和胃降逆方对卵清蛋白联合酸灌注诱导非糜烂性反流病大鼠食管黏膜中PAR2、SP、CGRP蛋白表达的影响

Effect of Hewei Jiangni recipe on PAR2/SP/CGRP protein expression in esophageal mucosa induced by ovalbumin combined with acid perfusion in rats with non-erosive reflux disease

目的观察和胃降逆方对卵清蛋白联合酸灌注诱导的非糜烂性反流病大鼠食管黏膜中特异性激活蛋白激酶2(specific activated protein kinase 2,PAR2)、P物质(substance P,SP)、降钙素基因相关肽(calcitonin gene-related peptide,CGRP)等相关蛋白表达的影响。方法雄性SD大鼠适应性喂养1周后,随机分为空白对照组5只和实验组25只。实验组采用腹腔注射卵清蛋白致敏剂诱导内脏高敏感,24小时后随机分为5组,模型组、和胃降逆方(高、中、低)剂量组、奥美拉唑组,每组5只。和胃降逆方(高、中、低)剂量组及奥美拉唑组分别给予相应的和胃降逆方水溶液及奥美拉唑水溶液灌胃,空白对照组、模型组均予等体积蒸馏水灌胃。干预2周后,悬尾实验评价大鼠内脏高敏感。验证成模后,联合弱酸灌注诱导非糜烂性反流病大鼠模型,通过透射电镜观察大鼠食管黏膜病理学变化,Western blotting方法检测大鼠食管黏膜PAR2、SP、CGRP蛋白表达,免疫荧光双标技术观察食管黏膜中与PAR2、SP、CGRP激活相关的肥大细胞(mast cell,MC)及辣椒素受体1(transient receptor potential V1,TRPV1)蛋白的共表达。结果(1)悬尾实验不动时间比较:与空白对照组相比,模型组大鼠不动时间显著减少(P<0.01);与模型组相比,和胃降逆方中剂量组、奥美拉唑组大鼠不动时间明显增多(P<0.01),和胃降逆方低剂量组大鼠不动时间增多(P<0.05)。(2)Western blotting方法检测PAR2、SP、CGRP表达比较:与空白对照组相比,模型组大鼠食管黏膜的PAR2、CGRP蛋白表达显著升高(P<0.01),SP蛋白表达升高明显(P<0.05);与模型组相比,各用药组PAR2的蛋白表达显著下降(P<0.01),各用药组SP的蛋白表达下降明显(P<0.05),各用药组CGRP的蛋白表达显著下降(P<0.01)。(3)MC与TRPV1共表达比较:与空白对照组相比,模型组大鼠食管黏膜的MC与TRPV1的共表达有一定程度的增多;与模型组相比,各用药组MC与TRPV1的共表达均不明显。结论和胃降逆方可通过降低PAR2、SP、CGRP的表达,改善非糜烂反流病大鼠的病理变化及内脏高敏感状态。

Objective To observe Hewei Jiangni recipe on specific activated protein kinase 2(PAR2)and substance P(SP)and calcitonin gene-related peptide(calcitonin gene-related peptide,CGRP)on the expression of calcitonin gene-related peptide in esophageal mucosa of SD rats with non-erosive reflux disease induced by ovalbumin combined with acid perfusion.Methods After one week of adaptive feeding,male SD rats were randomly divided into blank control group(n=5)and experimental groups(n=25).Visceral hypersensitivity was induced by intraperitoneal injection of ovalbumin sensitizer in the experimental group.24 hours later,the rats were randomly divided into five groups:model group,Hewei Jiangni prescription(high,middle and low)dose group and omeprazole group with 5 rats in each group.The high,middle and low dose groups and omeprazole group were given corresponding drugs respectively,while the blank control group and model group were given the same volume of distilled water.After 2 weeks of intervention,tail suspension test was used to evaluate the visceral hypersensitivity of rats.After the establishment of the model,the rat model of non-erosive reflux disease was induced by weak acid perfusion.The pathological changes of rat esophageal mucosa were observed by transmission electron microscope.Western blotting method was used to detect the expression of PAR2,SP and CGRP in rat esophageal mucosa.The co-expression of mast cell(MC)and transient receptor potential V1(TRPV1)proteins related to PAR2/SP/CGRP activation in esophageal mucosa was detected by immunofluorescence double labeling technique.Results(1)Comparison of immobility time in tail suspension test:compared with the blank control group,the immobility time in the model group was significantly lower than that in the blank control group(P<0.01).Compared with the model group,the immobility time in the middle dose group and omeprazole group was significantly higher than that in the model group(P<0.01),and that in the low dose group was significantly higher than that in the model group(P<0.05).(2)Comparison of PAR2,SP and CGRP expression by Western blotting:compared with the blank control group,the expression of PAR2 and CGRP protein in the model group was significantly higher than that in the blank control group(P<0.01).The expression of SP protein in the model group was significantly higher than that in the blank control group(P<0.05).Compared with the model group,the protein expression of PAR2 in each treatment group decreased significantly(P<0.01),the protein expression of SP in each treatment group decreased significantly(P<0.05),and the protein expression of CGRP in each treatment group decreased significantly(P<0.01).(3)Comparison of co-expression of MC and TRPV1:compared with the blank control group,the co-expression of MC and TRPV1 in the esophageal mucosa of the model group increased to a certain extent,but compared with the model group,the co-expression of MC and TRPV1 was not obvious in each treatment group.Conclusion Hewei Jiangni recipe can improve the pathological changes and visceral hypersensitivity of NERD rats by reducing the expression of PAR2,SP and CGRP.