Protective Effect of Artocarpus heterophyllus Lam. (Jackfruit) Polysaccharides on Liver Injury Induced by Cyclophosphamide in Mice

In recent years, Artocarpus heterophyllus Lam. (jackfruit) polysaccharides (namely JFP-Ps) have attracted much attention due to their multiple biological activities. This study aimed to explore the protective effects and the underlying mechanisms of JFP-Ps on cyclophosphamide (Cp)-induced liver damage. The protective effect of JFP-Ps was evaluated using HE staining, antioxidant testing, enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (RT-qPCR), Western blot and ultra-performance liquid chromatography equipped with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) metabolomics analysis. The results showed that Cp caused pathological liver damage, activated oxidative stress and downregulated cytokine expression, while JFP-Ps treatment was found to exert antioxidant effects and play immune regulatory roles through mitogen-activated protein kinase/nuclear factor-κB (MAPK/NF-κB) related inflammation and cell apoptosis pathways to protect the Cp-induced liver injury. Metabolomic results showed that the liver-protective effects of JFP-Ps were mainly related to aminoacyl transfer ribonucleic acid (tRNA) biosynthesis, sphingolipid metabolism, purine metabolism and the citrate cycle. These results indicate that JFP-Ps have great potential application in alleviating liver injury.


Introduction
The liver is an important metabolic organ and is responsible for the synthesis, decomposition, transformation and excretion of many molecules and other metabolic processes [1].Therefore, the liver is the main target of drugs and toxins and is damaged through drugassociated adverse effects [2].Cyclophosphamide (Cp), a cell cycle non-specific drug, is widely used in antitumor therapy due to its ability to inhibit the proliferation of cancer cells [3].However, Cp is hepatotoxic and can damage normal hepatocytes.It has been reported that Cp can be metabolized into active substances, such as phosphoramidic mustard and acrolein, under the action of cytochrome P450, which catalyzes the alkylation reaction between these metabolites and the guanine base of DNA [4], inhibiting the synthesis of DNA and RNA and causing DNA damage, leading to oxidative stress and cytotoxic effects [5].
Polysaccharides are natural polymer compounds that are widespread in animals, plants and microorganisms.Some exhibited antioxidant, anti-inflammatory, immune regulation and liver protection activities and have become a research hotspot in various

Histopathological Observation
The liver samples were fixed in 4% paraformaldehyde and then dehydrated with alcohol, embedded in paraffin, cut into 4 µm slices and stained with hematoxylin and eosin (H&E).The samples were observed under a light microscope (Nikon, Tokyo, Japan) and photographed.

Determination of Biochemical Indicators
Liver samples were homogenized in phosphate-buffered saline (PBS, pH 7.4) and centrifuged at 2500× g for 20 min to collect supernatants for further determination of the contents of MDA, TNF-α, IL-6, IL-2 and IFN-γ, as well as the activities of SOD, CAT and GSH-Px, according to the manufacturer's instructions.

Real-Time Quantitative PCR
Total RNA in the liver was extracted with Trizol and cDNA was prepared using BeyoRT TM III first-strand synthesis kit (Beyotime Biotechnology Co., Ltd., Shanghai, China).A three-step approach was used for target genes amplification.Relative mRNA expression level was calculated using the 2 −∆∆Ct method, with β-actin used for normalization.The primer information is listed in Table 1.
Mass spectrometry (MS) data were obtained using an Agilent 6530B Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) in positive and negative electrospray ionization (ESI) modes.The drying gas temperature was 325 • C; wavelength was 250 nm; cone voltage was 65 V; attenuation was 1000 mAU; draw speed was 100 µL/min; eject speed was 400 µL/min; MS scan range was from 50 to 1200 m/z; primary mass spectrometry scan was 2 spectra/s and MS scan time was 4 spectra/s.

Statistical Analysis
Results are presented as the mean ± SEM.Data were analyzed using one-way ANOVA followed by Duncan's multiple range test using SPSS Statistics 26 (IBM Inc., Armonk, New York, NY, USA).A p value < 0.05 indicated a significant difference.
The UPLC-Q-TOF-MS data were collected using Masshunter (Agilent Technologies, Inc.), processed using Profinder (Agilent Technologies, Inc.) and normalized and filtered using Mass Profiler Professional (MPP, Agilent Technologies, Inc.).Principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) were used to identify the differences in metabolites of different groups based on FC (Fold change) > 2 and p < 0.05.The identified metabolites were determined through searching databases and confirmed using UPLC-Q-TOF-MS/MS.

Effects of JFP-Ps on the Histopathology of the Liver
Histopathological examination shows that Cp causes liver damage.As shown in Figure 1, the hepatocytes in the NC group show normal histological structure.However, in the MC group, hepatocytes are scattered and structurally abnormal, with necrotic hepatocytes having small fragmented nuclei and inflammatory-infiltrating hepatocytes.A large number of fat droplets were deposited in the portal vein, and the small spaces in the portal vein became congested and inflamed.After treatment with different doses of JFP-Ps, the morphology and structure of the liver cells were alleviated to varying degrees compared to those of the MC group.Inflammation and cavitation were restored, and portal vein fat deposition was reduced.

Effect of JFP-Ps on Oxidative Stress in the Liver
The effects of JFP-Ps on oxidative stress were studied through measuring the level of MDA and the activities of SOD, CAT and GSH-Px in the liver homogenate.As shown in Table 2, compared with the NC group, the content of MDA in the MC group was significantly increased (p < 0.05), while the activities of SOD, CAT and GSH-Px were significantly decreased (p < 0.01).The contents of MDA in the liver homogenate of the JFP-Ps groups were lower than those of the MC group, but there were no significant differences (p > 0.05).The activities of SOD, CAT and GSH-Px tended to increase compared to those in the MC group (p < 0.05).These results suggest that JFP-Ps may regulate oxidative stress responses and attenuate liver damage.

Effect of JFP-Ps on Oxidative Stress in the Liver
The effects of JFP-Ps on oxidative stress were studied through measuring the level of MDA and the activities of SOD, CAT and GSH-Px in the liver homogenate.As shown in Table 2, compared with the NC group, the content of MDA in the MC group was significantly increased (p < 0.05), while the activities of SOD, CAT and GSH-Px were significantly decreased (p < 0.01).The contents of MDA in the liver homogenate of the JFP-Ps groups were lower than those of the MC group, but there were no significant differences (p > 0.05).The activities of SOD, CAT and GSH-Px tended to increase compared to those in the MC group (p < 0.05).These results suggest that JFP-Ps may regulate oxidative stress responses and a enuate liver damage.

Effect of JFP-Ps on the Level of Cytokines in the Liver
The level of cytokines in the liver homogenate was measured using ELISA.Compared with the NC group, the level of IL-6 was significantly decreased in the MC group (p < 0.05) and the levels of IL-2 and TNF-α also decreased in the MC group, but there was no significant difference (p > 0.05, Table 3).The level of IFN-γ in the MC group was higher than that in the NC group (p < 0.05).After intervention with different doses of JFP-Ps, the levels of IL-2, IL-6, TNF-α and IFN-γ were reversed to varying degrees compared with those in the MC group.As shown in Figure 2, compared with the NC group, the expression levels of IFNγ and IL-2 mRNA in the MC group were significantly increased (p < 0.01), while the expression levels of TNF-α, IL-6 and IL-10 mRNA were significantly reduced (p < 0.05).After treatment with JFP-Ps at different doses, the mRNA expression levels of IL-2 and IFN-γ were significantly decreased compared to those in the MC group (p < 0.05), while the mRNA expression levels of TNF-α, IL-6 and IL-10 were significantly increased compared to those in the MC group (p < 0.05).The increased expression of IFN-γ and IL-2 mRNA and the decreased expression of IL-10 mRNA in the MC group indicate that liver tissue may be damaged via inflammation.
In addition, the mRNA expression levels of p65, p38 and JNK proteins were measured.The results showed that their mRNA expression levels in the MC group were significantly downregulated (p < 0.05) compared with those in the NC group.After the JFP-Ps intervention, the mRNA expression levels were restored compared to those in the MC group (p < 0.05).
group (p < 0.05) and the levels of IL-2 and TNF-α also decreased in the MC group, but there was no significant difference (p > 0.05, Table 3).The level of IFN-γ in the MC group was higher than that in the NC group (p < 0.05).After intervention with different doses of JFP-Ps, the levels of IL-2, IL-6, TNF-α and IFN-γ were reversed to varying degrees compared with those in the MC group.As shown in Figure 2, compared with the NC group, the expression levels of IFN-γ and IL-2 mRNA in the MC group were significantly increased (p < 0.01), while the expression levels of TNF-α, IL-6 and IL-10 mRNA were significantly reduced (p < 0.05).After treatment with JFP-Ps at different doses, the mRNA expression levels of IL-2 and IFN-γ were significantly decreased compared to those in the MC group (p < 0.05), while the mRNA expression levels of TNF-α, IL-6 and IL-10 were significantly increased compared to those in the MC group (p < 0.05).The increased expression of IFN-γ and IL-2 mRNA and the decreased expression of IL-10 mRNA in the MC group indicate that liver tissue may be damaged via inflammation.
In addition, the mRNA expression levels of p65, p38 and JNK proteins were measured.The results showed that their mRNA expression levels in the MC group were significantly downregulated (p < 0.05) compared with those in the NC group.After the JFP-Ps intervention, the mRNA expression levels were restored compared to those in the MC group (p < 0.05).

Effect of JFP-Ps on Protein Expression of the Proteins Involved in Inflammation-Related Pathways in the Liver
The results of the Western blot showed that JFP-Ps intervention downregulated the expression levels of p-p65/p65 protein and phosphorylated p-p38/p38 protein (p < 0.05), while upregulating IκB-α protein and p-JNK/JNK protein expression levels (p < 0.05) (Figure 3).These results indicate that JFP-Ps can regulate the MAPK apoptosis pathway and inhibit the NF-κB/p65 inflammatory pathway to protect the liver.

Effect of JFP-Ps on Protein Expression of the Proteins Involved in Inflammation-Related Pathways in the Liver
The results of the Western blot showed that JFP-Ps intervention downregulated the expression levels of p-p65/p65 protein and phosphorylated p-p38/p38 protein (p < 0.05), while upregulating IκB-α protein and p-JNK/JNK protein expression levels (p < 0.05) (Figure 3).These results indicate that JFP-Ps can regulate the MAPK apoptosis pathway and inhibit the NF-κB/p65 inflammatory pathway to protect the liver.

Effect of JFP-Ps on Protein Expression of the Proteins Involved in Inflammation-Related Pathways in the Liver
The results of the Western blot showed that JFP-Ps intervention downregulated the expression levels of p-p65/p65 protein and phosphorylated p-p38/p38 protein (p < 0.05), while upregulating IκB-α protein and p-JNK/JNK protein expression levels (p < 0.05) (Figure 3).These results indicate that JFP-Ps can regulate the MAPK apoptosis pathway and inhibit the NF-κB/p65 inflammatory pathway to protect the liver.UPLC-Q-TOF-MS was used to collect data from the liver extract (Figure 4).The peak numbers, positions, and intensities of the five different treatment groups showed significant differences, indicating significant changes in the types and quantities of metabolites in the liver samples from different groups.Quality control was conducted on

Metabolic Profile of UPLC-Q-TOF-MS in the Liver
UPLC-Q-TOF-MS was used to collect data from the liver extract (Figure 4).The peak numbers, positions, and intensities of the five different treatment groups showed significant differences, indicating significant changes in the types and quantities of metabolites in the liver samples from different groups.Quality control was conducted on the obtained primary data using t-test (Table 4).A total of 227 substances were found in the positive mode (ESI+) (p < 0.05), while 398 substances were found in the negative mode (ESI−) (p < 0.05).The contents of these substances in the treatment groups were visualized using heat maps.

Multivariate Statistical Analysis
The PCA and OPLS-DA showed good intergroup differences and intragroup clustering among liver metabolites in different treatment groups (Figure 5).The R 2 and Q 2 values of the PCA model in ESI+ were 0.544 and 0.335, respectively, while the R 2 X, R 2 Y and Q 2 values of the OPLS-DA model were 0.527, 0.894 and 0.718, respectively.The R 2  To make the results more reliable, OPLS-DA was used for analysis.As shown in the OPLS-DA map, although the separation between the LG and MG groups and the MC group was still not significant in ESI+, there were significant intergroup differences and intragroup clustering in ESI−.These results indicate that there are certain differences in metabolites among the different groups.The metabolites in the liver under the Cp induced liver injury group were different from those in the normal liver group and could be regulated through JFP-Ps intervention.High doses of JFP-Ps may have a significant impact on the changes in these liver metabolites.

Identification of Metabolites
All detected metabolites were screened for differential metabolites based on FC (Fold change) > 2 and p < 0.05 (Figure 6).It was found that there were 92 differential metabolites in ESI+ and 154 in ESI− between the NC and MC groups.There were 52 differential compounds in ESI+ and 81 in ESI− between the MC and LG groups, 59 differential compounds in ESI+ and 92 in ESI− between the MC and MG groups and 91 differential compounds in ESI+ and 113 in ESI− between the MC and HG groups.Through analyzing these differential compounds using a Veen map, it was found that there were seven common differentials in each group in ESI+, 42 unique differentials in NC vs. MC, four unique differentials in MC vs. LG, eight unique differentials in MC vs. MG and 28 unique differentials in MC vs. HG; In ESI−, there were 12 common differences in each group, 83 unique differences in NC vs. MC, eight unique differences in MC vs. LG, 10 unique differences in MC vs. MG and 22 unique differences in MC vs. HG.These results indicate that the liver metabolites are significantly altered in Cp-induced liver injury mice, and JFP-Ps intervention can improve metabolic disorders in the liver caused by Cp.The MS/MS information was extracted, and the differential metabolites were further confirmed using the METLIN and HMDB databases.A total of 31 differential metabolites were identified in the liver of mice with Cp-induced liver injury in both ESI+ and ESI− (Tables 5 and 6).These substances mainly include amino acids, spermidine, niacinamide, xanthine, hypoxanthine, adenine, deoxyguanosine, glutathione, sphinganine, sphingosine, phytosphingosine, glucosylceramide, indole-3-carboxaldehyde and succinic acid.

Metabolic Pathway Analysis
Metabolic pathway analysis was conducted on the screened differential metabolites.As shown in Figure 7 and Table 7, the metabolic pathways in the liver related to JFP-Ps intervention in Cp-induced liver injury mice mainly include aminoacyl tRNA biosynthesis, sphingolipid metabolism, purine metabolism, glutathione metabolism, arginine biosynthesis, arginine and proline metabolism and the citrate cycle.

Metabolic Pathway Analysis
Metabolic pathway analysis was conducted on the screened differential metabolites.As shown in Figure 7 and Table 7, the metabolic pathways in the liver related to JFP-Ps intervention in Cp-induced liver injury mice mainly include aminoacyl tRNA biosynthesis, sphingolipid metabolism, purine metabolism, glutathione metabolism, arginine biosynthesis, arginine and proline metabolism and the citrate cycle.

Discussion
Cp induced hepatotoxicity including liver necrosis, inflammation and oxidative damage.MDA is the final product of lipid peroxidation; SOD is an antioxidant enzyme that can promote the disproportionation of superoxide into hydrogen peroxide to resist oxygen free radicals; and GSH-Px and CAT can protect cell structure and membrane function via catalyzing the decomposition of hydrogen peroxide [12].The present study showed that the MDA level in the MC group was increased, while the activities of SOD, CAT and GSH-Px were decreased.However, the oxidative stress indicators in the JFP-Ps groups were alleviated and approached those of the NC group, which may be related to the free radical scavenging activity of JFP-Ps.
The inflammatory cytokines reflect the immune state to a certain extent and play an essential role in the immune response [13].TNF-α is a multifunctional cytokine that stimulates the expression of a series of inflammatory mediators to regulate inflammation [14].IFN-γ is mainly secreted by natural killer cells and has antiviral, immune regulation and other active functions [15].IL-2 is a necessary cytokine for T cell proliferation and has an inductive effect on cells and memory cell generation [16].IL-6 plays a key regulatory function in immunity and is involved in the development, maturation, and sustained antibody production of B cells [17].It has been reported that Lycium ruthenicum Murr.polysaccharide enhanced serum cytokine expression in immunocompromised mice [18], which is consistent with our results, suggesting that JFP-Ps may exert regulatory effects on inflammation and immune function through regulating the secretion of these cytokines in the liver of Cp-exposed mice.
Liver damage is induced via the release of inflammatory cytokines, activation of apoptosis pathway, etc. [19].The NF-κB signaling pathway is related to immunity, inflammation and cell apoptosis [20].Cui et al. [21] reported that the polysaccharides from Caulis spatholobi had a protective effect mainly through regulating NF-κB signaling pathway in the intestinal mucosa of Cp-exposed chickens.The MAPK family, including ERK, JNK and p38, plays an important role in regulating cell apoptosis, cell cycle, cell growth inhibition and differentiation, as well as in mediating autophagy [22].The activation of p38 signaling has been shown to induce the expression of pro-inflammatory cytokines, while JNK signaling under stress stimulation can induce inflammation or cell apoptosis [23].The present study found that the liver damage was associated with the degradation of IκB-α protein and activation of the NF-κB inflammatory pathway, while JFP-Ps exerted a protective effect on the liver through inhibiting the p-p65/p65 and p-p38/p38 pathways and activating the p-JNK/JNK pathway.
Aminoacyl tRNA is the substrate for translation and is synthesized via matching amino acids with tRNA containing corresponding anticodons through aminoacyl tRNA synthase (ARS) [24].ARS participates in translation and serves as a signaling molecule in the development of immune cells in various immune diseases to mediate immune responses [25].Inflammatory signals can induce the phosphorylation of ARS and regulate the cascade reaction of various cytokines and MAPK and other cellular signaling pathways [26].Ascophyllum nodosum polysaccharide has been reported to inhibit the progression of inflammation mainly through regulating inflammation-related signals, including phenylalanine, tryptophan biosynthesis, and aminoacyl tRNA biosynthesis [27].Consistent with a previous study, JFP-Ps treatments altered the metabolism of various amino acids, including histidine, phenylalanine, methionine, isoleucine, lysine, proline and tryptophan, which are mainly related to the aminoacyl tRNA biosynthesis.
Arginine serves as a component of protein synthesis, and acts as a metabolic substrate for immune cells [28].Arginine can be metabolized to nitric oxide (NO) by nitric oxide synthase (NOS), and the abnormal synthesis of NO can induce tissue damage, while arginase could act as a competitive enzyme to compete with arginine to prevent the generation of NO [29].Arginine can modulate innate immune responses through modulating the MAPK signaling pathway [30].Our results show that the metabolic product of arginine biosynthesis is imbalanced in the liver of Cp-exposed mice.
Sphingolipids are common components of eukaryotic cell membranes.Extracellular stimuli, such as cytokines and cellular stress, can disrupt normal cell homeostasis and disrupt sphingolipid metabolism.Ceramide and sphingosine are metabolites of sphingolipids that act as key signaling molecules in immunity and inflammation [31].Changes in ceramide level may affect the interaction between lipids and proteins within the membrane, thereby affecting the transmission of intracellular signals [32].Polysaccharides obtained from Suanzaoren decoction have been reported to reduce the concentrations of phytosphingosine, sphingosine and ceramide, inducing neuronal cell death as a mechanism of immune deficiency [33].An abnormal sphingolipid metabolism was found in Cp-exposed mice, and JFP-Ps attenuated the expression of sphingosine, sphinganine, phytosphingosine and ceramide in the present study.
In general, purine nucleotides are regenerated through recycling pathways by hypoxanthine guanine phosphoribosyltransferase and adenine phosphoribosyltransferase [34].Hypoxanthine can be oxidized by xanthine oxidase to form xanthine and guanine, which in turn can respectively generate hypoxanthine mononucleotide and guanylate to fulfill the purine requirements within cells [35].We analyzed the metabolites (xanthine, hypoxanthine, adenine, guanosine and inosine) involved in purine metabolism.It was confirmed that Cp treatment caused oxidative damage to mouse liver, leading to an imbalance in purine metabolism, and JFP-Ps were beneficial for restoring this damage.
Glutathione (GSH) is a tripeptide mainly synthesized in the liver and acts as an antioxidant [36].In addition, GSH is essential for activating T lymphocytes and multiple signaling pathways, including NF-κB, p38 and JNK [37].The citric acid (TCA) cycle has emerged as an energy metabolism hub and a regulator of immune responses in most eukaryotes.Mitochondria are critically involved in cell proliferation, death, differentiation and immunity via driving macrophage polarization through IL-6, and via activating mitochondrial signaling through inflammatory factors like TNF-α [38].Succinate, which is formed in the TCA cycle, accumulates under inflammatory or stress conditions, and is involved in macrophage activation [39].Fumarate is considered anti-inflammatory, and its degradation is detrimental to the host and abrogates trained immunity [39].In the present study, JFP-PS attenuated liver injury, which may be associated with the TCA cycle.

Conclusions
JFP-Ps have a hepatoprotective effect on Cp-induced liver injury via inhibiting peroxidative damage and regulating the expression of related genes.UPLC-Q/TOF-MS/MSbased metabolomics results showed that the liver-protective effects of JFP-Ps were mainly related to aminoacyl tRNA biosynthesis, sphingolipid metabolism, purine metabolism, glutathione metabolism, arginine biosynthesis, arginine and proline metabolism and the citric acid cycle.The results of this Cp model indicate that JFP-Ps may have the potential to alleviate liver injury.Whether this effect can be transferred to other liver noxae and whether it can be applied to Cp therapy without impeding its therapeutic goals, needs to be evaluated in further studies.
Institutional Review Board Statement: All animal care and procedures were strictly conducted according to the protocols approved by the Animal Ethical Committee of Hainan Medical University (Permit # HYLL-2023-462).
Informed Consent Statement: Not applicable.
and Q 2 values of the PCA model in ESI− were 0.531 and 0.345, respectively, while the R 2 X, R 2 Y and Q 2 values of the OPLS-DA model were 0.51, 0.938 and 0.75, respectively.The results suggest that the interpretability and predictability of the model were acceptable.The OPLS-DA model was tested for 200 times using displacement tests.The R 2 Y and Q 2 Y values of ESI+ were 0.576 and −0.394, respectively, and the R 2 Y and Q 2 Y values of ESI− were 0.656 and −0.36, respectively, indicating that the model did not overfit and the results are reliable.The PCA maps show a good separation between the NC group and the MC group.

Figure 6 . 6
Figure 6.Volcano map (A: NC vs. MC in ESI+, B: NC vs. MC in ESI−, C: MC vs. LG in ESI+, D: MC vs. LG in ESI−, E: MC vs. MG in ESI+, F: MC vs. MG in ESI−, G: MC vs. HG in ESI+, H: MC vs. HG in ESI−) and Veen map ((I) ESI+; (J) ESI−).Red: Metabolites which pass both log2 Fold Change > 1.0 and p < 0.05 cut-offs and are up-regulated.Blue: Metabolites which pass both log2 Fold Change < −1.0 and p < 0.05 cut-offs and are down-regulated.Green: Metabolites which pass the p < 0.05 cut-off and fail to pass the |log2 Fold Change| > 1.0 cut-off.Grey: Metabolites which neither pass the p < 0.05 cut-off nor the |log2 Fold Change| > 1.0 cut-off

3 EFigure 7 .
Figure 7. Pathway analysis.(A) metabolic pathway diagram; (B) Aminoacyl-tRNA biosynthesis; (C) Sphingolipid metabolism; (D) Purine metabolism; (E) Glutathione metabolism.The colors from yellow to red represent metabolites with different level of significance, red being more significant and yellow less significant respectively.The compounds highlighted in red font represent the metabolic markers in the metabolic pathways.

Table 1 .
The primer information in RT-qPCR.

Table 2 .
Effects of JFP-Ps on the content of MDA and activities of SOD, CAT and GSH-Px.

Table 2 .
Effects of JFP-Ps on the content of MDA and activities of SOD, CAT and GSH-Px.GroupMDA (nmol/g) SOD (U/g) CAT (µmol/min/g)

Table 3 .
Effects of JFP-Ps on the levels of cytokines.

Table 3 .
Effects of JFP-Ps on the levels of cytokines.

Table 4 .
Results of data quality control using t-test.

Table 5 .
The significant metabolites in the liver (ESI+).

Table 5 .
The significant metabolites in the liver (ESI+).

Table 5 .
The significant metabolites in the liver (ESI+).

Table 5 .
The significant metabolites in the liver (ESI+).

Table 5 .
The significant metabolites in the liver (ESI+).

Table 5 .
The significant metabolites in the liver (ESI+).

Table 5 .
The significant metabolites in the liver (ESI+).

Table 5 .
The significant metabolites in the liver (ESI+).

Table 5 .
The significant metabolites in the liver (ESI+).

Table 5 .
The significant metabolites in the liver (ESI+).

Table 5 .
The significant metabolites in the liver (ESI+).

Table 6 .
The significant metabolites in the liver (ESI−).

Table 6 .
The significant metabolites in the liver (ESI−).

Table 6 .
The significant metabolites in the liver (ESI−).

Table 6 .
The significant metabolites in the liver (ESI−).

Table 6 .
The significant metabolites in the liver (ESI−).

Table 6 .
The significant metabolites in the liver (ESI−).

Table 6 .
The significant metabolites in the liver (ESI−).

Table 6 .
The significant metabolites in the liver (ESI−).

Table 6 .
The significant metabolites in the liver (ESI−).

Table 6 .
The significant metabolites in the liver (ESI−).

Table 6 .
The significant metabolites in the liver (ESI−).

Table 6 .
The significant metabolites in the liver (ESI−).