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仙味保金方治疗过敏性支气管哮喘的代谢组学研究
【作者】 陈馨馨；
【导师】 李友林；
【作者基本信息】 北京中医药大学 ， 中医内科学， 2011， 博士
【摘要】 目的：在建立家兔过敏性支气管哮喘模型的基础上,通过动物实验,引入代谢组学,确定与过敏性支气管哮喘相关联的有意义代谢产物,完善仙味保金方治疗过敏性支气管哮喘机理的研究方法,探索过敏性支气管哮喘动物模型代谢物组变化研究的新模式。方法：将家兔适应性饲养7天无异常后,模型组家兔和仙味保金方组家兔予10%卵蛋白(OVA)生理盐水溶液腹腔注射,剂量为1.2ml/kg,对照组家兔予等剂量生理盐水腹腔注射。每只家兔均注射一次。14天后,对模型组、仙味保金方组家兔用1%0VA生理盐水溶液雾化激发；对照组用生理盐水雾化激发。连续雾化7日,每日一次,每次反复操作直至家兔出现腹肌抽搐、烦躁、唇鼻紫绀、二便失禁等表现。雾化激发结束后第四天进行灌胃治疗,考虑到中药复方药效缓和但药效持久的特点,共给药3天。根据人与动物体表面积换算公式计算得到每只家兔每日的灌胃量为：0.35g药粉。具体灌胃操作如下：第一步：先将0.35g药粉用温开水溶解定容至20ml药液,每只家兔20ml药液。第二步：将家兔固定于兔盒内,经灌胃管注入20ml药液。(对照组与模型组家兔则以同样方法注入等剂量生理盐水。)雾化激发结束后采取家兔耳缘静脉血、支气管肺泡灌洗液标本,用于嗜酸性粒细胞计数检测；灌胃治疗结束后采取家兔耳缘静脉血、支气管肺泡灌洗液和尿液标本,用于代谢组学检测。嗜酸性粒细胞计数统计分析应用SASV8软件,经方差齐性检验及正态性检验后,采用wilcoxon秩和检验,计量资料采用x±s表示；代谢产物统计分析采用单因素方差分析,计量资料采用x±s表示。结果：1.血清和支气管肺泡灌洗液嗜酸性粒细胞计数统计分析结果：在模型组计数与对照组相比均呈高表达(P<0.05)。2.家兔血清样本代谢产物统计学分析结果：异柠檬酸：对照组与模型组有差别(P<0.05),模型组与仙味保金方组有差别(P<0.05)；果糖：对照组与模型组有差别(P<0.05),模型组与仙味保金方组有差别(P<0.05)；葡萄糖酸：对照组与模型组无差别(P>0.05),模型组与仙味保金方组有差别(P<0.05)；6-脱氧吡喃甘露糖：对照组与模型组有差别(P<0.05),模型组与仙味保金方组有差别(P<0.05)；硬脂酸：对照组与模型组无差别(P>0.05),模型组与仙味保金方组有差别(P<0.05)；半乳糖醛酸：对照组与模型组有差别(P<0.05),模型组与仙味保金方组无差别(P>0.05)。3.家兔支气管肺泡灌洗液样本代谢产物统计学分析结果：甘氨酸：对照组与模型组有差别(P<0.05)、模型组与仙味保金方组有差别(P<0.05)；天冬氨酸：对照组与模型组有差别(P<0.05)、模型组与仙味保金方组有差别(P<0.05)；十四酸：对照组与模型组有差别(P<0.05)、模型组与仙味保金方组有差别(P<0.05)；半乳糖：对照组与模型组有差别(P<0.05)、模型组与仙味保金方组有差别(P<0.05)。4.家兔尿液样本代谢产物统计学分析结果：对羟基苯乙酸：对照组与模型组无差别(P>0.05),模型组与仙味保金方组有差别(P<0.05)；木糖醇：对照组与模型组无差别(P>0.05),模型组与仙味保金方组有差别(P<0.05)；3,5-二羟基苯甲酸：对照组与模型组无差别(P>0.05),模型组与仙味保金方组有差别(P<0.05)；硬脂酸：对照组与模型组有差别(P<0.05)、模型组与仙味保金方组有差别(P<0.05)结论：1.卵蛋白致敏新西兰家兔可成功建立过敏性支气管哮喘模型。2.支气管肺泡灌洗液样本代谢产物中的甘氨酸、天冬氨酸、十四酸、半乳糖含量在模型组升高,说明与过敏性支气管哮喘病理变化有关。3.支气管肺泡灌洗液样本代谢产物中的甘氨酸、天冬氨酸、十四酸、半乳糖含量在仙味保金方组的含量比模型组明显降低,说明仙味保金方具有修复代谢网络的作用。4.血清样本代谢产物中的异柠檬酸、半乳糖醛酸、果糖、6-脱氧吡喃甘露糖在模型组含量改变,说明这些产物与过敏性支气管哮喘具有相关性。5.血清样本代谢产物中的异柠檬酸、果糖、葡萄糖酸、硬脂酸、6-脱氧吡喃甘露糖含量在仙味保金方组发生变化,说明这些产物与仙味保金方中药成分发挥作用有关。6.尿液样本代谢产物中的硬脂酸在模型组含量改变,说明与过敏性支气管哮喘具有相关性。7.尿液样本代谢产物中的对羟基苯乙酸、木糖醇、3,5-二羟基苯甲酸和硬脂酸在仙味保金方组含量改变,说明这些产物与仙味保金方中药成分发挥作用有关。
【Abstract】 Objective:We confirm significative metabolic product related allergic bronchial asthma by metabolomics based on New Zealand rabbits asthma model.The objective is consummate the research method of the asthma’s mechanism that xianweibaojin medication treating and exploring the new pattern of the reaserch in asthma model’s metabolites group variation.Method:After 7 days with New Zealand Rabbits by adaptive breeding we give model group and treatment group rabbits 10% Ovalbumin(1.2ml/kg) by Celiac injecting. Normal group rabbits are given equivalent doses of physiological saline.Every rabbit is injected one time.After 14 days we atomize model group and treatment group rabbits with 1% OVA solution by physiological saline. Normal group rabbits is atomized with physiological saline.We can’t stop atomizing until rabbits express abdominal muscle twitching, fidgety, nose and lips cyanosis, urinary and fecal incontinence. The atomization lasting 7 days and 1 time everyday.The fuorth day after atomizing we use irrigation stomach method to treat asthma rabbits.We determine 3 days to irrigate stomach because of the characteristics of the moderated and lasting efficacy of Chinese medicine compound. According to the conversion formula with people to animal we get the powder quantity of irrigation stomach is 0.35g.The specific operation:1.We give treatment group rabbits 15ml solution that containing 0.35g powder and 14.65ml warm water.2. Fixing the treatment group rabbits in the Rabbit box,then injecting the 15ml solution through gastric tube. (Normal group and model group rabbits are given the same dose physiological saline by the same method.)After irrigating stomach we gather Venous blood, bronchial alveolar lavage fluid and urine to detect eosinophil and metabolic product.We choose SASV8 software to analyse eosinophil count by wilcoxon and to analyse metabolic product by Oneway ANOVA.The measurement data are expressed by x±s.Result:1.The eosinophil count of blood serum and bronchial alveolar lavage fluid in model group are higher than normal group (P<0.05).2. The metabolic product of blood serum sample’s statistics analysis resaults: isocitric acid:Compared with the model group, the isocitric acid expression was significantly higher than normal group (P<0.05).fructose:Compared with the model group, the fructose expression was significantly lower than normal group (P<0.05); Compared with the model group, the fructose expression was significantly lower than treatment group (P<0.05)glucose sour:Compared with the model group, the glucose sour expression was significantly lower than normal group (P<0.05); Compared with the model group, the glucose sour expression was significantly lower than treatment group (P<0.05)6-deoxidizing pyranoid mannose:Compared with the model group, the 6-deoxidizing pyranoid mannose expression was significantly lower than normal group (P<0.05).stearic acid:Compared with the treatment group, the stearic acid expression was significantly lower than model group (P<0.05); Compared with the normal group, the stearic acid expression was no difference to model group (P>0.05).half of lactose hyaluronic acid:Compared with the model group, the half of lactose hyaluronic acid expression was significantly higher than normal group (P<0.05); Compared with the treatment group, the half of lactose hyaluronic acid expression was no difference to model group (P>0.05).3. The metabolic product of bronchial alveolar lavage fluid sample’s statistics analysis resaults:glycine:Compared with the model group, the glycine expression was significantly higher than normal group (P<0.05); Compared with the model group, the glycine expression was significantly higher than treatment group (P<0.05)L-aspartic acid:Compared with the model group, the L-aspartic acid expression was significantly higher than normal group (P<0.05); Compared with the model group, the L-aspartic acid expression was significantly higher than treatment group (P<0.05).tetradecaoic acid:Compared with the model group, the tetradecaoic acid expression was significantly higher than normal group (P<0.05); Compared with the model group, the tetradecaoic acid expression was significantly higher than treatment group (P<0.05)half of lactose::Compared with the model group, the half of lactose expression was significantly higher than normal group (P<0.05); Compared with the model group, the half of lactose expression was significantly higher than treatment group (P<0.05)4. The metabolic product of urine sample’s statistics analysis resaults:serine:Compared with the normal group, the serine expression was no difference to model group (P>0.05). hydroxyl ethyl benzene:Compared with the treatment group, the hydroxyl ethyl benzene expression was significantly lower than model group(P<0.05); Compared with the model group, the hydroxyl ethyl benzene expression was no difference to normal group (P>0.05).xylitol:Compared with the treatment group, the xylitol expression was significantly lower than model group (P<0.05); Compared with the model group, the xylitol expression was no difference to normal group (P>0.05).5-bis hydroxy benzoic acid:Compared with the treatment group, the5-bis hydroxy benzoic acid expression was significantly lower than model group (P<0.05); Compared with the model group, the5-bis hydroxy benzoic acid expression was no difference to normal group (P>0.05).stearic acid:Compared with the treatment group, the stearic acid expression was significantly lower than model group (P<0.05); Compared with the normal group, the stearic acid expression was significantly higher than model group (P<0.05).Conclusion:1.We Simulate New Zealand Rabbits allergic allergic bronchial asthma model with injecting ovalbumin in celiac successfully.2.The content of glycine, L-aspartic acid, tetradecaoic acid and half of lactose of bronchial alveolar lavage fluid in the model group higher than normal group.We confirm these four metabolic products have the close correlation to allergic bronchial asthma.3.These four different metabolic products’ content of bronchial alveolar lavage fluid in the treatment group below the model group obviously.The treatment group’s content is close to normal group. The description above explain that xianweibaojin medication can repair the Metabolic network and recovery allergic bronchial asthma by specific change.4.The content of isocitric acid, fructose,6-deoxidizing pyranoid mannose.half of lactose hyaluronic acid of blood serum are changed when the rabbits catch allergic bronchial asthma. We confirm these four metabolic products have the close correlation to allergic bronchial asthma.5.The content of isocitric acid and stearic acid of blood serum in the treatment group are lower than the model group. The content of fructose, glucose sour and 6-deoxidizing pyranoid mannose of blood serum in the treatment group are higher than the model group. We confirm these five metabolic products have the close correlation to xianweibaojin compound medicine.6. The content of stearic acid of urine in the model group are lower than the normal group. We confirm stearic acid have the close correlation to allergic bronchial asthma.7. The content of serine, hydroxyl ethyl benzene, xylitol and 5-bis hydroxy benzoic acid of urine in the treatment group are lower than the model group. We confirm these five metabolic products have the close correlation to xianweibaojin compound medicine.