| 摘要: |
| 目的 探讨微RNA（miRNA）-144抑制剂能否减轻大鼠反流性食管炎（RE）食管黏膜的损伤及可能机制。方法 取18只雄性SD大鼠，随机分为假手术组、RE组、miRNA-144抑制剂组，每组6只。RE组、miRNA-144抑制剂组采用前胃结扎联合幽门限制法建立慢性RE大鼠模型，造模后miRNA-144抑制剂组大鼠通过尾静脉注射miRNA-144 antagomir抑制体内miRNA-144的表达。应用qPCR法检测miRNA-144抑制剂的有效性及各组大鼠食管组织中Toll样受体4（TLR4）、NF-κB、核因子κB抑制蛋白α（IκBα） mRNA的表达变化。采用ELISA法检测大鼠食管组织中IL-6、IL-8、TNF-α的含量，蛋白质印迹法检测密封蛋白3（CLDN3）的表达，免疫组织化学染色检测cleaved caspase 3、Ki-67蛋白的表达，TUNEL法检测食管黏膜细胞凋亡情况。结果 与假手术组相比，RE组大鼠食管组织中TLR4、NF-κB、IκBα mRNA表达水平升高（P＜0.05，P＜0.01），炎症因子IL-6、IL-8、TNF-α的含量（P均＜0.05）及凋亡相关蛋白cleaved caspase 3的表达增加（P＜0.01），食管黏膜细胞凋亡增加（P＜0.01），食管黏膜屏障指标CLDN3蛋白的表达减少（P＜0.01）。与RE组相比，抑制miRNA-144表达可降低大鼠食管组织中TLR4、NF-κB、IκBα mRNA的表达（P均＜0.05），减少炎症相关因子IL-6、IL-8、TNF-α的含量（P均＜0.05），抑制RE大鼠食管黏膜细胞凋亡（P＜0.05），并增加CLDN3蛋白的表达（P＜0.01）。但抑制miRNA-144不影响食管黏膜细胞的增殖，miRNA-144抑制剂组Ki-67阳性细胞数与RE组相比差异无统计学意义（P＞0.05）。结论 miRNA-144抑制剂可减轻RE大鼠食管黏膜的损伤，这种作用可能主要通过TLR4/NF-κB信号通路来实现。 |
| 关键词: 微RNA-144 反流性食管炎 Toll样受体4 核因子κB 炎症 细胞凋亡 |
| DOI：10.16781/j.CN31-2187/R.20210667 |
| 投稿时间：2021-07-03修订日期：2021-10-26 |
| 基金项目:中国人民解放军联勤保障部队第九〇〇医院院立课题(2019L04),福建省自然科学基金面上项目(2020J011140). |
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| MicroRNA-144 inhibitor alleviates esophageal mucosal injury in rats with reflux esophagitis through Toll-like receptor 4/nuclear factor κB signal pathway |
| WANG Bao-shan,LIU Gang,WANG Wen,LIN Yan-fang,YAO Li-jia* |
(Department of Gastroenterology, 900th Hospital of Joint Logistics Support Force of PLA, Fuzhou 350025, Fujian, China
*Corresponding author) |
| Abstract: |
| Objective To investigate whether microRNA (miRNA)-144 inhibitor can attenuate the injury of esophageal mucosa in rats with reflux esophagitis (RE) and its possible mechanism.Methods Eighteen male SD rats were randomly divided into sham group,RE group,and miRNA-144 inhibitor group,with 6 rats in each group.Rats in the RE group and miRNA-144 inhibitor group were recruited to establish RE animal models by tying the proximal stomach and constricting proximal pylorus.Rats in the miRNA-144 inhibitor group were administered with miRNA-144 antagomir to inhibit the expression of miRNA-144 in vivo by tail vein injection.The efficacy of miRNA-144 inhibitor and the mRNA expression of Toll-like receptor 4(TLR4),nuclear factor κB (NF-κB) and inhibitor of nuclear factor κB α(IκBα) in esophageal tissues in each group were detected by quantitative polymerase chain reaction (qPCR).The contents of interleukin (IL)-6,IL-8 and tumor necrosis factor α(TNF-α) in rat esophageal tissues were determined by enzyme-linked immunosorbent assay (ELISA).The protein expression of claudin 3(CLDN3) was detected by Western blotting,the protein expression of cleaved cysteine aspartic acid specific protease (caspase 3) and Ki-67 was detected by immunohistochemistry staining,and the apoptosis of esophageal mucosal cells was examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay.Results Compared with the sham group,the mRNA expression levels of TLR4,NF-κB and IκBα in esophageal tissues of rats in the RE group were significantly increased (P< 0.05,P<0.01),the contents of inflammatory factors (IL-6,IL-8 and TNF-α) and the expression of cleaved caspase 3(an apoptosis-related protein) were significantly increased (all P<0.05 in the former ones and P<0.01 in the latter one),the apoptosis of esophageal mucosal cells was increased (P<0.01),and the expression of CLDN3 protein (an index of esophageal mucosal barrier) was decreased (P<0.01).Compared with the RE group,inhibition of miRNA-144 significantly reduced the mRNA expression levels of TLR4,NF-κB and IκBα(all P<0.05) and the contents of inflammatory factors (IL-6,IL-8 and TNF-α)(all P<0.05),and then inhibited the apoptosis of mucosal cells in the esophagus of the RE rats (P<0.05) and elevated the expression of CLDN3 protein (P<0.01).Whereas,there was no impact on the proliferation of esophageal mucosal cells by inhibiting the expression of miRNA-144,and there was no significant difference in the number of Ki-67 positive cells between the miRNA-144 inhibitor group and RE group (P>0.05).Conclusion miRNA-144 inhibitor can attenuate the injury of esophageal mucosa in RE rats,and the effect may be mainly through TLR4/NF-κB signal pathway. |
| Key words: microRNA-144 reflux esophagitis Toll-like receptor 4 nuclear factor κB inflammation apoptosis |
