Effect of Huoxue Tongjiang Decoction on esophageal visceral hypersensitivity and lipopolysaccharide/Toll-like receptor 4/mast cell pathway in rats with reflux esophagitis
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摘要: 目的 研究活血通降方对反流性食管炎(reflux esophagitis，RE)模型大鼠脂多糖(lipopolysaccharide，LPS)/Toll样受体4(Toll-like receptor 4，TLR4)/肥大细胞(mast cell，MC)通路的影响，探讨其对食管内脏高敏感的疗效机制。方法 将50只Wistar大鼠随机分为对照组、模型组、西药组、中药高剂量组及中药低剂量组5个组，每组10只。对照组行腹腔开关术，其余各组均采用改良部分贲门肌切开术+外置幽门部分结扎术联合束缚应激建立RE大鼠模型。造模7 d后，各组分别给予相应剂量药物或相同体积生理盐水灌胃，连续给药14 d，2次/d。苏木精-伊红染色评价食管黏膜病理表现及评分；采用行为学评分和观察肌电图变化评价大鼠食管的内脏敏感性；采用甲苯胺蓝染色检测食管组织中MC数目；采用免疫组织化学法检测食管组织类胰蛋白酶(tryptase，TPS)和结肠组织中紧密连接蛋白ZO-1及Occludin的表达。采用酶联免疫吸附法检测血清LPS、IL-8含量，采用蛋白免疫印迹法检测食管组织中TLR4的表达，结果 与对照组比较，模型组大鼠滴酸刺激时的行为学评分及颈斜方肌肌电图曲线下面积(area under curve，AUC)比值均明显增高，差异有统计学意义(P < 0.01)；与模型组比较，西药、低剂量及高剂量中药干预后，各组大鼠行为学评分及肌电图AUC比值均降低，差异有统计学意义(P < 0.01)。其中，与西药组比较，中药各组肌电图AUC比值降低更为显著，差异有统计学意义(P < 0.05)。与对照组比较，模型组血清LPS、IL-8的含量、食管黏膜病理评分和TLR4表达水平、食管组织中MC数目及TPS表达水平均明显升高，差异有统计学意义(P < 0.05)；结肠组织中Occludin与ZO-1的表达均显著下降，差异有统计学意义(P < 0.01)。给予药物干预后，中药低剂量组及高剂量组大鼠血清LPS、IL-8含量、食管黏膜病理评分和TLR4表达、食管组织中MC数目及TPS的表达水平均显著降低，差异有统计学意义(P < 0.01)；结肠组织中Occludin与ZO-1的表达均上调，差异有统计学意义(P < 0.05)。中药高剂量组对上述指标的作用均优于西药组，差异有统计学意义(P < 0.05)。结论 活血通降方可改善RE大鼠食管黏膜炎症和内脏高敏感，机制可能与保护肠黏膜屏障功能，降低LPS水平，抑制LPS/TLR4/MC通路，减少MC脱颗粒和TPS的释放有关。Abstract: Objective To study the effect of Huoxue Tongjiang Decoction on lipopolysaccharide(LPS)/Toll-like receptor 4(TLR4)/mast cell(MC) pathway in reflux esophagitis(RE) model rats, and to explore the therapeutic mechanism of Huoxue Tongjiang Decoction on esophageal visceral hypersensitivity.Methods A total of 50 Wistar rats were randomly divided into control group, model group, Western Medicine group, high dose Chinese Medicine group and low dose Chinese Medicine group, with 10 rats in each group. The control group underwent an abdominal switch surgery, and the other four groups were treated with modified partial cardiac myotomy + external pylorus partial ligation and restraint stress to establish the RE model rats. After seven days of modeling, each group was given corresponding doses of drugs or the same volume of physiological saline by gavage for 14 consecutive days, twice a day. HE staining was used to evaluate the pathological manifestations and scoring of esophageal mucosae. Evaluation of visceral sensitivity of rat esophagus using behavioral scoring and observation of electromyographic changes. Toluidine blue staining was used to detect the number of MC in esophageal tissues. The expression of TPS in esophageal tissues and the tight junction proteins ZO-1 and Occludin in colon tissues were detected by immunohistochemistry. The serum levels of LPS and IL-8 were detected by enzyme-linked immunosorbent assay. The expression of TLR4 in esophageal tissues was detected by Western blot.Results Compared with the control group, the behavioral scores and area under curve(AUC) ratio of cervical trapezius muscle electromyography in the model group were significantly increased(P < 0.01). Compared with the model group, after the intervention of western medicine, low dose and high dose of Traditional Chinese Medicine, the behavioral scores and AUC ratio of EMG of rats in each group were decreased(P < 0.01). Among them, compared with the Western Medicine group, the AUC ratio of EMG in the Chinese Medicine group decreased more significantly(P < 0.05). Compared with the control group, the serum levels of LPS and IL-8, the pathological score and expression of TLR4 in esophageal mucosa, the number of MC and the expression of TPS in esophageal mucosa in the model group were significantly increased(P < 0.05). The expressions of Occludin and ZO-1 in colonic mucosa were significantly decreased(P < 0.01). After drug intervention, the serum LPS and IL-8 content, TLR4 expression in esophageal mucosa, the number of MC and TPS expression in esophageal mucosa of the low dose and high dose of Chinese Medicine group were significantly decreased(P < 0.01); the expressions of ZO-1 and Occludin in colonic mucosa were up-regulated(P < 0.05). The effects of high dose of Traditional Chinese Medicine on the above indicators were better than those of Western Medicine group(P < 0.05).Conclusion Huoxue Tongjiang Decoction can improve esophageal mucosal inflammation and visceral hypersensitivity in RE rats, which may be related to protecting the intestinal mucosal barrier function, reducing LPS levels, inhibiting the LPS/TLR4/MC pathway, reducing MC degranulation and TPS release.
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表 1 各组大鼠行为学评分及颈斜方肌肌电图的AUC比较
X±S 组别 例数 行为学评分/分 AUC /% 对照组 5 1.80±0.45 183.02±21.22 模型组 5 3.67±0.241) 350.35±37.391) 西药组 5 2.27±0.432) 245.96±25.271)2) 中药低剂量组 5 2.00±0.242) 208.39±20.892)3) 中药高剂量组 5 1.87±0.382) 197.10±33.242)3) 与对照组比较，1) P < 0.01；与模型组比较，2) P < 0.01；与西药组比较，3) P < 0.05。 
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表 2 各组大鼠食管黏膜MC数目及TPS的表达水平
X±S 组别 例数 MC数目 TPS表达(MOD值) 对照组 5 5.00±1.58 0.19±0.03 模型组 5 17.40±0.891) 0.31±0.031) 西药组 5 10.40±1.511)2) 0.23±0.032) 中药低剂量组 5 7.80±0.841)2)3) 0.18±0.042)3) 中药高剂量组 5 8.20±0.841)2)3) 0.18±0.022)3) 与对照组比较，1) P < 0.01；与模型组比较，2) P < 0.01；与西药组比较，3) P < 0.05。
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表 3 各组大鼠结肠黏膜中ZO-1及Occludin的表达
MOD值，X±S 组别 例数 ZO-1表达 Occludin表达 对照组 5 0.32±0.03 0.33±0.03 模型组 5 0.24±0.021) 0.25±0.041) 西药组 5 0.27±0.021)2) 0.29±0.04 中药低剂量组 5 0.30±0.012)3) 0.30±0.042) 中药高剂量组 5 0.30±0.022)3) 0.33±0.042) 与对照组比较，1) P < 0.05；与模型组比较，2) P < 0.05；与西药组比较，3) P < 0.05。
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