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. 2022 Jul 22;14(3):422–431. doi: 10.1016/j.chmed.2022.05.003

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

Wurihan a,b, Aodungerle b, Bilige b, Lili b, Sirguleng c, Aduqinfu d, Meirong Bai a,b,⁎
PMCID: PMC9476469  PMID: 36118012

Abstract

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.

Keywords: garidi-5, kidney, liver, metabonomics, subacute toxicity

1. 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.

2. Methods and materials

2.1. 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.

2.2. 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).

2.3. 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).

2.4. 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.

2.5. 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.

2.6. 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.

2.7. 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.

3. Results

3.1. 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.

Fig. 1

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

3.2. Multivariate statistical analysis

3.2.1. 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.

Fig. 2

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

Fig. 3.

Fig. 3

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

Fig. 4.

Fig. 4

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

3.2.2. 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).

3.3. Screening of differential metabolites

3.3.1. 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 l-norvaline, Glu-Gln, glutamine (D), l-pipecolic acid, fructose, kaempferol-3-O-rutinoside, l-pyroglutamic acid, l-leucine, dl-pipecolinic acid, dl-3-aminoisobutyric acid, l-(+)-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, l-arginine, sebacic acid, butyryl carnitine (isomer of 920), 4-hydroxypyridine, γ-aminobutyric acid and glutathione decreased significantly; Methionine sulfoxide, N-α-acetyl-l-ornithine, dimethylphthalate, 5-aminovaleric acid, l-citrulline, indole-3-carbinol, trimethylamine N-oxide, thiamine monophosphate, glucose, 4-methyl-5-thiazoleethanol, spermidine, hydroxy butyric acid, N-acetylhistamine, cytidine and d-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
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3.3.2. Screening of liver differential metabolites

Sixty-four differential metabolites were identified in liver samples. Compared with Z, GG group, tyrosine, creatine, γ-glutamylleucine, d-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, dl-pipecolinic acid, α-methylhistidine, N-acetylglutamate, 2-amino-3-[hydroxy-[3-octadecanoyloxy-2-[octadec-11-enoyl]oxypropoxy]phosphoryl]oxypropanoic acid, β-nicotinamide mononucleotide, l-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, l-pyroglutamic acid, anserine, glycodeoxycholic acid, glycochenodeoxycholic acid, uridine-5-monophosphate and dimethyl sulfoxide increased significantly; Thiazolidine-4-carboxylic acid, dl-3-aminoisobutyric acid, ndoleacetic acid, 2-hydroxyisocaproic acid, l-(−)-phenylalanine, l-leucine, pyrrole-2-carboxylic acid, cytosine, (S)-3-amino-4-phenylbutyric acid, propionic acid, rac-glycerol 3-phosphoate, 5-aminovaleric acid, d-arabinose-5-phosphate disodium salt, adipic acid, (S)-3-amino-5-methylhexanoic acid, 3-indoxyl sulfate, l-5-oxoproline, 2-hydroxy-4-methylpentanoate, (3R,4S)-3-amino-4-methylhexanoic acid, phytosphingosine, d-(+)-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
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3.3.3. Screening of renal differential metabolites

Fifty-four differential metabolites were found in kidney samples. Compared with Z, GG group l-pipecolic acid, ethanolamine, trimethylamine N-oxide, dl-3-aminoisobutyric acid, d-(+)-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-dl-alanine increased significantly; S-adenosyl-l-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, l(+)-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, l-pyroglutamic acid, (S)-3-amino-4-phenylbutyric acid, stearic acid, threonic acid, 2-hydroxyisobutyric acid, ascorbic acid, palmitoylcarnitine, resibufogenin, N-acetyl-l-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
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3.4. 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.

Fig. 5

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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.

4. 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.

4.1. 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, l-leucine, l-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). l-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). l-Arginine (l-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, l-leucine and l-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.

4.2. 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, l-glutamate acid, S-adenosine-l-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. l-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-l-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 l-glutamate acid were up-regulated and the expression of S-adenosine-l-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.

4.3. 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).

4.4. Same differential metabolites in serum, liver and kidney

This study found that trimethylamine N-oxide, l-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.

5. Conclusion

In conclusion, garidi-5 can change the expression of metabolites such as trimethylamine N-oxide, l-pyroglutamic acid, glycine-betaine, xanthine, glutathione, l-leucine, l-arginine, cytidine, spermidine, urea, 5-aminovaleric acid, creatine, l-glutamic acid, S-adenosyl-l-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.

Acknowledgements

This work was supported by the Mongolian Medicine Safety Evaluation and Innovation Team Project (No. MY20190003) and “Innovation Team Development Plan” Project of Institutions of Higher Learning in Inner Mongolia (No. NMGIRT2216).

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