基于代谢组学的琼玉膏延缓衰老作用评价及机制研究
发布时间：2018-08-13 17:38
【摘要】：中医药在干预衰老方面具有独特的优势,古籍中流传下很多抗衰名方,琼玉膏就是其中之一,被历代养生家所推崇。然而关于琼玉膏延缓衰老作用机制的现代研究还停留在初级阶段,限制了它的应用与推广。因此,应当建立一套能够体现中医药特点,将系统论与还原论相结合的研究方法。 目的： 本课题拟通过对琼玉膏干预后的衰老模型大鼠血清、尿液进行代谢组学检测,并与正常组、模型组、对照组相比较,观察代谢产物的变化,寻找代谢路径,分析琼玉膏延缓衰老的作用机制；并与公认的抗衰剂维生素E和经典中成药六味地黄丸进行对比,评价其抗衰老效果。为深入评价琼玉膏延缓衰老的功效,探讨琼玉膏延缓衰老可能的代谢组学机制提供依据；为琼玉膏的进一步开发应用奠定基础。 方法： 将60只SD大鼠,雌雄各半,随机分为5组：空白对照组、模型组、琼玉膏组、西药对照组(维生素E)、中药对照组(六味地黄丸),每组12只。空白对照组每天给予蒸馏水灌胃10ml·kg-1,皮下注射生理盐水2.5ml·kg-1；模型组每天给予蒸馏水灌胃10m1·kg-1,皮下注射D-半乳糖2.5m1·kg-1；琼玉膏组每天给予琼玉膏灌胃10ml·kg-1,皮下注射D-半乳糖2.5ml·kg-1；中药对照组每天给予六味地黄丸灌胃10ml·kg-1,皮下注射D-半乳糖2.5m1·kg-1；西药对照组每天给予VitE灌胃2ml·kg-1,皮下注射D-半乳糖2.5ml·kg-1.连续用药60d,通过形态观察和行为学测试(Morris水迷宫)检测造模是否成功。 末次给药后用代谢笼收集24h尿液,加1滴质量体积比为1/100(w/v)的叠氮化钠,备用；将大鼠用10%的水合氯醛按照3ml·kg-1的剂量腹腔注射麻醉,行腹主动脉采血至10ml采血管,血浆静置后,使用低速离心机取血清,备用。将血清、尿液分别加血清缓冲液、尿液缓冲液,低温高速离心取上清液置于核磁管,利用NMR技术进行代谢组学检测。使用TopSpin软件(V3.0)对谱图进行预处理；使用SIMCA-P+软件(V11.0)对归一化后的数据进行模式识别多变量分析,如主成分分析(PCA)、偏最小二乘法-判别分析(PLS-DA)、正交偏最小乘-判别分析等；使用matlab7.0软件对个别组内代谢物的相关性进行分析,最后将获得的差异代谢物输入KEGG数据库查找相关代谢通路。 结果： 1.一般情况及模型验证：造模前后各组大鼠间的体重比较,P0.05无统计学意义,分组体重无差异。Morris水迷宫结果为,与空白对照组相比,模型组、琼玉膏组、中药对照组、西药对照组在前5天均有明显差异(P0.05),第6天仅模型组有差异(P0.05),其他组间差异不明显(P0.05)。与模型组相比,从第4天开始,各组均有明显差异(P0.05)。 2.NMR谱图主成分分析：PCA分析图显示,血清各组样本分布总体较为清晰,R2X=80.6%,Q2=0.718,有显著差异；而尿液各组样本分布较为分散,R2X=59.8%,Q2=0.475,差异不大。 3.琼玉膏抗衰老作用评价：经PLS-DA分析,三个用药组,即琼玉膏组、西药对照组、中药对照组,均与模型组有明显差别。尤其西药对照组和模型组区别最明显(R2Y=0.935,Q2=0.799)；琼玉膏组和模型组的区别也很明显(R2Y=0.931,Q2=0.826),且西药对照组和琼玉膏组没有显著差别(R2Y=0.755,Q2=0.002)。 4.差异代谢物筛选：本次研究共筛选、鉴定出血清46种、尿液18种差异代谢物,相关系数|r|0.576有统计学意义。其中在衰老动物模型血清样本中变化显著的有1-甲基组氨酸、醋酸盐、丙酮、乳酸盐、柠檬酸、谷氨酸、丙酮酸、琥珀酸盐、亮氨酸、异亮氨酸、缬氨酸、赖氨酸等33种差异代谢物,而琼玉膏可以调节其中27种代谢物的变化。尿液样本中变化显著的有乙酰乙酸、马尿酸盐、二甲基甘氨酸、琥珀酸盐等9种差异代谢物,琼玉膏可以调节其中3种差异代谢物的变化。其中醋酸盐、乳酸盐、琥珀酸盐、谷氨酸等代谢物与衰老变化联系较为紧密。 5.代谢通路分析：差异代谢物主要涉及糖代谢、氨基酸代谢、能量代谢、核苷酸代谢、辅酶因子与维生素代谢、脂代谢等多条代谢通路。 结论： 1.模型组找到平台的时间明显长于其他组,造模成功；琼玉膏等用药组对D-半乳糖引起的学习记忆能力下降有改善作用。 2.在本次研究中,琼玉膏抗衰老作用主要表现于血清,而对尿液的影响较小。 3.琼玉膏抗衰老作用评价：琼玉膏能够调节衰老大鼠体内代谢物的变化,其有效性与公认抗衰剂维生素E相似,优于六味地黄丸的抗衰功效。 4.明确琼玉膏延缓衰老作用机制相关的主要差异代谢物有醋酸盐、乳酸盐、琥珀酸盐、谷氨酸等。 5.琼玉膏延缓衰老的作用机制与多个代谢通路有关,尤其是糖代谢中的丙酮酸代谢、能量代谢中的氧化磷酸化以及涉及谷氨酸的氨基酸代谢。提示琼玉膏延缓衰老的作用机制可能与线粒体损伤与脑退化有关。
[Abstract]:Qiongyu ointment is one of the famous anti-aging prescriptions circulated in ancient books. It has been highly praised by health-preserving families in the past dynasties. However, the modern research on the mechanism of Qiongyu ointment's anti-aging effect is still in its infancy, which limits its application and promotion. Characteristics of Chinese medicine, the combination of system theory and reductionism research methods.
Objective:
In this study, the serum and urine of aging model rats after intervention with Qiongyu ointment were detected by metabonomics, and compared with normal group, model group and control group, the changes of metabolites were observed, the metabolic pathway was found, and the mechanism of Qiongyu ointment in delaying aging was analyzed. To evaluate the effect of Qiongyu ointment on delaying senility and to explore the possible metabolomic mechanism of Qiongyu ointment on delaying senility, and to lay a foundation for further development and application of Qiongyu ointment.
Method:
Sixty SD rats, half male and half female, were randomly divided into five groups: blank control group, model group, Qiongyu ointment group, western medicine control group (vitamin E), Chinese medicine control group (Liuwei Dihuang pill), 12 rats in each group. Subcutaneous injection of D-galactose 2.5m1 6550 -1. was continuously administered with 60d, and morphological examination and behavioral test (Morris water maze) were used to detect whether the model was successful.
24 hours urine was collected with a metabolic cage after the last administration, and a drop of sodium azide with a mass-volume ratio of 1/100 (w/v) was added for reserve. Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 3 ml Buffer, urine buffer and supernatant were centrifuged at low temperature and high speed and placed in NMR tube for metabolomics detection. TopSpin software (V3.0) was used to pretreat the spectrogram. SIMCA-P+ software (V11.0) was used to analyze the normalized data for multivariate pattern recognition, such as principal component analysis (PCA), partial least squares method-discriminant. Analysis (PLS-DA), orthogonal partial least multiplication-discriminant analysis, etc. The correlation of metabolites in individual groups was analyzed by MATLAB 7.0 software, and the metabolites were input into KEGG database to find the related metabolic pathways.
Result:
1. General situation and model validation: Before and after modeling, there was no significant difference in body weight between groups, P 0.05 was not statistically significant, and there was no difference in body weight between groups. There was no significant difference between the other groups (P 0.05). Compared with the model group, there was significant difference between the two groups from the 4th day (P 0.05).
2. Principal component analysis of NMR spectrogram: PCA analysis showed that the distribution of serum samples in each group was relatively clear, R2X = 80.6%, Q2 = 0.718, with significant difference, while urine samples in each group were dispersed, R2X = 59.8%, Q2 = 0.475, with little difference.
3. Evaluation of the anti-aging effect of Qiongyu ointment: Through PLS-DA analysis, three medication groups, namely Qiongyu ointment group, western medicine control group, Chinese medicine control group, were significantly different from the model group. Especially the western medicine control group and model group were most distinct (R2Y = 0.935, Q2 = 0.799); Qiongyu ointment group and model group were also distinct differences (R2Y = 0.931, Q2 = 0.826), and Western medicine control group. There was no significant difference between group and Qiong Yu ointment group (R2Y=0.755, Q2=0.002).
4. Differential metabolites screening: 46 kinds of hemorrhagic serum and 18 kinds of urine metabolites were identified, and the correlation coefficient | R | 0.576 was statistically significant. There are 33 different metabolites, such as amino acid, valine, lysine and so on. Qiongyu ointment can regulate the changes of 27 of them. Urine samples have 9 different metabolites, such as acetoacetic acid, hippurate, dimethylglycine, succinate and so on. Qiongyu ointment can regulate the changes of 3 different metabolites. Succinate, glutamic acid and other metabolites are closely related to aging.
5. Metabolic pathway analysis: Differential metabolites mainly involve glucose metabolism, amino acid metabolism, energy metabolism, nucleotide metabolism, coenzyme factor and vitamin metabolism, lipid metabolism and other metabolic pathways.
Conclusion:
1. The time for model group to find the platform was significantly longer than that of other groups, and the modeling was successful.
2. in this study, the anti-aging effect of Qiong Yu ointment is mainly manifested in serum, but has little effect on urine.
3. Evaluation of anti-aging effect of Qiongyu ointment: Qiongyu ointment can regulate the changes of metabolites in aging rats. Its efficacy is similar to that of vitamin E, which is better than that of Liuwei Dihuang pill.
4. Major metabolites related to the mechanism of Qiongyu Ointment in delaying senility are acetate, lactate, succinate, glutamate and so on.
5. The mechanism of Qiongyu ointment in delaying senility is related to many metabolic pathways, especially pyruvate metabolism in glucose metabolism, oxidative phosphorylation in energy metabolism and amino acid metabolism involving glutamate.
【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】：2014
【分类号】：R285.5
本文编号：2181709
[Abstract]:Qiongyu ointment is one of the famous anti-aging prescriptions circulated in ancient books. It has been highly praised by health-preserving families in the past dynasties. However, the modern research on the mechanism of Qiongyu ointment's anti-aging effect is still in its infancy, which limits its application and promotion. Characteristics of Chinese medicine, the combination of system theory and reductionism research methods.
Objective:
In this study, the serum and urine of aging model rats after intervention with Qiongyu ointment were detected by metabonomics, and compared with normal group, model group and control group, the changes of metabolites were observed, the metabolic pathway was found, and the mechanism of Qiongyu ointment in delaying aging was analyzed. To evaluate the effect of Qiongyu ointment on delaying senility and to explore the possible metabolomic mechanism of Qiongyu ointment on delaying senility, and to lay a foundation for further development and application of Qiongyu ointment.
Method:
Sixty SD rats, half male and half female, were randomly divided into five groups: blank control group, model group, Qiongyu ointment group, western medicine control group (vitamin E), Chinese medicine control group (Liuwei Dihuang pill), 12 rats in each group. Subcutaneous injection of D-galactose 2.5m1 6550 -1. was continuously administered with 60d, and morphological examination and behavioral test (Morris water maze) were used to detect whether the model was successful.
24 hours urine was collected with a metabolic cage after the last administration, and a drop of sodium azide with a mass-volume ratio of 1/100 (w/v) was added for reserve. Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 3 ml Buffer, urine buffer and supernatant were centrifuged at low temperature and high speed and placed in NMR tube for metabolomics detection. TopSpin software (V3.0) was used to pretreat the spectrogram. SIMCA-P+ software (V11.0) was used to analyze the normalized data for multivariate pattern recognition, such as principal component analysis (PCA), partial least squares method-discriminant. Analysis (PLS-DA), orthogonal partial least multiplication-discriminant analysis, etc. The correlation of metabolites in individual groups was analyzed by MATLAB 7.0 software, and the metabolites were input into KEGG database to find the related metabolic pathways.
Result:
1. General situation and model validation: Before and after modeling, there was no significant difference in body weight between groups, P 0.05 was not statistically significant, and there was no difference in body weight between groups. There was no significant difference between the other groups (P 0.05). Compared with the model group, there was significant difference between the two groups from the 4th day (P 0.05).
2. Principal component analysis of NMR spectrogram: PCA analysis showed that the distribution of serum samples in each group was relatively clear, R2X = 80.6%, Q2 = 0.718, with significant difference, while urine samples in each group were dispersed, R2X = 59.8%, Q2 = 0.475, with little difference.
3. Evaluation of the anti-aging effect of Qiongyu ointment: Through PLS-DA analysis, three medication groups, namely Qiongyu ointment group, western medicine control group, Chinese medicine control group, were significantly different from the model group. Especially the western medicine control group and model group were most distinct (R2Y = 0.935, Q2 = 0.799); Qiongyu ointment group and model group were also distinct differences (R2Y = 0.931, Q2 = 0.826), and Western medicine control group. There was no significant difference between group and Qiong Yu ointment group (R2Y=0.755, Q2=0.002).
4. Differential metabolites screening: 46 kinds of hemorrhagic serum and 18 kinds of urine metabolites were identified, and the correlation coefficient | R | 0.576 was statistically significant. There are 33 different metabolites, such as amino acid, valine, lysine and so on. Qiongyu ointment can regulate the changes of 27 of them. Urine samples have 9 different metabolites, such as acetoacetic acid, hippurate, dimethylglycine, succinate and so on. Qiongyu ointment can regulate the changes of 3 different metabolites. Succinate, glutamic acid and other metabolites are closely related to aging.
5. Metabolic pathway analysis: Differential metabolites mainly involve glucose metabolism, amino acid metabolism, energy metabolism, nucleotide metabolism, coenzyme factor and vitamin metabolism, lipid metabolism and other metabolic pathways.
Conclusion:
1. The time for model group to find the platform was significantly longer than that of other groups, and the modeling was successful.
2. in this study, the anti-aging effect of Qiong Yu ointment is mainly manifested in serum, but has little effect on urine.
3. Evaluation of anti-aging effect of Qiongyu ointment: Qiongyu ointment can regulate the changes of metabolites in aging rats. Its efficacy is similar to that of vitamin E, which is better than that of Liuwei Dihuang pill.
4. Major metabolites related to the mechanism of Qiongyu Ointment in delaying senility are acetate, lactate, succinate, glutamate and so on.
5. The mechanism of Qiongyu ointment in delaying senility is related to many metabolic pathways, especially pyruvate metabolism in glucose metabolism, oxidative phosphorylation in energy metabolism and amino acid metabolism involving glutamate.
【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】：2014
【分类号】：R285.5
【参考文献】
相关期刊论文 前10条
1 颜德馨,胡泉林,王平平,周晓燕,宓哲伟,周良彦;气虚血瘀是人体衰老的主要机制[J];中国医药学报;1989年02期
2 徐舒;陈合兵;李洪;张琪;蔡红兵;颜贤忠;吕志平;;“肝郁证”大鼠模型的建立及代谢组学的初步研究[J];中华中医药杂志;2009年06期
3 于健,吴乐正,徐林;老年黄斑变性患者线粒体DNA突变及氧化磷酸化功能检测与分析[J];第四军医大学学报;2003年09期
4 颜晗;李国林;印大中;;脂肪代谢:联系生殖与衰老的纽带[J];国外医学(老年医学分册);2009年03期
5 田翠平,瞿伟菁,张雯,汪虹,李明娟;甜茶素对小鼠糖异生作用和血脂代谢的影响[J];广西中医药;2001年04期
6 葛红山;李晓和;张帆;陈华;习海涛;黄检英;朱春芳;吕杰强;;抑制线粒体氧化磷酸化活性对卵子体外成熟、受精及发育的影响[J];北京大学学报(医学版);2013年06期
7 梁国荣;曾瑞峰;温丹婷;李俊;;心衰心阳虚证血浆氨基酸(缬、苯丙、异亮、亮氨酸)代谢指纹图谱研究[J];中华中医药杂志;2013年12期
8 WellnerD;MeisterA;刘道生;;人类氨基酸代谢和转运的遗传性紊乱[J];国外医学(分子生物学分册);1982年06期
9 李茜;;肥胖与衰老的相关性及体育锻炼对其影响的探讨[J];当代体育科技;2013年35期
10 任汉阳;王玉英;张瑜;薛春苗;曹俊岭;李寒冰;任慧玲;;老年脾虚动物模型的建立及脾虚与衰老相关性研究[J];河南中医;2006年07期
，本文编号：2181709
本文链接：https://www.wllwen.com/yixuelunwen/mazuiyixuelunwen/2181709.html
最近更新
教材专著
