基于代謝組學的瓊玉膏延緩衰老作用評價及機制研究
發(fā)布時間：2018-08-13 17:38
【摘要】：中醫(yī)藥在干預衰老方面具有獨特的優(yōu)勢,古籍中流傳下很多抗衰名方,瓊玉膏就是其中之一,被歷代養(yǎng)生家所推崇。然而關于瓊玉膏延緩衰老作用機制的現(xiàn)代研究還停留在初級階段,限制了它的應用與推廣。因此,應當建立一套能夠體現(xiàn)中醫(yī)藥特點,將系統(tǒng)論與還原論相結(jié)合的研究方法。 目的： 本課題擬通過對瓊玉膏干預后的衰老模型大鼠血清、尿液進行代謝組學檢測,并與正常組、模型組、對照組相比較,觀察代謝產(chǎn)物的變化,尋找代謝路徑,分析瓊玉膏延緩衰老的作用機制；并與公認的抗衰劑維生素E和經(jīng)典中成藥六味地黃丸進行對比,評價其抗衰老效果。為深入評價瓊玉膏延緩衰老的功效,探討瓊玉膏延緩衰老可能的代謝組學機制提供依據(jù)；為瓊玉膏的進一步開發(fā)應用奠定基礎。 方法： 將60只SD大鼠,雌雄各半,隨機分為5組：空白對照組、模型組、瓊玉膏組、西藥對照組(維生素E)、中藥對照組(六味地黃丸),每組12只�？瞻讓φ战M每天給予蒸餾水灌胃10ml·kg-1,皮下注射生理鹽水2.5ml·kg-1；模型組每天給予蒸餾水灌胃10m1·kg-1,皮下注射D-半乳糖2.5m1·kg-1；瓊玉膏組每天給予瓊玉膏灌胃10ml·kg-1,皮下注射D-半乳糖2.5ml·kg-1；中藥對照組每天給予六味地黃丸灌胃10ml·kg-1,皮下注射D-半乳糖2.5m1·kg-1；西藥對照組每天給予VitE灌胃2ml·kg-1,皮下注射D-半乳糖2.5ml·kg-1.連續(xù)用藥60d,通過形態(tài)觀察和行為學測試(Morris水迷宮)檢測造模是否成功。 末次給藥后用代謝籠收集24h尿液,加1滴質(zhì)量體積比為1/100(w/v)的疊氮化鈉,備用；將大鼠用10%的水合氯醛按照3ml·kg-1的劑量腹腔注射麻醉,行腹主動脈采血至10ml采血管,血漿靜置后,使用低速離心機取血清,備用。將血清、尿液分別加血清緩沖液、尿液緩沖液,低溫高速離心取上清液置于核磁管,利用NMR技術進行代謝組學檢測。使用TopSpin軟件(V3.0)對譜圖進行預處理；使用SIMCA-P+軟件(V11.0)對歸一化后的數(shù)據(jù)進行模式識別多變量分析,如主成分分析(PCA)、偏最小二乘法-判別分析(PLS-DA)、正交偏最小乘-判別分析等；使用matlab7.0軟件對個別組內(nèi)代謝物的相關性進行分析,最后將獲得的差異代謝物輸入KEGG數(shù)據(jù)庫查找相關代謝通路。 結(jié)果： 1.一般情況及模型驗證：造模前后各組大鼠間的體重比較,P0.05無統(tǒng)計學意義,分組體重無差異。Morris水迷宮結(jié)果為,與空白對照組相比,模型組、瓊玉膏組、中藥對照組、西藥對照組在前5天均有明顯差異(P0.05),第6天僅模型組有差異(P0.05),其他組間差異不明顯(P0.05)。與模型組相比,從第4天開始,各組均有明顯差異(P0.05)。 2.NMR譜圖主成分分析：PCA分析圖顯示,血清各組樣本分布總體較為清晰,R2X=80.6%,Q2=0.718,有顯著差異；而尿液各組樣本分布較為分散,R2X=59.8%,Q2=0.475,差異不大。 3.瓊玉膏抗衰老作用評價：經(jīng)PLS-DA分析,三個用藥組,即瓊玉膏組、西藥對照組、中藥對照組,均與模型組有明顯差別。尤其西藥對照組和模型組區(qū)別最明顯(R2Y=0.935,Q2=0.799)；瓊玉膏組和模型組的區(qū)別也很明顯(R2Y=0.931,Q2=0.826),且西藥對照組和瓊玉膏組沒有顯著差別(R2Y=0.755,Q2=0.002)。 4.差異代謝物篩選：本次研究共篩選、鑒定出血清46種、尿液18種差異代謝物,相關系數(shù)|r|0.576有統(tǒng)計學意義。其中在衰老動物模型血清樣本中變化顯著的有1-甲基組氨酸、醋酸鹽、丙酮、乳酸鹽、檸檬酸、谷氨酸、丙酮酸、琥珀酸鹽、亮氨酸、異亮氨酸、纈氨酸、賴氨酸等33種差異代謝物,而瓊玉膏可以調(diào)節(jié)其中27種代謝物的變化。尿液樣本中變化顯著的有乙酰乙酸、馬尿酸鹽、二甲基甘氨酸、琥珀酸鹽等9種差異代謝物,瓊玉膏可以調(diào)節(jié)其中3種差異代謝物的變化。其中醋酸鹽、乳酸鹽、琥珀酸鹽、谷氨酸等代謝物與衰老變化聯(lián)系較為緊密。 5.代謝通路分析：差異代謝物主要涉及糖代謝、氨基酸代謝、能量代謝、核苷酸代謝、輔酶因子與維生素代謝、脂代謝等多條代謝通路。 結(jié)論： 1.模型組找到平臺的時間明顯長于其他組,造模成功；瓊玉膏等用藥組對D-半乳糖引起的學習記憶能力下降有改善作用。 2.在本次研究中,瓊玉膏抗衰老作用主要表現(xiàn)于血清,而對尿液的影響較小。 3.瓊玉膏抗衰老作用評價：瓊玉膏能夠調(diào)節(jié)衰老大鼠體內(nèi)代謝物的變化,其有效性與公認抗衰劑維生素E相似,優(yōu)于六味地黃丸的抗衰功效。 4.明確瓊玉膏延緩衰老作用機制相關的主要差異代謝物有醋酸鹽、乳酸鹽、琥珀酸鹽、谷氨酸等。 5.瓊玉膏延緩衰老的作用機制與多個代謝通路有關,尤其是糖代謝中的丙酮酸代謝、能量代謝中的氧化磷酸化以及涉及谷氨酸的氨基酸代謝。提示瓊玉膏延緩衰老的作用機制可能與線粒體損傷與腦退化有關。
[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.
【學位授予單位】：廣州中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R285.5
本文編號：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.
【學位授予單位】：廣州中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R285.5
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