Use of lactobacillus murinus in preparation of product for relieving fructose-induced non-alcoholic fatty liver

Abstract

The application belongs to the field of microbial preparation, and particularly relates to application of Lactobacillus murinus in preparation of a product for relieving fructose-induced non-alcoholic fatty liver. The animal experiment proves that the Lactobacillus murinus can be used as a medicine and can be used for relieving fructose-induced non-alcoholic fatty liver.

Description

Technical Field

[0001] This invention belongs to the field of microbial preparation preparation, specifically involving the application of Lactobacillus murineis in the preparation of products that alleviate fructose-induced non-alcoholic fatty liver disease. Background Technology

[0002] Different types of fatty liver require significantly different treatments, primarily in terms of etiological treatment, lifestyle interventions, and medication selection. Personalized treatment strategies are needed for different causes and risk factors. For alcoholic fatty liver disease, caused by long-term excessive alcohol consumption (more than 80-160 grams per day), harmful substances produced by alcohol metabolism damage liver cells; complete abstinence is the most crucial measure. A high-protein, low-fat diet, supplementation with vitamins B, C, K, and folic acid, and the use of hepatoprotective, enzyme-lowering, and choleretic drugs are also recommended. For diabetic fatty liver disease related to obesity, due to excessive fat or sugar intake, active treatment of diabetes is necessary, choosing a low-sugar, low-fat, low-calorie, and high-protein diet to control blood sugar levels. For non-alcoholic fatty liver disease (NAFLD) caused by obesity and overweight, insulin resistance, metabolic syndrome, or hypertension, hyperlipidemia, and hyperglycemia, a dietary control approach is adopted, using a low-sugar, low-fat balanced diet; and 150-250 minutes of moderate-intensity aerobic exercise per week to control blood sugar and blood pressure. Medications such as Resmetirox (Rezdiffra) can be used for treatment. High-fat fatty liver disease is caused by a long-term high-fat diet (HFD), which consists of foods high in oil and fried foods, with the oil composed of various saturated and unsaturated fatty acids. Researchers fed mice an HFD diet (60% fat, 20% protein, and 20% carbohydrates) for 80 weeks to promote obesity, collecting liver, colon, adipose tissue, and feces for histological and molecular evaluation. Results showed that long-term HFD feeding led to obesity and insulin resistance, lipid accumulation in liver tissue, hepatocellular damage, and inflammatory responses. Histological analysis of the livers of HFD mice revealed steatosis, cellular damage, portal and lobular inflammation, and fibrosis.

[0003] In recent years, with the widespread consumption of processed foods and sugary drinks, fructose intake has increased significantly. Fructose is a monosaccharide widely found in high-fructose corn syrup, candy, soft drinks, and many processed foods. Scientific studies have found that long-term high fructose intake can lead to health problems such as insulin resistance, obesity, and chronic inflammation. Fructose is primarily metabolized in the liver and small intestine. Unlike glucose, which is phosphorylated by glucokinase GK, fructose is phosphorylated by hexokinase KHK, with a faster catalytic rate. The intermediate metabolites generated are mainly used for the de novo synthesis of DNL (diethyltoluene) lipids in the liver. Therefore, high fructose intake in the diet easily leads to hepatic fat accumulation and inflammatory responses, and is considered a significant factor contributing to the rapid increase in non-alcoholic fatty liver disease (NAFLD). In-depth exploration of the underlying mechanisms by which fructose induces NAFLD can provide potential intervention strategies for the prevention and treatment of NAFLD, and has significant clinical application value.

[0004] L-arginine (L-arg) is a multifunctional amino acid and a major intestinal metabolite in mammals and microorganisms, playing a crucial role in host-microbe interactions. In mammals, L-arg serves as a precursor to multiple metabolic pathways, participating in the regulation of cell division and growth. Studies have found that arginine metabolism is closely related to glucose and lipid metabolism, thus playing a regulatory role in the progression of diabetes and obesity. Research has also shown that in a rat model of colitis, fructose-induced gut microbiota dysbiosis is associated with arginine metabolism disorders. Whether gut microbiota-derived arginine affects fructose-induced NAFLD progression remains unclear. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides the application of *Lactobacillus murineis* in the preparation of products for alleviating fructose-induced non-alcoholic fatty liver disease (NAFLD), which solves the technical problem of the difficulty in treating fructose-induced NAFLD.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0009] In a first aspect, the present invention provides the application of *Lactobacillus murineis* in the preparation of products that alleviate fructose-induced non-alcoholic fatty liver disease.

[0010] Optionally, the product is a drug.

[0011] Optionally, the relief of fructose-induced non-alcoholic fatty liver includes reducing the histological scores of hepatic steatosis, hepatocyte ballooning degeneration, and lobular inflammation.

[0012] Optionally, the *Lactobacillus murineis* is *Lactobacillus murineis* ATCC 35020.

[0013] The gut microbiota-mediated "gut-hepatic axis" plays a crucial regulatory role in the progression of NAFLD. Dysbiosis of the gut microbiota may lead to abnormal hepatic lipid metabolism, thereby promoting the development and progression of NAFLD. Studies have found that NAFLD patients typically have low gut microbiota diversity, and the abundance of certain specific bacteria may be correlated with the occurrence and severity of NAFLD. The gut microbiota directly or indirectly affects hepatic metabolic function through metabolites such as short-chain fatty acids (SCFAs). Probiotics such as *Bifidobacterium* and *Saccharomyces boulardii* have been reported to significantly improve obesity and NAFLD symptoms. Among these, *Lactobacillus* has been the most extensively studied, with *L. rhamnosus*, *L. acidophilus*, and *L. plantarum* all confirmed to be closely related to NAFLD. Adjusting the gut microbiota structure and using probiotics have become new strategies for the prevention and treatment of NAFLD.

[0014] This study, based on a mouse model of NAFLD induced by a long-term high-fructose diet, found through 16-second microbiome analysis that *Lactobacillus murineis* (L. murinus) levels were significantly reduced after a high-fructose diet. Screening using intestinal contents metabolomics and serum metabolomics revealed significantly reduced levels of arginine metabolites in both fecal and serum samples. Microbiome-metabolomics analysis showed a significant correlation between *L. murinus* and arginine metabolites. Further experiments confirmed that exogenous supplementation with *L. murinus* significantly increased serum arginine levels and significantly alleviated high-fructose-induced NAFLD symptoms. In conclusion, *L. murinus* or arginine may play a beneficial role in alleviating fructose-induced NAFLD.

[0015] (III) Beneficial Effects

[0016] Experiments have shown that exogenous supplementation with Lactobacillus murinus can significantly increase serum arginine levels and significantly alleviate symptoms of non-alcoholic fatty liver disease (NAFLD) induced by high fructose.

[0017] Lactobacillus murinus or arginine play a beneficial role in alleviating fructose-induced NAFLD. Attached Figure Description

[0018] Figure 1 This is a correlation diagram showing the effects of high fructose on hepatic steatosis and liver function impairment as described in this invention.

[0019] Figure 2 This is a graph showing the effects of a high-fructose diet or high-fructose water on the gut microbiota of C57 mice according to the present invention.

[0020] Figure 3 This is a diagram illustrating the correlation between high fructose alteration of L. murinus and disruption of host-microbial arginine metabolism according to the present invention;

[0021] Figure 4 The accompanying diagram illustrates how L. murinus improves hepatic steatosis and restores liver function in NAFLD mice, supplementing the present invention.

[0022] Figure 5 This is a diagram illustrating the protective effect of L-arginine against NAFLD in high-fructose mice according to the present invention. Detailed Implementation

[0023] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] Experimental Procedure: Thirty-six male C57BL / 6J mice were purchased from Spiefol (Beijing) Biotechnology Co., Ltd. All mice were housed in a specific pathogen-free (SPF) environment at 25°C, 60% relative humidity, and a 12-hour light-dark cycle. After one week of acclimatization, all mice were randomly divided into six groups (n=6 per group): the normal diet group (RD group) was fed standard laboratory diet; the high-fructose diet group (HFrD group) was fed a high-fructose diet (20% fructose diet); and the high-fructose drinking water group was fed high-fructose drinking water (30% fructose water).

[0025] Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (TC), and triglycerides (TG) were measured using the corresponding detection kits (Beijing Ruierda Biotechnology Co., Ltd.) and an L100 semi-automatic biochemical analyzer. Liver total cholesterol and triglycerides were detected using an ELISA kit, with *Lactobacillus murineis* ATCC 35020 (purchased from Taisto Biotechnology, catalog number TS378659) used.

[0026] Example 1

[0027] Mice were fed a normal diet and a high-fructose diet, respectively, and observed for 12 weeks. At 12 weeks, mice in the HFrD group showed significantly enlarged and pale yellow livers, while mice in the RD group had normal liver size and color. Figure 1 As shown in A and 1B.

[0028] After the experiment, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. Blood was collected under anesthesia using the enucleation method and placed in centrifuge tubes without anticoagulants. The collected blood was allowed to stand at room temperature for 30 minutes, then centrifuged at 3000 rpm for 10 minutes at 4°C. The resulting serum was stored at -80°C. The collected liver tissue was divided into three parts: the first part was fixed with 10% neutral formalin solution for HE staining analysis; the second part was frozen in liquid nitrogen and stored at -80°C for biochemical analysis; and the third part was frozen in liquid nitrogen for Oil Red staining.

[0029] Frozen liver tissue was cut into 10 μm thick sections, and the sections were stained with Oil Red O to evaluate lipid deposition. Finally, the lipid deposition area was quantified using ImageJ software (nnn), thereby calculating the percentage of lipid deposition area.

[0030] like Figure 1 As shown in C and 1D, hematoxylin and eosin (H&E) staining revealed that the histochemical scores for hepatic steatosis, hepatocyte ballooning degeneration, and lobular inflammation in the HFrD group mice were approximately 2.0, 1.4, and 0.9, respectively. The HFrD group mice exhibited significant fatty liver disease, manifested as hepatic steatosis, hepatocyte ballooning degeneration, and lobular inflammation. The non-alcoholic fatty liver activity score (NAS) in the HFrD group mice was significantly elevated, approaching 3.

[0031] like Figure 1 As shown in E, other significant morphological features of hepatic steatosis were also observed in the livers of HFrD mice, including giant mitochondria, glycogenotyped nuclei, eosinophilic bodies, atypical mitosis, Mallory-Denk bodies, and microvesicular steatosis.

[0032] Giant mitochondria: They are quite large, with a pale white matrix and marginal cristae.

[0033] Eosinophilic bodies: In viral hepatitis, hepatocytes lose water in the cytoplasm and become concentrated, resulting in enhanced eosinophilic staining and the disappearance of cytoplasmic granules. If the disease progresses further, the cytoplasm becomes even more concentrated, and the nucleus also becomes concentrated and eventually disappears. Finally, only small, round bodies with uniform, deep red staining remain.

[0034] Atypical mitosis is an abnormal cell division. Due to this abnormal cell division, the newly produced cells receive unequal amounts of genetic material (DNA), thus failing to function properly.

[0035] Mallory-Denk bodies: Irregular, refractive structures within the cytoplasm. They often appear as beaded structures or as C-shaped structures surrounding the nucleus.

[0036] Microvesicular steatosis: The cytoplasm of hepatocytes is filled with tiny lipid vesicles, which are relatively uniform in size, with a diameter of 1-3 μm, and the cell nucleus is still located in the center of the cell.

[0037] like Figure 1 As shown in F, Figure 1 The color of the right side of F is darker, and the area exceeds 10%, indicating that Oil Red O staining further confirms that lipid accumulation in the liver of HFrD group mice is significantly increased.

[0038] like Figure 1 As shown in G, the content of triglycerides and cholesterol in liver tissue was measured. The results showed that the triglyceride content in the liver of HFrD mice was close to 30 mg / g, and the cholesterol content in the liver of HFrD mice was more than 10 μmol / ml. The triglyceride content in the liver of RD mice was 5 mg / g, and the cholesterol content in the liver of RD mice was 5 μmol / ml. The content of both triglycerides and cholesterol in the liver of HFrD mice was significantly higher than that in RD mice.

[0039] like Figure 1 As shown in Figure H, the levels of triglycerides and cholesterol in serum were measured. The results showed that the triglyceride level in the serum of HFrD mice was close to 2 mmol / L, and the cholesterol level in the serum of HFrD mice was close to 8 mmol / L. The triglyceride level in the serum of RD mice exceeded 1 mmol / L, and the cholesterol level in the serum of RD mice was close to 5 mmol / L. The levels of triglycerides and cholesterol in the serum of HFrD mice were significantly higher than those in RD mice.

[0040] like Figure 1 As shown in Figure I, biochemical liver function marker analysis revealed that in the high-fructose diet group, the serum alanine aminotransferase (ALT) level was close to 60 U / L and the aspartate aminotransferase (AST) level was close to 150 U / L; in the normal diet group, the serum ALT level was over 30 U / L and the AST level was close to 80 U / L; the serum ALT and AST levels in the high-fructose diet group were significantly elevated.

[0041] The above results indicate that a high-fructose diet induces lipid accumulation in the liver and impairs liver function.

[0042] Example 2

[0043] Fresh fecal samples from mice were collected in 1.5 ml sterile centrifuge tubes, rapidly frozen in liquid nitrogen, and stored at -80°C for subsequent 16S RNA sequencing analysis.

[0044] The specific steps for 16SRNA sequencing analysis are as follows:

[0045] Total DNA was extracted from fecal samples using a DNA extraction kit (DNeasy PowerSoil Kit, QIAGEN), and PCR amplification of the 16S rRNA gene V3-V4 region was performed using specific primers. The PCR products were then purified using a purification kit (QIAquick PCR Purification Kit, QIAGEN) and quantified using a quantitation analyzer (Qubit 4 Fluorometer, Thermo Fisher Scientific). The purified PCR products were then subjected to high-throughput sequencing on the Illumina MiSeq platform. The sequencing data were analyzed using the QIIME2 software package and R language (version 4.3.0) for quality control, operational taxonomic unit (OTU) clustering, species taxonomic annotation, and microbial community diversity analysis. Alpha diversity was assessed using the Shannon and Simpson diversity indices and Chao1 richness, while β diversity indicators, including Bray-Curtis, unweighted UniFrac, and weighted UniFrac distances, were used to examine structural differences in the microbial communities between groups.

[0046] The inventors of this invention first established a mouse model of high-fructose drinking water (30% fructose water) to verify the alterations in gut microbiota composition caused by high fructose intake. 16S rRNA gene sequencing was used to study the effects of high fructose intake on the abundance and diversity of the gut microbiota. Figure 2 As shown in Figure A, principal coordinate analysis (PCoA) was performed based on the obtained data to assess structural changes in the gut microbiota under different dietary conditions. The results showed that the high fructose intake group (HFrD group, which consumed 30% fructose water) exhibited significantly different clustering distributions compared to the RD group, indicating that a high fructose diet may alter the composition and diversity of the mouse gut microbiota at the OTU level.

[0047] like Figure 2 As shown in B, after mice were fed a normal diet and a high-sugar water diet, at the genus level, Alistipes, Lactobacillus, Akkermansia, and Lachnospiraceae_NK4A136 were found to be the major gut microbiota in mice; when mice were fed 30% fructose water, the relative abundance of Lactobacillus decreased in the HFrD group.

[0048] like Figure 2As shown in D, the statistical t-test results showed that after ingesting 30% fructose water, the levels of Lachnospiraceae_NK4A136, Lactobacillus, and Odoribacter in the intestines of mice in the HFrD group were significantly reduced compared to mice on a normal diet; the average abundance of Lactobacillus decreased from approximately 10% to 2%.

[0049] like Figure 2 As shown in C, at the species level, the relative abundance of Ileibacterium_valens, L. murinus, and Akkermansia_muciniphila also showed significant changes between the HFrD and RD groups.

[0050] Figure 2 A-2D analysis of taxonomic distribution at the genus and species levels further clarifies the specific changes in the abundance of gut microbiota in the HFrD group of mice.

[0051] The inventors of this application then established a mouse model of a high-fructose diet (a diet consisting of 20% fructose) to verify the effects of the high-fructose diet on the composition of the gut microbiota, and used 16S rRNA sequencing technology to analyze the gut microbiota. For example... Figure 2 As shown in E, the PCoA results indicate that the HFrD group and the RD group have significant differences in their microbial community structure.

[0052] like Figure 2 As shown in F-2H, through overlay plot analysis and t-test at the genus level, it was found that Lactobacillus was the main bacterium that changed, with its relative abundance decreasing from 50% to 22%; in particular, the abundance of L. murinus, L. gasseri, and L. reuteri decreased significantly; the average abundance of Lactobacillus decreased from 50% to 22%.

[0053] In summary, gut microbiota dysbiosis plays a crucial role in the pathogenesis of hepatic steatosis induced by a high-fructose diet. Combining 16S sequencing results from mice drinking high-fructose water and on a high-fructose diet, long-term high-fructose intake significantly reduced the relative abundance of Lactobacillus species, especially Lactobacillus murineis (L. murinus), which is a common and important factor contributing to changes in the gut microbiome profile induced by either a high-fructose diet or water intake.

[0054] Example 3

[0055] High fructose levels disrupt host-microbe arginine metabolism in *L. murinus* by altering its metabolism.

[0056] Intestinal metabolomics analysis experimental steps:

[0057] Intestinal contents samples were added to the extract (methanol, acetonitrile, and water in a volume ratio of 2:2:1; internal standard concentration of 2 mg / L), vortexed for 30 s, then magnetic beads were added, and the mixture was ground at 45 Hz for 10 min, followed by sonication in an ice-water bath for 10 min. After standing at -20°C for 1 hour, the mixture was centrifuged at 4°C and 12,000 rpm for 15 min. The supernatant was carefully transferred to centrifuge tubes, and the extract was dried in a vacuum concentrator. The extract was added to the dried metabolites (acetonitrile and water in a volume ratio of 1:1), redissolved, vortexed for 30 s, sonicated in an ice-water bath for 10 min, and centrifuged at 4°C and 12,000 rpm for 15 min. 10 μL of each sample was taken for machine analysis. A hybrid quadrupole Orbitrap mass spectrometer was coupled to an ultra-high performance liquid chromatography (UHPLC) system to perform untargeted metabolomics analysis. Chromatographic separation was performed using a column from Waters Corporation (Milford, Massachusetts, USA) with 0.1% formic acid aqueous solution (solvent A) and 0.1% formic acid ethanol solution (solvent B). The resolution of MS1 and MS1-related MS2 spectra was 37,500 m / z. Data were analyzed using Progenesis QI software (Waters), the Human Metabolome Database (HMDB), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database for metabolite identification.

[0058] Serum metabolomics analysis experimental steps:

[0059] 100 μl of serum was mixed with pre-chilled 80% methanol, incubated on ice for 5 min, and centrifuged at 15000 × g for 20 min at 4 °C. The resulting supernatant was diluted with LC-MS grade water to a final concentration containing 53% methanol. The sample was then transferred to a new centrifuge tube and centrifuged again at 15000 × g for 20 min at 4 °C. Finally, UHPLC-MS / MS analysis was performed using a Vanquish UHPLC system (Thermo Fisher, Karlsruhe, Germany) and an Orbitrap Q Exactive™ HF mass spectrometer. The supernatant was injected into the UHPLC-MS / MS system (Thermo Fisher, Karlsruhe, Germany) for analysis. The sample was loaded onto a Hypesil Gold column (100 × 2.1 mm, 1.9 μm, Waters, Milford, MA, USA) and subjected to a linear gradient flow at 0.2 mL / min for 17 min. The eluent consisted of eluent A (0.1% FA aqueous solution) and eluent B (methanol). The solvent gradient program included the following steps: starting with 2% B for 1.5 minutes, increasing to 100% B within 12.0 minutes, maintaining 100% B for 14.0 minutes, decreasing to 2% B at 14.1 minutes, and finally ending at 2%. The mass spectrometer operating conditions were: spray voltage of 3.2 kV, capillary temperature of 320 °C, sheath gas flow rate of 40 arb, and auxiliary gas flow rate of 10 arb.

[0060] The metabolic activities of the gut microbiota significantly influence host metabolism through host-microbe interactions. To assess the impact of high fructose on metabolism, metabolomics techniques were used to analyze metabolites in colonic contents and serum. First, partial least squares discriminant analysis (PLS-DA) was used to assess overall metabolic differences between different samples; such as... Figure 3 As shown in A and 3C, the PLS-DA results showed that, compared with the RD control group, the HFrD group exhibited significant clustering in both colonic contents and serum.

[0061] like Figure 3 As shown in Figure B, by comparing the colonic contents of the RD group and the HFrD group, a total of 127 metabolites showed significant changes (P < 0.05, change > 1.5-fold), including 75 upregulated and 52 downregulated metabolites. Figure 3 As shown in D, serum metabolomics analysis revealed significant changes in 33 metabolites between the two groups (P < 0.05, change > 1.5-fold), including 4 upregulated metabolites and 29 downregulated metabolites.

[0062] To explore the potential relationship between gut microbiome metabolism and serum metabolism, further analysis was conducted based on significantly altered metabolites, such as... Figure 3 As shown in E, the metabolite that is simultaneously altered in colonic contents and serum is arginine. Figure 3 As shown in Figure F, in the intestinal metabolome, the relative abundance of RD arginine reached 1, while the relative abundance of HFrD arginine decreased to 0.4; in the serum metabolome, the relative abundance of RD arginine reached 1, while the relative abundance of HFrD arginine decreased to 0.6. Therefore, it can be concluded that in both the HFrD and RD groups, only one arginine metabolite showed a significant decrease in relative abundance in both colonic contents and serum.

[0063] Existing technologies indicate that arginine is crucial for host-microbe metabolic interactions. To investigate the link between arginine and alterations in *Lactobacillus murineis*, the inventors conducted a joint analysis of the gut microbiome and metabolome, and used Pearson correlation analysis to assess significant changes in the gut microbiota and metabolites. Figure 3 As shown in G, at the genus level, arginine is positively correlated with the genus *Lactobacillus*. Figure 3 As shown in H, at the species level, arginine is also positively correlated with Lactobacillus murineis.

[0064] like Figure 3 As shown in Figure I, the ELISA experiment showed that serum arginine levels in HFrD mice were restored after supplementation with L. murinus, increasing from nearly 10 mmol / L to 15 mmol / L, further confirming the association between arginine and L. murinus.

[0065] The above data collectively reveal that fructose-induced host arginine metabolism disorder is closely related to the decline in intestinal L. murinus abundance caused by long-term high fructose intake.

[0066] Example 4

[0067] Mice in the *Lactobacillus murineis* intervention group (HFrD+L. murinus group) were given a high-fructose diet and were supplemented daily by gavage with *Lactobacillus murineis* solution (1×10⁻⁶). 8 (CFU / mL), add 100μl daily for 60 days;

[0068] To investigate the role of *L. murinus* in high-fructose-induced NAFLD, a model was constructed by supplementing *L. murinus* with this model. Figure 4 As shown in Figure A.

[0069] like Figure 4As shown in B and 4C, H&E staining results revealed that the histochemical scores for hepatic steatosis, hepatocyte ballooning degeneration, and lobular inflammation in the HFrD group mice were close to 0, 2.0, and 1, respectively, indicating significant fatty liver disease in the HFrD group mice, manifested as hepatic steatosis, hepatocyte ballooning degeneration, and lobular inflammation. The non-alcoholic fatty liver activity score (NAS) in the HFrD group mice was significantly increased, approaching 3. Compared with the HFrD group mice, HFrD mice supplemented with L. murinus for 8 weeks (HFrD+L. murinus) showed significantly reduced non-alcoholic fatty liver symptoms, manifested as decreased histological scores for hepatic steatosis, hepatocyte ballooning degeneration, and lobular inflammation; the non-alcoholic fatty liver activity score (NAS) was also significantly reduced.

[0070] like Figure 4 As shown in D and 4E, Oil Red O staining confirmed that lipid accumulation in the liver of mice supplemented with L. murinus was significantly reduced. Figure 4 The color of the right side of D is reduced, and the area will decrease from nearly 15% to 5%.

[0071] like Figure 4 As shown in F, the contents of triglycerides and cholesterol in liver tissue were measured. The results showed that the cholesterol content in the liver of HFrD mice was close to 50 μmol / g, the triglyceride content in the liver of HFrD mice was 18 mg / g, the cholesterol content in the liver of HFrD+L. murinus mice was 20 μmol / g, and the triglyceride content in the liver of HFrD+L. murinus mice was 12 mg / g. The contents of triglycerides and cholesterol in the liver of HFrD mice were significantly higher than those in HFrD+L. murinus mice.

[0072] like Figure 4 As shown in G, the levels of triglycerides and cholesterol in serum were measured. The results showed that the serum cholesterol level of HFrD mice was close to 13 mmol / L, and the serum triglyceride level of HFrD mice was greater than 1 mmol / L. The serum cholesterol level of HFrD+L. murinus mice was close to 7 mmol / L, and the serum triglyceride level of HFrD+L. murinus mice was greater than 0.5 mmol / L. The levels of triglycerides and cholesterol in the serum of HFrD mice were significantly higher than those in HFrD+L. murinus mice. Therefore, it can be concluded that the levels of triglycerides and cholesterol in the serum and liver tissue of HFrD mice supplemented with L. murinus were significantly reduced.

[0073] like Figure 4As shown in Figure H, biochemical liver function marker analysis revealed that in the high-fructose diet group, serum alanine aminotransferase (ALT) was close to 40 U / L and aspartate aminotransferase (AST) exceeded 150 U / L; in the HFrD+L. murinus group, serum ALT was close to 30 U / L and AST was close to 100 U / L. The serum ALT and AST levels in the high-fructose diet group were significantly higher than those in the HFrD+L. murinus group.

[0074] The above results indicate that, compared with the HFrD control group, supplementation with L. murinus improved liver function in mice, as evidenced by a significant decrease in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0075] Example 5

[0076] L-arginine exhibits protective effects against NAFLD in high-fructose mice.

[0077] Mice in the arginine intervention group (HFrD+Arginine) were fed HFrD for 30 days, and then arginine (2 mg / g) was administered by gavage daily for another 30 days.

[0078] To investigate the potential protective effect of arginine against high-fructose-induced non-alcoholic fatty liver disease, such as... Figure 5 As shown in Figure A, mice with high-fructose-induced non-alcoholic fatty liver disease underwent exogenous L-arginine supplementation for 4 weeks, and several indicators associated with non-alcoholic fatty liver disease were evaluated.

[0079] like Figure 5 As shown in B and 5C, the H&E staining results of the liver showed that the histochemical scores of hepatic steatosis, hepatocyte ballooning degeneration, and lobular inflammation in the HFrD group mice were close to 0, 2.0, and 1.5, respectively. The HFrD group mice had obvious fatty liver disease, manifested as hepatic steatosis, hepatocyte ballooning degeneration, and lobular inflammation. Arginine treatment significantly improved steatosis, ballooning degeneration, and lobular inflammation in HFrD mice. The non-alcoholic fatty liver activity score (NAS) of the HFrD group mice was close to 3.5, and arginine treatment in HFrD mice led to a significant decrease in the NAS score.

[0080] like Figure 5 As shown in D and 5E, the Oil Red O staining experiment also confirms this. Figure 5 The color of D right decreased to lighter and the area decreased to 4%. Compared with HFrD group mice, the number of lipid droplets in the liver of HFrD mice treated with arginine was also significantly reduced.

[0081] like Figure 5As shown in F, the contents of triglycerides and cholesterol in liver tissue were measured. The results showed that the cholesterol content in the liver of HFrD mice was close to 35 μmol / g, the triglyceride content in the liver of HFrD mice was 20 mg / g, the cholesterol content in the liver of HFrD+Arginine mice was 8 μmol / g, and the triglyceride content in the liver of HFrD+Arginine mice was 10 mg / g. The contents of triglycerides and cholesterol in the liver of HFrD mice were significantly higher than those in HFrD+Arginine mice.

[0082] like Figure 5 As shown in G, the levels of triglycerides and cholesterol in serum were measured. The results showed that the serum cholesterol level of HFrD mice was close to 13 mmol / L, and the serum triglyceride level of HFrD mice was over 1.2 mmol / L. The serum cholesterol level of HFrD+Arginine mice was close to 7 mmol / L, and the serum triglyceride level of HFrD+Arginine mice was 0.8 mmol / L. The levels of triglycerides and cholesterol in the serum of HFrD mice were significantly higher than those in HFrD+Arginine mice. Therefore, it can be concluded that the levels of triglycerides and cholesterol in the serum and liver of HFrD mice supplemented with arginine were significantly reduced.

[0083] like Figure 5 As shown in Figure H, biochemical liver function marker analysis revealed that in the high-fructose diet group, serum alanine aminotransferase (ALT) was 40 U / L and aspartate aminotransferase (AST) exceeded 120 U / L; in the HFrD+Arginine group, serum ALT was close to 30 U / L and AST was close to 100 U / L. The serum ALT and AST levels in the high-fructose diet group were significantly higher than those in the HFrD+Arginine group. These results indicate that, compared with the HFrD control group, arginine supplementation reduced serum ALT and AST levels, suggesting improved liver function in the mice.

[0084] In summary, *L. murinus* significantly alleviated high-fructose-induced non-alcoholic fatty liver disease. A significant positive correlation was found between *L. murinus* and arginine, suggesting that *L. murinus* exerts a protective effect against fructose-induced non-alcoholic fatty liver disease through arginine.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Lactobacillus murineis ( Lactobacillus murinus Application of ATCC 35020 in the preparation of drugs to alleviate fructose-induced non-alcoholic fatty liver disease.

2. The application according to claim 1, characterized in that, The relief of fructose-induced non-alcoholic fatty liver disease includes reducing the histological scores of hepatic steatosis, hepatocellular ballooning degeneration, and lobular inflammation.

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