Metabonomics study of liver and kidney subacute toxicity induced by garidi-5 in rats.

Objective: Metabonomics was used to analyze and explore the biomarkers and possible mechanisms of liver and kidney subacute toxicity induced by garidi-5 in rats.
Methods: Taking garidi-5 as the target drug and SD rats as the research objects, each administration group except the normal group was intragastric administration of the corresponding drug solution for 28 d. The serum, liver and kidney samples of rats were detected by metabolomics and characterized by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) to identify the sensitive markers and metabolic pathways of liver and kidney subacute toxicity.
Results: Metabolomics analysis showed that compared with the normal group (Z), the 52, 64 and 54 different metabolites were identified in the serum, liver and kidney samples of garidi-5 high dose group (GG), which were mainly enriched in ABC transporters, arginine and proline metabolism, nicotinate and nicotinamide metabolism, central carbon metabolism in cancer pathways.
Conclusion: The preliminarily suggested that garidi-5 can damage the liver and kidney by affecting the ABC transporters, arginine and proline metabolism, nicotinate and nicotinamide metabolism pathways, etc. Trimethylamine N-oxide, l-pyroglutamic acid, glycine-betaine, xanthine, glutathione, l-leucine, cytidine, l-arginine, spermidine, urea, 5-aminovaleric acid, creatine, l-glutamic acid, 1-methylnicotinamide and S-adenosyl-l-methionine can be used as potential biomarkers of liver and kidney toxicity sensitivity.

Introduction

The mongolian medicine garidi-5, synonymed chong'a and zhachong'ava, originated from the “Terminalia chebula Beaded prescription”, and is still used as the regional first prescription for eliminating sticky and dry yellow water diseases in Mongolian medicine of all dynasties. It has the effect of eliminate sticky, relieve pain, drying xieriwusu, detumescence etc. The major functions of it are sticky stinging pain, sticky epidemic, diphtheria, erysipelas, anthrax, Heruhu, Wuyaman, Taolai, Xieriwusu disease, swelling, etc. (Inner Mongolia Health Department, 1984). However, because of its toxicity, it is listed as the category of drugs with caution, and so far there is no new understanding and research report on the toxicity of garidi-5. However, according to reviewing relevant research findings, it was found that all the five medicinal of garidi-5 compounds have certain toxicity, and Aconiti Kusnezoffii Radix also has gastrointestinal, reproductive and genetic toxicity in addition to cardiotoxicity and neurotoxicity (Wurihan et al., 2021). Musk has embryotoxicity and cytotoxicity (Chen et al., 2014). Terminalia chebula Retz. has obvious liver toxicity (Wang et al., 2016). Acorus tatarinowii Schott has cardiotoxicity and neurotoxicity (Yang, 2018). Aucklandia has embryotoxicity, hepatotoxicity and nephrotoxicity (He et al., 2016). It can be seen that the garidi-5 toxicity evaluation is an urgent research work.

Metabolomics can provide such a method for toxicity assessment and mechanism study. It can sensitively monitor the abnormal metabolic changes caused by drugs through dynamic analysis of endogenous metabolites, and clarify the toxic effect and mechanism of drugs (Liu et al., 2010). The rapid and efficient detection technology of metabonomics provides a strong basis for promoting toxicological research, clarifying the potential markers of drug toxicity and safe drug use. Therefore, in this study, LC–MS technology was used to analyze the changes of metabolites in the liver and kidney of Mongolian medicine garidi-5, and to explore the subacute toxicity and mechanism of garidi-5 on the liver and kidney of rats from the perspective of endogenous metabolites, so as to lay a foundation for its in-depth research and safe drug use.

Methods and materials

Animals

SD male rat, SPF grade, weight (200 ± 20) g, provided by Liaoning Changsheng Biotechnology Co., Ltd. (SCXK (Liao) 2015-0001). Feeding conditions: room temperature (23 ± 1) °C, light for 12 h, free drinking and feeding. Animal welfare and experimental process are in accordance with the provisions of the Medical Ethics Committee of the Affiliated Hospital of Inner Mongolia University for Nationalities.

Drugs and reagents

Garidi-5 is provided by Mongolian medicine preparation room of Affiliated Hospital of Inner Mongolia Minzu University. Alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), UREA nitrogen (UREA), creatinine (CREP), creatine kinase (CK), creatine kinase isoenzyme (CKMB) kits were purchased from Shanghai Roche Diagnostic Products Diagnostic Products Co., Ltd.; purified water (Watsons Group Co., Ltd.), formic acid (Sigma-Aldrich Co.), mass spectrometry acetonitrile (Thermo Fisher Scientific Co., USA).

Instruments

Pipette (EppendorfN13462C, Eppendorf, Germany); Mass ectrometer (TripleTOF5600+, AB SCIEX™); Table-top High-speed Refrigerated Centrifuge (Mikro 220R, Hettich); Roche automatic biochemical analyzer (cobasc 311, Shanghai Roche diagnostic products Diagnostic Products Co., Ltd.); Comminution grinder (8R, Shanghai Anbai Biotechnology Co., Ltd.) Chromatographic UHPLC (Nexera UHPLC LC-30A, SHIMADZU).

Animal grouping and sampling

Thirty-two SD rats were randomly divided into normal group (Z), garidi-5 high-dose group (0.3429 g/kg, clinical equivalent 4 times, GG), garidi-5 medium-dose group (0.0857 g/kg, clinical equivalent, GZ) and garidi-5 low-dose group (0.0214 g/kg, clinical equivalent 1/4 times, GD) according to body weight, with eight rats in each group. From the first day of the experiment, each administration group was intragastric administration of the corresponding drug solution for 28 d, while the normal group was intragastric administration of the equal volume normal saline. After the last administration, anesthetize the animals, the serum was centrifuged at 3500 r/min for 10 min to separate, and part of the supernatant was taken to measure the contents of AST, ALT, CK, CKMB, UREA, CREP and ALP. Collect 1/2 liver and kidney of rats in each group and store them in refrigerator at −80 °C for test.

Metabolite extraction

The serum sample was thawed at 4 °C, 100 µL was taken, 300 µL of methanol was added, the shock extraction was extracted at 12,000 r/min, centrifuged at 4 °C for 10 min, the supernatant was taken over a 0.22 µm organic membrane, and 10 µL was injected, and detected with UPLC-MS.

The Kidney and the liver samples were weighed 0.3 g, 1000 µL of methanol was added to the PE tube, ground to a homogenate with a glass grinder, centrifuged for 15 min (4 °C, 12,000 r/min), and the supernatant was taken 10 µL.

UPLC-MS detection

Chromatographic conditions: SHIMADZU InerSustain C18 chromatographic column (100 mm × 2.1 mm, 2 µm). Mobile phase A: acetonitrile, B: 0.1% formic acid aqueous solution, column temperature 35 °C, flow rate 0.3 mL/min, injection volume 10 μL. Elution gradient: 95 %–30% B, 0–7 min; 30%–0% B, 7–13 min; 0%–95% B, 13–16 min.

Mass spectrum parameter conditions: Electrospray ionization (ESI) positive and negative ion modes were used for detection. ESI source conditions are as follows: ion source Gas1:50, ion source Gas2:50, curtain gas (CUR): 25, interface heating temperature: 500 °C (positive ion) and 450 °C (negative ion), ion spray voltage floating (ISVF) 5500 V (positive ion) and 4400 V (negative ion), TOF mass scanning range: 100–1200 Da, product ion scanning range: 50–1000 Da, TOF MS scanning cumulative time 0.2 s, product ion scanning cumulative time 0.01 s, the secondary mass spectrum was obtained by Information Dependent Acquisition (IDA) and high sensitivity mode. Declustering Potential (DP): ± 60 V, collision energy: (35 ± 15) eV.

Data processing

SPSS 22.0 was used for statistical analysis, and the biochemical indexes of each group were expressed as Mean ± SEM. T-test, rank sum test and one-way ANOVA were used for comparison between groups, with P < 0.05 as the difference, which was statistically significant. The difference was statistically significant with P < 0.05.

Use the Analysis Base File Converter software to convert the obtained original data into ABF format, and then import MS-Dial 4.60 software to extract the peak information. Compare with MassBank, Respect and GNPs databases. Multivariate analysis (PCA and PLS-DA) was used to analyze the samples of each group, and P < 0.05, FC > 1.5 (or < 0.5) and VIP > 1 were screened as differential metabolites. Metabolic pathways were analyzed using MBRole 2.0.

Results

Evaluation of serum biochemical indexes in each group

Compared with Z, the contents of ALT, UREA, CK and CKMB in GG group were significantly increased (P < 0.05), the contents of UREA, CREP, CKMB and ALP in GZ and GD groups were significantly decreased (P < 0.05, < 0.01), and the content of AST in the GD group was significantly decreased (P < 0.05). It is suggested that garidi-5 has certain toxicity to heart, liver and kidney, as shown in Fig. 1.

Fig. 1

Effects of garidi-5 on biochemical indexes (blood extraction from orbit) of rats. *P < 0.05, **P < 0.01 vs normal group (Z).

Multivariate statistical analysis

PCA analysis results

PCA classifies the data of each group according to the main new variables, removes the heavy abnormal and outlier samples, and can observe the aggregation and dispersion of the samples. As shown in Fig. 2, serum, liver and kidney metabolites of rats in Z and GG group showed a separation trend, indicating that there were certain differences in metabolites. Metabo Analyst was used for cluster analysis, and the difference between groups was analyzed by clustering results of heat map. The color depth indicates the level of metabolites, red is increased and blue is decreased. As shown in Fig. 3, there was significant difference in the relative abundance of metabolites between Z and GG group. The results showed that GG could induce certain changes the metabolites of serum, liver and kidney metabolites in rats (Fig. 4).

Fig. 2

PCA of metabolites in serum (A), liver (B) and kidney (C).

Fig. 3

Heatmap of metabolites in serum (A), liver (B) and kidney (C).

Fig. 4

PLS-DA score plot (A: serum; B: liver; C: kidney).

PLS-DA analysis results

The PLS-DA analysis method can distinguish the difference between groups more efficiently. Compared with the Z, the metabolites in serum, liver and kidney of rats in GG group can be completely separated, indicating a significant difference in metabolite spectrum. The PLS-DA model was verified by parameter test, which has a good explanatory and predictive degree (parameters R2Y and Q2 are both greater than 0.5).

Screening of differential metabolites

Screening of serum differential metabolites

To search for potential differential metabolites, the criteria of VIP > 1, P < 0.05 and fold change > 1.5 (or < 0.5) in OPLS-DA were used for screening, and retrieval analysis through MassBank, Respect and GNPS databases. Fifty-two differential metabolites were screened from serum samples of GG group. Compared with Z, GG group -norvaline, Glu-Gln, glutamine (D), -pipecolic acid, fructose, kaempferol-3-O-rutinoside, -pyroglutamic acid, -leucine, -pipecolinic acid, -3-aminoisobutyric acid, -(+)-lysine, 3-indoxyl sulfate, glutamic acid, deoxycanitine, 1-imidazoleacetic acid, adipic acid, 3-(4-hydroxyphenyl)lactic acid, glycine-betaine, pyridoxamine, gentisinic acid, fumaric acid, N-acetylneuraminic acid, 4-hydroxyphenyllactic acid, phytosphingosine, 3-hydroxyvaleric acid, xanthine, methionine, histidine, trans-vaccenic acid, N-epsilon-acetyllysine, 1-amino-1-cyclopentanecarboxylic acid, -arginine, sebacic acid, butyryl carnitine (isomer of 920), 4-hydroxypyridine, γ-aminobutyric acid and glutathione decreased significantly; Methionine sulfoxide, N-α-acetyl--ornithine, dimethylphthalate, 5-aminovaleric acid, -citrulline, indole-3-carbinol, trimethylamine N-oxide, thiamine monophosphate, glucose, 4-methyl-5-thiazoleethanol, spermidine, hydroxy butyric acid, N-acetylhistamine, cytidine and alloisoleucine increased significantly, as shown in Table 1.

Table 1

Identification of serum differential metabolites.

tR (min)	Metabolite names	Formula	Reference (m/z)	Adduct types	Trends (compared with normal)	GG vs Z
						VIP	Log2FC	P
6.777	l-Norvaline	C5H11NO2	116.0717	[M−H]−	↓	2.05	−3.63	0.000
9.603	Glu-Gln	C10H17N3O6	276.1190	[M+H]+	↓	2.04	−3.46	0.000
7.844	Methionine sulfoxide	C5H11NO3S	166.0532	[M+H]+	↑	2.04	2.62	0.000
7.859	N-α-acetyl-l-ornithine	C7H14N2O3	173.0931	[M−H]−	↑	1.99	2.76	0.000
1.208	Dimethyl phthalate	C10H10O4	195.0651	[M+H]+	↑	1.97	3.16	0.000
6.670	5-Aminovaleric acid	C5H11NO2	116.0717	[M−H]−	↑	1.93	3.60	0.000
8.056	l-Citrulline	C6H13N3O3	174.0884	[M−H]−	↑	1.93	3.82	0.000
7.559	Glutamine (D)	C5H10N2O3	145.0618	[M−H]−	↓	1.86	−4.05	0.000
10.36	l-pipecolic acid	C6H11NO2	130.0862	[M+H]+	↓	1.76	−1.85	0.000
2.878	Fructose	C6H12O6	179.0561	[M−H]−	↓	1.75	−0.85	0.000
0.526	Kaempferol-3-O-rutinoside	C27H30O15	593.1511	[M−H]−	↓	1.74	−1.03	0.000
7.439	l-pyroglutamic acid	C5H7NO3	130.0498	[M+H]+	↓	1.71	−1.10	0.000
6.571	l-leucine	C6H13NO2	130.0873	[M−H]−	↓	1.69	−3.81	0.000
10.162	dl-pipecolinic acid	C6H11NO2	130.0862	[M+H]+	↓	1.67	−1.28	0.000
8.027	dl-3-aminoisobutyric acid	C4H9NO2	102.0560	[M−H]−	↓	1.66	−7.19	0.000
1.256	Indole-3-carbinol	C9H9NO	130.0651	[M+H-H2O]+	↑	1.66	1.23	0.000
10.373	L-(+)-lysine	C6H14N2O2	147.1128	[M+H]+	↓	1.59	−3.11	0.000
0.647	3-Indoxyl sulfate	C8H7NO4S	212.0023	[M−H]−	↓	1.58	−0.72	0.000
8.001	Glutamic acid	C5H9NO4	148.0604	[M+H]+	↓	1.58	−1.39	0.000
9.246	Deoxycanitine	C7H15NO2	146.1175	[M+H]+	↓	1.58	−1.42	0.000
7.627	1-Imidazoleacetic acid	C5H6N2O2	127.0502	[M+H]+	↓	1.57	−2.03	0.000
8.133	Trimethylamine N-oxide	C3H9NO	76.0757	[M+H]+	↑	1.56	1.48	0.000
12.826	Thiamine monophosphate	C12H18N4O4PS	345.0775	[M]+	↑	1.55	2.16	0.000
2.099	Adipic acid	C6H10O4	145.0506	[M−H]−	↓	1.54	−3.02	0.000
8.254	4-Methyl-5-thiazoleethanol	C6H9NOS	144.0477	[M+H]+	↑	1.53	2.38	0.000
2.326	Glucose	C6H12O6	179.0561	[M−H]−	↑	1.53	3.02	0.000
5.623	Spermidine	C7H19N3	146.1651	[M+H]+	↑	1.52	3.04	0.000
3.058	3-(4-Hydroxyphenyl)lactic acid	C9H10O4	181.0506	[M−H]−	↓	1.50	−4.40	0.000
7.482	Glycine-betaine	C5H11NO2	117.0784	[M]+	↓	1.50	−1.34	0.000
10.154	Pyridoxamine	C8H12N2O2	169.0971	[M+H]+	↓	1.48	−1.06	0.000
7.203	Gentisinic acid	C7H6O4	155.0338	[M+H]+	↓	1.48	−1.11	0.000
2.508	Fumaric acid	C4H4O4	115.0036	[M−H]−	↓	1.46	−3.47	0.000
2.960	Hydroxy butyric acid	C4H8O3	103.0400	[M−H]−	↑	1.46	3.22	0.004
6.675	N-acetylneuraminic acid	C11H19NO9	310.1132	[M+H]+	↓	1.45	−1.09	0.000
2.719	4-Hydroxyphenyllactic acid	C9H10O4	181.0506	[M−H]−	↓	1.43	−1.97	0.000
3.049	Phytosphingosine	C18H39NO3	318.2994	[M+H]+	↓	1.41	−3.61	0.006
2.922	3-Hydroxyvaleric acid	C5H10O3	117.0557	[M−H]−	↓	1.39	−4.37	0.000
1.090	Xanthine	C5H4N4O2	153.0407	[M+H]+	↓	1.39	−1.62	0.000
6.475	Methionine	C5H11NO2S	150.0580	[M+H]+	↓	1.37	−2.65	0.000
10.152	Histidine	C6H9N3O2	156.0767	[M+H]+	↓	1.37	−1.99	0.000
0.972	trans-Vaccenic acid	C18H34O2	281.2480	[M−H]−	↓	1.35	−0.65	0.000
7.820	N-epsilon-acetyllysine	C8H16N2O3	189.1233	[M+H]+	↓	1.35	−1.53	0.000
10.321	1-Amino-1-cyclopentanecarboxylic acid	C6H11NO2	130.0862	[M+H]+	↓	1.33	−1.11	0.000
8.103	l-arginine	C6H14N4O2	173.1044	[M−H]−	↓	1.30	−4.06	0.000
5.868	N-acetylhistamine	C7H11N3O	154.0974	[M+H]+	↑	1.28	1.71	0.000
1.490	Sebacic acid	C10H18O4	201.1132	[M−H]−	↓	1.24	−0.96	0.000
7.925	Butyryl carnitine (isomer of 920)	C11H21NO4	232.1553	[M+H]+	↓	1.23	−0.94	0.000
5.930	4-Hydroxypyridine	C5H5NO	96.0443	[M+H]+	↓	1.21	−0.96	0.000
6.061	d-alloisoleucine	C6H13NO2	132.1019	[M+H]+	↑	1.21	1.88	0.000
7.590	γ-Aminobutyric acid	C4H9NO2	104.0706	[M+H]+	↓	1.11	−0.87	0.000
10.367	Glutathione	C10H17N3O6S	308.0910	[M+H]+	↓	1.11	−1.42	0.000
3.744	Cytidine	C9H13N3O5	244.0928	[M+H]+	↑	1.08	1.49	0.000

Screening of liver differential metabolites

Sixty-four differential metabolites were identified in liver samples. Compared with Z, GG group, tyrosine, creatine, γ-glutamylleucine, -2-aminoadipic acid, glutamine, 3-pyridylacetic acid, deoxycarnitine, N-acetylneuraminic acid, glutamic acid, asparagine, methionine sulfoxide, Gly-Leu, alanine betaine, 1-methylnicotinamide, aspartate, trimethylamine N-oxide, 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-[[(2S,3R,4S,5R)-3,4,5-trihydroxyoxan-2-yl]oxymethyl]oxan-2-yl]oxychromen-4-one, morpholine, sebacic acid, (3S)-5-[(1S,8aR)-2,5,5,8a-tetramethyl-4-oxo-4a,6,7,8-tetrahydro-1H-naphthalen-1-yl]-3-methylpentanoic acid, urea, butyryl carnitine (isomer of 920), glutathione, β-nicotinamide adenine dinucleotide, protoporphyrin IX, dehydroisoandrosterone sulfate, glutamic acid, -pipecolinic acid, α-methylhistidine, N-acetylglutamate, 2-amino-3-[hydroxy-[3-octadecanoyloxy-2-[octadec-11-enoyl]oxypropoxy]phosphoryl]oxypropanoic acid, β-nicotinamide mononucleotide, -carnosine, (2S)-2-[(2R,3S,7R,8R,8aS)-2,3,4′-trihydroxy-4,4,7,8a-tetramethyl-6′-oxospiro[2,3,4a,5,6,7-hexahydro-1H-naphthalene-8,2′-3,8-dihydrofuro[2,3-e]isoindole]-7′-yl]-3-methylbutanoic acid, N-acetylmethionine, pyroglutamic acid, anserine, glycodeoxycholic acid, glycochenodeoxycholic acid, uridine-5-monophosphate and dimethyl sulfoxide increased significantly; Thiazolidine-4-carboxylic acid, -3-aminoisobutyric acid, ndoleacetic acid, 2-hydroxyisocaproic acid, -(−)-phenylalanine, -leucine, pyrrole-2-carboxylic acid, cytosine, (S)-3-amino-4-phenylbutyric acid, propionic acid, rac-glycerol 3-phosphoate, 5-aminovaleric acid, arabinose-5-phosphate disodium salt, adipic acid, (S)-3-amino-5-methylhexanoic acid, 3-indoxyl sulfate, -5-oxoproline, 2-hydroxy-4-methylpentanoate, (3R,4S)-3-amino-4-methylhexanoic acid, phytosphingosine, -(+)-malic acid, FA 18:2 + 2O and glutathione (oxidized) decreased significantly, as shown in Table 2.

Table 2

Identification of liver differential metabolites.

tR (min)	Metabolite name	Formula	Reference (m/z)	Adduct type	Trend (compared with normal)	GG vs Z
						VIP	Log2FC	P
5.684	Thiazolidine-4-carboxylic acid	C4H7NO2S	134.0270	[M+H]+	↓	1.73	−6.32	0.000
7.794	dl-3-aminoisobutyric acid	C4H9NO2	102.0560	[M−H]−	↓	1.70	−1.30	0.000
6.202	Tyrosine	C9H11NO3	182.0811	[M+H]+	↑	1.67	2.09	0.000
6.108	Ndoleacetic acid	C10H9NO2	176.0706	[M+H]+	↓	1.66	−4.63	0.000
10.413	2-Hydroxyisocaproic acid	C6H12O3	131.0713	[M−H]−	↓	1.65	−1.81	0.000
7.702	Creatine	C4H9N3O2	132.0767	[M+H]+	↑	1.65	1.53	0.000
7.563	γ-Glutamylleucine	C11H20N2O5	261.1445	[M+H]+	↑	1.61	1.61	0.000
7.570	D-2-Aminoadipic acid	C6H11NO4	162.0760	[M+H]+	↑	1.61	1.28	0.000
7.255	Glutamine	C5H10N2O3	147.0764	[M+H]+	↑	1.60	1.57	0.000
7.665	3-Pyridylacetic acid	C7H7NO2	138.0549	[M+H]+	↑	1.59	1.40	0.000
9.237	Deoxycarnitine	C7H15NO2	146.1175	[M+H]+	↑	1.59	1.17	0.000
6.169	L-(−)-phenylalanine	C9H11NO2	164.0717	[M−H]−	↓	1.57	−0.80	0.000
6.694	N-acetylneuraminic acid	C11H19NO9	310.1132	[M+H]+	↑	1.57	1.27	0.000
7.681	l-glutamic acid	C5H9NO4	148.0604	[M+H]+	↑	1.57	1.64	0.000
7.117	Asparagine	C4H8N2O3	133.0607	[M+H]+	↑	1.55	1.31	0.000
7.845	Methionine sulfoxide	C5H11NO3S	166.0532	[M+H]+	↑	1.53	2.01	0.000
7.222	Gly-Leu	C8H16N2O3	189.1233	[M+H]+	↑	1.50	2.44	0.000
6.145	l-leucine	C6H13NO2	130.0873	[M−H]−	↓	1.48	−4.34	0.000
1.325	Pyrrole-2-carboxylic acid	C5H5NO2	112.0393	[M+H]+	↓	1.47	−6.01	0.000
9.822	Alanine betaine	C6H13NO2	132.1019	[M+H]+	↑	1.47	1.44	0.000
6.841	1-Methylnicotinamide	C7H9N2O	137.0703	[M+H]+	↑	1.39	2.08	0.000
1.972	Cytosine	C4H5N3O	112.0505	[M+H]+	↓	1.38	−4.81	0.000
1.368	(S)-3-amino-4-phenylbutyric acid	C10H13NO2	180.1019	[M+H]+	↓	1.38	−1.29	0.000
2.116	Propionic acid	C3H6O2	73.0295	[M−H]−	↓	1.37	−2.89	0.000
5.790	Aspartate	C4H7NO4	132.0302	[M−H]−	↑	1.36	3.80	0.000
8.173	Trimethylamine N-oxide	C3H9NO	76.0757	[M+H]+	↑	1.35	1.99	0.001
7.170	Rac-glycerol 3-phosphoate	C3H9O6P	171.0063	[M−H]−	↓	1.34	−0.94	0.001
7.654	(3,4-Dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-[[(2S,3R,4S,5R)-3,4,5-trihydroxyoxan-2-yl]oxymethyl]oxan-2-yl]oxychromen-4-one	C26H28O16	595.1304	[M−H]−	↑	1.34	1.94	0.001
7.956	Morpholine	C4H9NO	88.0756	[M+H]+	↑	1.33	1.68	0.001
1.432	Sebacic acid	C10H18O4	201.1132	[M−H]−	↑	1.32	2.52	0.001
1.203	(3S)-5-[(1S,8aR)-2,5,5,8a-tetramethyl-4-oxo-4a,6,7,8-etrahydro-1H-naphthalen-1-yl]-t3-methylpentanoic acid	C20H32O3	321.2424	[M+H]+	↑	1.32	1.44	0.001
6.587	5-Aminovaleric acid	C5H11NO2	116.0717	[M−H]−	↓	1.30	−0.58	0.001
1.832	Urea	CH4N2O	61.0396	[M+H]+	↑	1.26	2.36	0.001
7.921	Butyryl carnitine (isomer of 920)	C11H21NO4	232.1553	[M+H]+	↑	1.26	1.02	0.003
10.378	Glutathione	C10H17N3O6S	308.0910	[M+H]+	↑	1.25	1.01	0.003
9.980	β-Nicotinamide adenine dinucleotide	C21H27N7O14P2	664.1163	[M+H]+	↑	1.25	2.28	0.003
6.965	d-arabinose-5-phosphate disodium salt	C5H11O8P	229.0118	[M−H]−	↓	1.22	−0.85	0.004
2.231	Adipic acid	C6H10O4	145.0506	[M−H]−	↓	1.22	−3.22	0.004
1.721	(S)-3-amino-5-methylhexanoic acid	C7H15NO2	146.1175	[M+H]+	↓	1.20	−2.51	0.005
1.770	Protoporphyrin IX	C34H34N4O4	563.2636	[M+H]+	↑	1.19	3.35	0.006
0.697	Dehydroisoandrosterone sulfate	C19H28O5S	367.1584	[M−H]−	↑	1.18	4.04	0.006
7.759	Glutamic acid	C5H9NO4	148.0604	[M+H]+	↑	1.18	1.17	0.006
0.632	3-Indoxyl sulfate	C8H7NO4S	212.0023	[M−H]−	↓	1.17	−1.07	0.007
11.437	dl-pipecolinic acid	C6H11NO2	130.0862	[M+H]+	↑	1.16	1.01	0.007
9.182	α-Methylhistidine	C7H11N3O2	170.0924	[M+H]+	↑	1.16	1.79	0.008
11.502	L-5-oxoproline	C5H7NO3	130.0498	[M+H]+	↓	1.12	−1.78	0.011
6.652	N-acetylglutamate	C7H11NO5	188.0564	[M−H]−	↑	1.10	1.07	0.013
5.770	2-Amino-3-[hydroxy-[3-octadecanoyloxy-2-[octadec-11-enoyl]oxypropoxy]phosphoryl]oxypropanoic acid	C42H80NO10P	788.5440	[M−H]−	↑	1.09	5.64	0.014
9.607	β-Nicotinamide mononucleotide	C11H15N2O8P	335.0638	[M+H]+	↑	1.08	3.77	0.015
10.660	l-carnosine	C9H14N4O3	227.1138	[M+H]+	↑	1.08	1.89	0.015
8.134	(2S)-2-[(2R,3S,7R,8R,8aS)-2,3,4′-trihydroxy-4,4,7,8a-tetramethyl-6′-oxospiro[2,3,4a,5,6,7-hexahydro-1H-naphthalene-8,2′-3,8-dihydrofuro[2,3-e]isoindole]-7′-yl]-3-methylbutanoic acid	C28H39NO7	502.2773	[M+H]+	↑	1.07	1.13	0.017
5.168	N-acetylmethionine	C7H13NO3S	192.0688	[M+H]+	↑	1.07	1.61	0.017
5.949	l-pyroglutamic acid	C5H7NO3	30.0498	[M+H]+	↑	1.06	1.06	0.017
9.863	2-Hydroxy-4-methylpentanoate	C6H12O3	131.0713	[M−H]−	↓	1.06	−4.35	0.018
11.353	Anserine	C10H16N4O3	239.1149	[M−H]−	↑	1.06	1.00	0.018
5.639	Glycodeoxycholic acid	C26H43NO5	472.3030	[M+Na]+	↑	1.05	4.27	0.019
1.775	(3R,4S)-3-amino-4-methylhexanoic acid	C7H15NO2	146.1175	[M+H]+	↓	1.04	−1.45	0.021
5.634	Glycochenodeoxycholic acid	C26H43NO5	450.3210	[M+H]+	↑	1.04	2.04	0.022
6.744	Uridine-5-monophosphate	C9H13N2O9P	323.0280	[M−H]−	↑	1.03	1.92	0.022
2.015	Dimethyl sulfoxide	C2H6OS	79.0212	[M+H]+	↑	1.02	1.93	0.025
5.214	Phytosphingosine	C18H39NO3	318.2994	[M+H]+	↓	1.02	−1.40	0.025
1.258	d-(+)-malic acid	C4H6O5	133.0142	[M−H]−	↓	1.01	−1.91	0.027
0.999	FA 18:2+2O	C18H32O4	311.2221	[M−H]−	↓	1.00	−0.58	0.027
11.461	Glutathione (oxidized)	C20H32N6O12S2	613.1592	[M+H]+	↓	1.00	−1.46	0.027

Screening of renal differential metabolites

Fifty-four differential metabolites were found in kidney samples. Compared with Z, GG group -pipecolic acid, ethanolamine, trimethylamine N-oxide, -3-aminoisobutyric acid, -(+)-malic acid, pyroglutamic acid, 4-methyl-5-thiazoleethanol, kynurenic acid, glycohyodeoxycholic acid, pantothenate, (E,6S)-7-hydroxy-2-methyl-6-[(10S,13S,14S,17S)-4,4,10,13,14-pentamethyl-3-oxo-1,2,5,6,7,11,12,15,16,17-decahydrocyclopenta[a]phenanthren-17-yl]hept-2-enoic acid, 2-piperidone, phenylacetylglycine, uridine 5′-diphospho-N-acetylglucosamine, biliverdin, rac-glycerol 3-phosphoate, 4-methyldaphnetin, N-methyl--alanine increased significantly; S-adenosyl--methionine, salicylic acid, 5-fluorouracil, methyl heptadecanoic acid, argininosuccinic acid, 5-methylcytidine, targinine, (3S)-5-[(1S,8aR)-2,5,5,8a-tetramethyl-4-oxo-4a,6,7,8-tetrahydro-1H-naphthalen-1-yl]-3-methylpentanoic acid, (+)-cystathionine, cinnamic acid, arachidonic acid, neoandrographolide, gluconic acid, Tyr, carnosine, citric acid, 2-hydroxyisocaproic acid, 9-nitro-20(S)-camptothecin, (4S,5Z,6S)-4-(2-methoxy-2-oxoethyl)-5-[2-[(E)-3-phenylprop-2-enoyl]oxyethylidene]-6-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4H-pyran-3-carboxylic acid, quinaldic acid, proline, ectoine, -pyroglutamic acid, (S)-3-amino-4-phenylbutyric acid, stearic acid, threonic acid, 2-hydroxyisobutyric acid, ascorbic acid, palmitoylcarnitine, resibufogenin, N-acetyl--leucine, mefenamic acid, p-coumaraldehyde, cysteine S-sulfate, spermidine decreased significantly, as shown in Table 3.

Table 3

Identification of renal differential metabolites.

tR (min)	Metabolite names	Formula	Reference (m/z)	Adduct type	Trend (compared with normal)	GG vs Z
						VIP	Log2FC	P
11.421	l-pipecolic acid	C6H11NO2	130.0862	[M+H]+	↓	2.39	−2.18	0.000
6.517	Ethanolamine	C2H7NO	62.0600	[M+H]+	↑	2.28	0.91	0.000
7.096	Trimethylamine N-oxide	C3H9NO	76.0757	[M+H]+	↑	2.15	0.99	0.000
11.061	S-adenosyl-l-methionine	C15H22N6O5S	399.1445	[M+H]+	↓	1.98	−1.82	0.000
7.777	dl-3-aminoisobutyric acid	C4H9NO2	102.0560	[M−H]−	↑	1.95	1.74	0.000
7.211	d-(+)-malic acid	C4H6O5	133.0142	[M−H]−	↑	1.92	1.80	0.000
8.677	Salicylic acid	C7H6O3	137.0244	[M−H]−	↓	1.88	−6.87	0.000
4.881	5-fluorouracil	C4H3FN2O2	129.0105	[M−H]−	↓	1.81	−3.00	0.000
1.034	Methyl heptadecanoic acid	C18H36O2	283.2642	[M−H]−	↓	1.79	−0.75	0.000
10.993	Argininosuccinic acid	C10H18N4O6	291.1299	[M+H]+	↓	1.74	−0.81	0.000
5.958	Pyroglutamic acid	C5H7NO3	128.0353	[M−H]−	↑	1.74	1.55	0.000
9.718	Targinine	C7H16N4O2	189.1346	[M+H]+	↓	1.69	−0.73	0.000
4.305	5-Methylcytidine	C10H15N3O5	258.1084	[M+H]+	↓	1.69	−0.88	0.000
1.377	4-Methyl-5-thiazoleethanol	C6H9NOS	144.0477	[M+H]+	↑	1.68	1.62	0.000
1.165	(3S)-5-[(1S,8aR)-2,5,5,8a-tetramethyl-4-oxo-4a,6,7,8-tetrahydro-1H-naphthalen-1-yl]-3-methylpentanoic acid	C20H32O3	321.2424	[M+H]+	↓	1.67	−1.03	0.000
9.747	l(+)-cystathionine	C7H14N2O4S	221.0601	[M−H]−	↓	1.67	−3.55	0.000
4.931	Kynurenic acid	C10H7NO3	190.0498	[M+H]+	↑	1.65	2.05	0.000
6.174	Cinnamic acid	C9H8O2	147.0451	[M−H]−	↓	1.65	−0.70	0.000
1.039	Arachidonic acid	C20H32O2	303.2329	[M−H]−	↓	1.65	−1.00	0.000
5.770	Glycohyodeoxycholic acid	C26H43NO5	448.3070	[M−H]−	↑	1.65	2.03	0.000
3.041	Pantothenate	C9H17NO5	220.1179	[M+H]+	↑	1.63	1.90	0.000
4.722	Gluconic acid	C6H12O7	195.0510	[M−H]−	↓	1.62	−4.62	0.000
8.095	(E,6S)-7-hydroxy-2-methyl-6-[(10S,13S,14S,17S)-4,4,10,13,14-pentamethyl-3-oxo-1,2,5,6,7,11,12,15,16,17-decahydrocyclopenta[a]phenanthren-17-yl]hept-2-enoic acid	C30H46O4	471.3468	[M+H]+	↑	1.57	5.04	0.010
10.592	Neoandrographolide	C26H40O8	503.2615	[M+Na]+	↓	1.57	−2.35	0.010
6.009	Tyr	C9H11NO3	180.0666	[M−H]−	↓	1.55	−2.20	0.010
1.628	2-piperidone	C5H9NO	100.0757	[M+H]+	↑	1.54	1.67	0.010
10.782	Carnosine	C9H14N4O3	225.0993	[M−H]−	↓	1.53	−1.62	0.010
8.392	Citric acid	C6H8O7	191.0197	[M−H]−	↓	1.53	−2.68	0.010
9.896	2-Hydroxyisocaproic acid	C6H12O3	131.0713	[M−H]−	↓	1.49	−1.74	0.010
11.648	9-Nitro-20(S)-camptothecin	C20H15N3O6	394.1033	[M+H]+	↓	1.48	−1.34	0.010
11.428	(4S,5Z,6S)-4-(2-methoxy-2-oxoethyl)-5-[2-[(E)-3-phenylprop-2-enoyl]oxyethylidene]-6-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4H-pyran-3-carboxylic acid	C15H22O2	233.1546	[M−H]−	↓	1.47	−1.37	0.020
3.544	Quinaldic acid	C10H7NO2	174.0549	[M+H]+	↓	1.46	−2.70	0.020
9.820	Proline	C5H9NO2	116.0706	[M+H]+	↓	1.44	−1.57	0.020
4.345	Phenylacetylglycine	C10H11NO3	194.0811	[M+H]+	↑	1.43	1.87	0.020
8.391	Ectoine	C6H10N2O2	143.0820	[M+H]+	↓	1.42	−3.92	0.020
7.279	Uridine 5′-diphospho-N-acetylglucosamine	C17H27N3O17P2	606.0742	[M−H]−	↑	1.42	2.55	0.020
7.164	l-pyroglutamic acid	C5H7NO3	130.0498	[M+H]+	↓	1.41	−0.86	0.020
2.122	Biliverdin	C33H34N4O6	583.2551	[M+H]+	↑	1.39	2.50	0.020
6.886	Rac-glycerol 3-phosphoate	C11H15N5O4	282.1196	[M+H]+	↑	1.39	2.10	0.020
1.173	(S)-3-amino-4-phenylbutyric acid	C10H13NO2	180.1019	[M+H]+	↓	1.38	−0.87	0.030
0.456	Stearic acid	C18H36O2	283.2642	[M−H]−	↓	1.37	−1.27	0.030
4.363	Threonic acid	C4H8O5	135.0298	[M−H]−	↓	1.37	−3.06	0.030
5.614	4-Methyldaphnetin	C10H8O4	191.0351	[M−H]−	↑	1.36	2.07	0.030
2.408	2-Hydroxyisobutyric acid	C4H8O3	103.0400	[M−H]−	↓	1.36	−2.51	0.030
2.566	N-methyl-dl-alanine	C4H9NO2	104.0706	[M+H]+	↑	1.35	2.07	0.030
1.368	Ascorbic acid	C6H8O6	175.0248	[M−H]−	↓	1.33	−2.37	0.030
11.757	Resibufogenin	C24H32O4	429.2282	[M+FA-H]-	↓	1.33	−1.28	0.030
6.700	Palmitoylcarnitine	C23H45NO4	400.3421	[M+H]+	↓	1.32	−0.59	0.040
4.324	N-acetyl-l-leucine	C8H15NO3	172.0979	[M−H]−	↓	1.32	−2.43	0.040
3.300	Mefenamic acid	C15H15NO2	242.1175	[M+H]+	↓	1.30	−2.89	0.040
5.946	p-Coumaraldehyde	C9H8O2	147.0451	[M−H]−	↓	1.30	−1.54	0.040
3.505	Cysteine S-sulfate	C3H7NO5S2	199.9692	[M−H]−	↓	1.29	−1.71	0.040
5.774	Spermidine	C7H19N3	146.1651	[M+H]+	↓	1.29	−1.05	0.040

Pathway enrichment analysis

The differential metabolites screened above were introduced into MBRole 2.0 to analyze its metabolic pathway. Serum differential metabolites involve ABC transporters, arginine and proline metabolism, central carbon metabolism in cancer, nicotinate and nicotinamide metabolism, glutathione metabolism, phenylalanine metabolism, cAMP signaling pathway, metabolic pathways, GABAergic synapse, β-alanine metabolism (Fig. 5A).

Fig. 5

Metabolic pathway (A: serum; B: hepatic; C: renal).

Liver differential metabolites are mainly related to ABC transporters, arginine and proline metabolism, central carbon metabolism in cancer, arginine biosynthesis, nicotinate and nicotinamide metabolism, protein digestion and absorption, metabolic pathways, alanine, aspartate and glutamate metabolism, histidine metabolism and aminoacyl-tRNA biosynthesis are significantly related, as shown in Fig. 5B.

Renal differential metabolites are related to metabolic pathways, ABC transporters, amino acid biosynthesis, arginine and proline metabolism, central carbon metabolism in cancer, nicotinate and nicotinamide metabolism, alanine, aspartate and glutamate metabolism, purine metabolism, histidine metabolism and glutathione metabolism pathways, as shown in Fig. 5C. It was found that serum, liver and kidney metabolites had the strongest correlation with ABC transporters, followed by arginine and proline metabolism, nicotinate and nicotinamide metabolism and central carbon metabolism in cancer.

Discussion

Drug-induced liver and kidney injury and acute and chronic liver and kidney failure are increasing year by year, which has become another thorn seriously restricting clinical safe drug use (Liu et al., 2010). Garidi-5 is the first prescription for eliminate viscosity and dry yellow water disease in Mongolian medicine clinical of past dynasties. But the composition of the prescription of five drugs have a certain toxicity, is listed as the category of drugs with caution. Liver and kidney are not only important places for drug metabolism and excretion, but also susceptible target organs for drug toxicity. The metabolic disorder of liver and kidney has become the main cause of liver and kidney toxicity. In this study, take garidi-5 as an example, detected its endogenous metabolites 28 d after intragastric administration, screened and identified the abnormal metabolic changes caused by garidi-5, and provided experimental basis for exploring its liver and kidney toxicity and mechanism.

Garidi-5 disordered ABC transporters metabolic pathway

This study found that the metabolism of ABC transporters in serum, liver and kidney of rats after administration of garidi-5 was disordered, such as abnormal changes in the contents of lycine-betaine, xanthine, glutathione, -leucine, -arginine, cytidine, spermidine and urea. ABC transporters need to consume ATP and participate in the transmembrane transport of amino acids, glucose, metabolites, drugs and charged ions (Wu et al., 2020), which is extremely important in the disposal of small molecules (endogenous metabolites, uremic toxins and drugs) in blood, kidney, liver, intestine and other organs. In chronic kidney disease (CKD), ABC transporters in renal and non-renal tissues are directly or indirectly affected by various small molecular metabolites, leading to abnormal inter organ communication (Torres et al., 2021).

Studies have shown that glycine-betaine is a quaternary amine alkaloid, mainly metabolized in mitochondria of liver and kidney cells. It can provide active methyl for the body and participate in protein and lipid metabolism. It can protect liver and kidney, inhibit inflammation, antitumor and play a positive role in the prevention and treatment of obesity, diabetes, cardiovascular disease and non-alcoholic fatty liver disease (NAFLD) (Liao et al., 2014, An et al., 2021). Xanthine is a purine base widespread in the body. It is also the product of purine metabolism, which is catalyzed by xanthine oxidase and further oxidized to uric acid. Liver and kidney are the main sites of purine metabolism. When they are damaged, xanthine oxidase decreases, interferes with the metabolism of xanthine into uric acid, causes too little formation of blood uric acid, and affects the excretion of xanthine (Bao et al., 2020). Glutathione (GSH) is mainly synthesized in the liver and transported to other tissues and organs through blood and bile. It is involved in glucose and lipid metabolism in the body and can activate various metabolic enzymes, and plays an important role in improving liver and kidney damage and sepsis (Piecha et al., 2007, Hu et al., 2019). -Leucine is the main amino acid for hydrolysis, conversion and synthesis of protein, as well as one of the branch chain amino acids, which plays an important role in liver diseases and is used for liver diseases with reduced bile secretion, poisoning, lipid metabolism and glucose metabolism disorders, etc. (Jin et al., 2019). -Arginine (-Arg) is one of the essential amino acids. It has antioxidation and immune regulation function. It promotes the synthesis of nitric oxide (NO) in the body and participates in intracellular signal transduction. It plays an important role in maintaining liver and kidney function, and has a certain antagonistic effect on the myocardial, liver and kidney injury and alcoholic fatty liver changes in diabetic rats (Zhang et al., 2020, Saka et al., 2021). Cytidine is an important component of ribonucleic acid, which plays a physiological role in cells mainly in the form of cytidylic acid. Spermidine is a polyamine compound that is critical in cell proliferation, autophagy and development, and its elevated level is related to many tumors and cancers (Novita et al., 2021). The catabolism of protein can determine the urea production. Urea is mainly synthesized by the liver and excreted from the kidney (He, 2014). Decreased renal function can lead to accumulation of urea (Feng et al., 2019). It is also found that the level of urea increases after administration of garidi-5. The results showed that garidi-5 could down-regulate the expression of glycine-betaine, xanthine, glutathione, -leucine and -arginine, and up-regulate the expression of spermidine, cytidine and urea.

It is speculated that garidi-5 may affect the expression of the above metabolites and disorder the metabolic pathway of ABC transporters in serum, liver and kidney of rats, leading to abnormal protein metabolism and pathological changes of liver and kidney to produce toxicity in rats.

Garidi-5 disorder arginine and proline metabolism pathway

This study found that the metabolism of arginine and proline in serum, liver and kidney of rats after administration of garidi-5 was disordered, such as 5-aminovaleric acid, creatine, urea, -glutamate acid, S-adenosine--methionine, spermidine etc. metabolites have abnormal changes. Arginine is one of the essential amino acids, participating in the urea cycle, mainly synthesizing proteins, polyamines and creatine. It can also enter the tricarboxylic acid cycle as a substrate to provide energy (Guo et al., 2019). Arginine is mainly synthesized in renal tubular epithelial cells, and abnormal can induce renal inflammation, fibrosis, CKD and the growth and proliferation of some tumor cells (Levillain, 2012, Xiao et al., 2013). Proline is synthesized from arginine and glutamate, and is an important functional amino acid participate in cell proliferation and differentiation, protein synthesis, metabolism, antioxidant and immune response (Chen et al., 2017). Proline is mainly metabolized in the small intestine, liver and kidney, and is catalyzed by ornithine in liver to generate polyamines, including spermidine, putrescine and spermidine, which are necessary metabolites for protein synthesis and cell growth, proliferation and differentiation (Tan et al., 2015). Studies have shown that 5-aminovaleric acid is an intermediate product produced by l-lysine catabolism, which can inhibit the oxidation of fatty acids (Olli KArkkAinen et al., 2020). Creatine mainly exists in skeletal muscle, brain, myocardium, kidney, liver and other organs. Long-term intake of high concentration of creatine can cause muscle spasm, gastrointestinal diseases, and have adverse effects on liver, kidney and cardiovascular system (Liu, 2017).

The increase of urea level is closely related to chronic kidney disease. -glutamate acid is mainly involved in the metabolism of amino acids in the body, and a variety of metabolites such as proline, arginine and γ-aminobutyric acid are synthesized which are closely related to resistance (Galili et al., 2001). S-Adenosyl--methionine (SAM) is a key metabolite that regulates hepatocyte growth, death, and differentiation. The biosynthesis of SAM is inhibited, which will aggravate liver injury. SAM depletion in the liver is closely related to hepatocyte necrosis and liver fibrosis (Tao et al., 2017). Spermidine has anti-inflammatory, antioxidant and anti-atherosclerotic effects, and participates in lipid metabolism (Gao, 2019). The results showed that the expressions of creatine, urea and -glutamate acid were up-regulated and the expression of S-adenosine--methionine was down-regulated after administration of garidi-5. The expression of spermidine contained in serum and kidney and 5-aminovaleric acid metabolite contained in serum and liver were disordered. It is suggested that garidi-5 can disorder the metabolic pathways of arginine and proline in serum, liver and kidney of rats by dysregulating the above metabolites, resulting in the imbalance of amino acid metabolism in rats, thus causing liver and kidney toxicity.

Garidi-5 disorder nicotinate and nicotinamide metabolism

Nicotinic acid (NA) is a component of dehydrogenase coenzyme I and coenzyme II, which is converted into nicotinamide (NAM) in vivo. It plays an important role in maintaining lipid metabolism, glucose glycolysis, protein, carbohydrate and amino acid metabolism in the body (Chen and Jiang, 2015). This study found that the metabolism of nicotinate and nicotinamide metabolism in serum, liver and kidney of rats were disordered after administration of garidi-5, such as the increase of the content of 1-methylnicotinamide. 1-methylnicotinamide (MNA) is the main metabolite of nicotinamide, which has antithrombosis and anti-inflammatory effects. Nicotinamide (NA) is transformed into MNA and N-methyltransferase (NNMT), and its up-regulated expression is closely related to liver cirrhosis (Magdalena et al., 2010).

Same differential metabolites in serum, liver and kidney

This study found that trimethylamine N-oxide, -pyroglutamic acid and DL-3-aminoisobutyric acid were common differential metabolites in serum, liver and kidney of GG group. Elevated plasma trimethylamine N-oxide (TMAO) level can induce inflammatory response, increase intracellular calcium release, activate autonomic nervous system, aggravate ventricular remodeling, affect cardiac structure and function, and promote the occurrence of cardiovascular diseases (Tan et al., 2019). The increase of TMAO content can also cause dysfunction of vascular endothelial cells in rats with chronic kidney disease (Organ et al., 2016). The contents of TMAO in serum, liver and kidney of GG group were significantly increased, indicating that TMAO is a biomarker of liver and kidney toxicity of GG.

Pyroglutamic acid exists in free form in blood, tissue fluid and various organs. Pyroglutamic acid is an intermediate product of γ-glutamyl cycle, which can interconvert with glutamate and antagonize glutamate receptors in vivo, and is associated with a series of genetic diseases. When lipid metabolism is abnormal, the level of pyroglutamate decreases (Kumar & Bachhawat, 2012). The content of pyroglutamic acid in GG group was significantly decreased in serum, but significantly increased in liver and kidney, suggesting that the occurrence of liver and kidney toxicity in GG may be closely related to the abnormal metabolism of glutamine.

Conclusion

In conclusion, garidi-5 can change the expression of metabolites such as trimethylamine N-oxide, -pyroglutamic acid, glycine-betaine, xanthine, glutathione, -leucine, -arginine, cytidine, spermidine, urea, 5-aminovaleric acid, creatine, -glutamic acid, S-adenosyl--methionine and 1-methylnicotinamide in rats, and disorder ABC transporters, arginine and proline metabolism, nicotinate and nicotinamide metabolic pathways in serum, liver and kidney of rats, so as to aggravate the abnormal metabolism of protein and amino acid in rats, leading to liver and kidney toxicity.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.