纤维化因子及NADPH氧化酶在心理应激诱导小鼠的食管纤维化中的表达及意义

The esophageal expression of fibrotic cytokines and NADPH oxidase in stress induced esophageal fibrosis and its significance

目的研究胶原蛋白1(colla-1)、转化生长因子β1(TGF-β1)、α-平滑肌肌动蛋白(α-SMA)及还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(NOX-4)在慢性束缚应激(CRS)模型小鼠食管中的表达水平,探讨心理应激诱导食管纤维化的发生及氧化应激所产生的影响。方法 20只雄性SPF级昆明小鼠随机分2组,即CRS组和对照组。CRS组小鼠每天在自制式束缚器中限制活动2 h,其余时间两组小鼠在相同环境中自由饮水摄食,实验持续14 d。通过Masson染色在电子显微镜下观察食管组织纤维化改变。采用免疫组化、实时定量RT-PCR、ELISA法检测NOX-4在小鼠食管中的表达,并通过实时定量RT-PCR法检测食管组织中纤维化因子colla-1、TGF-β1及α-SMA mRNA的表达量。结果与对照组小鼠相比,CRS组小鼠体重增加量低于对照组[(8.75±1.69)g vs (12.69±3.16)g],差异有统计学意义(t=3.11,P<0.05)。Masson染色镜下观察可见:CRS组小鼠食管黏膜上皮细胞间明显的纤维化表现,而对照组未见明显异常;免疫组化结果显示:NOX-4阳性着色细胞在大部分标本中均可见到,CRS组小鼠食管组织中阳性着色较对照组染色深且丰富;ELISA法检测结果表明,CRS组小鼠血清中NOX-4表达量明显高于对照组(1.42±0.05 vs 0.49±0.08),差异有统计学意义(t=-27.32,P<0.01)。实时定量RT-PCR实验结果表明,CRS组纤维化标志物(colla-1,TGF-β1,α-SMA)及NOX-4 mRNA表达水平是对照组的(2.43±0.36、2.78±0.13、2.24±0.18、2.48±0.40)倍,差异有统计学意义(t=-11.19、-38.86、-19.90、-10.37,P<0.01)。结论 colla-1、TGF-β1、α-SMA参与心理应激诱导食管纤维化的形成,而氧化应激在食管纤维化产生中具有重要的作用。

Objective To detect the expression levels of collagen1 (colla-1), transforming growth factor-β1 (TGF-β1),α-smooth muscle actin (α-SMA) and nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX-4) in mouse esophagus submitted to chronic restraint stress (CRS), in order to discuss stress-induced esophageal fibrosis and the role of oxidative stress. Methods 20 male Kunming mice were randomly divided into two groups, CRS and normal control (NC). The mice in CRS group were submitted to 2 h per day of restraint stress using home-made device for a period of 14 days, and the mice in both group were treated the same at rest of the time. Fibrotic changes of esophageal tissue were observed using Masson staining. The expression levels of NOX-4 and related fibrotic cytokines in esophageal tissues were detected by several methods such as immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (qRT-PCR). Results Body weight in CRS group was significantly lower than NC group (8.75±1.69 vs 12.69±3.16), with statistically significant difference (t=3.11, P<0.05). Masson staining revealed that CRS mice showed distinct fibrosis of epithelial interstitium, while there was no distinct changes observed in NC mice. Immunohistochemical staining revealed intense staining for NOX-4 in epithelial, mucosal and submucosal layers of esophagi in CRS mice. ELISA showed that the serum level of NOX-4 in CRS mice was higher than NC mice (1.42±0.05 vs 0.49±0.08), with statistically significant difference (t=-27.32, P<0.01). Real-time PCR results showed that the expression of colla-1, TGF-β1,α-SMA and NOX-4 in CRS mice were as (2.43±0.36, 2.78±0.13, 2.24±0.18, 2.48±0.40) times higher than NC mice, with statistically significant difference (t=-11.19,-38.86,-19.90,-10.37, P<0.01). Conclusions Fibrotic cytokines such as colla-1, TGF-β1 and α-SMA may participate in formation of stress induced esophageal fibrosis, and oxidative stress may play crucial role in the process of esophageal fibrosis.