逍遥散调节慢性束缚应激大鼠代谢组学的实验研究

作者	单位
罗和古	北京中医药大学中医诊断系
陈家旭	北京中医药大学中医诊断系
岳广欣	北京中医药大学中医诊断系
丁杰	北京中医药大学中医诊断系
颜贤忠	国家生物医学分析中心

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中文摘要:

目的通过检测慢性束缚应激大鼠血浆和逍遥散干预引起的大鼠内源性代谢物的变化,确定与之相关的"代谢组学特征"的小分子标志化合物,探讨慢性束缚应激大鼠代谢网路改变和逍遥散作用机制。方法选用雄性SD大鼠36只,随机分为3组。即:正常对照组、模型组及逍遥散治疗组,其中每组又分为7、21天组,每组6只。以慢性束缚方法制作应激大鼠模型,每天束缚3 h。自造模第1天开始,逍遥散治疗组每日在束缚前1 h灌服逍遥散,给药量为3.854 g/kg,灌胃容积为1 mL/100 g体重。正常对照组、模型组灌服等体积的蒸馏水。分别于第8、22天采集血浆后取上清300μL,在27℃的条件下,在Varian UNITYINO- VA 600MHz超导傅立叶变换核磁共振波谱仪上分别调用弛豫编辑脉冲序列(CPMG)、扩散编辑脉冲序列(LED),采用预饱和方式抑制水峰,自由感应衰减(FID)信号经过32k点傅立叶变换得到一维NMR谱图。以TSP为化学位移参考峰的位置,调用VNMR软件中的程序将~1H谱中从4.5～0.5 ppm(CPMG)以及6.0～0 ppm(LED)范围内的谱峰,按每段为0.04 ppm,进行分段积分。将积分数据归一化之后,将积分值进行中心化和定标,用SIMCA-P 10.0软件包进行主成分分析(PCA),必要时进行判别分析(PLS-DA)。结果(1)正常组与模型组之间代谢产物有明显的不同,两组比较,存在代谢网路的改变。模型组与治疗组各对照组间基本能分开,不同组别之间代谢产物存在差异、代谢网路有所不同,给予逍遥散后可以干预代谢物或代谢途径而致代谢终产物的改变。(2)慢性束缚应急大鼠血清中代谢产物乳酸(1.4、4.16)、胆碱(3.24)、N-乙酰半乳糖胺(NAC)、饱和脂肪酸(1-3)升高,而不饱和脂肪酸(1.99、4-5)、血糖(3-4)、HDL(0.83)等化合物含量降低。逍遥散可使乳酸、胆碱、NAC、饱和脂肪酸下降,血糖、不饱和脂肪酸、HDL、3.44 ppm等化合物含量升高,有明显的代谢终产物的调节效应。结论慢性束缚应激大鼠代谢表型为乳酸、胆碱、NAC、饱和脂肪酸含量上升,血糖、不饱和脂肪酸、HDL、3.44 ppm等化合物含量降低。其代谢产物标志物可能是乳酸、胆碱、NAC、饱和脂肪酸、血糖、不饱和脂肪酸、HDL、3.44 ppm等化合物。慢性束缚应激发生的代谢终产物的改变以脂类物质更明显。逍遥散对代谢终产物有明显的调节效应,但主要干扰何种代谢物或代谢途径而改变代谢终产物还需进一步研究确认。

英文摘要:

Objective To provide a scientific basis for systematic research on the mechanism of chronic im- mobilization stress (CIS) induced metabolic network change in rats,through detecting the changes of endogenous metabolites in rats with CIS,treated or un-treated with Xiaoyao Powder (XYP),for determining the small molecule marker compound that closely associated with the metabonomieal specificity of CIS and acting mechanism of XYP. Methods Thirty-six experimental male SD rats were divided into 3 groups,the normal control group,the model group and the XYP group.And all the three groups were subdivided into two subgroups respectively on day 7 and day 21 of the experiment.The stress rat model of CIS was made by chronic restraining method for 3 h every day. Starting from the first day of modeling,XYP 3.854 g/kg in volume of 1 mL/100 g body weight was administered lb before restraining via gastrogavage to rats in the XYP group,while equal volume of distilled water was given to rats in the other two groups instead.Blood samples were collected on the 8 th day and 22 th day for detection in the fol- lowing procedure:at 27℃,300μL supernate of blood plasma was taken,calling the Carr-Purcell-Meiboom-Gill (CPMG) and longitudinal eddy-delay (LED) sequence respectively on a Fourier variable nuclear magnetic reso- nance (NMR) spectrometer,pre-saturated inhibition of the water peak was performed;free induction decay (FID) signals were transferred via 32 k Fourier transformation to gain one-dimensional NMR spectrogram;by taking TSP as the chemical migration reference peak,the segmental integral calculus (0.04 ppm per segment) was performed from 4.5-0.5 ppm (CPMG) and 6.0-0 ppm (LED) within the peak ranges in ~1H spectra using the VNMR soft- ware ;after normalization,centering and scaling were conducted on data,then used for pattern recognition of princi- pal component analysis (PCA) using the SIMCA-P 10.0 software,or if necessary,the partial least squares discrim- inate analysis (PLS-DA) was performed.Results (1) The metabolites in the model group were significantly dif- ferent from those in the control group,suggesting that the animal model was successfully established with the meta- bolic network different to that of control.The model group and the XYP group could be differentiated from the con- trol group by the differences of metabolites and metabolic networks between groups ;XYP could intervene the metab- olites or the metabolic path to cause changes in final metabolites. (2) The serum contents of laetic acid ( 1.4, 4.16),choline (3.24),N-acetylgalactosamine (NAC) and saturated fatty acids (1-3) increased,but unsaturated fatty acids (1.99,4-5),blood sugar (3-4),HDL (0.83),etc.reduced in the CIS rats.XYP showed obvious regulatory effects on final metabolites,causing decrease of lactic acid,choline,NAC,saturated fatty acids and blood sugar, and increase of unsaturated fatty acids, blood sugar, HDL, 3.44 ppm compound, etc. Conclusions The metabolic phenotype in CIS rats includes the increase of lactic acid,choline,NAC,saturated fatty acid,and the decrease of blood sugar contents,unsaturated fatty acid,HDL,3.44 ppm compound,etc., these may be the markers of the metabolites.The final metabolites changes induced by CIS are primarily the lipid substances.XYP markedly regulates the contents of final metabolites,showing the regulatory effects on final metab- olites,but what is the metabolite or metabolic pathways it interferes to alter the final metabolites should be con- firmed by further studies.

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