Microbiome Metabolomic Analysis of the Anxiolytic Effect of Baihe Dihuang Decoction in a Rat Model of Chronic Restraint Stress

Figures & data

Figure 1 BDD improves anxiety behaviors. (A) Anxiety-like behaviors induced by CRS. (B and C) The movement trails of the rats in each group assessed using a video tracking software in the open field test (B) and elevated cross maze test (C). (D and E) Results of open field test were the center distance (D) and central enter times (E). (F and G) Results of elevated plus maze test were the proportion of time keeping the open arm (OT%) (F) and the proportion of entering the open arm (OE%) (G). The immobile times in the forced swimming test (H). Values are expressed as means ± SD. n=6 rats per group. **P<0.01, significant differences compared with the CON group; ^#P<0.05, ^##P<0.01, significant differences compared with the CRS group.

Table 1 Characterization of the Main Metabolites in BDD by UPLC-Q-TOF/MS

Time	Source	Identification	Molecular	Adduct	Detect	Expect	Error
0.89	RR	Stachyose	C24H42O21	M-H	665.2153	665.214	2.0
1.30	RR	Citric Acid	C6H8O7	M-H	191.0175	191.0192	−8.9
3.98	LB	Hopantenic acid	C10H19NO5	M+H	234.1323	234.1341	−7.7
4.69	LB	Regaloside K	C18H24O11	M-H	415.1248	415.1240	1.9
5.37	LB	Regaloside C	C18H24O11	M-H	415.1232	415.1240	−1.9
5.61	LB	Regaloside H	C18H24O10	M-H	399.1291	399.1291	0
6.34	LB	Regaloside A	C18H24O10	M-H	399.1324	399.1291	8.3
6.56	RR	Echinacoside	C35H46O20	M-H	785.2511	785.2504	0.9
6.65	LB	Regaloside D	C18H24O10	M-H	399.1298	399.1291	1.8
6.89	LB	Regaloside F	C19H26O11	M-H	429.1397	429.1390	−1.6
7.58	RR	Rehmapicrogenin	C10H16O3	M-H	183.1008	183.1021	−7.1
7.87	RR	Cistanoside A	C36H48O20	M-H	799.2665	799.2661	0.5
8.26	LB	Regaloside E	C20H26O12	M-H	457.1336	457.1346	−2.2
9.11	RR	Rehmaionoside A^#	C19H34O8	M+HCOO	435.2229	435.2230	−0.2
				M-H	389.2180	389.2175	1.3
10.43	RR	Acteoside^#	C29H36O15	M-H	623.1995	623.1976	3.0
10.94	LB	4-Acetyl-regaloside D	C20H26O11	M-H	441.1398	441.1397	0.2
11.23	LB	Regaloside B	C20H26O11	M-H	441.1440	441.1397	9.7
12.22	RR	Isoacteoside	C29H36O15	M-H	623.1981	623.1976	0.8
12.57	LB	Regaloside I	C20H26O11	M-H	441.1399	441.1397	0.5
12.71	RR	Sibirioside A	C21H28O12	M-H	471.1504	471.1503	0.2
15.28	LB	3,6′-O-Diferuloylsucrose/isomer	C32H38O17	M-H	693.2050	693.2031	2.7
19.82	RR	Martynoside/isomer	C31H40O15	M-H	651.2305	651.2289	2.5
20.99	LB	(25R)-furosta-5-ene-1β,3α,22α,26-tetrol-3,26-O-β-D-glucopyranoside/isomer	C39H64O15	M+Na	795.4156	795.4143	1.6
21.40	LB	(25S)-27-hydroxyspirost-5-en-3β-yl O-α-L-rha-(1→2) -O-[β-D-glu-(1→6)]-β-D-glucopyranoside/isomer	C45H72O18	M+Na	923.4617	923.4616	0.1
21.56	RR	Martynoside/isomer	C31H40O15	M-H	651.2297	651.2289	1.2
21.81	LB	(25R)-26-O-β-D-glu- furosta-5-ene-3β,20 α,26 -triol- 3 - O - α - L-rha (1→2) -β -D-glucopyranoside/isomer	C45H74O18	M+Na	925.4789	925.4773	1.7
23.90	LB	(25S)-27-hydroxyspirost-5-en-3β-yl O-α-L-rha-(1→2) -O-[β-D-glu-(1→6)]-β-D-glucopyranoside/isomer	C45H72O18	M+Na	923.4620	923.4616	0.4
24.42	LB	(25 R) −26-O -β-D-glu-furosta-5-ene- 3 β,20 α,26-triol- 3-O-α-L-rha (1→2) -β-D-glucopyranoside/isomer	C45H74O18	M+Na	925.4802	925.4773	3.1
25.06	LB	(25 R) −26 -O-β -D- glu- furosta-5-ene- 3 β,20 α,26 - triol −3 -O-α-L-rha (1→2) -β -D -glucopyranoside/isomer	C45H74O18	M+Na	925.4811	925.4773	4.1
25.33	LB	(25 R)-26-O-β-D- glucopyranosyl - furosta-5-ene-3 β,20 α,26 - triol- 3 - O - α -L-rha (1→2) -β - D - glucopyranoside/isomer	C45H74O18	M+Na	925.4846	925.4773	7.3
25.50	LB	(25R)-26-O-β-D-glu −5α-furosta-3β,20α,26-triol-3-O-α-L-rha (1→2)-β-D-glucopyranoside /isomer	C45H76O18	M+Na	927.4968	927.4929	4.2
25.80	LB	(25R)-26-O-β-D- glu-5α-furosta-3β,20α,26 triol-3-O-α-L-rha (1→2)-β-D- glucopyranoside /isomer	C45H76O18	M+Na	927.4902	927.4929	−2.9
27.60	LB	(25R)-3β,17α-dihydroxy-5α-spirostan-6-one 3-O-α-L-rha-(1→2)-β-D-glucopyranoside/isomer	C39H64O15	M+Na	795.418	795.4143	4.7
28.50	LB	(25R)-3β,17α-dihydroxy-5α-spirostan-6-one3-O-α-L-rha-(1→2)-β-D-glucopyranoside/isomer	C39H64O15	M+Na	795.4117	795.4143	−3.3
28.69	LB	(25S)-spirost-5-ene-3β,27-diol 3-O-α-L-rha-(1 →2) -O-[β-D-glu- (1→4)]-β-D-glucoside/isomer	C45H72O18	M+Na	923.4608	923.4616	−0.9
31.02	LB	(25R)-spirost-5-en-3β-yl-O-α-L-rha-(1→2)-O-[β-D-glu-(1→6)]-β-D glucopyranoside/isomer	C45H72O17	M+H	885.4874	885.4848	2.6
31.21	LB	26-O-β-D-glu-3β,26-dihydroxy-cholestan-16,22-dioxo-3-O-α-L-rha-(1→2)-β-D-glucopyranoside/isomer	C45H74O17	M+H	887.5031	887.5004	3.0
31.28	LB	(25R)-spirost-5-en-3β-yl-O-α-L-rha-(1→2)-O-[β-D-glu-(1→6)]-β-D glucopyranoside/isomer	C45H72O17	M+H	885.4871	885.4848	2.6
31.48	LB	26-O-β-D-glu-3β,26-dihydroxy-cholestan-16,22-dioxo-3-O-α-L-rha-(1→2)-β-D-glucopyranoside/isomer	C45H74O17	M+H	887.5043	887.5004	4.4
32.80	LB	(25R)-3β,17α-dihydroxy-5α-spirostan-6-one 3-O-α-L-rha-(1→2)-β-D-glucopyranoside/isomer	C39H62O14	M+HCOO	799.4147	799.4116	−3.5
				M-H	753.4098	753.4061	4.9
33.04	LB	(25R)-3β,17α-dihydroxy-5α-spirostan-6-one 3-O-α-L-rha-(1→2)-β-D-glucopyranoside/isomer	C39H62O14	M+HCOO	799.4134	799.4116	2.3
				M-H	753.4087	753.4061	3.5
33.45	LB	(25R)-3β,17α-dihydroxy-5α-spirostan-6-one 3-O-α-L-rha-(1→2)-β-D-glucopyranoside/isomer	C39H62O14	M+HCOO-	799.4146	799.4116	3.8
				M-H	753.4122	753.4061	8.1
33.65	LB	(25R)-3β,17α-dihydroxy-5α-spirostan-6-one 3-O-α-L-rha-(1→2)-β-D-glucopyranoside/isomer	C39H62O14	M+HCOO	799.4142	799.4116	3.3
				M-H	753.3997	753.4061	−8.5
34.55	LB	(25R)-3β-5α-spirostan-6-one-3-O-α-L-rha-(1→2)-β-Dglucopyranoside/isomer	C39H62O13	M+HCOO	783.4188	783.4167	2.7
				M-H	737.4135	737.4112	3.1
34.92	LB	(25R)-3β-5α-spirostan-6-one-3-O-α-L-rha-(1→2)-β-Dglucopyranoside/isomer	C39H62O13	M+HCOO	783.4193	783.4167	3.3
				M-H	737.4163	737.4112	6.9
35.00	RR	Linoleic acid	C18H32O2	M-H	279.2318	279.2324	−2.1

Notes: ^#Confirmed by comparison with the reference substance.

Abbreviations: LB, Lilii Bulbus; RR, Rehmanniae radix; Glu, glucopyransoyl; Rha, rhamnopyranosyl.

Figure 2 Representative total ion current profiles of BDD in positive (A) and negative (B) ion modes.

Figure 3 BDD reduces inflammatory factors, hippocampal damage, and microglia activation. (A–D) Levels of inflammatory factors in the hippocampus (n=6). (E) Representative HE staining of hippocampal tissue (200×). (F) Representative immunofluorescence staining of IBA1 (red) in the hippocampal tissue (100×). Values are expressed as means ± SD. (n=6). *P<0.05, **P<0.01, significant differences compared with the CON group; ^#P<0.05, ^##P<0.01, significant differences compared with the CRS group.

Figure 4 BDD reverses abnormal diversity of gut microbiota. (A) Alpha diversity analysis of the CON, the CRS, and BDDH groups. (B) Principal coordinate analysis. (C) Non-metric multidimensional scaling analysis of the CON, CRS, and BDDH groups based on the weighted UniFrac distance algorithm. (D) Samples distances heat map on OTU level. n=6 rats per group. **P<0.01, significant differences compared with the CON group; ^##P<0.01, significant differences compared with the CRS group; ns, no significant difference between two groups.

Figure 5 BDD ameliorates the abnormal abundance of potential microbial biomarkers at the phylum level. (A) Average relative abundances at the phylum level in the CON, the CRS, and BDD groups. (B–F) Comparison of relative abundances of potential microbial biomarkers at the phylum level in the CON, CRS, and BDD groups. n=6 rats per group. *P<0.05, **P<0.01, significant differences compared with the CON group; ^##P<0.01, significant differences compared with the CRS group; ns, no significant difference between two groups.

Figure 6 BDD ameliorates the abnormal abundance of potential microbial biomarkers at the genus level. (A) Average relative abundances at the genus level in the CON, the CRS, and BDDH groups. (B–K) Comparison of relative abundances of potential microbial biomarkers at the genus level in the CON, CRS, and BDDH groups. n=6 rats per group. **P<0.01, significant differences compared with the CON group. ^##P<0.01, significant differences compared with the CRS group.

Figure 7 BDD normalizes the disturbed metabolic profiles. (A and B) PCA score plots of fecal samples of rats in different groups in positive (A) mode and negative mode (B). (C and D) OPLS DA score plots fecal samples of rats in different groups in positive mode (C) and negative mode (D). (E and F) OPLS DA model validation plot of fecal samples of rats in different groups in positive mode (E) and negative mode (F). (G and H) The heat map of different metabolites both in positive (G) and in negative ion modes (H). R² and Q² represent the interpretation rate of the model to the matrix and the prediction ability of the model, respectively.

Table 2 Potential Biomarkers Associated with CRS and Their Variation Tendency After BDD Treatment Based on UPLC-Q-TOF/MS

NO.	Metabolite	RT (min)	m/z	Icon	VIP	Formula	CRS vs CON	BDDH vs CRS
1	Spermidine	0.51	146.1651	M+H	1.33	C7H19N3	↓^b	↑^b
2	1,4-Diaminobutane	0.54	89.1077	M+H	1.05	C4H12N2	↓^a	↑^b
3	D-Ornithine	0.56	133.0971	M+H	1.04	C5H12N2O2	↓^b	↑^b
4	N1-Acetylspermidine	0.60	188.1756	M+H	1.23	C9H21N3O	↓^b	↑^b
5	L-Arginine	0.60	173.1037	M-H	1.32	C6H14N4O2	↓^b	↑^b
6	L-Glutamine	0.63	145.0611	M-H	1.00	C5H10N2O3	↓^b	↑^b
7	Taurine	0.64	124.0065	M-H	1.20	C2H7NO3S	↓^b	↑^b
8	(S)-Glutamic acid	0.66	146.0451	M-H	1.07	C5H9NO4	↓^b	↑^b
9	N-Acetylputrescine	0.74	131.1179	M+H	1.10	C6H14N2O	↓^a	↑^b
10	Urocanic acid	2.11	139.0502	M+H	1.12	C6H6N2O2	↓^b	—
11	Pantothenic Acid	2.79	218.103	M-H	1.18	C9H17NO5	↓^b	↑^b
12	L-Kynurenine	2.83	191.0814	M+H	1.99	C10H12N2O3	↑^b	↓^b
13	Cortisol	3.50	363.216	M+H	2.47	C21H30O5	↑^b	↓^b
14	LysoPC (14:1(9Z))	3.92	486.2568	M+Na-2H	1.69	C22H44NO7P	↓^b	↑^b
15	N’-Formylkynurenine	4.12	271.0472	M+H	1.27	C11H12N2O4	↑b	↓^b
16	5-Hydroxyindoleacetic acid	4.37	190.0504	M-H	1.10	C10H9NO3	↓^a	↑^b
17	9(S)-HODE	6.65	295.2276	M-H	1.06	C18H32O3	↓^b	↑^b
18	Arachidonoyl Ethanolamide	6.81	348.2871	M-H	1.02	C22H37NO2	↓^b	↑^b
19	Palmitoylethanolamide	6.93	300.2893	M+H	1.98	C18H37NO2	↓^b	↑^b

Notes: ↑, upregulated; ↓, downregulated; ^aP<0.05; ^bP<0.01; —: no significant differences.

Abbreviations: RT, retention time; VIP, variable importance in the projection in OPLS-DA model.

Figure 8 BDD significantly recovers neurotransmitter dysfunction. (A) PCA score plots of neurotransmitters. (B) OPLS DA score plots of neurotransmitters. (C) Heat map of neurotransmitters. (D) Levels of neurotransmitters in the hippocampus. n= 6 rats per group. *P<0.05, **P<0.01, significant differences compared with the CON group; ^#P<0.05, ^##P<0.01, significant differences compared with the CRS group.

Figure 9 Metabolic pathways and network analysis. (A and B) Summary of pathway analysis in fecal (A) and hippocampus (B) samples with MetaboAnalyst. (C) Metabolic network analysis. Red up arrow or red down arrow means that the hippocampal metabolite is significantly increased or decreased compared with the CRS group; green up arrow or green down arrow means that the fecal metabolite is significantly increased or decreased compared with the CRS group.

Figure 10 Correlation analysis between metabolites and microorganisms. Pearson correlation analysis among microbial biomarkers and differential metabolites in fecal samples (A). Pearson correlation analysis among microbial biomarkers and neurotransmitters in the hippocampus (B). The color key represents Pearson’s correlation coefficient. The green color indicates positive correlations, while the red color indicates negative correlations. n=6 rats per group, significance is indicated as *P<0.01.

Data Sharing Statement

Raw sequencing data can be identified in the NCBI Sequence Read Archive (accession number PRJNA1047763). The datasets during the current study available from the corresponding author on reasonable request.