疏肝和胃方对胃食管反流病大鼠食管中PAR-2、Claudin1、Claudin4和炎性因子的影响

Effect of Shugan Hewei Formula(疏肝和胃方)on Expressions of PAR-2,Claudin1,Claudin4 and Inflammatory Factors in Esophageal Tissue of GERD Rats

目的探讨疏肝和胃方对胃食管反流病(GERD)大鼠食管组织中蛋白酶激活受体2(PAR-2)、炎性因子和紧密连接蛋白(Claudin)表达的影响。方法采用完全随机法将SD大鼠随机分为4组(正常组、模型组、中药组及西药组),并行下食管括约肌切开术加十二指肠半结扎术建立GERD模型。中药组予疏肝和胃方灌胃;西药组予雷贝拉唑灌胃;正常组及模型组予等量的生理盐水灌胃。予HE染色观察食管组织病理改变,予免疫组织化学和Western Blot检测食管组织中PAR-2、Claudin1及Claudin4蛋白表达,ELISA检测食管组织中血小板活化因子(PAF)、白细胞介素-1β(IL-1β)、白细胞介素-8(IL-8)表达。结果正常组大鼠黏膜及肌层无病理改变;模型组大鼠食管下段见鳞状上皮增生,黏膜下见中性粒细胞及淋巴细胞,固有层见乳头节,食管肌层也可见不同程度炎性细胞浸润;与模型组相比,中药组大鼠食管黏膜结构轻度改变,未见明显局部糜烂及鳞状上皮增生,仅见少量炎性细胞浸润;西药组可见炎性细胞浸润且部分可见鳞状上皮增生及糜烂。与正常组相比,PAR-2、IL-8和IL-1β显著增加(P<0.05),PAF呈增加趋势(P>0.05),Claudian1和Claudin4显著降低(P<0.05);与模型组相比,中药组PAR-2、IL-8和IL-1β显著降低(P<0.05),PAF呈降低趋势(P>0.05),Claudian1和Claudin4表达显著增加(P<0.05),西药组PAR-2、IL-8和IL-1β显著降低(P<0.05),PAF呈下降趋势(P>0.05),Claudian1和Claudin4呈增加趋势(P>0.05);西药组PAR-2、IL-8、IL-1β、PAF表达高于中药组,Claudian1和Claudin4表达显著低于中药组。结论GERD通过PAR-2激活、食管黏膜炎症传导的关联途径,影响紧密连接蛋白表达,导致食管黏膜损伤。疏肝和胃方通过调控PAR-2传导途径和作用靶点,可一定程度上减轻GERD模型大鼠食管黏膜损伤,为临床治疗GERD提供了可靠的实验依据。

Objective To investigate the effect of Shugan Hewei Formula(疏肝和胃方)on expressions of protease activated receptor-2(PAR-2),inflammatory factors and tight junction protein(Claudin)in esophageal tissue of gastroesophageal reflux disease(GERD)rats.Methods SD rats were randomly divided into 4 groups(normal group,model group,traditional Chinese medicine group and western medicine group),and the GERD model was established by lower esophageal sphincterotomy and half duodenum ligation.In the traditional Chinese medicine group,Shugan Hewei Formula was given to the stomach;in the Western medicine group,Rabeprazole was given by gavage.In the normal group and the model group,the normal saline was given by gavage.The pathological changes of the esophagus were observed by HE staining and the expressions of PAR-2,Claudin1 and Claudin4 in the esophageal tissues were detected by immunohistochemistry and Western Blot.The expressions of platelet activating factor(PAF),interleukin-1β(IL-1β)and interleukin-8(IL-8)in esophageal tissues were detected by ELISA.Results There were no pathological changes in the mucosa and muscularis in the normal group.There was squamous cell hyperplasia in the lower esophagus,neutrophil and lymphocyte in the submucosa and papillary segment in the lamina propria in the model group.Compared with those of the model group,there were slight changes in the structure of esophageal mucosa,no obvious local erosion and squamous cell hyperplasia,and only a few inflammatory cells infiltration.In the Western medicine group,there were inflammatory cells infiltrating and some squamous epithelial hyperplasia and erosion.Compared with those of the normal group,the expressions of PAR-2,IL-8 and IL-1β were increased significantly(P<0.05),while PAF increased(P>0.05),Claudin1 and Claudin4 decreased significantly(P<0.05).The expressions of PAR-2,IL-8 and IL-1β decreased significantly(P<0.05),the expressions of Claudin1 and Claudin4 increased significantly(P<0.05),the expressions of PAR-2,IL-8 and IL-1β decreased significantly(P<0.05),the expressions of PAR-2,IL-8,IL-1β and PAF in the Western medicine group were higher than those in the traditional Chinese medicine group and the expressions of Claudian1 and Claudin4 were significantly lower than those in the traditional Chinese medicine group.Conclusion Through PAR-2 activation and inflammation conduction in esophageal mucosa,GERD can affect the expression of connexin and lead to esophageal mucosa injury.By regulating PAR-2 pathway and action target,Shugan Hewei Formula can reduce esophageal mucosa injury in GERD model rats to some extent,which provides reliable experimental evidence for clinical treatment of GERD.