5味山姜属中药对胃溃疡寒证大鼠温胃散寒药效及作用机制研究

发布时间：2017-12-30 23:20

本文关键词：5味山姜属中药对胃溃疡寒证大鼠温胃散寒药效及作用机制研究　出处：《广州中医药大学》2016年博士论文　论文类型：学位论文

【摘要】：目的：筛选冰乙酸灌胃制作胃溃疡寒证大鼠模型的最佳造模方法,观察5味山姜属中药对冰乙酸灌胃型胃溃疡寒证的行为体征、胃液量、胃酸、胃蛋白酶、胃组织病理变化,阐释此5味山姜属中药的温胃散寒功效本质；测定胃组织PGE2、EGF、TGF-a,血清N0、IL-8、TNF-α、NT,血浆ET,脑组织AchE、5-HT、NE的含量,探索5味山姜属中药治疗胃溃疡寒证的作用机理。方法：设置5%、10%、15%、20%、25%5个冰乙酸组,加4℃知母水煎液制作胃溃疡寒证大鼠模型,比较溃疡面积、溃疡指数、致死性、病理组织变化以及自然修复情况,确定出最佳的冰乙酸灌胃浓度。最佳灌胃浓度确定后,设置空白对照组、病证模型对照组、2个阳性对照组、10个受试药物组,每组10只。除空白对照组外,其余各组采用最终确定的冰乙酸浓度加4℃知母水煎液造模,造模成功后,空白对照组、模型对照组灌服蒸馏水,其余各组分别灌服各受试药物。观察胃溃疡寒证大鼠行为表征的变化(进食量、体重、毛色、大便、精神状态)；测定胃液量、胃酸、胃蛋白酶活力；HE染色观察胃组织病理变化情况；采用酶联免疫法测定PGE2、EGF、TGF-α、IL-8、 TNF-β、NT、血浆ET、5-HT、NE的含量；硝酸还原法测定N0含量；蛋白定量法测定AchE含量。采用SPSS 19.0进行统计学处理,以P0.05为有统计学意义。结果：一、寒凉药加冰乙酸灌胃建立胃溃疡寒证大鼠模型①15%冰乙酸浓度效果最好,形成的溃疡大,未见致死性,且自然恢复的慢。②冰乙酸灌胃复制胃溃疡寒证大鼠模型的最佳造模方法为：给予大鼠灌服4℃冰知母水煎液和15%冰乙酸溶液,各1次/d,8：00灌服冰知母,18：00灌服冰乙酸,连续4 d。室内环境设定温度16℃,湿度60%。二、5味山姜属中药对胃溃疡寒证大鼠温胃散寒药效研究(一)对胃溃疡寒证大鼠行为体征的影响：①对胃溃疡寒证大鼠体重的影响：与模型组比较,10个受试药物组大鼠体重均增加(P0.05或P0.01)。②对胃溃疡寒证大鼠进食量的影响：与模型组比较,10个受试药物组大鼠进食量均增加,增加率升高明显。③对胃溃疡寒证大鼠毛色评分值的影响：与模型组比较,10个受试药物组,除大高良姜低剂量组外,各组大鼠毛色评分值均降低(P0.05或P0.01)。④对胃溃疡寒证大鼠大便、精神状态的影响：受试药物大鼠大便量均减少,较干爽,成形；精神状态有所恢复,行动较灵活。(二)对胃液量、胃酸、胃蛋白酶活力的影响与模型组比较,大高良姜高剂量、高良姜高剂量、高良姜低剂量、草豆蔻高剂量、草豆蔻低剂量、红豆蔻高剂量、红豆蔻低剂量、益智高剂量能减少胃液的分泌量(P0.05或P0.01)；大高良姜高剂量、高良姜高剂量、高良姜低剂量、草豆蔻高剂量、草豆蔻低剂量、红豆蔻高剂量、红豆蔻低剂量、益智高剂量、益智低剂量组pH升高分泌和胃蛋白酶活力降低(P0.05或P0.01)。(三)对胃组织病理组织变化的影响与模型组比较,大高良姜高剂量组炎细胞浸润减少,粘膜表面渗出物减少,肉芽组织增多,粘膜表层出现新生上皮细胞排列；高良姜高剂量组表面坏死组织减少,肌层、粘膜下层粘膜肌层结构清晰,肉芽组织增多,粘膜表面上皮组织整齐排列；草豆蔻高剂量组炎细胞浸润减少,表面渗出物减少,肉芽组织增多,表面开始出现上皮修复；红豆蔻高剂量组炎细胞浸润减少,肉芽组织均匀分布,粘膜表面渗出物消失,新生上皮组织逐渐覆盖粘膜表面；益智高剂量组炎细胞浸润减少,部分粘膜表面出现上皮修复；大高良姜低剂量组粘膜表面渗出物减少,肉芽组织增多,粘膜表面有新生腺体覆盖；高良姜低剂量组粘膜表面渗出物减少,粘膜层覆盖新生上皮组织；草豆蔻低剂量组肌层、粘膜下层结构清晰,断裂的粘膜肌层开始修复,粘膜表面覆盖上皮组织；红豆蔻低剂量组粘膜表面渗出物减少,炎细胞浸润减少,肉芽组织增多,粘膜肌层开始修复；益智低剂量组肌层结构完整,粘膜下层布满肉芽组织。三、5味山姜属中药对胃溃疡寒证大鼠温胃散寒作用机制研究(一)对胃溃疡寒证大鼠胃组织PGE2含量变化的影响与模型组比较,高良姜高剂量、草豆蔻高剂量组大鼠胃组织PGE2含量明显升高,结果有显著性差异(P0.01)；大高良姜高剂量、草豆蔻低剂量、红豆蔻高剂量、红豆蔻低剂量组大鼠胃组织PGE2含量明显升高,结果有统计学意义(P0.05)；大高良姜低剂量、高良姜低剂量、益智高剂量、益智低剂量组大鼠胃组织PGE2含量变化不明显(P0.05)。(二)对胃溃疡寒证大鼠胃组织EGF含量的影响与模型对照组比较,大高良姜高剂量、草豆蔻高剂量、益智高剂量组大鼠胃组织EGF含量明显升高,结果有显著性差异(P0.01)；高良姜高剂量、草豆蔻低剂量、红豆蔻高剂量组胃组织EGF含量升高,结果有统计学意义(P0.05)；大高良姜低剂量、红豆蔻低剂量、益智低剂量组胃组织EGF变化无差异(P0.05)。(三)对胃溃疡寒证大鼠胃组织TGF-a含量的影响与模型组比较,大高良姜高剂量、大高良姜低剂量、高良姜高剂量组、草豆蔻高剂量、红豆蔻高剂量、益智高剂量组大鼠胃组织TGF-α含量明显升高,结果有显著性差异(P0.01)；高良姜低剂量、草豆蔻低剂量、红豆蔻低剂量、益智低剂量组大鼠胃组织TGF-α含量变化不明显(P0.05)。(四)对胃溃疡寒证大鼠血清N0含量的影响与模型组比较,红豆蔻高剂量以及附子理中丸、西咪替丁组大鼠血清N0含量明显升高,结果有显著性差异(P0.01)；大高良姜高剂量、高良姜高剂量、草豆蔻高剂量、红豆蔻低剂量、益智高剂量组大鼠胃组织N0含量升高,结果有统计学意义(P0.05)；大高良姜低剂量组、高良姜低剂量、草豆蔻低剂量、益智低剂量组变化不明显(P0.05)。(五)对胃溃疡寒证大鼠血清IL-8含量的影响与模型组比较,草豆蔻高剂量、红豆蔻高剂量、益智高剂量组以及附子理中丸、西咪替丁组大鼠胃组织IL-8含量明显降低,结果有显著性差异(P0.01)；大高良姜高剂量、大高良姜低剂量、高良姜高剂量、高良姜低剂量、草豆蔻低剂量、红豆蔻低剂量、益智低剂量组大鼠胃组织IL-8含量升高,结果有统计学意义(P0.05)。(六)对胃溃疡寒证大鼠血清TNF-α含量的影响与模型组比较,大高良姜高剂量、大高良姜低剂量、高良姜高剂量、高良姜低剂量、草豆蔻高剂量、草豆蔻低剂量、红豆蔻高剂量、红豆蔻低剂量、益智高剂量、益智低剂量组大鼠血清TNF-α含量变化均无差异(P0.05)。(七)对胃溃疡寒证大鼠血清NT含量的影响与模型组比较,大高良姜高剂量、大高良姜低剂量、高良姜高剂量、高良姜低剂量、草豆蔻高剂量、草豆蔻低剂量、红豆蔻高剂量、红豆蔻低剂量、益智高剂量组大鼠血清NT含量明显升高(P0.01)。益智低剂量组大鼠血清NT含量升高,结果有统计学意义(P0.05)。(八)对胃溃疡寒证大鼠血浆ET含量的影响与模型组比较,高良姜高剂量、草豆蔻高剂量、草豆蔻低剂量、红豆蔻高剂量、红豆蔻低剂量组大鼠血浆ET含量明显降低(P0.01)。大高良姜高剂量、大高良姜低剂量、高良姜低剂量、益智高剂量、益智低剂量组大鼠血浆ET含量降低,结果有统计学意义(P0.05)。(九)对胃溃疡寒证大鼠脑组织AchE含量的影响与模型对照组比较,高良姜高剂量、草豆蔻高剂量、红豆蔻高剂量组大鼠脑组织AchE含量明显升高(P0.01)；大高良姜高剂量、高良姜低剂量、红豆蔻高剂量组大鼠脑组织AchE含量升高,结果有统计学意义(P0.05)；大高良姜低剂量、草豆蔻低剂量、红豆蔻低剂量、益智低剂量组大鼠脑组织AchE含量变化无差异(P0.05)。(十)对胃溃疡寒证大鼠脑组织5-HT含量的影响与模型对照组比较,大高良姜高剂量、高良姜高剂量、草豆蔻高剂量、红豆蔻高剂量、红豆蔻低剂量、益智高剂量、益智低剂量组大鼠脑组织5-HT含量明显降低(P0.01)。草豆蔻低剂量组大鼠脑组织5-HT含量降低,结果有统计学意义(P0.05)；大高良姜低剂量、高良姜低剂量组大鼠脑组织5-HT无明显变化(P0.05)。(十一)对胃溃疡寒证大鼠脑组织NE含量的影响与模型对照组比较,草豆蔻高剂量、红豆蔻高剂量、益智高剂量组大鼠脑组织NE含量明显降低(P0.01)。红豆蔻低剂量组大鼠脑组织NE含量降低,结果有统计学意义(P0.05)；大高良姜高剂量、大高良姜低剂量、高良姜高剂量、高良姜低剂量、草豆蔻低剂量、益智低剂量组大鼠脑组织NE无明显变化(P0.05)。结论：一、冰乙酸灌胃型大鼠胃溃疡寒证大鼠模型的最佳冰乙酸浓度为15%。二、冰乙酸灌胃型胃溃疡寒证与PGE2、EGF、TGF-α、NT、NO、AchE含量降低有关；与 IL-8、TNF-α、ET、5-HT、NE升高含量有关。三、5味山姜属中药治疗冰乙酸型胃溃疡寒证的作用机理可能与减少胃液分泌,降低胃酸酸度、胃蛋白酶活力有关；与升高PGE2、EGF、TGF-α、NT、NO、AchE含量有关；与降低IL-8、血浆ET、5-HT、NE含量有关。四、存在亲缘关系的大高良姜、高良姜、草豆蔻、红豆蔻、益智这5味中药,在温胃散寒功效上存在相似的药理效应。
[Abstract]:Objective: the best method for screening acetic acid made mould made by filling the stomach ulcer with cold syndrome rat model, observation of 5 kinds of Alpinia herbs on acetic acid gastric ulcer gastric perfusion cold syndrome behavioral signs, volume of gastric juice, gastric acid, pepsin, pathological changes of gastric tissue, warm the stomach illustrates this 5 kinds of traditional Chinese medicine for dispelling cold Alpinia effect of nature; the gastric tissue was measured by PGE2, EGF, TGF-a, serum N0, IL-8, TNF- alpha, NT, ET in plasma, brain AchE, 5-HT, NE content, explore the mechanism of 5 kinds of traditional Chinese medicine in the treatment of gastric ulcer in Alpinia cold syndrome. Methods: 5%, 10%, 15%, 20%, 25%5 ice acetic acid group, plus 4 degrees Zhimu Decoction made cold syndrome rat model of gastric ulcer, ulcer area and ulcer index, comparison, lethality, histopathological changes and natural restoration, determined the optimal concentration of acetic acid by gavage. The best concentration was determined after intragastric administration, blank control group, disease model group. 2 positive controls Group, 10 subjects in drug group, 10 rats in each group. Except control group, other groups were finally determined the concentration of acetic acid plus 4 DEG C Zhimu Decoction model, after the success of the model, the blank control group, model control group with distilled water, the other groups were fed with the drugs. To observe the change of gastric ulcer of rats with cold behavior (food intake, body weight, hair color, stool, mental state); Determination of the amount of gastric juice, gastric acid, pepsin activity; to observe the pathological changes of gastric tissue HE staining; the determination of PGE2 by enzyme-linked immunosorbent assay EGF, TGF- alpha, IL-8 beta, TNF-, NT. Plasma ET, 5-HT, NE content; N0 content was determined by nitrate reduction method; Determination of AchE protein quantitative method. SPSS 19 was used for statistical treatment, P0.05 had statistical significance. Results: a cold medicine, ice acetic acid gastric ulcer was established by intragastric infusion of cold syndrome rats model of 15% acetic acid concentration had the best effect, The formation of ulcer, no fatal, and natural recovery is slow. The acetic acid filling optimum method of gastric ulcer with cold copy rat model for rats were given orally 4 C water ice Zhimu Decoction and 15% acetic acid, 1 times /d, 8:00 fed with ice Anemarrhena, 18:00 perfusion of ice acetic acid, 4 consecutive D. indoor environment setting temperature of 16 degrees Celsius, humidity 60%. two, cold efficacy of powder on gastric ulcer of rats with cold warm stomach 5 taste medicine Alpinia (1) effect on gastric ulcer of rats with cold behavioral signs: effect on body weight of gastric ulcer: comparison with cold syndrome rats the model group, 10 subjects weight rats in drug group were increased (P0.05 or P0.01). The effect on food intake of gastric ulcer of cold syndrome rats: compared with model group, 10 subjects in drug group rats increased food intake, increase rate increased significantly. The score of gastric ulcer with cold rat hair color: with the model group Comparison of 10 subjects in the drug group, low dose group galangal, rats hair color scores were lower (P0.05 or P0.01). The stool of gastric ulcer of cold syndrome rats, effects of mental status: tested drugs rats stool volume decreased, relatively dry, forming; mental state has recovery action is flexible. (two) the volume of gastric juice, gastric acid, pepsin activity effect compared with the model group, high dose of galangal Alpinia galanga, high dose, low dose of katsumada galangal, high dose, low dose of katsumada galangal fruit, high dose, low dose galanga, high dose can reduce the secretion of puzzle the gastric juice (P0.05 or P0.01); Ag high dose, high dose and low dose of Alpinia katsumada galangal,, high dose, low dose katsumada galangal fruit, high dose, Galanga low dose, high dose puzzle, the puzzle low dose group pH l hypersecretion and pepsin activity decreased (P0.0 5 or P0.01). (three) compared with the model group effect on the pathological changes of gastric tissue, reduce the infiltration of inflammatory cells in high dose group from the mucosal surface, less exudation, granulation tissue increased, mucosal surface appears new epithelial cells arranged; necrotic tissue in high dose group decreased Gaoliangjiang, muscle layer, submucosa mucosa the muscle layer structure is clear, granulation tissue increased, the mucosal surface epithelium lined up; reduce caodoukou high dose group and inflammatory cell infiltration, reduce surface exudation, granulation tissue increased, began to appear on the surface epithelial repair; reduce red nutmeg high dose group and inflammatory cell infiltration, granulation tissue distribution, mucosal surface exudate disappeared, the epithelium the organization gradually covered the mucosal surface; reduce the puzzle of high dose group and inflammatory cell infiltration, epithelial repair part of the mucosal surface; low dose group Ag mucosal exudation reduced, The granulation tissue increased, the mucosal surface of new gland cover; galangal low dose group mucosal exudation reduced, mucous layer covering new epithelial tissue; caodoukou low dose group muscle layer, submucosa structure clear, muscularis mucosa rupture began to repair, covering the surface of mucosal epithelial tissue; galanga low dose group mucosal surface exudation to reduce, reduce the infiltration of inflammatory cells, granulation tissue increased, muscularis mucosa began to repair; puzzle low dose group muscle layer structure is complete, full of submucosa granulation tissue. Three, study cold mechanism of 5 kinds of Alpinia herbs on gastric ulcer of rats with stomach cold temperature (a) compared with the model group influence on the change of PGE2 content the gastric tissue of rats with cold gastric ulcer, galangal high dose of PGE2 in gastric tissue of rats in high dose group caodoukou increased significantly, the results have significant difference (P0.01); high dose AG, katsumada Low dose, high dose galanga, PGE2 in gastric tissue in low dose group rats galanga increased significantly, the results were statistically significant (P0.05); Ag low dose, low dose and high dose of galangal, puzzle, PGE2 in gastric tissue in low dose group rat puzzle did not change significantly (P0.05). (two) effect with the model of EGF in gastric tissue of rats with gastric ulcer in the control group, the high dose of Alpinia galanga, katsumada high dose of EGF in gastric tissue of rats in high dose group puzzle was significantly increased, results there was significant difference (P0.01); high dose katsumada galangal, low dose, increase the content of EGF in gastric tissue the high dose group of galanga, the results were statistically significant (P0.05); Ag galanga low dose, low dose, no difference in intelligence gastric tissue of low dose group EGF changes (P0.05). (three) on gastric ulcer of rats with cold stomach group affected TGF-a content compared with the model group, large From high dose, low dose of galangal Alpinia galanga, high dose group, high dose of katsumada galangal fruit, high dose, puzzle of gastric tissue of rats in high dose group TGF- was significantly increased. Results there was significant difference (P0.01); low dose katsumada galangal, low dose, low dose of red cardamom, puzzle of gastric tissue low dose group rat alpha TGF- content did not change significantly (P0.05). (four) compared with the model group of gastric ulcer cold syndrome rats serum N0 content, Galanga high dose and Fuzilizhong pills, the content of serum N0 of cimetidine group rats increased significantly. Results there was significant difference (P0.01); Alpinia galanga high dose, high dose of katsumada galangal, high dose, low dose galanga, increase the content of N0 in gastric tissue of rats in high dose group puzzle, the results were statistically significant (P0.05); Ag low dose group, low dose katsumada galangal, low dose, low intelligence agent The amount of group did not change significantly (P0.05). (five) compared with the model group of gastric ulcer cold syndrome rats serum IL-8 content, katsumada galanga high dose, high dose, high dose group and the puzzle of Fuzilizhong pills, IL-8 in gastric tissue in rats of cimetidine decreased obviously, the result was significant the difference (P0.01); high dose Ag AG, low dose, high dose and low dose of Alpinia katsumada galangal, low dose, low dose, Galanga, increase the content of IL-8 in gastric tissue in low dose group rat puzzle, the results were statistically significant (P0.05). (six), cold syndrome of gastric ulcer the serum level of TNF- alpha in model group and Odaka Yoshijiangta Ag dose, low dose, high dose and low dose of Alpinia katsumada galangal,, high dose, low dose katsumada galangal fruit, high dose, low dose galanga, puzzle puzzle of high dose low dose group rats serum T There were no differences in changes of NF- content of alpha (P0.05). (seven) comparison of gastric ulcer of cold syndrome rats serum NT model group and Odaka Yoshijiangta Ag dose, low dose, high dose and low dose of Alpinia katsumada galangal,, high dose, low dose of red cardamom cardamom grass, high dose, low galanga dose puzzle high dose group rats serum NT content increased significantly (P0.01). Increased intelligence low dose group rats serum NT content, the results were statistically significant (P0.05). (eight) effect on gastric ulcer of rats with cold plasma ET content compared with the model group, high dose of katsumada galangal, high dose katsumada, low dose, high dose galanga, Galanga low dose group rat plasma ET content decreased significantly (P0.01). From high dose, low dose of galangal Alpinia galanga, low dose, high dose puzzle, reduce the content of plasma ET in the low dose group rat puzzle, statistical results Significance (P0.05). (nine) effect and model of AchE in the brain tissue of rats with gastric ulcer in the control group, the high dose of katsumada galangal, high dose, AchE content of brain tissue in rats of high dose galanga increased significantly (P0.01); Ag high dose, low dose of ginger Gao Liang increased. The content of AchE in brain tissue of rats with high dose galanga, the results were statistically significant (P0.05); Ag low dose, low dose of katsumada galangal fruit, low dose, no difference in changes of AchE content in brain tissue in rats of low dose puzzle (P0.05). (ten) effect and model of 5-HT in the brain tissue of cold rats with gastric ulcer compared to the control group, Odaka Yoshijiangta dose, high dose of katsumada galangal, high dose, high dose of galanga galanga, low dose, high dose 5-HT puzzle, in the brain tissue of low dose group of rats decreased significantly (P0.01). Educational caodoukou low dose group rat brain The contents of 5-HT decreased, the results were statistically significant (P0.05); Ag low dose group rat brain tissue from low dose of 5-HT had no significant change (P0.05). (eleven) effect and model of NE in the brain tissue of rats with gastric ulcer in the control group, katsumada high dose, high dose of red nutmeg NE, the content of brain tissue in rats of high dose significantly reduced the puzzle (P0.01). To reduce the content of NE in brain tissue of rats with low dose galanga, the results were statistically significant (P0.05); high dose Ag AG, low dose, high dose and low dose of Alpinia katsumada galangal, and low dose group the rat puzzle low dose of NE had no significant change (P0.05). Conclusion: the optimum concentration of acetic acid, acetic acid gavage type gastric ulcer in rat model of cold syndrome rats for two 15%., intragastric administration of acetic acid gastric ulcer cold syndrome and PGE2, EGF, NT, TGF- alpha, NO, decreased the content of AchE TNF-; and IL-8, ET, 5-HT, alpha, NE content increased. In three, the mechanism of 5 kinds of traditional Chinese medicine in the treatment of Alpinia acetic acid gastric ulcer with cold syndrome may reduce gastric secretion, reduce gastric acidity, pepsin activity; with the increase of PGE2, EGF, NT, NO, TGF- alpha, AchE content related; with the decrease of IL-8, plasma ET, 5-HT, NE content. Four, kinship of galangal, galangal, caodoukou, Galanga, 5 herbs in this puzzle, warm the stomach for dispelling pharmacological effects similar to the existence of the cold effect.

【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R259

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