目的:探讨黄龙汤对脓毒症大鼠肠黏膜上皮紧密连接蛋白Claudin-1和Occludin的mRNA及蛋白表达水平的影响。方法将55只Wistar大鼠按随机数字表法分为假手术组(n=10)、模型组(n=15)、西药治疗组(n=15)和中西药联合组(n=15)。采用盲肠结扎穿孔术(CLP)复制脓毒症模型,假手术组只手术不行CLP。假手术组和模型组灌胃2 mL生理盐水,西药治疗组腹腔注射美罗培南每次50 mg;中西药联合组在西药治疗基础上灌胃中药黄龙汤〔生大黄(后下)15 g,芒硝10 g,枳实15 g,厚朴10 g,人参10 g,当归20 g,黄芩15 g,甘草10 g煎浓成150 mL〕每次2 mL(含生药0.68 g/mL),4组均每日2次。给药4 d后处死大鼠,取临近回盲部的部分回肠组织约10 cm,用反转录-聚合酶链反应(RT-PCR)检测肠组织Claudin-1和Occludin的mRNA表达水平,用蛋白质免疫印迹试验(Western Blot)检测Claudin-1和Occludin的蛋白表达水平。结果与假手术组比较,模型组、西药治疗组及中西药联合组大鼠小肠上皮Claudin-1和Occludin的mRNA〔积分吸光度(IA)比值〕及蛋白表达水平(灰度值比值)均明显下降(Claudin-1 mRNA:0.327±0.029、0.387±0.035、0.420±0.053比0.561±0.035,Occludin mRNA:0.364±0.058、0.422±0.033、0.442±0.048 比0.616±0.036,Claudin-1蛋白:0.307±0.015、0.337±0.017、0.355±0.027 比0.426±0.017,Occludin 蛋白:0.326±0.028、0.355±0.017、0.366±0.025比0.455±0.018,P<0.05或P<0.01);与模型组比较,西药治疗组和中西药联合组Claudin-1、Occludin的mRNA及蛋白表达显著上调(P<0.05或P<0.01);且以中西药联合组升高更显著(均P<0.05)。结论黄龙汤对脓毒症大鼠肠黏膜屏障的保护作用可能是通过上调脓毒症模型大鼠小肠组织中Claudin-1和Occludin的mRNA及蛋白表达水平而实现的。
Objective To investigate the effects of Huanglong decoction on mRNA and protein expression levels of intestinal epithelial tight junction protein Claudin-1 and Occludin in rat with sepsis. Methods Fifty-five Wistar rats were randomly divided into sham operation group (n=10), model group (n=15), western medicine treatment group (15 rats) and combined traditional Chinese and western medicine group (n=15). In sepsis model group, cecal ligation with puncture (CLP) was reproduced. In the sham operation group, the steps of operation performed were similar to those in the model group, but without CLP. Normal saline 2 mL was given to sham operation group and model group by gavage;meropenem 50 mg was given to the western medicine treatment group by intra-peritoneal injection; in the combined traditional Chinese and western medicine treatment group, besides meropenem 50 mg was injected intra-peritoneally, 2 mL Huanglong decoction (containing raw herbal medicine 0.68 g/mL) was administered by gavage [ingredients of the decoction: rhubarb 15 g (put into water the latest), Glauber salt 10 g, fructus aurantii immaturus 15 g, mangnoliae officinalis 10 g, ginseng 10 g, Angelica sinensis 20 g, Scutellaria baicalensis 15 g, Radix Glycyrrhizae 10 g, concentrated to a decoction 150 mL], in the four groups, the respective drugs or normal saline were given twice a day. After 4 days, the rats were sacrificed, and a partial ileal tissue close to the ileocecal region about 10 cm in length was taken; and mRNA expression levels of Claudin-1 and Occludin were detected by reverse transcription-polymerase chain reaction (RT-PCR), and protein expression levels of Claudin-1 and Occludin were detected by Western Blot. Results Compared with the sham operation group, the expression levels of Claudin-1 and Occludin mRNA [integrated absorbance (IA) ratio] and protein (gray value ratio) were significantly lowered in model group, western medicine group and combined therapy group (Claudin-1 mRNA: 0.327±0.0