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目的探讨miR-199a在2,4,6-三硝基苯磺酸(TNBS)/乙醇诱导的溃疡性结肠炎(UC)大鼠中的表达情况及雷公藤多苷(TWP)对其的作用。方法采用2,4,6-三硝基苯磺酸(TNBS)/乙醇灌肠法建立溃疡性结肠炎动物模型。对大鼠结肠组织进行大体形态损伤评分和镜下结肠损伤评分;采用芯片分析、Real-time PCR技术评估各组结肠黏膜组织中miR-199a的表达水平;针对milwalk数据库中预测的下游靶基因mRNA用表达谱进一步筛选分析靶基因mRNA,并用Real-time PCR技术验证靶基因mRNA在各组中的表达,最后在DAVID数据库进行pathway分析,以分析miR-199a参与UC炎症活动时所调节的靶基因mRNA。结果 TWP高剂量组结肠黏膜大体形态损伤评分、组织病理损伤评分与模型对照组间差异有统计学意义(P<0.01)。芯片分析显示,模型对照组中miR-199a较正常对照组上调(P<0.01),在AZA组表达较模型对照组下调(P<0.01,P<0.05)。miR-199a-3p在TWP中剂量组,miR-199a-5p在TWP高剂量组中较模型对照组均下调(P<0.05)。miR-199a的Real-time PCR结果显示,模型对照组miR-199a的表达水平较正常对照组明显上调(P<0.01);TWP的中、高剂量组和AZA组中miR-199a-3p的表达水平较模型对照组均下调(P<0.05);TWP中剂量组miR-199a-5p的表达水平较模型对照组下调(P<0.05)。表达谱分析显示,FASL为miR-199a的靶基因。模型对照组中靶基因FASL表达水平较正常对照组上调(P<0.05),TWP组、AZA组中靶基因FASL表达水平较模型对照组下调,其中以AZA组的下调为显著(P<0.01)。靶基因FASL的Real-time PCR结果显示,模型对照组中靶基因FASL表达水平较正常对照组明显上调(P<0.01);TWP中剂量组、高剂量组和AZA组中靶基因FASL表达水平较模型对照组下调(P<0.01)。结论 miR-199a在TNBS/乙醇UC大鼠中有上调表达,FASL是miR-199a的下游靶基因。TWP能改善UC的炎症活动,对UC活动时上调的miR-199a具有下调作用;FASL在UC活动时呈上调表达,TWP对miR-199a下游靶基因FASL具有下调作用。
Objective To investigate the expression of miR-199a in 2,4,6-trinitrobenzene sulfonic acid (TNBS) / ethanol-induced ulcerative colitis (UC) rats and the effect of TWP . Methods The animal model of ulcerative colitis was established by 2,4,6-trinitrobenzene sulfonic acid (TNBS) / ethanol enema. The gross morphological damage score and microscopic colon injury score of the colon tissue of rats were scored. The expression of miR-199a in colonic mucosa of each group was evaluated by real-time PCR and the expression of downstream target gene mRNA predicted by milwalk database The target gene mRNA was further screened by expression profiling and Real-time PCR was used to verify the target gene mRNA expression in each group. Finally, a pathway analysis was performed in the DAVID database to analyze the target genes regulated by miR-199a in UC inflammatory activity mRNA. Results The morphological damage scores, histopathological damage scores and the control group of TWP high dose group were statistically significant (P <0.01). Chips analysis showed miR-199a was up-regulated in the model control group (P <0.01), and down-regulated in the AZA group compared with the model control group (P <0.01, P <0.05). miR-199a-3p in TWP middle dose group, miR-199a-5p in the TWP high-dose group than the model control group were down (P <0.05). Real-time PCR results of miR-199a showed that the expression of miR-199a in model control group was significantly higher than that in normal control group (P <0.01); The expression of miR-199a-3p in middle and high dose TWA group and AZA group (P <0.05). The expression level of miR-199a-5p in TWP middle-dose group was lower than that in model control group (P <0.05). Expression profiling showed that FASL was the target gene of miR-199a. The expression of FASL in the model control group was higher than that in the normal control group (P <0.05). The expression of FASL in TWP group and AZA group was lower than that in the model control group (P <0.01) . Real-time PCR results of FASL target gene showed that the expression level of FASL in the model control group was significantly higher than that in the normal control group (P <0.01). The expression levels of FASL in TWP middle dose group, high dose group and AZA group The model control group was down-regulated (P <0.01). Conclusion miR-199a is upregulated in TNBS / ethanol-induced UC rats, and FASL is a downstream target of miR-199a. TWP can down-regulate the inflammatory activity of UC and downregulate miR-199a up-regulated in UC activity. FASL up-regulates in UC activity and TWP down-regulates miR-199a downstream target gene FASL.