A Comparative Study on Fecal Metabonomics in Depressed Rats Intervened by Sinisan Containing Bupleuri Radix and Paeoniae Radix Alba before and after Vinegar-Processing
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摘要: 目的 应用1H-NMR代谢组学技术探究慢性不可预知温和应激(CUMS)抑郁大鼠粪便代谢组学的变化，评价柴胡和白芍醋炙前后组方四逆散的抗抑郁作用，并探讨四逆散中君药柴胡和臣药白芍醋炙的增效机制及炮制内涵。方法 利用CUMS程序建立大鼠抑郁模型，以1H-NMR为技术手段研究不同给药情况下抑郁大鼠粪便代谢组学的变化。结果 CUMS抑郁大鼠模型复制成功。代谢组学结果表明，与空白组比较，模型组大鼠中共有17种代谢物水平发生变化。与模型组比较，四逆散组及柴胡和白芍醋炙后组方的四逆散组干预后的抑郁大鼠代谢发生显著变化。与四逆散组比较，柴胡和白芍醋炙后组方的四逆散组干预的抑郁大鼠粪便中乳酸、α-葡萄糖、β-葡萄糖、甲酸、乙酸、苏氨酸6种代谢物的水平发生变化，同时对丙酮酸代谢、糖酵解与糖质新生代谢、甲烷代谢、乙醛酸和二羧酸代谢、甘氨酸-丝氨酸-苏氨酸代谢、氨酰合成代谢共6条代谢通路产生影响。结论 柴胡和白芍醋炙后组方的四逆散抗抑郁作用更佳，这可能与能量代谢、氨基酸代谢、免疫调控及肠道菌群有关。Abstract: OBJECTIVE To explore the changes of fecal metabonomics in the depressed rats with chronic unpredictable mild stress (CUMS) by 1H-NMR metabonomics technology, evaluate the antidepressant effects of Sinisan containing Bupleuri Radix and Paeoniae Radix Alba before and after vinegar-processing, and discuss the effect enhancement mechanism and processing connotation of Sinisan containing vinegar-processed Bupleuri Radix and vinegar-processed Paeoniae Radix Alba.METHODS The depressed rat model was established using the CUMS program, and the changes of fecal metabonomics in depressed rats under different administration conditions were investigated by 1H-NMR technology.RESULTS The CUMS depressed rat model was successfully replicated. The results of metabonomics showed that, compared to the control group, a total of 17 metabolites were changed in the rats of the model group. Compared to the model group, there were significant changes in the depressed rats intervened by Sinisan and Sinisan containing Bupleuri Radix and Paeoniae Radix Alba after vinegar-processing. Compared to the Sinisan group, a total of 6 metabolites, including lactic acid, α-glucose, β-glucose, formic acid, acetic acid, and threonine, were changed in the feces of the rats intervened by Sinisan containing Bupleuri Radix and Paeoniae Radix Alba after vinegar-processing. Meanwhile, a total of 6 metabolic pathways, including pyruvate metabolism, glycolysis or gluconeogenesis, methane metabolism, glyoxylate and dicarboxylate metabolism, glycine, serine and threonine metabolism, aminoacyl-tRNA biosynthesis, were influenced as well.CONCLUSION Sinisan containing Bupleuri Radix and Paeoniae Radix Alba after vinegar-processing shows better antidepressant effect, which may be related to energy metabolism, amino acid metabolism, immune regulation, and intestinal flora.
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Key words:
- Bupleuri Radix /
- Paeoniae Radix Alba /
- vinegar-processing /
- Sinisan /
- depression /
- metabonomics
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表 1 大鼠行为学指标结果
组别 体质量/g 糖水偏好率/% 强迫游泳时间/s 造模前 造模后 造模前 造模后 造模前 造模后 空白组 260.41±9.87 323.76±9.63 73.83±9.72 83.73±4.76 70.12±25.92 55.67±29.61 模型组 247.70±15.95 286.16±10.04** 68.51±29.28 41.08±8.05** 46.22±10.86 109.59±18.63** 四逆散组 254.07±15.86 315.16±9.25## 80.63±16.62 64.61±10.65## 54.20±28.04 92.01±38.34## 醋炙品组方四逆散组 258.00±15.86 314.29±10.51## 78.28±12.41 74.73±2.34## 56.23±23.51 70.36±18.46## 阳性药组 256.00±13.80 309.73±8.78## 83.05±16.58 72.12±4.48## 51.98±18.92 68.47±38.36## 注：与空白组比较, **P < 0.01；与模型组比较, ##P < 0.01。 
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表 2 内标及大鼠粪便1H-NMR数据归属
编号 代谢物 δ1H 1 DSS 0(s), 0.62(dd), 1.75(m), 2.91(dd) 2 丁酸 0.88(t), 1.55(m), 2.16(t) 3 亮氨酸 0.94(d), 0.95(d), 1.67(m), 1.70(m), 1.74(m), 3.73(m) 4 缬氨酸 0.98(d), 1.03(d), 2.26(m), 3.60(d) 5 异亮氨酸 0.93(t), 1.00(d), 1.25(m), 1.46(m), 1.97(m), 3.66(d) 6 丙酸 1.04(t), 2.17(q) 7 乳酸 1.32(d), 4.11(q) 8 苏氨酸 1.32(d), 3.58(d), 4.24(m) 9 乙偶姻 1.37(d), 2.21(s), 4.42(q) 10 丙氨酸 1.47(d), 3.77(q) 11 乙酸 1.91(s) 12 谷氨酸 2.34(dt), 2.04(m), 2.11(m), 3.75(m) 13 琥珀酸 2.40(s) 14 天冬氨酸 2.67(dd), 2.80(dd), 3.89(m) 15 赖氨酸 1.43(m), 1.49(m), 1.71(m), 1.86(m), 1.92(m), 3.01(t), 3.75(t) 16 甲醇 3.35(s) 17 甘氨酸 3.55(s) 18 β-阿拉伯糖 4.51(d), 3.51(dd), 3.66(m) 19 α-阿拉伯糖 5.26(d), 4.11(m), 3.83(dd) 20 β-木糖 4.57(d), 3.22(dd), 3.31(t), 3.45(m), 3.92(dd), 3.62(m) 21 α-木糖 5.19(d), 3.52(dd), 3.43(t), 3.67(m), 3.60(dd), 3.64(m) 22 β-葡萄糖 4.64(d), 3.24(dd), 3.48(m), 3.41(m), 3.75, 3.90(dd), 3.48(m) 23 α-葡萄糖 5.23(d), 3.52(dd), 3.71(m), 3.74(m), 3.74, 3.81(dd), 3.84(m) 24 尿嘧啶 5.79(d), 7.53(d) 25 延胡索酸 6.51(s) 26 酪氨酸 3.04(dd), 3.18(dd), 3.93(dd), 6.89(d), 7.18(d) 27 苯丙氨酸 3.12(dd), 3.27(dd), 3.98(dd), 7.32(m), 7.36(m), 7.42(m) 28 色氨酸 7.19(t), 7.27(t), 7.53(d), 7.72(d) 29 尿刊酸 6.39(d), 7.28(d), 7.35(s), 7.86(s) 30 黄嘌呤 7.91(s) 31 次黄嘌呤 8.18(s), 8.20(s) 32 甲酸 8.44(s) 33 烟酸 7.51(m), 8.24(m), 8.60(dd), 8.93(s) 注：s.单重峰；d.双重峰；t.三重峰；q.四重峰；dd.双重双峰；m.多重峰
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表 3 空白组与模型组存在显著性差异的代谢物及其相关系数
代谢物 δ1H r P 变化倍数 次黄嘌呤 8.18(s), 8.20(s) -0.834 0 3.99E-04 0.485 8 黄嘌呤 7.91(s) -0.905 7 1.99E-05 0.371 8 苯丙氨酸 3.12(dd), 3.27(dd), 3.98(dd), 7.32(m), 7.36(m), 7.42(m) -0.769 4 2.08E-03 0.523 3 酪氨酸 3.04(dd), 3.18(dd), 3.93(dd), 6.89(d), 7.18(d) -0.739 8 3.78E-03 0.443 4 延胡索酸 6.51(s) 0.800 7 1.02E-03 2.380 4 尿嘧啶 5.79(d), 7.53(d) -0.829 3 4.59E-04 0.343 2 α-阿拉伯糖 5.26(d), 4.11(m), 3.83(dd) -0.989 2 7.49E-02 0.919 8 α-木糖 5.19(d), 3.52(dd), 3.43(t), 3.67(m), 3.60(dd), 3.64(m) 0.794 6 1.17E-03 2.478 5 β-木糖 4.57(d), 3.22(dd), 3.31(t), 3.45(m), 3.92(dd), 3.62(m) 0.761 0 2.52E-03 2.206 9 甘氨酸 3.55(s) -0.712 6 6.19E-03 0.604 4 甲醇 3.35 (s) -0.824 4 5.05E-04 0.414 9 谷氨酸 2.34(dt), 2.04(m), 2.11(m), 3.75(m) -0.671 3 1.20E-02 0.603 4 乙酸 1.91 (s) -0.946 6 8.59E-07 0.319 1 丙氨酸 1.47(d), 3.77(q) -0.838 3 3.33E-04 0.429 4 丙酸 1.04(t), 2.17(q) -0.898 2 2.84E-05 0.606 4 异亮氨酸 0.93(t), 1.00(d), 1.25(m), 1.46(m), 1.97(m), 3.66(d) -0.802 5 9.39E-05 0.377 1 丁酸 0.88(t), 1.55(m), 2.16(t) -0.929 4 1.12E-09 0.201 9
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表 4 四逆散组和醋炙品组方四逆散组存在显著性差异的代谢物及其相关系数
代谢物 δ1H r P 变化倍数 甲酸 8.44(s) 0.693 034 0.010 20 7.349 7 α-葡萄糖 5.23(d), 3.52(dd), 3.71(m), 3.74(m), 3.74, 3.81(dd), 3.84(m) -0.654 767 0.015 46 0.498 0 β-葡萄糖 4.64(d), 3.24(dd), 3.48(m), 3.41(m), 3.75, 3.90(dd), 3.48(m) -0.767 294 0.002 46 0.270 2 乙酸 1.91(s) 0.728 564 0.004 11 1.345 0 苏氨酸 1.32(d), 3.58(d), 4.24(m) 0.639 519 0.015 96 1.576 9 乳酸 1.32(d), 4.11(q) 0.627 454 0.020 30 1.457 5
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