【作者】 刘晓雯；

【导师】 杨牧祥；

【作者基本信息】 河北医科大学 ， 中医诊断学， 2008， 硕士

【摘要】 目的:胃炎饮由柴胡、香附、川楝子、元胡、炒白术、茯苓、炙甘草、川连、陈皮、枳壳、姜半夏、姜竹茹等药物组成,具有疏肝利胆,健脾益气,和胃降逆的功效,为导师多年临床观察筛选的效方,对胆汁反流性胃炎有较好的治疗效果。本研究采用反流液空腹灌胃法复制胆汁反流性胃炎大鼠模型,给药治疗后,通过观察各组大鼠胃黏膜病理形态、血浆胃动素（MTL）、胃黏膜P物质（SP）、胃黏膜前列腺素E₂（PGE₂）、血清胃泌素（GAS）等指标的变化,以验证胃炎饮的药效,并从调节胃肠肽角度探讨该药治疗胆汁反流性胃炎的作用机制。方法第一部分:胃炎饮对胆汁反流性胃炎大鼠胃黏膜病理形态学的影响健康wistar大鼠40只,雌雄各半,随机分为5组,每组8只,即正常组、模型组、胃炎饮高剂量组（予21.4g生药/Kg体重灌胃）、胃炎饮低剂量组（予10.7g生药/Kg体重灌胃）、吗叮啉对照组（予2.78mg/Kg体重灌胃）。胃炎饮高、低剂量组和吗叮啉对照组从造模开始后第二周至第五周造模结束每天给药,正常组和模型组同时灌服等容积生理盐水,每日1次,连续28天。除正常组外,其余各组以15ml/kg体重反流液空腹灌胃,每日1次,连续35天。最后一次灌胃后禁食24h,无菌操作剖腹取胃,沿胃大弯剪开,用冰生理盐水漂洗胃内容物,滤纸吸干,先肉眼观察胃黏膜的大体形态,再剪取胃窦部组织2块,每块约1mm3,4%多聚甲醛固定,逐级酒精脱水,常规石蜡包埋,切片,分别用HE染色和AB/PAS染色,光镜下观察胃黏膜病理形态学变化。在HE染色下做各组炎细胞半定量分析。在AB/PAS染色下,用测微尺在光镜100倍下测量胃黏膜AB/PAS阳性层厚度,自窦、体交界500um处开始,每张切片取平均间隔测量10个部位后取平均值。第二部分:胃炎饮对胆汁反流性胃炎大鼠血浆胃动素和胃黏膜P物质含量的影响动物分组、用药情况同前,每组8只。最后一次灌胃后禁食24h,无菌操作断头取血5ml,加入100g/L EDTA-2Na 0.2ml,在塑料管中混匀,3500 r/min离心15min,取血浆-20℃以下保存,待测MTL。无菌操作剖腹取胃,沿胃大弯剪开,用冰生理盐水漂洗胃内容物,滤纸吸干,用载玻片刮取胃窦部黏膜,称重后,冰浴下制备10%的组织匀浆,3500r/min离心15min,取上清,-20℃以下保存,留作SP含量检测。第三部分:胃炎饮对胆汁反流性胃炎大鼠胃黏膜前列腺素E₂和血清胃泌素含量的影响动物分组、用药情况同前,每组8只。最后一次灌胃后禁食24h,无菌操作断头取血5ml,3500 r/min离心15min,取血清-20℃以下保存,留作GAS含量检测。无菌操作剖腹取胃,沿胃大弯剪开,用冰生理盐水漂洗胃内容物,滤纸吸干,用载玻片刮取胃窦部黏膜,称重后,冰浴下制备10%的组织匀浆,3500r/min离心15min,取上清,-20℃以下保存,留作PGE₂含量检测。结果第一部分:胃炎饮对胆汁反流性胃炎大鼠胃黏膜病理形态学的影响1 HE染色正常组大鼠胃黏膜表面光滑,未见破损,脱落,极少见炎细胞;模型组大鼠胃黏膜出现明显破损,脱落,黏膜下可见大量炎细胞浸润;与模型组比较,胃炎饮高、低剂量组和吗叮啉对照组胃黏膜破损,脱落较轻,炎细胞浸润较少,胃炎饮高剂量组效果最佳,与正常组相近（Fig.1-5）。炎细胞半定量分析结果显示:与正常组比较,模型组炎细胞浸润明显（P<0.01）;与模型组比较,胃炎饮高、低剂量组和吗叮啉对照组炎细胞浸润均减轻（P<0.01或P<0.05）;与吗叮啉对照组比较,胃炎饮高剂量组炎细胞浸润较轻,具有统计学意义（P<0.05）。见Table 1。2 AB/PAS染色正常组大鼠胃黏膜AB/PAS阳性层较厚,连续性好（Fig.6）。模型组大鼠胃黏膜AB/PAS阳性层明显变薄,甚至缺失（Fig.7）。胃炎饮高、低剂量组和吗叮啉对照组大鼠胃黏膜AB/PAS阳性层变薄、缺失较轻,其中以胃炎饮高剂量组最佳（Fig.8-10）。AB/PAS阳性层厚度测量结果:与正常组比较,模型组大鼠AB/PAS阳性层厚度明显下降（P<0.01）;与模型组比较,胃炎饮高、低剂量组及吗叮啉对照组AB/PAS阳性层厚度均明显增加（P<0.01或P<0.05）;胃炎饮高、低剂量组优于吗叮啉对照组,具有统计学意义（P<0.01或P<0.05）。见Table 2。第二部分:胃炎饮对胆汁反流性胃炎大鼠血浆胃动素和胃黏膜P物质含量的影响1各组大鼠血浆MTL含量的比较与正常组比较,模型组大鼠血浆MTL含量明显下降,具有统计学意义（P<0.01）;与模型组比较,胃炎饮高、低剂量组和吗叮啉对照组MTL含量均明显升高,具有统计学意义（P<0.01或P<0.05）;胃炎饮高、低剂量组MTL含量均明显高于吗叮啉对照组（P<0.05）。见Table 3。2各组大鼠胃黏膜SP含量的比较与正常组比较,模型组大鼠胃黏膜SP含量明显下降,具有统计学意义（P<0.01）;与模型组比较,胃炎饮高、低剂量组和吗叮啉对照组SP含量均明显升高,具有统计学意义（P<0.01或P<0.05）;与吗叮啉对照组比较,胃炎饮高、低剂量组SP含量虽较高但无统计学意义（P>0.05）。见Table 4。第三部分:胃炎饮对胆汁反流性胃炎大鼠胃黏膜前列腺素E₂和血清胃泌素含量的影响1各组大鼠胃黏膜PGE₂含量的比较与正常组比较,模型组大鼠胃黏膜PGE₂含量明显下降,具有统计学意义（P<0.01）;与模型组比较,胃炎饮高、低剂量组和吗叮啉对照组PGE₂含量均明显升高,具有统计学意义（P<0.01或P<0.05）;胃炎饮高、低剂量组PGE₂含量明显高于吗叮啉对照组,具有统计学意义（P<0.01或P<0.05）。见Table 5。2各组大鼠血清GAS含量的比较与正常组比较,模型组大鼠血清GAS含量明显下降,具有统计学意义（P<0.01）;与模型组比较,胃炎饮高、低剂量组和吗叮啉对照组GAS含量均明显升高,具有统计学意义（P<0.01或P<0.05）;胃炎饮高、低剂量组GAS含量明显高于吗叮啉对照组,具有统计学意义（P<0.01或P<0.05）。见Table 6。结论1胃炎饮疗效确切,能显著改善胆汁反流性胃炎大鼠胃黏膜病理形态学变化,减轻胃黏膜损伤,减少炎细胞浸润,增加AB/PAS阳性层厚度,具有保护胃黏膜的作用。2胃炎饮可以显著升高胆汁反流性胃炎大鼠血浆MTL和胃黏膜SP含量,增强胃肠动力,调节MMC,促进胃排空,加快反流液的清除,从而使得反流液对胃黏膜的攻击机会减少,减轻胃黏膜损伤。3胃炎饮可显著升高胆汁反流性胃炎大鼠胃黏膜PGE₂和血清GAS含量,从而加强黏液-碳酸氢盐屏障,改善胃黏膜血流,促进黏膜修复,具有保护和营养胃黏膜的作用。更多还原

【Abstract】 Objective: Weiyanyin is composed of bupleurum chinense, nutgrass galingale rhizome, fructus meliae toosendan, corydalis tuber, atractylodes macrocephala, hoelen, prepared radlx glycyrrhizae, rhizoma coptidis, aurantii nobilis pericarpium, aurantii fructus, prepared rhizoma pinelliae with ginger, stir-baked caulis banbusae in teaniam with ginger juice, et al. In traditional Chinese medicine, it has the effects to disperse the depressed liver-energy, cholagogue, invigorate the spleen, tonifying didney pneuma, harmony stomach, and calm the adverse-rising energy. It is an effective prescription sieved by professor after many years clinical practice, which has good therapeutic efficacy on treating bile reflux gastritis. This research applies reflux liquid to duplicate the model of bile reflux gastritis rats by intragastric administration. The aim is to verify the curative effect of Weiyanyin by observing the changes of pathological morphology of gastric mucosa, motilin （MTL） in blood plasma, substance P （SP） in gastric mucosa, prostaglandin E2 （PGE2） in gastric mucosa and gastrin （GAS） in blood serum of bile reflux gastritis rats. And then discuss the functional mechanism of Weiyanyin curing bile reflux gastritis from the perspective on adjustment of gastrointestinal peptide. MethodsPart 1: The effect of Weiyanyin on pathological morphology of gastric mucosa of bile reflux gastritis rats40 healthy wistar rats,half male and half female, were randomly divided into 5 groups, 8 rats every group, named normal group, model group, high-dose group （21.4g/Kg avoirdupois Weiyanyin intragastric administration）, low-dose group （10.7g/Kg avoirdupois Weiyanyin intragastric administra- tion）, control group （2.78mg/Kg avoirdupois Domperidone intragastric administration）. Meanwhile normal group and model group were given equal volume physiological saline by intragastric administration from the second week to the fifth week, once a day Sustained 28 days. Except for normal group, each group was given reflux liquid 15ml/kg avoirdupois by intragastric administration once a day, for 35 days. After the last time administration, we stopped giving the rats food and drink for 24 hours. We cut the belly open to obtain stomach by aseptic technique, then sheared the stomach along arcus major ventriculi, rinsed gastric contents with ice-physiological saline, blotted it with filter paper and macroscopic observed it. At last, two pieces of tissues of sinus ventriculi were scissorsed, each piece about 1mm³. The tissues were fixed with 4 percent paraform, dehydrolysised with alcohol, paraffin imbedding, and cut into sheets. And the sheets were dyed by HE and AB/PAS dyeing respectively to observe pathological changes of gastric mucosa with light microscope. Detected inflammatory cells of every group by semiquantitative analysis through HE dyeing and measured the AB/PAS positive layer thickness with micrometer though AB/PAS dyeing. In every sheet, we measured 10 times in different parts and each interval was the same, and then obtained average value.Part 2: The effect of Weiyanyin on MTL in blood plasma and SP in gastric mucosa of bile reflux gastritis ratsThe dividing groups and medication were same to above. After the last time intragastric administration, stopped giving the rats food and drink for 24 hours and put them to death by decapitation and obtained blood 5ml. Added 100g/L EDTA-2Na 0.2ml to the blood and jolted in plastic tube. Centrifuged the blood （3500 r/min, 15min） and conserved the plasma under -20℃to examine the content of MTL later. We cut the belly open to obtain stomach by aseptic technique, sheared the stomach along arcus major ventricul, rinsed gastric contents with ice-physiological saline, and blotted the tissue with filter paper. Scraped mucosal tissues of sinus ventriculi with microscope slide, weighed them then put them into homogenizer, added physiological saline into them, mixed them, made them into 10 percent of homogenate in ice bath. Centrifuged the homogenate （3500 r/min, 15min） then took clear supernatant liquid into cryogenic box to conserve under -20℃for checking the content of SP later.Part 3: The effect of Weiyanyin on PGE2 in gastric mucosa and GAS in blood serum of bile reflux gastritis rats: The dividing groups and medication used the same way as above. After the last time administration, fasted the rats for 24 hours and put them to death by decapitation and obtained blood 5ml. Centrifuged the blood （3500 r/ min, 15 min） to make serum separated and conserved the serum under -20℃to detect the content of GAS. We cut the belly open to obtain stomach by aseptic technique, sheared the stomach along arcus major ventricul, and then rinsed gastric contents with ice-physiological saline, and blotted the gastric mucosa with filter paper. Mucosal tissues of sinus ventriculi were scraped by microscope slide and weighted. And 10% tissue bomogenate was prepared in ice bath, which centrifugated immediately to take supernatant （3500r/min, 15 min） and conserved under -20℃to examine PGE2 later.ResultsPart 1: The effect of Weiyanyin on Pathological morphology of gastric mucosa of bile reflux gastritis rats:1 HE dyeingMucosal epidermis of rats in normal group was smooth, without breakage, amotic and few inflammation cells could be seen. Mucosal epidermis of rats in model group was sparseness, attenuation. Epidermis cells appeared breakage, desquamate, and a lot of inflammation cells could be seen. Compared with model group, epidermis cells in mucosa of high-dose group, low-dose group and control group were better, and less inflammation cells could be seen. The mucosa of high-dose group was similar to normal group. （Fig. 1-5） Semi-Quantitative Analysis showed that, compared with normal group, inflammatory cell infiltration in mucosa of model group was much more obviously （P<0.01）; Compared with model group, inflammatory cell infiltration of high-dose group, low-dose group and control group was less （P<0.01 or P<0.05）; And inflammatory cell infiltration of high-dose group was less than control group （P<0.05）. （Table 1）2 AB/PAS dyeingAB/PAS positive layer thickness of normal group was thick and consecutive. It was thin even absent in model group. AB/PAS positive layer thickness of high-dose group, low-dose group and control group was thin slightly, and high-dose group was the best among the three groups. （Fig. 6-10）Measurements of AB/PAS positive layer thickness showed: Compared with normal group, AB/PAS positive layer thickness in mucosa of model group was much thinner （P<0.01）; The figures of high-dose group, low-dose group and control group were lower than model group （P<0.01 or P<0.05）; High-dose group and low-dose group were higher than control group （P<0.01 or P<0.05）. （Table 2）Part 2: The effect of Weiyanyin on MTL in blood plasma and SP in gastric mucosa of bile reflux gastritis rats1 The comparison of the contents of MTL in blood plasma among groupsThe table 3 showed: Compared with normal group, the content of MTL in model group was lower, which had statistical significance （P<0.01）; Compared with model group, the contents of MTL in high-dose group, low-dose group and control group were higher, which had statistical significance （P<0.01 or P<0.05）; Compared with control group, the contents of MTL in high-dose group and low-dose group were higher （P<0.05）.2 The comparison of the contents of Substance P in gastric mucosa among groupsThe table 4 showed: Compared with normal group, the content of SP in model group was lower, which had statistical significance （P<0.01）; Compared with model group, the contents of SP in high-dose group, low-dose group and control group were higher, which had statistical significance （P<0.01 or P<0.05）; Compared with control group, the contents of SP in high-dose group and low-dose group were higher but had no statistical significance （P>0.05）.Part 3: The effect of Weiyanyin on PGE₂ in gastric mucosa and GAS in blood serum of bile reflux gastritis rats1 The comparison of the contents of PGE₂ in gastric mucosa among groupsThe table 5 showed: Compared with normal group, the content of PGE₂ in model group was lower, which had statistical significance （P<0.01）; Compared with model group, the contents of PGE₂ in high-dose group, low-dose group and control group were higher, which had statistical significance （P<0.01 or P<0.05）; The contents of PGE₂ in high-dose group and low-dose group were higher than control group, which had statistical significance （P<0.01 or P<0.05）.2 The comparison of the contents of GAS in blood serum among groupsThe table 6 showed: Compared with normal group, the content of GAS in model group was lower, which had statistical significance （P<0.01）; The contents of GAS in high-dose group, low-dose group and control group were higher than model group, which had statistical significance （P<0.01 or P<0.05）; Compared with control group, the contents of GAS in high-dose group and low-dose group were higher, which had statistical significance （P<0.01 or P<0.05）.Conclusion1 Weiyanyin has a certain curative effect. It could improve the changes of pathomorphology in gastric mucosa, relieve the damnification and inflammatory cell infiltration of gastric mucosa, and increase AB/PAS positive layer thickness. It has the function of protecting gastric mucosa of bile reflux gastritis rats effectively.2 Weiyanyin could increase the content of MTL in blood plasma and SP in gastric mucosa of bile reflux gastritis rats. It could enhance dynamia of stomach intestine, accommodate MMC and promote gastric emptying, speed up the clearance of reflux liquid. So it could decrease the attack chance of reflux liquid, relieve the damage of stomach mucosa.3 Weiyanyin could increase the content of PGE₂ in gastric mucosa and GAS in blood serum of bile reflux gastritis rats. It could strengthen barrier of stomach mucosa, improve blood flow, and encourage the recovery of mucosa. It has the function of protecting and nourishing gastric mucosa.更多还原