蛋白激酶受体2在慢性束缚应激小鼠食管中的表达及其意义

The esophageal expression of PAR-2 and its significance in a murine model of chronic restraint stress

目的研究蛋白激酶受体2(proteinase-activated receptor-2,PAR-2)在慢性束缚应激(chronic restraint stress,CRS)模型小鼠食管中的表达水平,探讨PAR-2在心理应激诱导食管高敏感性及食管炎症中的作用。方法20只雄性无特定病原体(specific pathogen free,SPF)昆明小鼠随机分2组(每组10只),即CRS组和正常对照(normal control,NC)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HE染色在光学显微镜下观察食管组织病理学改变并进行组织损伤评分。采用免疫组化,Western blot法,逆转录PCR(reverse transcription-PCR,RT-PCR)方法检测蛋白激酶受体2(proteinase-activated receptor-2,PAR-2)在小鼠食管中的表达,并通过RT-PCR法检测食管组织中相关炎症因子mRNA的表达量。结果CRS组小鼠体重增加量显著低于NC组,差异有统计学意义(t=4.28,P<0.05)。HE染色镜下观察发现CRS组小鼠食管粘膜上皮层增生,中性粒细胞及浆细胞浸润等炎症性改变,NC组未见明显异常;此外CRS组小鼠上皮细胞损伤及粘膜下炎症细胞评分均高于NC组,两组差异有统计学意义(t值分别为-4.16,-3.58,P值均<0.05);NC组与CRS组粘膜下水肿评分差异无统计学意义。免疫组化结果显示,PAR-2阳性着色细胞在大部分标本中均可见到,但CRS组小鼠组织中阳性着色较NC组染色深且丰富。Western blot法检测结果表明,CRS组食管标本中PAR-2蛋白质表达量显著高于NC组,差异有统计学意义(t=-19.67,P<0.05)。RT-PCR实验结果表明,CRS组PAR-2 mRNA表达水平高于NC组,差异有统计学意义(t=-15.40,P<0.05);与NC组相比,CRS组炎症相关因子白细胞介素(interleukin,IL)-1β、白IL-8及肿瘤坏死因子(tumor necrosis factor,TNF)-αmRNA表达水平升高,差异有统计学意义(t值分别为-7.94,-17.82,-25.54,P值均<0.05)。通过Pearson相关性分析得知,CRS组小鼠食管中PAR-2 mRNA相对表达倍数分别与炎症细胞因子IL-1β,IL-8,TNF-α之间有正相关关系,相关性具有统计学意义(r值分别为0.90、0.95、0.98,P值均<0.05)。结论心理应激可能通过致敏PAR-2受体介导食管高敏感性的形成,并引起食管炎症的发生。

Objective To detect the expression level of proteinase-activated receptor-2(PAR-2)in mouse esophagus by establishing chronic restraint stress(CRS),and to discuss the role of PAR-2 in esophageal hypersensitivity and esophageal inflammation.Methods Total of 20 male Kunming mice were randomly divided into two groups:CRS and normal control(NC)group with 10 mice in each group.Mice in CRS group were subjected to 2 h per day of restraint stress using home-made device for a period of 14 d.Histopathological changes of esophageal tissue were observed using HE staining and histological damage scoring system.The expression levels of PAR-2 and related cytokines in esophageal tissues were detected by several methods such as immunohistochemistry,Western blotting and reverse transcription-PCR(RT-PCR).Result Compared to NC group,the body weight of CRS group were significantly decreased(t=4.28,P<0.05).The epithelial hyperplasia,neutrophil infiltration and plasma cell infiltration were observed in the esophageal mucosa of mice in CRS group by HE staining,but no obvious abnormality was observed in NC group.In addition,histological damage scores for epithelial damage and submucosal inflammatory cells were higher in CRS group(t values were-4.16 and-3.58 respectively,both P<0.05).For submucosal edema,no significant differences were found between the two groups(t=-0.95,P>0.05).Immunohistochemical results showed that PAR-2 positive staining cells were found in most specimens,but the positive staining in CRS group was deeper and more abundant than that in NC group.Western blotting showed that the expression level of PAR-2 protein in esophageal specimens of CRS group was significantly higher than that of NC group(t=-19.67,P<0.05).RT-PCR results showed that the expression level of PAR-2 in CRS group was higher than that in NC group(t=15.40,P<0.05).Compared with NC group,the levels of inflammatory related factors interleukin(IL)-1β,IL-8 and tumor necrosis factor(TNF)-αmRNA in CRS group were significantly higher than those in NC group(t values were-7.94,-17.82 and-25.54 respectively,both P<0.05).Pearson correlation analysis showed that the relative expression multiples of PAR-2 in esophagus of CRS mice were positively correlated with IL-1β,IL-8 and TNF-α(r values were 0.90,0.95,and 0.98 respectively,both P<0.05).Conclusion Chronic stress may result in esophageal hypersensitivity through activation of PAR-2,and it may also cause esophageal inflammation.