Blueberry malvidin-3-galactoside modulated gut microbial dysbiosis and microbial TCA cycle KEGG pathway disrupted in a liver cancer model induced by HepG2 cells

Zhn Chng,Ji Lin,Ningxuan Gao,Xiyun Sun,Xianjun Mng,Ruihai Liu,d,Yunn Liu,Wishng Wang,Bin Li,*,1,Yuhua Wang,*,1

a College of Food Science,Shenyang Agricultural University,Liaoning,China

b National R&D Professional Center For Berry Processing,Shenyang Agricultural University,Liaoning,China

c Key Laboratory of Healthy Food Nutrition and Innovative Manufacturing of Liaoning,China

d Department of food Science,Cornell University,Ithaca,NY,USA

e Emergency Medicine Department of General Hospital of Shenyang Military Command,Laboratory of Rescue Center of Severe Trauma PLA,Liaoning,China

f Shenyang Blueberry Industry Co.LTD.,Liaoning,China

ABSTRACT HCC (Hepatocellular Carcinoma) is a critical health issue worldwide.Our previous animal experiment has confirmed that blueberry malvidin-3-galactoside(M3G)can regulate the progression of HCC.In this study,feces samples from the same batch of mice were collected to explore the regulatory mechanism of M3G on intestinal microbiota and microbial TCA cycle metabolism KEGG pathway in HCC mice based on 16S rRNA sequencing and metagenomics.Our results showed that blueberry M3G increased the microbial diversity and regulated the structure of intestinal microbiota in mice,such as increasing the abundance of Clostridia(butyric acid-producing bacteria),Oscillospira and Ruminococcus,and reducing the abundance of pathogenic Erysipelotrichi.Compared with the group of liver cancer and 5-fluorouracil,blueberry M3G significantly regulated microbial TCA cycle KEGG pathway via improving the expression of key proteins(porA,DLAT,aceE,PC and OGDH).Additionally,we found which the abundance of Muribaculum intestinale increased by blueberry M3G may be an important factor affecting the microbial TCA cycle KEGG pathway via the pearson correlation(R)analysis of protein and microbiota.Taken together,these results demonstrate that the blueberry M3G has the potential to be an intestinal microbiota regulator and an adjuvant to HCC therapy.

Keywords:Blueberry malvidin-3-galactoside HCC metagenomics microbial TCA cycle KEGG pathway

1.Introduction

Hepatocellular carcinoma(HCC)is the sixth most common cancer and the fourth most lethal malignant tumor in the world, and the pathogenesis is extremely complex[1].The fundamental causes of liver cancer are hepatitis B or C virus infection and excessive alcohol consumption [2,3].HCC has a poor prognosis and high mortality due to the early diagnosis rate of less than 30% [4].At present, the treatment approach of HCC is still the combination of surgical resection and chemotherapy [5], and targeted therapy and immunotherapy have not been really implemented.In recent years, the strong relationship between liver cancer and intestinal microbiota has been demonstrated,which may become an important target for the treatment of liver cancer.Ma et al.[6]found that a high-fat diet increased the abundance of Clostridium in the gut and promoted the development of liver cancer.Additionally, the level of microbial metabolite such as deoxycholic acid also directly affects the progression of HCC[7].It can be seen that the gut microbiota and microbial metabolites may fuel the development of HCC.Therefore,how to better prevent and treat HCC is still a worldwide problem worthy of attention.

Blueberry anthocyanin (BA) has been shown to have many beneficial physiological functions.In terms of anticancer activity, our previous study has proved that malvidin-3-galactoside(M3G) inhibited the proliferation of HepG2 cells in vitro and in vivo in a concentration dependent manner [8].In another study, it was also confirmed that the anti-cancer ability of BA in HepG2 cells was related to its concentration [9].In addition,BA also has many other functions such as anti-obesity, inhibits angiogenesis, improve cardiovascular health and so on [10-12].Large amounts of anthocyanin monomers in blueberries play an important role in human health, among which malvidin-3-Ogalactoside, malvidin-3-O-glucoside and petunidin-3-O-glucoside have the highest content [13].Study has proved that these three monomers can regulate the diversity of the gut microbiota[13],but the mechanism of how M3G regulates the microbial KEGG pathway in HCC mice is still unclear.

TCA cycle plays an important role in cancer as a target for linking numerous metabolic pathways[14].Intermediate product such as succinic acid produced in the TCA cycle has an important position in the metabolism of the body and succinic acid has anti-obesity effect [15].Meanwhile, the complete TCA cycle can promote the utilization of succinate [16].It is well known that the intestinal dysbiosis and the microbial metabolic pathway damage in HCC patients.Increased pathogenic microbiota and colonization can also down-regulate microbial TCA cycle metabolic pathway [17].Therefore,the regulatory effect of blueberry M3G on microbial TCA cycle KEGG pathway and its intermediate metabolites may be a potential strategy for the treatment of HCC assisted by functional food.

The effect of blueberry M3G on the structure of intestinal microbiota and the microbial TCA cycle KEGG pathway in HepG2 cells-induced HCC mice is unknown yet.In this study, we investigated the effects of M3G on intestinal microbiota diversity and the regulation of microbial TCA cycle metabolic kegg pathway in a liver cancer animal model via 16S rRNA sequencing and metagenomics.

2.Materials and methods

2.1.Chemicals

Blueberry M3G monomer (reference standard, purity ≥95%,soluble in ddH2O) was obtained from the Crown Blueberry Biotechnology co., LTD (Shenyang, Liaoning).24 C57BL/6 J male mice were purchased from the Vital River Laboratory Animal Technology Co., Ltd (Beijing, China).The reagents needed for PCR amplification and fluorescence quantification were Q5 highfidelity DNA polymerase, 5*Reaction Buffer, 5*High GC Buffer,dNTP (2.5 mmol/L), DNA Template, Forwardprimer (10 μmol/L),Reverseprimer(10 μmol/L)and ddH2O,RNA 6000 Nano kit and Agilent High Sensitivity DNA Kit(Agilent,5067-1511 and 5067-4626)were provided by the Personalbio Technology co.,LTD (Shanghai,China).

2.2.Animal experiments

The Animal Ethics Committee of Shenyang Agricultural University approved all experimental procedures in China (license number: SYXK ＜Liao＞2011-0001).C57BL/6 J mice of aged 6-8 weeks were purchased from the Vital River Laboratory Animal Technology Co., Ltd (Beijing, China).The mice were individually housed in a SPF animal breeding room and 12 h of light/dark cycle for a week.The indoor temperature and humidity were controlled at(25±2)°C and 50%±2%respectively.During this time,the mice were free access to water and the diet(GB 14924.3-2001,National Rat Diet Standard,China).The HCC murine model was constructed by injecting HepG2 cells into the subcutaneous tissue of the liver region of C57BL/6 J mice (1×106HepG2 cells/per, SPF animal lab,human HepG2 cells derived from Chinese Academy of Sciences(Shanghai)76 cells)).After one week stabilization,mice were randomly divided into four groups (six mice in each group): liver cancer control group(LC,0 mg/kg M3G),low-dose blueberry M3G(LM3G, 40 mg/kg body weight (bw)), high-dose blueberry M3G(HM3G, 80 mg/kg body weight (bw)) and 5-fluorouracil positive group(5 F,20 mg/kg body weight(bw)).Blueberry M3G was administered by oral gavage once daily and mice in the group of 5 F were given once daily with chemotherapeutic drugs by subcutaneous injection for 21 days.The diet was supplemented with fresh diet daily.Feces were collected on days 7,14 and 21,respectively.The collection environment and operation process were strictly controlled to reduce unnecessary pollution.The feces of mice were stored in sterile centrifuge tubes.The samples were rapidly frozen and storage at -80°C in liquid nitrogen.The roadmap of research design is shown in Fig.1.

Fig.1.Test roadmap.

Fig.2.Effect of blueberry M3G on intestinal microbiota diversity in HepG2 cells-reduced HCC mice(A)alpha diversity(B)beta diversity(*,P ＜0.05;**,P ＜0.01;***,P ＜0.001).

2.3.DNA extraction

Each stool sample was collected at 180-220 mg and 1.4 mL of ASL buffer was added to the sample and vortexes for 1 minute until the sample was completely homogeneous.Heat the sample at 70°C for 5 min.The sample was vortex for 15 s before centrifugation.The supernatant was taken for subsequent centrifugation.During DNA extraction, QIAamp rotating column was used to transfer the sample solution after multiple centrifuges to a new 1.5 mL centrifuge tube and incubated for 1 minute at(25±2)°C.Finally, centrifuge quickly for one minute to elute the DNA.

2.4.PCR quantification and amplification

In this experiment, the inserted fragment range was about 250 bp, and the sequencing region was the standard bacterial V3-V4 region.The selected specific primer was 520 F (5’-barcode+aytgggydtaaagng-3’) and 802R (5’-TACNVGGGTATCTAATCC-3’).Barcode in the front primer is an oligonucleotide sequence of 7 bases used to distinguish different samples in the same library.PCR amplification was performed using NEB Q5 DNA high fidelity polymerase.Finally, 2% agarose gel was used for cataphoresis.The target fragment was cut and recovered with Axygen gel recovery kit.PCR products were quantified by quant-it PicoGreen dsDNA Assay Kit on Microplate reader(BioTek,FLx800).

2.5.16S rRNA sequencing

2×300 BP double-ended sequencing was performed using miseq machine and miseq kit V3 (600 cycles).First, the gradient of the libraries on the computer will be diluted to 2 nmol/L, and then mixed in proportion to the amount of data required.Mixed libraries were transformed into single-stranded libraries by 0.1 N NaOH for computer sequencing.

2.6.Metagenome Shotgun Sequencing

This project is based on Illumina HiSeq high-throughput sequencing platform,using Whole Genome Shotgun.The total DNA of metagenomics was randomly interrupted into short fragments and inserted fragment libraries of suitable length were constructed.Paired-end sequencing was performed on these libraries.The content and expression of A/T/C/G four bases were observed to determine the preparation process of sequencing library and to provide basis for subsequent analysis.Sequences of good quality were used to build sequences.Functional annotation of genes was carried out according to the constructed library.

2.7.Data Access

All raw sequences were deposited in the NCBI Sequence Read Archive under accession number PRJNA509691 (16S rRNA) and PRJNA561083(metagenomics).

2.8.Statistical analysis

Results were expressed as mean±SEM.SPSS v.16.0 software(SPSS, Inc., Chicago, IL, USA).Significance was controlled by oneway analysis of variance and multiple comparisons of graphs.The polyline plots and bar charts were calculated by GraphPad Prism 8.0.1.244×86 (GraphPad software Inc., CA, USA) (*, P ＜0.05; **,P ＜0.01;***,P ＜0.001).

Fig.3.Effect of blueberry M3G on intestinal microbiota structure in HepG2 cells-reduced HCC mice(A)intestinal microbiota structure at class level(B)microbiota with a significant change in abundance at class level(C)intestinal microbiota structure at genus level(D)microbiota with a significant change in abundance at class level(*,P ＜0.05;**,P ＜0.01;***,P ＜0.001).

Fig.3.(Continued)

The SciPy library of Python software was used to carry out T test on the samples,and the statistical analysis result of different significance was obtained.FDR was controlled by Benjamini-Hochberg(P ＜0.05).R software was used for cluster analysis of the difference samples.Beta analysis of the composition spectrum of metagenomics samples was performed by R software and QIIME software respectively.

3.Results

3.1.Effect of blueberry M3G on intestinal microbiota diversity in HepG2 cells-reduced HCC mice

As shown in Fig.2A,both LM3G and HM3G improved the diversity(Simpson)and abundance(ACE)of intestinal microbiota in HCC mice,whereas HM3G showed a more significant effect(P ＜0.01).It is interesting to note that ACE index of LC group also increased,the abundance of some pathogenic microbiota could not be excluded.As shown in Fig.2B, we found that in the first 7 days, the distance between and within four groups were relatively close,which indicated that there was little difference in intestinal microbiota structure.With the passage of time, the distance between each group was scattered, LC and 5 F were far away from LM3G and HM3G on day 21,explaining that the structure of intestinal microbiota in HCC mice was changed.

3.2.Effect of blueberry M3G on intestinal microbiota structure in HepG2 cells-reduced HCC mice

As shown in Fig.3A, we found that Bacteroidia, Clostridia and Verrucomicrobiae were dominant in the structure of intestinal microbiota in HCC mice.The abundance of Bacteroidia and Clostridia was decreased significantly with time in intestinal of LC mice(P ＜0.05),but LM3G,HM3G and 5 F(P ＜0.01)controlled the decline trend of the former and increased the abundance of the latter.It is also noted that the abundance of pathogenic Erysipelotrichi has an increasing trend in LC mice, but decreased significantly after supplementation of LM3G(P ＜0.05),HM3G(P ＜0.01)and 5 F(P ＜0.05)(Fig.3B).

Fig.4.Effect of blueberry M3G on the overall microbial carbohydrate metabolism in HepG2 cells-reduced HCC mice.

As shown in Fig.3C, Unclassified S24-7, Unclassified Clostridiales and Bacteroides are significantly enriched in intestinal of HCC mice.The abundance of Clostridiales and Oscillospira in LC mice was decreased, whereas the other three groups produced opposite results.Moreover, the level of Ruminococcus in LC mice was lower than that from other three groups.After treatment,the abundance of Ruminococcus was significantly enriched,especially mice supplemented with blueberry M3G(Fig.3D).

Table 1 Effect of blueberry M3G on microbial TCA cycle KEGG pathway in HepG2 cells-reduced HCC mice

3.3.Effect of blueberry M3G on the overall microbial carbohydrate metabolism in HepG2 cells-reduced HCC mice

As shown in Fig.4,the expression of amino sugar and nucleotide sugar metabolism, fructose and mannose metabolism, galactose metabolism and inositol phosphate metabolism in LC and 5fincreased first and then decreased,while LM3G and HM3G shown an upward trend in the end.In the other three microbial kegg pathways(starch and sucrose metabolism,butanoate metabolism and c5-branched dibasic acid metabolism), HM3G significantly increased the expression level compared with the other three groups.In addition,the expression level of microbial TCA cycle kegg pathway in HM3G and 5 F was higher than LC and LM3G.

3.4.Effect of blueberry M3G on microbial TCA cycle KEGG pathway in HepG2 cells-reduced HCC mice

As shown in Fig.5, the microbial TCA cycle KEGG pathway was divided into four modules.In the fatty and biosynthesis and fatty acid elongation in mitochondria pathway (#module1), the abundance of porA in the synthesis of acetyl-coa and pyruvate in intestinal of LC mice was significantly reduced, no statistical differences could be found in HCC mice among treatment groups.It is interesting to note that LM3G,HM3G and 5 F may prevent oversynthesis of acetyl-coa via regulating the level of KorA(Fig.5A).

The abundance of DLAT synthesized by acetyle-coa decreased significantly on 21 days compared with 7 days in LC group in the#module2 KEGG pathway(Valine,Leucine&,isoleucine degradation and Fatty acid metabolism),but blueberry M3G balanced the expression level of the DLAT and 5 F enriched it.In addition,blueberry M3G could regulate the pathway of #module2 by enriching the aceE of synthetic Thpp and pyruvate(Fig.5B).

In the#module3(Alanine,aspartate and glutamate metabolism and Clyaxylate and dicarboxylate), the abundance of PC in HM3G group was decreased, whereas LC, LM3G and 5 F enriched it(Fig.5C).In LC and 5 F mice, the level of OGDH synthesized from Thpp and 3-carboxy-1-hydroxypropyl-Thpp was decreased, but mice supplemented with blueberry M3G could regulate the expression of OGDH(#module4,Ascorbate and aldarate metabolism and D-Gln&D-Glu metabolism).Moreover,KorA was overexpressed in HCC mice, but the level of KorA was regulated after treatment via blueberry M3G and 5 F(Fig.5D).

3.5.Correlation analysis(R)between functional proteins with significant changes in microbial TCA cycle KEGG pathway and corresponding intestinal microbiota

As shown in Table 1, we found that proteins #494675 and#833268 were decreased in LC(compared to day 7 and day 21)and increased in LM3G,HM3G and 5 F.Proteins#3190,#3191,#3368,#3369, #63417, #3367, #12019, #146350, #2695988, #1241186 and #15267 were enriched in LC and LM3G mice, while supplementation of HM3G and 5 F reduced them.In addition,among the other three proteins (#5594, #1377786 and #93504), only HM3G reduced their expression.

As shown in Fig.6, the expression of protein #93504 and the abundance of Barnesiella viscericola were significantly negatively correlated in the gut of HM3G (R=-0.754, P=0.456) and 5 F (R=-0.984, P=0.113) mice, but opposite in the LC (R=0.974, P=0.146)and LM3G (R=0.998, P=0.038) mice (Fig.6A).In addition, the expression of a large number of proteins was negatively correlated with the abundance of pathogenic Muribaculum intestinale in LM3G and HM3G mice,which revealed that M.intestinale may be an important intestinal microbiota involved in microbial TCA cycle kegg pathway,and blueberry M3G could enriched M.intestinale in the gut of mice.

4.Discussion

BA can improve intestinal microbiota and liver metabolism abnormalities in mice via targeting the gut-liver axis[18].Liu et al.[13] found that BA monomers such as malvidin-3-O-galactoside,malvidin-3-O-glucoside and petunidin-3-O-glucoside could significantly improve the diversity of intestinal microbiota.In this study,we also found that blueberry M3G increased the alpha diversity(ACE and Simpson) and significantly regulated the beta diversity(Fig.2) of intestinal microbiota in mice.Previous study showed that Clostridia is a beneficial symbiotic microbiota that can promote regulatory T cell differentiation and alleviate colitis in mice by producing butyrate [19].It has also been found in human trial that the abundance of Erysipelotrichi in the gut was positively correlated with the development of fatty liver and predictive of the development of fatty liver in patients[20].Our study showed that blueberry M3G could improve the abundance of Clostridia and decrease Erysipelotrichi in the gut of mice (Fig.3B).In addition,blueberry M3G also has a good effect in regulating the abundance of Oscillospira and Ruminococcus which are proportional to human health(Fig.3D).Taken together,we believe that blueberry M3G has an advantage in regulating intestinal microbiota structure in HCC mice.Therefore, compared with the anticancer drugs with strong side effects such as 5 F,blueberry M3G has potential intestinal regulation in HCC patients.

The liver can receive blood from multiple organs, such as the intestinal tract, and plays a core role in many metabolic pathways,microbial metabolic synthesis or transformation of biomass by microbiota,and physiological functions such as bile acid regulation by microbiota[21].During the development of liver diseases,the structure of intestinal microbiota changes in a characteristic way.Toxins produced by intestinal dysbiosis and metabolites such as secondary bile acids, fatty acids and lipopolysaccharides play a mediating role in microbial metabolism KEGG pathways,and endotoxin enter the gut-liver circulation through intestinal barrier and portal vein,thus aggravating the development of liver diseases[22].In this study,we found that the expression level of microbial carbohydrate metabolism KEGG pathway was changed due to the liver cancer, and blueberry M3G regulated this pathway to a certain extent(Fig.4).We focused on the microbial TCA cycle KEGG pathway and found that blueberry M3G has certain advantages over 5fin the regulation of most functional proteins (Fig.5).Qin et al.[23] demonstrated that the excessive increase of acetyl-coA may aggravate the metastasis of cancer cells.In this study, blueberry M3G avoids the excessive synthesis of acetyl-coA via increasing the abundance of porA and regulating the expression of korA (#module 1).Mohamadzadeh et al.[24]found that Propionibacterium can increase the content of Th17 cells in the gut by increasing the expression of DLAT[24].In this study,the abundance of DLAT was not significantly different in mice supplemented with blueberry M3G, but decreased in LC mice (#module2).An animal study has shown that PC is an important intermediate product in the maintenance of TCA cycle,which can improve the antioxidant capacity and redox metabolism of liver cycle[25].However,we found PC was significantly enriched in LC group, LM3G and 5 F, while the abundance in HM3G was decreased (#module3).The causes of above phenomenon may include differences between mice species and disease types.In addition,compared with metabonomics,metagenomics can’t determine the content of metabolites, and can only judge the change of metabolic function and the key gut microbiota behind it, which is also the limitation of this study.In summary,from the overall regulatory effect, blueberry M3G still regulates the microbial TCA cycle KEGG pathway in a positive direction,and further studies on the more profound molecule mechanism can be conducted on this basis.

Fig.5.Effect of blueberry M3G on microbial TCA cycle KEGG pathway in HepG2 cells-reduced HCC mice(A)fatty acid biosynthesis and fatty acid elongation in mitochondria(B)valine,leucine&,isoleucine degradation and fatty acid metabolism(C)alanine,aspartate and glumate metabolism and clyaxylate and dicarboxylate(D)ascorbate and aldarate metabolism and D-Gln&D-Glu metabolism.

Fig.5.(Continued)

Based on above results, we conclude that 5 F and blueberry M3G can regulate intestinal microbiota structure and microbial TCA cycle KEGG pathway in HCC mice to some extent.However,blueberry M3G has an advantage in regulating the abundance of certain microbiota and functional proteins.In conclusion, combined with our previous study,we believe that M3G,a blueberry anthocyanin(BA) monomer, could be a potential candidate and an intestinal microbiota regulator for treating liver cancer.

Declaration of Competing Interest

There are no conflicts to declare.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31972090), LiaoNing Revitalization Talents Program (XLYC1807127), Liaoning BaiQianWan Talents Program(2018-B-21), Tianzhu Mountain Scholar Support Project (2018),the Innovative Talent Support Program for Institution of Higher Learning of Liaoning Province (LR2017038) and the project of“double hundred” for major scientific and technological achievements transformating of Shenyang Science and Technology Bureau(Z19-3-012).The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

Fig.6.Correlation analysis (R) between functional proteins with significant changes in microbial TCA cycle KEGG pathway and corresponding intestinal microbiota (A)correlation between positive and negative is not obvious(B)positive and negative correlation is obvious(*,R ＜0.5;**,0.5 ＜R ＜0.8;***,R ＞0.8).

Fig.6.(Continued)