1. Introduction

Polygoni Multiflori (PM, Heshouwu in Chinese), derived from the roots of Polygonum multiflorum Thunb., is a widely used Chinese medicinal material in clinic. Raw PM (RPM) has the effects of detoxicating, eliminating carbuncle, cutting off malaria, loosening bowel, and relieving constipation. A few of ancient books of traditional Chinese medicine, such as “ben cao hui yan,” recorded that RPM is poisonous [1]. To reduce toxicity and enhance tonic effect, RPM was usually processed by black bean sauce or by nine cycles of steaming and sun drying to obtain the processed PM (PMP) [2]. PMP demonstrates the functions of nourishing the liver, kidney, and blood, strengthening the muscles and bones, and blackens hair [3–6]. Interestingly, RMP steamed with black soybeans for nine cycles was safe. However, modern processing technologies, usually steamed with the black soybean juice once, still exhibit obvious hepatotoxicity [7, 8]. PMP-related liver injury has been reported in many countries and regions, such as South Korea, Japan, and UK [9]. Recently, the National Medical Products Administration in China has also warned of the risk of liver injury from the extracts of RPM and PMP [10]. Unfortunately, although extensive experiments have been performed in recent years, the potential toxic mechanisms that cause liver injury remain unclear. Therefore, it is particularly urgent and important to establish a method for the early warning of liver injury of PMP.

Metabolome is located downstream of gene regulation and protein network and could provide terminal information of biology. Metabolomics is the study of endogenous metabolite alterations associated with maintaining the normal function and development of organisms after the stimulation or disturbance of biological systems [11]. It has been widely applied in various fields of life sciences and represents a powerful tool for interpreting life phenomena and exploring mechanisms of disease [12]. Chromatography combined with mass spectrometry (such as LC-MS and GC-MS) and nuclear magnetic resonance (NMR) spectroscopy is widely applied in metabolomics studies, and LC-MS is the most popular used methodology in untargeted metabolomics research. Ultrahigh performance liquid chromatography-mass spectrometry technology provides a rapid, concise, and high-throughput approach for the analysis of biological fluids or tissues [13]. Combined with multivariate statistical analysis methods, it provides an effective method for the discovery of metabolic pathways. The targeted and untargeted methods in MS-based metabolomics were used to detect changes in the processing technologies of Polygoni Multiflori Radix [14]. In conclusion, metabolomics has been extensively used for the discovery of biomarkers and the research on the metabolic mechanism [15].

The hepatotoxicity components of PMP have always been the subject of controversy. In our previous study [16], we performed a comparative study on the chemical components in the aqueous extract and 60% ethanol extract of PMP (PMPE). The results showed that the different components were attributed to anthraquinones, such as emodin, rhein, and physcion. Furthermore, the pharmacokinetics characters of these main components in rats were studied. The t_1/2(h) of those components of the PMPE was longer compared to the aqueous extract of PMP and increased with the increasing of dosage of PMPE. Anthraquinone components (such as emodin) which has higher content in PMPE are regarded as potential liver damage components [16]. Based on our previous research and literature review, the PMPE showed a degree of hepatotoxicity [4, 16, 17]; therefore, we selected PMPE to investigate the components and mechanism of potential liver injury of PMP. The study aimed to further explore the potential hepatotoxicity mechanism of PMPE using the LC-MS-based metabolomics method. The reliability of discriminated metabolite was further screened by receiver operating characteristic (ROC) curve analysis, and the related metabolic pathways were clarified. Combined with biochemical indicators, our work could further clarify the potential mechanism of PMPE-induced liver injury.

2. Materials and Methods

3. Results

3.1. Clinical Observation after Treatment of PMPE

Compared with the BC group, the weekly growth of the body weight in PMPE-treated rats significantly reduced (Figure 1(a)), whereas the liver index of PMPE-treated rats significantly increased (p < 0.05) (Figure 1(b)). Liver pathology revealed liver cell degeneration, necrosis, and inflammatory cell infiltration after administration PMPE (Figure 1(c)).

3.2. Hepatotoxicity Assessment of the PMPE

Compared with the BC group, the levels of ALP, TAB, and TBIL increased significantly (p < 0.05), and the content of ALT, TG, and AST also showed increasing tendency (Figure 2).

3.5. Identification of Potential Liver Injury Biomarker and Pathway Analysis

PCA was used for visualizing group clustering and visualizing possible outliers. The BC group and PMPE group were obviously separated in the positive and negative ion mode (positive: R² = 0.433, Q² = 0.177; negative: R² = 0.559, Q² = 0.344), indicating that there was a significant difference between the BC and the PMPE groups (Figure 3). PLS-DA and permutation analysis were performed to evaluate the reliability of the model (positive: R² = 0.487, Q² = 0.96; negative: R² = 0.605, Q² = 0.99). The model validation of PLS-DA was performed at 200 permutations analysis (Supplementary Figure S1), and the results indicated that the PLS-DA model was credible and had no overfitting. OPLS-DA was carried out to reveal the differences of metabolites among groups as a supervised classification analysis method (positive: R² = 0.568, Q² = 0.952; negative: R² = 0 .605, Q² = 0.987). Scatter plots and S-plot are shown in Figure 4. Variable important in projection (VIP) was used to evaluate the strength and explanatory power of the expression pattern of metabolites on the classification and discrimination of each group of samples. Differential metabolites were screened according to the VIP (VIP > 1.0) and P values of Student’s t-test (p < 0.05). A total of 342 differential characteristic ions from positive and negative ion patterns were selected. Based on the exact molecular ion mass, retention time of reference compounds, and metabolites database (METLIN, LIPID MAPS, and HMDB database), a total of 39 metabolites were identified as biomarker candidates of PMPE-induced liver injury finally. All candidate biomarkers were presented in Supplementary Table S2.

The ROC curves were performed for the analysis of sorting out the potential diagnostic metabolites with high specificity and sensitivity to distinguish between the BC and PMPE groups. The diagnostic values were assessed by the area under the ROC curve (AUC), which was carried out using GraphPad Prism (version 8.0 San Diego, USA). The AUCs of candidate liver injury biomarkers which were higher than 0.8 were regarded as potential biomarkers (Figure 5 and Supplementary Table S3). The results manifested that these biomarkers had better discrimination ability for healthy rats and liver injury rats. A total of 16 potential biomarkers were reconfirmed from the positive and negative ion patterns in rat plasma (8 for negative and 8 for positive). Potential biomarkers are summarized in Table 1. Six metabolites were significantly decreased in the PMPE group compared with those in the BC group, including kynurenine, lactic acid, pyruvate, sphinganine, phytosphingosine, and indoleacrylic acid (Figure 6).

Table 1. Biomarker candidates of PMPE-induced liver injury.

	VIP	FC	m/z	Formula	Biomaker	HMDB	KEGG	P-value
Pos	4.98	0.72	188.06905	C11H9NO2	Indoleacrylic acid	HMDB00734	-	5.62E-05
	1.99	0.47	209.09043	C10H12N2O3	Kynurenine ∗	HMDB0000684	C00328	2.84E-04
	1.41	0.79	302.30336	C18H39NO2	Sphinganine	HMDB0000269	C00836	4.11E-02
	1.62	0.76	318.29862	C18H39NO3	Phytosphingosine	HMDB0004610	C12144	3.47E-02
	1.62	1.82	454.29073	C21H44NO7P	LysoPE(0 : 0/16 : 0)	HMDB0011473	-	1.10E-02
	2.62	2.58	468.30649	C22H47NO7P	LysoPC(14 : 0)	HMDB0010379	C04230	2.33E-03
	1.07	1.65	482.32192	C23H48NO7P	LysoPC(15 : 0)	HMDB0010381	C04230	4.79E-03
	3.74	2.35	494.32224	C24H49NO7P	LysoPC(16 : 1(9Z))	HMDB0010383	C04230	4.65E-02
Neg	1.39	0.30	87.00888	C3H4O3	Pyruvate ∗	HMDB0000243	C00022	3.10E-07
	5.7	0.47	89.02465	C3H6O3	Lactic acid	HMDB0000190	C00256	1.02E-02
	2.49	1.54	187.00644	C7H8O4S	p-Cresol sulfate	HMDB11635	C06677	4.23E-02
	2.05	2.38	464.30076	C26H43NO6	Glycocholic acid	HMDB0000138	C01921	1.22E-02
	1.14	2.30	465.30223	C27H46O4S	Cholesterol sulfate	HMDB0000653	C18043	1.42E-02
	2.33	1.80	496.27305	C26H45NO7S	Taurocholic acid	HMDB0000036	C05122	3.02E-02
	3.8	2.23	498.28706	C26H45NO6S	Taurochenodeoxycholic acid	HMDB0000951	C05465	3.30E-02
	1.05	1.50	524.27794	C27H44NO7P	LysoPE (22 : 6/0 : 0)	HMDB0011526	-	6.14E-03

• Pos: positive; Neg: negative; FC: fold change; HMDB: human metabolome database; KEGG: Kyoto encyclopedia of genes and genomes.  ^∗: compared with the reference standards.

To further elucidate the metabolic pathways of specific metabolites associated with PMPE-induced liver injury, we used the MetaboAnalyst4.0 (https://www.metaboanalyst.ca/) for enrichment and topological analysis of the KEGG signaling pathway. The most affected metabolic pathways were primary bile acid biosynthesis, sphingolipid metabolism, energy metabolism, glycerophospholipid metabolism, and tryptophan metabolism (Figure 7 and Supplementary Figure S2). In order to exhibit the results clearly, we constructed a network of potential metabolic pathways based on biomarkers in rat plasma (Figure 8).

4. Discussion

Biliary cholestasis and bile duct injury are the main clinical manifestations of cholestasis caused by drugs. Serum biochemical parameters ALP is preferred in clinical diagnosis. According to the above results, the levels of ALP, TBA, and TBIL showed significantly increased accompanied by liver cell degeneration, necrosis, and inflammatory cell infiltration, indicating that the PMPE-induced hepatotoxicity may be related to cholestasis. Meanwhile, the levels of taurochenodeoxycholic acid, taurocholic acid, and glycocholic acid were significantly increased in plasma of hepatotoxicity rats. Pathway enrichment and topology analysis also indicated that the biosynthesis pathway of bile acids was disrupted or interfered. In the process of generation, formation, transportation, and discharge of bile acid, any failure in those steps could cause cholestasis. Bile acid, especially hydrophobic toxic bile acids accumulation in liver cells, could induce inflammatory reactions by damaging mitochondria and lead to endoplasmic reticulum stress. These changes could directly destroy membranes of cell and organelle by virtue of detergent action and lead to hepatocyte injury finally. When hepatocytes are damaged, bile acid metabolism is impaired and reversely flows into blood [18, 19]. These explain the mechanisms of serum TBA increased in rat with PMPE-induced liver injury.

As the endogenous ligand of FXR (farnesoid X receptor), bile acid is not only an important substance in lipid digestion and absorption but also serves as a signal molecule to transmit information through FXR-mediated signaling pathways and participate in the regulation of lipid metabolism in the body. FXR could monitor the metabolism of triglycerides and fatty acids through a variety of mechanisms. Generally, when FXR was activated by bile acids, SREPB-1c (sterol regulatory element-binding protein-1c), which is a major transcription factor involved in fat synthesis genes, was downregulated by SHP (recombinant small heterodimer partner) signal pathway in order to inhibit lipid synthesis. The activity of LDLR (low-density lipoprotein receptor) and VLDLR (very low-density lipoprotein receptor) was upregulated to increase lipid oxidation consumption and reduced lipid accumulation in the liver thereby [20]. Thus, lipids will accumulate in the liver when the activity of FXR was downregulated in the condition of disordered intrahepatic bile acid metabolism.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE), two most affluent glycerophospholipids, play a pivotal biological function in regulating lipoprotein metabolism [21]. They can be metabolized by phospholipase A2 (PLA2) into LPC (lysophosphatidylcholine), LPE (lysophosphatidylethanolamine), and arachidonic acid, which plays a very important role in lipid inflammation [22]. Metabolic disorders of these lipids result in changes in membrane lipid composition and affect the physical properties and functional integrity of the membrane. These could lead to hepatocellular apoptosis, inflammation, and the progression of liver disease. In addition, studies have reported that LPC has a certain proinflammatory function and is a new type of inflammatory lipids [23]. The experimental studies in vivo have shown that LPC can induce hepatitis which is harmful to the liver. It can also induce hepatocyte lipid apoptosis by activating GPCRs (G protein-coupled receptors) and depolarization of mitochondrial membrane [24]. Many researches have shown that the incubation of primary hepatocytes with LPC could induce cell death by apoptosis [25]. In our study, LPC and LPE were the most increased lipids in the oral administration group of PME, and the accumulation of these metabolites caused hepatocyte dysfunction. Sphingolipids are an important ingredient of the biofilm structure. As the important active molecules in organisms, sphingolipids and their metabolites have been identified as new biomarkers of chronic hepatopathy disease and hepatocellular carcinoma (HCC) [26].

As the site of gluconeogenesis and glycogen synthesis, the liver plays an important role in regulating blood sugar levels. Bile acids could regulate glucose metabolism through FXR-SHP or bile acid-TGR5 (Takeda G protein-coupled receptor 5) signal pathway [27]. Some representative important metabolites related to energy metabolism and tryptophan metabolism were detected in this study, such as pyruvic acid, lactic acid, kynurenine, and indoleacrylic acid. These metabolites were significantly reduced. The low level of kynurenine is closely related to abnormal liver function and energy metabolism. It has been reported that drug-induced liver injury is related to the mitochondrial function and energy homeostasis [28]. Moreover, the decrease of indoleacrylic acid is related to the disorder of tryptophan metabolism in the liver. The parallels with our findings are striking. These results indicated that liver injury affected the disorder of these metabolic pathways. In conclusion, all results indicated that PMP affected metabolic disorder of bile acids, lipids, and glucose metabolism; impaired liver function; and could induce liver injury finally.

Compared with the control group, the content of p-cresol sulfate in the PME group was significantly increased. Cholesterol sulfate is a representative substance of uremic toxin produced by tyrosine. The results suggested that the kidney may be damaged in different degrees, which may be attributed to the aggravation of kidney metabolism after liver injury. In addition, the disorder of bilirubin metabolism and blood circulation could also lead to renal function decline (hepatorenal syndrome) [29].

5. Conclusion

In summary, LC-MS-based plasma metabolomics analysis combined with ROC curve analysis method provides an integrated view of the metabolic features of PMP-induced hepatoxicity. The differential metabolites were identified. Metabolic pathway analysis indicated that the bile acid biosynthesis pathway was the main pathways affected by PMP, which may be the cause of liver injury. The sphingolipid metabolism, energy metabolism, glycerophospholipid metabolism, and tryptophan metabolism pathway were also involved. These discriminating metabolites may help to understand the pathogenesis of liver injury and provide a good prospect for the clinical diagnosis of PMPE-induced liver injury.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Liming Wang and Zhida Wang contributed equally to this work. They performed the experiment and wrote the manuscript. Yanchao Xing analyzed the data. Erwei Liu and Xiumei Gao revised the manuscript. Manuscript editing and review were performed by Linlin Wang and Zhifei Fu. All authors read and approved the final manuscript.