Jiongshan Zhang,Yue Li,Gemin Xiao,Yongwei Li,Heping Xie,Yan Chen

Department of Traditional Chinese Medicine,The Third Affiliated Hospital of Sun Yat-sen University,Guangzhou,China

ABSTRACT

Background:Non-alcoholic fatty liver disease(NAFLD)is a frequently occurring clinical disease,which could be treated by traditional Chinese medicine.However,the effects and mechanisms of xiao-zhi-fang(XZF)alone or in combination with fructooligosaccharides(FOS)on NAFLD remains unclear.

Methods:This study established the NAFLD mouse model by feeding with a high-fat diet and by administering with XZF and/or FOS.Moreover,the fat content in the liver tissues was evaluated by oil red O and hematoxylin and eosin staining,and the liver function indices were examined using a biochemical analyzer.Changes in the intestinal flora were monitored using 16S rRNA gene sequencing.The results of the current study showed that the alanine aminotransferase,aspartate aminotransferase,total triglyceride,total cholesterol levels,and the amount of lipid accumulation were markedly elevated in NAFLD mice.

Results:XZF alone or in combination with FOS significantly reduced lipid accumulation in the liver and blood lipid levels in NAFLD mice.Moreover,XZF alone or in combination with FOS notably altered the intestinal flora structure in NAFLD mice.Consequently,17 operational taxonomic units were decreased and nine were increased in the XZF and/or FOS groups.

Conclusions:FOS may accelerate the intestinal microecological balance in NAFLD mice after treatment with XZF by promoting intestinal flora growth,thereby enhancing the therapeutic effect of XZF on NAFLD.

Keywords:

Fructooligosaccharides(FOS)

High-fat diet(HFD)

Intestinal flora

Non-alcoholic fatty liver disease(NAFLD)

Xiao-zhi-fang(XZF)

1.Background

Non-alcoholic fatty liver disease(NAFLD)is defined as the presence of＞5% hepatic steatosis due to lack of secondary causes of hepatic fat accumulation(e.g.,long-term use of a steatogenic medication,significant alcohol consumption,or monogenic hereditary disorders).1-4The global prevalence of NAFLD is estimated to be approximately 24-25%.Moreover,it is estimated to be 29.2% in China.2,4-6However,detecting this disease early is difficult because of its insidious onset.The NAFLD disease spectrum includes non-alcoholic hepatic steatosis,non-alcoholic steatohepatitis,and hepatocellular carcinoma.7Dietary fat consumption,genetic predisposition,excessive accumulation of visceral fat,insulin resistance,and increased levels of proinflammatory mediators are NAFLD risk factors.7-9The synergistic effects of these factors can lead to hepatic steatosis,chronic hepatitis,lipid toxicity,and hepatocyte damage,which can progress to chronic hepatitis and cirrhosis.10The common drugs currently used for NAFLD treatment include vitamin K and metformin,among others.11The nondrug treatments include weight loss,exercise,and control of related risk factors(e.g.,body weight,blood glucose,and blood lipid levels).12However,no safe and effective methods exist for NAFLD treatment.

NAFLD is a modern disease that may be treated by using traditional Chinese medicine(TCM).13,14Based on the pathogenic factors and clinical symptoms,the following categories of the TCM syndrome can be attributed to the disease-liver distension,accumulation,abdominal mass,epigastric fullness,and meteorism.15,16The NAFLD treatment methods in TCM currently include polyjuice potion,a single Chinese herb,Chinese patent medicine,acupuncture,and acupoint catgut embedding.17,18However,the TCM therapeutic effect and pathogenesis of NAFLD are poorly understood.

Xiao-zhi-fang(XZF),a formulated compound based on TCM,has been clinically used in treating NAFLD.19Moreover,fructooligosaccharides(FOS)is primarily derived from microbial fermentation,20,21and can improve blood glucose,strengthen the immune system,and promote nutrient absorption.22,23FOS has been shown to enhance the therapeutic effect of silybin on NAFLD.24However,whether the therapeutic effect of XZF on NAFLD can be enhanced by FOS is unclear and,if so,what is the key mechanism involved.This study established the NAFLD mouse model by feeding a highfat diet(HFD)to investigate the effects of XZF alone or in combination with FOS on NAFLD.Furthermore,the regulatory effects of XZF and FOS on the intestinal flora were evaluated through 16S rRNA sequencing technology to reveal the possible XZF and FOS mechanisms in NAFLD.

2.Materials and methods

2.1.Ethical approval

The animal experiments were approved by the Institute of Animal Care and Use Committee of Forevergen Medical Laboratory Animal Center(IACUC-G16029).Moreover,the principles of animal welfare were strictly followed throughout the experiments.

2.2.Drugs

XZF(patent number:China,201110416602.1)is a TCM compound that is used to treat fatty liver in China.It contains 30 g of Fructus Crataegi,20 g of Radix Polygoni Multiflori,20 g of Senmen Cassiae,30 g of Radix Salviae Miltiorrhizae,15 g of Rhizoma Polygoni Cuspidati,15 g of Rhizoma Atractylodis Macrocephalae,15 g of Rhizoma Alismatis,and 10 g of Radix Bupleuri(The Third Affiliated Hospital of Sun Yat-sen University,Guangzhou,China).The compound was concentrated into a water decoction containing 3.4 g/mL of crude drugs.

2.3.HFD-induced NAFLD mice model and treatment protocol

The current study purchased 24 male-specific-pathogen-free C57BL/6 mice weighing 12-15 g each from Forevergen Medical Laboratory Animal Center(Guangzhou,China).The mice were adaptively fed with adequate and clean food and water for 7 days at room temperature(15-20°C)under humidity conditions of 60-70% and 12 h of light per day.

The mice were randomly divided into normal control(NC;n=6)and NAFLD model(n=18)groups.Mice in the NC group were fed a conventional diet(Medicine,Jiangsu,China;catalog number:MD12062)for 8 weeks,whereas those in the NAFLD model group were fed an HFD(ReadyDietech,Shenzhen,China;catalog number:D12492)for 8 weeks.Three mice per group were randomly chosen and assessed by oil red O staining,hematoxylin and eosin(H&E)staining,and liver function index to confirm the establishment of the NAFLD mouse model after feeding for 8 weeks.Then,the NAFLD mice were further divided into three subgroups:NAFLD(n=5),NAFLD+XZF(n=5),and NAFLD+XZF+FOS(n=5)groups.XZF and FOS were dissolved in normal saline and administered at 21.5 g/kg and 0.4 g/kg by gavage,respectively,and mice in the NAFLD group were given an equal volume of normal saline,once a day for 8 weeks.

2.4.Oil red O staining and H&E staining

The mice were made to fast for 12 h and then sacrificed by dislocation.Next,liver tissues were quickly removed and cut into sections(thickness,5μm)that were fixed using 4% formaldehyde for oil red O staining.The sections were stained with 0.5 g oil Red O(Sigma-Aldrich,St.Louis,MO,USA;catalog number:O0625)in darkness overnight after washing in 100% propylene glycol.Subsequently,the sections were sealed with glycerin gelatin and observed under a microscope.In addition,the liver sections embedded in paraffin were stained with H&E as previously described.4

2.5.Detection of liver function index

All mice were made to fast for 12 h and anesthetized using 2% sodium pentobarbital(0.3 mL/100 g).Blood was collected from the femoral artery.After serum separation,the AU480 automatic biochemical analyzer(Beckman,CA,USA)was used to confirm the levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),total triglyceride(TG),and total cholesterol(TC).

2.6.DNA extraction and identification

Mice feces were aseptically collected before the end of the experiment and immediately stored at-80°C.The specimens were tested at the Beijing Genomics Institute(Shenzhen,China).Genomic DNA from the collected feces was extracted using the QIAamp Fast DNA Stool Mini Kit(QIAGEN GmbH,Germany;catalog number:51604).In addition,DNA purity and concentration were confirmed using 2.0% agarose gel electrophoresis.

2.7.16S rRNA detection

Qualified DNA samples were applied for the construction of a library,which was analyzed using the Ion Plus Fragment Library Kit(Thermo Fisher Scientific,Waltham,MA,USA),according to the manufacturer's instructions.Furthermore,the constructed library was sequenced using the Ion S5XL System(Thermo Fisher Scientific)after Qubit quantification.

2.8.Statistical analysis

The data were presented as mean±standard deviation from at least three independent experiments.Statistical significance(P＜0.05)was calculated by using a one-way analysis of variance and Student’s t-test with SPSS 20.0 software(IBM,Armonk,NY,USA).The available data on 16S rRNA were obtained by splicing the raw data obtained from sequencing.The UPARSE software(V8 10.0.240,http://www.drive5.com/usearch/)was used to cluster the sequences into operational taxonomic units(OTUs)based on the available data.Specie annotation was analyzed using Mothur and the SSUrRNA database with the SILVA132 database as the reference(threshold,0.8-1.0).Alpha diversity indices(abundance-based coverage estimator(ACE),Shannon)were analyzed using the R software(v3.6.0),and the principal coordinates analysis(PCoA)was conducted based on the unweighted UniFrac distances.The differences between groups were compared and analyzed using the LEfSe software(https://www.r-project.org/)and t-test.P＜0.05 indicated that the difference was statistically significant.

3.Results

3.1.Establishment of the NAFLD mouse model

The NAFLD mouse model was established by feeding an HFD for 8 weeks to explore the possible therapeutic drugs for NAFLD.Oil red O and H&E staining revealed a large number of lipid droplets in the NAFLD mouse model compared with the mice in the NC group,thereby indicating that the fat content in the liver tissues was markedly elevated in these mice(Fig.1A).Furthermore,serum ALT,AST,TC,and TG levels were significantly increased in the NAFLD mice(Fig.1B-E;P＜0.0001),confirming the establishment of the NAFLD mouse model.

Fig.1.Establishment and identification of the NAFLD mouse model.C57BL/6 mice were fed with an HFD or NC diet for 8 weeks.The fat in the liver tissues was determined in the NC and NAFLD groups via(A)oil red O and H&E staining;scale bar 100μm.The(B)ALT,(C)AST,(D)TC,and(E)TG levels were confirmed using a biochemical analyzer(n=3 per group);****P＜0.0001.Abbreviations:ALT,alanine aminotransferase;AST,aspartate aminotransferase;H&E,hematoxylin and eosin;HFD,high-fat diet;NAFLD,non-alcoholic fatty liver disease;NC,normal control;TC,total cholesterol;TG,total triglyceride.

3.2.The effects of XZF and the combination of XZF and FOS against NAFLD

The mice were administered with XZF alone(21.5 g/kg/day)or in combination with FOS(0.4 g/kg/day).The ALT,AST,TC,and TG levels were notably reduced in the NAFLD-XZF and NAFLDXZF+FOS groups compared with the NAFLD group(Fig.2A-D).Moreover,FOS enhanced the protective effects of XZF in the NAFLD mouse model(Fig.2A-D).The oil red O and H&E staining showed a mass of lipid droplets in the liver tissues of the NAFLD group,whereas marked improvements in the number and size of lipid droplets were observed in the NAFLD-XZF and the NAFLDXZF+FOS groups(Fig.2E).Overall,XZF alone or in combination with FOS had significant inhibitory effects on hepatic lipid accumulation and blood lipid level in NAFLD mice.

3.3.The composition of intestinal microbiomes influenced by XZF and FOS

The results of the 16S rRNA gene sequencing revealed 754,772,and 624 OTUs in the NAFLD,NAFLD-XZF,and NAFLD-XZF+FOS groups,respectively.The abundant intestinal flora distribution was also displayed using the heat map at the phylum level.The intestinal flora primarily consisted of the Tenericutes,Cyanobacteria,Actinobacteria,Verrucomicrobia,Bacteroidetes,Proteobacteria,Firmicutes species of microorganisms(Fig.3A).Simultaneously,the current study analyzed the species composition and phylogenetic OTU in three samples.The results showed that the bacterial communities in the NAFLD group were primarily distributed in Bacteroidetes,Cyanobacteria,Firmicutes,Proteobacteria,Tenericutes,and Verrucomicrobia,whereas the bacterial communities in the NAFLDXZF and NAFLD-XZF+FOS group were primarily distributed in Bacteroidetes,Firmicutes,Proteobacteria,and Verrucomicrobia(Fig.3B).These results demonstrated that,in general,the composition of the intestinal microbiomes was influenced by XZFand FOS.

3.4.Intestinal flora structure analysis in the NAFLD mice after XZF and/or FOS administration

The current study analyzed the correlations of the three groups to further evaluate the potential impacts of XZF and FOS in the intestinal flora during the development of NAFLD.First,UniFracbased PCoA was applied to compare the entire microbial communities.Fig.4A shows that PC1 and PC2 accounted for 75% and 24%,respectively,and the sample points can be completely separated among the three groups,indicating that XZF alone or in combination with FOS could significantly alter the overall structure and composition of intestinal flora.Second,the beta diversity analysis was adopted to compare the differences in the intestinal flora distribution among the three groups(Fig.4B).The differences in the structure of the intestinal flora were high between the NAFLD and NAFLD-XZF groups as well as the NAFLD-XZF+FOS group,whereas little difference was noted between the NAFLD-XZF and NAFLDXZF+FOS groups.Moreover,513 common OTUs were found among the three groups using a Venn diagram(Fig.4C).

3.5.The impacts of XZF and FOS on the abundance of the common intestinal flora

Seventeen OTUs(Anaeroplasma,Helicobacter,and Prevotella,among others)were markedly decreased,whereas nine OTUs(Akkermansia,Bacteroides,Parabacteroides,Blautia,and Clostridium,among others)were significantly increased in the NAFLD-XZF and the NAFLD-XZF+FOS groups,respectively,relative to the NAFLD group(Fig.5).Therefore,the current study speculated that these OTUs may be crucial strains of XZF and FOS that can improve the intestinal flora in NAFLD mice.

4.Discussion

NAFLD is a chronic metabolic disease with complex pathogenesis.25Previous studies confirmed that the pathogenesis of NAFLD is primarily associated with intrahepatic lipid accumulation,insulin resistance,oxidative stress,inflammation,and other factors.26-28Moreover,the dysregulation of intestinal microecology is closely related to NAFLD.29The current study successfully established the NAFLD mouse model by feeding an HFD for 8 weeks.Furthermore,this study demonstrated that XZF alone or in combination with FOS could significantly lower hepatic lipid accumulation and blood lipid levels in NAFLD mice.In addition,XZF with or without FOS can prominently improve intestinal flora disorder in these mice.

In TCM,fatty liver is primarily caused by greasy food,a surfeit of flavors,and a fondness of liquor,which can cause liver and spleen injury.30,31XZF includes Fructus Crataegi(which dissipates phlegm and stasis),Rhizoma Alismatis(which clears and disinhibits wetness-heat of liver and gallbladder syndrome),Radix Salviae Miltiorrhizae(which removes blood stasis to promote tissue regeneration),Rhizoma Atractylodis Macrocephalae(which nourishes qi to invigorate the spleen),Senmen Cassiae(which dispels wind and heat from the body),Rhizoma Polygoni Cuspidati(which dispels wind to eliminate dampness),Radix Polygoni Multiflori(which nourishes blood and liver),and Radix Bupleuri(which disperses and rectifies the liver).In addition,modern medicine has shown that Salvia miltiorrhiza can improve blood circulation, reduce inflammation, and prevent fibrosis.32Furthermore,Bupleurum has anti-inflammatory and anti-fatty effects on the liver.13XZF may have certain curative effects according to TCM pharmacology.However,the XZF therapeutic effect and mechanism of action in NAFLD have not been elucidated thus far.The present study has shown that XZF can markedly reduce hepatic lipid accumulation and blood lipid levels in the NAFLD mice.

FOS is an indigestible oligosaccharide that can integrally reach the colon.21In addition,it is primarily fermented by Bifidobacterium and can produce lactic acid and short-chain fatty acids(e.g.,acetic acid,propionic acid,and butyric acid).33FOS is utilized by colon cells or transported to the liver through the portal vein.34Moreover,it can selectively promote the growth of beneficial microorganisms(Bifidobacteria)and inhibit the growth of harmful microorganisms(Salmonella and Escherichia coli)as a nutrient matrix.35,36Furthermore,FOS can prevent the colonization and invasion of pathogenic bacteria,improve intestinal micro flora,and reduce serum cholesterol and neutral fat.37To the best of our knowledge,the present study is the first to show that the combination of XZF and FOS can aggravate the reductions in hepatic lipid accumulation as well as blood lipid levels in NAFLD mice.

The intestinal tract is the primary site for the digestion and absorption of various nutrients.38,39The intestinal flora can participate in multiple biological pathways,such as energy storage,intestinal endocrine,immunity,metabolism,and nutrition.40The intestinal flora and host organism maintain a dynamic balance under normal physiological states.However,it may lead to the occurrence of major human diseases,such as type 2 diabetes,obesity,fatty liver,irritable bowel syndrome,and tumors,when the balance state changes.41Based on its functions,the intestinal flora can be generally divided into three types of pathogens:probiotics(Bifidobacteria and Lactobacillus), opportunistic pathogens(Enterococcus and Enterobacteria),and pathogenic bacteria(Clostridium difficile and pathogenic E.coli).42Studies published in recent years have reported that the balance of the intestinal microecology is disturbed in NAFLD patients with a reduction in the number of beneficial bacteria and an increase in harmful bacteria.42,43Therefore,the adjustment of the structure of the intestinal flora is crucial during NAFLD treatment.However,whether XZF and FOS can optimize the intestinal microecological balance in these patients is unclear.This study showed that XZF alone or in combination with FOS notably reduces the abundances of 17 OTUs(Anaeroplasma,Helicobacter,and Prevotella,among others)and elevates the abundances of 9 OTUs(Akkermansia,Bacteroides,Parabacteroides,Blautia,and Clostridium,among others).Among them,Akkermansia is known to improve conditions such as obesity,inflammation,insulin resistance,and glucose tolerance;participate in the regulation of the immune response;and maintain the metabolic balance.44,45Bacteroides,as obligate anaerobes,play a vital role in maintaining the balance of the intestinal flora.46These findings indicated that XZF and FOS can significantly improve disorders of the intestinal flora in HFD-induced NAFLD mice.

Fig.3.The composition of intestinal microbiomes in the NAFLD mouse model administered with XZF and/or FOS.(A)16S rRNA gene sequencing was carried out after XZF and/or FOS were administered in the NAFLD mouse model.The heat map was adopted to exhibit the distribution of the intestinal flora at the phylum level.The vertical column represents the group and the horizontal column represents the species.The middle square denotes the relative abundance of the species,and the red denotes the low abundance of the phylum between groups.(B)OTUs phylogenetic analysis of the three groups(n=3 per group).Abbreviations:FOS,fructooligosaccharides;NAFLD,non-alcoholic fatty liver disease;OTUs,operational taxonomic units;XZF,xiao-zhi-fang.

5.Conclusions

The findings of the present study indicate that FOS can enhance the therapeutic effect of XZF on NAFLD by adjusting intestinal flora.In addition,these findings may provide the theoretical basis for the efficient treatment of NAFLD and contribute to the clinical application of XZF and FOS.

Authors’contributions

J.Zhang and Y.Chen conceived and designed the experiments.J.Zhang,Y.Li,G.Xiao,Y.Li,and H.Xie performed the experiments and analyzed the data.J.Zhang and Y.Chen drafted the manuscript.All authors read and approved the final manuscript.

Fig.4.The intestinal flora structure in the NAFLD mice model administered with XZF and/or FOS.(A)The entire comparison of microbial communities was exhibited using UniFrac-based PCoA.(B)Differences in the abundance distribution of the intestinal flora in the NAFLD mouse model administered with XZF and/or FOS were compared using the beta diversity analysis.Blue indicates short distance and high similarity,and red indicates long distance and low similarity.(C)A Venn diagram was created to analyze the common and unique OTUs among the three groups(n=3 per group).Abbreviations:FOS,fructooligosaccharides;NAFLD,non-alcoholic fatty liver disease;XZF,xiao-zhi-fang.

Fig.5.The influence of XZF and FOS on the abundance of the common intestinal flora.(A)The heat map exhibited the relative abundance of common OTUs.(B)The absolute abundance of common OTUs was clustered by applying the histogram analysis(n=3 per group).Abbreviations:FOS,fructooligosaccharides;NAFLD,non-alcoholic fatty liver disease;OTUs,operational taxonomic units;XZF,xiao-zhi-fang.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by the Natural Science Foundation of Guangdong province of China(grant number 2017A030313738)to J.Zhang.