槲皮素对慢性混合反流性食管炎大鼠食管黏膜的保护作用及对核转录因子-κB/白细胞介素-6信号通路的影响

摘要

目的 观察槲皮素对慢性混合反流性食管炎大鼠食管黏膜损伤的保护作用及对核转录因子κB (NF-κB)/白细胞介素(IL)-6信号通路的影响.方法 采用贲门结扎术+食管十二指肠吻合术建立慢性混合反流性食管炎大鼠模型.48只健康清洁级SD雄性大鼠采用随机数字表法分为正常对照组、假手术组、模型对照组、奥美拉唑组、低剂量槲皮素组和高剂量槲皮素组,每组8只,分别腹腔注射生理盐水2 ml(正常对照组、假手术组和模型对照组)、奥美拉唑20 mg/kg(奥美拉唑组)、槲皮素100 mg/kg(低剂量槲皮素组)和槲皮素200 mg/kg(高剂量槲皮素组),每日1次.6周后处死,光镜下观察食管组织的形态学改变并评分,分别采用免疫组织化学染色及酶联免疫吸附试验检测NF-κB p65、IL-6蛋白在大鼠食管黏膜及血清中的表达,各组进行比较.结果 正常对照组、假手术组、模型对照组、奥美拉唑组、低剂量槲皮素组、高剂量槲皮素组大鼠的食管黏膜病理评分分别为0.250±0.463、0.250±0.463、2.625±0.518、1.500±0.535、1.250±0.463、1.375±0.518;食管黏膜NF-κB p65蛋白评分分别为0.500±0.535、0.625±0.518、3.500±0.535、1.875±0.649、1.750±0.707、2.000±0.535;食管黏膜IL-6蛋白评分分别为1.125±0.641、1.125±0.835、5.375±0.518、2.375±0.518、2.000±0.535、2.250±0.463;血清NF-κB p65蛋白含量分别为(68.618±18.500)、(77.824±22.228)、(184.882±49.165)、(106.693±45.312)、(76.215±16.588)、(108.207±42.107) pg/ml;血清IL-6蛋白含量分别为(24.826±4.008)、(23.599±4.351)、(51.378±9.697)、(32.370±11.657)、(23.085±4.660)、(26.243±4.955) pg/ml.假手术组与正常对照组比较,以上5项指标差异均无统计学意义(P=1.000、P=0.642、P=1.000、P=0.518、P=0.673);正常对照组、假手术组、奥美拉唑组、低剂量槲皮素组和高剂量槲皮素组与模型对照组比较,差异均有统计学意义(P <0.001、P<0.001、P<0.001、P=0.002、P=0.001;P<0.001、P<0.001、P<0.001、P =0.004、P=0.002;P=0.001、P<0.001、P<0.001、P=0.025、P=0.023;P均<0.001;P<0.001、P<0.001、P<0.001、P=0.023、P<0.001);低剂量槲皮素组、高剂量槲皮素组与奥美拉唑组比较,差异均无统计学意义(P =0.334、P=0.717、P=0.176、P=0.121、P=0.074;P=0.642、P =0.678、P=0.619、P=0.949、P=0.225);低剂量槲皮素组与高剂量槲皮素组比较,差异均无统计学意义(P =0.619、P=0.438、P=0.334、P=0.086、P=0.243).大鼠食管黏膜病理评分与黏膜NF-κB p65、IL-6蛋白含量呈明显正相关(r=0.803,P<0.001;r=0.758,P<0.001),与血清NF-κB p65、IL-6蛋白含量亦呈明显正相关(r=0.486,P=0.004;r=0.544,P=0.001).结论 大鼠食管黏膜损伤程度越大,黏膜及血清NF-κB p65、IL-6蛋白含量越高.槲皮素能降低反流性食管炎大鼠食管黏膜的损伤程度,其作用机制可能与下调反流性食管炎大鼠NF-κB、IL-6水平,减轻食管炎症有关.%Objective To observe the protective effects of quercetin on esophageal mucosa in chronic mixed reflux esophagitis (RE) rats and the effect of quercetin on nuclear factor (NF)-κB/interleukin (IL)-6 signaling pathway.Methods Mixed RE model was successfully induced by cardia ligation and esophagoduodenostomy.48 healthy male Sprague-Dawley rats were equally divided into the following 6 groups using random number table method:normal control group,sham-operation group,model control group,omeprazole group,low-dose quercetin group,and high-dose quercetin group.The 6 groups were treated with peritoneal injection of 2 ml normal saline (normal control,sham-operation,model control groups),20 mg/kg omeprazole,100 mg/kg quercetin (low-dose) and 200 mg/kg quercetin (high-dose) once daily,respectively.The rats were sacrificed after 6 weeks of intervention.The microscopic pathological changes of esophageal mucosa were scored.NF-κB p65 and IL-6 protein levels in esophageal mucosa and serum were assessed using immunohistochemistry and enzyme-linked immunosorbent assay,respectively.Results In normal control group,shamoperation group,model control group,omeprazole group,low-dose quercetin group and high-dose quercetin group,the pathological scores of esophageal mucosa were 0.250 ± 0.463,0.250 ± 0.463,2.625 ± 0.518,1.500 ±0.535,1.250 ±0.463,and 1.375 ±0.518; the NF-κB p65 protein scores in esophageal mucosa were 0.500±0.535,0.625 ±0.518,3.500 ±0.535,1.875 ±0.649,1.750 ±0.707,and 2.000 ±0.535; the IL-6 protein scores in esophageal mucosa were 1.125 ± 0.641,1.125 ± 0.835,5.375 ± 0.518,2.375 ± 0.518,2.000 ±0.535,and 2.250 ±0.463; the serum NF-κB p65 protein levels were (68.618 ± 18.500),(77.824 ± 22.228),(184.882 ± 49.165),(106.693 ± 45.312),(76.215 ± 16.588),and (108.207 ± 42.107) pg/ml; the serum IL-6 protein levels were (24.826 ±4.008),(23.599 ±4.351),(51.378 ± 9.697),(32.370 ± 11.657),(23.085 ± 4.660),and (26.243 ± 4.955) pg/ml.In terms of the 5 indicators,there were no statistically significant differences between the normal control group and the sham-operation group (P =1.000,P =0.642,P =1.000,P =0.518,P =0.673) ; the results in the normal control,shamoperation,omeprazole,low-dose quercetin,and high-dose quercetin groups were significantly different from those in the model control group (P < 0.001,P < 0.001,P < 0.001,P =0.002,P =0.001 ; P < 0.001,P < 0.001,P<0.001,P=0.004,P=0.002; P=0.001,P<0.001,P<0.001,P=0.025,P=0.023; all P <0.001 ; P <0.001,P <0.001,P <0.001,P =0.023,P <0.001) ; there were no statistically significant differences between low-dose quercetin group and omeprazole group,nor between high-dose quercetin group and omeprazole group (P=0.334,P=0.717,P=0.176,P=0.121,P =0.074; P =0.642,P=0.678,P=0.619,P =0.949,P =0.225); there were no statistically significant differences between low-dose quercetin group and high-dose quercetin group (P =0.619,P =0.438,P =0.334,P =0.086,P =0.243).The microscopic pathological score of esophageal mucosa was positively correlated with NF-κB p65 and IL-6 protein scores in esophageal mucosa (r =0.803,P < 0.001 ; r =0.758,P < 0.001),also positively correlated with serum NF-κB p65 and IL-6 protein levels (r=0.486,P=0.004; r=0.544,P=0.001).Conclusions The expression levels of NF-κB p65 and IL-6 protein in esophageal mucosa and serum increase with the severity of esophageal mucosal injury.Quercetin can reduce the severity of esophageal mucosal injury in RE,possibly through down-regulating NF-κB and IL-6 expression and mitigatng esophageal inflammatory status.