Fecal microbiota transplantation alleviates high-fat and high-sugar diet-induced fatty liver in mice

Fecal microbiota transplantation alleviates high-fat and high-sugar diet-induced fatty liver in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Fecal microbiota transplantation alleviates high-fat and high-sugar diet-induced fatty liver in mice Fangxia Mi, Jinglu Guo, Wentao Zheng, Hua Ye This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6242383/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Around the world, Nonalcoholic fatty liver disease (NAFLD) has become the most frequent chronic liver disease. Fecal microbiota transplantation (FMT) is a successful method for rebuilding gut flora and has been applied in treating and researching various microbiome-related conditions like inflammatory bowel disease. FMT is considered a breakthrough medical development in recent years, but further research is needed in NAFLD-related areas. Mice were randomized into control, high-fat and high-sugar diet (HFCS) and HFCS + FMT groups. A mouse model of NAFLD was established on a high-fat and high-sugar diet for 20 weeks, followed by FMT for 8 weeks. After 8 weeks of FMT initiation, serum, liver tissue specimens and feces of mice were collected for biochemical experiments, histopathology and molecular biology to obtain experimental data and statistical analysis. Our results showed that Firmicutes and Bacteriae significantly increased and bifidobacteria significantly decreased in mice fed HFCS. After FMT treatment, the abundance of the above bacteria was changed, and the composition of the above bacteria in the gut was close to that of the normal diet group. FMT reduced body weight in mice and improved serum alanine aminotransferase (ALT) and total cholesterol (TC) levels. The significant decrease of intrahepatic proinflammatory cytokines and liver pathology showed that hepatitis was relieved after FMT. These data indicate that High-fat and high-sugar diet can induce NAFLD in mice and change the structure of intestinal flora. NAFLD was alleviated by correcting intestinal flora with FMT. Biological sciences/Microbiology Health sciences/Diseases/Metabolic disorders Gut-liver axis NAFLD Fecal microbiota transplantation high-fat and high-sugar diet Figures Figure 1 Figure 2 1. Introduction Nonalcoholic liver disease (NAFLD) includes various conditions characterized by the buildup of fat in the liver, including simple fatty liver, nonalcoholic steatohepatitis, cirrhosis, and possibly liver cancer [ 1 , 2 ] . NAFLD's worldwide prevalence has been increasing each year, with 25% in 2016 and over 30% in 2019 [ 3 , 4 ] . Recently, there have been numerous calls to rename NAFLD as metabolic dysfunction-related fatty liver disease (MAFLD), which would help clinicians focus more on the disease and enhance patient management [ 5 ] . Studies have shown that Asians and Westerners are more likely to suffer from obesity-related diseases, such as NAFLD, even if they have the same BMI, which may be related to excessive visceral fat deposition [ 6 ] . There are currently no drugs with proven efficacy to treat NAFLD, and it is important to prevent NAFLD through a healthy lifestyle and weight loss [ 7 ] . The human colon provides a nutrient-rich environment for its microbiome: it is densely populated and home to an estimated 40–100 trillion bacteria, with different resident microbes competing fiercely with each other for nutrients and space, but also working collaboratively to digest complex polysaccharides and other polymer molecules [ 8 ] . Positive balance shutdown between intestinal flora and host is beneficial to the normal development of biological metabolic system [ 9 ] . Intestinal microecosystem can promote the metabolism, and has the functions of flora barrier, immune regulation, cancer prevention and cancer suppression, etc. It is the most complex one of the most important microecosystems, such as Lactobacillus, bifidobacterium and other bacteria, can not only synthesize vitamins necessary for human health, but also use protein residues to synthesize essential amino acids that are essential for body growth and development [ 10 ] . Recent research has shown that an imbalance in gut bacteria is associated with the onset of NAFLD [ 11 , 12 ] . Fecal microbiota transplantation (FMT) refers to the correction of ecological disorders through the management of the transplantation of feces collected from the donor into the recipient's intestine, thus treating underlying diseases [ 13 ] . More and more studies have discovered that FMT is effective in treating inflammatory bowel disease, constipation, autism, severe infections and other conditions [ 14 , 15 ] . Probiotics are advantageous to the host by changing the microbiota composition and have been employed as a promising treatment for human NAFLD [ 16 ] . FMT can provide patients with more diversity and abundance of symbiotic bacteria and other microorganisms than probiotics and synbiotic therapies, which is conducive to maintaining the ecological balance of intestinal microorganisms [ 17 ] . Therefore, we speculated that restoring the homeostasis of the gut-liver axis in the course of NAFLD by regulating intestinal microorganisms such as FMT would promote the establishment of a virtuous cycle between the restoration of intestinal microecological homeostasis and the improvement of NAFLD. 2. Methods 2.1 Animals experiments All procedures were approved by the Ethics Committee on Animal Research of the Ningbo University School of Medicine (Approval number: AEWC-NBU20210012). All methods were performed in accordance with relevant guidelines and regulations and in accordance with ARRIVE guidelines. Male C57BL/6 mice (20 ± 2g) aged 5–6 weeks were placed in a 21–22◦C light-dark cycle SPF chamber for 12/12 h and fed adaptive for one week. There were three groups in this experiment: control group, HFCS group and HFCS + FMT group, with five mice in each group. The Control group was given a normal diet, while the HFCS group and HFCS + FMT group were given a high-fat and high-sugar diet, that is, a high-fat diet with 60% fat content and 42g/L sugar water mixed with 55% fructose and 45% sucrose by weight. The whole experiment was carried out for 28 weeks, after 8 weeks, the Control group and HFCS group were given normal saline administration, and the HFCS + FMT group was given FMT. Feces from mice on a normal diet were collected before each FMT. The specific procedure was to add 200mg of fresh feces to 4ml of normal saline, rotate for 5min, stand gauze and filter for 2min, and collect fecal suspension in a sterile test tube [ 18 ] . Sterile normal saline and fecal bacteria solution were injected into the oral cavity of mice according to groups, the dose was 200µL/ time, 3 times/week, a total of 24 times. Each mouse was weighed once a week, and the fur color and exercise status of the mice were observed. On the night before the mice were killed, a sterile frozen storage tube was prepared, and 2–3 mice feces were collected for 16S sequencing and analysis of bacterial microbial diversity. After 28 weeks, the mice were anesthetized by inhaling 40 percent carbon dioxide gas and having blood drawn from their eyeballs. Mouse liver tissue samples were collected, half of the largest liver leaf tissue was cut and soaked in a centrifuge tube containing 5 mL10% formalin solution for fixed preservation for hematoxylin eosin (H&E) staining. The remaining liver tissue was placed in labeled freezer tubes. 2.2 Histological analysis of the livers The collected liver tissue specimens were soaked in formalin buffer solution for at least 24h. Half of the largest liver lobe was cut into paraffin wax and sliced for HE staining. The sections of each group under different multiples of field of view were observed under microscope, and the appropriate liver histopathology pictures were taken. 2.3 Biochemical analysis Serum AST and TC levels were measured according to the kit instructions. (Ningbo MedicalSystem Biotechnology Co., Ltd., Ningbo, China). 2.4 Quantitative real-time PCR (qRT-PCR) Liver tissue was cut on dry ice to extract total RNA, and the concentration of RNA in the sample was obtained by spectrophotometry (Denovix, America). cDNA was prepared from 1 µg of total RNA using a TransScript® All-in-One First-Strand cDNA Kit (TransGen Biotech, Beijing, China). The expression value of the blank control group was normalized as 1, and the expression level of the target gene in other groups was expressed as a relative multiple, and then the expression level of the target gene was compared between different groups. 2.5 Gut microbiota analysis The DNA of each group was extracted using the QIAamp Fast DNA Stool Mini Kit (Cat. No. 51604) according to the instructions. Primers 341F 5′-CCTACGGGRSGCAGCAG-3′ and 806R 5′-GGACTACVVGGGTATCTAATC-3′ with specifc barcodes were used to amplify the V3–V4 region of the bacterial ribosomal RNA gene to obtain a ~ 500-bp product. Sample extraction, database construction, sequencing and analysis services are completed by Shanghai Ruiyi Biotechnology Co., Ltd. (Shanghai, China). 2.6 Statistical analysis Data were expressed as mean ± standard error, with statistical analysis conducted using SPSS statistics Version 26.0 software (IBM, China), and the results were illustrated using GraphPad Prism9.0 software. To compare differences between groups, a one-way ANOVA was conducted, followed by Tukey's test for pairwise comparisons. A P-value of less than 0.05 indicates statistical significance. 3. Results 3.1 Impact of FMT on body weight and serum transaminase levels. The HFCS group mice were obese, unresponsive, inactive, coarse, and dull hair. After receiving fecal suspension FMT intervention in the normal group for 8 weeks, the mice were symmetrical, responsive, active, with soft hair and better gloss, and their body weight decreased significantly compared with that in the HFCS group (Fig. 1 A). The HFCS group had significantly higher ALT and TC levels compared to the control group. Mice treated with FMT showed improvements in ALT and TC compared to HFCS (Fig. 1 B,C). 3.2 Improvement of intestinal flora by FMT. After 28 weeks, we sequenced the 16S rDNA baseline in the stool. PCoA is used to compare and analyze the microbial community structure between different samples being measured. The three groups of mice were grouped separately, and the control group was obviously separated from the HFCS group, indicating that the intestinal flora of NAFLD mice had obvious changes, and the FMT intervention group was close to the normal group. The HFCS group had significantly higher ALT and TC levels compared to the control group (Fig. 2 A). According to the number of OUT and the species represented by OUT, the corresponding distribution maps of each sample at phylum, class and genus level were obtained. After 28 weeks of feeding HFCS, there were extensive changes in gut microbial community structure at all levels compared to the control group. The HFCS group, when compared to the control group, demonstrated significant phylum-level changes, including an increase in Firmicutes and a decrease in Bacteroidetes, leading to a higher Firmicutes to Bacteroidetes ratio (Fig. 2 B). On the level of class, compared with the control group, bacilli in HFCS + NS group increased significantly (Fig. 2 C). At the genus level, Bifidobacterium was significantly decreased compared with the control group (Fig. 2 D). The abundance of the specified bacteria in mice on a high-fat and high-sugar diet changed after FMT treatment, bringing their gut composition closer to that of the normal diet group. The marker species analyzed by LefSe indicate that FMT can interfere with intestinal flora (Fig. 2 E). 3.3 FMT treatment attenuated HFCS-induced steatohepatitis. The result showed that the hepatocytes of the control group were of the same size without steatosis. In HFCS group, the structure of hepatic lobule was disordered, and lymphocytes and neutrophils were infiltrated. After FMT treatment, hepatocyte swelling was reduced, fat vacuoles were less and hepatocyte arrangement was more orderly (Fig. 1 D). Following the FMT intervention, the liver NAS score in the HFCS + FMT group decreased significantly compared to the HFCS group, which had a score much higher than the control group (Fig. 1 E). 3.4 Effect of FMT on the intrahepatic immune. Compared to the control group, the HFCS group had elevated IL-1β mRNA levels in the liver, which were decreased by FMT intervention (Fig. 1 F). The IL-17α protein level in liver tissue was higher compared to the control group. The FMT intervention effectively reversed this imbalanced immune factor, thereby reducing IL-17α in the liver (Fig. 1 G). The findings indicated that FMT treatment might lower the levels of inflammatory factors in NAFLD. 4. Discussion Numerous specialists suggested renaming NAFLD to MAFLD, indicating fatty liver disease linked to metabolic issues across the body [ 19 ] . With the change of modern diet structure, the prevalence of metabolic syndrome caused by high-fat and high-sugar diet is increasing worldwide. And The new definition of MAFLD may improve the diagnostic rate of the disease [ 20 ] . A mouse model of NAFLD with high fat and high sugar was established in this study, and the pathogenesis of human NAFLD was simulated by using a 42g/L sugar water mixed with a high fat diet with 60% fat content and a weight ratio of 55% fructose and 45% sucrose. A study demonstrated that the colonization of intestinal microbiota in NASH patients can worsen liver steatosis and inflammation in germ-free mice on a high-fat diet, confirming the crucial role of intestinal microbiota dysfunction in the disease's pathogenesis [ 21 ] . However, in the research experiments on FMT for NAFLD, there were several ways to pretreat mice before FMT, including antibiotic pretreatment to cause enteric sterility, acid-inhibiting agents such as omeprazole to resist the effect of gastric acid, and intestinal fecal bacteria transplantation on the occurrence and development of NAFLD. Few experiments have shown whether FMT can be transferred to models that are already colonized, or to what extent replacement of natural microorganisms is necessary to induce phenotypic changes [ 22 ] . Using broad-spectrum antibiotics before FMT might lead to side effects and pose ethical and antibiotic management challenges [ 23 ] . Therefore, in this experiment, We selected the method with the least influence, that is, no antibiotic pretreatment, and tested the effects of FMT on liver pathology, liver enzymes and intestinal flora of mice by gavage. Therefore, a mouse model of NAFLD was established through a high-fat and high-sugar diet, and the intestinal sterility of mice was caused by no antibiotic pretreatment before FMT. The weight of NAFLD mice can be reduced and the pathological changes of liver can be relieved after receiving normal mouse faecal bacteria solution. Q-PCR results showed that FMT intervention could reduce liver inflammation in mice with NAFLD, which was manifested by down-regulation of IL-1β and IL-17A mRNA expression in liver. The above implantation signals were encouraging. However, we did not find statistically significant differences in serum ALT and TC between NAFLD mice and NAFLD mice after receiving normal mouse faecal bacteria solution, but the trend was improved, which we considered to be related to the small sample size in this experiment. The ratio of firmicutes and Bacteriotes in the intestinal tract of mice in the high-fat and high-sugar diet group was increased, while FMT intervention could significantly reduce the abundance of firmicutes and the ratio of firmicutes to bacteriotes in the intestinal flora. A high-quality study has shown that Firmicutes are linked to weight gain [ 24 ] . Several studies have identified a close relationship between the Firmicutes to Bacilli ratio and obesity, suggesting it as a potential obesity marker [ 25 , 26 ] . Altering the balance between firmicutes and bacteriae in the gut microbiota can protect metabolism and help prevent weight gain [ 27 ] . The result showed that bifidobacteria abundance decreased significantly in NAFLD model mice at the genus level, and the above bacterial abundance was successfully reversed after FMT intervention. Bifidobacterium is a well-known intentional group of bacteria that can be applied as probiotics in the daily human diet [ 28 ] . Research indicates that bifidobacterium can successfully prevent the growth and settlement of harmful bacteria, preserve the gastrointestinal barrier's integrity, and suppress the creation and release of inflammatory cytokines [ 29 ] . Our preliminary study showed that fecal transplantation alleviated high-fat, high-sugar induced NAFLD through a beneficial effect on the microbiome. Declarations Author Contribution Fangxia Mi and Jinglu Guo wrote the main manuscript text. Fangxia Mi and Wentao Zheng performed all the experiments together, and Hua Ye directed the methodology and funding of the research. All authors reviewed the manuscript Data Availability The datasets used and analysed during the current study available from the corresponding author on reasonable request. References NASSIR F. NAFLD: Mechanisms, Treatments, and Biomarkers. Biomolecules[J] , 12 (6): (2022). SHARPTON, S. R. et al. Gut microbiome-targeted therapies in nonalcoholic fatty liver disease: a systematic review, meta-analysis, and meta-regression. Am. J. Clin. Nutr[J] . 110 (1), 139–149 (2019). POUWELS, S. et al. Non-alcoholic fatty liver disease (NAFLD): a review of pathophysiology, clinical management and effects of weight loss. BMC Endocr. 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The severity of nonalcoholic fatty liver disease is associated with gut dysbiosis and shift in the metabolic function of the gut microbiota. Hepatology[J] 63 (3), 764–775 (2016). YUAN, J. et al. Fatty Liver Disease Caused by High-Alcohol-Producing Klebsiella pneumoniae. Cell. Metab[J] . 30 (4), 675–688e677 (2019). MATSUOKA K. Fecal microbiota transplantation for ulcerative colitis. Immunol. Med[J] . 44 (1), 30–34 (2021). GUPTA, S. & MULLISH BH and ALLEGRETTI JR. Fecal Microbiota Transplantation: The Evolving Risk Landscape. Am. J. Gastroenterol[J] . 116 (4), 647–656 (2021). ZHANG, F. et al. Microbiota transplantation: concept, methodology and strategy for its modernization. Protein Cell[J] . 9 (5), 462–473 (2018). HU, H. et al. Intestinal microbiome and NAFLD: molecular insights and therapeutic perspectives. J. Gastroenterol[J] . 55 (2), 142–158 (2020). CHEN, J. and VITETTA L. Gut Microbiota Metabolites in NAFLD Pathogenesis and Therapeutic Implications. Int. J. Mol. Sci[J] , 21 (15): (2020). MI, F. & WANG, X. Effects of Different Preparation Methods on Microbiota Composition of Fecal Suspension. Mol. Biotechnol[J] . 65 (6), 871–880 (2023). GOFTON, C. et al. MAFLD: How is it different from NAFLD? Clin. Mol. Hepatol[J] . 29 (Suppl), S17–s31 (2023). XIAN, Y. X., XU, F. & WENG JP and MAFLD vs. NAFLD: shared features and potential changes in epidemiology, pathophysiology, diagnosis, and pharmacotherapy. Chin. Med. J. (Engl)[J] . 134 (1), 8–19 (2020). CHIU, C. C. et al. Nonalcoholic Fatty Liver Disease Is Exacerbated in High-Fat Diet-Fed Gnotobiotic Mice by Colonization with the Gut Microbiota from Patients with Nonalcoholic Steatohepatitis. Nutrients[J] , 9 (11): (2017). YU, E. W. et al. Fecal microbiota transplantation for the improvement of metabolism in obesity: The FMT-TRIM double-blind placebo-controlled pilot trial. PLoS Med[J] . 17 (3), e1003051 (2020). SINGH, P. et al. Effect of antibiotic pretreatment on bacterial engraftment after Fecal Microbiota Transplant (FMT) in IBS-D. Gut Microbes[J] . 14 (1), 2020067 (2022). INDIANI, C. et al. Childhood Obesity and Firmicutes/Bacteroidetes Ratio in the Gut Microbiota: A Systematic Review. Child. Obes[J] . 14 (8), 501–509 (2018). ZOU, Y. et al. Rice bran attenuated obesity via alleviating dyslipidemia, browning of white adipocytes and modulating gut microbiota in high-fat diet-induced obese mice. Food Funct[J] . 11 (3), 2406–2417 (2020). BERVOETS, L. et al. Differences in gut microbiota composition between obese and lean children: a cross-sectional study. Gut Pathog[J] . 5 (1), 10 (2013). PINTO FCS, SILVA AAM and SOUZA SL. Repercussions of intermittent fasting on the intestinal microbiota community and body composition: a systematic review. Nutr. Rev[J] . 80 (3), 613–628 (2022). TURRONI, F. & VAN SINDEREN D Genomics and ecological overview of the genus Bifidobacterium. Int. J. Food Microbiol[J] . 149 (1), 37–44 (2011). AZAD MAK, SARKER, M. et al. Probiotic Species in the Modulation of Gut Microbiota: An Overview. Biomed Res Int[J], 2018: 9478630 (2018). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6242383","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":437272102,"identity":"1ca36650-49ce-4ff7-9350-586126c1a4b7","order_by":0,"name":"Fangxia Mi","email":"","orcid":"","institution":"Ningbo Hangzhou Bay Hospital","correspondingAuthor":false,"prefix":"","firstName":"Fangxia","middleName":"","lastName":"Mi","suffix":""},{"id":437272104,"identity":"3a38a2a7-3f7b-4e3f-ab37-9235633502a7","order_by":1,"name":"Jinglu Guo","email":"","orcid":"","institution":"Ningbo Hangzhou Bay Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jinglu","middleName":"","lastName":"Guo","suffix":""},{"id":437272105,"identity":"2ed58784-c5f5-4d8d-9bfb-1cff8cdecb55","order_by":2,"name":"Wentao Zheng","email":"","orcid":"","institution":"The Affiliated Yanming Hospital of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Wentao","middleName":"","lastName":"Zheng","suffix":""},{"id":437272108,"identity":"88363e5c-34b0-48d4-bd23-e3d85b015abf","order_by":3,"name":"Hua Ye","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqElEQVRIiWNgGAWjYDACCRDBZsPDz99AmpY0GckZB0jTctjGoCGBSB0GtxtYN3woO89jwHCA8cPHHGK03DnAdnPGuds85swNzJIztxGj5Ub+t9u8bbd5LBsOsDHzEqclge3237ZzPAYHEkjRwth2gAQtkkAtN3vOJfNIzjjYTJxf+IBabvwos7Pn528++OEjMVoUDsCZjA1EqAcCeSLVjYJRMApGwUgGAErAOP7X58FIAAAAAElFTkSuQmCC","orcid":"","institution":"The Affiliated Lihuili Hospital of Ningbo University","correspondingAuthor":true,"prefix":"","firstName":"Hua","middleName":"","lastName":"Ye","suffix":""}],"badges":[],"createdAt":"2025-03-17 08:08:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6242383/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6242383/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81053978,"identity":"3f960d10-392b-40cb-a4d7-4509063041c6","added_by":"auto","created_at":"2025-04-21 16:56:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":184490,"visible":true,"origin":"","legend":"\u003cp\u003eFMT interferes with HFCS in mice. \u003cstrong\u003e(A) \u003c/strong\u003eBody weight at 28th week. \u003cstrong\u003e(B,C) \u003c/strong\u003eSerum ALT and TC. \u003cstrong\u003e(D) \u003c/strong\u003eHE staining of liver. \u003cstrong\u003e(E) \u003c/strong\u003eNAS score. \u003cstrong\u003e(F,G)\u003c/strong\u003e IL-17α and IL-1β expression levels in the liver. *P\u0026lt;0.05, **P\u0026lt;0.01 and, ****P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6242383/v1/ecd8f48dd58a2ee51d71a7da.png"},{"id":81053979,"identity":"f8248751-7dc1-47d5-b9b2-9a9ed19e24fe","added_by":"auto","created_at":"2025-04-21 16:56:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":142713,"visible":true,"origin":"","legend":"\u003cp\u003eImprovement of intestinal flora by FMT. \u003cstrong\u003e(A) \u003c/strong\u003ePrincipal coordinate analysis (PCoA).\u003cstrong\u003e (B)\u003c/strong\u003eBacterial composition of the different communities at phylum level.\u003cstrong\u003e (C) \u003c/strong\u003eBacterial composition of the different communities at the class level.\u003cstrong\u003e (D) \u003c/strong\u003eBacterial composition of the different communities at the genus level. \u003cstrong\u003e(E) \u003c/strong\u003eHistogram of the linear discriminant analysis (LDA) scores. (n=5 mice per group).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6242383/v1/43a7bbff305ec91b49c003c9.png"},{"id":83815748,"identity":"54f89a6d-7177-4269-bdc4-36b8edf42017","added_by":"auto","created_at":"2025-06-03 07:39:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":865618,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6242383/v1/c77c1ebd-ab36-4aaa-ab35-63d60210e044.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fecal microbiota transplantation alleviates high-fat and high-sugar diet-induced fatty liver in mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNonalcoholic liver disease (NAFLD) includes various conditions characterized by the buildup of fat in the liver, including simple fatty liver, nonalcoholic steatohepatitis, cirrhosis, and possibly liver cancer\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. NAFLD's worldwide prevalence has been increasing each year, with 25% in 2016 and over 30% in 2019\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Recently, there have been numerous calls to rename NAFLD as metabolic dysfunction-related fatty liver disease (MAFLD), which would help clinicians focus more on the disease and enhance patient management\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Studies have shown that Asians and Westerners are more likely to suffer from obesity-related diseases, such as NAFLD, even if they have the same BMI, which may be related to excessive visceral fat deposition\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThere are currently no drugs with proven efficacy to treat NAFLD, and it is important to prevent NAFLD through a healthy lifestyle and weight loss\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. The human colon provides a nutrient-rich environment for its microbiome: it is densely populated and home to an estimated 40\u0026ndash;100 trillion bacteria, with different resident microbes competing fiercely with each other for nutrients and space, but also working collaboratively to digest complex polysaccharides and other polymer molecules\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Positive balance shutdown between intestinal flora and host is beneficial to the normal development of biological metabolic system\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Intestinal microecosystem can promote the metabolism, and has the functions of flora barrier, immune regulation, cancer prevention and cancer suppression, etc. It is the most complex one of the most important microecosystems, such as Lactobacillus, bifidobacterium and other bacteria, can not only synthesize vitamins necessary for human health, but also use protein residues to synthesize essential amino acids that are essential for body growth and development\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Recent research has shown that an imbalance in gut bacteria is associated with the onset of NAFLD\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFecal microbiota transplantation (FMT) refers to the correction of ecological disorders through the management of the transplantation of feces collected from the donor into the recipient's intestine, thus treating underlying diseases\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. More and more studies have discovered that FMT is effective in treating inflammatory bowel disease, constipation, autism, severe infections and other conditions \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Probiotics are advantageous to the host by changing the microbiota composition and have been employed as a promising treatment for human NAFLD\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. FMT can provide patients with more diversity and abundance of symbiotic bacteria and other microorganisms than probiotics and synbiotic therapies, which is conducive to maintaining the ecological balance of intestinal microorganisms\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Therefore, we speculated that restoring the homeostasis of the gut-liver axis in the course of NAFLD by regulating intestinal microorganisms such as FMT would promote the establishment of a virtuous cycle between the restoration of intestinal microecological homeostasis and the improvement of NAFLD.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals experiments\u003c/h2\u003e \u003cp\u003e All procedures were approved by the Ethics Committee on Animal Research of the Ningbo University School of Medicine (Approval number: AEWC-NBU20210012). All methods were performed in accordance with relevant guidelines and regulations and in accordance with ARRIVE guidelines. Male C57BL/6 mice (20\u0026thinsp;\u0026plusmn;\u0026thinsp;2g) aged 5\u0026ndash;6 weeks were placed in a 21\u0026ndash;22◦C light-dark cycle SPF chamber for 12/12 h and fed adaptive for one week.\u003c/p\u003e \u003cp\u003eThere were three groups in this experiment: control group, HFCS group and HFCS\u0026thinsp;+\u0026thinsp;FMT group, with five mice in each group. The Control group was given a normal diet, while the HFCS group and HFCS\u0026thinsp;+\u0026thinsp;FMT group were given a high-fat and high-sugar diet, that is, a high-fat diet with 60% fat content and 42g/L sugar water mixed with 55% fructose and 45% sucrose by weight. The whole experiment was carried out for 28 weeks, after 8 weeks, the Control group and HFCS group were given normal saline administration, and the HFCS\u0026thinsp;+\u0026thinsp;FMT group was given FMT. Feces from mice on a normal diet were collected before each FMT. The specific procedure was to add 200mg of fresh feces to 4ml of normal saline, rotate for 5min, stand gauze and filter for 2min, and collect fecal suspension in a sterile test tube\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Sterile normal saline and fecal bacteria solution were injected into the oral cavity of mice according to groups, the dose was 200\u0026micro;L/ time, 3 times/week, a total of 24 times.\u003c/p\u003e \u003cp\u003eEach mouse was weighed once a week, and the fur color and exercise status of the mice were observed. On the night before the mice were killed, a sterile frozen storage tube was prepared, and 2\u0026ndash;3 mice feces were collected for 16S sequencing and analysis of bacterial microbial diversity. After 28 weeks, the mice were anesthetized by inhaling 40 percent carbon dioxide gas and having blood drawn from their eyeballs. Mouse liver tissue samples were collected, half of the largest liver leaf tissue was cut and soaked in a centrifuge tube containing 5 mL10% formalin solution for fixed preservation for hematoxylin eosin (H\u0026amp;E) staining. The remaining liver tissue was placed in labeled freezer tubes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Histological analysis of the livers\u003c/h2\u003e \u003cp\u003eThe collected liver tissue specimens were soaked in formalin buffer solution for at least 24h. Half of the largest liver lobe was cut into paraffin wax and sliced for HE staining. The sections of each group under different multiples of field of view were observed under microscope, and the appropriate liver histopathology pictures were taken.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Biochemical analysis\u003c/h2\u003e \u003cp\u003eSerum AST and TC levels were measured according to the kit instructions. (Ningbo MedicalSystem Biotechnology Co., Ltd., Ningbo, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Quantitative real-time PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eLiver tissue was cut on dry ice to extract total RNA, and the concentration of RNA in the sample was obtained by spectrophotometry (Denovix, America). cDNA was prepared from 1 \u0026micro;g of total RNA using a TransScript\u0026reg; All-in-One First-Strand cDNA Kit (TransGen Biotech, Beijing, China). The expression value of the blank control group was normalized as 1, and the expression level of the target gene in other groups was expressed as a relative multiple, and then the expression level of the target gene was compared between different groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Gut microbiota analysis\u003c/h2\u003e \u003cp\u003eThe DNA of each group was extracted using the QIAamp Fast DNA Stool Mini Kit (Cat. No. 51604) according to the instructions. Primers 341F 5\u0026prime;-CCTACGGGRSGCAGCAG-3\u0026prime; and 806R 5\u0026prime;-GGACTACVVGGGTATCTAATC-3\u0026prime; with specifc barcodes were used to amplify the V3\u0026ndash;V4 region of the bacterial ribosomal RNA gene to obtain a\u0026thinsp;~\u0026thinsp;500-bp product. Sample extraction, database construction, sequencing and analysis services are completed by Shanghai Ruiyi Biotechnology Co., Ltd. (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eData were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error, with statistical analysis conducted using SPSS statistics Version 26.0 software (IBM, China), and the results were illustrated using GraphPad Prism9.0 software. To compare differences between groups, a one-way ANOVA was conducted, followed by Tukey's test for pairwise comparisons. A P-value of less than 0.05 indicates statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Impact of FMT on body weight and serum transaminase levels.\u003c/h2\u003e \u003cp\u003eThe HFCS group mice were obese, unresponsive, inactive, coarse, and dull hair. After receiving fecal suspension FMT intervention in the normal group for 8 weeks, the mice were symmetrical, responsive, active, with soft hair and better gloss, and their body weight decreased significantly compared with that in the HFCS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The HFCS group had significantly higher ALT and TC levels compared to the control group. Mice treated with FMT showed improvements in ALT and TC compared to HFCS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB,C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Improvement of intestinal flora by FMT.\u003c/h2\u003e \u003cp\u003eAfter 28 weeks, we sequenced the 16S rDNA baseline in the stool. PCoA is used to compare and analyze the microbial community structure between different samples being measured. The three groups of mice were grouped separately, and the control group was obviously separated from the HFCS group, indicating that the intestinal flora of NAFLD mice had obvious changes, and the FMT intervention group was close to the normal group. The HFCS group had significantly higher ALT and TC levels compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e According to the number of OUT and the species represented by OUT, the corresponding distribution maps of each sample at phylum, class and genus level were obtained. After 28 weeks of feeding HFCS, there were extensive changes in gut microbial community structure at all levels compared to the control group. The HFCS group, when compared to the control group, demonstrated significant phylum-level changes, including an increase in Firmicutes and a decrease in Bacteroidetes, leading to a higher Firmicutes to Bacteroidetes ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). On the level of class, compared with the control group, bacilli in HFCS\u0026thinsp;+\u0026thinsp;NS group increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). At the genus level, Bifidobacterium was significantly decreased compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The abundance of the specified bacteria in mice on a high-fat and high-sugar diet changed after FMT treatment, bringing their gut composition closer to that of the normal diet group. The marker species analyzed by LefSe indicate that FMT can interfere with intestinal flora (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 FMT treatment attenuated HFCS-induced steatohepatitis.\u003c/h2\u003e \u003cp\u003eThe result showed that the hepatocytes of the control group were of the same size without steatosis. In HFCS group, the structure of hepatic lobule was disordered, and lymphocytes and neutrophils were infiltrated. After FMT treatment, hepatocyte swelling was reduced, fat vacuoles were less and hepatocyte arrangement was more orderly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Following the FMT intervention, the liver NAS score in the HFCS\u0026thinsp;+\u0026thinsp;FMT group decreased significantly compared to the HFCS group, which had a score much higher than the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effect of FMT on the intrahepatic immune.\u003c/h2\u003e \u003cp\u003eCompared to the control group, the HFCS group had elevated IL-1β mRNA levels in the liver, which were decreased by FMT intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). The IL-17α protein level in liver tissue was higher compared to the control group. The FMT intervention effectively reversed this imbalanced immune factor, thereby reducing IL-17α in the liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). The findings indicated that FMT treatment might lower the levels of inflammatory factors in NAFLD.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eNumerous specialists suggested renaming NAFLD to MAFLD, indicating fatty liver disease linked to metabolic issues across the body\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. With the change of modern diet structure, the prevalence of metabolic syndrome caused by high-fat and high-sugar diet is increasing worldwide. And The new definition of MAFLD may improve the diagnostic rate of the disease\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. A mouse model of NAFLD with high fat and high sugar was established in this study, and the pathogenesis of human NAFLD was simulated by using a 42g/L sugar water mixed with a high fat diet with 60% fat content and a weight ratio of 55% fructose and 45% sucrose.\u003c/p\u003e \u003cp\u003eA study demonstrated that the colonization of intestinal microbiota in NASH patients can worsen liver steatosis and inflammation in germ-free mice on a high-fat diet, confirming the crucial role of intestinal microbiota dysfunction in the disease's pathogenesis\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. However, in the research experiments on FMT for NAFLD, there were several ways to pretreat mice before FMT, including antibiotic pretreatment to cause enteric sterility, acid-inhibiting agents such as omeprazole to resist the effect of gastric acid, and intestinal fecal bacteria transplantation on the occurrence and development of NAFLD. Few experiments have shown whether FMT can be transferred to models that are already colonized, or to what extent replacement of natural microorganisms is necessary to induce phenotypic changes\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Using broad-spectrum antibiotics before FMT might lead to side effects and pose ethical and antibiotic management challenges\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Therefore, in this experiment, We selected the method with the least influence, that is, no antibiotic pretreatment, and tested the effects of FMT on liver pathology, liver enzymes and intestinal flora of mice by gavage. Therefore, a mouse model of NAFLD was established through a high-fat and high-sugar diet, and the intestinal sterility of mice was caused by no antibiotic pretreatment before FMT.\u003c/p\u003e \u003cp\u003eThe weight of NAFLD mice can be reduced and the pathological changes of liver can be relieved after receiving normal mouse faecal bacteria solution. Q-PCR results showed that FMT intervention could reduce liver inflammation in mice with NAFLD, which was manifested by down-regulation of IL-1β and IL-17A mRNA expression in liver. The above implantation signals were encouraging. However, we did not find statistically significant differences in serum ALT and TC between NAFLD mice and NAFLD mice after receiving normal mouse faecal bacteria solution, but the trend was improved, which we considered to be related to the small sample size in this experiment.\u003c/p\u003e \u003cp\u003eThe ratio of firmicutes and Bacteriotes in the intestinal tract of mice in the high-fat and high-sugar diet group was increased, while FMT intervention could significantly reduce the abundance of firmicutes and the ratio of firmicutes to bacteriotes in the intestinal flora. A high-quality study has shown that Firmicutes are linked to weight gain\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Several studies have identified a close relationship between the Firmicutes to Bacilli ratio and obesity, suggesting it as a potential obesity marker\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Altering the balance between firmicutes and bacteriae in the gut microbiota can protect metabolism and help prevent weight gain\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe result showed that bifidobacteria abundance decreased significantly in NAFLD model mice at the genus level, and the above bacterial abundance was successfully reversed after FMT intervention. Bifidobacterium is a well-known intentional group of bacteria that can be applied as probiotics in the daily human diet\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Research indicates that bifidobacterium can successfully prevent the growth and settlement of harmful bacteria, preserve the gastrointestinal barrier's integrity, and suppress the creation and release of inflammatory cytokines\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Our preliminary study showed that fecal transplantation alleviated high-fat, high-sugar induced NAFLD through a beneficial effect on the microbiome.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eFangxia Mi and Jinglu Guo wrote the main manuscript text. Fangxia Mi and Wentao Zheng performed all the experiments together, and Hua Ye directed the methodology and funding of the research. All authors reviewed the manuscript\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNASSIR F. NAFLD: Mechanisms, Treatments, and Biomarkers. \u003cem\u003eBiomolecules[J]\u003c/em\u003e, \u003cb\u003e12\u003c/b\u003e(6): (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSHARPTON, S. R. et al. Gut microbiome-targeted therapies in nonalcoholic fatty liver disease: a systematic review, meta-analysis, and meta-regression. \u003cem\u003eAm. J. Clin. 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Rev[J]\u003c/em\u003e. \u003cb\u003e80\u003c/b\u003e (3), 613\u0026ndash;628 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTURRONI, F. \u0026amp; VAN SINDEREN D Genomics and ecological overview of the genus Bifidobacterium. \u003cem\u003eInt. J. Food Microbiol[J]\u003c/em\u003e. \u003cb\u003e149\u003c/b\u003e (1), 37\u0026ndash;44 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAZAD MAK, SARKER, M. et al. Probiotic Species in the Modulation of Gut Microbiota: An Overview. Biomed Res Int[J], 2018: 9478630 (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Gut-liver axis NAFLD Fecal microbiota transplantation high-fat and high-sugar diet","lastPublishedDoi":"10.21203/rs.3.rs-6242383/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6242383/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAround the world, Nonalcoholic fatty liver disease (NAFLD) has become the most frequent chronic liver disease. Fecal microbiota transplantation (FMT) is a successful method for rebuilding gut flora and has been applied in treating and researching various microbiome-related conditions like inflammatory bowel disease. FMT is considered a breakthrough medical development in recent years, but further research is needed in NAFLD-related areas. Mice were randomized into control, high-fat and high-sugar diet (HFCS) and HFCS\u0026thinsp;+\u0026thinsp;FMT groups. A mouse model of NAFLD was established on a high-fat and high-sugar diet for 20 weeks, followed by FMT for 8 weeks. After 8 weeks of FMT initiation, serum, liver tissue specimens and feces of mice were collected for biochemical experiments, histopathology and molecular biology to obtain experimental data and statistical analysis. Our results showed that Firmicutes and Bacteriae significantly increased and bifidobacteria significantly decreased in mice fed HFCS. After FMT treatment, the abundance of the above bacteria was changed, and the composition of the above bacteria in the gut was close to that of the normal diet group. FMT reduced body weight in mice and improved serum alanine aminotransferase (ALT) and total cholesterol (TC) levels. The significant decrease of intrahepatic proinflammatory cytokines and liver pathology showed that hepatitis was relieved after FMT. These data indicate that High-fat and high-sugar diet can induce NAFLD in mice and change the structure of intestinal flora. NAFLD was alleviated by correcting intestinal flora with FMT.\u003c/p\u003e","manuscriptTitle":"Fecal microbiota transplantation alleviates high-fat and high-sugar diet-induced fatty liver in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-21 16:56:12","doi":"10.21203/rs.3.rs-6242383/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4313ce80-a361-4140-ba11-8043fb226413","owner":[],"postedDate":"April 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46559215,"name":"Biological sciences/Microbiology"},{"id":46559216,"name":"Health sciences/Diseases/Metabolic disorders"}],"tags":[],"updatedAt":"2025-06-03T07:38:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-21 16:56:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6242383","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6242383","identity":"rs-6242383","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}