No abstract

Non-alcoholic fatty liver disease (NAFLD) is one of the most common metabolic diseases currently in the context of obesity worldwide, which contains a spectrum of chronic liver diseases, including hepatic steatosis, non-alcoholic steatohepatitis and hepatic carcinoma. In addition to the classical "Two-hit" theory, NAFLD has been recognized as a typical gut microbiota-related disease because of the intricate role of gut microbiota in maintaining human health and disease formation. Moreover, gut microbiota is even regarded as a "metabolic organ" that play complementary roles to that of liver in many aspects. The mechanisms underlying gut microbiota-mediated development of NAFLD include modulation of host energy metabolism, insulin sensitivity, and bile acid and choline metabolism. As a result, gut microbiota have been emerging as a novel therapeutic target for NAFLD by manipulating it in various ways, including probiotics, prebiotics, synbiotics, antibiotics, fecal microbiota transplantation, and herbal components. In this review, we summarized the most recent advances in gut microbiota-mediated mechanisms, as well as gut microbiota-targeted therapies on NAFLD.

Gut dysbiosis is heavily involved in the development of various human diseases. There are thousands of publications per year for investigating the role of gut microbiota in diseases. However, emerging evidence has indicated the frequent data inconsistency between different studies, which is largely overlooked. There are many factors that can cause data variation and inconsistency during the process of microbiota study, in particular, sample storage conditions and sequencing process. Here, we systemically evaluated the impacts of six fecal sample storage conditions (three non-commercial storage protocols, −80 • C, −80 • C with 70% ethanol (ET_−80 • C), 4 • C with 70% ethanol (ET_4 • C), and three commercial storage reagents, OMNIgeneGUT OMR-200 (GT) and MGIEasy (MGIE) at room temperature, and Longsee at 4 • C (LS) on gut microbiome profile based on 16S rRNA gene sequencing. In addition, we also investigated the impacts of storage periods (1 and 2 weeks, or 6 months) and sequencing platform on microbiome profile. The efficacy of storage conditions was evaluated by DNA yield and quality, α and β diversity, relative abundance of the dominant and functional bacteria associated with short-chain fatty acid (SCFA) production, and BAs metabolism. Our current study suggested that −80 • C was acceptable for fecal sample storage, and the addition of 70% ethanol had some benefits in maintaining the microbial community structure. Meanwhile, we found that samples in ET_4 • C and GT reagents were comparable, both of them introduced some biases in α or β diversity, and the relative abundance of functional bacteria. Samples stored in MGIE reagent resulted in the least variation, whereas the most obvious variations were introduced by LS reagents. In addition, our results indicated that variations caused by storage condition were larger than that of storage time and sequencing platform. Collectively, our study provided a multi-dimensional evaluation on the impacts of storage conditions, storage time periods, and sequencing platform on gut microbial profile.

23Gut dysbiosis contributes to the development of various human diseases. There are 24 thousands of publications per year for investigating the role of gut microbiota in 25 development of various diseases. However, emerging evidence has indicated data 26 inconsistency between different studies frequently, but gained very little attention by 27 scientists. There are many factors that can cause data variation and inconsistency during 28 the process of microbiota study, in particular, sample storage conditions and subsequent 29 sequencing process. Here, we systemically evaluated the impacts of six fecal sample 30 storage conditions (including -80 ℃, -80 ℃ with 70% ethanol (ET_-80 ℃), 4°C with 70% 31 ethanol (ET_4℃), and three commercial storage reagents including OMNIgene•GUT 32 OMR-200 (GT), MGIEasy (MGIE), and Longsee (LS)), storage periods (1, 2 weeks or 6 33 months), and sequencing platform on gut microbiome profile using 16S rRNA gene 34 sequencing. Our results suggested that -80℃ is acceptable for fecal sample storage, and 35 the addition of 70% ethanol offers some benefits. Meanwhile, we found that samples in 36 ET_4 ℃and GT reagents are comparable, both introduced multi-dimensional variations. 37The use of MGIE resulted in the least alteration, while the greatest changes were observed 38 in samples stored in LS reagents during the whole experiment. Finally, we also confirmed 39 that variations caused by storage condition were larger than that of storage time and 40 sequencing platform.41 42 3 IMPORTANCE 43 In the current study, we performed a multi-dimensional evaluation on the variations 44 introduced by types of storage conditions, preservation period and sequencing platform on 45 the basis of data acquired from 16S rRNA gene sequencing. The efficacy of preservation 46 methods was comprehensively evaluated by DNA yield and quality, α and β diversity, 47 relative abundance of the dominant bacteria and functional bacteria associated with SCFAs 48 producing and BAs metabolism. Our results confirmed that variations introduced by 49 storage condition were larger than that of storage periods and sequencing platform.50 Collectively, our study provided a comprehensive view to the impacts of storage conditions, 51 storage times, and sequencing platform on gut microbial profile. 52 53 KEYWORDS: storage conditions, storage periods, sequencing platform, microbial profile 54 55 4 56The mammalian gastrointestinal tract is the main site for commensal bacteria (1, 2), 57 which contains at least 100-times as many genes as host genome (3). In recent years, the 58 passion on gut microbiota-related research is overwhelming due to the involvement of gut 59 dysbiosis in development of various human diseases including obesity, diabetes mellitus, 60 nonalcoholic fatty liver diseases, cardiovascular disease, and even cancers (4-7). Emerging 61 high-throughput sequencing technologies including 16S rRNA gene and metagenomics lay 62 the solid foundation for investigating the role of gut microbiota in human diseases (8, 9). 63 Ther...

Gut dysbiosis contributes to nonalcoholic fatty liver disease (NAFLD) formation. However, the underlying molecular mechanism is not fully understood. Here, we report a novel therapeutic target for NAFLD, the hepatic adenosine receptor A1 (ADORA1) that is inhibited by gut microbiota-derived acetic acid from Astragalus polysaccharides (APS). APS supplement attenuated hepatic steatosis by reversing gut dysbiosis in high-fat diet fed mice, and reduced hepatic ADORA1 expression. Patients with hepatic steatosis showed increased expression of hepatic ADORA1, and specific ADORA1 antagonist ameliorated hepatic steatosis as well. Meanwhile, the metabolic benefits of APS were microbiota-dependent due to the production of acetic acid, which improved hepatic steatosis by suppressing ADORA1 both in vitro and in vivo resulting to the inhibition of rate-limiting enzyme for fatty acid de novo synthesis, fatty acid synthase. Our results highlight the critical role of gut microbiota-acetic acid-hepatic ADORA1 axis in NAFLD development and reveal the novel mechanism underlying the metabolic benefits of APS.(APS) are extracted from Astragalus membranaceus, a frequently used herbal medicine with established efficacy in lowering plasma lipids, improving insulin sensitivity 12,13 , and ameliorating metabolic risk in metabolically stressed transgenic mice 14 . Given the non-absorptive properties of polysaccharides in the gastrointestinal tract, the metabolic benefits of most plant polysaccharides are associated with modulation of gut dysbiosis such as the anti-obesity effects of the polysaccharides extracted from Ganoderma lucidum and Hirsutella sinensis 11,15 . However, the gut microbiota-involved molecular mechanisms underlying the metabolic benefits of most plant polysaccharides are still largely unknown.In the present study, we explored the gut microbiota-involvement in the molecular mechanisms underlying NAFLD in high-fat diet (HFD)-induced NAFLD mice with or without APS supplement by using integrated hepatic transcriptomics, metagenomics, and targeted metabolomics approaches. Our results are the first to demonstrate that APS attenuates hepatic steatosis in HFD-fed mice by normalizing their gut dysbiosis. The hepatic target protein, adenosine receptor A1 (ADORA1), was screened and validated in both human patients and mice with hepatic steatosis. Further, our results indicated that the suppression of hepatic ADORA1 prevented NAFLD formation through gut microbiota-derived acetic acid, which is enriched with APS supplementation.Collectively, our current study highlights hepatic ADORA1 as a novel target for NAFLD prevention or therapy. Results APS attenuates hepatic steatosis in HFD-fed miceTo confirm the anti-NAFLD effect of APS, we fed mice with chow diet, or HFD with or without APS supplement for 12 weeks. We observed that 12 weeks of HFD resulted in obvious increases in body weight, epididymal fat mass and index, formation of hepatic steatosis, and stimulation of pro-inflammatory cytokines expression (Il-1β and Il-6) in liver an...