实时定量PCR检测冬胃颗粒对寒邪客胃型胃溃疡大鼠胃黏膜细胞ECE-1表达的影响

Effects of Dongwei Granule on the ECE-1 gene expression in gastric mucosa of acetic acid-induced gastric ulcer rats with cold stimulation by real-time PCR

[目的]实时定量PCR检测冬胃颗粒对寒邪客胃型胃溃疡大鼠胃黏膜细胞ECE-1表达的影响,在基因水平探讨冬胃颗粒防治寒性胃溃疡的作用机制。[方法]采用乙酸注射法制备大鼠胃溃疡模型,随机分为正常对照组、溃疡模型组、寒性溃疡组、西药治疗组、中药治疗组。寒性溃疡组及治疗组制成溃疡模型后采用寒冷因素刺激复制寒邪客胃型动物模型,前3天用0℃冰水浸泡,每次10min,后3天用0℃冰水灌胃,治疗组同时给予药物灌胃。测定大鼠胃黏膜溃疡指数(UI)及溃疡抑制率,应用ELISA法检测胃黏膜ET-1含量及实时定量PCR法检测ECE-1mRNA转录表达水平。[结果]中药治疗组溃疡指数明显低于溃疡模型组与寒性溃疡组(P<0.01),及西药治疗组(P<0.05),其溃疡抑制率为54.22%,明显高于西药治疗组的45.40%。溃疡模型组及寒性溃疡组大鼠胃黏膜ET-1水平与正常对照组比较明显增加(P<0.01)。中药治疗组胃黏膜ET-1水平明显降低,且接近正常对照组,与溃疡模型组及寒性溃疡组比较差异均有统计学意义(P<0.05,P<0.01)。相关性分析显示:胃黏膜ET-1水平与UI呈显著正相关(r=0.89,P<0.01)。溃疡模型组及寒性溃疡模型组大鼠胃黏膜ECE-1mRNA的转录表达水平明显增加(P<0.05,P<0.01),药物治疗可下调胃黏膜ECE-1mRNA的转录表达水平,冬胃颗粒治疗组优于西药治疗组(P<0.05),与胃黏膜组织中ET-1水平变化趋势一致,二者呈显著正相关(r=0.91,P<0.01)。[结论]寒性刺激可增加乙酸性胃溃疡大鼠胃黏膜损害,胃黏膜ET-1水平及ECE-1mRNA转录表达水平与胃溃疡的形成关系密切,冬胃颗粒对寒性胃溃疡具有较好的预防和治疗作用。

[Objective]To study the effects of Dongwei Granule（DG） on the ECE-1 gene expression in gastric mucosa of acetic acid-induced gastric ulcer rats with cold stimulation by real-time PCR.[Methods] The rat model of gastric ulcer was constructed by injection of acetic acid.The rats were randomly divided into normal control group,model of gastric ulcer group,gastric ulcer model of cold stimulation group,Ranitidine treatment group and DG treatment group.The rats of gastric ulcer model of cold stimulation group and the two treatment groups were stimulated by cold,filled the stomach with 0℃ ice water for 3 days after soaked with 0℃ ice water for 3 days.The rats of the treatment groups were administered Chinese or western medicine.The ulcer sizes were recorded to calculate the ulcer index （UI） and ulcer inhibition rate.The levels of ET-1 were detected by ELISA,and the gene expression of ECE-1 was detected by real-time PCR.[Results]Compared with gastric ulcer model and model of cold stimulation group,the UI of DG treatment group was reduced significantly （P＜0.01）.The ulcer inhibition rate of DG treatment group （54.55％） was better than Ranitidine treatment group （45.40％）.Compared with the normal control group the levels of gastric mucosal ET-1 of model of gastric ulcer group and gastric ulcer model of cold stimulation group were increased significantly （P＜0.01）,and DG treatment obviously reduced the gastric mucosal ET-1 level（P＜0.05,0.01） closing to the normal control group.The correlation analysis showed that gastric mucosal ET-1 levels correlated with the UI（r=0.89,P＜0.01）.The ECE-1 mRNA expression levels of the gastric ulcer group and the gastric ulcer model of cold stimulation group were increased significantly compared with the normal control group,and DG treatment obviously reduced the ECE-1 mRNA expression level （P ＜0.05,0.01）,superior to the western medicine treatment （P＜0.05）.The ECE-1 mRNA expression level change trend was consistent with gastric mucosal ET 1 level,both were significantly positive correlation （r =0.91,P＜0.01）.[Conclusion]Cold stimulation can aggravate the damage of gastric mucosa of gastric ulcer rats.Both gastric mucosal ET-1 level and ECE-1 mRNA expression levels are closely related to the formation of gastric ulcers.DG can protect the gastric mucosa of gastric ulcer rats induced by cold stimulation.