Yuzhen Hong, Mingyue Shen, Lixin Huang, Ting Wu, Jianhua Xie*

State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China

Keywords:

Gut microbiota

Liver damage

Oxidative stress

Polysaccharide

A B S T R A C T

There are a number of health benefits of Mesona chinensis Benth polysaccharide (MP), but little is known about its hepatoprotective effect and effect on gut microbiota composition in mice with liver damage induced by cyclophosphamide (CTX).This study indicated that MP supplementation effectively inhibited the production of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT), enhanced liver antioxidant capacity and repaired liver damage in mice caused by CTX.The release of inflammatory cytokines in liver and the concentration of lipopolysaccharide (LPS) in serum were decreased, and the level of short chain fatty acids (SCFAs) in colon was increased after MP administration.Those effects may be correlated with the regulation of the gut microbiota.Importantly, MP restrained liver inflammatory responses induced by CTX may via increasing the SCFAs-producing bacteria family Ruminococcaceae and reduced LPS-producing bacteria genus Bacteroides.In short, the prevention of CTX-induced liver injury by supplementing MP is achieved at least in part by regulating the community structure of the gut microbiota, and MP is expected to be a potential prebiotic to treat and prevent liver diseases.

1.Introduction

Cyclophosphamide (CTX) is an alkylated chemotherapeutic drug widely used in the treatment of malignant tumors, which was produced in the late 1950s [1].However, it also has many side effects, including immunosuppression and liver toxicity [2,3].The liver is an important organ and plays an irreplaceable role in the body’s metabolic functions and the detoxification of exogenous substances.Exogenous hepatotoxic substances, such as bacteria,viruses, chemotherapy drugs and metabolites, could produce excess reactive oxygen species (ROS), causing inflammation, cirrhosis, and liver disease [4].Current data have indicated that liver disease is widespread and global, whether acute or chronic.At the same time,drug-induced liver injury is one of the most challenging acute and chronic liver diseases treated by physicians and a common cause of acute liver failure [5].In addition, oxidative stress plays an important role in mediating CTX-induced liver damage.After CTX enters the human body, it is converted into active metabolites such as acrolein and phosphoramide mustard by liver microsomal enzymes.Acrolein can easily lead to the decline of the body’s antioxidant defense system, a serious imbalance in the body’s oxidation and antioxidant balance system and the formation of a large number of free radicals,resulting in changes in the vital functions of cells and the destruction of the internal and external environment steady state [6,7].Therefore,it is necessary to find an effective natural antioxidant with few toxic to protect CTX-induced liver damage.

In recent years, polysaccharides extracted from plants, animals and fungi have attracted widespread attention in the fields of biochemistry and medicine for their high efficiency and nontoxic properties [8-11].In addition, polysaccharides with a variety of biological activities have been widely studied to prevent liver toxicity caused by CTX[12,13].Mesona chinensisBenth is a commonly used medicinal and edible plant resource in China, which has high nutritional value and potential health care function [14].M.chinensisBenth polysaccharide(MP) is the main active ingredient ofM.chinensisBenth, which has anti-oxidant, hypoglycemic, hypolipidemic, hypotensive, and antiviral activities [15,16].Our earlier studies showed that MP could alleviate CCl4-induced liver injury [17].

Gut microbiota, also known as the microbial organ, is a huge metabolic organ in the human body, responsible for the host’s health and disease [18].Due to the presence of the gut-liver axis, the gut and liver interact with each other.When the liver is damaged, the intestinal microbial composition and the integrity of intestinal epithelial cells could be destroyed, and abnormal changes in the gut microbiota can cause a wide range of chronic inflammatory diseases.Ecological disorders can lead to increased levels of lipopolysaccharide (LPS) in the system, eventually leading to low-level systemic inflammation [19].Recent studies have shown that elevated plasma endotoxin is associated with intestinal LPS transport and endotoxemia is prone to occur in patients with acute liver failure [20].Since dietary fiber and polysaccharides are difficult to be digested by enzymes in the human digestive tract, but they can be fermented by the intestinal flora to short chain fatty acids (SCFAs) [21,22].As essential nutrient sources,SCFAs can be used to maintain intestinal homeostasis, which are of great significance in improving chronic inflammatory diseases, protecting the intestinal mucosa and promoting colon cell health [23].Therefore,we speculate that MP may have potential therapeutic effects on CTX-induced liver injury, and the liver-protective effect of MP changes in histological and biochemical parameters of CTX-induced liver injury mice.In addition, MP can reduce liver damage by stimulating the number and activity of specific intestinal bacteria.Further, there are few studies on the effect of MP on the gut microbiota.

In this study, the therapeutic effects of MP on liver in mice with CTX poisoning were investigated.Meanwhile, the effect of MP on the structure and composition of intestinal flora of CTX poisoning mice was also studied, which could offer instructive guide for the treatment of cyclophosphamide poisoning byM.chinensisBenth.The theoretical evidence presented here provides insights into the interactions between MP and gut microbiota, especially as far as their liver protection is concerned.

2.Materials and methods

2.1 Materials and reagents

DryM.chinensisBenth was obtained from Jiangxi, China.Cyclophosphamide (CTX) was purchased from Sigma-Aldrich(Sigma, St.Louis, MO, USA).Levamisole (LMS) was purchased from Shandong Renhetang pharmaceutical Co., Ltd.(Linyi,Shandong, China).Endotoxin Detection Matrix Limulus Kit was purchased from Xiamen Limulus Reagent Factory Co., Ltd.(Xiamen,Fujian, China).The assay kits for aspartate aminotransferase (AST)and alanine aminotransferase (ALT) as well as enzyme-linked immunosorbent assay (ELISA) kits for interleukin (IL)-6 and tumor necrosis factor (TNF)-α were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).The assay kits for superoxidase dismutase (SOD), malondialdehyde (MDA), glutathione(GSH) and glutathione peroxidase (GSH-Px) were purchased from Beyotime Biotechnology (Shanghai, China).Acetic acid and valeric acid were produced by Merck (Darmstadt, Germany).Propionic acid was produced by Janssen Chimica (Belgium), while butyric acid was produced by Sigma (St.Louis, MO, USA).

2.2 Extraction of polysaccharides from M.chinensis Benth

The polysaccharide was extracted fromM.chinensisBenth according to our previous studies in our laboratory [14].Briefly,dryM.chinensisBenth was crushed into powder then soaked in ethanol remove most of alcohol soluble substances.Afterwards,the residue was extracted with distilled water at 100 °C for 2 h.Then the combined aqueous extracts were filtrated and precipitated in 95% ethanol (V/V) overnight.The precipitate was re-dissolved with distilled water, followed by deproteinized by sevage method.Finally, it was frozen at –80 °C and lyophilized to obtain MP.MP consisted of 39.01% total sugar, 29.30% uronic acid and 27.30% protein.The average molecular weight of MP is 157 kDa composing of glucose, xylose, galactose and galacturonic acid in a molar of 1.49 : 2.54 : 0.68 : 6.33 [14].

2.3 Animals and experimental design

Male BALB/c mice ((20 ± 2) g) were purchased from Hunan Slake Scene of Laboratory Animal (SJA) Co., Ltd (Changsha, China,certificate number: SCXK (Xiang) 2016-0002).All mice were kept under the circumstance of (22 ± 1) °C in a 12 h light-dark cycle and were freely given access to water and standard chow.All animal experimental protocol was executed on the basis of Nanchang University Medical College Animal Care Review Committee.

As shown in Fig.1A, after one week of adaptation, all mice were randomly divided into two parts, 6 mice in the normal control group (NC) treated with physiological saline (0.9% NaCl)only.The rest of all mice were intraperitoneally injected with 100 mg/kg body weight (bw) CTX for 3 consecutive days, then divided into 5 groups for intragastric treatment for 7 days (6 mice per group)as follow: the model control group (MC) treated with 0.9% NaCl,the positive control group (LMS) treated with 40 mg/kg·bw LMS,the MP 50 group treated with 50 mg/kg·bw MP, the MP 100 group treated with 100 mg/kg·bw MP and the MP 200 group treated with 200 mg/kg·bw MP.All the mice were weighed daily during the experiment.At the end of the drug administration, all mice were fasted for 24 h.Fecal samples were collected before animals were sacrificed.Blood samples were taken from the orbital vascular plexus and stranded for 4 h, serum was separated by centrifugation at 3 000gat 4 °C for 10 min.The colonic contents were collected and liver tissues were weighted.All collected samples were immediately stored at –80 °C for further analysis.The liver index was calculated as follow:

Fig.1 Effect of MP on hepatic damaged in mice induced by CTX.(A) Sterile saline-treated mice (NC) and CTX-induced liver injury mice were given different doses of MP or levamisole (LMS, 40 mg/kg·bw); (B) Body weight; (C) Liver index; (D) Activity of AST and ALT in serum.Data are presented as the mean ± SEM(n = 6).##P < 0.01, compared with MC group; **P < 0.01, compared with the first day (B).Different letters (a-c or A-D) in the same column represent significant differences among treatments when P < 0.05 (C-D).

2.4 Determination of serum biomarkers

According to the corresponding instruction manual, the levels of AST, ALT in serum were determined using a commercial kit.

2.5 Determination of liver oxidative stress indicators

One hundred mg liver was added into 9-times cold phosphate buffer solution and homogenate to obtain 10% liver homogenate, then centrifuged at 10 000gat 4 °C for 15 min to obtain supernatant.The indexes of SOD, MDA, GSH and GSH-Px in the supernatant were measured according to the manufacturer’s instructions for each kit.

2.6 Determination of cytokine and LPS

The contents of cytokines IL-6 and TNF-α in the liver homogenate were measured using a commercial ELISA kit.The concentration LPS in serum was detected using endotoxin detection kit.

2.7 Histological analysis

The liver of the mice were immobilized with 10% medium formalin then prepared to be embedded in paraffin following conventional histology.The samples were cut to a thickness of 5.0 μm and then stained with hematoxylin and eosin (H&E).Finally,the tissues sections were examined using Zeiss microscope.

2.8 Measurement of SCFAs

One hundred mg colonic contents of mice was added with deionized water at a ratio of 1:9 (m/V), then fully vortexed and ultrasound was performed at 4 °C for 5 min.After ice-water bath for 20 min, the mixture was centrifuged at 13 000gat 4 °C for 15 min then the supernatant was transfered to another clean centrifuge tube and centrifuge again under the same conditions.Subsequently, the supernatants were filtered with a 0.22 μm hydrophilic membranes and analyzed by Agilent 6890 gas chromatograph (GC) system.

2.9 Fecal total genomic DNA extraction and gut microbiota 16S rRNA sequencing

Total genomic DNA from fecal samples was extracted used the Mag-Bind Soil DNA kit (Omega, Norcross, Georgia, USA) according to the kit’s instructions.The extracted DNA was quantified using a UV spectrophotometer (Thermo Science, Waltham, MA, USA),and the quality of DNA extraction was detected by 0.8% agarose gel electrophoresis.

The primers 338F (5’-ACTCCTACGGGAGGCAGCAG-3’)and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) were used to amplify the V3 and V4 regions of the 16S rRNA gene in a polymerase chain reaction (PCR) system.PCR products were separated by gel electrophoresis, purified by AxyPrep DNA gel extraction kit (Axygen),and a TruSeq Nano DNA LT library Prep kit was used to construct a DNA library.All barcoded V3–V4 PCR amplicons were sequenced by the Illumina MiSeq platform.

2.10 Statistical analysis

All analyses were performed using SPSS software version 21(IBM software, NY, USA) and the data were expressed as mean ±SEM (standard error of mean).Difference among various groups was analyzed by one-way analysis of variance (ANOVA) using Duncan’s multiple range test and the results withP< 0.05 were regarded as statistically significant.

3.Results

3.1 Effects of MP on body weight and liver index in mice

The effects of MP on body weight in liver damage mice induced by CTX are shown in Fig.1B.The body weight of NC group showed a normal rising trend during the experiment, after treated with CTX,mice in the MC group showed an extremely significant weight loss(P< 0.01), it manifested that CTX damaged the mice severely.However,mice showed better weight recovery after MP administration, suggesting that MP relieved the body weight loss induced by CTX.The same phenomenon was observed for liver index (Fig.1C), liver index in the MC group were significantly reduced compared to other treatment groups(P< 0.05).While treatment with different doses of MP significantly promoted the increased liver index (P< 0.05).

3.2 Effect of MP on the markers of liver function

Serum AST and ALT are specific indicators of liver damage,as presented in Fig.1D, contrasted with the NC group, the serum levels of AST and ALT in MC group were significantly increased(P< 0.05), indicating that the model of drug-induced liver injury in mice with CTX was successfully established.In contrast, there was no significant difference between LMS and the NC group(P> 0.05).Furthermore, at the dose of 200 mg/kg·bw, MP administration significantly suppressed the levels of AST and ALT in MP 200 group as compared with those in MC group (P< 0.05).A slight decrease was also seen in the content of ALT, but it did not show significant difference at doses of 50 and 100 mg/kg·bw, relative to MC group (P< 0.05).These results indicate that MP plays a protective role in liver function.

3.3 Histopathological analysis of the liver

Histopathological studies were performed to further confirm the protective effect of MP on liver, H&E-stained liver sections in the NC group showed normal hepatic lobule structure and clearly defined, and hepatocytes are radially arranged with the central vein as the center (Fig.2A).On the contrary, hepatocyte structure was abnormal, intense cellular degeneration, central vein and hepatic sinus congestion were obviously seen, which were accompanied by a large number of inflammatory cells infiltration in the MC group(Fig.2B).After that, supplementation of LMS significantly protected liver histopathological changes caused by CTX (Fig.2C).However,compared with the MC group, liver histopathological severity was improved to different degrees by MP at different doses.There was also a small amount infiltration of inflammatory cells and central venous congestion in MP 50 and MP 100 groups, while MP 200 group recovered to be close to the NC group, indicating that MP of high dose confers a better protective effect on liver injury induced by CTX (Fig.2D–2F).

Fig.2 Effect of MP on histological imaged of liver injury induced by CTX in mice.(A) NC group, (B) MC group, (C) LMS group, (D) MP 50 group, (E) MP 100 group, (F) MP 200 group.Scale bar, 100 μm.× 200 magnification and 50 μm.× 400 magnification, respectively.

3.4 Oxidative stress in the liver

The activity of SOD and GSH-Px in MC group was significantly declined as compared with the NC group (P< 0.05).After supplementation of MP, the activity of SOD and GSH-Px markedly increased compared with the MC group (P< 0.05) (Fig.3).Similarly,the levels of GSH in the liver of MC group were lower compared with the NC group (P< 0.05).However, the levels of GSH were markedly raised with increment of MP concentration (P< 0.05).In the MC group, the concentration of MDA (the end product of lipid peroxides) exhibited a significantly increasing trend (P< 0.05)as compared to the NC group of mice, but administration of MP produced a significant decrease in MDA levels in the liver compared to MC group.Furthermore, the levels of SOD, GSH-Px, GSH and MDA in LMS group were not significantly difference when compared with the NC group (P> 0.05).All these results indicated that MP had obvious protective effects on CTX-induced liver injury, which may be mediated through increasing antioxidant capacity and reducing the production of lipid peroxides.

Fig.3 Effect of MP on indicator of oxidative stress of liver injury induced by CTX in mice.(A) SOD, (B) GSH-Px, (C) GSH and (D) MDA in liver, respectively.Data are presented as the mean ± SEM (n = 6).Different letters (a-d) represent significant differences among treatments when P < 0.05.

3.5 inflammatory cytokines and LPS concentrations

Inflammatory cytokines are key signaling molecules of the intestinal immune system and play an important role in the occurrence of hepatitis [24].The effect of MP on the levels of IL-6 and TNF-α in liver were shown in Figs.4A, 4B.Administration of CTX significantly (P< 0.05) increased the levels of IL-6 and TNF-α in the liver as compared to those of NC group.However, compared with those in MC group, the levels of inflammatory cytokine were significantly decreased in LMS group (P< 0.05).Meanwhile, the levels of IL-6 and TNF-α in MP 50, MP 100 and MP 200 groups were significantly lower than those in MC group (P< 0.05), showing a dosedependent relationship.These results give evidence that MP could reverse mouse liver inflammatory cytokines increase caused by CTX.

As a metabolite of intestinal bacteria, LPS promotes the accumulation of toxic substances.The MC group significantly increased the concentration of LPS compared with the NC groups.However, LMS and MP supplementation could effectively reverse and approach the NC group, reducing the LPS concentration in serum, especially high-dose MP (P< 0.05, Fig.4C).All those results indicated that 200 mg/kg·bw MP had better protection against CTX-induced liver injury by reducing the release of pro-inflammatory cytokine and LPS.

Fig.4 Effect of MP on inflammatory cytokines production in liver and the concentration of LPS in serum induced by CTX in mice.(A) IL-6, (B) TNF-α,(C) LPS.Data are presented as the mean ± SEM (n = 6).Different letters (a-d) represent significant differences among treatments when P < 0.05.

3.6 SCFAs in colonic contents in mice

SCFAs, including acetic acid, propionic acid, butyric acid,and valeric acid, were the main metabolites of polysaccharides fermented by intestinal microorganism in the gut, which had a beneficial effect on human health.The concentration of total SCFAs were increased after MP treatment, which was mainly caused by the increased concentration of acetic acid, propionic acid, butyric acid and valeric acid (Table 1).When compared with the NC group, the concentration of acetic acid in the MC group was significantly reduced (P< 0.05).However, the administration of MP at 200 mg/kg·bw significantly improved the reduction in the concentrations of acetic acid, propionic acid, butyric acid and valeric acid caused by CTX (P< 0.05).Compared with the MC group, a slight increase was seen in the concentration of butyric acid and valeric acid, but it did not show significant difference at doses of 50 and 100 mg/kg·bw (P> 0.05).All these results showed that the higher dose of MP was more helpful to enhance the production of SCFAs.Therefore, the MP 200 group was selected for further investigation.

Table 1Effect of MP on SCFAs in colonic contents induced by CTX in mice.

3.7 Intestinal microbial composition and diversity

More and more researches show that polysaccharides can regulate the structure of intestinal microorganisms to prevent diseases [25].In this study, Illumina MiSeq was used as a sequencing platform,and the effect of MP on the composition of mouse gut microbiota was investigated by 16S rRNA sequencing.Based on 97% sequence similarity, the effective sequences of all samples were clustered into operational taxonomic units (OTUs) to facilitate the study of species diversity information.The abundance grade curve (Fig.5A) demonstrated that most OTUs had high abundance in the gut microbiota.Interestingly, the abundance of OTUs is the lowest in the NC group and the highest in the MP 200 group (Table 2).In addition, the richness of the ACE and Chao1 indexes did not change significantly among the groups, but there was an upward trend in the MP 200 group (P> 0.05).However, compared with the NC group, the Shannon diversity index was significantly decreased in the MC group(P> 0.05), which was increased significantly with the high-dose MP supply (P< 0.05).These results suggest that MP supplementation increases the diversity of the gut microbiota.

Fig.5 Effect of MP on the abundance grade curve and diversity of gut microbiota in mice induced by CTX.(A) The abundance grade curve from each samples.(B) NMDS analysis based on unweighted UniFrac distances.(C) Hierarchical clustering results analysis based on unweighted UniFrac distances.(D) PLS-DA analysis.MPH, 200 mg/kg·bw MP.

Table 2Effect of MP on OTUs and α-diveristy index.

β-Diversity was analyzed to evaluate the similarities between the groups.Nonmetric multidimensional scaling (NMDS) analysis may be more stable for the ranking of complex structured flora data.Furtherβ-diversity analysis of the NMDS using an unweighted UniFrac distance matrix revealed that MC group treated with CTX was separated from NC group, and a clear separation of the MC group and the MP 200 group was also observed, indicating that MP could change the composition of gut microbiota (Fig.5B).Hierarchical clustering results showed that the NC and MC groups had distinct microbial composition as they were located on two separated branches.Whereas the clustered MP 200 group was closer to the NC group (Fig.5C).These observations further confirmed that MP could alter the composition of fecal flora in CTX-induced mice.Moreover,partial least squares discriminant (PLS-DA) analysis showed that after CTX treatment, the composition of intestinal microorganisms of mice in the MC group deviated from that in the NC group, and after MP treatment, they were close to that in the NC group.The aggregation of samples indicated the similarity between each group and the independence among the three groups.These data also supported successful modeling (Fig.5D).

To determine the specific taxa associated with MP, relative abundance was assessed at the phylum, family, and genus levels.At the level of the phylum, the structures of three groups of microbial communities were mainly composed of Firmicutes and Bacteroidetes,followed by Proteobacteria(Fig.6A).Compared with that of the NC group, the abundance of Firmicutes and Bacteroidetes did not change significantly in the MC group (P> 0.05).After supplementation with MP, the abundance of Firmicutes was significantly increased(P< 0.05), while that of Bacteroidetes was significantly decreased(P< 0.05), indicating that MP had a significant influence on the gut microbiota composition of mice.However, the abundance of Proteobacteria was not significantly altered among the three groups(Fig.6B).

Fig.6 Effect of MP on gut microbiota composition in mice induced by CTX.(A) Gut microbiota composition at the level of phylum.(B) Relative abundance of gut microbiota on phylum level.(C) Gut microbiota composition at the level of family.(D) Relative abundance of gut microbiota on family level.(E) Heatmap analysis at the genus level in each group.(F) Relative abundance of gut microbiota on genus level.Different letters (a-b) represent significant differences among treatments when P < 0.05.MPH, 200 mg/kg·bw MP.

Fig.6(Continued)

Alternations of gut microbiota at family level in all groups are shown in Fig.6C.The predominant families included unclassified Clostridiales, S24-7, Ruminococcaceae, Lactobacillaceae,Lachnospiraceae, Bacteroidaceae and Helicobacteraceae.Unclassified Clostridiales was not significantly different between the NC combination and the MC group (P< 0.05), and increased significantly in the MP 200 group (P> 0.05).Compared with the NC group, CTX treatment significantly reduced the abundance of Ruminococcaceae and increased the abundance of Bacteroidaceae(P< 0.05).However,they were reversed by MP administration.In particular, the abundance of Lactobacillaceae was significantly higher in the MC group than in the NC group, and the administration of MP at 200 mg/kg·bw was significantly reduced (P< 0.05) (Fig.6D).

To validate the differences in the gut microbiota across all groups, several fecal microbiotas at the genus level were analyzed and compared.As shown in Fig.6E, the dominant bacteria of NC group and MP 200 group were relatively similar, but different from MC group.Consistent with the family level, the abundance ofLactobacillusandBacteroidesin the MC group were higher than in the NC group.Furthermore, oral MP supplementation could effectively recover the above bacterial changes caused by CTX.Besides, the abundances ofOscillospirain the NC group and the MP 200 group were similar and higher than the MC group, but there were no significant difference (Fig.6F).A linear discriminant analysis effect size (LEFSe) analysis was performed to identify the specifically altered bacterial phenotypes.LEfSe indicated that Bacteroidia and Bacilli were the predominant contributors to the gut microbiota of NC group and MC group, respectively, whereasFirmicutes was the predominant contributor to the gut microbiota of MP 200 group.In short, MP supplementation mainly increased 12 bacterial taxa induced by CTX in mice (Fig.7).

Fig.7 Effect of MP on structure and key phylotypes of gut microbiota.(A) Cladogram for taxonomic classification of gut microbiota.(B) The taxonomic histograms show the LDA scores calculated for characteristics at the OTU level.MPH, 200 mg/kg·bw MP.

4.Discussion

In this study, MP could significantly reverse the reduction in body weight and liver index of mice caused by CTX.When the liver is under stress or damaged, the permeability of the cell membrane increases, and enzymes in the cytoplasm and mitochondria overflow the cells into the blood, which will cause a pathological increase in blood enzyme activity.Therefore, ALT and AST are commonly used for early assessment of liver injury biochemical markers [26].However, MP can significantly reduce the serum AST and ALT activities, suggesting that MP has a protective effect on CTX-induced liver injury.These results are consistent with those ofMangifera indicapolysaccharide [27].

Different polysaccharides have different chemical structures,physical-chemical properties and monosaccharide composition [12].The structure and chemical composition of polysaccharides may be an important factor affected the antioxidant activity of polysaccharides.The higher content of uronic acid and protein is consistent with the better antioxidant activity of polysaccharides.In addition, it was reported that compounds with antioxidant activity contain the following two or more than two functional groups, such as —OH,—C＝O, —COOH, —NR2and —SH [17,28].Our previous research showed that MP contains 29.30% of uronic acid and 27.30% of protein and is an acidic glycoprotein compound.Moreover, the content of galacturonic acid in the monosaccharide composition is the highest.Fourier transform infrared spectroscopy (FT-IR) analysis further confirmed MP contains —OH, —COOH and C＝O groups [14,17].Therefore, MP has superior antioxidant properties to relieve oxidative stress.

Current research has revealed that CTX-induced liver injury includes excessive formation of ROS and increased production of lipid peroxides, thereby reducing antioxidant enzyme activity [7].As a free radical scavenger, GSH is one of the most abundant tripeptide nonenzyme antioxidants in the liver and plays a vital role in protecting cells from oxidative damage and functional integrity [29].We found a significant reduction in GSH content in the liver of CTX-treated mice,suggesting that CTX severely damaged liver.Antioxidant enzymes such as SOD and GSH-Px play an important protective role in oxidative stress induced by superoxide anion, intraperitoneal injection of CTX in mice for three consecutive days significantly reduced liver SOD and GSH-Px activity in the MC group, which indicated that CTX severely promoted oxidative stress in liver.In addition,MDA is one of fatty acid peroxidation products, and its content can reflect the degree of lipid peroxidation [27,30].In this study, MDA concentration was significantly increased in CTX-treated liver-injured mice.MP treatment could dramatically increase the activity of GSH,SOD, and GSH-Px in liver, reduce MDA production, and showed a dose-dependent relationship.Histopathological observation of liver directly supports this conclusion.In summary, it was shown that MP has significant hepatoprotective function, and its liver protective effect may be related to reducing oxidative stress.

When the liver is damaged by harmful substances, the intestinal permeability and the degree of mucosal damage increase and the large amount of LPS enters the liver through the portal vein, not only the direct damage hepatocytes but also activate the toll like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling pathway,which in turn releases pro-inflammatory cytokins including IL-6,TNF-α, and damages liver cells [31].Therefore, the metabolite LPS of microorganisms has potential liver toxicity and is an effective inducer of inflammation.In this study, CTX promoted a significant increase the levels of IL-6 and TNF-α in liver, as well as serum LPS concentrations.MP treatment significantly inhibited the release of IL-6,TNF-α, and LPS in CTX-induced mice, which suggested that MP can significantly reduce CTX-induced inflammation and reduce LPS-mediated liver damage.

SCFAs can intervene in the regulation of pro-inflammatory and anti-inflammatory mediators through a variety of ways, which is of great significance in improving chronic inflammatory diseases and promoting colon cell health.Propionic acid and butyric acid can inhibit high levels of pro-inflammatory factors secreted by macrophages though inhibiting NF-κB [32,33].Further, the protective effect of SCFAs on the intestinal mucosa is beneficial to prevent LPS [34],a harmful metabolite of microorganisms in the intestine, from entering the bloodstream, thereby damaging the liver.In this study,CTX significantly reduced the production of SCFAs.Fortunately,MP can greatly promote the production of SCFAs, uronic acid can induce the production of acetic acid, and the disappearance of xylose often has a greater impact on the production of propionic acid and butyric acid [35].Our previous research showed that MP is primarily composed of glucose, xylose, galactose and galacturonic acid [14], further indicating that MP can be effectively used by intestinal microorganisms to exert their beneficial effects, preventing toxic metabolites from entering the blood to further damage liver.Therefore, it is necessary to further explore the effect of MP on intestinal flora composition of mice with CTX-induced liver injury.

The gut microbiota plays a vital role in the physiology and pathology of the host.At present, the relationship between gut microbiota and various diseases has become a hotspot for scientists.Imbalance of gut microbiota leads to changes in immune response and is related to various liver diseases, including non-alcoholic fatty liver disease (NAFLD), and liver fibrosis [36,37].Here, MP intake was found to increase gut microbiota diversity and promote the growth of some special bacterial genus.Similarly,βdiversity analysis demonstrated a clear separation of the CTX-treated group from the normal group.Subsequently, the MP intervention deviated from the CTX treatment group, indicating that MP can indeed regulate the intestinal microbial community to develop toward the NC group.Previous research has found that the higher the proportion of Firmicutes and the lower the proportion of Bacteroidetes were conducive to the decomposition of polysaccharides [38].In this research showed that MP causes a significant increase in Firmicutes and a significant decrease in Bacteroidetes.These results indicate that MP is more fully degraded by intestinal microorganisms.

Obviously, at the family level, the administration of MP reversed the decrease in the abundance of Ruminococcaceae caused by CTX, while the Ruminococcaceae were mainly responsible for the degradation of polysaccharides and the production of SCFAs, and were negatively related to increased alcoholic cirrhosis and intestinal permeability [39,40], so it may also degrade MP and increase the production of SCFAs.Surprisingly, Bacteroidaceae can mediate the inflammatory response of the host by promoting the secretion of cytokines, stimulating the neutrophil excitement process [41].The results of this study showed that abundance of Bacteroidaceae was significantly increased in CTX-induced liver injury mice, while MP administration was slightly down-regulated.In conclusion, it is further explained that the protective effect of MP on CTX-induced liver injury in mice may be due to changing the composition of the gut microbiota, then increasing the production of SCFAs, and reducing the inflammatory response.Interestingly, the abundance ofLactobacillusat the genus level was significantly increased in CTX-induced liver injury mice and significantly reduced to normal levels after MP replenishment, which was consistent with the study of [42].The abundance ofLactobacillusin the intestinal flora of patients with liver injury was significantly increased.Research also shows that lower levels ofLactobacilluscan reduce obesity symptoms in diet-induced obese mice [43].Furthermore,the abundance ofOscillospira, a class of SCFAs-producing bacteria, increased after MP replenishment.LPS is mainly secreted byBacteroidesand is considered to be an inflammatory stimulus[44].In this study, the abundance ofBacteroidesdecreased after MP treatment, which suggested that MP relieve liver damage by inhibiting the proliferation of these bacteria and weakening the secretion of inflammatory factors.Our results clearly show that MP supplementation can partially improve liver damage by regulating the gut microbiota.

Microorganisms in the intestine can affect the liver through different mechanisms, such as the production of metabolites (SCFAs)and immunomodulation (immune response of the liver to intestinal source factors, e.g., LPS).After binding to the TLR4 receptor,LPS activates NF-κB and produces inflammatory cytokines and chemokines that cross the intestinal barrier through the portal system to the liver under physiological conditions, triggering low-level inflammation.This suggests that LPS plays a key role between gut microbiota and host inflammation.In contrast to the CTX-treated group, MP treatment could inhibit the increase of LPS levels and thus the inflammation of liver tissue, which is consistent with the reduction of LPS-producing bacteria.Related research shows that butyric acid inhibits neutrophil release, reduces NF-κB activation, and inhibits the adhesion of monocytes and lymphocytes to endothelial cells, suggesting that SCFAs may play an anti-inflammatory role by inhibiting the expression of inflammatory cytokines [45].Namely,MP can promote the production of SCFAs, which is consistent with the increase of SCFAs-producing bacteria.By reducing the level of LPS, increasing the content of SCFAs brings benefits to the body.Therefore, it is important to better understand the role of MP in regulating gut microbiota.

5.Conclusion

In summary, this study revealed that MP has protective effects on CTX-induced liver injury, which could reduce oxidative stress and inflammatory responses.At the same time, MP restored the SCFAs content and reduced the increase of LPS levels caused by CTX treatment.Supplementary MP could improve gut microbiota composition in CTX-induced liver injury mice.Furthermore, MP could increase SCFAsproducing bacteria and decrease LPS-producing bacteria.In other words, MP effectively impeded CTX-induced liver injury development by elevating antioxidant status, inhibiting inflammatory responses and modulating gut microbiota.These findings provide a new perspective on the health effects of MP as functional foods.

Conflicts of Interest

The authors declare no conflict of interest.

Acknowledgement

This study was supported by grants from the National Natural Science Foundation of China (21566024), and the Natural Science Foundation of Jiangxi province, China (20181ACB20013).