Nature Reviews Gastroenterology and Helpatology Volume 15 October 2019 589

Objective. Recently, huckleberry has been identified as a candidate for the treatment of liver fibrosis. Given the role of gut-liver centrality in liver fibrosis and the importance of the gut microbiota homeostasis to the maintenance of the intestinal epithelial bulwark, this report aimed to investigate whether blueberry could benumb liver fibrosis and protect the intestinal epithelial barrier by maintaining the homeostasis of the gut microbiota. Method. A CCl_four-induced rat liver fibrosis model was used to detect the roles of blueberry in liver fibrosis and intestinal epithelial barrier. The liver weight and torso weight were measured, the liver function was monitored by ALT and AST activity, protein and mRNA were determined by western absorb and RT-qPCR, and the gut microbiome was detected by Miseq. Results. The results showed that huckleberry could reduce the rate of liver weight/body weight gain ( ), ALT ( ) and AST ( ) activity, and the expression of collagen I ( ), collagen IV ( ), and α-SMA ( ) expression in CCl₄-induced rat liver. CCl₄ impaired the abdominal epithelial barrier and decreased the expression of the tight junction poly peptide. Blueberry restored the abdominal epithelial barrier and increased the expression of the tight junction protein. The gut microbiota homeostasis was impaired past CCl_iv, but after handling with blueberry, the intestinal flora returned to normal. Conclusion. Blueberry attenuated liver fibrosis, protected intestinal epithelial barrier, and maintained the homeostasis of the gut microbiota in a CCl_four-induced injury rat model.

one. Introduction

Liver fibrosis refers to the excessive aggregating of extracellular matrix (ECM) proteins, including hepatic collagen, which results from chronic liver injury and occurs in most chronic liver diseases [ane]. The main causes of liver fibrosis include chronic HCV/HBV infection, alcohol abuse, and nonalcoholic steatohepatitis (NASH) [i]. Advanced liver fibrosis leads to liver cirrhosis, resulting in portal hypertension and liver failure and oft requires liver transplantation. Liver fibrosis is also a key risk gene for hepatocellular carcinoma progression [2]. The liver injury model induced by CCl_four has been widely used in the study of liver fibrosis, liver necrosis, and medicinal found extract assessments [3, iv]. Continuous CCl_iv treatment increases hepatic prison cell damage and results in pathological processes of hepatocytes, which undergo apoptosis and regeneration and gradually render to normal. However, when necrotic liver cells exceed their regenerative capacity, they can lead to liver prison cell damage and fifty-fifty liver failure [5].

The abdominal tract and the liver are anatomically and physiologically connected. The relationship between the two has been called the "gut-liver centrality," and the effects of intestinal metabolites on the liver are essential to the onset and progression of liver diseases [6–8]. The gut microbiota, in particular, has recently emerged as an important gut-liver axis-mediated factor [9–11]. Alterations of the abdominal microbial composition could induce liver damage and initiate liver fibrosis changes to the development of cirrhosis and associated complications [12]. The tight junctions (TJ) inside the gut epithelium represent a natural barrier to bacteria and their metabolic products [xiii]. Breaking downward the bulwark allows harmful components to injure the liver. Therefore, the gut-liver axis is an important focus for the treatment of liver diseases.

Blueberry (BB) is a flowering plant that belongs to Vaccinium spp. of the family Ericaceae. The Human Nutrition Enquiry Center (Mayer, Usa) has conducted a serial of in-depth studies on blueberries. These studies have indicated that blueberries contain anthocyanins, polyphenols, and flavonoids and appear to have the highest antioxidant chapters among common fruits and vegetables [fourteen, fifteen]. Blueberries may besides have anti-inflammatory and antitumor furnishings [sixteen, 17]. Recently, BB has been found to be a potential candidate for the treatment of liver fibrosis [xviii–21]. However, the underlying mechanisms remain unclear.

In this study, we aimed to investigate the preventative effects of BB treatment on CCl₄-induced liver fibrosis, intestinal epithelial barrier disruption, and gut microbiota imbalance. The results demonstrated that treatment with BB improved liver fibrosis, intestinal epithelial barrier residual, and gut microbiota homeostasis.

two. Materials and Methods

two.1. Chemicals and Materials

CCl_iv and other chemicals were supplied by Chengdu Jinshan Chemical Reagent Co., Ltd. CCl_iv was dissolved in vegetable oil.

2.2. Animals and Experimental Blueprint

Male Sprague-Dawley rats weighing 200–250 m were selected for apply in the study. They were obtained from the Laboratory Animal Heart of the Third Military Medical University of the People's Liberation Army. Rats were housed in a low-cal- and temperature-controlled room on a 12/12 h light/nighttime cycle. The animals were allowed costless admission to food and water and were kept in SPF. The whole experiment was conducted in accordance with the guidelines of the Animal Care and Employ Committee of Guizhou Medical University.

Rats were randomly divided into two groups equally follows: Ctrl group and CCl₄ group. The rats in the Ctrl grouping were injected with vehicle every other day for viii weeks, while the rats in the CCl₄ group were injected every other 24-hour interval with CCl₄ (3 ml/kg forty% CCl₄; i.p.) for viii weeks. The Ctrl grouping was randomly subdivided into two groups every bit follows: (1) Ctrl (command) group (n = half dozen): animals were fed with 10 ml/kg saline daily by gavage and (2) BB (blueberry) group (n = 6): rats were perfused daily with x ml/kg BB juice by gavage. The CCl₄ grouping was also randomly subdivided into two groups: (three) CCl_four (north = half-dozen): rats were injected with CCl₄ (3 ml/kg xl% CCl₄; i.p.) once a week, and 10 ml/kg saline was administered daily by gavage and (iv) CCl₄ + BB group (n = 6): rats were injected with CCl_iv (three ml/kg xl% CCl_four; i.p.) once a week, and 10 ml/kg BB juice was administered daily by gavage. The rats were fed for some other 8 weeks for a total of 16 weeks.

2.3. Blood and Tissue Samples

At the end of the treatment, all rats were sacrificed past cardiac puncture under sodium thiopental anesthesia (l mg/kg, i.p.). Claret was nerveless in dry tubes. The livers and colon tissues were rapidly removed, washed in 0.9% NaCl, and stored in ice. The materials were stored at −80°C until the master assay.

2.4. Determinations in Serum

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) measurements were performed on a Siemens ADVIA-2400 automatic biochemical analyzer.

two.v. Western Blot Analysis

Total protein was extracted from the rat liver and colon with RIPA buffer. Protein concentration was determined via BCA assay. Equal amounts of protein and 5x SDS loading buffer were mixed and boiled for v min. Proteins (30μg per lane) were separated via SDS-Folio on a 10% gel and transferred onto polyvinylidene difluoride membranes (Zhongshan Jinqiao Biological science and Technology Co., Ltd., Beijing, Communist china). Membranes were blocked with 5% nonfat milk for 1 h at room temperature. The membranes were incubated with primary antibodies against Collagen I (3241980-1, Abcam), Collagen IV (AA06195689, Bioss), α-SMA (GR282976-35, Abcam), Claudin1 (ab15098, Abcam), Claudin2 (ab53032, Abcam), ZO1 (21773-1-AP, Proteintech Group, Inc), and β-actin overnight at 4°C, respectively. Following primary incubation, membranes were incubated with horseradish peroxidase-labeled goat antimouse or antirabbit immunoglobulin 1000 secondary antibiotic (A0208, Beyotime Biotechnology) for one h at room temperature. Poly peptide bands were visualized using an ECL Prime Western Blotting Detection Reagent (RPN2232SK, Shanghai Haoran Biotechnology Co., Ltd.).

ii.half-dozen. Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)

Full RNA was extracted from the colon using TRIzol reagent (TAKARA). Total RNA (twoμthousand) was reverse transcribed into cDNA using the BestarTM qPCR RT kit (DBI). To find the mRNA expression levels of cluadin1, cluadin2, and ZO1, qPCR was performed using the BestarTM qPCR MasterMix (DBI) on an ABI 7900 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The post-obit primer pairs were used for qPCR: claudin1: forward, 5′-GGACAACATCG TGACTGCTC-3′ and contrary, v′-CCCAGCAGG ATGCCAATTAC-three′; claudin2: forward, 5′-GCTGTAGTGG GTGGAGTCTT-three′ and reverse, five′-GGCCTGGTAG CCATCATAGT-3′; ZO1: forrard, five′-CACCTCGCACG TATCACAAG-3′ and reverse, v′-GGCAATGACAC TCCTTCGTC-3′; GAPDH: forward, 5′-CCTCGTCTCAT AGACAAGATGGT-three′ and reverse, 5′-GGGTAGAGTCATA CTGGAACATG-3′. The thermocycling conditions for qPCR were every bit follows: initial denaturation at 95°C for 10 sec and forty cycles of 95°C for 10 sec and sixty°C for threescore sec. The expression was quantified using the 2^−ΔΔCq method and normalized to the internal reference factor GAPDH.

ii.7. Histological Analysis

The liver and colon tissues were alkane-embedded after fixation and cut into 5μm thick sections. Afterwards, the haematoxylin and eosin (H&Due east) stain was used to stain sections of liver and colon for histological assay according to standard instructions. According to the standard instructions, Masson's stain was also performed, with the results showing reticulin fibers in the fibrotic areas. In order to randomly select microscopic areas in liver sections for exam, a Ti-S inverted fluorescence microscope (Nikon, Tokyo, Nihon) was used.

2.8. 16S rRNA Gene V4 Amplification, Quantitation, and Sequencing

The sample was subjected to targeted metagenomic analysis by sequencing the V4 region of the 16S rRNA factor. Primers modified from Caporaso et al. [22] were utilized to conduct V4 amplification. As a brief introduction, these primers were designed to amplify the 16S rRNA gene from 515 to 806, which includes a barcode and an adapter for annealing to the Illumina flow jail cell. The primers in this report differed from those in Caporaso et al. considering both of the primers independent a 12 bp barcode and not just the reverse primer (Dataset S25). This makes it possible to pool many samples using a unique combination of barcodes, overcoming the dependence on a large number of reverse primers with unique barcodes. The PCR reaction mixture was prepared using Qiagen HotStar HiFidelity polymerase. Qiagen HotStar HiFidelity polymerase was employed to set the PCR reaction mixture. Each mixture had a volume of 25μL and was polymerized using 0.fiveμ50 HotStar 92 polymerase, iμ50 sample Deoxyribonucleic acid, two.fiveμL reverse primer (10μM), 2.5 91μ50 forward primer (10μGrand), 5μL HotStar PCR Buffer, and xivμ50·H₂O. A touchdown PCR programme was utilized on a Biometra TProfessional Bones Gradient thermocycler: 95°C for v min, followed by 7 cycles of 95°C for 45 sec, 65°C for 1 min (decreasing at 2°C/94 cycle), and 72°C for 90 sec, followed by 30 cycles of 95°C for 45 sec, 50°C for 30 sec, and 72°C for 95 sec. A final extension at 72°C was carried out for 10 min, and the reactions were held at 4°C. The reactions were run on a ane% agarose gel in order to ensure the success of the amplification. Unsuccessful reactions were followed past 1 more than trial, failure of which would atomic number 82 to removal from the experiment. The amplicon libraries were diluted 40x. To quantify the amplicons, either an Agilent Bioanalyzer (greenhouse libraries) or a Caliper LabChip GX (field experiment libraries) was used at the Deoxyribonucleic acid Technologies Cadre at the Genome Center, UC Davis. The libraries were then pooled into 4 pooled libraries (ii for the greenhouse experiment and ii for the field experiment) at equivalent concentrations. To remove any primer dimer from the polymerized amplicon libraries, the 4 pooled libraries were run on 1.8% 5 agarose gels and a 400 bp band was extracted. Every bit a final check of quality, the bands were subjected to purification (Macherey-Nagel Nucleospic Gel and 104 PCR Cleanup kit) and bioanalysis. Later, each library was submitted to the UC Davis DNA Technologies core for 250 × 250 paired end and dual index sequencing on an Illumina MiSeq instrument.

ii.ix. Sequence Assay

According to the barcode sequences, a customized Perl script based on the principle of exact matching was utilized to decompile the sequences obtained from the MiSeq runs. MOTHUR'south command brand.contigs was adopted to form contiguous reads by overlapping these sequences [23]. A read containing any base of ambiguity, forth with any read exceeding 275 bp was so discarded. The sequences were then clustered into operational taxonomic units (OTUs) past UCLUST [24] based on 97% pairwise identity. This was performed using QIIME's [25] open reference OTU picking strategy based on the Greengenes 16S rRNA database (13_5 release) as a reference [26]. Using default parameters, the QIIME's version of the Ribosomal Database Project's classifier [27] was pitted against the Greengenes 16S rRNA database (13_5 release) to develop a taxonomy of the representative sequences for each OTU. All OTUs identified as belonging to chloroplast and mitochondria were deleted from the data set. PyNAST [28] in QIIME was adopted for alignment of the representative sequences for each OTU. QIIME's implementation of ChimeraSlayer [29] was performed to identify the Chimeric OTUs, which were so removed from the OTU table and OTU representative sequence file. FastTree [30] generated a phylogenetic tree from the alignment file.

2.10. Statistical Analysis

Data were reported as mean ± standard difference. All statistical analyses were performed using GraphPad Prism (Version 8; GraphPad Software, Inc., La Jolla, CA, USA). The ii-way ANOVA test was used to analyze differences among the four groups. Statistical significance was obtained at .

iii. Results

iii.1. Blueberry Improved CCl_iv-Induced Liver Weight Loss and Liver Function

To define the furnishings of BB on CCl_iv-induced liver injury, rats were injected with CCl₄ for viii weeks and then subjected to treatment with BB by gavage for another 8 weeks. Throughout the entire procedure, the rats' body and liver weight were constantly monitored. The results demonstrated that no significant difference in body weight was institute between the control group and the experimental group (Figure 1(a)). The administration of CCl_four increased liver weight, and handling with BB prevented CCl₄-induced liver weight gain ( ) (Figure 1(b)). A significant increase in the serum activities of ALT and AST, ii reliable indicators of liver injury [21], is bear witness of hepatocellular damage. Treatment with CCl₄ indeed contributed to a significant increment in serum ALT and AST. Information technology was observed that these enzyme activities decreased in CCl_four-treated rats due to BB handling (Figures 1(c) and i(d)). The differences of ALT and AST were statistically meaning ( for ALT, for AST). These data indicated that blueberry could amend CCl₄-induced liver damage.

3.two. Blueberry Attenuated CCl_iv-Induced Liver Fibrosis

Then, the role of BB in CCl₄-induced liver fibrosis was examined. After HE and Masson's staining, there were signs of CCl₄-induced liver collagen fiber regeneration, diffuse steatosis, hepatic plate disorder, and lobular construction destruction (Figures 2(a) and 2(b)). Treatment with BB improved the dysfunction induced by CCl₄ (Figures two(a) and ii(b)). Western absorb was conducted in each group to notice the fibrotic poly peptide collagen I, collagen IV, and α-SMA. The results showed that handling with BB reduced the expression of collagen I, collagen Iv, and α-SMA induced past CCl_iv (Figures 2(c)–2(f)). The differences were statistically meaning ( ). These data also indicated that BB could alleviate CCl₄-induced liver fibrosis.

iii.3. Blueberry Protected Intestinal Epithelial Barrier Breakdown Induced by CCl_four

The tight junctions (TJ) within the gut epithelium stand for a natural bulwark to leaner and their metabolic products and play a key protective role in preventing liver injury [xiii]. Then, we tested whether BB could mitigate liver impairment past maintaining the balance of the intestinal epithelial barrier. CCl₄ disrupted the intestinal epithelial barrier and reduced the expressions of TJ protein (Effigy iii). Treatment with BB could improve the structure of colon (Figure iii(a)) and increase the poly peptide (Figures three(b) and 3(c)) and mRNA levels (Figure 3(d)) of claudin1, claudin2, and ZO1 reduced by CCl₄. The differences were statistically meaning ( for protein and for RNA). These data indicated that BB could improve the intestinal epithelial barrier.

3.4. Effects of BB on the Alterations of the Gut Microbiota Induced by CCl₄

An aggregate of 1,934,046 sequences was procured from the 24 faecal samples, with an average of 438.86 bp per sample. A full of 1352 OTUs (similarity greater than 97%) were adamant later quality and chemical composition analysis. As Figures four(a)–iv(c) suggest, a pregnant decrease in firmicutes and bacteroidetes was observed following CCL₄ treatment in the dominant phyla of the identified bacterial phyla, including Bacillus and Bacteroides. Later BB handling, the gut microbiota showed a recovery trend, suggesting that administration of BB helped foreclose CCL₄-induced hardness and changes in the number of bacteroides. After handling with BB, the gut microbiome showed a trend of recovery, suggesting that BB administration contributed to the prevention of CCl₄-induced alterations in the abundance of firmicutes and bacteroidetes.

In lodge to compare the differences betwixt groups with differing taxon affluence, a linear discriminant analysis effect size (LEFSE) method was adopted, with a higher score indicating greater consistency. Low-density lipoprotein A showed unlike taxa in the gut microbiota between different groups. LEFSE analysis was performed on the sequence of each sample to generate a cladogram. Through the cladogram, the dissimilar microbial communities of each group could be observed at dissimilar levels. Each circle represents the classification level from phylum to species. The diameter of the small circumvolve is proportional to the relative affluence of gut microbiota. Every bit shown in Figure 5, at that place were 22 largest differences in the intestinal flora (from phylum to species) of the four groups: the Ctrl grouping, the CCl₄ group, the BB group, and the CCl_iv + BB group (Figures 5(a) and five(b)). These data suggest that oral administration of BB considerably contributed to the prevention of CCl₄-induced alterations in the abundance of gut microbiota.

(a)

(b)

4. Word

The main cause of liver fibrosis is chronic liver injury, which is a reversible process in its early stage [31]. Previous studies have demonstrated that BB has a strong antiliver injury effect [18–21]. Withal, the underlying mechanisms of BB against liver fibrosis remain obscure. Therefore, this written report sought to elucidate these mechanisms of BB in liver fibrosis. In this study, the liver fibrosis model was induced by CCl₄ in rats. The serum ALT and AST enzymes' activities markedly increased after CCl₄ administration, indicating serious hepatic injury; treatment with BB significantly decreased the activity of these enzymes (Figures 1(c) and 1(d)). The H&East and Masson's staining showed that CCl₄ administration acquired severe histological damage of liver tissue; even so, treatment with BB notably attenuated the caste of liver injury (Figures 2(a) and 2(b)). These findings demonstrate that BB has a protective consequence against CCl_four-induced liver injury.

To ascertain the underlying mechanisms of BB in liver fibrosis, we first investigated the intestinal epithelial barrier. The abdominal epithelial barrier consists of multiple defense mechanisms, which can be divided into an epithelial (i.e., the mucus layer and epithelial cells) and an immunological barrier (i.eastward., the epithelial secretions and immune cells). In the electric current written report, our principal focus was on the epithelial barrier, specially the monolayer epithelial cells, which are interconnected by junctional complexes. On the one hand, the epithelium promotes the absorption of nutrients, water, and electrolytes in the lumen. On the other mitt, it acts as a barrier preventing the transport of potentially harmful substances through the extracellular and paracellular compartment transport [32]. Paracellular transport is regulated by the apical junctional complex and consists of the tight junction (TJ) and the subjacent adherent junction (AJ) [33].

The TJs seal the paracellular space and course a selective barrier that allows transport via at least two pathways: a loftier-capacity, charge-selective pore pathway for small ions and uncharged molecules and a depression-capacity leak pathway for larger molecules, regardless of accuse [34]. TJs are considered highly dynamic, opening and endmost continuously in response to various stimuli [35]. They consist of several transmembrane proteins, such as occludin, members of the claudin family, and junction adhesion molecules, as well as cytoplasmic plaque proteins, such as the zonula occludens proteins (i.eastward., ZO-one, ZO-two, and ZO-3), which connect the transmembrane proteins with the perijunctional actomyosin ring [33, 36]. Contraction of this ring is important in regulating paracellular permeability and is mainly mediated by activation of myosin light concatenation kinase (MLCK), which phosphorylates myosin 2 regulatory light chain (MLC) [33, 36]. In this study, we demonstrated that BB could improve the intestinal epithelial barrier cleaved downward by CCl₄ (Figure 3).

Now normally referred to equally "a new virtual metabolic organ," the gut microbiota (GM) is a diverse ecosystem of archaea, leaner, fungi, protozoa, and viruses that produces pathogenesis of the gastrointestinal tract, liver, respiratory, cardiovascular, endocrine, and many other diseases [37]. The gut-liver centrality, which has received increasing attention in recent years, is the consequence of close anatomical and functional bidirectional interactions between the alimentary canal and the liver, primarily through the portal vein [38]. The symbiotic human relationship between GM and the liver is regulated and stabilized by a complex network of interactions, including metabolism, immunity, and neuroendocrine crosstalk between them [39]. Excessive intake of tissue-subversive foods, such equally alcohol, CCl_iv, and/or loftier-fat diets (HFD), weakens the intestinal barrier function and produces large amounts of intestinal microbial components (then-called microbial-associated molecular patterns (MAMPs)), bacterial metabolites, and even intestinal microbiota, which are easy targets of transfer to the liver. Equally a outcome, this can lead to serious liver diseases, such as liver inflammation, fibrosis, and cancer [6]. Therefore, these intestinal microbial components and metabolites not only touch on the abdominal tract where the gut microbes are located but as well the organs that are located away from the gut through their systemic circulation [forty, 41]. Our data demonstrated that BB could foreclose CCl_four-induced alterations in the abundance of gut microbiota (Figures four and 5).

In decision, the current study has demonstrated that BB prevented CCl₄-induced gut microbiota alterations to attenuate liver fibrosis and maintain the balance of the abdominal epithelial bulwark.

Data Availability

The information used to back up the findings of this study are available from the corresponding writer upon asking.

Conflicts of Involvement

All authors declare no potential conflicts of involvement about this commodity.

Acknowledgments

This written report was supported past the National Natural Science Foundation of China (no. 81460484). The authors would like to thank Annoroad Gene Technology Co., Ltd. for the technical aid with 16S rDNA sequencing and analysis.

Copyright

Copyright © 2019 Zhiqiang Yan et al. This is an open up access commodity distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Source: https://www.hindawi.com/journals/cjgh/2019/5236149/

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