不同灸量隔姜灸对脾虚证大鼠胃组织丝裂原细胞外激酶和细胞外调节蛋白激酶表达的影响

不同灸量隔姜灸对脾虚证大鼠胃组织丝裂原细胞外激酶和细胞外调

节蛋白激酶表达的影响

目的观察不同灸量隔姜灸对脾虚证大鼠胃组织丝裂原细胞外激酶（MEK）1/2和细胞外调节蛋白激酶（ERK）1/2表达的影响，探讨隔姜灸治疗脾虚证的可能作用机制及量效特征。方法将75只SD大鼠按随机数字表法分为空白对照组、模型组、隔姜灸3壮组、隔姜灸6壮组、隔姜灸9壮组，每组15只。200%大黄浓缩液4 ℃灌胃制作脾虚证大鼠模型。造模成功后，隔姜灸组选取“足三里”“中脘”予不同灸量治疗，连续8 d。HE染色镜下观察大鼠胃组织病理学改变，免疫组化检测大鼠胃组织MEK1/2及ERK1/2的蛋白表达。结果与空白对照组比较，模型组大鼠胃黏膜损伤明显，可见较大的破损、脱落；与模型组比较，隔姜灸3壮组大鼠胃黏膜表面有部分脱落、破损情况改善，隔姜灸6壮组和隔姜灸9壮组大鼠胃黏膜表面较完整、脱落及破损明显改善。与空白对照组比较，模型组胃组织MEK1/2及ERK1/2蛋白表达明显升高（P＜0.01）；与模型组比较，隔姜灸各组胃组织MEK1/2及ERK1/2蛋白明显表达升高（P＜0.01）；与隔姜灸3壮组比较，隔姜灸6壮组和隔姜灸9壮组胃组织MEK1/2及ERK1/2蛋白表达明显升高（P＜0.01），但二者作用效果相当，差异无统计学意义（P>0.05）。结论隔姜灸通过提高胃组织MEK1/2及ERK1/2的蛋白表达，激活MEK/ERK信号转导通路，进而促进脾虚证大鼠胃黏膜的修复。

Abstract：Objective To observe effects of different dosages of moxibustion with ginger-separated moxibustion on expressions of mitogen extracellular kinase （MEK）1/2 and extracellular regulated protein kinase （ERK）1/2 of gastric tissue in rats with spleen deficiency；To explore the possible mechanism and the dose-effect relationship. Methods Seventy-five SD rats were randomly divided into blank control group，model group，ginger-separated moxibustion for three zhuang group，six zhuang group and nine zhuang group according to random digits table method，with fifteen rats in each group. The rat model of spleen deficiency was established by intragastric administration with 200% Rhei Radix et Rhizoma infusion at 4 ℃. Ginger-separated moxibustion groups were treated with different dosage of moxibustion at “Zusanli”，“Zhongwan” for eight days after the modeling. Pathological changes of gastric tissue by HE staining were observed under light microscope，and immunohistochemistry was used to detect the expressions of MEK1/2 and ERK1/2 protein in gastric tissue of rats. Results Compared with the blank

control group rats，the gastric mucosa injury in the model group was obvious，which showed that the damage and abscission was more serious；compared with the model group，the gastric mucosa of rats was partly exfoliated and the damage was improved in three zhuang group，and the surface of gastric mucosa of rats was more complete and damage was improved obviously in six zhuang group and nine zhuang group；compared with the blank control group，the expressions of MEK1/2 and

ERK1/2 protein in gastric tissue increased obviously in other groups （P＜0.01）；compared with three zhuang group，the expressions of MEK1/2 and ERK1/2 protein in gastric tissue increased in six zhuang group and nine zhuang group （P＜0.01），but the effects of the two group were similar，without statistical significance （P>0.05）. Conclusion Ginger-separated moxibustion can repair gastric mucosa in rats with spleen deficiency，which may be closely associated with its effect in increasing the expressions of MEK1/2 and ERK1/2 protein in gastric tissue and activating the MEK/ERK signal transduction pathway.Keywords：dosage of moxibustion；ginger-separated moxibustion；spleen deficiency；proliferation and restoring of gastric mucosa；mitogen activated protein kinase；extracellular regulated protein kinase；rats

脾虚证是指脾气虚弱导致脾失运化的病证，是消化性溃疡、慢性胃炎等消化系统疾病的常见中医证型。有研究发现，脾虚大鼠胃底部黏膜缺损、炎细胞浸润、血管扩张充血、黏膜下层水肿[1]。丝裂原活化蛋白激酶（mitogen activated protein kinase，MAPK）信号传导通路作用于核转录因子，与细胞增殖和凋亡关系密切，并参与胃黏膜炎症及损伤过程，其中丝裂原细胞外激酶（MEK）/细胞外调节蛋白激酶（ERK）是MAPK信号传导通路中的关键环节[2-3]。临床研究显示，针灸治疗脾胃虚寒证胃炎疗效确切，尤其是艾灸疗法的作用更加明显[4]。前期研究表明，隔姜灸不仅能改善脾胃虚寒证患者的临床症状，还具有抗氧化等良性调节作用[5]。本实验在前期研究的基础上，观察不同灸量隔姜灸对MEK1/2及ERK1/2蛋白表达的影响，从MEK/ERK信号通路角度探讨不同灸量隔姜灸对脾虚证大鼠胃黏膜的保护作用及量效特征，以期为隔姜灸治疗脾虚证的临床运用提供依据。

1 材料与方法

1.1 动物及分组

雌性健康SD大鼠75只，SPF级，月龄2～3月，体质量200～250 g，湖南斯莱克景达实验动物有限公司，动物许可证号SCXK（湘）2011-0003。饲养于温度20～25 ℃、相对湿度50%～70%环境。75只SD大鼠按随机数字表法分为空白对照组、模型组、隔姜灸3壮组、隔姜灸6壮组、隔姜灸9壮组，每组15只。

1.2 主要试剂与仪器

艾条，南阳市卧龙汉医艾绒厂；生姜片，厚2～3 mm，直径1.8 cm；ERK1/2试剂盒（美国Proteintech Group公司），MEK1/2试剂盒（英国Abcam公司），石蜡、中性树胶（美国Sigma公司），苏木素、PBS、枸橼酸盐缓冲液、二步法试剂盒（Wellbio公司），DAB试剂盒（北京中杉金桥生物技术有限公司），常规化学试剂（国药控股上海生物医药有限公司）。摇床（其林贝尔仪器制造有限公司），恒温箱（北京六一仪器厂），微波炉（美的集团），切片刀（德国莱卡），包埋机（常州中威电子仪器有限公司），精密pH计（雷磁上海仪电科学仪器股份有限