A GC–MS-Based Metabolomic Strategy to Investigate the Protective Effects of Mulberry Polysaccharide on CCl4-Induced Acute Liver Injury in Mice

Mulberry (Morus alba) fruits of the woody mulberry tree (family: Moraceae Morus) is a type of mulberry fruit grown in the southern Xinjiang region, which polysaccharides have antioxidant and liver protective effects. This article further preliminary study on the protective effects of mulberry polysaccharide (MP) on liver. A detection kit was used to assess serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), liver malondialdehyde (MDA), superoxide dismutase (SOD) and other indicators. Liver tissue sections were stained with hematoxylin and eosin (H&E) and observed under a microscope. The entire endogenous metabolite profiling was acquired via metabolomics strategy using gas chromatography-mass spectrometry (GC–MS) to assess the underlying protective mechanisms of MPs. Results indicated that MPs exerted a hepatoprotective effect on acute liver injury by decreasing serum ALT and AST levels, hepatic MDA, and restored hepatic SOD glutathione peroxidase (GSH-Px) activities. A total of 33 possible endogenous metabolites associated with lipid, glucose, and energy metabolism including amino acids, sugars, and fatty acids, were found. The results of the present study provide a reference for elucidating the protective mechanisms of MPs against acute liver injury.


Introduction
Acute liver injury is characterized by hepatocyte damage and inflammation. Studies have found that the induction of ethanol, high-fat diet, CCl4 and drugs may cause fatty liver and then cause liver damage [1]. The main mechanism of liver injury is the generation of free radicals and lipid peroxidation. Excessive production of free radicals may lead to damage to the membrane structure and function, causing severe toxicity to liver cells [2] and induce malignant tumors such as liver cancer and liver cirrhosis disease [3]. However, there are currently limited reports on its pathogenesis. Natural products, an abundant resource to be utilized in drug development, have received an increased attention due to their specific functions. A number of polysaccharides with hepatoprotective effects have been reported, such as Crassostrea gigas, Mori Fructus, Anoectochilus roxburghii.According to traditional Oriental medicine, mulberry fruits can protect against liver and kidney damage [4].In Korea, mulberry has been used as a traditional medicine due to its beneficial health effects stemming from its anti-inflammatory, antioxidant,and hepatoprotective properties [5,6].The medicinal mulberry Morus L. black mulberry (Morus nigra Linn.) was a type of mulberry fruit grown in the southern Xinjiang region and used as an edible medicinal material by local residents.Hu Junping, a professor in the department of Pharmacognosy, Xinjiang Medical University, identified it as xinjiang medicinal mulberry, and the research group also conducted animal pharmacodynamics research on its anti-inflammatory and hepatoprotective effects. Lee [7] et al. explored the effects of medicinal mulberry on pneumonia caused by bronchi and respiratory tract infections by establishing in vitro and in vivo models. In previous studies, the polysaccharide content in medicinal mulberries in Xinjiang was shown significantly higher than in common mulberry varieties, indicating the medicinal mulberry has a higher utilization value [8]. Based on our preliminary research regarding the 1 3 effects of polysaccharides in the medicinal mulberry and literature review [9], investigating the protective mechanistic effects of mulberry polysaccharides (MPs) on acute liver injury is important.
Metabolomics is used to assess pathological states in humans and animals by monitoring small molecular changes [10], which can provide a more in-depth understanding of the biochemical effects of drugs and help further determine the pathological process of diseases. Metabolomics research showed compounds can be biotransformed into hydrophilic molecules [11], which are easily excreted through the kidneys in urine. When liver injury occurs, the metabolic capacity of the liver is weakened and the levels of most metabolites change. Therefore, detecting changes in metabolites to evaluate the protective effects of drugs on liver injury is feasible.
Substantial modern technical advances have enabled robust and comprehensive profiling of molecular markers in the study of human health and disease. Numerous metabolomics technologies, including high-resolution nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-MS (LC-MS) [12][13][14]have been applied for disease diagnosis, metabolic marker exploration, toxicology research, new drug development, and drug screening by analyzing biological samples such as urine, blood, and tissue. In addition, GC-MS is considered the standard technique for metabolomics research [15].
In the present study, the protective effects and different doses of medicinal MPs on CCl 4 -induced acute liver injury in mice were investigated. In addition, biochemical indicators and metabolic markers in liver pathological conditions were analyzed using metabolomics with GC-MS technology.

Preparation of Mulberries
The mulberries (Morus nigra Linn.) were purchased from the local market in Aksu, Xinjiang, and only the uncrushed fruits were kept at room temperature until extraction. After extraction of mulberry polysaccharide by water extraction and alcohol precipitation, monosaccharide was derived from 1-phenyl-3-methyl-5-pyrazolone (PMP) by ultrasonicrelated cellulose method under optimized conditions. High performance liquid chromatography was used to separate and identify monosaccharide components. The mobile phase was ammonium formate (A) and acetonitrile (B) with pH = 5.5, the injection volume was 10 μL, the flow rate was 1 mL/min, and the detector wavelength was set to 245 nm [16]. The final MP consisted of Man, Rah, GalUA, Glc, Gal and Arab with a molar ratio of 2.96:1:1.57:3.92:2.83:1.08.

Liver Injury Model Construction and Treatment
The hepatoprotective effects in vivo were evaluated using CCl 4 -induced hepatotoxicity model [17,18]. Male Kunming mice with body weight (BW) of 20 ± 2 g were provided by the Experimental Animal Center of Xinjiang Medical University, Urumqi, China. Mice were housed in cages at 21 ± 1 °C with 50-60% relative humidity under 12 h light/ dark cycle conditions. Food and water were available ad libitum. All animal experiments were approved by the Animal Ethics Committee of Xinjiang Medical University (No. A-20100920002).
Mice were randomly divided into six groups of 11 animals each. The mice in the control group were treated with saline (10 mL/kg b.w. i.g.) and the mice in the positive control group were treated with silymarin by gavage (100 mg/ kg b.w. i.g.). The treatment groups were administered MP at 50, 100, or 200 mg/kg per day by gavage. All groups were treated for 2 weeks. On the 14th day, all mice except those in the normal control group,model group were intraperitoneally injected with 0.3% CCl 4 dissolved in olive oil (10 ml/ kg b.w.), and mice in the normal group were injected with equal amount of olive oil instead of CCl 4 [19].

Determination of ALT, AST, SOD, GSH-Px and MDA Levels
All mice were starved for 16 h and then sacrificed. Blood was taken from the eyeball and centrifuged at 3,000 g for 10 min at 4 °C to extract serum. The liver was immediately removed, washed, and homogenized in ice-cold physiological saline to prepare a 10% (w/v) homogenate. The obtained homogenate was centrifuged at 4,000 g and 4 °C for 10 min to remove cellular debris and the supernatant was collected 1 3 for analysis. The serum ALT and AST levels and the SOD, GSH-Px, MDA, and protein levels in liver were determined using the reagent test kits.

Histopathological Observation
Part of the liver tissue was fixed in 10% formalin solution and embedded in paraffin. Sections (5 μm in thickness) were cut, stained with hematoxylin and eosin (H&E), and examined under a Nikon Eclipse E600 microscope (Nikon Corp., Tokyo, Japan) at 200x magnification.

Metabolic profiling analysis
References [20,21] part of the method Homogenize liver samples frozen at -80 C. Acetonitrile solution (250 μL) was added into the sample (100 μL), which was then vortexed for 1.5 min, placed into an ultrasonic bath for 10 min, and finally centrifuged at 10,000 r/min and 4 °C for 10 min. The supernatant was removed and placed into a new Eppendorf tube and evaporated using N 2 . Oximation was performed at 70 °C for 1 h after the addition of 50 μL methoxyamine pyridine solution (15 g/L). Trimethylsilylation was performed at 70 °C for 1 h after addition of 50 μL derivatization reagent MSTFA (1% TMCS). Then, 150 μL heptanoic acidcontaining acetonitrile solution (0.9 mg/mL) was added as a reference and centrifuged (10,000 r/min, 10 min, 4 °C). Finally, the supernatant was transferred into the inner tube of the microinjection bottle for GC-MS analysis.
The sample was analyzed with Agilent 7890B/5977A GC-MS equipped with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J & W Scientific, Inc., Folsom, CA, USA). Helium was used as the carrier gas with a constant flow rate of 1.0 mL/min. The temperature program was as follows: the initial temperature was 85 °C, followed by holding for 5 min, then elevated to 300 °C at a rate of 10 °C/min, and maintained for 6 min. The temperatures of the injector, transfer line, and ion source were set to 270 °C, 270 °C, and 230 °C, respectively. The mass range (50 − 600 m/z) in a full-scan mode for electron impact ionization (70 eV) was applied.

Date Processing and Statistical Analysis
The peaks and representative peaks shared by the GC-MS TIC map were retrieved and each peak area was integrated. The retention time, each peak area, and internal standard peak were imported into the Excel table for use. The ratio of the peak area of the sample to the internal standard peak (relative peak area) indicates the number of metabolites [22], and SPSS 17.0 software was used to analyze the difference between the groups based on t-test. Pattern recognition multivariate analysis of normalized data was performed using the SIMCA-P12.0 software, using principal component analysis (PCA), and partial least squares-discrimination analysis (PLS-DA), and maximizing the difference between groups [21]. Finally, data were presented as means ± standard deviation (SD) for test drug groups using SPSS 17.0. A P-value < 0.05 was considered statistically significant and highly significant when P < 0.01.

Effects of MP on Body Weight and Relative Liver Weight
MP had no effect on body weight and relative liver weight ( Table 1). Body weight was not significantly different among the groups (P > 0.05). However, compared with the normal group, the relative liver weight in the CCl 4 model group significantly increased (P < 0.05), indicating CCl 4 could induce hypertrophy of liver tissues in mice. Conversely, the treatment with silymarin (100 mg/kg b.w.) or MP (100 mg/kg) significantly decreased relative liver weight compared with CCl 4 model group (P < 0.05).

Effects of MP on ALT and AST Levels
Serum ALT, AST levels and other enzyme levels are quantitative markers of liver cell injury. As shown in Table 2, compared with the normal control group, the serum ALT and AST levels in the CCl4 group were significantly increased (P < 0.05), which was indicative of hepatic failure. However, the treatment with different dosages of MP (100 and 200 mg/kg) in mice significantly reduced the ALT and AST levels compared with the CCl 4 treatment group (P < 0.05); the effects were similar to the silymarin group. Table 1 Effects of MP on body weight and relative liver weight in mice (n = 6) 50 mg/mL was for the low-dosed group(L), 100 mg/mL was for the medium-dosed group(M), and 200 mg/mL was for the high-dosed group(H) *P < 0.05 and **P < 0.01 when compared with control; △ P < 0.05 and △△ P < 0.01 when compared with model

Effects of MP on the Activities of Hepatic Antioxidant Enzymes
SOD, MDA, and GSH-Px are the main antioxidant enzymes in liver tissues and can protect against oxidative damage. As shown in Table 3, compared with the normal control group, the SOD and GSH-Px levels in the CCl 4 model group were significantly decreased (P < 0.05). However, MDA levels in the CCl 4 treatment groups were significantly higher than in the normal control group when mice were pretreated with silymarin (P < 0.05). MP dosages of 100 mg/mL and 200 mg/mL significantly lowered the MDA levels compared with the model group (P < 0.05).

Histopathological Observations
Histological liver slices in the normal control group showed general liver parenchyma, including hepatic lobules, and blood sinuses surrounding normal hepatocytes in hepatic lobules were distributed radially towards the central lobe veins (Fig. 1A). However, a massive inflammation and infiltration area around the central veins was observed in mice in the CCl 4 model group. Focal central vein congestion, vacuolization, and necrosis with inflammation was observed (Fig. 1B). Compared with mice in the CCl4 group, mice with 100 mg/kg MP had moderate liver injury and the area of inflammation and infiltration was decreased. Liver sections showed mild central vein congestion, vocalization, and necrosis with sinusoidal dilatation (Fig. 1C). In mice that received an increased MP dosage, liver sections showed absence of vacuolization, inflammatory cells, and regeneration of hepatocytes around the central veins, however, slight congestion was observed in the central veins and the liver sections were almost similar to normal liver architecture and had higher hepatoprotective activities. The effects of high MP dosage was similar to silymarin ( Fig. 1D-F).

Identification of Each Metabolite Map in the NIST Map
A total of 11 liver tissue metabolite peaks were extracted using GC-MS and their peak areas manually integrated. The TIC chromatograms obtained from the control, model, low-dose, middle-dose, high-dose, and positive drug groups are shown in Fig. 2.Liver tissue sample data collection was performed using the XCMS Online software and typical TIC maps for each group were obtained; 33 common metabolites were identified based on MS fragment information and HMDB online data. Then, the peak areas extracted from the TIC map in the NIST library were compared.The relative value of the normalized peak area was analyzed using SPSS22.0. The results showed that compared with the normal group, the model group had significant differences in metabolites such as benzoic acid, glucuronide amide, D-leucine, hexadecanoic acid, D-glucose, linoleic acid, and proline (P < 0.05).Compared with the model group, there were significant differences in metabolites such as ergosterol, glycine, benzoic acid, hexadecanoic acid, and glucuronamide treated with high-dose MP(P < 0.05);There were significant differences in metabolites such as glycine, D-glucose, octadecanoic acid, and arachidonic acid treated with medium-dose MP(P < 0.05).There were significant differences in metabolites such as glucuronamide、D-leucine, linoleic acid, glycine, palmitic acid, and D-glucose treated with low-dose MP(P < 0.05).There were significant differences in metabolites such as linoleic acid, hexadecanoic acid, ergosterol, and glycine in the positive drug group (P < 0.05).

PCA of Model
An obvious separating tendency was observed in the PCA (Fig. 3A) and Validate Model (Fig. 3B) of the two groups and the R 2 Y = 0.927 and Q 2 Y = 0.678. The results indicate the models were valid and reliable.

PLS-DA of Metabolites
PLS-DA was performed to discriminate differences among groups. Differential metabolites contributing to the group separation were identified using VIP and P-values. In addition, the metabolites that matched the condition (VIP > 1.0 and P < 0.05) were recognized as candidate compounds. Generally, a threshold of VIP > 1 and P < 0.05 in the Mann-Whitney U test is considered statistically significant. In summary, 14 discriminating metabolites were filtered and recognized as candidate metabolites which could directly perform antioxidant and liver protective effects (Table 4) or activate the scavenging peroxide mechanism to enhance the antioxidant function of the tissue in vivo.
The normalized peak area relative values were imported into SIMCA-P12.0 statistical software for PLS-DA. To determine the accuracy of the data of each group and the difference among all treatments, PCA was performed using SIMCA.As shown in Fig. 4, each group of mice was dispersed according to different metabolite patterns. There were significant differences among the three-dimensional map groups, with obvious clustering characteristics, indicating that the data of each group are reliable. The liver injury induced by CCl4 may affect the physiological environment and metabolite metabolism of mouse liver tissue [23].
The metabolites contributing to the group separation are shown in the Figure and were recognized as key metabolites with antioxidant ability.The PLS-DA model (construction of variable importance in projection, VIP) was combined to reflect metabolite differences in groups and to search for possible endogenous metabolites (Fig. 5). Based on the PLS-DA results ( Fig. 5 and Table 4), compared with the normal group, a total of eight metabolites were upregulated and seven were downregulated in the model group and each MP group, involving amino acids, amides, sugars and arachidonic acid. These potential endogenous metabolites (VIP > 1) were considered potential biomarkers. Therefore, six potential biomarkers that could provide novel clues for subsequent studies of their protective effects on liver damage were identified. However, these potential biomarkers need further verification and investigation.
Variable importance in projection (VIP) > 1 was used to filter the biomarker ↑ or ↓ compared with normal

Pathway Analysis and Biological Interpretation
The integration of metabolic pathway metabolites were further performed. The significant relevant pathways affected the protective effects of mulberry polysaccharide on CCl4-induced acute liver injury were analyzed using MetaboAnalyst 3.0 (www. metab oanal yst. ca), a web-based server that supports pathway analysis, integrated analysis, and pathway enrichment topology. As shown in Fig. 6, elevated purine, amino sugar, and nucleotide sugar metabolism were the most relevant pathways affected by MPs with the most significant impact value. Furthermore, MP-induced pathways were associated with phenylalanine, thiamine, and tyrosine metabolism and glycolysis. Among the pathways induced by MPs, tyrosine metabolism was the most significant. In addition, a metabolic network was built using the Kyoto Encyclopedia of Genes and Genomes (KEGG, http:// www. kegg. jp) database (Fig. 6.). The data indicate that changes in the comprehensive metabolic profile of mice with MP-induced liver protection were mainly associated with phenylalanine and tyrosine metabolism. Consequently, amino acid metabolism was identified as the key metabolic pathway stimulated by MPs indicating MPs mainly influence the biosynthesis of amino acids in liver tissue with specific biochemical metabolites. In addition, MPs promoted the biosynthesis of starch and sucrose which also reflects the influence of MPs on the liver tissue to some extent.

Discussion
MPs are a natural resource combining medicinal and nutritional properties and exert antioxidant effects due to specific biochemical metabolites. Reportedly, the hypoglycemic and lipid-lowering effects are mainly due to the MP antioxidant activity [24]. Water-soluble polysaccharides can play an important role in free radical scavengers and prevent many free radical-mediated chronic diseases such as acute liver damage [25]. In the present study, the in vitro antioxidant activity of MPs was determined using four free radical assays. The results indicated that MPs have a protective effect on CCl 4 -induced liver toxicity in mice which could potentially be used to treat liver disease in humans.
In the early stage of our research group, the polysaccharides of mulberry were obtained by water extraction and alcohol precipitation, and the yield of polysaccharides was determined by the phenol-sulfuric acid method [26]. active. Therefore, the research group established an acute liver injury model again to observe the protective effect of mulberry polysaccharides on liver injury, and analyzed the composition of monosaccharides by HPLC pre-column derivatization method, and obtained the ratio of 7 monosaccharides. Activity is not only determined by the content of polysaccharides, but also closely related to its microstructural properties. It has been reported in the literature [27] that the polysaccharide samples containing GLcU and xyl have better antioxidant capacity, so the subject will continue to pay more attention to the monosaccharide composition and biological activity of polysaccharides at all levels of mulberry.
CCl 4 , a commonly used chemical for inducing liver damage, can significantly increase the serum ALT and AST levels. The serum ALT and AST levels were elevated in the CCl 4 group (Table 2) compared with the normal control  In addition, the liver SOD, MDA, and GSH-Px levels were evaluated in each group. The SOD and GSH-Px levels in the silymarin and MP treatment groups were significantly increased (Table 3). This result could also be improved by the same treatment (Journal of China Institute of Pharmacy). The liver enzyme antioxidant levels in the 200 mg/kg MP group were the same as in the silymarin treatment group, indicating that MPs can effectively enhance the antioxidant ability of liver tissue.
Metabolomics is a popular metabolite analysis technology and has been rapidly developed and applied. Metabolomics is a new technology developed after genomics and   Figure. The color of the circle represents the significance and the size of circle represents the impact factor of each pathway. Red represents significant and blue non-significant (P < 0.05) proteomics and used in many fields, including NMR, MS, and HPLC/GC. GC-MS is commonly used to detect metabolites of steroid-containing biological samples [28] (urine, serum, and tissue). Shiman [29] et al. used capillary electrophoresis (CE)-MS/MS combined with GC time-of-flight (TOF)-MS to identify potential biomarkers of hepatotoxicity. In the present study, the acute liver injury caused by CCl 4 was explored using GC-MS metabolomic technology. The results indicated significant differences among various MP doses. Furthermore, a total of 14 possible endogenous substances were found that involved amino acid, glucose, and lipid metabolism. The use of metabolomics shows that combining physiological and metabolite information with drug action can provide a new methodology for exploring clinical and basic research which has been applied in some studies [30].
The MPs significantly affected amino acid metabolism. In particular, the most notable changes were observed in phenylalanine and thiamine metabolism. Phenylalanine was predominant in 13 altered metabolites and the levels were increased in the treated groups compared with the control group. In addition, the most relevant pathways affected by MPs were amino acid metabolism pathways such as phenylalanine and tyrosine. Furthermore, phenylalanine, tyrosine, and tryptophan biosynthesis showed MPs, to a certain extent, exert protective effects by mainly influencing amino acid metabolism.
Studies have found the inhibitory effect of phenylalanine and its metabolites on glycolysis, while the catabolic process of amino acids is mainly in the liver [31]. The reduced phenylalanine-to-tyrosine conversion ratio in blood could promote secondary effects. In addition, liver damage changes the activity of many enzymes. For example, MDA changes can lead to reduced hepatic lipid and glucose metabolism. Changes in phenylalanine and tyrosine metabolism were observed in the present study, indicating MPs protect the liver with their hepatoprotective activities. The tricarboxylic acid (TCA) cycle is involved in aerobic oxidation of glucose and is the main process for lipid and amino acid metabolism, thus, inhibition of the TCA cycle may lead to organ degeneration [32]. Some altered saccharide metabolites, including fructose, glucose, and galactose, which are involved in glucose metabolism as well as the TCA cycle pathway [33], were shown to be closely related to TCA cycling and energy metabolism in the MP-treated group in this study. In epidemiological and clinical studies, stearic acid has been found to have a higher ability to lower LDL cholesterol than other saturated fatty acids [34]. In conclusion, the results of the present work showed MPs had a hepatoprotective effect on CCl 4 -induced liver injury in mice. In addition, 13 metabolites were identified as biomarkers and need further verification. Using a multiple integrated metabolomics method, the biomarkers associated with liver damage were identified and further investigated using PLS-DA with a panel of the integrated characteristic metabolites. These newly identified pathways could help further analyze the mechanisms associated with liver damage and explore novel potential therapeutic targets, which should be investigated in future studies.
Funding This study was supported by funds (No. 81760753) from the National Natural Science Foundation of China.
Data Availability All the authors in the manuscript have published the usability of the data and a declaration of consent for publication in this journal.