摘要目的 探讨中药黄连抑制多耐药尿道致病性大肠埃希菌作用的分子机制. 方法 应用RNA-seq技术分析黄连对尿道致病性大肠埃希菌( uropathogenic Escherichia coli,UPEC)临床分离株( NB8株)转录组的影响. 琼脂稀释法测定黄连水煎液对UPEC临床分离株( NB8株)的最低抑菌浓度( MIC) ,检测黄连水煎液对病原菌生长曲线的影响;用10倍MIC的黄连水煎液作用于UPEC NB8株菌30 min后,提取UPEC NB8株菌总RNA,去除rRNA,反转录合成cDNA,在HiSeq2000测序平台上进行转录组测序,通过分析软件(tophat+cufflinks)构建转录本并计算表达量,并将得到的表达谱进行差异表达、GO功能富集以及KEGG代谢通路分析,另使用生理盐水处理UPEC NB8株菌为阴性对照.结果 黄连水煎液对UPEC NB8株的MIC为12.5 mg/ml. UPEC NB8株菌黄连处理组有3665个基因表达,生理盐水对照组有3430个基因表达,差异表达基因共有1428个,其中上调基因921个、下调基因507个,差异基因主要富集在结合与催化模块中,细胞黏附、凋亡及多细胞过程中的基因上调,参与酶活性调节的基因下调;KEGG代谢通路富集分析发现,脂多糖合成通路相关基因呈显著上调、与DNA复制相关的修复聚合酶Ⅲ呈现较显著的上调趋势,脂肪酸代谢、组氨酸代谢、硫胺代谢及叶酸代谢、核糖体合成的铁载体组呈现整体下调趋势. 结论 获得了黄连水煎液作用于UPEC的表达谱,阐述了黄连抑制UPEC生长的分子机制,主要机制在于破坏UPEC的细胞壁、影响DNA的复制以及蛋白质的转录和翻译等,说明黄连水煎液对于UPEC的抑制是多方面多层次的.

abstractsObjective To investigate the molecular mechanism of the inhibitory effects of rhizoma coptidis on multi-drug resistant uropathogenic Escherichia coli.Methods High-throughput RNA sequencing ( RNA-seq ) was performed to investigate the transcriptome in a multi-drug resistant uropathogenic Escherichia coli strain (NB8) treated with water decoction of rhizoma coptidis .Agar dilution test was used to determine the minimal inhibitory concentration ( MIC) of water decoction of rhizoma coptidis against the NB 8 strain.A growth curve was drawn to evaluate the effects of water decoction of rhizoma coptidis on the growth of NB8 strain.Total RNAs were extracted from the NB 8 strain after treated with the water decoction of rhizo-ma coptidis for 30 minutes and then synthetized to cDNA by reverse transcription after screening out the rRNAs.The HiSeq 2000 sequencing system was used for transcriptome sequencing .The TopHat software was used to map and analyze the RNA-Seq reads, and then Cufflinks was run to assemble transcripts and es-timate their abundances .The differential expression , GO enrichment and KEGG metabolic pathway were fur-ther analyzed .The NB8 strain dealt with normal saline was used as negative control .Results The MIC of water decoction of rhizoma coptidis to NB 8 strain was 12.5 mg/ml.There were 3665 genes expressed in NB8 strain treated with water decoction of rhizoma coptidis and 3430 genes expressed in NB8 strain treated with normal saline .The number of differentially expressed genes was 1428 including 921 up-regulated genes and 507 down-regulated genes .Those differentially expressed genes mainly enriched in the modules of binding and catalysis.The genes concerning cell adhesion , apoptosis and multicellular process were up-regulated, while those concerning the regulation of enzyme activities were down-regulated.Results of the KEGG meta-bolic pathway enrichment analysis showed that the genes concerning synthetic pathway of LPS were signifi -cantly up-regulated as well as those encoding the repair polymerase Ⅲthat was involved in DNA replication . However , the genes concerning fatty acid metabolism , histidine metabolism , thiamine metabolism , folate metabolism and iron carrier in ribosome synthesis showed overall down-regulation.Conclusion The tran-scriptome in uropathogenic Escherichia coli strain treated with rhizoma coptidis was profiled .The main mo-lecular mechanism of the inhibitory effects of rhizoma coptidis on uropathogenic Escherichia coli was to de-stroy the cell wall of Escherichia coli, affect the replication of DNA and regulate the transcription and transla-tion of proteins .This study illustrated that the inhibitory effects of rhizoma coptidis on uropathogenic Esche-richia coli were achieved in multiple levels .