黄芪建中汤干预脾虚型慢性萎缩性胃炎大鼠胃黏膜表皮生长因子含量、诱导型一氧化氮合成酶和表皮生长因子受体基因的表达

Effects of huangqi jianzhong decoction on the expression of epidermal growth factor,mRNA of inducible Nitric-oxide synthase and epidermal growth factor receptor in stomach mucosa of chronic atrophic gastritis rats with syndrome of spleen deficiency

目的:观察黄芪建中汤对造模脾虚型慢性萎缩性胃炎大鼠的治疗作用及机制,并且为临床应用提供试验依据。方法:实验于2003-12/2005-09在河南中医学院实验动物中心和郑州大学细胞分子生物学研究中心完成。雄性Wistar大鼠30只,体质量(170±10)g,饲养1周后按体质量编号完全随机法分为4组,即正常对照组6只:不做任何处理;慢性萎缩性胃炎组8只:以2%的水杨酸钠灌胃8周造成胃黏膜损伤,后4周结合饥饱失常、劳倦过度使大鼠致虚;维酶素组8只:造模后,给予7.9%维酶素2mL灌胃,1次/d,连续21d;黄芪建中汤组8只:造模后,给予2.5kg/L黄芪建中汤水箭剂2mL灌胃,1次/d,连续21d。末次给药后所有大鼠再逐只处死,平行胃小弯取小块胃窦部胃壁组织,手工制成胃黏膜组织芯片,用原位杂交的方法检测诱导型一氧化氮合成酶-mRNA和表皮生长因子受体-mRNA的表达量。从剩余胃黏膜组织中精确称取2g,置于玻璃匀浆器中研磨制成组织匀浆,按放免试剂盒说明测定其表皮生长因子含量。结果:纳入大鼠30只,造模过程中死亡1只,造模结束后抽样处死5只,24只进入结果分析。①原位杂交结果:诱导型一氧化氮合成酶-mRNA和表皮生长因子受体-mRNA基因表达的阳性部位呈现紫蓝色颗粒,前者以慢性萎缩性胃炎组显色最强,后者主要以胃小凹底部和胃腺中上部显色突出。②与正常对照组大鼠相比,慢性萎缩性胃炎组大鼠胃黏膜组织的上述指标均显著增高[(333.00±56.71),(453.67±68.25);(0.73±0.21)(3.14±0.77)μg/L;(124.36±26.13),(318.42±45.38),(P<0.01,P<0.05)],经黄芪建中汤治疗21d后,恢复至接近正常水平,其中对诱导型一氧化氮合成酶-mRNA和表皮生长因子受体-mRNA表达量的恢复作用显著优于维酶素。结论:黄芪建中汤对慢性萎缩性胃炎有良好的治疗作用,其机制与下调慢性萎缩性胃炎大鼠胃黏膜表皮生长因子含量、诱导型一氧化氮合成酶-mRNA及表皮生长因子受体-mRNA的表达有关,其疗效优于维酶素。

AIM： To observe the therapeutic mechanism of huangqi jianzhong decoction in the established rat model of chronic atrophic gastritis with spleen deficiency, so as to provide experimental data for clinical application. METHODS： The experiment was conducted in the Cell-Molecular Biology Research Center of Zhengzhou University from December 2003 to September 2005. Thirty male Wistar rats of （170±10） g were selected and randomly divided into 4 groups according to the body mass： normal control group （n=6）： without any treatment; chronic atrophic gastritis group （n=8）： The rats models of chronic gastritis were established by infusing 2% saccylic acid for 8 weeks combined with disorder administration and exhaustion for 4 weeks; Vitacoenzyme group （n=8）： After modeling, the rats were infused with 2 mL 7.9% Vitacoenzyme once per day for 21consecutive days; huangqi jianzhong decoction group （n =8）： After modeling, the rats were infused with 2 mL 2.5 kg/L huangqi jianzhong decoction （double distilled water） once daily for 21 consecutive days. After administration for the last time, all the rats were executed to cut down the sinus ventriculi gastric wall tissue on a parallel with lesser curvature of stomach, which were made into tissue array by handiwork. The expressions of iNOS- mRNA and epidermal growth factor receptor （EGFR）-mRNA in stomach mucosa were examined, respectively by in situ hybridization. 2 g surplus gastric mucosa was weighed and put into homogenizer to grind into tissue homogenate; its EGF content was detected according to the Farr test kit. RESULTS： Thirty rats were involved in the experiment. One rat died during modeling and five rats were sampled, respectively from the chronic atrophic gastritis group, Vitacoenzyme group and huangqi jianzhong decoction group after rat models were established. Finally, 24 rats were involved in the result analysis. （1） Result of in situ hybridization The positive part of iNOS-mRNA and EGFR-mRNA expression showed hyaeinthine bead; the former coloration was shown strongest in the chronic atrophic gastritis group and the latter was mainly shown at the bottom of the gastric pits and the medio-superior part of the gastric gland. （2） Compared with the control group, all above parameters in the chronic atrophic gastritis group were increased significantly [（333.00±56.71）, （453.67±68.25）; （0.73±0.21）, （3.14±0.77）; （124.36±26.13）, （318.42±45.38）μg / L, P 〈 0.01, P 〈 0.05]. After treated with huangqi jianzhortg decoction for 21 days, each symptom and sign nearly recovered to the normal level, The huangqi jianzhong decoction had superior effect on the expression of iNOS gene and EGFR gene compared with Vitacoenzyme. CONCLUSION： Huangqi jianzhong decoction has protective effect on chronic atrophic gastritis, which predominates over Vitacoenzyme. Its mechanism might be related to down-regulating the expressions of EGF, iNOS-mRNA and EGFR-mRNA.