Establishment method and application of mouse model for metabolism-related fatty liver disease

Abstract

The invention belongs to the technical field of animal experiment models, and discloses an establishment method and application of a mouse model for metabolism-related fatty liver disease, a female mouse exposes different doses of amoxicillin through oral intragastric administration every day from the thirteenth day (GD13) of pregnancy to the seventh day (PND7) after birth of offspring, a pregnant mouse naturally delivers, and a baby mouse has conventional diet after 28 (PND28) weaning after birth. Each group is divided into two exposed subgroups thirteen weeks after the offspring is born, one half continues to be subjected to conventional diet for 6 weeks, and the other half is subjected to high-fat diet for 6 weeks. The research shows that perinatal amoxicillin exposure increases the susceptibility to liver lipid accumulation induced by high fat diet by destroying early intestinal flora colonization. The reduction of lactobacillus colonization inhibits the transformation from primary bile acid to secondary bile acid, reduces the secondary bile acid, and enhances the intestinal lipid absorption and liver lipid deposition in the adult period. Lactobacillus is supplemented in the lactation period, so that early intestinal flora colonization can be improved, and liver lipid accumulation caused by high-fat diet in the adult period is relieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal experimental models, and particularly relates to a method for establishing a mouse model of metabolic associated fatty liver disease and its application. Background Art

[0002] Metabolic Associated Fatty Liver Disease (MAFLD) is a chronic liver disease characterized by hepatic lipid deposition, accompanied by overweight or obesity, type 2 diabetes, and metabolic disorders. It is estimated that MAFLD affects more than one-third of the world's population and has become one of the most important public health problems. It is generally believed that sedentary lifestyle, high-sugar or high-fat diet, advanced age, and dyslipidemia are all risk factors for MAFLD. Abnormal accumulation of hepatic lipids plays a central role in the development of MAFLD. Recently, the Developmental Origins of Health and Disease theory suggests that MAFLD develops throughout the life cycle but originates in early life. Increasing data indicate that adverse environmental exposures in early life increase the risk of adult MAFLD. However, the exact mechanism has not been elucidated. Many studies have shown that alterations in epigenetic and metabolic reprogramming are key mechanisms underlying susceptibility to adult MAFLD. Recently, two studies have shown that early-life exposure to toxins can lead to adult steatosis by altering the hydroxymethylation reprogramming of hepatic β-oxidation genes. On the other hand, increasing evidence suggests a specific association between gut microbiota dysbiosis and MAFLD. Gut microbiota colonization refers to the process by which the microbial community in the host intestine gradually and successfully establishes and finally forms a relatively stable and diverse microbial ecosystem from an initially sterile or low-diversity state through a series of complex ecological processes. It is well known that gut microbiota colonizes in early life. Several studies have shown that early gut microbiota colonization affects health throughout the life cycle. In this study, we hypothesized that adverse factors in early life increase susceptibility to adult MAFLD by affecting early gut microbiota colonization. Amoxicillin (AM) is a commonly used antibiotic during the perinatal period, usually used to treat premature rupture of membranes to reduce the incidence of maternal and infant infections. In this study, we aimed to construct a mouse model with altered early gut microbiota colonization using perinatal AM exposure, and then investigated the effect of altered early gut microbiota colonization on the susceptibility to high-fat diet (HFD)-induced MAFLD. Our results showed that perinatal AM exposure altered early gut microbiota colonization and increased susceptibility to HFD-induced hepatic lipid deposition in adulthood. We provided evidence that early gut microbiota colonization affects susceptibility to adult hepatic lipid deposition by disrupting gut bile acid (BA) metabolism and dietary lipid absorption.

[0003] In view of the above analysis, the problems and defects of the prior art are as follows: it is necessary to construct a mouse model of metabolic associated fatty liver disease in adulthood and reveal the role of increased intestinal lipid absorption caused by changes in intestinal flora colonization in early life in the susceptibility of offspring to metabolic associated fatty liver disease in adulthood induced by perinatal amoxicillin exposure. Summary of the Invention

[0004] In view of the problems of the prior art, the present invention provides a method for establishing a mouse model of metabolic associated fatty liver disease and its application.

[0005] The present invention is implemented as follows. A method for establishing a mouse model of metabolic associated fatty liver disease, the method comprising:

[0006] C57BL / 6J mice at 8 weeks of age, female mice: 18 - 20 g; male mice: 22 - 24 g. They were housed in a SPF-level Laboratory Animal Room at a temperature of 22 ± 3°C and a humidity of 50 ± 5%, following a 12-hour light / dark cycle; with free access to food and water. A 2-week adaptation period was carried out before the start of the breeding protocol. Female and male mice at 10 weeks of age were paired overnight at a ratio of 4:2. From 8 pm to 7 am the next morning, if the female mouse had a vaginal plug after mating, it was considered pregnant and marked as gestational day 0 (GD0). Maternal antibiotic exposure was mainly used to prevent infection and treat complications, which usually occurred around birth. In our mouse model, from GD13 to postnatal day 7 (PND7), AM was administered to the mother mice to closely mimic the clinical exposure time. On GD13, the mother mice were divided into three groups: control group (Ctrl), low AM group (AML, 20 mg / kg / d), and high AM group (AMH, 200 mg / kg / d). AM was dissolved in sterile water. The control group mice received sterile water by gavage, while the AML and AMH groups received the corresponding doses of AM from GD13 to PND7. The human dose range is 500 to 1500 mg / day (https: / / www.drugs.com / dosage / amoxicillin.html). Calculated according to body surface area, the mouse dose is approximately 50 - 150 mg / kg / d. The offspring weaned at PND28 were fed a normal diet (3.44 kcal / g: 24% protein, 13% fat, 63% carbohydrate). At postnatal week 13 (PNW13), the offspring mice were randomly divided into two subgroups of normal diet (NC) and high-fat diet (HFD). HFD included 14% protein, 42% fat, 44% carbohydrate, and 0.2% cholesterol (TP 26304, TrophicDiet, Nantong, China) for 6 weeks. During the whole study, the offspring were weighed regularly and fecal samples were collected.

[0007] The present invention provides a method for evaluating metabolic associated fatty liver disease, which method comprises:

[0008] a. Randomly divide the mother mice into different groups at GD13, and administer sterile water or AM by gavage to the mother mice daily from GD13 to PND7; the offspring are naturally delivered. At the 13th week of the offspring, each group is randomly divided into two subgroups, and after continuing the normal diet or high-fat diet exposure for six weeks, a metabolic cage experiment is carried out and samples are taken, and relevant specimens are retained for the next step of detection.

[0009] b. Analyze the data of the metabolic cage experiment, weigh the collected tissues, calculate the organ coefficients, conduct biochemical analysis tests, HE staining, Oil Red O staining, liver lipidomics, liver transcriptomics, RT-PCR and other experiments to evaluate the effect of perinatal AM exposure on metabolic-related fatty liver disease in adult offspring mice.

[0010] The present invention also provides a method for evaluating the impact of gut microbiota disorder on lipid absorption, which includes:

[0011] a. Animal experiment 1: Male and female C57BL / 6J mice at 12 weeks of age were randomly divided into two groups. In the control group, mice were gavaged with an equal volume of sterile water. In the AMH group, mice were force-fed with AM (AMH, 200 mg / kg / d) for 6 days. After fasting for 4 hours, baseline blood samples were collected from the mice by tail bleeding. Next, olive oil was gavaged to the mice at a dose of 10 μl / g body weight. Blood was drawn by tail bleeding at 1 hour, 2 hours, 4 hours, and 6 hours after the oil bolus. To ensure sufficient yield for downstream analysis, 30 - 40 μL of whole blood was collected, and serum was separated by centrifugation at 3500 rpm for 15 minutes at 4°C to obtain approximately 10 μL of serum. Throughout the experiment, mice had access to water but not to food. Serum TG was detected using a kit to evaluate lipid absorption;

[0012] b. Animal experiment 2: The same AM exposure as in animal experiment 1 was given. After 6 days of AM exposure, the animals were fasted overnight, refed with 60% HFD for 2 hours, and then euthanized. Intestinal tissues were collected and stored in paraformaldehyde for further analysis. Serum TG and TC were detected using a kit, and HE staining of intestinal tissues was performed to evaluate intestinal lipid absorption;

[0013] c. Animal experiment 3: The same AM exposure as in animal experiment 1 was given. Briefly, after 6 days of AM exposure, the mice were fasted for 4 hours and administered BODIPY 500 / 510 C1, C12 FA (2 μg / g body weight) and olive oil (10 μl / g body weight) for 2 hours, and then euthanized. The small intestinal tissues were embedded with OCT embedding medium and sectioned for fluorescence distribution analysis to evaluate intestinal fatty acid absorption;

[0014] d. Based on the construction of the animal model, we detected the effect of cecal content bile acid metabolism on intestinal lipid absorption.

[0015] The present invention also provides a method for evaluating the alteration of gut microbiota colonization in early life, which includes:

[0016] a. Randomly divide pregnant mice on GD13 into different groups, and intragastrically administer sterile water or AM to the pregnant mice daily from GD13 to PND7; collect the feces of the pregnant mice at GD18 during delivery. The offspring are delivered naturally, and the feces of the offspring at PND28 and PND84 are collected.

[0017] b. Use metagenomics to detect the composition and function of the intestinal flora of the offspring at PND84; use 16S rRNA sequencing to detect the composition and function of the intestinal flora at different times, including GD18 of the pregnant mice, the 28th day and the 84th day after the birth of the offspring, and evaluate the colonization of the intestinal flora.

[0018] The present invention also provides a method for evaluating the improvement effect of supplementing Lactobacillus murinus during lactation on the intestinal flora colonization and MAFLD susceptibility caused by AM. The method includes:

[0019] a. On GD13, randomly divide pregnant mice into two groups: a control group (Ctrl) or a high-AM group (AMH, 200 mg / kg / d). From GD13 to PND7, intragastrically expose to sterile water or AM (200 mg / kg / d). On PND8, the male offspring of the control group are randomly divided into two subgroups: half as the Ctrl group, and the other half are intragastrically administered Lactobacillus murinus as the LM group. Similarly, the male offspring of the amoxicillin group are randomly divided into two subgroups: half as the AMH group, and the other half are gavaged with Lactobacillus murinus and designated as the AMH+LM group. On PNW13, all male offspring are fed a HFD for 6 weeks. Throughout the study, all offspring are regularly weighed and fecal samples are collected.

[0020] c. Use 16S rRNA sequencing to detect the composition and function of the intestinal flora of the offspring on the 28th day after birth, and evaluate the improvement of the intestinal flora colonization by supplementing Lactobacillus murinus.

[0021] b. Conduct experiments such as weighing the collected tissues, calculating the organ coefficients, biochemical analysis detection, HE staining, oil red O staining, and liver lipidomics to evaluate the protective effect of supplementing Lactobacillus murinus on metabolic associated fatty liver disease in adult offspring mice.

[0022] c. Detect bile acids in cecal contents to evaluate the improvement effect of supplementing Lactobacillus murinus on intestinal bile acid metabolism in adult offspring mice.

[0023] Another object of the present invention is to provide an application of a mouse model of metabolic associated fatty liver disease induced by changes in intestinal flora colonization in screening drugs for metabolic associated fatty liver disease.

[0024] Combined with the above technical solutions and the solved technical problems, the advantages and positive effects of the technical solutions to be protected by the present invention are:

[0025] This study investigated the effect of perinatal AM exposure on the susceptibility to HFD-induced MAFLD in adulthood. The main innovative findings are as follows: First, perinatal AM exposure exacerbates liver lipid deposition induced by HFD in adulthood; Second, perinatal AM exposure promotes dietary lipid absorption by disrupting intestinal BA metabolism; Third, perinatal AM exposure interferes with early gut microbiota colonization and continuously disrupts the structure and function of the gut microbiota in adulthood; Finally, supplementation with Lactobacillus during lactation can improve gut microbiota colonization and intestinal BA metabolism and reduce HFD-induced liver lipid deposition. These findings provide evidence that early gut microbiota colonization affects the susceptibility to MAFLD in adulthood.

[0026] The results of this study have important translational value in clinical practice. In fact, Lactobacillus is a major gut microbiota encoding bile salt hydrolase, which converts primary BA into secondary BA. This study shows that Lactobacillus not only decreases in AM-exposed weaned pups but also in adult offspring. We used metagenomic screening to identify the most contributing strain, Lactobacillus murinus, and supplemented this strain during PND8 to PND28 to directly observe its effects on gut microbiota colonization and dietary lipid absorption. The results showed that supplementation with Lactobacillus murinus not only improved early gut microbiota colonization but also improved the susceptibility to bile acid metabolism and liver lipid deposition in adulthood. An earlier study found that maternal supplementation with Lactobacillus during lactation can reduce respiratory syncytial virus-induced goblet cell hypertrophy and mucus production in offspring. A recent report showed that supplementation with Lactobacillus during pregnancy can prevent offspring from developing dextran sulfate sodium-induced colitis. These results suggest that supplementation with Lactobacillus during lactation may be a potential method to improve early gut microbiota colonization and adult HFD-induced liver lipid deposition.

[0027] At the technical level, the present invention has the following remarkable features:

[0028] First, by exposing pregnant mice to the commonly used antibiotic amoxicillin during the perinatal period from GD13 to PND7, the clinical antibiotic use time and exposure method were simulated. This exposure method has high repeatability and controllability, which can ensure consistent conditions for each experiment, thereby improving the accuracy and reliability of experimental results.

[0029] Second, the present invention uses small animal metabolic cages to evaluate the basal metabolism of mice, using basic indicators: mouse body weight gain, food intake, liver / fat weight and organ coefficients, biochemical analysis detection, HE staining, Oil Red O staining, liver lipidomics, liver transcriptomics, RT-PCR, etc. to comprehensively evaluate the MAFLD-like phenotype of mice; combining multiple omics to comprehensively evaluate the role of gut microbiota colonization in metabolic associated fatty liver disease.

[0030] Next, the present invention found that AM exposure during pregnancy induced increased intestinal lipid absorption, which exacerbated hepatic lipid deposition after a high-fat diet, revealing an extrahepatic mechanism of MAFLD from the perspective of intestinal lipid absorption.

[0031] Next, the present invention found that perinatal AM exposure disrupted the composition and function of the offspring's adult intestinal microbiota and traced it back to the altered intestinal microbiota colonization in early life, revealing the developmental origin mechanism of adult MAFLD from the perspective of intestinal microbiota colonization.

[0032] Next, this study found that the supplementation of probiotic Lactobacillus murinus improved the susceptibility of offspring to MAFLD after a high-fat diet in adulthood caused by perinatal AM exposure. This method has clinical and preventive significance and lays a theoretical foundation for the clinical application of probiotic Lactobacillus murinus in preventing developmental diseases caused by disrupted intestinal microbiota colonization in early life.

[0033] In addition, the present invention also emphasizes the natural delivery process of pregnant mice, which helps to exclude the influence of the delivery method on the experimental results and makes the model closer to the actual physiological situation.

[0034] In terms of technical effects, the metabolic-associated fatty liver disease model constructed by the present invention has the following advantages:

[0035] First, the model has high stability. Due to the use of standardized prenatal drug exposure and probiotic Lactobacillus murinus supplementation, as well as multi-omics to prove the MAFLD model. This model shows high consistency under different experimental conditions, which is beneficial for subsequent research and application.

[0036] Second, it has broad application prospects. This model can not only be used for basic research on MAFLD, but also for research on clinical Lactobacillus murinus intervention, providing new ideas and directions for the clinical treatment of MAFLD.

[0037] The method for constructing a mouse model of metabolic-associated fatty liver disease proposed by the present invention, through multi-omics combined with innovative perinatal drug intervention and MAFLD evaluation methods, successfully constructed a stable, reliable and promising animal model, providing a new powerful tool for the research and treatment of MAFLD. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0039] The following are two specific embodiments provided by the present invention, describing how to construct a mouse model of metabolic-associated fatty liver disease according to the above claims:

[0040] Figure 1 It is a flow chart of a method for establishing a mouse model of metabolic dysfunction-related fatty liver provided by an embodiment of the present invention;

[0041] Figure 2 It is a detailed flow chart of a method for establishing a mouse model of metabolic dysfunction-related fatty liver provided by an embodiment of the present invention;

[0042] Figure 3 It is the effect of perinatal AM exposure on fat weight and blood lipids of adult offspring fed with HFD provided by an embodiment of the present invention;

[0043] Figure 4 It is a diagram of the effect of perinatal AM exposure on lipid metabolism of adult offspring fed with HFD provided by an embodiment of the present invention;

[0044] Figure 5 It is a diagram of the effect of perinatal AM exposure on liver lipid accumulation and lipid profile of adult offspring fed with HFD provided by an embodiment of the present invention;

[0045] Figure 6 It is a diagram of the effect of perinatal AM exposure on energy balance of adult offspring fed with HFD provided by an embodiment of the present invention;

[0046] Figure 7 It is a diagram of the effect of perinatal AM exposure on liver lipid metabolism genes of adult offspring fed with HFD provided by an embodiment of the present invention;

[0047] Figure 8 It is a diagram of the effect of AM exposure on intestinal lipid absorption provided by an embodiment of the present invention;

[0048] Figure 9 It is a diagram of the effect of perinatal AM exposure on fecal BA composition of adult offspring fed with HFD provided by an embodiment of the present invention;

[0049] Figure 10 It is a diagram of the effect of perinatal AM exposure on the intestinal flora at the species level of adult offspring provided by an embodiment of the present invention;

[0050] Figure 11 It is a diagram of the effect of perinatal AM exposure on the intestinal flora composition of maternal mice, weaned pups and adult offspring provided by an embodiment of the present invention;

[0051] Figure 12 It is a diagram of the effect of perinatal AM exposure on the richness and diversity of the intestinal flora of maternal mice, weaned pups and adult offspring provided by an embodiment of the present invention;

[0052] Figure 13 It is a functional diagram of predicting bacterial microbiota in the feces of 12-week-old male and female offspring mice by combining PICRUSt2 with the eggNOG database provided by an embodiment of the present invention;

[0053] Figure 14 It is a diagram showing the effects of Lactobacillus murinus supplementation provided by an embodiment of the present invention on the intestinal flora composition and fecal bile acid metabolism of male offspring;

[0054] Figure 15 It is a diagram showing the effects of Lactobacillus murinus supplementation provided by an embodiment of the present invention on the liver lipid profile of adult male offspring fed a high-fat diet (HFD);

[0055] Figure 16 It is a diagram showing the effects of Lactobacillus murinus supplementation provided by an embodiment of the present invention on liver lipid accumulation in male offspring induced by HFD;

[0056] Figure 17 It is a schematic diagram of the animal experiment design provided by an embodiment of the present invention. Detailed implementation manners

[0058] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0059] As Figure 1 、 Figure 2 shown, a method for establishing a mouse model of metabolic dysfunction-related fatty liver provided by an embodiment of the present invention is characterized in that the method includes:

[0060] S1: Animal experiment:

[0061] S2: Metagenomic sequencing;

[0062] S3: Liver lipidomics analysis;

[0063] S4: Bile acid quantitative analysis;

[0064] S5: Statistical analysis.

[0065] S1 provided by an embodiment of the present invention specifically includes:

[0066] Obtain 8-week-old C57BL / 6J mice, female: 18 - 20 g; male: 22 - 24 g; the mice are given a 2-week adaptation period before the start of the breeding program; they are housed in a specific pathogen-free (SPF) environment at a temperature of 22 ± 3 °C and a humidity of 50 ± 5%, following a 12-hour light / dark cycle; food and water are freely available, and for breeding, female and male mice are paired overnight at a ratio of 4:2, from 8 pm to 7 am the next morning, and mating is confirmed by the presence of a vaginal plug, marked as day 0 of pregnancy;

[0067] At GD13, pregnant mice were divided into three groups: a control group, a low-dose amoxicillin group AML at 20 mg / kg / d, and a high-dose amoxicillin group AMH at 200 mg / kg / d; mice in the control group were given sterile water by gavage, while the amoxicillin groups were given the corresponding dose of amoxicillin from GD13 to postnatal day 7; the human dose range was 500 to 1500 mg / day, and the mouse dose was approximately 50 - 150 mg / kg / d according to body surface area calculation; amoxicillin was dissolved in sterile water; the pups were weaned at PND28 and fed a normal diet, 3.44 kcal / g: 24% protein, 13% fat, 63% carbohydrate; at 13 weeks after birth, the pups were divided into normal diet NC and high-fat diet HFD subgroups; the HFD contained 14% protein, 42% fat, 44% carbohydrate, and 0.2% cholesterol for 6 weeks; the body weights of the mice were weighed regularly, and fecal samples were collected throughout the study period.

[0068] S2 provided in the embodiment of the present invention specifically includes:

[0069] 700 ng of DNA was prepared for each sample; PCR amplification and purification were performed using the AMPure XP system; the DNA concentration was measured using the Qubit DNA Assay Kit on a Qubit 2.0 fluorometer and diluted to 2 ng / mL; the library insert size was evaluated using the Agilent Bioanalyzer 2100 system; the accurate concentration of the library > 3 nM was verified by qPCR; the library preparation was sequenced on the Illumina HiSeq 4000 platform to generate 150 bp paired-end reads.

[0070] S3 provided in the embodiment of the present invention specifically includes:

[0071] The liver tissue was thawed on ice, mixed for about 10 seconds, and then centrifuged at 3000 rpm for 5 minutes at 4°C; each 20 mg sample was homogenized with 1 mL of a mixture containing methanol, MTBE, and an internal standard mixture, and then mixed for 15 minutes; 200 μL of water was added to the mixture and stirred for 1 minute, and then centrifuged at 12,000 rpm for 10 minutes at 4°C; after centrifugation, 500 μL of the supernatant was extracted and concentrated; the concentrated powder was dissolved in 200 μL of solvent B for LC-MS / MS analysis.

[0072] S4 provided in the embodiment of the present invention specifically includes:

[0073] Mix 20 mg of cecal content with 1 μg / mL of internal standard mixture solution (10 μL) and 200 μL of methanol / acetonitrile (v / v = 2:8) and homogenize; vortex the sample at 2,500 rpm for 10 minutes and store it at -20 °C for 10 minutes to promote protein precipitation; finally, centrifuge at 12,000 rpm for 10 minutes at 4 °C; transfer the supernatant to a new plastic microcentrifuge tube and concentrate it using a concentrator; after concentration, reconstitute the sample in 100 μL of 50% methanol-aqueous solution for subsequent LC-MS / MS analysis using an Applied Biosystems 6500 triple quadrupole; the HPLC column used is a Waters ACQUITY UPLC HSS T3 C18, 00 mm × 2.1 mm, 1.8 μm.

[0074] S5 provided in the embodiment of the present invention specifically includes:

[0075] Statistical analysis was performed using SPSS 23.0, and the data were expressed as mean ± standard error; a two-tailed Student's t-test was used for comparison between two groups, and one-way ANOVA was used for comparison among multiple groups; when significant differences were found, a post hoc least significant difference test was used to compare the control group and the AM exposure group; the Kruskal-Wallis H test was used to analyze non-normally distributed data; a two-way ANOVA was used for mouse body weight assessment; GraphPad Prism 8.0 was used for graphical representation and visualization; a p-value less than 0.05 indicated statistical significance.

[0076] The mice provided in the embodiment of the present invention specifically include: female: 18 - 20 g; male: 22 - 24 g.

[0077] Solvent B provided in the embodiment of the present invention is specifically acetonitrile: isopropanol with a volume ratio of 1:9, containing 10 mM ammonium formate and 0.1% formic acid.

[0078] The normal diet provided in the embodiment of the present invention is 3.44 kcal / g: 24% protein, 13% fat, 63% carbohydrates.

[0079] The following is a specific embodiment provided by the present invention, describing how to construct a mouse model of metabolic associated fatty liver disease according to the above claims:

[0080] 1. Selection and treatment of pregnant mice:

[0081] At GD13, select a group of healthy pregnant mice and ensure they have suitable environmental and feeding conditions during the experiment.

[0082] These pregnant mice were gavaged daily with 200 mg / kg of AM treatment from GD13 to PND7. The offspring were weaned at 28 days after birth and sacrificed at 84 days after birth after being fed a high-fat diet for 6 weeks for subsequent analysis.

[0083] The materials and methods used in the present invention:

[0084] Amoxicillin (sodium salt, HY-B0467, CAS No. 34642-7-8; ≥98.23%) was sourced from Med ChemExpress (USA). Biochemical assay kits for alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were from Zhejiang Elikang Biotechnology Co., Ltd. The triglyceride assay kit (BC0625) was purchased from Beijing Solarbio Science & Technology Co., Ltd. TRIzol (Cat 15596026) reagent was purchased from Thermo Fisher Scientific (USA). Reverse transcriptase (Catalog 10109118001) and RT-PCR (Catalog 04887352001) reagents were from Roche Diagnostics Ltd. (Switzerland).

[0085] Chemicals for lipid analysis, including methanol (Catalog 106007), acetonitrile (Catalog 113358), formic acid (98%, Catalog 00940), and isopropanol (Catalog 101040), were all from Merck KGaA (Germany). Ammonium formate (Catalog A11550) and dichloromethane (Catalog AC6100500) were purchased from Fisher Chemicals (USA). Standard substances of 12:0Lyso PC (Catalog 855475), Cer(d18:1 / 4:0) (Catalog 860524), PC(13:0 / 13:0) (Catalog 850340), DG(12:0 / 12:0) (Catalog 800812), and TG(17:0 / 17:0 / 17:0) (Catalog 860903) were from Avanti Polar Lipids (USA).

[0086] Animal experiments in the present invention:

[0087] Eighteen-week-old C57BL / 6J mice (female: 18 - 20 g; male: 22 - 24 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. Before the start of the breeding protocol, the mice underwent a 2-week acclimation period. They were housed in a specific pathogen-free (SPF) environment with a 12-hour light / dark cycle at 22 ± 3 °C and 50 ± 5% humidity. Food and water were provided ad libitum. For breeding, females and males were paired overnight at a ratio of 4:2 from 8 p.m. to 7 a.m., and mating was confirmed by the presence of a vaginal plug, marking gestational day (GD) 0. Maternal antibiotic exposure was mainly used to prevent infection and treat complications, which usually occur around birth. In our mouse model, from GD13 to postnatal day 7 (PND), AM was administered to the dams to closely mimic the clinical exposure time.

[32] At GD13, the maternal mice were divided into three groups: control group (Ctrl), low AM group (AML, 20 mg / kg / d), and high AM group (AMH, 200 mg / kg / d). Control group mice received sterile water by gavage, while the AML and AMH groups received the corresponding doses of AM from GD13 to PND7. AM was dissolved in sterile water. The human dose range is 500 to 1500 mg / day (https: / / www.drugs.com / dosage / amoxicillin.html). Based on body surface area calculation, the mouse dose is approximately 50 - 150 mg / kg / d. The pups weaned at PND28 were fed normal food (3.44 kcal / g: 24% protein, 13% fat, 63% carbohydrate). At postnatal week 13 (PNW), the pups were randomly divided into normal diet (NC) and high-fat diet (HFD) subgroups. The HFD consisted of 14% protein, 42% fat, 44% carbohydrate, and 0.2% cholesterol (TP 26304, TrophicDiet, Nantong, China) for 6 weeks. Throughout the study, the offspring were weighed regularly, and fecal samples were collected.

[0088] Experiment 2 Twelve-week-old male and female C57BL / 6J mice were randomly divided into two groups. In the control group, the mice were given an equal volume of sterile water by gavage. In the AMH group, the mice were gavaged with AM (AMH, 200 mg / kg / d) for 6 days. After a 4-hour fast, baseline blood samples were collected from the mice by tail bleeding. Next, the mice were gavaged with olive oil at a dose of 10 μl / g body weight. Blood was drawn by tail bleeding at 1 hour, 2 hours, 4 hours, and 6 hours after the oil bolus. To ensure sufficient yield for downstream analysis, 30 - 40 μL of whole blood was collected, and serum was separated by centrifugation at 3500 rpm for 15 minutes at 4 °C to obtain approximately 10 μL of serum. Throughout the experiment, the mice had access to water but not to food.

[0089] In Experiment 3, the same AM exposure as in Animal Experiment 2 was given. Six days after AM exposure, the animals were fasted overnight, refed with 60% HFD for 2 hours, and then euthanized. Intestinal tissues were collected and stored in paraformaldehyde for further analysis.

[0090] In Experiment 4, the same AM exposure as in Animal Experiment 2 was given. The experimental procedure was carried out as described in the literature. Briefly, six days after AM exposure, the mice were fasted for 4 hours and administered BODIPY 500 / 510 C1, C12 FA (2 μg / g body weight) and olive oil (10 μl / g body weight) for 2 hours, and then euthanized. The excised small intestine was embedded and frozen at cutting temperature (OCT) for further analysis.

[0091] For Experiment 5, the procurement and housing conditions of the animals were the same as in Experiment 1. At GD13, the maternal mice were randomly divided into two groups: control group (Ctrl) or high AM group (AMH, 200 mg / kg / d). From GD13 to PND7, the mother mice were gavaged with an equal volume of sterile water or AM (200 mg / kg / d). At PND8, the male offspring of the control group were randomly divided into two subgroups: half as the control group and the other half were gavaged with Lactobacillus murinus, designated as the LM group. Similarly, the male offspring of the AMH group were randomly divided into two subgroups: half as the AMH group and the other half were gavaged with Lactobacillus murinus, designated as the AMH+LM group. At PNW13, all male offspring were fed HFD for 6 weeks. Throughout the study, all offspring were weighed regularly and fecal samples were collected.

[0092] 16S rRNA sequencing

[0093] Microbial genomic DNA was extracted from fecal samples using the QIAamp Fast DNA Stool Mini Kit (Qiagen GmbH, Hilden, Germany). The V3-V4 region of the 16S rRNA gene was amplified using the universal primers 341F and 806R. The raw sequences were analyzed using QIIME 2 (version 2020.2). The initial reads were qualitatively filtered, denoised, and assembled. The optimized HiFi reads were clustered into operational taxonomic units (OTUs) using UPARSE7.1 at a sequence similarity level of 97%. The most abundant sequence of each OTU was selected as the representative sequence. PICRUSt2 predicted the functional potential of the gut microbiota. This test was assisted by Majorbio Biopharm Technology Co., Ltd. All data were presented on the Majorbio I-Sanger cloud platform (https: / / cloud.majorbio.com / ).

[0094] Metagenomic sequencing

[0095] This experiment was described in the literature.

[35] Briefly, each sample was prepared with 700 ng of DNA. PCR amplification and purification were performed using the AMPure XP system. DNA concentration was measured on a Qubit 2.0 fluorometer (Life Technologies, CA, USA) using the Qubit DNA Assay Kit and diluted to 2 ng / mL. The insert size of the library was evaluated using the Agilent Bioanalyzer 2100 system. The accurate concentration of the library (>3 nM) was verified by qPCR. After cluster generation, the library preparation was sequenced on the Illumina HiSeq 4000 platform to obtain 150-bp paired-end reads. Diversity analysis: Alpha diversity (Shannon and Sobs indices) and beta diversity (Bray-Curtis dissimilarity) were calculated using QIIME2. Principal coordinate analysis (PCoA) was used to visualize sample clustering. Differential abundance: The differential abundance of species between groups was evaluated using DESeq2, with the significance threshold set at p < 0.05 and fold change ≥2. This test was assisted by Mariobio Pharmaceutical Technology Co., Ltd.

[0096] Liver Lipidomics Analysis

[0097] The liver tissue was thawed on ice, mixed for about 10 seconds, and then processed at 3000 rpm for 5 minutes at 4 °C using a centrifuge. Each 20 mg sample was homogenized with 1 mL of a mixture containing methanol, MTBE, and an internal standard mixture, and then mixed for 15 minutes. 200 μL of water was added to the mixture and stirred for 1 minute, and then centrifuged at 12000 rpm for 10 minutes at 4 °C. 500 μL of the supernatant was extracted and concentrated after centrifugation. The concentrated powder was dissolved in 200 μL of solvent B (acetonitrile:isopropanol, volume ratio 1:9, using 10 mM ammonium formate and 0.1% formic acid) for LC-MS / MS analysis. Multivariate analysis: Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed using MetaboAnalyst 5.0 to identify lipid species contributing to group separation. Univariate analysis: Student's t-test or analysis of variance was used to identify significantly altered lipids (p < 0.05, fold change ≥2). This detection was assisted by Metware Biotechnology Co., Ltd.

[0098] Quantitative Analysis of Bile Acids

[0099] The cecal content (20 mg) was mixed with 10 μL of internal standard mixed solution (1 μg / mL) and 200 μL of methanol / acetonitrile (v / v = 2:8), and then homogenized. The sample was vortexed at 2500 rpm for 10 minutes and stored at -20 °C for 10 minutes to facilitate protein precipitation. Finally, the mixture was centrifuged at 12000 rpm for 10 minutes at 4 °C. The supernatant was transferred to a new plastic microtube and concentrated using a concentrator (CentriVap, LABCONCO, USA). After concentration, the sample was redissolved in 100 μL of 50% methanol aqueous solution for subsequent LC-MS / MS analysis using an Applied Biosystems 6500 triple quadrupole (QTRAP 6500+, SCIEX, USA). The HPLC column used was a Waters ACQUITY UPLC HSS T3 C18 (100 mm × 2.1 mm, 1.8 μm). Quantification: BA was quantified using a standard curve and normalized to the internal standard. Detection was assisted by Meiwell Biotechnology Co., Ltd.

[0100] Bacterial strains and growth conditions

[0101] The freeze-dried powder of Lactobacillus murinus (Shanghai Baocun Biotechnology Center, Shanghai, China) was reconstituted in 0.5 mL of Man Rogosa Sharpe (MRS) medium (Shanghai Ruichu Biotechnology Co., Ltd., Shanghai, China, China). The resulting bacterial suspension was spread on blood agar, and colonies formed within approximately 24 hours. Then, individual colonies were transferred to MRS medium and incubated at 37 °C under anaerobic conditions. After growing for 12 - 16 hours, the optical density (OD600) of the culture at 600 nm was evaluated, ranging from 0.64 to 0.8, corresponding to a bacterial concentration of 1×10^9 CFU / mL determined by plate counting. Subsequently, the culture was centrifuged, washed, and resuspended in saline. The experimental group was given live bacteria at a dose of 1×10^8 organisms per mouse per day, while the control group received an equal volume of saline. The stock solution of L. murinus was stored in MRS medium containing 25% glycerol at -80 °C for future use.

[0102] Indirect calorimetry

[0103] In the PNW 17, groups of 5 - 6 mice were placed in individual metabolic cages (TSE Systems, Germany) to monitor various metabolic parameters. Each mouse was acclimated to its chamber for 48 hours. After the acclimation period, various metabolic parameters were measured at 25 °C and a 12 - hour light / dark cycle. Throughout the experiment, all mice had continuous access to food and water. After the initial acclimation, body activity, fluid consumption, food intake, body weight, and respiratory function (oxygen consumption and carbon dioxide production) were recorded every 2 minutes. The respiratory exchange ratio (RER) was calculated using the formula VCO2 / VO2. Energy expenditure (EE) (Kcal / h) = (3.815 + 1.232 × RER) × VO2 (ml / min). Food consumption was specifically measured in the metabolic chamber. Over a 2 - day continuous monitoring period, all the collected data were averaged and presented for the dark and light phases.

[0104] Tissue and fecal sample collection

[0105] At specified time intervals, the mice were anesthetized and tissues were carefully removed. The collected tissue samples were weighed and divided into two groups: some samples were fixed with 4% paraformaldehyde for subsequent histological examination, while the other samples were snap - frozen plasma fecal pellets and intestinal contents in liquid nitrogen, quickly frozen in liquid nitrogen, and stored at - 80 °C until analysis was required.

[0106] Biochemical parameter measurement

[0107] For biochemical parameters, including plasma ALT, AST, TG, TC, HDL - C, and LDL - C, blood samples were centrifuged to separate plasma. Biochemical parameters were measured using an automatic biochemical analyzer (Di Rui CS - T300, Changchun, China).

[0108] Liver TG determination

[0109] Liver tissue was homogenized in a 1:1 mixture of n - heptane and isopropanol. Then the liver homogenate was centrifuged at 4 °C, 8000g for 10 minutes. The supernatant was separated and the TG content was detected using a commercial TG kit (Beijing Solarbio Science & Technology Co., Ltd.).

[0110] Histological examination and Oil Red O staining

[0111] Mouse liver tissue blocks were immersed in 4% paraformaldehyde for 24 hours, then dehydrated and embedded in paraffin. Sections were stained successively with hematoxylin and eosin. Two researchers blindly scored the tissues, evaluating ballooning degeneration (0 - 2 points), steatosis (0 - 3 points), and lobular inflammation (0 - 3 points) [2]. Oil Red O staining scans were performed on 10 - μm liver tissue sections and tissue photographs using a pathological slide scanner.

[0112] Real-time fluorescence quantitative reverse transcription polymerase chain reaction

[0113] Total RNA was extracted from liver tissues using TRIzol reagent. The total RNA was treated with RNase-free DNase and then reverse transcribed with reverse transcriptase. Real-time RT-PCR was performed using the LightCycler 480 SYBR Green qPCR master mix. All primers are listed in the Key Resources Table.

[0114] High-throughput RNA sequencing

[0115] Total RNA was isolated from matched samples using TRIzol reagent. RNA-seq libraries were generated using the VAHTS mRNA seq V3 library preparation kit. Quality control procedures included using a BioAnalyzer and Qubit. Sequencing of all libraries was performed on the Illumina NovaSeq 6000 system. After quality control, sequence alignment was performed using STAR. The raw read counts of annotated genes were normalized and differential expression analysis was performed using DEseq2 v1.40.2, applying a threshold of p < 0.05 and fold change ≥ 2. We used Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.

[0116] Enrichment analysis was performed using the clusterProfiler package in R. The testing and analysis were assisted by Shanghai Personal Biotechnology Co., Ltd.

[0117] Postprandial triglyceride response assay

[0118] The postprandial triglyceride response assay was performed. In Animal Experiment 2, serum TG was measured using the above kit.

[0119] Statistical analysis

[0120] Statistical analysis was performed using SPSS 2-3.0, and the data were reported as mean ± SEM. The two-tailed Student's t-test was used to compare two groups, while one-way ANOVA was used for multiple groups. When significant differences were found, the post hoc LSD test was used to compare the control group and the AM exposure group.

[0121] The Kruskal-Wallis H test was used to analyze data with non-normal distribution. Two-way ANOVA was used to evaluate the body weight of mice. GraphPad Prism 8.0 was used for graphical representation and visualization. A p-value less than 0.05 indicated statistical significance.

[0122] Evidence related to the technical effects obtained in the embodiments of the present invention.

[0123] (1) Perinatal AM exposure exacerbated hepatic lipid deposition in male offspring fed a HFD

[0124] Before HFD feeding, body weight changes were compared. As Figure 3 shown in A and 3C, there were no differences among the groups. Six weeks after HFD feeding, the body weight of AMH-exposed male pups increased ( Figure 3 B), while no differences were observed between AMH-exposed female offspring and the control group ( Figure 3 D). Food intake was measured, and there were no differences among the different groups ( Figure 3 E and 3G). Brown adipose tissue (BAT), subcutaneous white adipose tissue (sWAT), perirenal white adipose tissue, and gonadal white adipose tissue were measured. As Figure 3 shown in F, gWAT increased in HFD-fed male offspring exposed to AMH, and there were no differences in other tissues ( Figure 3 F and 3H). ALT increased in both male and female offspring ( Figure 3 I and 3L), but AST increased only in male offspring ( Figure 3 J and 3M). As Figure 3 shown in N-3T, there were no differences in plasma TC, LDL, and HDL. Interestingly, plasma TG was higher in HFD-fed male offspring exposed to AM than in HFD-fed male offspring ( Figure 3 K). Next, liver lipids were evaluated. H&E staining showed that the NAFLD activity score increased in HFD-fed male ( Figure 4 A and 4C) and female offspring ( Figure 4 B and 4E) exposed to AMH. Oil Red-O staining showed that the liver of HFD-fed male offspring fed AMH had more lipid droplets than that of HFD-fed male offspring ( Figure 5 A and 5C). No differences in liver lipid droplets were observed between AMH-exposed HFD-fed female offspring and HFD-fed female offspring ( Figure 5 B and 5F). Liver weight and coefficient were analyzed, and the results showed that the liver weight and index increased in HFD-fed male offspring exposed to AMH ( Figure 5 D and 5D), but not in female offspring ( Figure 5 F and 5G). Finally, liver TG content was measured, and the results showed that liver TG content increased in HFD-fed male offspring exposed to AMH, while it did not increase in female offspring ( Figure 5 E and 5H).

[0125] (1) Perinatal AM exposure alters the liver lipid profile of HFD-fed male offspring

[0126] Lipidomics was used to analyze the liver lipid profile. OPLS-DA score plots ( Figure 5I and 5K) represent different lipid metabolites between different groups. The hepatic lipid profiles of HFD-fed male offspring exposed to AMH were different from those of male offspring exposed to HFD ( Figure 5 I), while there were no differences between HFD-fed female offspring exposed to AMH and those exposed to HFD ( Figure 5 K). Volcano plots were used to analyze different lipid metabolites ( Figure 5 J and 5L). Four lipids increased and nine lipids decreased in AMH-exposed male offspring. In addition, 637 lipids increased and 248 lipids decreased in HFD-fed male offspring. Interestingly, 108 lipids were elevated and only 2 lipids were decreased in AMH-exposed HFD-fed male offspring compared with HFD-fed male offspring ( Figure 5 J). In AMH-exposed female offspring, only 2 lipids increased and 9 lipids decreased. In addition, 345 lipids increased and 248 lipids decreased in HFD-fed female offspring. Compared with HFD-fed female offspring, only 4 lipids increased and 4 lipids decreased in AMH-exposed HFD-fed female offspring ( Figure 5 L). Lipids were classified into eight major categories: fatty acyls (FA), glycerolipids (GL), glycerophospholipids (GP), sphingolipids (SP), sterol lipids (ST), prenol lipids (PR), glycosphingolipids (SL), and polyketides (PK). The elevation of glycerolipids is one of the main characteristics of hepatic lipid deposition. Detailed lipidomic analysis showed that total lipids, FA, GL, GP, and ST increased in male offspring fed HFD. Heatmaps showed obvious differences between different groups ( Figure 4 G and 4H). In AMH-exposed HFD-fed male offspring, the elevation of total lipids, FA, and GL induced by HFD was exacerbated ( Figure 5 M). Although total lipids, GL, SP, and ST increased, perinatal AM exposure did not exacerbate HFD-induced hepatic lipid elevation in female offspring ( Figure 5 N).

[0127] (2) Perinatal AM exposure does not affect the basal metabolism of adult offspring

[0128] Food intake was evaluated within 48 h. For males, there were no changes in food intake among different groups. ( Figure 6 A-6C). Food intake of AM-exposed females decreased within 48 h ( Figure 6 D and 6F). In addition, food intake of HFD-fed females fed AM was reduced compared with that of HFD-fed females ( Figure 6E and 6F). The respiratory exchange ratio (RER) was used to evaluate basal metabolism. In this experiment, an RER close to 1 indicates that the body mainly consumes carbohydrates, and an RER close to 0.7 indicates that the body mainly uses fat as energy. For males ( Figure 6 G-6I) and females ( Figure 6 J-6L), there were no differences in RER values among different groups within 48 hours. Next, locomotor activity was monitored within 48 hours. For males ( Figure 6 M-6O) and females ( Figure 6 P-6R), there were no differences in locomotor activity among different groups within 48 hours. Finally, energy expenditure (EE) was compared among different groups. For males ( Figure 6 S-6U) and females ( Figure 6 V-6X), there were no differences in energy expenditure among different groups within 48 hours.

[0129] (4) Perinatal AM exposure does not alter hepatic lipid metabolism gene expression in adult offspring

[0130] Transcriptomic analysis of genes related to hepatic lipid metabolism was performed ( Figure 7 A and 7C). Dynamic multi-group differential scatter plots were used to analyze differential gene expression. As Figure 7 shown in B, 76 genes were upregulated and 52 genes were downregulated in male offspring exposed to AMH. In addition, 315 genes were upregulated and 316 genes were downregulated in male offspring fed an HFD. Compared with male offspring fed an HFD, 91 genes were upregulated and 42 genes were downregulated in male offspring fed an HFD and exposed to AMH ( Figure 7 B). For female offspring exposed to AMH, 70 genes were upregulated and 24 genes were downregulated. In addition, 92 genes were upregulated and 50 genes were downregulated in female offspring fed an HFD. Compared with female offspring fed an HFD, only 25 genes were upregulated and 22 genes were downregulated in female offspring fed an HFD and exposed to AMH ( Figure 7 D). GO and KEGG enrichment analyses were used for functional annotation and enrichment analysis. ( Figure 7 E-7L). In the GO pathway enrichment analysis of differential genes, no lipid metabolism pathways were enriched. Similarly, in the KEGG pathway enrichment analysis of differential genes, no pathways related to lipid metabolism were enriched. Finally, real-time RT-PCR was used to detect genes related to hepatic lipid metabolism. There were no differences in genes related to hepatic lipid metabolism among different groups, including lipid synthesis, fatty acid-binding protein, β-oxidation, lipid degradation, and lipid transport ( Figure 7 M and 7N).

[0131] (5) AM exposure enhances intestinal lipid absorption in male mice

[0132] A postprandial TG response was performed to test intestinal lipid absorption. As Figure 8 shown in A, serum TG levels increased at all time points after olive oil administration in male animals exposed to AM. There was no difference in serum TG between AM-exposed females and the control group. Six hours after olive oil administration in female animals exposed to AM, serum TG levels decreased ( Figure 8 D). Next, serum and intestinal lipids were measured in mice that had fasted overnight and were then refed with 60% HFD for 2 h. Serum TG levels increased in AM-exposed males ( Figure 8 B), but not in AM-exposed females ( Figure 8 E). Serum TC did not increase in AM-exposed males and females ( Figure 8 C and 8F). Histology showed that enterocytes in the duodenum and jejunum contained numerous lipid droplets 2 h after AM-exposed males had fasted overnight and were refed with 60% HFD ( Figure 8 G). In AM-exposed HFD-fed females, only a slight increase in lipid droplets was shown ( Figure 8 H). Finally, BODIPY-labeled FA was administered to directly observe FA uptake by enterocytes. Numerous fluorescently labeled lipid droplets were shown in the intestine of AM-exposed male animals ( Figure 8 I), but not in female animals ( Figure 8 J).

[0133] (6) Perinatal AM exposure disrupts fecal BA composition in male offspring

[0134] Fecal BA composition was determined by UPLC-MS. Principal component analysis of fecal BA profiles showed a significant difference between AM-exposed male offspring and the control group ( Figure 9 A), but no difference between AM-exposed female offspring and the control ( Figure 9 D). A significant difference was observed between AM-exposed HFD-fed male offspring and HFD-fed male offspring ( Figure 9 A), but no difference between AM-exposed HFS-fed female offspring and HFD-fed female offspring ( Figure 9 D). Although primary BAs did not change, secondary BAs increased in HFD-fed male offspring ( Figure 9 B), but not in HFD-fed female offspring ( Figure 9 E). Thus, the ratio of non-12-OH BA to 12-OH BA increased in HFD-fed male offspring ( Figure 9 C), but not in HFD-fed female offspring ( Figure 9 F). Notably, perinatal AM exposure attenuated the increase in secondary BAs induced by HFD in male offspringFigure 9 B). Additionally, perinatal AM exposure alleviated the increase in the ratio of non-12-OH BA to 12-OH BA in male offspring fed an HFD ( Figure 9 C). The effect of perinatal AM exposure on fecal BA composition was further analyzed. Perinatal AM exposure had little effect on primary BAs in both males and females ( Figure 9 G and 9H). In AM-exposed HFD-fed males, secondary BAs, including ω-MCA, Tα-MCA, Tω-MCA, 6-keto LCA, IALCA, TUDCA, 3β-UDCA, THDCA, and 3β-HDCA, were decreased ( Figure 9 I), but not in female offspring ( Figure 9 J).

[0135] (7) Perinatal AM exposure disrupts the composition of the gut microbiota in adult offspring

[0136] The gut microbiota composition was evaluated using metagenomics. The Sobs index showed a significant decrease in species richness in both AM-exposed male and female offspring ( Figure 10 A and 10C). Principal coordinate analysis (PCoA) was performed to evaluate the β-diversity of gut microbiome characteristics, and significant differences were shown between AM-exposed offspring and the control group by Bray-Curtis distance ( Figure 10 B and 10D). Circos plots were used to show the top 10 most abundant bacterial strains. In the control group, Lactobacillus was clearly dominant (green). The abundance of Lactobacillus was significantly decreased in offspring exposed to AM ( Figure 10 E and 10F). Further analysis showed that at the species level, the gut microbiota of both AM-exposed males and females was significantly disrupted ( Figure 10 G and 10H). The linear discriminant analysis effect size (LEfSe) algorithm was used to analyze the bacterial composition, indicating that Lactobacillus murinus was dominant in the control group, while the abundance was very low in AM-exposed male and female offspring ( Figure 10 I and 10J).

[0137] (8) Perinatal AM exposure disrupts early gut microbiota colonization

[0138] To investigate the effect of perinatal AM exposure on gut microbiota colonization, the fecal microbiota structure was evaluated at different time points after birth, and the gut microbiota function was evaluated in adulthood. As Figure 11 shown in A-11E, the Sobs index decreased not only in maternal mice during parturition but also in weaned pups and adult offspring. Additionally, lower community richness was shown not only in maternal mice exposed to AM at parturition but also in weaned pups and adult offspring ( Figure 12A-12E). During parturition, weaning of pups, and adulthood of the offspring, the beta-diversity of the gut microbiome profiles of maternal mice was evaluated. There were significant differences between AM-exposed mice and the control group ( Figure 11 F-11J). Venn diagrams at the OTU level showed that the number of OTUs decreased not only in maternal mice exposed to AM during parturition but also in weaned pups and adult offspring ( Figure 12 F-12J). Community bar charts were used to visually illustrate the trends of species changes at different time points after birth. At the genus level, the top 10 most abundant bacterial genera were highlighted. The relative abundance of Lactobacillus decreased significantly not only in female mice exposed to AM during parturition but also in weaned pups and adult offspring ( Figure 11 K-11O). Differential tests were used to evaluate the changes in the microbial composition between AM-exposed mice and the control group. As Figure 11 shown in K-11O, the top 10 bacterial taxa showed significant differences not only between AM-exposed mice and the control group. Further analysis showed that the Lactobacillus population decreased significantly not only in maternal mice during parturition but also in weaned pups and adult offspring ( Figure 12 K-12O). Based on 16S amplicon sequencing data combined with the eggNOG database, we predicted the biological functions of bacteria ( Figure 8 ). Combining with the eggNOG database, the predicted functions of bacteria mainly involved metabolism, lipid transport, and metabolism of PNW12 offspring. ( Figure 13 A and 13B). Finally, the microbial changes between weaned pups and adult offspring were compared. As Figure 11 shown in P-11Q, there was no change in the relative abundance of the Lactobacillus population between AM-exposed mice and the control group.

[0139] (9) Supplementing Lactobacillus murinus can partially improve the composition of the gut microbiota in male offspring exposed to AM

[0140] The effect of supplementing Lactobacillus during lactation on the gut microbiota composition was evaluated in male offspring exposed to AM. The Sob and Shannon indices showed that supplementing Lactobacillus slightly restored community richness ( Figure 14 A), without affecting community diversity ( Figure 14 B). PCoA plots showed that the difference was minimal between the control group and the offspring supplemented with Lactobacillus murinus, while the difference was the largest between the control group and the offspring exposed to AM. Supplementing Lactobacillus murinus partially reduced the difference between the control group and the offspring exposed to AM ( Figure 14 C). Venn diagrams at the OTU level showed that supplementing Lactobacillus did not reverse the number of OTUs ( Figure 14 D). Community bar charts were used to evaluate the effect of supplementing Lactobacillus murinus on the 10 most abundant species at the genus and species levels in the gut microbiota of male offspring.Figure 14 E and 14G). The decrease in the proportion of Lactobacillus murinus induced by AM was partially reversed in the male offspring supplemented with Lactobacillus murinus ( Figure 14 F and 14H). The gut microbiota health index (GMHI) was used to further evaluate the gut health and dysbiosis of male offspring. As Figure 8 shown in I, supplementation with Lactobacillus reversed the decrease in GMHI of male offspring induced by AM.

[0141] (10) Supplementation with Lactobacillus murinus can partially improve BA metabolism in male offspring exposed to AM

[0142] The effect of adding Lactobacillus murinus on gut BA metabolism was analyzed. Principal component analysis of fecal BA profiles showed separation between different groups ( Figure 14 J). Therefore, in male offspring fed an HFD and exposed to AM, the ratio of non-12-OH BA to 12-OH BA decreased. In addition, supplementation with Lactobacillus murinus reversed the decrease in the ratio of non-12-OH BAs to 12-OH BA in male offspring fed an HFS and exposed to AM ( Figure 14 K). Although there were no differences in primary BAs, secondary BAs in male offspring fed an HFD and exposed to AM decreased ( Figure 14 L and 14N). Supplementation with Lactobacillus murinus can reverse the decrease in secondary BAs in male offspring fed an HFD and exposed to AM ( Figure 14 L and 14N). Next, the effect of supplementing Lactobacillus murinus on fecal BA composition was analyzed. There were no differences in primary BAs among different groups. In male offspring fed an HFD and exposed to AM, secondary BAs including MCA, UDCA, and HDCA decreased ( Figure 14 O). Supplementation with Lactobacillus murinus can reverse the decrease in secondary BAs in male offspring fed an HFD and exposed to AM ( Figure 14 O).

[0143] (11) Supplementation with Lactobacillus murinus can reduce liver lipid deposition in male offspring exposed to AM

[0144] Before feeding the HFD, there were no differences in body weight among different groups ( Figure 15 A). After six weeks of HFD feeding, the body weight of male offspring fed an HFD and exposed to AM increased. Supplementation with Lactobacillus can reduce the increase in body weight of male offspring ( Figure 15 B). Food intake was measured and showed no differences between different groups ( Figure 15 C). As expected, the gWAT of male offspring fed an HFD and exposed to AM increased. Supplementation with Lactobacillus can reduce the increase in gWAT of male offspring ( Figure 15 D). Although there were no differences in serum TC and LDL, the increase in serum ALT, AST, and TG was attenuated in male offspring administered Lactobacillus murinusFigure 15 E-15H and 15J). In male offspring administered with Lactobacillus, the decrease in serum HDL-C content was alleviated ( Figure 15 I). Compared with the AM-exposed HFD-fed control group, the NAFLD activity score of AMH-exposed HFD-fed mice supplemented with L. murinus was lower ( Figure 16 A and 16C). Determined by Oil Red O staining, male offspring of AMH-exposed HFD-fed mice supplemented with Lactobacillus murinus showed fewer hepatic lipid droplets compared with the AMH-exposed HFD-fed control group ( Figure 16 B and 16D). Although there was no effect on liver weight ( Figure 16 E), supplementation with Lactobacillus could alleviate the increase in liver coefficient in male offspring ( Figure 16 F). In addition, supplementation with Lactobacillus reversed the increase in hepatic TG content induced by AM-exposed HFD in male offspring ( Figure 16 G). Next, the effect of Lactobacillus supplementation on hepatic lipid profiles was analyzed. Volcano plots and OPLS-DA score plots showed differential lipid metabolites between HFD-fed and AMH-exposed HFD-fed male offspring ( Figure 15 K and 15N), between AMH-exposed HFD-fed and AM-exposed HFD-fed male offspring supplemented with L. murinus ( Figure 15 L and 15O), and between HFD-fed and HFD-fed male offspring supplemented with L. lactis ( Figure 15 M and 15P). The OPLS-DA score plot showed the differences between different groups ( Figure 15 Q). Qualitative analysis showed that supplementation with L. murinus could alleviate the increase in total lipids and glycerolipids in the liver of AM-exposed HFD-induced male offspring ( Figure 15 R). Finally, the effect of Lactobacillus supplementation on hepatic glycerolipids was analyzed. The circular heat map further showed that the increase in hepatic DG and TG content induced by AM-exposed HFD in male offspring was reversed in male offspring administered with Lactobacillus ( Figure 16 H and 16I). As expected, the hepatic DG and TG content increased in AM-exposed HFD-fed male offspring. Supplementation with Lactobacillus reversed the increase in hepatic DG and TG induced by AM-exposed HF in male offspring ( Figure 16 J). Further analysis of the TG structural diversity showed that in AM-exposed HFD-fed male offspring, TGs with low carbon numbers (0-10) ( Figure 16 K) and high double bond numbers (>50) ( Figure 16 H) were dominant. Supplementation with Lactobacillus could alleviate the increase in hepatic TG content induced by AM / HFD ( Figure 16 K and 16L).

[0145] Such asFigure 3 , Effects of perinatal AM exposure on fat weight and blood lipids in adult offspring fed a HFD

[0146] Such as Figure 5 , Diagram of the effects of perinatal AM exposure on lipid metabolism in adult offspring fed a HFD;

[0147] Such as Figure 6 , Diagram of the effects of perinatal AM exposure on energy balance in adult offspring fed a HFD;

[0148] Such as Figure 7 , Diagram of the effects of perinatal AM exposure on genes related to liver lipid metabolism in adult offspring fed a HFD;

[0149] Such as Figure 11 , Diagram of the effects of perinatal AM exposure on the richness and diversity of the gut microbiota in maternal mice, weaned pups, and adult offspring;

[0150] Such as Figure 13 , Diagram of the functions of bacterial microbiota predicted by PICRUSt2 combined with the eggNOG database in the feces of 12-week-old male and female offspring mice;

[0151] Such as Figure 16 , Diagram of the effects of Lactobacillus murinus supplementation on the liver lipid profile in adult male offspring fed a HFD;

[0152] Such as Figure 17 , Schematic diagram of the animal experiment design.

[0153] It should be noted that the embodiments of the present invention can be implemented by hardware, software, or a combination of software and hardware. The hardware part can be implemented using dedicated logic; the software part can be stored in a memory and executed by an appropriate instruction execution system, such as a microprocessor or dedicated designed hardware. Those of ordinary skill in the art can understand that the above devices and methods can be implemented using computer-executable instructions and / or included in processor control code, such as provided on a carrier medium such as a disk, CD, or DVD-ROM, a programmable memory such as read-only memory (firmware), or a data carrier such as an optical or electronic signal carrier. The devices and modules of the present invention can be implemented by hardware circuits of programmable hardware devices such as very large scale integrated circuits or gate arrays, semiconductors such as logic chips, transistors, etc., or programmable logic devices such as field programmable gate arrays, or can be implemented by software executed by various types of processors, or can be implemented by a combination of the above hardware circuits and software such as firmware.

[0154] The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be covered by the protection scope of the present invention.

Claims

1. A method for establishing a mouse model of metabolic associated fatty liver disease, characterized in that, The method includes: S1: Animal experiment: S2: Metagenomic sequencing; S3: Liver lipidomics analysis; S4: Bile acid quantitative analysis; S5: Statistical analysis.

2. The method for establishing a mouse model of metabolism-related fatty liver disease according to claim 1, wherein The specific content of S1 includes: Obtain 8-week-old C57BL / 6J mice, female: 18 - 20 g; male: 22 - 24 g; the mice have a 2-week adaptation period before the start of the breeding protocol; they are housed in a specific pathogen-free (SPF) environment at a temperature of 22 ± 3°C and a humidity of 50 ± 5%, following a 12-hour light / dark cycle; food and water are freely available. For breeding, female and male mice are paired overnight at a ratio of 4:2, from 8 pm to 7 am the next morning. Mating is confirmed by the presence of a vaginal plug, marked as day 0 of pregnancy; At GD13, pregnant mice are divided into three groups: a control group, a low-dose amoxicillin group AML, 20 mg / kg / d, and a high-dose amoxicillin group AMH, 200 mg / kg / d; mice in the control group are given sterile water by gavage, while the amoxicillin groups are given the corresponding dose of amoxicillin from GD13 to postnatal day 7; the human dose range is 500 to 1500 mg / day, and according to body surface area calculation, the mouse dose is approximately 50 - 150 mg / kg / d; amoxicillin is dissolved in sterile water; the pups are weaned at PND28 and fed a normal diet, 3.44 kcal / g: 24% protein, 13% fat, 63% carbohydrate; at 13 weeks after birth, the pups are divided into a normal diet NC and a high-fat diet HFD subgroup; the HFD contains 14% protein, 42% fat, 44% carbohydrate, and 0.2% cholesterol for 6 weeks; the body weights of the mice are regularly measured, and fecal samples are collected throughout the study period.

3. The method for establishing a mouse model of metabolic associated fatty liver disease according to claim 1, wherein The specific content of S2 includes: Prepare 700 ng of DNA for each sample; perform PCR amplification and purification using the AMPure XP system; measure the DNA concentration on a Qubit 2.0 fluorometer using the Qubit DNA Assay Kit and dilute it to 2 ng / mL; evaluate the library insert size using the Agilent Bioanalyzer 2100 system; verify the accurate concentration of the library > 3 nM by qPCR; sequence the library preparation on the Illumina HiSeq4000 platform to generate 150 bp paired-end reads.

4. The method for establishing a mouse model of metabolism-related fatty liver disease according to claim 1, wherein ​ ​ 5. The method for establishing a mouse model of metabolism-related fatty liver disease according to claim 1, wherein ​ Mix 20 mg of cecal contents with 10 μL of an internal standard mixture solution at 1 μg / mL and 200 μL of methanol / acetonitrile v / v = 2:8 and homogenize; vortex the sample at 2,500 rpm for 10 minutes and store at -20 °C for 10 minutes to facilitate protein precipitation; finally, centrifuge at 12,000 rpm for 10 minutes at 4 °C; transfer the supernatant to a new plastic microcentrifuge tube and concentrate using a concentrator; after concentration, reconstitute the sample in 100 μL of 50% methanol-aqueous solution for subsequent LC-MS / MS analysis on an Applied Biosystems 6500 triple quadrupole; the HPLC column used is a Waters ACQUITY UPLC HSS T3 C18, 00 mm × 2.1 mm, 1.8 μm.

6. The method for establishing a mouse model of metabolism-related fatty liver disease according to claim 1, characterized in that, The specific S5 includes: Statistical analysis was performed using SPSS 23.0, and the data were expressed as mean ± standard error; two-tailed Student's t-test was used for comparison between two groups, and one-way ANOVA was used for comparison among multiple groups; when significant differences were found, the post hoc least significant difference test was used to compare the control group and the AM exposure group; the Kruskal-Wallis H test was used to analyze non-normally distributed data; two-way ANOVA was used for mouse body weight assessment; GraphPad Prism 8.0 was used for graphical representation and visualization; a p-value less than 0.05 indicated statistical significance.

7. The method for establishing a mouse model of metabolism-related fatty liver disease according to claim 1, characterized in that, The specific mice include: female: 18 - 20 g; male: 22 - 24 g.

8. The method for establishing a mouse model of metabolism-related fatty liver disease according to claim 1, characterized in that The specific solvent B is acetonitrile: isopropanol with a volume ratio of 1:9, containing 10 mM ammonium formate and 0.1% formic acid.

9. The method for establishing a mouse model of metabolism-related fatty liver disease according to claim 1, wherein The normal diet is 3.44 kcal / g: 24% protein, 13% fat, 63% carbohydrates; the high-fat diet is 14% protein, 42% fat, 44% carbohydrates, and 0.2% cholesterol.

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