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Perspective
Intestinal barrier function-NAFLD interactions
and possible role of gut microbiota
yizhe cui, qiuju wang, renxu Chang, xiaocui zhou, and chuang xu
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00080 • Publication Date (Web): 25 Feb 2019
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Page 1 of 31 Journal of Agricultural and Food Chemistry

1 Intestinal barrier function-NAFLD interactions and possible role of gut microbiota

2 Yizhe Cui,† Qiuju Wang,† Renxu Chang,† Xiaocui Zhou,‡ and Chuang Xu†*

3 †College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, No.

4 2 Xinyang Road, New Development District, Daqing, Heilongjiang, China 163319;

5 ‡China Animal Health and Epidemiology Center, Laboratory of Zoonosis, Qingdao, China 266000.

6 Short running head: Promoting intestinal health to relieve NAFLD

7 Corresponding author:Chuang Xu

8 College of Animal Science and Veterinary, Heilongjiang Bayi Agricultural University, Daqing,

9 Heilongjiang Province, China 163319. Email: xuchuang7175@163.com & xuchuang@byau.edu.cn;

10 Telephone:86-13936967175; Fax number:86-0459-6819121

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23 ABSTRACT

24 Nonalcoholic fatty liver disease (NAFLD) is a metabolic stress liver injury that is closely related to

25 obesity, insulin resistance (IR), type 2 diabetes, atherosclerosis and metabolic syndrome. The

26 pathological features are diffuse hepatic vesicular steatosis, including nonalcoholic steatohepatitis, liver

27 fibrosis, and even liver cancer. A variety of pathological outcomes cause serious harm to human health.

28 At present, an increasing number of researchers are investigating the pathogenesis of NAFLD from the

29 perspective of changes in the function of the intestinal barrier. The physical, chemical, immunological

30 and microbiological barriers in the intestinal tract constitute the complete intestinal barrier, which plays

31 an important defensive role against the invasion of harmful substances from the intestines. Protecting

32 the function of the intestinal barrier is a new way to treat NAFLD and its related diseases. In this

33 review, we summarized the current knowledge of the role of the intestinal barrier in NAFLD.

34 Key words: barrier function, intestinal microflora, gut-liver axis, oxidative stress, NAFLD

35 Introduction

36 The pathogenesis of nonalcoholic fatty liver disease (NAFLD) is not yet fully understood; currently,

37 researchers are investigating the “two-hit” hypothesis.1 The “first hit” , which leads to the formation of

38 NAFLD, refers to an increase in the level of free fatty acid (FFA) in the adipocytes and a decrease in

39 the oxidation of FFAs in the liver, resulting in the excessive accumulation of fat in the liver cells. The

40 “second hit” refers to the release of inflammatory cytokines and the increase in the level of oxidative

41 stress in NAFLD and the consequent persistent damage to the liver. However, many other factors,

42 including the complex interplay of genetic, environmental, dietary and metabolic influences and the

43 intestinal microbiome, can generate the nonalcoholic steatohepatitis (NASH) phenotype, and NASH

44 may not always be preceded by NAFLD. In addition, because of the complex role of gut microflora in

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45 maintaining human health and disease formation, NAFLD is considered to be a typical intestinal

46 microflora-related disease. The theory of the gut-liver axis suggests that there is a relationship between

47 liver diseases and the intestinal tract.2 Changes in the function of the intestinal barrier are closely

48 related to the “second hit” and are involved in the development of NAFLD. The intestinal barrier is a

49 complex structure composed of the following four main barriers: microbiological, chemical, physical

50 and immunological. These barriers play important roles in maintaining the stability of the internal and

51 external environments (Fig 1). In the pathological conditions of hypoxia, inflammatory stimulation, and

52 bacterial overgrowth in the small intestine, the permeability of the intestinal mucosal barrier increases,

53 and intestinal bacteria and their products enter the circulation through the damaged intestinal mucosal

54 barrier and move to the liver. In many studies, alterations in the gut microbiota were reflected in

55 metabolic disorders, which involved a variety of metabolic diseases, such as type 2 diabetes (T2D),3

56 obesity,4 cardiovascular disease,5 and NAFLD.6 Based on these findings, a new treatment direction

57 involving the intestinal barrier was proposed. In this review, we described our current understanding of

58 the function of the intestinal barrier and the potential role of intestinal microbiota in NAFLD. In

59 addition, we briefly summarized the status of novel microbiota therapy in clinical and experimental

60 research.

61 Mechanistic advances in gut microbial-mediated NAFLD development

62 The intestinal microbial barrier is composed of the healthy intestinal flora. Because of a

63 complementary role between the gut microbiota and the liver in many ways, that is even called

64 “metabolic organ”.7 There are 100 times more genes in the intestinal flora found in the human body

65 than in the human genome. Under normal conditions, the intestinal microflora is in balance with the

66 internal and external environments, and it plays an irreplaceable role in activating the human immune

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67 system and helping the human body digest and absorb nutrients.8 Increasing numbers of studies have

68 shown that bacterial growth in the small intestine is involved in the development of NAFLD,9 mainly

69 due to the following aspects (Fig 2): 1) The endogenous ethanol produced by the intestinal microflora

70 can lead to bacterial translocation by disrupting the intestinal mucosal barrier and increasing intestinal

71 permeability, eventually increasing the level of lipopolysaccharides (LPSs) in the portal vein.10 2)

72 Choline has a high affinity for triglycerides, and intestinal microbes metabolize choline in the host diet,

73 promoting the formation of fatty phospholipids in the liver and reducing the deposition of fat in the

74 liver to prevent the occurrence of NAFLD. Intestinal bacteria convert choline into toxic dimethylamine

75 and trimethylamine by the enzymes.11 These poisonous amines are absorbed by the liver to produce

76 trimethylamine-N-oxide (TMAO), which causes liver disease and ultimately choline deficiency.

77 However, mice cannot secrete trimethylamine, which demonstrated the important role of intestinal

78 microflora in choline transformation.12 Choline is an important component of membrane phospholipids

79 and plays an important role in biochemical processes. Choline promotes fat transport in the form of

80 phospholipids, thus preventing abnormal accumulation of fat in the liver;13 when cells lack choline,

81 hepatocyte death and hepatic steatosis often occur.14 Choline deficiency may affect the composition of

82 gut microbiota and the potential development of fatty liver.15 Studies have shown that the composition

83 of intestinal microflora varies with dietary choline content. The relationship between choline

84 metabolism and intestinal microflora provides a new perspective for the development of targeted

85 therapy for intestinal microflora in NAFLD. 3) The release of LPSs from intestinal microbes can

86 activate inflammatory-related cytokines in intestinal epithelial cells (iECs) and promote the

87 development of NAFLD.16 Small intestinal bacterial overgrowth (SIBO) is characterized by excessive

88 production of gram-negative bacteria in the proximal part of the small intestine.17 Researchers found

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89 that under NASH pathological conditions, SIBO significantly increased the levels of tumor necrosis

90 factor α (TNF-α), alanine aminotransferase (ALT), and serum aspartate aminotransferase (AST).18 In

91 addition, the prevalence of SIBO and TNF-α was higher than that of the control group. 19 These

92 findings may be related to the disturbance of the gut microflora, which inhibits the expression of

93 specific tight junction proteins, i.e., obstructive proteins and ZO-1, leading to increased intestinal

94 permeability.20

95 In terms of bacterial structure, some researchers found that compared with the simple fatty liver (SFL)

96 group and healthy group, the NASH group had a lower proportion of Bacteroides (P<0.05).21 A recent

97 randomized controlled trial showed that the abundance of anaerobes, such as Lactobacillus and

98 Bifidobacterium, was reduced in the NASH group (P<0.05) compared with the healthy group and the

99 SFL group, while the abundance of Enterobacteriaceae and other aerobic bacteria was significantly

100 increased (P<0.05).16 Scientists speculated that the increased intestinal permeability of hereditary obese

101 mice leads to increased portal endotoxemia, which increased the sensitivity of hepatic stellate cells to

102 bacterial endotoxin.22 In NAFLD patients, increased intestinal permeability was five times more likely

103 than in the control group.23 Likewise, the mechanism of NAFLD fibrosis induced by endotoxin may be

104 closely related to intestinal permeability.24

105 In addition, metabolic disorders, nutritional factors, genetic factors and bacterial dysfunctions play

106 important roles in the development of NAFLD.25 In terms of metabolism, disordered lipid metabolism

107 can induce intestinal flora disorder, and intestinal flora imbalance can aggravate disordered lipid

108 metabolism. Both lipid metabolism and intestinal flora disorders are related to the development of fatty

109 liver.26 LPS can directly stimulate hepatic stellate cells and Kupffer cells (KCs) and induce

110 steatohepatitis.27 As an LPS sensor, Toll-like receptor 4 (TLR4) plays an important role in the

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111 regulating innate immune response and insulin resistance (IR).28

112 NAFLD also is associated with IR, diabetes and obesity. First, IR increases the mobilization of fatty

113 acids from adipose tissue that are transported to the liver and may serve as a substrate for excess

114 triglyceride (TG) production leading to steatosis. Second, a large number of harmful substances, such

115 as LPS, produced by SIBO, can induce bacterial translocation and liver oxidative stress, causing liver

116 damage and fibrosis and leading to the occurrence of NASH.29 LPS can promote the release of TNF-α

117 in macrophages in the liver and promote the development of fatty liver through the sensitization of

118 TNF-α. In addition, TNF-α is associated with susceptibility to IR. This cytokine plays an important role

119 in the interaction between lipid accumulation and the inflammatory reaction in the human body.30 In

120 conclusion, changes in intestinal bacteria may lead to IR, which indicates that maintaining intestinal

121 barrier integrity and using intestinal microbial population manipulation as a new therapeutic strategy

122 for NAFLD have potential significance.31

123 Role of intestinal chemical barrier in NAFLD development

124 The intestinal mucosal chemical barrier refers to the gastric secretion of gastric acid, mucus, mucin,

125 bile, glycoproteins, mucopolysaccharides, digestive enzymes, lysozymes and other chemical

126 substances. These substances can alter the attack sites of pathogenic bacteria and act as a chemical

127 barrier by destroying antigens in the diet and defending against microorganisms invading the small

128 intestine.32 The outer loose mucus layer contains a limited number of intestinal microbes, while the

129 inner mucus layer contains very few microbes. The mucus layer contains antimicrobial peptides, which

130 help prevent contact between bacteria and the epithelium (Fig 1).The chemical barrier can protect the

131 intestinal mucosa from erosion due to enzymes and acidic and alkali conditions. Gastric acid and bile

132 can inactivate bacteria. The pH of gastrointestinal mucus and digestive juice is not conducive to the

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133 growth of bacteria. Gastric acid is the best bactericide in the gastrointestinal tract.33 Bile salts can be

134 combined with endotoxin, cholic acid can degrade endotoxin molecules, and lysozyme can destroy

135 bacterial cell walls, destroying bacteria. The digestive juice secreted by the intestines can dilute the

136 toxin and clean the intestinal cavity, making it difficult for the potentially pathogenic bacteria to adhere

137 to the intestinal epithelium.34 Intestinal secretions not only protect the intestinal epithelium from

138 physical damage but also adhere to antigenic material, making it more susceptible to degradation by

139 proteases.35 In addition, there are some complementary components in the secretion, which can help

140 intestinal immune cells clear pathogens.36 Bile acid can not only regulate the balance of glucose and

141 lipid metabolism but also maintain the stability of the intestinal environment by inhibiting bacterial

142 growth and bacterial translocation in the small intestine.37 In addition to these well-known functions,

143 accumulating data identify bile acids as pleiotropic signaling molecules that control gut-liver

144 crosstalk.38 By activating the expression of farnesoid X receptor (FXR),39 bile acid negatively regulates

145 the expression of sterol regulatory element binding protein-1c (SREBP-1C)40 and reduces the

146 expression of fat-related genes (such as acetyl coenzyme A carboxylase 1, acetyl coenzyme A

147 carboxylase 2, fatty acid synthetase, and glucose-6-phosphate deaminase), reducing the occurrence of

148 NAFLD.41

149 Role of intestinal physical barrier in NAFLD development

150 The physical intestinal barrier is mainly composed of the intestinal mucous layer and epithelial cells.

151 The junction complexes between epithelial cells include tight junction, adhesion junction, desmosome

152 and gap junction (Fig 3). The iECs are composed of a layer of columnar epithelial cells that forms the

153 first line of defense between the intestinal lumen (intestinal bacteria and toxin macromolecules) and the

154 inner milieu.42 The intestinal epithelial tight junction, as a dynamic permeability barrier, has dual

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155 functions: preventing potential harmful substances or pathogens from entering the body while allowing

156 nutrients, ions and water to enter the body. Studies have shown that increased intestinal mucosal

157 inflammation and destruction43 of intestinal epithelial barrier lead to the possibility of translocation of

158 microbial products, thereby inducing NAFLD.31 In the pathological conditions of hypoxia and

159 inflammatory stimulation, tight junction proteins can produce contraction phenomena and move to the

160 cytoplasm. The cell pore is clearly expanded, and the permeability of the intestinal mucosa increases,

161 resulting in intestinal bacterial translocation and the release of metabolite LPSs into the blood and liver

162 through the portal system,31 stimulating liver KCs44 and releasing inflammatory factors, such as TNF-α

163 and IL-6. The inflammatory chemokines act on liver cells to make them liposomes.

164 Role of intestinal immunological barrier in NAFLD development

165 The immune barrier is mainly composed of immunoglobulin A (IgA) secreted by lymphocytes and

166 plasma cells.45 A study found that the intestinal epithelium can also express innate immunity by

167 regulating molecular TLRs.46 Because IgA has a special affinity for gram-negative bacteria in the

168 intestinal tract, the function of IgA is obviously inhibited when the intestinal mucosa is damaged, thus

169 promoting bacterial translocation in the intestine.47 The LPS produced by bacterial metabolism is

170 combined with the corresponding TLRs, producing pathogen-associated molecular patterns (PAMPs)

171 in the intestinal tract.48 The activation of nuclear factor kappa-B (NF-κB) and the mitogen-activated

172 protein kinase (MAPK) signal transduction pathway stimulates the formation of inflammatory

173 chemokines, such as TNF-α and IL-6, and leads to IR, which further promotes the development of

174 NAFLD (Fig 2).49

175 Abnormal intestinal barrier function can lead to disorder of digestion and absorption of nutrients, slow

176 growth, reduced disease resistance, and increased susceptibility to pathogenic microorganisms and lead

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177 to the occurrence of various diseases.50 When the intestinal epithelial tight junction changes, decreases

178 or is absent, the interstitial permeability of iEC increases. Bacteria, toxins and macromolecules can

179 enter the systemic circulation through tight junctions. For example, some intestinal inflammatory

180 diseases, such as inflammatory bowel syndrome (IBS),51 are characterized by increased iEC bypass

181 permeability. In the case of massive blood loss, intestinal blood supply is not a priority, and the

182 quantity and quality of lamina propria plasma cells decrease, which leads to the decline in of IgA and

183 the ability to match sIgA.52 The decrease in immunoglobulin secreted by B lymphocytes and the

184 decrease in sIgA content in the mucosal surface are conducive to the invasion of bacteria and toxins

185 into the body and can also cause excessive production of oxygen free radicals. The intestine contains a

186 microbial ecosystem in which the activity of microorganisms plays an important role in the function of

187 the intestinal immune system.53 Balanced microorganisms play an important role in the healthy

188 development of the intestinal mucosal immune system. Like antimicrobial peptides, probiotics,

189 glutamine and acidifiers, they can regulate animal intestinal health by improving intestinal pH;

190 activating endogenous enzymes; and mobilizing, stimulating and strengthening the autoimmune

191 system.54

192 Gut microbiota as a therapeutic target for NAFLD

193 Because of the close relationship between the liver and gastrointestinal tract, it is not surprising that

194 intestinal microflora disorders are associated with liver fat accumulation.55-58 Although the exact

195 mechanism of intestinal microflora in the development and progress of NAFLD is not yet clear, its

196 potential explanation is related to bacterial overgrowth, gut leakiness,57 increased endotoxemia

197 absorption,59-60 and inflammation. In recent years, knowledge of gut microbiota has increased, has

198 enhancing the understanding of the metabolic and immune potential and microbial-host interactions,

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199 mainly in the intestine, including the liver and other organs. The role and characteristics of metabolites

200 produced by microorganisms and their direct roles in the intestine and other body parts remain unclear.

201 However, there is growing evidence that the gut microbiota is the real goal of NAFLD interventions. In

202 view of the key role of the gut microbiota in the pathogenesis of metabolic diseases,61-62 the design of

203 gut microbiota regulation strategies to improve NAFLD may be considered as a new treatment option

204 for these patients. In this respect, probiotics, prebiotics and synbiotics have attracted wide attention.

205 Probiotics

206 Probiotics is a group of complex bacteriam.63 Commonly used probiotics include Lactobacillus,

207 Bifidobacterium and Polycoccus, which can inhibit the expansion of gram-negative pathogens and have

208 a wide range of beneficial effects on host metabolism.64-65 Some evidence has indicated that probiotics

209 have the ability to improve liver damage and reduce bacterial translocation.66 Early investigations of

210 probiotics have shown that it is possible to improve the pathophysiology and development of

211 NAFLD.67-68 A recent analysis showed that probiotics can reduce the levels of liver transaminase, total

212 hepatic sterol and TNF.69 Probiotic therapy of patients with NAFLD has been supported not only in

213 clinical practice but also in animal experiments.70 It can improve the immunity of the host, inhibit the

214 growth and translocation of harmful bacteria, protect the intestinal mucosal barrier, reduce the

215 absorption of toxins,71 significantly reduce the expression and secretion of proinflammatory factors in

216 the liver,72 improve IR and reduce oxidative stress in NAFLD patients; it can also assist in the

217 development and stability of the intestinal flora and prevent the propagation of pathogenic bacteria.

218 Probiotics can reduce intestinal endotoxemia by restoring the intestinal microecological balance,

219 reducing the formation of the “second hit” on hepatocytes73.

220 However, the exact mechanism of how probiotics do this is not fully understood. The possible

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221 mechanisms proposed include microbial interaction and competition with pathogenic bacteria, which

222 can eradicate pathogenic bacteria and promote the healthy balance of intestinal microflora.74-75 In

223 addition, probiotics can effectively improve the integrity of the epithelial barrier and stimulate the host

224 immune response.76-77 Furthermore, probiotics have synergistic effects with chemical drugs, such as

225 statins, in the treatment of NAFLD, which highlights the great potential of probiotics alone or in

226 combination with other drugs.78-79 However, the clinical efficacy of probiotics still needs further

227 verification with a larger number of participants, dose optimization and even product formulation.

228 Prebiotics

229 At present, probiotics and prebiotics play a key role in the treatment and prevention of NAFLD, but

230 there are often conflicting results; therefore, more rigorous experimental design, improvement in the

231 clinical research quality and confirmation of the therapeutic effects are necessary.75 In a meta-analysis,

232 the decrease of AST, ALT and gamma-glutamyl transferase (γ-GT) is related to prebiotics and

233 probiotics, so altering the composition and metabolism of intestinal microbial has potential protective

234 effect on NAFLD.80 Although the specific effects of probiotics on gut microbiota remain unclear, most

235 studies have shown that the increase in Bifidobacteria and levels of Faecalibacterium prausnitzii and

236 the decrease in Bacteroides intestinalis, Bacteroides vulgatus and Propionibacterium are related to

237 endotoxemia.81-83 While these studies suggest that probiotics may be used as potential drugs to treat

238 NAFLD, the overall changes in intestinal microflora need further study. In addition, there are no

239 reports on the effects of probiotics on liver lipids or liver histology, and there is no evidence linking

240 specific bacteria to the pathophysiology of NAFLD.

241 Synbiotics

242 Synbiotics are a complex of probiotics and prebiotics that act as a nutritional supplement to produce

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243 synergistic effects. Previous studies have reported that synbiotics have a potentially stronger role in

244 regulating gut microbiota than probiotics or prebiotics alone.85-87 In recent years, synbiotics have been

245 widely used in metabolic diseases.62, 88 There is abundant evidence that synbiotics can improve lipid

246 metabolism, IR, inflammatory mediators and liver enzyme markers by improving the form or function

247 of gut microbiota.87, 89-90 Scientists have elucidated the potential effects of synthetic compounds in

248 NASH models and found that they can regulate gut microbiota, reduce digestion and endotoxemia.61

249 However, the available evidence is inconsistent, and the amount of synbiotic data need to be

250 supplemented for clinical treatment of NAFLD patients. Drug therapy for NAFLD is still in its infancy.

251 Synbiotics might be beneficial for NAFLD, especially for those with metabolic comorbidities. In order

252 to verify the biochemical and histological efficacy of combined biotherapy in patients with NAFLD,

253 more participants are required to participate in further studies for longer treatment periods. However,

254 many discrepant results have been observed with respect to gut microbiota-targeted therapies,

255 especially in clinical studies. Probiotics have been proposed to constitute an effective preventive

256 treatment for antibiotic-induced dysbiosis and associated adverse effects in mice91 and in some,92 but

257 not all, human studies.93-94 Importantly, adverse effects associated with probiotic application may be

258 underreported in clinical trials,95 further complicating the efficacy debate. Similarly, the role of

259 probiotics in the treatment of infections or existing diseases,96 such as inflammatory bowel disease,

260 remains a controversial issue,97 and some studies have even reported probiotic-associated morbidity

261 and mortality.98 Therefore, medical institutions such as the European Food Safety Agency100 or the

262 Food and Drug Administration of the United States101 refused to approve probiotics as a form of

263 medical intervention.102 Given this probiotic effect, it is critically important to further elucidate

264 probiotic-induced factors contributing to the inhibition of indigenous microbiome reconstitution.103

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265 Conclusion and perspectives

266 In conclusion, the manifestations of intestinal barrier damage include the destruction of the intestinal

267 epithelium, the growth of bacteria in the small intestine, and an increase in the level of LPS. Intestinal

268 barrier damage is involved in the development of NAFLD. Intestinal barrier defects have been

269 associated with NAFLD and therefore constitute a new therapeutic target. Microorganisms have

270 potential applications in the treatment of NAFLD, but the limitations of NAFLD diagnosis and

271 biomarkers limit further research. At the same time, personalized microbial therapy is still challenging.

272 Probiotics, prebiotics and synbiotics have a central importance in maintaining a healthy intestinal

273 microflora balance. They assist in curbing endotoxemia by bringing about a massive decline in the

274 population of pathogenic bacteria, achieved by tweaking the flora. Based on the complex relationship

275 between intestinal microflora and NAFLD, targeted therapy of intestinal microflora using probiotics,

276 probiotics and synbiotics has become a promising method. However, the existing studies mainly focus

277 on the protection of bacterial communities and do not provide functional information between intestinal

278 microflora and host. Whether intestinal microflora is the cause of the disease or whether it has an effect

279 on the pathophysiology of the disease is still unclear. However, the relationship between intestinal

280 microflora and NAFLD should continue to be explored in the following aspects: (1) How to better

281 understand the specificity of patients; (2) Develop potential NAFLD biomarkers; (3) Deeply explore

282 the relationship between intestinal microflora and NAFLD; (4) Determine whether lifestyle interferes

283 with the intestinal tract; and (5) How to interact with intestinal microflora and the possible health

284 impact.

285 To better understand the complex interactions between intestinal microflora and hosts. In addition to

286 systematic biology methods, we also need to apply more technical methods, carry out necessary

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287 cooperative research, explore the diversity of biological samples, and investigate its pathogenesis and

288 treatment effect.

289 AUTHOR INFORMATION

290 Corresponding Author

291 * E-mail: xuchuang7175@163.com&xuchuang@byau.edu.cn Phone: +86 13936967175

292 ORCID

293 Yizhe Cui: 0000-0002-7877-8328

294 Funding

295 The work was financially supported in part by the National Key R&D Program of China [grant number

296 2017YFD0502200]; Group control technology and product development and demonstration of

297 important mass production disease groups in dairy cattle [grant number GA16B20]; China Postdoctoral

298 Science Foundation [grant number 2017M620124; 2018T110320]; Doctoral Program Foundation of

299 Heilongjiang Bayi Agricultural University of China [grant number XDB-2016-10]; Postdoctoral

300 Program Foundation of Heilongjiang Bayi Agricultural University [grant number 601038] and the

301 Natural Science Foundation of Heilongjiang Province [grant number C201444].

302 Notes

303 The authors declare no competing financial interest. YZC and QJW contributed equally to this paper;

304 hence, both are first authors.

305 ABBREVIATIONS USED

306 NAFLD, nonalcoholic fatty liver disease; IR, insulin resistance; FFA, free fatty acid; NASH,

307 nonalcoholic steatohepatitis; T2D, type 2 diabetes; LPSs, lipopolysaccharides; FMO1, flavin

308 monooxygenases 1; FMO3, flavin monooxygenases 3; TMAO, trimethylamine-N-oxide; SIBO, small

309 intestinal bacterial over growth; TNF-α, tumor necrosis factor α; ALT, alanine aminotransferase; AST,

310 serum aspartate aminotransferase; SFL, simple fatty liver; KCs, Kupffer cells; TLR4, Toll-like receptor
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311 4; FXR, farnesoid X receptor; SREBP-1C, sterol regulatory element binding protein-1c; PAMPs,

312 pathogen-associated molecular patterns; NF-κB, nuclear factor kappa-B; MAPK, mitogen-mediated

313 protein kinase; γ-GT, gamma-glutamyl transferase; iEC, intestinal epithelial cells; iIEL, intestinal

314 intraepithelial lymphoid tissue; LPLs, lamina propria lymphocytes; PPs, Peyer’s patches; sIgA,

315 secretory immunoglobulin A; IBS, bowel syndrome.

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600 (101) Saldanha, L. G. US Food and Drug Administration regulations governing label claims for food

601 products, including probiotics. Clin Infect Dis. 2008, 46 Suppl 2, S119-121; discussion S144-151.

602 (102) Zmora, N.; Zilberman-Schapira, G.; Suez, J.; Mor, U.; Dori-Bachash, M.; Bashiardes, S.; Kotler,

603 E.; Zur, M.; Regev-Lehavi, D.; Brik, R. B.; Federici, S.; Cohen, Y.; Linevsky, R.; Rothschild, D.; Moor, A. E.;

604 Ben-Moshe, S.; Harmelin, A.; Itzkovitz, S.; Maharshak, N.; Shibolet, O.; Shapiro, H.; Pevsner-Fischer,

605 M.; Sharon, I.; Halpern, Z.; Segal, E.; Elinav, E. Personalized Gut Mucosal Colonization Resistance to

606 Empiric Probiotics Is Associated with Unique Host and Microbiome Features. Cell. 2018, 174 (6),

607 1388-1405 e1321.

608 (103) Suez, J.; Zmora, N.; Zilberman-Schapira, G.; Mor, U.; Dori-Bachash, M.; Bashiardes, S.; Zur, M.;

609 Regev-Lehavi, D.; Ben-Zeev Brik, R.; Federici, S.; Horn, M.; Cohen, Y.; Moor, A. E.; Zeevi, D.; Korem, T.;

610 Kotler, E.; Harmelin, A.; Itzkovitz, S.; Maharshak, N.; Shibolet, O.; Pevsner-Fischer, M.; Shapiro, H.;

611 Sharon, I.; Halpern, Z.; Segal, E.; Elinav, E. Post-Antibiotic Gut Mucosal Microbiome Reconstitution Is

612 Impaired by Probiotics and Improved by Autologous FMT. Cell. 2018, 174 (6), 1406-1423 e1416.

613 Figure legends

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614

615 Fig 1. Four components of the intestinal barrier. The microbiological barrier refers to the normal

616 intestinal flora, the interdependence of various strains on each other, and the formation of the host

617 biological defense by beneficial bacteria through biological antagonism and immune function. The

618 chemical barrier refers to the gastric secretion of gastric acid, mucus, mucin, bile, glycoproteins,

619 mucopolysaccharides, various digestive enzymes, lysozymes and other chemicals. The physical barrier

620 consists of a layer of columnar epithelial cells between the intestinal lumen and the inner milieu.

621 Between the epithelial cells are intercellular junctional complexes, including tight junctions, adherens

622 junctions, desmosomes and gap junctions. The immune barrier is composed of iEC, intestinal

623 intraepithelial lymphoid tissue (iIEL), lamina propria lymphocytes (LPLs), Peyer’s patches (PPs),

624 mesenteric lymph nodes, other intestinal tissues and the secretory immunoglobulin A (sIgA) of

625 intestinal plasma cells.

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626

627 Fig 2. Small intestinal bacterial over growth (SIBO) and Toll-like receptor (TLR4) signaling in

628 NAFLD. Bacterial translocation occurs due to the overgrowth of intestinal bacteria or the disruption of

629 the function of intestinal barriers. Translocated intestine-derived pathogen-associated molecular

630 patterns (PAMPs) activate TLR4 signaling cascades in KCs that regulate the inflammatory response.

631 TLR4 activation in KCs induces the production of various cytokines, such as IL-1β and TNF-α, that

632 subsequently induce hepatocyte lipid accumulation and apoptosis. Downstream signaling events

633 include the MyD88-dependent activation of NF-κB.

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634

635 Fig 3. Intestinal physical barrier. The protein complexes between intestinal epithelial cells include

636 tight junctions, adherens junctions, desmosomes and gap junctions. Figure adapted from Ulluwishewa

637 et al. (2011).42

638 TOC Graphic

639

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