Ulinastatin improves rat liver metabolism after reduced-size liver transplantation

doi:10.3969/j.issn.2095-4344.0594

Abstract

Abstract:

BACKGROUND: Effects of ulinastatin in anti-inflammatory, viscera protection and improving microcirculation after liver resection or liver transplantation have been widely researched. However, the role of microRNA regulation mechanism of ulinastatin has not yet been reported.
OBJECTIVE: To discover the variation of microRNA expression profile and the change of proteomics in rats after reduced-size liver transplantation using ulinastatin, and to investigate if there is a lien between differentially expressed miroRNAs and proteins, so as to provide an in-depth theoretical evidence for clinical application of ulinastatin.
METHODS: Rat models of reduced-size liver transplantation were established using the Kamada double cuff method and then divided into two groups. Rats in the experimental and control groups were given intraperitoneal injection of ulinastatin (100 U/g) and normal saline (2 mL) respectively at 0, 12, 24, 36, and 48 hours after surgery. miroRNA chip and proteomic analysis were performed at 24 and 48 hours after transplantation. Then results were then imported into mirTarBase software for target prediction.
RESULTS AND CONCLUSION: Compared with the control group, in the experimental group, there were 19 differentially expressed (> two-fold) proteins and 17 differentially expressed (> 1.5 fold) proteins in rats. A miRNA regulation channel: miR-181-a-5p and Gpx1 were found. To conclude, after reduced-size liver transplantation in rats, use of ulinastatin alters miR-181-a-5p expression, which improves liver metabolism through regulating Gpx1 expression. This may be one mechanism of action of ulinastatin.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: MicroRNAs, Mass Spectrometry, Liver Transplantation, Reperfusion Injury, Tissue Engineering

CLC Number:

R446

Gao Hongqiang, Liu Jing, Li Zhiqiang, Wang Hailei, Zhao Xiongqi, Zhang Shengning, Ran Jianghua, Li Li . Ulinastatin improves rat liver metabolism after reduced-size liver transplantation [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(3): 435-440.

Figures/Tables 2

2.1 miRNAs芯片检测结果及数据分析将miRNAs芯片检测结果输入QIAGEN公司为产品提供的在线分析表(www.SABiosciences.com/pcrarraydataanalysis.php)，所得结果见图1，2。与对照组大鼠肝组织相比，减体积肝移植术后24 h乌司他丁组肝脏组织中表达上调超过2倍的miRNAs有6个，分别为miR-208a-3p、miR-34a-5p、 miR-375-3p、let-7b-3p、miR-7a-5p、miR-15b-5p；表达下调超过2倍的miRNA有6个，miR-134-5p、miR-146-5p、miR-150-5p、miR-181a-5p、miR-199a-5p、miR-107-3p、miR-204-5p。减体积肝移植后48 h乌司他丁组肝脏组织中表达上调超过2倍的miRNAs有miR-21-5p、miR-423-5p，表达下调超过2倍的miRNA有8个：miR-222-3p、miR-100a-5p、miR-31a-5p、miR-375-3p、miR-181a-5p、miR-199a-5p、miR-15b-5p、miR-107-3p。说明大鼠减体积肝移植后乌司他丁的使用导致了miRNA表达谱的变化，为后续靶基因预测提供了依据。 2.2 蛋白质质谱分析结果双向电泳后凝胶扫描结果发现与对照组相比，大鼠减体积肝移植术后24 h乌司他丁组有143个蛋白丰度变化超过1.5倍，术后48 h乌司他丁组有106个蛋白丰度变化超过1.5倍，见图3所示。在这些差异表达的蛋白中，通过质谱仪进行质谱分析来明确所鉴定的蛋白质为何种蛋白质。利用德国布鲁克(Bruker Dalton)Ultraflex III TOF/TOF质谱仪，UV波长为355 nm，重复速率为200 Hz，加速电压为20 000 V，最优质量分辨率为1 500 u。扫描质量范围为700-3 200 u，收集信号，胰酶自切峰为内标校正质谱仪。所有实验样品的质谱图均以用默认模式获得。数据库检索：利用软件flexAnalysis(Bruker Dalton)过滤基线峰、识别信号峰。利用BioTools(Bruker Dalton)软件搜索NCBI数据库，寻找匹配的相关蛋白质，同时查询其功能，结果在大鼠减体积肝移植术后两个时间点鉴定出17种差异表达的蛋白质，见图4，5；表1，这些蛋白质的功能将是作者后续研究的重点。 2.3 靶基因预测结果登录靶基因预测软件mirTarBase进入操作界面；点击advanced research按钮进入界面；选择种属species为大鼠，Rat；输入差异表达的miRNAs (24，48 h所有差异表达的miRNAs)以及鉴定出的蛋白质名称(表1所列蛋白质)0，用“,”隔开；点击确定“submit”，弹出对话框，见图6，通过靶基因预测软件发现了miR-181a相关联的蛋白为Gpx1和 Tgm2，其中Gpx1为强力证据推荐，说明该预测具有非常高的可信度，点击Gpx1条目，可见支持该预测的文献[12]。"

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