旋覆代赭汤加味对胃食管反流性咽喉炎大鼠咽喉黏膜紧密连接的影响

Effect of modified Xuanfu Daizhe Decoction on tight junction of throat mucosa in rat model of gastroesophageal reflux laryngopharyngitis

目的观察旋覆代赭汤加味对胃食管反流性咽喉炎(LPRD)大鼠咽喉黏膜紧密连接的影响.方法选择80只健康Wistar大鼠,根据随机数字表法分为假手术组、LPRD模型组、西药对照组和中药治疗组,每组20只.采用改进的不全幽门缝扎+食管下括约肌切开术制备LPRD模型;假手术组开腹15 min后即关腹,不进行其他操作.假手术组和LPRD模型组均给予生理盐水10 mL/kg灌胃,每日2次;西药对照组给予5 mL/kg奥美拉唑混悬液,以10 mL/kg体质量灌胃,每日2次;中药治疗组给予旋覆代赭汤加味浓缩药液10 mL/kg灌胃,每日2次.观察大鼠一般情况,于治疗14 d处死大鼠,取咽部以及食管组织进行检测.观察各组大鼠咽部及食管组织大体形态变化和病理学改变,电镜下观察各组大鼠咽喉黏膜超微结构以及紧密连接claudin蛋白表达情况.结果与LPRD模型组相比,西药对照组和中药治疗组大鼠的体质量增长较明显,毛色光亮程度有所恢复,精神好转,进食量逐渐增多,排泄量增多.LPRD模型组咽部黏膜病理改变最明显;西药对照组和中药治疗组较LPRD模型组咽部黏膜病理改变程度轻,假手术组无咽部黏膜病理改变.电镜观察显示,假手术组大鼠咽部黏膜的上皮细胞侧面可见紧密连接,且大部分细胞胞膜或胞质内出现棕褐色颗粒,蛋白表达较强;LPRD模型组表达最弱,上皮细胞可见紧密连接蛋白表达薄弱,极少部分细胞可见阳性表达;西药对照组和中药治疗组claudin蛋白表达均强于LPRD模型组,但弱于假手术组,部分细胞可见蛋白表达.与假手术组相比,LPRD模型组大鼠claudin蛋白表达评分明显降低(分:1.33±0.48比3.35±0.67,P<0.05);西药对照组和中药治疗组claudin蛋白表达均较LPRD模型组明显升高(分:2.42±0.51、2.74±0.45比1.33±0.48,均P<0.05),且以中药治疗组升高最明显.结论旋覆代赭汤可有效抑制胃食管反流,减轻LPRD大鼠咽部黏膜病理改变,并有效保护紧密连接,增加claudin蛋白表达,有效防御反流液损伤咽部黏膜.

Objective To observe the effect of modified Xuanfu Daizhe Decoction on the tight junction of laryngopharyngeal mucosa in the rat model of gastroesophageal reflux laryngopharyngitis disease(LPRD).Methods Eighty healthy Wistar rats were randomly divided into 4 groups:sham operation group,LPRD model group,traditional Chinese medicine treatment group and Western medicine control group(20 rats in each group).The LPRD model was established by the improved method of"incomplete pylorus suture ligation+lower esophageal sphincterotomy".Laparotomy was performed in the rat of sham operation group for 15 minutes,and then the abdomen was closed without any other operative procedure being done.Each rat in sham operation group and LPRD model group was given 10 mL/kg body weight of normal saline by gavage,twice a day;each rat in Western medicine control group was given 5 mL/kg omeprazole suspension,with 10 mL/kg body weight for gavage,twice a day;each rat in the traditional Chinese medicine treatment group was given modified concentrated Xuanfu Daizhe Decoction 10 mL/kg body weight by gavage,twice a day.The general situations of rats in various groups were observed,and after 14 days,the rats in each group were sacrificed,and the pharynx and esophageal tissues were taken out for investigations.The general morphological and pathological changes of pharyngeal and esophageal tissues were observed,and under the electron microscope,the ultrastructure and tight junction claudin protein expression situation of pharyngeal and laryngeal mucosa in various groups were investigated.Results Compared with LPRD model group,the body weight of rats increased significantly,the brightness of hair color was recovered,the spirit was improved,the food intake was elevated gradually and the excreta amount increased in the Western medicine control group and traditional Chinese medicine treatment group.The pathological changes of the pharyngeal mucosa in the LPRD model group were the most obvious;the pathological changes of the pharyngeal mucosa in the traditional Chinese medicine treatment group and the Western medicine control group were lighter than those in the LPRD model group,and there was no pathological change of the pharyngeal mucosa in the sham operation group.The electron microscopic examinations showed that the tight junctions could be seen on the epithelial cell lateral sides of the pharyngeal mucosa in the sham operation group,and the brown granules with relatively high expression appeared in the membrane or cytoplasm of most of the cells;in LPRD model group,the tight junction protein expression in the epithelial cells was the lowest,and only in a few cells its expression was positive;in Western medicine control group and traditional Chinese medicine treatment group,the claudin protein expression could be seen which was higher than that in LPRD model group,but lower than that in sham operation group,protein expression could be observed in some cells.The expression of claudin protein in the LPRD model group was lower than that in the sham operation group(1.33±0.48 vs.3.35±0.67,P<0.05),the expressions of claudin protein in the traditional Chinese medicine treatment group and the Western medicine control group were higher than those in the LPRD model group(2.42±0.51,2.74±0.45 vs.1.33±0.48,both P<0.05),and the most obvious increase was seen in the traditional Chinese medicine treatment group.Conclusion The modified Xuanfu Daizhe Decoction can effectively inhibit gastroesophageal reflux,reduce pathological changes of pharyngeal mucosa in rats,validly protect the mucosal epithelial cell tight junction,increase the expression of claudin protein,and efficiently prevent reflux liquid from damaging pharyngeal mucosa.