to a certain extent after sevoflurane treatment. Compared with the control group, the relative expression of TNF-α and IL-6 mRNA in the sevoflurane-treated group increased significantly while IL-4 decreased significantly (P < 0.05). Although MCP-1 showed an upward trend, the difference was not significant (P > 0.05). Compared with the control group, the ratio of Bcl-2/Bax in the sevoflurane treatment group decreased significantly (P < 0.05), indicating that the apoptosis after sevoflurane treatment increased significantly. The miRanda Predictive analysis of the target genes of miR-214 with the two prediction databases of Target Scan showed that the number of target genes in the two databases was 4652 and 395, respectively. The target genes of the two databases were selected through the intersection of Wayne diagrams, and a total of 319 identical genes were used for follow-up research. In this study, the KEGG pathway analysis of the DAVID platform was used to predict the target gene Pathway of miR-214. The P value was used for sorting and selection, and the results showed that the natural killer cell mediated cytotoxicity pathway ranked first.Conclusion This study found that sevoflurane treatment can cause a certain degree of damage to the liver through the construction of animal models in vivo, and miR-214 plays an important role in this process. Therefore, the possibility of miR-214 as a biomarker of hepatic injury after sevoflurane treatment has been verified to a certain extent, providing a theoretical basis for early detection of liver injury.