南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (8): 1119-1125.doi: 10.12122/j.issn.1673-4254.2022.08.02

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敲低WDSUB1通过抑制NF-κB通路减轻实验性小鼠结肠炎

王少鑫，崔立红，刘新尧，罗 哲，李 辉，浦 江   

  1. 解放军总医院第一医学中心 消化内科，北京 100853
  • 出版日期:2022-08-20 发布日期:2022-09-05

WDSUB1 knockdown alleviates dextran sulfate sodium-induced colitis in mice by inhibiting nuclear factor-κB signaling pathway

WANG Shaoxin, CUI Lihong, LIU Xinyao, LUO Zhe, LI Hui, PU Jiang   

  1. Department of Gastroenterology, First Medical Center of Chinese PLA General Hospital, Beijing 100853, China
  • Online:2022-08-20 Published:2022-09-05

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摘要： 目的 采用体内转染小干扰RNA（siRNA）和口服葡聚糖硫酸钠（DSS）的方法构建小鼠结肠炎模型，研究WDSUB1对小鼠结肠炎症损伤的影响并探讨可能的机制。 方法 在小鼠成纤维细胞L929中转染不同的WDSUB1 siRNA序列，采用Western blotting (WB)检测的方法，选择WDSUB1敲低效果最佳的siRNA序列进行小鼠体内转染实验。选择12只雄性C57BL/6小鼠随机进行尾静脉注射siRNA（siWDSUB1/siControl），口服2.5% DSS饮用水，构建两组DSS诱导的结肠炎小鼠模型，根据siRNA不同，分为WDSUB1敲低组和对照组，6只/组。通过WB和RT-PCR方法验证两组小鼠结肠组织中WDSUB1的表达水平，记录实验过程中结肠炎小鼠的体质量及粪便性状，并进行临床症状评分。采用RT-PCR和WB方法，分别检测两组结肠炎恢复期小鼠结肠组织中炎性因子IL-6，COX-2，TNFα的mRNA表达水平，以及NF-κB通路中IκBα和P65的表达变化。结果 L929细胞中转染不同WDSUB1 siRNA 序列后，选择敲低效果最佳的WDSUB1 2#完成体内转染，构建结肠炎小鼠模型。RT-PCR和WB结果均表明，WDSUB1敲低组小鼠的结肠组织中WDSUB1 mRNA和蛋白表达均明显低于对照组（P<0.05）。在分析两组小鼠结肠炎症状时，WDSUB1 敲低组结肠炎小鼠的体质量丢失明显少于对照组，腹泻和便血程度较对照组也明显减轻（P<0.05）。WDSUB1敲低组小鼠结肠组织中COX-2，IL-6和TNFα mRNA均明显低于对照组（P<0.05），且IκBα表达明显受抑制，而p65表达无明显变化。结论 敲低WDSUB1能够减轻实验性小鼠结肠炎症程度，此过程可能是通过抑制NF-κB通路和炎性因子表达实现的。

关键词: WDSUB1；葡聚糖硫酸钠；结肠炎；NF-κB

Abstract: Objective To explore the effect of WDSUB1 on dextran sulfate sodium (DSS)-induced inflammatory colon injury in mice and the underlying mechanism. Methods Different WDSUB1 siRNA sequences were transfected into mouse fibroblast L929 cells and the optimal sequence was selected by Western blotting. Twelve male C57BL/6 mice were randomized into two groups for injection of siWDSUB1 or siControl via the caudal vein, followed by treatment with 2.5% DSS in drinking water to establish mouse models of DSS- induced colitis (n=6). The expression level of WDSUB1 in the colon tissue of the mice was detected with Western blotting and RT-PCR, the changes in body weight and fecal condition were recorded, and the clinical symptoms of the mice were evaluated. The mRNA expression levels of IL-6, COX-2 and TNF-α and the protein expression of IκBα and P65 in the colon tissues were detected with RT- PCR and Western blotting, respectively. Results The mRNA and protein expressions of WDSUB1 in the colon tissues were significantly lower in colitis mice with WDSUB1 knock-down than in the control mice. Compared with the control mice, the mice receiving siWDSUB1 injection showed obviously milder weight loss, diarrhea and hematochezia with significantly lower mRNA expressions of COX2, IL- 6 and TNFα (P<0.05) and protein expression of IκBα but without obvious changes in P65 expression in the colon tissue. Conclusion WDSUB1 knockdown can alleviate DSS-induced colitis in mice possibly by inhibiting the NF-κB signaling pathway and decreasing the expression of inflammatory factors in the colon tissues.

Key words: WDSUB1; dextran sodium sulfate; colitis; nuclear factor-κB