药学学报

Group	CN	MD	ALP	SZRD
Occludin	165.54±10.63	128.34±9.52^##	178.55±24.65^***	178.56±17.82^***
Claudin 1	184.29±21.78	128.19±6.96^###	185.49±13.44^***	171.827 2±15.08^***
ZO-1	179.44±19.97	120.97±13.87^###	182.15±19.62^***	164.64±8.07^***

Group	CN	MD	ALP	SZRD
Occludin	165.54±10.63	128.34±9.52^##	178.55±24.65^***	178.56±17.82^***
Claudin 1	184.29±21.78	128.19±6.96^###	185.49±13.44^***	171.827 2±15.08^***
ZO-1	179.44±19.97	120.97±13.87^###	182.15±19.62^***	164.64±8.07^***

Type	Metabolite	Formula	Adduct	t_R/min	MW/Da	MS/MS fragments	MD vs CN	ALP vs MD	SZRD vs MD
Amino acid	L-Valine	C₅H₁₁NO₂	[M-H]^-	1.159	116.072 6	91.058 4, 65.041 3	↑^#	↓^*	↓^***
L-Tyrosine	C₉H₁₁NO₃	[M-H]^-	1.305	180.067 6	163.043 7, 119.050 8, 93.032 8, 72.008 6	↑^###	↓^*	↓^**
Phenylalanine	C₉H₁₁NO₂	[M+H]⁺	1.983	166.086 6	166.083 8, 120.082 3, 77.043 5, 52.035 0	↑^###	-	↓^*
L-Kynurenine	C₁₀H₁₂N₂O₃	[M+H]⁺	1.907	209.092 6	174.055 1, 146.058 6, 120.043 3, 91.057 2	↑^##	-	↓^*
Leucylproline	C₁₁H₂₀N₂O₃	[M+H]⁺	1.344	229.152 7	55.024 1, 70.068 3	↓^##	↑^**	↑^**
Proline	C₅H₉NO₂	[M+H]⁺	0.900	116.072 5	116.072 7, 70.074 1	↑^#	-	-
Methionine	C₅H₁₁NO₂S	[M+H]⁺	1.160	150.057 3	133.030 2, 74.025 9, 61.016 6	↑^###	↑^*	↓^*
5-oxo-L-Proline	C₅H₇NO₃	[M+H]⁺	1.170	130.047 9	130.062 1, 103.058 4, 84.038 6, 56.053 5	↑^#	-	-
Isoleucine	C₆H₁₃NO₂	[M+H]⁺	1.390	132.102 5	86.097 2, 69.072 1, 56.055 3	↑^##	-	↓^*
Norvaline	C₅H₁₁NO₂	[M+H]⁺	3.000	118.064 6	55.063 5, 89.035 1, 91.071 9	↑^#	-	↓^*
Tryptophan	C₁₁H₁₂N₂O₂	[M+H]⁺	3.030	205.098 1	170.060 2, 143.073 3, 118.064 6, 91.056 5	↑^#	-	↓^*
Arginine	C₆H₁₄N₄O₂	[M+H]⁺	0.82	175.117 9	130.093 3, 116.071 4, 70.069 8	↑^##	-	-
Lysine	C₆H₁₄N₂O₂	[M+H]⁺	0.763	147.111 8	55.057 9, 67.057 0, 84.082 9	↑^##	↑^*	↑^***
Organic acid	β-Hydroxyisovaleric acid	C₅H₁₀O₃	[M-H]^-	2.904	117.056 4	117.056 4, 69.040 9	↑^###	↓^*	↓^*
3-Methyl-2-oxovaleric acid	C₆H₁₀O₃	[M-H]^-	4.110	129.055 6	51.026 6, 67.023 4, 84.764 8	↑^###	-	↓^*
3-Methyl-2-oxovalerate	C₆H₁₀O₃	[M-H₂O-H]^-	3.825	111.044 6	50.870 8, 70.499 7, 101.065 8	↑^###	-	-
Succinate	C₄H₆O₄	[M-H]^-	4.862	248.959 1	60.988 4, 72.467 0, 112.987 3	↑^##	↑^*	↑^***
γ-Linolenic acid	C₁₈H₃₀O₂	[M-H]^-	11.127	277.216 5	107.045 7, 121.027 3, 165.062 0	↑^##	-	↑^**
Stearic acid	C₁₈H₃₆O₂	[M-H]^-	12.996	283.262 6	283.258 7	↑^##	-	-
Urate	C₅H₄N₄O₃	[M-H]^-	0.886	357.029 0	69.011 2, 97.006 1, 136.917 9, 161.002 1	↓^###	-	-
Uric acid	C₅H₄N₄O₃	[M+H]⁺	0.947	169.036 6	53.027 8, 69.011 4, 96.016 2	↓^###	-	-
Citrate	C₆H₈O₇	[M+H]⁺	1.164	215.015 9	72.114 5, 77.039 7	↑^##	-	↓^*
3-Hydroxybutyric acid	C₄H₈O₃	[M-H]^-	1.430	103.041 0	59.011 2	↑^##	↓^*	↓^*
Citric acid	C₆H₈O₇	[M-H]-	1.188	191.020 2	67.018 6, 57.035 4	↑^#	-	↓^*
L-Pipecolic acid	C₆H₁₁NO₂	[M+H]⁺	0.763	130.086 8	55.055 1, 56.053 2, 67.058 5	↑^#	-	-
Creatine	C₄H₉N₃O₂	[M+H]⁺	0.891	132.077 0	55.033 4, 58.059 5, 69.046 0, 72.058 9	↓^#	-	↑^*
Xanthosine	C₁₀H₁₂N₄O₆	[M-H]^-	1.904	283.067 2	151.025 2, 108.019 8	↑^##		↑^*
Indole-3-propionic acid	C₁₁H₁₁NO₂	[M-H]^-	6.063	188.070 3	59.016 8, 116.049 0, 142.064 4	↓^#	↑^**	↑^***
Amide	N-Isovalerylglycine	C₇H₁₃NO₃	[M-H]^-	3.618	158.082 5	74.024 4, 158.082 5	↑^##	-	↓^*
Nicotinamide	C₆H₆N₂O	[M+H]⁺	1.160	123.044 4	96.044 8, 80.052 8, 52.032 8	↓^#	-	-
Lipid	PE (16:1-22:6)	C₄₃H₇₂NO₈P	[M-H]^-	9.367	760.487 9	96.971 9, 164.963 9, 253.220 3	↑^##	-	-
PC 35:1	C₄₃H₈₄NO₈P	[M-H]^-	11.205	832.594 4	168.042 9, 267.148 8, 281.246 6	↑^##	-	↓^*
SM (18:2-C23:0)	C₄₆H₉₁N₂O₆P	[M-H]^-	9.246	857.671 9	78.960 4, 168.032 0, 236.058 6	↑^###	↑^*	↑^**
SM 33:1	C₃₈H₇₇N₂O₆P	[M-H]^-	9.982	747.561 0	78.961 3, 123.000 2, 168.040 3	↓^#	-	↑^***
PC 33:2	C₄₁H₇₈NO₈P	[M-H]^-	10.098	802.557 8	78.961 2, 145.462 4, 168.039 5	↓^###	↑^*	↑^**
PC 39:6	C₄₇H₈₂NO₈P	[M-H]^-	10.585	878.587 6	78.962 4, 229.199 0, 313.172 4, 508.336 6	↓^#	↑^*	↑^*
Ceramide (d18:1/16:0)	C₃₄H₆₇NO₃	[M+H]⁺	10.413	560.508 1	87.058 3, 133.102 4	↑^#	↑^***	↑^***
PI (16:0/22:6)	C₄₃H₇₂NO₈P	[M-H]^-	9.367	760.494 6	96.972 1, 253.220 3	↑^#	↑^*	↑^***
SM (18:1/C23:1)	C₄₆H₉₁N₂O₆P	[M-H]^-	9.87	857.678 7	78.960 3, 241.010 4	↑^##	-	↑^*
SM d41:1	C₄₆H₉₃N₂O₆P	[M-H]^-	9.246	859.682 1	78.960 6, 799.676 4	↑^##	↑^*	↑^***
PI 38:4	C₄₇H₈₃O₁₃P	[M-H]^-	14.537	885.542 2	78.958 8, 241.008 1	↑^##	-	↑^*
Arachidonic acid	C₂₀H₃₂O₂	[M-H]^-	11.438	303.233 2	59.018 2	↑^###	-	↑^***
Indoles	3-Formylindole	C₉H₇NO	[M+H]⁺	3.000	146.060 6	117.055 6, 91.054 6, 65.041 5	↑^##	-	↓^*
Indoline	C₈H₉N	[M+H]⁺	1.980	120.081 4	50.022 1, 55.937 3, 65.063 2	↑^###	-	-
Others	Choline	C₅H₁₄NO	[M]⁺	0.870	104.106 9	88.093 1, 58.063 2	↑^#	-	-
Betaine	C₅H₁₁NO₂	[M+H]⁺	0.900	118.085 9	81.254 3, 58.072 0	↑^#	-	-
Bilirubin	C₃₃H₃₆N₄O₆	[M+H]⁺	10.330	585.270 2	89.068 2, 299.141 3	↑^#	-	-
Taurohyodeoxycholic acid	C₂₆H₄₅NO₆S	[M+H-H₂O]⁺	5.847	482.292 2	464.258 1, 339.256 8, 126.021 3	↑^#	-	↓^*
Taurocholic acid	C₂₆H₄₅NO₇S	[M+H]⁺	5.906	516.297 8	462.272 4, 337.247 4, 126.021 9	↑^#	↓^*	↓^*
Glycocholate	C₂₆H₄₃NO₆	[M+H]⁺	6.359	466.316 3	55.059 4, 93.073 4, 237.159 9	↓^#	↓^*	-
Taurine	C₂H₇NO₃S	[M-H]^-	0.820	124.008 2	79.957 7, 124.008 2	↓^#	-	-
Estrone-3-sulfate	C₁₈H₂₂O₅S	[M-H]^-	9.555	349.109 2	61.992 5, 96.958 9, 265.145 6	↑^###	↓^**	↓^***

Type	Metabolite	Formula	Adduct	t_R/min	MW/Da	MS/MS fragments	MD vs CN	ALP vs MD	SZRD vs MD
Amino acid	L-Valine	C₅H₁₁NO₂	[M-H]^-	1.159	116.072 6	91.058 4, 65.041 3	↑^#	↓^*	↓^***
L-Tyrosine	C₉H₁₁NO₃	[M-H]^-	1.305	180.067 6	163.043 7, 119.050 8, 93.032 8, 72.008 6	↑^###	↓^*	↓^**
Phenylalanine	C₉H₁₁NO₂	[M+H]⁺	1.983	166.086 6	166.083 8, 120.082 3, 77.043 5, 52.035 0	↑^###	-	↓^*
L-Kynurenine	C₁₀H₁₂N₂O₃	[M+H]⁺	1.907	209.092 6	174.055 1, 146.058 6, 120.043 3, 91.057 2	↑^##	-	↓^*
Leucylproline	C₁₁H₂₀N₂O₃	[M+H]⁺	1.344	229.152 7	55.024 1, 70.068 3	↓^##	↑^**	↑^**
Proline	C₅H₉NO₂	[M+H]⁺	0.900	116.072 5	116.072 7, 70.074 1	↑^#	-	-
Methionine	C₅H₁₁NO₂S	[M+H]⁺	1.160	150.057 3	133.030 2, 74.025 9, 61.016 6	↑^###	↑^*	↓^*
5-oxo-L-Proline	C₅H₇NO₃	[M+H]⁺	1.170	130.047 9	130.062 1, 103.058 4, 84.038 6, 56.053 5	↑^#	-	-
Isoleucine	C₆H₁₃NO₂	[M+H]⁺	1.390	132.102 5	86.097 2, 69.072 1, 56.055 3	↑^##	-	↓^*
Norvaline	C₅H₁₁NO₂	[M+H]⁺	3.000	118.064 6	55.063 5, 89.035 1, 91.071 9	↑^#	-	↓^*
Tryptophan	C₁₁H₁₂N₂O₂	[M+H]⁺	3.030	205.098 1	170.060 2, 143.073 3, 118.064 6, 91.056 5	↑^#	-	↓^*
Arginine	C₆H₁₄N₄O₂	[M+H]⁺	0.82	175.117 9	130.093 3, 116.071 4, 70.069 8	↑^##	-	-
Lysine	C₆H₁₄N₂O₂	[M+H]⁺	0.763	147.111 8	55.057 9, 67.057 0, 84.082 9	↑^##	↑^*	↑^***
Organic acid	β-Hydroxyisovaleric acid	C₅H₁₀O₃	[M-H]^-	2.904	117.056 4	117.056 4, 69.040 9	↑^###	↓^*	↓^*
3-Methyl-2-oxovaleric acid	C₆H₁₀O₃	[M-H]^-	4.110	129.055 6	51.026 6, 67.023 4, 84.764 8	↑^###	-	↓^*
3-Methyl-2-oxovalerate	C₆H₁₀O₃	[M-H₂O-H]^-	3.825	111.044 6	50.870 8, 70.499 7, 101.065 8	↑^###	-	-
Succinate	C₄H₆O₄	[M-H]^-	4.862	248.959 1	60.988 4, 72.467 0, 112.987 3	↑^##	↑^*	↑^***
γ-Linolenic acid	C₁₈H₃₀O₂	[M-H]^-	11.127	277.216 5	107.045 7, 121.027 3, 165.062 0	↑^##	-	↑^**
Stearic acid	C₁₈H₃₆O₂	[M-H]^-	12.996	283.262 6	283.258 7	↑^##	-	-
Urate	C₅H₄N₄O₃	[M-H]^-	0.886	357.029 0	69.011 2, 97.006 1, 136.917 9, 161.002 1	↓^###	-	-
Uric acid	C₅H₄N₄O₃	[M+H]⁺	0.947	169.036 6	53.027 8, 69.011 4, 96.016 2	↓^###	-	-
Citrate	C₆H₈O₇	[M+H]⁺	1.164	215.015 9	72.114 5, 77.039 7	↑^##	-	↓^*
3-Hydroxybutyric acid	C₄H₈O₃	[M-H]^-	1.430	103.041 0	59.011 2	↑^##	↓^*	↓^*
Citric acid	C₆H₈O₇	[M-H]-	1.188	191.020 2	67.018 6, 57.035 4	↑^#	-	↓^*
L-Pipecolic acid	C₆H₁₁NO₂	[M+H]⁺	0.763	130.086 8	55.055 1, 56.053 2, 67.058 5	↑^#	-	-
Creatine	C₄H₉N₃O₂	[M+H]⁺	0.891	132.077 0	55.033 4, 58.059 5, 69.046 0, 72.058 9	↓^#	-	↑^*
Xanthosine	C₁₀H₁₂N₄O₆	[M-H]^-	1.904	283.067 2	151.025 2, 108.019 8	↑^##		↑^*
Indole-3-propionic acid	C₁₁H₁₁NO₂	[M-H]^-	6.063	188.070 3	59.016 8, 116.049 0, 142.064 4	↓^#	↑^**	↑^***
Amide	N-Isovalerylglycine	C₇H₁₃NO₃	[M-H]^-	3.618	158.082 5	74.024 4, 158.082 5	↑^##	-	↓^*
Nicotinamide	C₆H₆N₂O	[M+H]⁺	1.160	123.044 4	96.044 8, 80.052 8, 52.032 8	↓^#	-	-
Lipid	PE (16:1-22:6)	C₄₃H₇₂NO₈P	[M-H]^-	9.367	760.487 9	96.971 9, 164.963 9, 253.220 3	↑^##	-	-
PC 35:1	C₄₃H₈₄NO₈P	[M-H]^-	11.205	832.594 4	168.042 9, 267.148 8, 281.246 6	↑^##	-	↓^*
SM (18:2-C23:0)	C₄₆H₉₁N₂O₆P	[M-H]^-	9.246	857.671 9	78.960 4, 168.032 0, 236.058 6	↑^###	↑^*	↑^**
SM 33:1	C₃₈H₇₇N₂O₆P	[M-H]^-	9.982	747.561 0	78.961 3, 123.000 2, 168.040 3	↓^#	-	↑^***
PC 33:2	C₄₁H₇₈NO₈P	[M-H]^-	10.098	802.557 8	78.961 2, 145.462 4, 168.039 5	↓^###	↑^*	↑^**
PC 39:6	C₄₇H₈₂NO₈P	[M-H]^-	10.585	878.587 6	78.962 4, 229.199 0, 313.172 4, 508.336 6	↓^#	↑^*	↑^*
Ceramide (d18:1/16:0)	C₃₄H₆₇NO₃	[M+H]⁺	10.413	560.508 1	87.058 3, 133.102 4	↑^#	↑^***	↑^***
PI (16:0/22:6)	C₄₃H₇₂NO₈P	[M-H]^-	9.367	760.494 6	96.972 1, 253.220 3	↑^#	↑^*	↑^***
SM (18:1/C23:1)	C₄₆H₉₁N₂O₆P	[M-H]^-	9.87	857.678 7	78.960 3, 241.010 4	↑^##	-	↑^*
SM d41:1	C₄₆H₉₃N₂O₆P	[M-H]^-	9.246	859.682 1	78.960 6, 799.676 4	↑^##	↑^*	↑^***
PI 38:4	C₄₇H₈₃O₁₃P	[M-H]^-	14.537	885.542 2	78.958 8, 241.008 1	↑^##	-	↑^*
Arachidonic acid	C₂₀H₃₂O₂	[M-H]^-	11.438	303.233 2	59.018 2	↑^###	-	↑^***
Indoles	3-Formylindole	C₉H₇NO	[M+H]⁺	3.000	146.060 6	117.055 6, 91.054 6, 65.041 5	↑^##	-	↓^*
Indoline	C₈H₉N	[M+H]⁺	1.980	120.081 4	50.022 1, 55.937 3, 65.063 2	↑^###	-	-
Others	Choline	C₅H₁₄NO	[M]⁺	0.870	104.106 9	88.093 1, 58.063 2	↑^#	-	-
Betaine	C₅H₁₁NO₂	[M+H]⁺	0.900	118.085 9	81.254 3, 58.072 0	↑^#	-	-
Bilirubin	C₃₃H₃₆N₄O₆	[M+H]⁺	10.330	585.270 2	89.068 2, 299.141 3	↑^#	-	-
Taurohyodeoxycholic acid	C₂₆H₄₅NO₆S	[M+H-H₂O]⁺	5.847	482.292 2	464.258 1, 339.256 8, 126.021 3	↑^#	-	↓^*
Taurocholic acid	C₂₆H₄₅NO₇S	[M+H]⁺	5.906	516.297 8	462.272 4, 337.247 4, 126.021 9	↑^#	↓^*	↓^*
Glycocholate	C₂₆H₄₃NO₆	[M+H]⁺	6.359	466.316 3	55.059 4, 93.073 4, 237.159 9	↓^#	↓^*	-
Taurine	C₂H₇NO₃S	[M-H]^-	0.820	124.008 2	79.957 7, 124.008 2	↓^#	-	-
Estrone-3-sulfate	C₁₈H₂₂O₅S	[M-H]^-	9.555	349.109 2	61.992 5, 96.958 9, 265.145 6	↑^###	↓^**	↓^***

基于血清代谢组学探讨酸枣仁汤改善睡眠剥夺大鼠焦虑样行为的作用机制

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孙亚禹 ¹ , 刘嘉颖 ¹ , 任剑峰 ² , 杜晨晖 ²^,^* , 闫艳 ¹^,^*

药学学报 | 研究论文 2026,61(6): 2180-2191

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药学学报 |研究论文 2026 , 61 (6) : 2180 -2191

基于血清代谢组学探讨酸枣仁汤改善睡眠剥夺大鼠焦虑样行为的作用机制

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孙亚禹¹, 刘嘉颖¹, 任剑峰², 杜晨晖²^,^*, 闫艳¹^,^*

作者信息

¹山西大学中医药现代研究中心, 山西太原 030006

²山西中医药大学中药与食品工程学院, 山西太原 030619

通讯作者:

杜晨晖, E-mail: dch@sxtcm.edu.cn

闫艳, Tel: 86-351-7018379, E-mail: yanyan520@sxu.edu.cn

作者简介:

作者贡献:

杜晨晖、闫艳负责研究思路的提出; 孙亚禹、刘嘉颖参与实验实施及数据处理; 孙亚禹撰写文章草稿并负责修改; 任剑峰负责论文写作指导。

Mechanistic insights from serum metabolomics into how Suanzaoren Decoction alleviates anxiety-like behavior in sleep-deprived rats

Ya-yu SUN¹, Jia-ying LIU¹, Jian-feng REN², Chen-hui DU²^,^*, Yan YAN¹^,^*

Affiliations

¹Modern Research Center for Traditional Chinese Medicine, Shanxi University, Taiyuan 030006, China

²College of Traditional Chinese Medicine and Food Engineering, Shanxi University of Chinese Medicine, Taiyuan 030619, China

出版时间: 2026-06-12 doi: 10.16438/j.0513-4870.2025-1378

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摘要

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采用血清代谢组学技术探讨酸枣仁汤(Suanzaoren Decoction, SZRD) 改善大鼠焦虑样行为的作用机制。采用改良多平台水环境法制备失眠模型。与空白组比, 每天睡眠剥夺18 h, 造模21天后, 模型组大鼠运动总距离和中心区域活动时间均显著增加(P < 0.01); 进入开臂时间占比显著减小(P < 0.001), 进入开臂次数百分比明显降低(P < 0.001), 表明睡眠剥夺大鼠出现焦虑样行为。与模型组相比, SZRD能显著降低睡眠剥夺大鼠总运动距离和中心区域停留时间(P < 0.01); 并显著增加进入开臂的时间比例以及次数占比(P < 0.001), 显著改善睡眠剥夺大鼠失眠伴焦虑样行为。病理切片及免疫荧光结果显示, 与空白组比, 模型组大鼠肠道黏膜上皮细胞出现炎性浸润; 结肠组织ZO-1、occludin、claudin-1平均荧光强度均显著降低; 肝脏红色脂滴增多, SZRD组能够改善肠道与肝脏损伤, 且增加结肠occludin、claudin-1、ZO-1平均荧光强度。此外, GC-MS测定结果表明, 睡眠剥夺大鼠结肠内容物中乙酸、丙酸、异丁酸、戊酸、异戊酸和异己酸(P < 0.05) 含量显著降低, SZRD显著增加了6种短链脂肪酸含量。为进一步探明机制, 采用UPLC-Q-TOF-MS/MS技术进行血清代谢组学研究, 与正常大鼠相比, 模型组共扰动53个差异代谢物; 而SZRD显著回调其中的36个差异代谢物, 包括氨基酸类、有机酸类、脂质类等。KEGG通过分析显示, SZRD主要影响了苯丙氨酸、酪氨酸与色氨酸生物合成, 色氨酸代谢、酪氨酸代谢、三羧酸循环等代谢通路。SZRD改善了睡眠剥夺导致的大鼠肠道和肝脏炎性损伤, 降低了肠道通透性, 主要通过调控氨基酸代谢和三羧酸循环发挥抗失眠伴焦虑的作用。研究结果将为阐明SZRD治疗“虚劳虚烦不得眠”的作用特点提供科学依据。本实验获得山西中医药大学动物伦理委员会批准(批准号: AWE202310391)。

关键词

酸枣仁汤 / 睡眠剥夺 / 焦虑样行为 / 血清代谢组学 / 短链脂肪酸

Abstract

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Serum metabolomics reveals the mechanism of Suanzaoren Decoction (SZRD) in ameliorating anxiety-like behaviors in rats. Insomnia was induced by the modified multiple platform method. Compared with the control group, rats subjected to 18-hour daily sleep deprivation for 21 days exhibited significantly increased total locomotor distance and center zone exploration time (P < 0.01), alongside markedly reduced open arm entry time percentage and frequency (P < 0.001), indicating anxiety-like behaviors. SZRD administration significantly decreased locomotor activity and center zone duration (P < 0.01), while increasing open arm entry time and frequency (P < 0.001), thereby alleviating insomnia-associated anxiety. Pathological sections and immunofluorescence results revealed that, compared with the control group, the model group exhibited inflammatory infiltration in the intestinal mucosal epithelial cells, along with significantly decreased mean fluorescence intensities of ZO-1, occludin, and claudin-1 in the colonic tissue, together with increased hepatic lipid accumulation. SZRD ameliorated intestinal and hepatic damage while enhancing tight junction protein expression. GC-MS analysis demonstrated that the significantly reduced levels of acetic acid, propionic acid, isobutyric acid, valeric acid, isovaleric acid, and isocaproic acid in the colonic contents of sleep-deprived rats (P < 0.05) were significantly reversed by SZRD. UPLC-Q-TOF-MS/MS-based serum metabolomics identified 53 differential metabolites in model rats compared to controls; SZRD significantly reversed 36 of these, encompassing amino acids, organic acids, and lipids. KEGG pathway analysis indicated that SZRD primarily modulated phenylalanine, tyrosine and tryptophan biosynthesis, tryptophan metabolism, tyrosine metabolism, and the tricarboxylic acid cycle. These findings demonstrate that SZRD ameliorates intestinal and hepatic inflammatory injury, reduces intestinal permeability, and exerts anti-insomnia and anxiolytic effects primarily through regulating amino acid metabolism and the tricarboxylic acid cycle, providing scientific evidence for elucidating the therapeutic characteristics of SZRD in treating "consumptive disease with restlessness and insomnia". This experiment was approved by the Animal Ethics Committee of Shanxi University of Chinese Medicine (Approval No.: AWE202310391).

Key words

Suanzaoren Decoction / sleep deprivation / anxiety-like behavior / serum metabolomics / short-chain fatty acid

引用本文

孙亚禹, 刘嘉颖, 任剑峰, 杜晨晖, 闫艳. 基于血清代谢组学探讨酸枣仁汤改善睡眠剥夺大鼠焦虑样行为的作用机制. 药学学报, 2026 , 61 (6) : 2180 -2191 . DOI: 10.16438/j.0513-4870.2025-1378

Ya-yu SUN, Jia-ying LIU, Jian-feng REN, Chen-hui DU, Yan YAN. Mechanistic insights from serum metabolomics into how Suanzaoren Decoction alleviates anxiety-like behavior in sleep-deprived rats[J]. Acta Pharmaceutica Sinica, 2026 , 61 (6) : 2180 -2191 . DOI: 10.16438/j.0513-4870.2025-1378

正文

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失眠是以频繁而持续的睡眠起始或维持困难并导致睡眠满意度不足为特征的睡眠障碍, 常伴有疲劳、情绪低落和认知障碍等日间功能损害。《2025中国健康睡眠白皮书》显示, 我国65%的人群存在睡眠紊乱症状^[1]。临床治疗失眠的药物包括苯二氮䓬类、非苯二氮䓬类及新兴食欲素受体拮抗剂苏沃雷生。尽管这些药物能够改善失眠症状, 但其使用常伴随成瘾性、嗜睡、头晕及乏力等不良反应^[2]。

酸枣仁汤(Suanzaoren Decoction, SZRD) 出自东汉末年张仲景《金匮要略·血痹虚劳病脉证并治》, “虚劳虚烦不得眠, 酸枣汤主之”, 是治疗失眠第一方。临床研究显示^{[3, 4]}, SZRD加减方能延长失眠伴焦虑症患者睡眠时间, 提高睡眠质量, 并能改善患者烦躁易怒、多梦易醒、心烦失眠等症状。动物实验研究表明^{[5, 6]}, SZRD具有改善对氯苯丙氨酸诱导失眠模型大鼠焦虑样行为作用, 且能显著降低改良多平台水环境法睡眠剥夺模型大鼠兴奋性行为。本课题组前期研究^[7]显示SZRD能显著降低慢性束缚应激焦虑模型小鼠焦虑指数, 且能延长焦虑小鼠睡眠时间。综上, 临床和动物研究均显示SZRD在改善睡眠的同时, 兼具缓解焦虑样情绪的作用。然而, SZRD改善睡眠障碍所致焦虑样行为的作用机制研究尚属空白。

代谢组学作为发现组学, 可定性和半定量分析生物体内代谢物水平的变化, 是研究生物体潜在病理生理机制和生物标志物的重要手段^{[8, 9]}。非靶向代谢组学旨在尽可能广泛地检测、识别和相对定量生物样本中所有代谢物, 具有高灵敏、高通量和实时性等特点, 能够无偏向性地揭示生物体系内整体的、动态的代谢物变化规律。

本研究旨在通过制备改良多平台水环境法睡眠剥夺模型, 以旷场和高架十字迷宫实验, 进行SZRD改善睡眠剥夺焦虑样行为药效学评价。利用苏木精-伊红(HE) 染色、AB-PAS (Alcian blue - periodic acid Schiff, AB-PAS) 染色、油红染色考察睡眠剥夺对肝脏和肠道损伤程度; 并采用自建的GC-MS法测定结肠内容物中短链脂肪酸(乙酸、丙酸、异丁酸、异戊酸、戊酸以及异己酸) 的含量; 采用UPLC-Q-TOF-MS血清代谢组学和多元统计分析技术, 发现血清差异代谢物, 并富集相关代谢通路; 最后, 采用Pearson相关性分析短链脂肪酸与差异代谢物, 揭示SZRD的作用特点以及可能的作用途径。本研究为阐明SZRD的作用机制提供新的方向与途径。

材料与方法

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动物 SPF级雄性SD大鼠(8周) 龄, 28只(180 ± 20 g), 购自北京维通利华实验动物技术有限公司, 动物许可证号SCXK (京) 2021-0006。动物饲养于湿度50% ± 10%、温度(25 ± 2) ℃、12 h昼夜循环的环境中, 自由进食饮水。动物实验经山西中医药大学动物伦理委员会批准(批准号AWE202310391)。

药品与试剂 酸枣仁(批号: 20220430, 山西灵石县福禄堂农业科技有限公司)、茯苓(批号: 200501, 亳州市京皖中药饮片厂)、知母(批号2020110451, 安徽盛海堂中药饮片有限公司)、川芎(批号200713005, 北京仟草中药饮片有限公司) 和甘草(批号201202, 亳州市京皖中药饮片厂) 饮片。经山西中医药大学中药鉴定教研室杜晨晖教授鉴定: 酸枣仁为鼠李科酸枣Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H.F. Chou的干燥成熟种子; 茯苓为多孔菌科真菌茯苓Poriacocos (Schw.) Wolf.的干燥菌核; 知母为百合科植物知母Anemarrhena asphodeloides Bunge.的干燥根茎; 川芎为伞形科植物川芎Ligusticum chuanxiong Hort.的干燥根茎; 甘草为豆科植物甘草Glycyrrhiza uralensis Fisch的干燥根和根茎。

阿普唑仑(货号H31021282) 购自上海上药信谊药厂有限公司; 质谱级乙腈(货号A801556)、甲醇(货号M813903)、甲酸(货号F874924) 购自美国ThermoFisher Scientific公司; 二甲苯(货号X823028)、DAPI染液(货号G1012) 购自北京索莱宝科技有限公司; Triton-100 (货号GC204003)、抗荧光淬灭封片剂(货号G1401) 购自武汉赛维尔生物科技有限公司。

仪器自制睡眠剥夺箱(高70 cm, 宽80 cm, 长110 cm), 内部放有15根直径为6.5 cm的不锈钢柱。OFT-100型自主活动测试仪(成都泰盟科技有限公司)。Olympus双光子共聚焦倒置荧光显微镜(型号FV1200MPE)。Agilent 1290 Infinity Ⅱ超高效液相色谱系统、Agilent 7890 B气相质谱仪、Agilent 5977质量选择检测器(Agilent公司), 5600⁺Q-TOF型高分辨质谱仪(AB SCIEX公司), 全自动氮吹浓缩仪(Biotage公司), 离心浓缩仪(DNA120, 赛默飞世尔科技有限公司); Milli-Q型超纯水系统(Millipore公司)。

SZRD改善睡眠剥夺大鼠失眠伴焦虑药效学评价

SZRD制备依据第5版《方剂学》教材, 称取酸枣仁180 g、茯苓100 g、知母100 g、川芎50 g、甘草30 g, 全方共计460 g, 粉碎过筛。加10倍量水, 浸泡1 h后, 煮沸30 min后用8层纱布过滤, 药渣再加8倍水煎煮, 沸后20 min, 纱布过滤, 合并滤液, 减压浓缩至2.4 g·mL^-1。HPLC-DAD-ELSD测定SZRD提取物中木兰花碱、洋川芎内酯、斯皮诺素、阿魏酸、6'''-阿魏酰斯皮诺素、藁本内酯、酸枣仁皂苷A、酸枣仁皂苷B的平均含量分别为2.77、0.056、0.35、0.12、0.19、0.024、0.16、0.12 mg·g^-1。

分组及给药 28只大鼠随机分为4组(n = 7), 即为空白组(control, CN)、模型组(model, MD)、阳性药[阿普唑仑(alprazolam, ALP), 0.25 mg·kg^-1]、酸枣仁汤组(SZRD, 18.36 g·kg^-1)。于每天13:00灌胃给予各组相应药物, CN组和MD组灌胃给药等量生理盐水, 每天1次, 边造模边给药, 连续21天。

睡眠剥夺模型制备 CN组大鼠放在无水的睡眠剥夺箱中, 并将其余大鼠放入睡眠剥夺箱中, 睡眠剥夺3周(21天), 每天剥夺18 h (下午2:00至次日8:00), 每日8:00换水。控制水箱中温度为30 ℃, 大鼠可自由饮食、饮水。

旷场实验(open field test, OFT) 于给药后第21天, 安静环境下, 在长50 cm、宽50 cm、高50 cm旷场箱内进行实验。旷场箱底部分为16个相等的小正方形。将大鼠置于中心方格内适应2 min后, 记录10 min大鼠总运动距离和中心区域停留时间。

高架十字迷宫实验(elevated plus maze, EPM) 第22天, 进行EPM实验, 记录5 min内大鼠进入开臂时间占比以及进入开臂次数占比, 实验完成后将大鼠取出, 将两臂清理干净, 喷洒酒精除去气味。

样本收集于末次给药前1天, 所有大鼠禁食不禁水。第23天, 给药1 h后, 使用异氟烷麻醉, 腹主动脉取血, 静置1 h, 4 ℃、3 500 r·min^-1离心10 min, 取血清, 于-80 ℃保存。处死后, 在冰上迅速取出肝脏和结肠。结肠组织样本经4%多聚甲醛固定、石蜡包埋后切片用于免疫荧光操作。并将结肠内容物置于冻存管, 于-80 ℃备用。

结肠HE及AB-PAS染色 HE染色与AB-PAS染色均委托武汉赛维尔生物科技有限公司完成。染色后, HE切片及AB-PAS切片于光学显微镜下观察并拍照。

结肠屏障蛋白测定将石蜡切片进行脱蜡复水, 浸入抗原修复液中, 95 ℃水浴加热15 min; 透化剂透化15 min后, 血清封闭; 在组织上滴加兔ZO-1抗体(1∶400)、兔occludin抗体(1∶400)、兔claudin 1抗体(1∶400), 4 ℃孵育过夜。避光滴加Alexa Fluor^® 488标记山羊抗兔荧光二抗(1∶400), 室温避光孵育2 h, 经DAPI染核后滴加抗荧光淬灭封片剂, 覆上盖玻片进行封片。激光共聚焦显微镜以波长405、488 nm激发检测。10倍物镜观察, 利用Olympus Fluoview FV1000软件捕捉和分析图像。

肝脏HE染色及油红染色肝脏组织经4%多聚甲醛固定后, 分别制备石蜡切片(用于HE染色) 和冰冻切片(用于油红O染色)。所有染色操作均委托武汉赛维尔生物科技有限公司。染色后切片均使用光学显微镜进行观察与图像采集。

血清代谢组学分析

血清样品处理移取血清样本50 μL于1.5 mL EP管中。加入200 μL预冷甲醇沉淀蛋白。涡旋后静置10 min。4 ℃条件下, 以14 000 ×g离心15 min, 上清液离心浓缩后, 用100 μL 20%甲醇水溶液复溶, 进行LC-MS分析。

质控(QC) 样品的处理为了评估UPLC-Q-TOF-MS整个分析过程的稳定性和重复性, 将所有待测血清样品各取10 μL混合制备QC样本。进样前, 连续进8针QC; 进样时, 每走完1组样本, 插入1针QC样本。

色谱质谱条件正离子模式下, 以ACQUITY UPLC HSS T3柱(2.1 mm×150 mm, 1.8 μm) 进行分析, 流动相为0.1%甲酸水溶液(A) 和乙腈(B), 梯度洗脱条件为: 0~1 min, 2% B; 1~3 min, 2%~50% B; 3~14 min, 50%~60% B; 14~18 min, 60%~90%。负离子模式下, 以ACQUITY UPLC^® HSS T3柱(2.1 mm×150 mm, 1.8 μm) 进行分析, 流动相为含0.1%甲酸水溶液(A) 及乙腈(B), 梯度洗脱条件为: 0~1 min, 2% B; 1~3 min, 2%~50% B; 3~14 min, 50%~60% B; 14~18 min, 60%~90% B。流速均为0.35 mL·min^-1, 柱温50 ℃, 进样量5 μL。

离子源为ESI源, 正、负离子模式下分别扫描。毛细管电压5 500 V、-4 500 V, 离子源温度450 ℃; 辅助气N₂; 气帘气、雾化气及辅助气压力分别为30、55、55 psi; 去簇电压为60、-60 V; 碰撞能量采用35、-35 eV。TOF MS扫描范围设定为100~1 500 Da, product ion扫描范围设定为50~1 500 Da, 并开启动态背景扣除(DBS) 以减少干扰。

数据处理 采用MS-DIAL软件对LC-MS数据进行解卷积、峰对齐、峰校准和归一化等数据预处理。将数据导入悟空网站(https://www.omicsolution.com/wkomics/wkold/), 使用K值近邻法, 将缺失值先替换为该变量检出样本的最小值的20%, 再执行KNN算法, 处理缺失值。将归一化峰表数据导入SIMCA 14.0软件偏最小二乘法判别分析(PLS-DA) 和正交偏最小二乘法判别分析(OPLS-DA)。结合OPLS-DA分析中的变量投影重要性(VIP) > 1及t检验(P < 0.05), 筛选差异代谢物。结合二级碎片离子和在线数据库HMDB对差异代谢物进行鉴定。通过MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/) 对差异代谢物进行代谢通路分析。结肠屏障蛋白结果使用Image J软件统计屏障蛋白密度。

统计学分析 实验数据用GraphPad Prism 8进行分析, 结果用x±s值表示, 组间差异显著性分析用独立样本t检验和one-way ANOVA分析, P < 0.05表示差异有统计学意义。

结果

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1　行为学实验

在OFT中, 如图1A、B所示, 与CN组相比, 造模21天后, MD组大鼠运动总距离和中心区域活动时间均显著增加(P < 0.01); 与MD组相比, ALP与SZRD组均能显著降低MD大鼠的自主活动能力(P < 0.001) 和中心区域活动时间(P < 0.05)。

在EPM实验中, 如图1C、D所示, 与CN组比, MD组大鼠进入开臂时间占比显著减小(P < 0.001), 进入开臂的次数百分比明显降低(P < 0.001), MD组大鼠出现焦虑样行为。与MD组相比, 给药SZRD与ALP后均能增加MD组大鼠进入开臂的次数及时间百分比(P < 0.05, P < 0.01), SZRD显著降低了睡眠剥夺大鼠的焦虑水平。

2　结肠HE与AB-PAS染色

如图2A所示, 与CN组相比, MD组黏膜上皮破损, 隐窝减小(红色箭头); 黏膜层遭到破坏(黑色箭头) 并伴随可见的炎症细胞浸润(绿色箭头)。与MD组相比, ALP和SZRD可明显改善结肠组织, 黏膜上皮未出现破损并且杯状细胞形态完整, 排列整齐; 炎症浸润范围减少。AB-PAS染色可将结肠中杯状细胞分泌的酸性黏液染成蓝色, 糖原、中性黏液质染成红色, 酸性和中性混合黏液质染成紫蓝色。如图2B所示, 与CN组相比, MD组大鼠结肠杯状细胞中蓝色、红色显著减少, 表明MD大鼠结肠杯状细胞的酸性黏液以及糖原、中性黏液质显著减少, 提示MD组大鼠杯状细胞黏液屏障受损。与MD组相比, ALP及SZRD组蓝色、红色显著增加, 能够修复结肠杯状细胞黏液屏障。

3　大鼠结肠中屏障蛋白分析

如图2C与表1所示, 与CN组比, MD组大鼠结肠组织ZO-1、occludin、claudin-1平均荧光强度均显著降低; 表明睡眠剥夺大鼠肠道屏障蛋白出现损伤。与MD组相比, SZRD大鼠结肠组织中occludin、claudin-1及ZO-1荧光强度显著升高, SZRD可以显著改善由于睡眠剥夺引起的肠道屏障蛋白的减少, 具有保护肠道的作用。

4　大鼠肝脏HE与油红染色

肝脏HE染色结果显示(图3A), 与CN组相比, MD组大鼠肝脏肝细胞排列紊乱, 肝索扩张。与MD组相比, ALP和SZRD明显改善细胞排列整齐。油红染色结果显示(图3B), 与CN组相比, MD组大鼠肝脏红色脂滴增多。与MD组相比, ALP与SZRD组均可以显著降低肝脏中红色脂滴水平。

5　短链脂肪酸分析

短链脂肪酸是肠道微生物群代谢物。与CN组相比, MD组大鼠结肠内容物中乙酸(P < 0.01, 图4A)、丙酸(P < 0.001, 图4B)、异丁酸(P < 0.01, 图4C)、戊酸(P < 0.05, 图4D)、异戊酸(P < 0.001, 图4E) 和异己酸(P < 0.01, 图4F) 的含量显著下降。与MD组相比, ALP组能够显著回调乙酸、丙酸、异戊酸和异己酸的含量(P < 0.05); SZRD可回调以上6种短链脂肪酸的含量(P < 0.05)。

6　LC-MS血清代谢组学分析

在正、负离子模式下, QC原始峰表中峰面积RSD值小于或等于30%占所有峰的90%以上, 即说明QC样品聚集良好, 该检测系统稳定可靠, 所得差异代谢物能反映样本间生物学差异(图5A、B)。

PLS-DA显示CN组与MD组能显著分开, ALP与SZRD组接近CN组, 提示给药后对模型具有一定回调作用(图5C、D)。为进一步寻找CN和MD组的差异代谢物, 采用有监督的OPLS-DA模型进行分析。如图5E、F所示, 在正、负离子模式下, CN组与MD组沿t[1]轴分开, 且在正离子模式下, R²Y = 0.999, Q² = 0.992; 负离子模式下R²Y = 0.998, Q² = 0.908, R²Y接近1, Q² > 0.5, 表明模型具有较高的解释率及预测率。随机排列检验(n = 200) 显示(图5G、H), Q²与Y轴交于负半轴, 表明该模型没有过拟合, 模型有效。以VIP > 1和t检验(P < 0.05) 结合S-plot图筛选潜在差异性代谢物正离子模式下182个, 负离子模式下242个(图5I、J)。

通过HMDB数据库, 筛选CN组与MD组的差异代谢物。在正离子模式下鉴定了26个, 负离子模式下鉴定了27个, 共53个差异代谢物, 包括13个氨基酸类, 15个有机酸类, 2个酰胺类, 13个脂肪酸类, 2个吲哚类, 8个其他类。与MD组比, ALP影响差异代谢物共有18个, 包括5个氨基酸类, 4个有机酸类, 6个脂肪酸类, 3个其他类。与MD组相比, SZRD显著回调了36个代谢物, 包括10个氨基酸类, 10个有机酸类, 11个脂质类, 1个酰胺类, 1个吲哚类, 3个其他类(表2)。

7　代谢通路分析

将CN组和MD组筛选到的53个差异代谢物输入MetaboAnalyst 5.0数据库中进行代谢通路分析。如图6A所示, 共富集到代谢通路共有11条, 包括苯丙氨酸、酪氨酸与色氨酸的生物合成, 苯丙氨酸代谢, 牛磺酸和次牛磺酸代谢, 色氨酸代谢, 酪氨酸代谢, 烟酸盐和烟酸胺代谢, 半胱氨酸和蛋氨酸代谢, 一碳代谢, 三羧酸循环, 甘油磷脂代谢, 鞘脂代谢。与MD组相比, ALP组主要回调苯丙氨酸、酪氨酸与色氨酸的生物合成, 鞘脂代谢, 色氨酸代谢, 半胱氨酸与蛋氨酸代谢等4条代谢通路(图6B)。与MD组相比, SZRD组回调了6条代谢通路苯丙氨酸、酪氨酸与色氨酸的生物合成, 苯丙氨酸代谢, 半胱氨酸和蛋氨酸代谢, 色氨酸代谢, 酪氨酸代谢和三羧酸循环(图6C)。

8　相关性分析

将SZRD回调的30个差异代谢物与结肠粪便中SZRD显著回调的6种短链脂肪酸进行Spearman相关性分析。如图7所示, 乙酸与10种差异代谢物呈现负相关(P < 0.05), 与3种正相关(P < 0.05), 丙酸与14种差异代谢物负相关(P < 0.05), 与4种代谢物呈现正相关(P < 0.05)。异丁酸与15种差异代谢物负相关(P < 0.05), 与5种差异代谢物正相关(P < 0.05), 异戊酸与11种差异代谢物负相关(P < 0.05), 2种差异代谢物正相关(P < 0.05)。戊酸与18种差异代谢物负相关(P < 0.05), 2种代谢物正相关(P < 0.05); 异己酸与18种差异代谢物负相关(P < 0.05), 与1种差异代谢物正相关(P < 0.05)。

讨论

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睡眠不足会增加患心血管疾病、糖尿病和肥胖慢性疾病以及抑郁和焦虑等心理疾病的风险^{[10, 11]}。研究表明, 长期睡眠剥夺导致下丘脑-垂体-肾上腺轴的激活进一步引起皮质酮水平升高^[12], 而皮质酮的增加会直接增加肠上皮凋亡、减少黏液分泌, 削弱屏障^[13], 屏障蛋白的损伤导致LPS等病原分子进入体内, 进一步引发全身炎症, 会引起焦虑抑郁样行为^{[14, 15]}。SZRD作为临床上治疗失眠的经典名方, 在改善患者睡眠质量的同时, 能显著缓解焦虑样情绪^[16]。

本研究采用改良多平台水环境法复制失眠大鼠模型, 每天剥夺18 h, 持续剥夺21天。该模型属于快动眼(REM期) 睡眠剥夺, 即剥夺期间大鼠可进入NREM期睡眠, 但在进入REM期时, 由于全身肌肉放松会落入水中由此惊醒。研究显示^{[17, 18]}, 睡眠剥夺21天和自然恢复21天后, 大鼠出现稳定的失眠伴焦虑特征。本研究结果同样显示长期睡眠剥夺下, 大鼠总运动距离显著增加, 兴奋性增加, 并且进入开臂时间百分比以及进入次数百分比显著下降, 这些结果表明大鼠出现焦虑样行为。SZRD显著降低了失眠大鼠总运动距离、中心区域活动时间, 提示其可能通过调节中枢兴奋性发挥镇静作用^{[19, 20]}。SZRD可显著提高睡眠剥夺大鼠进入开臂的次数百分比以及在开臂停留时间百分比, 可显著降低由睡眠剥夺引起的焦虑样行为。

越来越多的研究显示, 改良多平台水环境法睡眠剥夺模型导致肠道损伤, 主要表现为occludin、claudin 1和ZO-1等结肠屏障蛋白表达量减少, 肠道屏障的通透性增加以及短链脂肪酸的合成减少^{[21, 22]}, 并且这种炎症反应会进一步增加失眠和焦虑样行为。Gao等^[21]研究表明长期慢性睡眠剥夺会导致肠道屏障蛋白和杯状细胞缺失。本研究结果显示, SZRD可增加occludin、claudin 1和ZO-1等屏障蛋白的平均荧光强度。屏障蛋白增加可有效减少有害物质的渗透和炎症反应, 肠道环境的改善和免疫反应的调节有助于改善睡眠剥夺诱导的大鼠焦虑样行为。另外, 肠道菌群产生的短链脂肪酸会促进肠道屏障蛋白的表达^[23]。本实验结果显示, 睡眠剥夺大鼠结肠内容物中乙酸、丙酸、异丁酸、戊酸、异戊酸、异己酸等短链脂肪酸含量显著降低, SZRD可显著提高短链脂肪酸的含量。SZRD改善睡眠剥夺大鼠肠道环境可能与其促进短链脂肪酸含量增加有关。

本研究进一步采用血清代谢组学探究SZRD改善睡眠剥夺导致焦虑样行为的作用机制。通路分析发现, SZRD主要影响氨基酸代谢和三羧酸循环代谢通路。三羧酸循环代谢通路是细胞氧化磷酸化的中心途径, 满足细胞生物能量、生物合成和氧化还原平衡要求^[24]。已有研究显示, 慢性睡眠剥夺会导致氨基酸代谢紊乱, 芳香族氨基酸(苯丙氨酸、色氨酸) 以及支链氨基酸(异亮氨酸以及缬氨酸) 的含量增加^[25-27]。研究表明^[28-30], 支链氨基酸增多会激活mTOR及其上游PI3K/AKT信号通路, 并促进胰岛素抵抗, 减少葡萄糖摄取和脂质氧化。而芳香族氨基酸苯丙氨酸、酪氨酸、色氨酸作为常见的生酮氨基酸生成乙酰辅酶A后进入三羧酸循环为机体供能^[31]。本研究结果表明, 睡眠剥夺大鼠血清中苯丙氨酸、酪氨酸以及色氨酸等芳香族氨基酸及缬氨酸、异亮氨酸支链氨基酸含量显著增加。相关性分析结果显示, 短链脂肪酸乙酸、异丁酸等与支链氨基酸呈负相关。睡眠剥夺导致肠道结构以及肠道菌群破坏, 短链脂肪酸生成减少, 导致短链脂肪酸参与三羧酸循环减少。支链氨基酸与芳香族氨基酸作为参与能量代谢的另一个重要来源, 在短链脂肪酸合成减少的情况下, 使得支链氨基酸与芳香族氨基酸的含量进一步增加。基于以上分析, 睡眠剥夺导致大鼠肠道菌产生的短链脂肪酸减少, 由于负反馈调节途径的存在, 血清中参与能量代谢的支链氨基酸以及芳香族氨基酸含量升高。睡眠剥夺期间维持清醒状态需要更高的能量消耗, 葡萄糖需求增加^[32], 迫使生酮氨基酸生成3-羟基丁酸, 提供额外ATP, 共同维持机体能量平衡^[33]。本研究中睡眠剥夺大鼠血清3-羟基丁酸含量显著升高, 提示睡眠剥夺大鼠体内能量代谢异常。SZRD降低芳香族氨基酸以及支链氨基酸含量的同时, 降低3-羟基丁酸, 表明SZRD可以调控由睡眠剥夺引起的氨基酸及能量代谢紊乱。

研究表明, 焦虑与循环游离脂肪酸水平相关^[34]。本研究肝脏HE与油红染色均显示睡眠剥夺大鼠肝脏结构出现损伤, 出现脂滴聚集, 提示睡眠剥夺大鼠出现脂质代谢紊乱, 与Liu等^[35]报道一致。

结论

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本研究表明SZRD能改善睡眠剥夺所致的焦虑样行为, 通过修复睡眠剥夺大鼠肝脏结构损伤, 增加肠道屏障蛋白occludin、claudin-1、ZO-1表达, 提高结肠内容物中短链脂肪酸的含量, 进而发挥改善失眠伴焦虑样行为作用。血清代谢组学结果显示, SZRD能够改善睡眠剥夺导致的氨基酸代谢、能量代谢和脂质代谢紊乱。研究结果为阐明SZRD改善睡眠障碍的作用特点提供科学依据, 为SZRD的特色用药提供实验基础。

参考文献

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2026年第61卷第6期

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doi: 10.16438/j.0513-4870.2025-1378

接收时间：2025-11-19
首发时间：2026-09-21
出版时间：2026-06-12

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收稿日期：2025-11-19
修回日期：2026-02-21

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¹山西大学中医药现代研究中心, 山西太原 030006

²山西中医药大学中药与食品工程学院, 山西太原 030619

通讯作者:

杜晨晖, E-mail: dch@sxtcm.edu.cn

闫艳, Tel: 86-351-7018379, E-mail: yanyan520@sxu.edu.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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TxT

Group	CN	MD	ALP	SZRD
Occludin	165.54±10.63	128.34±9.52^##	178.55±24.65^***	178.56±17.82^***
Claudin 1	184.29±21.78	128.19±6.96^###	185.49±13.44^***	171.827 2±15.08^***
ZO-1	179.44±19.97	120.97±13.87^###	182.15±19.62^***	164.64±8.07^***

Group

ALP

SZRD

Occludin

165.54±10.63

128.34±9.52^##

178.55±24.65^***

178.56±17.82^***

Claudin 1

184.29±21.78

128.19±6.96^###

185.49±13.44^***

171.827 2±15.08^***

ZO-1

179.44±19.97

120.97±13.87^###

182.15±19.62^***

164.64±8.07^***

Type	Metabolite	Formula	Adduct	t_R/min	MW/Da	MS/MS fragments	MD vs CN	ALP vs MD	SZRD vs MD
Amino acid	L-Valine	C₅H₁₁NO₂	[M-H]^-	1.159	116.072 6	91.058 4, 65.041 3	↑^#	↓^*	↓^***
L-Tyrosine	C₉H₁₁NO₃	[M-H]^-	1.305	180.067 6	163.043 7, 119.050 8, 93.032 8, 72.008 6	↑^###	↓^*	↓^**
Phenylalanine	C₉H₁₁NO₂	[M+H]⁺	1.983	166.086 6	166.083 8, 120.082 3, 77.043 5, 52.035 0	↑^###	-	↓^*
L-Kynurenine	C₁₀H₁₂N₂O₃	[M+H]⁺	1.907	209.092 6	174.055 1, 146.058 6, 120.043 3, 91.057 2	↑^##	-	↓^*
Leucylproline	C₁₁H₂₀N₂O₃	[M+H]⁺	1.344	229.152 7	55.024 1, 70.068 3	↓^##	↑^**	↑^**
Proline	C₅H₉NO₂	[M+H]⁺	0.900	116.072 5	116.072 7, 70.074 1	↑^#	-	-
Methionine	C₅H₁₁NO₂S	[M+H]⁺	1.160	150.057 3	133.030 2, 74.025 9, 61.016 6	↑^###	↑^*	↓^*
5-oxo-L-Proline	C₅H₇NO₃	[M+H]⁺	1.170	130.047 9	130.062 1, 103.058 4, 84.038 6, 56.053 5	↑^#	-	-
Isoleucine	C₆H₁₃NO₂	[M+H]⁺	1.390	132.102 5	86.097 2, 69.072 1, 56.055 3	↑^##	-	↓^*
Norvaline	C₅H₁₁NO₂	[M+H]⁺	3.000	118.064 6	55.063 5, 89.035 1, 91.071 9	↑^#	-	↓^*
Tryptophan	C₁₁H₁₂N₂O₂	[M+H]⁺	3.030	205.098 1	170.060 2, 143.073 3, 118.064 6, 91.056 5	↑^#	-	↓^*
Arginine	C₆H₁₄N₄O₂	[M+H]⁺	0.82	175.117 9	130.093 3, 116.071 4, 70.069 8	↑^##	-	-
Lysine	C₆H₁₄N₂O₂	[M+H]⁺	0.763	147.111 8	55.057 9, 67.057 0, 84.082 9	↑^##	↑^*	↑^***
Organic acid	β-Hydroxyisovaleric acid	C₅H₁₀O₃	[M-H]^-	2.904	117.056 4	117.056 4, 69.040 9	↑^###	↓^*	↓^*
3-Methyl-2-oxovaleric acid	C₆H₁₀O₃	[M-H]^-	4.110	129.055 6	51.026 6, 67.023 4, 84.764 8	↑^###	-	↓^*
3-Methyl-2-oxovalerate	C₆H₁₀O₃	[M-H₂O-H]^-	3.825	111.044 6	50.870 8, 70.499 7, 101.065 8	↑^###	-	-
Succinate	C₄H₆O₄	[M-H]^-	4.862	248.959 1	60.988 4, 72.467 0, 112.987 3	↑^##	↑^*	↑^***
γ-Linolenic acid	C₁₈H₃₀O₂	[M-H]^-	11.127	277.216 5	107.045 7, 121.027 3, 165.062 0	↑^##	-	↑^**
Stearic acid	C₁₈H₃₆O₂	[M-H]^-	12.996	283.262 6	283.258 7	↑^##	-	-
Urate	C₅H₄N₄O₃	[M-H]^-	0.886	357.029 0	69.011 2, 97.006 1, 136.917 9, 161.002 1	↓^###	-	-
Uric acid	C₅H₄N₄O₃	[M+H]⁺	0.947	169.036 6	53.027 8, 69.011 4, 96.016 2	↓^###	-	-
Citrate	C₆H₈O₇	[M+H]⁺	1.164	215.015 9	72.114 5, 77.039 7	↑^##	-	↓^*
3-Hydroxybutyric acid	C₄H₈O₃	[M-H]^-	1.430	103.041 0	59.011 2	↑^##	↓^*	↓^*
Citric acid	C₆H₈O₇	[M-H]-	1.188	191.020 2	67.018 6, 57.035 4	↑^#	-	↓^*
L-Pipecolic acid	C₆H₁₁NO₂	[M+H]⁺	0.763	130.086 8	55.055 1, 56.053 2, 67.058 5	↑^#	-	-
Creatine	C₄H₉N₃O₂	[M+H]⁺	0.891	132.077 0	55.033 4, 58.059 5, 69.046 0, 72.058 9	↓^#	-	↑^*
Xanthosine	C₁₀H₁₂N₄O₆	[M-H]^-	1.904	283.067 2	151.025 2, 108.019 8	↑^##		↑^*
Indole-3-propionic acid	C₁₁H₁₁NO₂	[M-H]^-	6.063	188.070 3	59.016 8, 116.049 0, 142.064 4	↓^#	↑^**	↑^***
Amide	N-Isovalerylglycine	C₇H₁₃NO₃	[M-H]^-	3.618	158.082 5	74.024 4, 158.082 5	↑^##	-	↓^*
Nicotinamide	C₆H₆N₂O	[M+H]⁺	1.160	123.044 4	96.044 8, 80.052 8, 52.032 8	↓^#	-	-
Lipid	PE (16:1-22:6)	C₄₃H₇₂NO₈P	[M-H]^-	9.367	760.487 9	96.971 9, 164.963 9, 253.220 3	↑^##	-	-
PC 35:1	C₄₃H₈₄NO₈P	[M-H]^-	11.205	832.594 4	168.042 9, 267.148 8, 281.246 6	↑^##	-	↓^*
SM (18:2-C23:0)	C₄₆H₉₁N₂O₆P	[M-H]^-	9.246	857.671 9	78.960 4, 168.032 0, 236.058 6	↑^###	↑^*	↑^**
SM 33:1	C₃₈H₇₇N₂O₆P	[M-H]^-	9.982	747.561 0	78.961 3, 123.000 2, 168.040 3	↓^#	-	↑^***
PC 33:2	C₄₁H₇₈NO₈P	[M-H]^-	10.098	802.557 8	78.961 2, 145.462 4, 168.039 5	↓^###	↑^*	↑^**
PC 39:6	C₄₇H₈₂NO₈P	[M-H]^-	10.585	878.587 6	78.962 4, 229.199 0, 313.172 4, 508.336 6	↓^#	↑^*	↑^*
Ceramide (d18:1/16:0)	C₃₄H₆₇NO₃	[M+H]⁺	10.413	560.508 1	87.058 3, 133.102 4	↑^#	↑^***	↑^***
PI (16:0/22:6)	C₄₃H₇₂NO₈P	[M-H]^-	9.367	760.494 6	96.972 1, 253.220 3	↑^#	↑^*	↑^***
SM (18:1/C23:1)	C₄₆H₉₁N₂O₆P	[M-H]^-	9.87	857.678 7	78.960 3, 241.010 4	↑^##	-	↑^*
SM d41:1	C₄₆H₉₃N₂O₆P	[M-H]^-	9.246	859.682 1	78.960 6, 799.676 4	↑^##	↑^*	↑^***
PI 38:4	C₄₇H₈₃O₁₃P	[M-H]^-	14.537	885.542 2	78.958 8, 241.008 1	↑^##	-	↑^*
Arachidonic acid	C₂₀H₃₂O₂	[M-H]^-	11.438	303.233 2	59.018 2	↑^###	-	↑^***
Indoles	3-Formylindole	C₉H₇NO	[M+H]⁺	3.000	146.060 6	117.055 6, 91.054 6, 65.041 5	↑^##	-	↓^*
Indoline	C₈H₉N	[M+H]⁺	1.980	120.081 4	50.022 1, 55.937 3, 65.063 2	↑^###	-	-
Others	Choline	C₅H₁₄NO	[M]⁺	0.870	104.106 9	88.093 1, 58.063 2	↑^#	-	-
Betaine	C₅H₁₁NO₂	[M+H]⁺	0.900	118.085 9	81.254 3, 58.072 0	↑^#	-	-
Bilirubin	C₃₃H₃₆N₄O₆	[M+H]⁺	10.330	585.270 2	89.068 2, 299.141 3	↑^#	-	-
Taurohyodeoxycholic acid	C₂₆H₄₅NO₆S	[M+H-H₂O]⁺	5.847	482.292 2	464.258 1, 339.256 8, 126.021 3	↑^#	-	↓^*
Taurocholic acid	C₂₆H₄₅NO₇S	[M+H]⁺	5.906	516.297 8	462.272 4, 337.247 4, 126.021 9	↑^#	↓^*	↓^*
Glycocholate	C₂₆H₄₃NO₆	[M+H]⁺	6.359	466.316 3	55.059 4, 93.073 4, 237.159 9	↓^#	↓^*	-
Taurine	C₂H₇NO₃S	[M-H]^-	0.820	124.008 2	79.957 7, 124.008 2	↓^#	-	-
Estrone-3-sulfate	C₁₈H₂₂O₅S	[M-H]^-	9.555	349.109 2	61.992 5, 96.958 9, 265.145 6	↑^###	↓^**	↓^***

Type

Metabolite

Formula

Adduct

t_R/min

MW/Da

MS/MS fragments

MD vs CN

ALP vs MD

SZRD vs MD

Amino acid

L-Valine

C₅H₁₁NO₂

[M-H]^-

1.159

116.072 6

91.058 4, 65.041 3

↑^#

↓^*

↓^***

L-Tyrosine

C₉H₁₁NO₃

[M-H]^-

1.305

180.067 6

163.043 7, 119.050 8, 93.032 8, 72.008 6

↑^###

↓^*

↓^**

Phenylalanine

C₉H₁₁NO₂

[M+H]⁺

1.983

166.086 6

166.083 8, 120.082 3, 77.043 5, 52.035 0

↑^###

↓^*

L-Kynurenine

C₁₀H₁₂N₂O₃

[M+H]⁺

1.907

209.092 6

174.055 1, 146.058 6, 120.043 3, 91.057 2

↑^##

↓^*

Leucylproline

C₁₁H₂₀N₂O₃

[M+H]⁺

1.344

229.152 7

55.024 1, 70.068 3

↓^##

↑^**

Proline

C₅H₉NO₂

[M+H]⁺

0.900

116.072 5

116.072 7, 70.074 1

↑^#

Methionine

C₅H₁₁NO₂S

[M+H]⁺

1.160

150.057 3

133.030 2, 74.025 9, 61.016 6

↑^###

↑^*

↓^*

5-oxo-L-Proline

C₅H₇NO₃

[M+H]⁺

1.170

130.047 9

130.062 1, 103.058 4, 84.038 6, 56.053 5

↑^#

Isoleucine

C₆H₁₃NO₂

[M+H]⁺

1.390

132.102 5

86.097 2, 69.072 1, 56.055 3

↑^##

↓^*

Norvaline

C₅H₁₁NO₂

[M+H]⁺

3.000

118.064 6

55.063 5, 89.035 1, 91.071 9

↑^#

↓^*

Tryptophan

C₁₁H₁₂N₂O₂

[M+H]⁺

3.030

205.098 1

170.060 2, 143.073 3, 118.064 6, 91.056 5

↑^#

↓^*

Arginine

C₆H₁₄N₄O₂

[M+H]⁺

0.82

175.117 9

130.093 3, 116.071 4, 70.069 8

↑^##

Lysine

C₆H₁₄N₂O₂

[M+H]⁺

0.763

147.111 8

55.057 9, 67.057 0, 84.082 9

↑^##

↑^*

↑^***

Organic acid

β-Hydroxyisovaleric acid

C₅H₁₀O₃

[M-H]^-

2.904

117.056 4

117.056 4, 69.040 9

↑^###

↓^*

3-Methyl-2-oxovaleric acid

C₆H₁₀O₃

[M-H]^-

4.110

129.055 6

51.026 6, 67.023 4, 84.764 8

↑^###

↓^*

3-Methyl-2-oxovalerate

C₆H₁₀O₃

[M-H₂O-H]^-

3.825

111.044 6

50.870 8, 70.499 7, 101.065 8

↑^###

Succinate

C₄H₆O₄

[M-H]^-

4.862

248.959 1

60.988 4, 72.467 0, 112.987 3

↑^##

↑^*

↑^***

γ-Linolenic acid

C₁₈H₃₀O₂

[M-H]^-

11.127

277.216 5

107.045 7, 121.027 3, 165.062 0

↑^##

↑^**

Stearic acid

C₁₈H₃₆O₂

[M-H]^-

12.996

283.262 6

283.258 7

↑^##

Urate

C₅H₄N₄O₃

[M-H]^-

0.886

357.029 0

69.011 2, 97.006 1, 136.917 9, 161.002 1

↓^###

Uric acid

C₅H₄N₄O₃

[M+H]⁺

0.947

169.036 6

53.027 8, 69.011 4, 96.016 2

↓^###

Citrate

C₆H₈O₇

[M+H]⁺

1.164

215.015 9

72.114 5, 77.039 7

↑^##

↓^*

3-Hydroxybutyric acid

C₄H₈O₃

[M-H]^-

1.430

103.041 0

59.011 2

↑^##

↓^*

Citric acid

C₆H₈O₇

[M-H]-

1.188

191.020 2

67.018 6, 57.035 4

↑^#

↓^*

L-Pipecolic acid

C₆H₁₁NO₂

[M+H]⁺

0.763

130.086 8

55.055 1, 56.053 2, 67.058 5

↑^#

Creatine

C₄H₉N₃O₂

[M+H]⁺

0.891

132.077 0

55.033 4, 58.059 5, 69.046 0, 72.058 9

↓^#

↑^*

Xanthosine

C₁₀H₁₂N₄O₆

[M-H]^-

1.904

283.067 2

151.025 2, 108.019 8

↑^##

↑^*

Indole-3-propionic acid

C₁₁H₁₁NO₂

[M-H]^-

6.063

188.070 3

59.016 8, 116.049 0, 142.064 4

↓^#

↑^**

↑^***

Amide

N-Isovalerylglycine

C₇H₁₃NO₃

[M-H]^-

3.618

158.082 5

74.024 4, 158.082 5

↑^##

↓^*

Nicotinamide

C₆H₆N₂O

[M+H]⁺

1.160

123.044 4

96.044 8, 80.052 8, 52.032 8

↓^#

Lipid

PE (16:1-22:6)

C₄₃H₇₂NO₈P

[M-H]^-

9.367

760.487 9

96.971 9, 164.963 9, 253.220 3

↑^##

PC 35:1

C₄₃H₈₄NO₈P

[M-H]^-

11.205

832.594 4

168.042 9, 267.148 8, 281.246 6

↑^##

↓^*

SM (18:2-C23:0)

C₄₆H₉₁N₂O₆P

[M-H]^-

9.246

857.671 9

78.960 4, 168.032 0, 236.058 6

↑^###

↑^*

↑^**

SM 33:1

C₃₈H₇₇N₂O₆P

[M-H]^-

9.982

747.561 0

78.961 3, 123.000 2, 168.040 3

↓^#

↑^***

PC 33:2

C₄₁H₇₈NO₈P

[M-H]^-

10.098

802.557 8

78.961 2, 145.462 4, 168.039 5

↓^###

↑^*

↑^**

PC 39:6

C₄₇H₈₂NO₈P

[M-H]^-

10.585

878.587 6

78.962 4, 229.199 0, 313.172 4, 508.336 6

↓^#

↑^*

Ceramide (d18:1/16:0)

C₃₄H₆₇NO₃

[M+H]⁺

10.413

560.508 1

87.058 3, 133.102 4

↑^#

↑^***

PI (16:0/22:6)

C₄₃H₇₂NO₈P

[M-H]^-

9.367

760.494 6

96.972 1, 253.220 3

↑^#

↑^*

↑^***

SM (18:1/C23:1)

C₄₆H₉₁N₂O₆P

[M-H]^-

9.87

857.678 7

78.960 3, 241.010 4

↑^##

↑^*

SM d41:1

C₄₆H₉₃N₂O₆P

[M-H]^-

9.246

859.682 1

78.960 6, 799.676 4

↑^##

↑^*

↑^***

PI 38:4

C₄₇H₈₃O₁₃P

[M-H]^-

14.537

885.542 2

78.958 8, 241.008 1

↑^##

↑^*

Arachidonic acid

C₂₀H₃₂O₂

[M-H]^-

11.438

303.233 2

59.018 2

↑^###

↑^***

Indoles

3-Formylindole

C₉H₇NO

[M+H]⁺

3.000

146.060 6

117.055 6, 91.054 6, 65.041 5

↑^##

↓^*

Indoline

C₈H₉N

[M+H]⁺

1.980

120.081 4

50.022 1, 55.937 3, 65.063 2

↑^###

Others

Choline

C₅H₁₄NO

[M]⁺

0.870

104.106 9

88.093 1, 58.063 2

↑^#

Betaine

C₅H₁₁NO₂

[M+H]⁺

0.900

118.085 9

81.254 3, 58.072 0

↑^#

Bilirubin

C₃₃H₃₆N₄O₆

[M+H]⁺

10.330

585.270 2

89.068 2, 299.141 3

↑^#

Taurohyodeoxycholic acid

C₂₆H₄₅NO₆S

[M+H-H₂O]⁺

5.847

482.292 2

464.258 1, 339.256 8, 126.021 3

↑^#

↓^*

Taurocholic acid

C₂₆H₄₅NO₇S

[M+H]⁺

5.906

516.297 8

462.272 4, 337.247 4, 126.021 9

↑^#

↓^*

Glycocholate

C₂₆H₄₃NO₆

[M+H]⁺

6.359

466.316 3

55.059 4, 93.073 4, 237.159 9

↓^#

↓^*

Taurine

C₂H₇NO₃S

[M-H]^-

0.820

124.008 2

79.957 7, 124.008 2

↓^#

Estrone-3-sulfate

C₁₈H₂₂O₅S

[M-H]^-

9.555

349.109 2

61.992 5, 96.958 9, 265.145 6

↑^###

↓^**

↓^***