Morchella importuna Polysaccharides Alleviate Carbon Tetrachloride-Induced Hepatic Oxidative Injury in Mice

This study aimed to investigate the effects of Morchella importuna polysaccharides (MIPs) on carbon tetrachloride (CCl4)-induced hepatic damage in mice. A total of 144 female mice were randomly assigned to four treatment groups, namely, control, CCl4, low-dose MIP (LMIP) group, and high-dose MIP (HMIP) group. After the 10-day experiment, serum and liver were sampled for biochemical and metabolomic analyses. The HMIPs markedly decreased the liver weight under CCl4 intoxication. Furthermore, the significantly elevated concentrations of five serum biochemical parameters, including alanine aminotransferase, aspartate aminotransferase, triglyceride, total cholesterol, and total bile acid under CCl4 treatment were subverted by MIP administration in a dose-dependent manner. Moreover, MIPs relieved the increased hepatic malonaldehyde and protein carbonyl content and the decreased superoxide dismutase and catalase contents caused by CCl4 intoxication. There was also a dose-dependent decrease in the CCl4-induced inflammatory indices, such as the levels of interleukin-1, interleukin-6, tumor necrosis factor-alpha, and myeloperoxidase, with MIP administration. Subsequent ultra-high performance liquid chromatography–tandem mass spectrometry-based serum metabolomics identified nine metabolites between the control and CCl4 groups and 10 metabolites between the HMIP and CCl4 groups, including some critical metabolites involved in flavonoid biosynthesis, amino acid metabolism, energy metabolism, and toxicant degradation. These novel findings indicate that MIPs may be of therapeutic value in alleviating the oxidative stress and inflammation caused by CCl4. Liquid chromatography-mass spectrometry-based metabolomics provides a valuable opportunity for identifying potential biomarkers and elucidating the protective mechanisms of medicinal mushrooms against hepatic oxidative injury.


INTRODUCTION
The liver is considered a vital organ of the body with critical functions in protein synthesis, glucose homeostasis, detoxification, and nutrient utilization (Gao et al., 2008;Al-Seeni et al., 2016). The high levels of environmental toxins, alcohol, heavy metals, and metabolic dysfunction of the liver are among various insults that may lead to acute or chronic hepatic injury, followed by progressive fibrosis, cirrhosis, and even carcinoma (Sun et al., 2011;Nwokocha et al., 2012;Su et al., 2019;Chan et al., 2020).
Although some medicines have been developed to protect the liver, hepatoprotective drugs have potentially adverse side effects . Hence, finding natural plant extracts effective for the protection of liver injury is of intense interest.
Medicinal mushrooms have a long history of application in traditional oriental therapies, and fungal extracts are increasingly being employed to treat a wide range of diseases (Nakahara et al., 2020;Tung et al., 2020). Morchella importuna is a rare edible and medicinal mushroom species of true morels (the genus Morchella), highly appreciated for its commercial value, potent bioactivities, culinary qualities, and taste (He et al., 2019;Wang et al., 2020). Polysaccharide, an important active constituent of M. importuna, possesses various bioactivities, including antibacterial, antioxidant, anti-inflammatory, and antitumor properties, and a cardiovascular protective effect (Meng et al., 2018;Hou et al., 2020;Mu et al., 2020;Tang et al., 2020;Xue et al. 2019). This study has indicated that some plant polysaccharides also have hepatoprotective activity against drugand toxicant-induced liver injuries. The polysaccharide of wild edible mushroom Russula vinosa protects the liver from CCl 4induced oxidative stress injury (Liu et al., 2014). Similarly, the antioxidant activities of Cyclocarya paliurus polysaccharide might be of therapeutic value in ameliorating the hepatic and renal oxidative stress caused by CCl 4 (Wu et al., 2020b).
Morchella importuna polysaccharides (MIPs), with a molecular weight (Mw) of 28.5 kDa, are composed of Nacetylglucosamine, galactose, glucose, and mannose with molar ratios of 1.00:14.95:1.53:10.51 (Wen et al., 2019). These MIPs show strong antioxidant, immune-enhancing, hepatoprotective, and lipid-lowering activities. Therefore, it is attractive to further investigate their bioactivities, which provides feasible ideas and insights for the potential curative application of MIPs.
Accordingly, this study was performed to explore the effects of MIP administration on alleviating CCl 4 -induced oxidative stress and inflammation in mice. Subsequent metabolomic analysis was performed to uncover the potential pathways that were disturbed by CCl 4 intoxication. These results provide partial theoretical evidence about the MIPs in preventing and treating oxidative injury in the liver.

Preliminary Characterization of MIPs
The Mw distribution of MIPs was determined by highperformance gel chromatography (HP-GPC) using a Waters 515 high-performance liquid chromatograph equipped with a laser detector, a differential refractive index, and a Shodex OHpak SB-806 gel chromatographic column (300 × 7.8 mm) set at 40 • C ± 0.1 • C. The mobile phase was 0.05 M NaH 2 PO 4 -NaH 2 PO 4 buffer with 0.02% NaN 3 (pH 6.7) and a flow rate of 0.5 ml/min. The volume of sample loaded was 500 µl. Polysaccharide standards with Mw values of 738, 5, 800, 1.22 × 10 4 , 2.37 × 10 4 , 4.8 × 10 4 , 1.0 × 10 5 , 1.86 × 10 5 , 3.8 × 10 5 , and 8.53 × 10 5 g/mol were dissolved in the mobile phase. After filtration through a 0.45µm filter, the samples and standards were analyzed under the abovementioned chromatographic conditions. Mw of the sample was calculated according to the plot of Mw vs. retention time of the standards.
The monosaccharide compositions of MIPs were analyzed by ion-exchange chromatography. After hydrolysis of the MIPs (5 mg) with 2 M of trifluoroacetic acid (TFA, 5 ml) at 120 • C for 6 h, excess TFA was removed by co-distillation with methanol. The hydrolysate was redissolved in deionized water (1 ml) and passed through a 0.45-µm filter. High-performance anionexchange chromatography-pulsed amperometric detection was carried out on a Dionex ICS-5000 system. Samples were eluted with a 91:9 (v/v) mixture of water and 200 mM NaOH.

Analysis of MIPs by Fourier-Transform Infrared Spectroscopy
Spectroscopically, pure KBr was grounded in an agate mortar, then oven-dried at 110-150 • C for 48 h, and

Animals
Female C57BL/6 mice (17 ± 0.28 g) were purchased from Dossy Experimental Animals Co., Ltd. (Chengdu, China). Before the experiment, all mice were conditioned for 1 week under a 12-h light/12-h dark cycle, at 22 ± 3 • C, and 50-70% relative humidity, with free access to water and food. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Sichuan Academy of Agricultural Sciences. All procedures followed the Guide for the Care and Use of Laboratory Animals (National Institutes of Health).

Experimental Design
In a 10-day experiment, 144 mice were randomly assigned to four treatments with six replications per treatment (six mice per replication) as follows: control (n = 36), orally administered saline once a day; CCl 4 (n = 36), orally administered saline once a day; LMIPs (n = 36), orally administered saline and MIPs at 100 mg/kg body weight (BW) once a day; HMIPs (n = 36), orally administered saline and MIPs (200 mg/kg BW) once a day. At 2 h after the last administration, all mice (except for the control group) were intraperitoneally injected with CCl 4 (0.2% CCl 4 /olive oil mixture) at 5 ml/kg BW. The mice in the control group were intraperitoneally injected only with the same volume of olive oil.

Sample Collection
All mice fasted for 16 h after treatment. One mouse with the average BW within each replication was anesthetized    Frontiers in Physiology | www.frontiersin.org by the intraperitoneal injection of 500 µl of pentobarbital sodium (8 mg/kg BW). Venous blood (1 ml) was collected and centrifuged at 3,000 g and 4 • C for 10 min to obtain serum. The liver tissues were harvested for biochemical or histopathological analysis.

Histopathological Analysis
The liver tissue was fixed in a 4% paraformaldehyde solution for 48 h, then dehydrated, and embedded in the wax block. It was cut into 5-µM thick sheets and stained with H&E. Histopathological sections were viewed under a light microscope at optical magnifications of 100 and 400×.

Serum Metabolomic Analysis
Sample Pre-treatment Serum (100 µl) and pre-chilled methanol (400 µl; Thermo Fisher, Massachusetts, USA) were mixed thoroughly with a vortex. The samples were incubated on ice for 5 min, and then centrifuged at 15,000 rpm and 4 • C for 5 min. Some supernatants were diluted with liquid chromatography-mass spectrometry (LC-MS)-grade water (Thermo Fisher) to obtain a final concentration of 60% methanol. These samples were subsequently transferred to a fresh Eppendorf tube with a 0.22-µm filter and then centrifuged at 15,000 g and 4 • C for 10 min. Finally, the filtrate was injected into the liquid chromatography-tandem mass spectrometry (LC-MS/MS) system.

Liquid Chromatography-Tandem Mass Spectrometry Analysis
The LC-MS/MS analysis was performed using a Vanquish UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher). Samples were injected onto a Hypersil Gold column (100 × 2.1 mm, 1.9 µm) and eluted by a 16-min linear gradient at a flow rate of 0.2 ml/min. The eluents for the positive polarity mode were eluent A (0.1% formic acid in water) and eluent B (methanol). The eluents for the negative polarity mode were eluent A (5 mM ammonium acetate, pH 9.0) and eluent B (methanol). The solvent gradient was set as follows: 2% B, 1.5 min; 2-100% B, 12.0 min; 100% B, 14.0 min; 100-2% B, 14.1 min; and 2% B, 16.0 min. The Q Exactive HF-X mass spectrometer was operated in a positive/negative polarity mode with a spray voltage of 3.2 kV, a capillary temperature of 320 • C, a sheath gas flow rate of 35 arb, and an aux gas flow rate of 10 arb.

Data Analysis
The raw data files generated by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) were processed using the Compound Discoverer 3.0 (CD3.0, Thermo Fisher) to perform peak alignment, peak picking, and quantitation for each metabolite.
These metabolites were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/), Human Metabolome Database (http://www.hmdb.ca/), and LIPID MAPS database (http:// www.lipidmaps.org/). Principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) were performed using metaX (a flexible and comprehensive software for processing metabolomic data). We applied univariate analysis (t-test) to calculate the statistical significance (P-value). The metabolites with variable importance in the projection (VIP) > 1, P < 0.05, and fold change (FC) ≥2 or ≤0.5 were considered to be differential metabolites. Volcano plots filtered out the metabolites of interest that met the VIP, log 2 (FC), and -log 10 (P-value) criteria.
For hierarchal clustering and heat map generation, the data were normalized using z-scores of the intensity areas of differential metabolites and plotted using the Pheatmap package in R software. The correlation between differential metabolites was analyzed using the cor() function in R (Pearson's method). Statistically significant (P < 0.05) correlations between differential metabolites were calculated using cor.mtest() function in R. Correlation plots were plotted using the corrplot package in R. The functions of these metabolites and metabolic pathways were studied using the KEGG database. The metabolic pathway enrichment analysis of differential metabolites was performed when the ratio was satisfied by x/n > y/N, and the metabolic pathway was considered significantly enriched when P < 0.05.

Statistical Analysis
All data were assessed by ANOVA and Duncan's multiple range test using SPSS 23.0 analysis software at P < 0.05 (IBM Corp., New York, USA). One mouse was selected from each replication as an experimental unit. Data were expressed as mean ± SD.

Determination of Mw and Monosaccharide Composition of MIPs
the MIPs were relatively pure because the polydispersity ratio was close to 1, and the Mw distribution was narrow. Further structural analysis showed that the monosaccharide molar ratios of the MIPs were 47.1:30.4:12.5:10.0 (Table 1).

Infrared Spectrum Analysis of MIPs
Infrared spectroscopy is a powerful technique for analyzing the structure of polysaccharides. From the infrared spectrum analysis (Figure 1), the absorption peaks at 3,100-3,000 cm −1 (C-H stretching vibration), 1,675-1,640 cm −1 (C=C stretching vibration), and 1,000-675 cm −1 (out-of-plane bending vibration of olefin C-H) were ascribed to the double bonds in the MIPs. The strong absorption peaks at 3,100-3,000 cm −1 (C-H stretching vibration on the aromatic ring), unequal absorption (C=C) at 1,600, 1,580, 1,500, and 1,450 cm −1 , and the absorption peaks at 1,000-675 cm −1 indicated that aromatic rings exist in the MIPs. The absorption peaks at 3,650-3,600 cm −1 , 1,300-1,000, and 769-659 characterized the existence of alcoholic hydroxyl and phenol groups. The absorption peaks at 1,750-1,700 cm −1 (C=O) and 2,820 and 2,720 (aldehyde group C-H) depicted the existence of the aldehyde groups. There was also a strong absorption peak at 1,715 cm −1 (keto group). Strong absorption at 3,300-2,500 cm −1 , C=O stretching absorption at 1,720-1,706, C-O stretching absorption at 1,320-1,210 cm −1 , and absorption at 920 cm −1 highlighted the existence of carboxyl groups.

Effects of MIP Administration on the Body and Liver Weights
The body and liver weights were determined to assess the effect of MIP administration following hepatic injury induced by CCl 4 . As shown in Figure 2A, there were no obvious changes in BW among the four groups. However, a significant (P < 0.01) increase in liver weight was observed in the CCl 4 -treated mice compared with the control group ( Figure 2B). HMIP administration significantly decreased liver inflammation and, in turn, liver weight (P < 0.05).

Effect of MIP Administration on Serum Biochemical Parameters
To clarify the effects of MIP administration on the CCl 4induced liver injury markers, we measured the biochemical indices of serum samples. The results (Figure 3) showed that the levels of serum AST, ALT, TG, TC, and TBA were elevated by CCl 4 intoxication. Conversely, pre-treatment with MIPs dosedependently alleviated the increase in AST, ALT, TG, and TBA, with a more significant (P < 0.01) result at a higher dose (200 mg/kg BW).

Effect of MIP Administration on Hepatic Antioxidant Activity
Biomarkers of membrane lipid peroxidation and protein oxidative injury (MDA and PC) were examined. As illustrated in Figures 4A,D, CCl 4 directly induced a significant increase in hepatic MDA and PC contents (P < 0.01), which were restored by MIP administration in a dose-dependent manner (P < 0.01). The activities of SOD and CAT in the experimental animals are shown in Figures 4B,C. Compared with the control group, the acute administration of CCl 4 to mice induced characteristic hepatotoxicity that affected hepatic antioxidant parameters, as indicated by a significant decrease in SOD and CAT levels (P < 0.01). These CCl 4 -induced declines were markedly attenuated by pre-treatment with LMIPs and HMIPs (P < 0.01).

Effect of MIP Administration on Hepatic Inflammation
We also assessed the effect of the MIP administration on the production of proinflammatory mediators (i.e., IL-1β, IL-6, and TNF-α) after CCl 4 intoxication. In this study, IL-1β (Figure 5A), IL-6 ( Figure 5B), and TNF-α ( Figure 5C) levels were markedly increased in the CCl 4 group. MIP administration attenuated the production of these cytokines in a dose-dependent manner. Additionally, MPO activity, the marker of neutrophilic infiltration, was assayed to evaluate the hepatic injury. CCl 4 intoxication led to an evident accumulation of MPO in the liver (Figure 5D). In contrast, MIP administration resulted in a dose-dependent diminution of MPO, with a more significant effect at a higher dose (200 mg/kg).

Histopathological Detection
In the control group, structurally ordered tissues and hepatocytes with prominent nuclei and uniform cytoplasm were observed under the optical microscope. Acute administration of CCl 4 induced substantial liver damage, as evidenced by massive  hepatocyte necrosis with inflammatory cell infiltration and hepatocyte ballooning degeneration. However, the abnormal status was ameliorated by the HMIP administration. Hepatic architecture distortion was relieved, as indicated by decreased cellular necrosis, inflammatory infiltration, and ballooning degeneration (Figure 6).

Metabolic Profiling Comparison Among the Control, CCl 4 , and HMIP Groups
The PCA and PLS-DA were used to study the differences in serum metabolite profiles among the control, CCl 4 , and HMIPs groups. The PCA score plots of the serumbased LC-MS data showed an obvious distinction among the three groups (Figure 7). The PLS-DA score plot for the three groups revealed visible separations of the serum metabolites (Figure 8), indicating that HMIP administration could partly regulate the metabolic disorders caused by CCl 4 .

Effect of HMIP Administration on the Metabolic Profiles
In the PLS-DA score plot, variables that significantly contributed to the clustering and discrimination were identified according to a VIP threshold of ≥1.0. When the P-value was lower than 0.05, the compounds were screened as candidate differential metabolites for one-way ANOVA analysis. The results were visualized in the form of a volcano plot (Figure 9). There were 9 differential metabolites between the control and CCl 4 groups and 10 differential metabolites between HMIP and CCl 4 groups (Tables 2, 3). For hierarchal clustering and heat map generation, the data were calculated and plotted in R software. It showed that the differential metabolite profiles differed among the three groups (Figure 10). These results indicate that HMIPs effectively prevent CCl 4 -induced liver injury via mitigating the altered serum metabolite profiles and regulating metabolic abnormalities.

Enrichment Analysis of Metabolic Pathway
The metabolic pathway analysis (MetPA) of the candidate metabolites in Tables 2, 3 was carried out to explore the relevance of the pathways linked to the differential metabolites. The bubble map of MetPA indicated that the dramatically disturbed metabolic pathways between the control and CCl 4 groups included flavonoid and isoflavonoid biosynthesis; steroid biosynthesis; alanine, aspartate, and glutamate metabolism; nicotinate and nicotinamide metabolism; and benzoate and aminobenzoate degradation (Figure 11). Furthermore, there were five such metabolic pathways between the HMIP and CCl 4 groups, namely, isoflavonoid biosynthesis, tryptophan metabolism, phenylalanine metabolism, naphthalene degradation, and benzoate and aminobenzoate degradation (Figure 12).

DISCUSSION
Finding natural sources of hepatoprotective medicines is of keen research interest in ensuring liver protection and thereby its roles in regulating numerous physiological processes, including metabolism, synthesis, and detoxification (Bing-Ya et al., 2015;Lu et al., 2016;Yang et al., 2019). M. importuna mushrooms are rich sources of many bioactive components, such as polysaccharides, dietary fiber, and proteins (Gursoy et al., 2009). In particular, polysaccharides from M. importuna have shown multiple pharmacological and biological activities, including antioxidant, antimicrobial, antiviral, and hepatoprotective activities (Heleno et al., 2013). However, how MIPs confer protection against oxidative injury remains unknown. The animal model of acute liver injury induced by CCl 4 is one of the classical models used to screen antioxidative medicines. Therefore, we aimed to evaluate the suitability as potent medicinal foods by examining the effects of LMIPs and HMIPs on CCl 4 -induced acute liver damage in mice. Weight loss after CCl 4 intoxication is considered to be caused by the direct toxicity of CCl 4 or the indirect toxicity associated with liver damage (Yang et al., 2010). However, BW was not decreased by CCl 4 intoxication and marginally increased by MIP administration in this study. Marked swelling of the CCl 4treated liver occurred, as evidenced by the enhancement of liver weight. In comparison, a significant decrease in liver weight was observed in all mice infused with HMIPs. Clinical assessment of liver injury involves measurement of the ALT, AST, TG, TC, and TBA, which in part, reflect the degree of hepatocyte damage and necrosis. This study demonstrated that the levels of these markers were elevated by CCl 4 intoxication. MIPs inhibited the leakage of these enzymes from the liver tissue into the bloodstream. CCl 4 is decomposed into numerous free radicals, such as trichloromethyl, after its metabolization by cytochrome P450 in hepatocytes (He et al., 2016). Due to the free radical-mediated destruction and increased permeability of the cellular membrane structure, high levels of ALT and AST enzymes infiltrate into the bloodstream, significantly promoting the activity of these enzymes in the serum. Concurrently, the trichloromethyl peroxyl radical is generated by the reaction of trichloromethyl radical with molecular oxygen and can attack liver microsomal lipid and hepatocyte membrane phospholipid molecules, leading to the initiation of the lipid peroxidation reaction (Bruinsma et al., 2015;Shi-Bin et al., 2015). High levels of TG and TC were deposited in hepatocytes, which was concomitant with an increase in the serum levels of TG and TC (Lykkesfeldt, 2007).
Lipid peroxidation resulting from oxidative stress produces a large amount of MDA, which can also contribute to hepatocyte damage, thereby aggravating the lesions (Tsikas, 2017). PC is the product of damage to amino acid side chains by oxygenfree radicals. With the aggravation of oxidative stress, the protein carbonylation becomes more severe, which impedes protein folding and promotes protein-protein cross-linking. Thus, PC is considered an effective index to evaluate the degree of protein oxidation. In contrast, SOD and CAT are important antioxidant enzymes, responsible for scavenging free radicals and inhibiting lipid peroxidation, thus protecting the hepatocytes against CCl 4 -mediated toxicity (Dmitry et al., 2017). In this study, the excessive increase in the hepatic MDA and PC contents in the CCl 4 group were markedly attenuated by HMIP administration. Furthermore, MIPs dosedependently alleviated the significantly decreased concentration of SOD caused by CCl 4 intoxication. Hence, MIP administration preserved hepatic health by attenuating the oxidative damage in the liver.
In addition, the inflammatory response observed during CCl 4 -induced hepatic damage may be accompanied by the release of several cytokines (i.e., IL-1, IL-6, and TNF-α), contributing to a degenerative scenario (Sha et al., 2020). The MPO activity serves as a marker for inflammatory cell infiltration and indicates the degree of inflammation . Our data showed a dramatic increase in the levels of IL-1β, IL-6, TNF-α, and MPO by CCl 4 intoxication. However, MIP administration led to a marked decrease in pro-inflammatory cytokines and MPO compared with the CCl 4 group. Moreover, H&E staining confirmed that CCl 4 intoxication induced acute liver damage, as indicated by vacuolation, the ballooning of hepatocytes, and inflammatory cell infiltration. HMIP administration mitigated these detrimental injuries, which further supported the aforementioned hepatoprotective effects. These results supported that MIPs possess potent hepatoprotective activity by inhibiting oxidative stress and inflammation.
Based on the more favorable inhibitory effects of HMIPs over LMIPs on CCl 4 -induced liver injury, a UHPLC-MS/MSbased metabolomic analysis was performed to evaluate the protective effect of HMIPs against CCl 4 -induced hepatic damage. The observed trends in serum biomarkers following CCl 4 intoxication or HMIP administration are shown in Tables 1, 2. These metabolites were mainly involved in flavonoid biosynthesis, amino acid metabolism, energy metabolism, and toxicant degradation. Numerous studies have indicated multifunctional flavonoids isolated from Chinese medicines and plants as potential therapeutic agents for the improvement of the liver tissue via antioxidant, anti-inflammatory, and antifibrotic mechanisms (Gou et al., 2020;Wu et al., 2020a). Serum metabolomic analysis revealed that metabolites (i.e., isoliquiritigenin and prunin) related to flavonoid biosynthesis were significantly reduced by CCl 4 intoxication. However, the ratio of prunin between the HMIP and CCl 4 groups was lower than 1.0, perhaps indicating that HMIP administration did not affect flavonoid biosynthesis.
The liver is a vital organ in regulating amino acid metabolism, and any dysfunction in the liver could cause a disturbance in the amino acid concentrations (Waters et al., 2005). The decreased ratio of γ-aminobutyric acid observed between the control and CCl 4 groups in this study indicated that CCl 4 infusion might disturb the alanine, aspartate, and glutamate metabolism because γ-aminobutyric is one of the metabolites in this pathway. Tryptophan is metabolized along the kynurenine and serotonin pathways, while phenylacetylglycine participates in phenylalanine metabolism (Kamiguchi et al., 2017;Li et al., 2017). In this study, amino acids in the serum were promoted by CCl 4 intoxication, perhaps because of decreased protein synthesis and increased protein catabolism (Guo et al., 2017). CCl 4 intoxication can disturb methylation pathways such as the hypomethylation of ribosomal RNA, leading to defective protein synthesis (Clawson et al., 1987). Moreover, protein catabolism plays an important role in regulating lipid and glucose metabolism, which could induce cytokine secretion under abnormal conditions (Sinha et al., 2020).

Trigonelline
(N-methylnicotinic acid) and 6hydroxynicotinic acid are intermediates of nicotinate and nicotinamide metabolism, respectively. Nicotinamide has been used as a metabolic precursor of nicotinamide adenine dinucleotide (NAD + ) biosynthesis to raise the intracellular NAD + pool for improvement of metabolic diseases involving glycolysis (French, 2015;Wan et al., 2019). Trigonelline showed a hepatoprotective effect against non-alcoholic fatty liver diseases by reducing oxidative stress and pro-apoptosis . In this study, we discovered that trigonelline was impeded by CCl 4 intoxication as evidenced by the ratio between the control and CCl 4 groups ( Table 1). However, the reduced levels of trigonelline could not be reversed by HMIP infusion. Additionally, it was reported that some commonly used medications, such as p-aminobenzoate, an antifibrotic agent, may unpredictably be the underlying cause of liver injury (Al Attar and Kilgore, 2018). In our study, catechol, a strong toxicant with carcinogenic properties, was decreased by HMIP administration. Thus, we speculated that HMIPs might beneficially facilitate toxicant degradation to protect the liver tissues. In brief, HMIPs showed hepatoprotective activity against liver injury by affecting amino acid metabolism, energy metabolism, and toxicant degradation. However, the underlying mechanisms for these findings remain unclear and require elucidation.

CONCLUSION
Taken together, our findings demonstrated that MIP administration alleviated CCl 4 -induced hepatic injury by promoting antioxidant and anti-inflammatory activities. Furthermore, the metabolomic analysis showed that HMIPs regulate the pathways of amino acid metabolism, energy metabolism, and toxicant degradation to subvert the disturbances induced by CCl 4 . This study provides a scientific basis for MIPs as a curative treatment for hepatic injury.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by the Ethics Review Committee for Animal Experimentation of Sichuan Academy of Agricultural Sciences.

AUTHOR CONTRIBUTIONS
YX, BG, and WP conceived and designed the experiments. YX, LX, JT, and YC conducted the experiments. YX, XH, ZZ, and JZ analyzed the data. YX and WP wrote the article. All authors have read and agreed to the published version of the manuscript.