慢性束缚应激对小鼠食管上皮细胞间距增宽及炎症发生的研究

Effects of chronic restraint stress on esophageal epithelial dilated intercellular space and inflammation in mice

目的探讨慢性束缚应激对食管上皮细胞间隙增宽以及炎症发生的影响。方法选用8周龄,雄性SPF级昆明小鼠20只,随机分为2组,每组10只。慢性束缚应激组小鼠每天在自制式束缚器中限制活动2 h,其余时间2组小鼠在相同环境中自由饮水摄食,实验持续14 d。通过HE染色在光学显微镜下观察食管上皮组织病理学改变并进行间距测量。通过酶联免疫吸附(ELISA)和实时定量RT-PCR法检测食管组织中氧化应激指标(Nox-4、8-OHdG、MDA)、抗氧化蛋白(Nrf-2、HO-1)、紧密连接蛋白重要指标(Claudin-1、Claudin-4、Occludin、ZO-1)以及炎症因子(MCP-1、IL-1β、TNF-α)mRNA的表达量。结果2组小鼠食管上皮细胞镜下测量的平均细胞间隙为对照组(0.64±0.04μm)、慢性束缚应激组(1.16±0.02μm);与对照组比较,慢性束缚应激组小鼠上皮细胞间距明显增宽,差异具有统计学意义(t=20.63,P<0.01)。ELISA结果显示,慢性束缚应激组小鼠血液中Nox-4[(9.72±1.80)mg/ml vs(4.54±0.46)mg/ml]、8-OHdG[(4.94±0.58)mg/ml vs(1.44±0.16)mg/ml]以及MDA[(3.56±0.41)mg/ml vs(1.02±0.12)mg/ml]的释放浓度明显高于对照组,且差异具有统计学意义(t=25.54,19.62,13.67,P<0.01)。RT-PCR实验结果显示,慢性束缚应激组食管组织中抗氧化蛋白Nrf-2和HO-1的mRNA转录水平显著低于对照组,差异具有统计学意义(t=-10.36,-9.28,P<0.05)。慢性束缚应激组紧密连接蛋白如Claudin-1、Claudin-4、Occludin及ZO-1的mRNA转录水平明显低于对照组,差异具有统计学意义(t=-10.23、-9.65、-7.81、-8.75,P<0.01)。慢性束缚应激组炎症因(MCP-1、IL-1β、TNF-α)的mRNA转录水平显著高于对照组,差异具有统计学意义(t=31.42、42.37、46.83,P<0.01)。结论慢性束缚应激可能直接使食管氧化/抗氧化系统平衡紊乱,进而导致食管上皮细胞间距增宽及验证的发生。

Objective To investigate the effects of chronic restraint stress on the esophageal epithelial cells dilated intercellular space and the occurrence of inflammation.Methods Twenty male SPF Kunming mice aged 8 weeks were randomly divided into two groups,namely the chronic restraint stress group(n=10)and the control group(n=10).Stress mice were restrained in self-made restraint device for 2 hours every day,experiment lasted 14 days.The esophageal epithelium were observed by HE staining under optical microscope and the spaces between cells was measured.The expression of oxidative stress(Nox-4,8-OHdG,MDA),antioxidant protein(Nrf-2,HO-1),tight junction protein(Claudin-1,Claudin-4,Occludin,ZO-1)and inflammatory factors(MCP-1,IL-1β,TNF-α)in esophageal tissues were detected by enzymelinked immunosorbent assay(ELISA)and real-time quantitative RT-PCR.Results The mean intercellur space in stress mice was(1.16±0.02μm),compare to control mice(0.64±0.04μm),it was significantly wider than control group(t=20.63,P<0.01).The results of ELISA showed that the blood concentrations of Nox-4[(9.72±1.80)mg/ml vs(4.54±0.46)mg/ml],8-OHdG[(4.94±0.58)mg/ml vs(1.44±0.16)mg/ml]and MDA[(3.56±0.41)mg/ml vs(1.02±0.12)mg/ml]in stress group were significantly higher than those in control group(t=25.54,19.62,13.67,P<0.01).RT-PCR results of antioxidant proteins Nrf-2 and HO-1 in stress group were significantly lower than those in control group(t=-10.36,-9.28,P<0.05).The transcription levels of tight junction proteins such as Claudin-1,Claudin-4,Occludin and ZO-1 in stress group were significantly lower than those in control group(t=-10.23,-9.65,-7.81,-8.75,P<0.01).The transcription levels of inflammatory factors(MCP-1,IL-1β,TNF-α)in stress group were significantly higher than those in control group(t=31.42,42.37,46.83,P<0.01).Conclusion Chronic restraint stress may directly disturb the balance of oxidation/antioxidant system in esophagus,which eventually lead to the esophageal epithelial dilated intercellular space and inflammation.