和胃降逆方对非糜烂性反流病大鼠食管黏膜组织中肥大细胞类胰蛋白酶及相关因子表达的影响

Effect of Hewei Jiangni recipe on the expression of mast cell tryptase in esophageal mucosa and related factors of rats with non-erosive reflux disease

目的探讨和胃降逆方对非糜烂性反流病(NERD)大鼠血清P物质(SP)、降钙素基因相关肽(CGRP)、特异性激活蛋白激酶2(PAR2)及食管黏膜组织中肥大细胞类胰蛋白酶(MCT)表达的影响。方法采用随机数字表法将48只无特定病原体级雄性Sprague Dawley大鼠分为空白组、模型组、奥美拉唑组、和胃降逆配方颗粒组(配方颗粒组)、和胃降逆方水煎剂组(水煎剂组)及和胃降逆方颗粒剂高、中、低剂量组(颗粒剂高、中、低剂量组),每组6只。模型组、奥美拉唑组、配方颗粒组、水煎剂组及和胃降逆方颗粒剂高、中、低剂量组大鼠腹腔注射卵清蛋白和氢氧化铝佐剂混悬液,构建内脏高敏感性NERD大鼠模型;空白组大鼠腹腔注射等量生理盐水;24 h后,奥美拉唑组、配方颗粒剂组、水煎剂组及和胃降逆方颗粒剂高、中、低剂量组大鼠按照10 mL·kg^-1分别给予840 mg·L^-1奥美拉唑溶液、850 g·L^-1和胃降逆配方颗粒溶液、850 g·L^-1和胃降逆方水煎剂溶液及440、220、140 g·L^-1和胃降逆方颗粒剂溶液灌胃,空白组、模型组大鼠给予10 mL·kg^-1的蒸馏水灌胃,每日1次,连续2周。灌胃后第14天,模型组及各干预组大鼠给予弱酸食管滴注,空白组大鼠给予蒸馏水食管滴注,然后处死大鼠,取血清及食管黏膜组织;苏木精-伊红染色观察大鼠食管黏膜鳞状上皮层病理变化,酶联免疫吸附试验法检测大鼠血清SP、CGRP、PAR2的表达,免疫组织化学法检测大鼠食管黏膜组织中MCT的表达。结果空白组大鼠食管黏膜鳞状上皮细胞间隙紧密,无明显炎性细胞浸润、鳞状上皮层增生等表现;模型组大鼠食管黏膜鳞状上皮细胞间隙明显增宽,鳞状上皮层存在少量炎性细胞浸润,乳突出现一定延长;奥美拉唑组及各中药干预组大鼠可见增宽的细胞间隙不同程度减小或恢复,炎性细胞及鳞状上皮层增生均不明显。与空白组比较,模型组大鼠血清SP、CGRP蛋白表达水平和食管黏膜组织中MCT相对表达量显著升高(P<0.01),2组大鼠血清PAR2蛋白表达水平比较差异无统计学意义(P>0.05)。与模型组比较,奥美拉唑组、颗粒中剂量组大鼠血清SP蛋白表达水平下降(P<0.05),其余各用药组大鼠血清SP蛋白表达水平与模型组比较差异无统计学意义(P>0.05)。与模型组比较,奥美拉唑组及颗粒剂高、中剂量组大鼠血清CGRP蛋白表达水平显著下降(P<0.01),其余各用药组大鼠血清CGRP蛋白表达水平与模型组比较差异无统计学意义(P>0.05)。与模型组比较,颗粒剂高剂量组大鼠血清PAR2蛋白表达水平升高(P<0.05),其余各用药组大鼠血清PAR2蛋白表达水平与模型组比较差异无统计学意义(P>0.05)。与模型组比较,奥美拉唑组、水煎剂组及颗粒剂中、低剂量组大鼠食管黏膜组织中MCT相对表达量显著降低(P<0.01),配方颗粒剂组、颗粒剂高剂量组大鼠食管黏膜组织中MCT相对表达量与模型组比较差异无统计学意义(P>0.05)。其余各用药组大鼠血清SP蛋白表达水平与奥美拉唑组比较差异无统计学意义(P>0.05)。水煎剂组和颗粒剂低剂量组大鼠血清CGRP蛋白表达水平高于奥美拉唑组(P<0.05,P<0.01),其余各用药组大鼠血清CGRP蛋白表达水平与奥美拉唑组比较差异均无统计学意义(P>0.05)。和胃降逆方各用药组大鼠血清PAR2蛋白表达水平与奥美拉唑组比较差异均无统计学意义(P>0.05)。配方颗粒剂组、颗粒剂高剂量组大鼠食管黏膜组织中MCT相对表达量与奥美拉唑组比较显著增高(P<0.01);水煎剂组及颗粒剂中、低剂量组大鼠食管黏膜组织中MCT相对表达量与奥美拉唑组比较差异无统计学意义(P>0.05)。结论和胃降逆方可抑制大鼠食管黏膜组织中肥大细胞激活,降低血清神经肽类物质SP、CGRP表达,一定程度减轻NERD大鼠食管黏膜组织炎性增生及细胞间隙增宽状态,从而调整内脏高敏感状况;其中220 g·L^-1和胃降逆方颗粒剂对NERD大鼠的改善效果最佳。

Objective To investigate the effects of Hewei Jiangni recipe on the expression of substance P(SP),calcitonin gene-related peptide(CGRP),specific activated protein kinase 2(PAR2)in serum and mast cell tryptase(MCT)in esophageal mucosa in rats with non-erosive reflux disease(NERD).Methods Forty-eight male specific pathogen free Sprague Dawley rats were randomly divided into blank group,model group,omeprazole group,Hewei Jiangni ormula granule group(formulated granule group),Hewei Jiangni decoction group(decoction group)and the high,middle and low dose of Hewei Jiangni granule group,with 6 rats in each group.The rats in the model group,omeprazole group,formulated granule group,decoction group and the high,middle and low dose of Hewei Jiangni granule group were intraperitoneally injected with ovalbumin and aluminum hydroxide adjuvant suspension to establish the visceral hypersensitivity NERD rat model;the rats in the blank group were intraperitoneally injected with the same amount of normal saline.Twenty-four hours later,the rats in the omeprazole group,formula granule group,decoction group and the high,middle and low dose of Hewei Jiangni granule group were given 840 mg·L^-1 omeprazole solution,850 g·L^-1 formula granule solution,850 g·L^-1 decoction and 440,220,140 g·L^-1 Hewei Jiangni granule solution respectively according to the amount of 10 mL·kg^-1;the rats in the blank group and model group were given distilled water(10 mL·kg^-1),once a day for two weeks.On the 14 th day after intragastric administration,the rats in the model group and the intervention group were given weak acid esophagus drip,and the rats in the blank group were given distilled water esophagus drip.Then the rats were executed,and the serum,esophageal mucosal tissue were collected.The pathological changes of esophageal squamous epithelium were observed by hematoxylin-eosin staining.The expressions of SP,CGRP and PAR2 in serum of rats were detected by enzyme-linked immunosorbent assay.The expression of MCT in esophageal mucosa of rats was detected by immunohistochemistry.Results In the blank group,there was a tight space between squamous epithelial cells in esophageal mucosa of rats,and no obvious inflammatory cell infiltration and squamous epithelial hyperplasia.In the model group,the squamous epithelial cell space of esophageal mucosa was significantly widened,a small amount of inflammatory cell infiltration was found in the squamous epithelial layer,and the mastoid process was prolonged to a certain extent.In the omeprazole group and all traditional Chinese medicine intervention group,the widened cell space decreased or recovered to varying degrees,and the proliferation of inflammatory cells and squamous epithelium were not obvious.The levels of SP and CGRP protein in serum and the relative expression of MCT in esophageal mucosa of rats in the model group were significantly higher than those in the blank group(P<0.01),but there was no significant difference in the level of PAR2 protein in serum between the model group and the blank group(P>0.05).The level of SP protein in serum of rats in omeprazole group and middle dose of Hewei Jiangni granule group was lower than that in the model group(P<0.05),and there was no significant difference in the level of SP protein in serum between all intervention groups and model group(P>0.05).The level of CGRP protein in serum of rats in the omeprazole group and the high,middle dose of Hewei Jiangni granule group was significantly lower than that in the model group(P<0.01),and there was no significant difference in the level of CGRP protein in serum between all intervention groups and the model group(P>0.05).The level of PAR2 protein in the high dose of Hewei Jiangni granule group was significantly higher than that in the model group(P<0.05),and there was no significant difference in the level of PAR2 protein in serum between all intervention groups and the model group(P>0.05).The relative expression of MCT in esophageal mucosa of rats in omeprazole group,decoction group and the middle,low dose of Hewei Jiangni granule group was significantly lower than that in the model group(P<0.01),and there was no significant difference in the relative expression of MCT between the formula granule group,the high dose of Hewei Jiangni granule group and the model group(P>0.05).There was no significant difference in the level of SP protein in serum of rats between the intervention groups(P>0.05).The level of CGRP protein in serum of rats in the decoction group and the low dose of Hewei Jiangni granule group was higher than that in the omeprazole group(P<0.05,P<0.01);there was no significant difference in the level of CGRP protein in serum of rats between the other intervention groups and omeprazole group(P>0.05).There was no significant difference in the level of CGRP protein in serum of rats between the Hewei Jiangni recipe groups and the omeprazole group(P>0.05).The relative expression of MC protein in esophageal mucosa of rats in the formula granule group and the high dose of Hewei Jiangni granule group was significantly higher than that in the omeprazole group(P<0.01).There was no significant difference in the relative expression of MCT in esophageal mucosa of rats between the(P>0.05).Conclusion Hewei Jiangni recipe can inhibit the activation of mast cell in the esophageal mucosa tissue of rats,reduce the levels of SP and CGRP in serum,also reduce the inflammatory proliferation and the widening of cell space in the esophageal mucosa tissue of NERD rats to a certain extent,so as to adjust the visceral hypersensitivity of NERD rats.The dose of 220 g·L^-1 Hewei Jiangni recipe granule has the best effect on NERD rats.