轮状病毒双链RNA诱导大鼠肠上皮细胞凋亡并削弱伤口修复能力。

Rotavirus double-stranded RNA induces apoptosis and diminishes wound repair in rat intestinal epithelial cells.

作者信息

Sato Akiko, Iizuka Masahiro, Nakagomi Osamu, Suzuki Manabu, Horie Yasuo, Konno Shiho, Hirasawa Fujiko, Sasaki Kenji, Shindo Kenichi, Watanabe Sumio

机构信息

Department of Internal Medicine, Akita University School of Medicine, Akita, Japan.

出版信息

J Gastroenterol Hepatol. 2006 Mar;21(3):521-30. doi: 10.1111/j.1440-1746.2005.03977.x.

DOI:10.1111/j.1440-1746.2005.03977.x

PMID:16638093

Abstract

BACKGROUND

Recent studies have shown that toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA). Rotaviruses, having a dsRNA genome, infect intestinal epithelial cells (IEC) and cause acute gastroenteritis in young children. The aim of the present study was to clarify the pathophysiological function of rotavirus dsRNA in IEC.

METHODS

Expression of TLR3 mRNA or protein in IEC cell lines (IEC-6, HT-29, Caco-2) was assessed by reverse transcription polymerase chain reaction (RT-PCR), Western blot analysis or immunohistochemistry. Induction of cytokines (TNF-alpha, interferon-beta, interleukin-6) mRNA and activation of signal proteins (ERK1/2 MAPK and IkappaB-alpha) in IEC after stimulation with rotavirus dsRNA were assessed by RT-PCR or Western blot analysis. IEC-6 cells were wounded and cell migration into wound areas after stimulation with rotavirus dsRNA (1-25 microg/mL) was assessed. Induction of apoptosis after stimulation with rotavirus dsRNA was also assessed.

RESULTS

Expression of TLR3 mRNA and TLR3 protein was detected in IEC. Expression of TLR3 mRNA in IEC-6 tended to be up-regulated by exposure to IFN-gamma. Induction of cytokine mRNA and activation of the signal proteins were detected after stimulation with rotavirus dsRNA. Apoptosis was induced and epithelial migration into the wound area was dose-dependently diminished (44.1-94.4%, P < 0.01) by exposure to rotavirus dsRNA. Diminishment of wound repair was suppressed by anti-TLR3 antibody or caspase inhibitor.

CONCLUSION

Rotavirus dsRNA induces severe apoptosis and diminishes wound repair in IEC through TLR3, which might be involved in the pathogenesis of rotavirus-induced enteritis.

摘要

背景

最近的研究表明，Toll样受体3（TLR3）可识别双链RNA（dsRNA）。轮状病毒具有dsRNA基因组，可感染肠道上皮细胞（IEC）并导致幼儿急性胃肠炎。本研究的目的是阐明轮状病毒dsRNA在IEC中的病理生理功能。

方法

通过逆转录聚合酶链反应（RT-PCR）、蛋白质印迹分析或免疫组织化学评估IEC细胞系（IEC-6、HT-29、Caco-2）中TLR3 mRNA或蛋白的表达。通过RT-PCR或蛋白质印迹分析评估轮状病毒dsRNA刺激后IEC中细胞因子（肿瘤坏死因子-α、干扰素-β、白细胞介素-6）mRNA的诱导及信号蛋白（ERK1/2 MAPK和IκB-α）的激活。对IEC-6细胞进行划痕损伤，并评估轮状病毒dsRNA（1-25μg/mL）刺激后细胞向伤口区域的迁移情况。还评估了轮状病毒dsRNA刺激后细胞凋亡的诱导情况。

结果

在IEC中检测到TLR3 mRNA和TLR3蛋白的表达。IEC-6中TLR3 mRNA的表达在暴露于干扰素-γ后有上调趋势。轮状病毒dsRNA刺激后检测到细胞因子mRNA的诱导及信号蛋白的激活。暴露于轮状病毒dsRNA可诱导细胞凋亡，并使上皮细胞向伤口区域的迁移呈剂量依赖性减少（44.1-94.4%，P<0.01）。抗TLR3抗体或半胱天冬酶抑制剂可抑制伤口修复的减少。