Application of Lactobacillus plantarum strain A21094 in preparing products for preventing and / or treating non-alcoholic fatty liver disease

Abstract

The present invention provides the application of plant lactobacillus strain A21094 in the preparation of products for preventing and / or treating non-alcoholic fatty liver disease, and belongs to the field of biotechnology. The application of plant lactobacillus strain A21094 in the preparation of products for preventing and / or treating non-alcoholic fatty liver disease, the deposit number of the plant lactobacillus strain A21094 is GDMCCNo:62961. The plant lactobacillus strain A21094 achieves the therapeutic purpose of non-alcoholic fatty liver disease by inhibiting cholesterol synthesis, promoting bile acid pathway, regulating fatty acid synthesis pathway, regulating inflammatory signaling pathways such as interleukin 1β (IL-1β), interleukin 17 (IL-17), tumor necrosis factor (TNF-α), and regulating intestinal flora structure, thereby providing a new approach for the preparation of drugs or probiotic preparations.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of Lactobacillus plantarum strain A21094 in preparing products for preventing and / or treating non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) refers to a clinical pathological syndrome characterized by diffuse macrovesicular steatosis of hepatocytes, excluding alcohol and other clear liver-damaging factors, including simple fatty liver and its evolved steatohepatitis (NASH) and cirrhosis. With the increasing incidence of obesity, hyperlipidemia, type II diabetes and metabolic syndrome, it seriously endangers human health.

[0003] The hallmark of NAFLD is the accumulation of neutral lipids in the liver, which is derived from an imbalance in lipid acquisition and clearance. Disturbance of cholesterol metabolism and accumulation of free cholesterol in hepatocytes are associated with the pathological mechanisms of NAFLD / NASH. Although the pathophysiological mechanisms of NAFLD are still unclear, intestinal flora, its metabolites and genetic factors are considered to be important factors. Intestinal flora dysbiosis often causes harmful effects on the liver of NAFLD patients through the gut-liver axis.

[0004] Many studies have shown that controlling the intestinal microbiota through the use of probiotics or FMT may be an effective strategy for preventing and treating NAFLD. At present, patents such as CN108641988B, CN113786419A, CN114164142A, and CN115089619A have reported the effects of different strains of Lactobacillus plantarum on alleviating non-alcoholic fatty liver disease, but the lipid-lowering effect of the above strains is not ideal, the mechanism of action is not clear, and there is no comparison with commercial strains on the market, so it is impossible to explain whether the application is feasible; at the same time, the strains do not have tolerance function, and it is impossible to explain whether they can colonize in the intestine. Summary of the invention

[0005] In view of this, the object of the present invention is to provide the use of Lactobacillus plantarum strain A21094 in the preparation of a medicament for preventing and / or treating non-alcoholic fatty liver disease. Lactobacillus plantarum A21094 activates the rate-limiting enzyme for cholesterol synthesis and bile acid synthase, inhibits cholesterol synthesis, promotes bile acid synthesis, and regulates the structure and function of intestinal flora to achieve the treatment of non-alcoholic fatty liver disease. At the same time, the strain has good gastrointestinal tolerance and good intestinal colonization performance.

[0006] The invention provides application of a Lactobacillus plantarum strain A21094 in preparing a product for treating non-alcoholic fatty liver disease. The preservation number of the Lactobacillus plantarum strain A21094 is GDMCC No: 62961.

[0007] The invention provides application of a Lactobacillus plantarum strain A21094 in preparing a product for inhibiting cholesterol synthesis in non-alcoholic fatty liver disease. The preservation number of the Lactobacillus plantarum strain A21094 is GDMCC No: 62961.

[0008] The invention provides application of a Lactobacillus plantarum strain A21094 in preparing a product for promoting bile acid synthesis in non-alcoholic fatty liver disease. The deposit number of the Lactobacillus plantarum strain A21094 is GDMCC No: 62961.

[0009] The invention provides application of a Lactobacillus plantarum strain A21094 in preparing a product for regulating fatty acid synthesis in non-alcoholic fatty liver disease. The deposit number of the Lactobacillus plantarum strain A21094 is GDMCC No:62961.

[0010] The invention provides application of Lactobacillus plantarum strain A21094 in preparing a product for enhancing oral glucose tolerance in non-alcoholic fatty liver disease and / or improving insulin resistance in non-alcoholic fatty liver disease. The deposit number of the Lactobacillus plantarum strain A21094 is GDMCC No: 62961.

[0011] The invention provides application of a Lactobacillus plantarum strain A21094 in preparing a product for inhibiting inflammatory response in non-alcoholic fatty liver disease. The deposit number of the Lactobacillus plantarum strain A21094 is GDMCC No: 62961.

[0012] Preferably, the product is a product that inhibits the expression of inflammatory factors such as interleukin 6 (IL-6), interleukin 1β (IL-1β), interleukin 17 (IL-17), and tumor necrosis factor (TNF-α) and reduces the level of lipopolysaccharide in serum.

[0013] The invention provides application of a Lactobacillus plantarum strain A21094 in preparing a product for regulating the intestinal flora structure in non-alcoholic fatty liver disease. The preservation number of the Lactobacillus plantarum strain A21094 is GDMCC No: 62961.

[0014] Preferably, the regulation of the intestinal flora structure in non-alcoholic fatty liver disease is to increase the abundance of Lactobacillales and Akkermansia in the intestines of patients with non-alcoholic fatty liver disease, and reduce the abundance of Turicibacter and Allobaculum.

[0015] Preferably, the Lactobacillus plantarum strain A21094 comprises a lyophilized powder of the Lactobacillus plantarum strain A21094;

[0016] Preferably, the product is a medicine;

[0017] Preferably, the freeze-dried powder of the Lactobacillus plantarum strain A21094 comprises inoculating the activated Lactobacillus plantarum strain A21094 into an MRS liquid culture medium for anaerobic fermentation to obtain a seed solution;

[0018] amplifying and culturing the seed solution to obtain a large amount of bacterial solution;

[0019] The large amount of bacterial liquid is freeze-dried from the separated bacterial cells to obtain freeze-dried powder.

[0020] The present invention provides an application of Lactobacillus plantarum strain A21094 in preparing a product for treating non-alcoholic fatty liver disease, and the deposit number of the Lactobacillus plantarum strain A21094 is GDMCC No: 62961. The Lactobacillus plantarum strain A21094 treats non-alcoholic fatty liver disease by inhibiting cholesterol synthesis, promoting bile acid pathways, regulating fatty acid synthesis pathways, regulating inflammatory signaling pathways such as IL-1β, IL-17 and TNF-α, regulating biological circadian rhythms, and regulating intestinal flora structure. It can be seen that the present invention provides a new means for treating non-alcoholic fatty liver disease.

[0021] At the same time, the plant lactobacillus A21094 is a microorganism derived from the intestines of centenarians. It has strong acid resistance, bile salt resistance, cell adhesion, self-hydrophobicity and antibacterial activity, better growth ability, and can better pass through the stomach to reach the intestines for colonization, thereby exerting its function of improving intestinal flora imbalance, which is conducive to maintaining the therapeutic function for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the phylogenetic tree analysis result of strain A21094;

[0023] Figure 2 The results of TC, TG, HDL-C and LDL-C determination in mouse serum;

[0024] Figure 3 This is the result of H&E staining analysis of mouse liver tissue sections;

[0025] Figure 4 The results of NAS scoring and analysis of H&E staining of mouse liver tissue sections;

[0026] Figure 5 Results of AST, ALT and total bile acid tests in mouse serum;

[0027] Figure 6 The results of inflammatory factor detection in mouse serum and liver tissue;

[0028] Figure 7Results of OGTT and ITT tests for mice;

[0029] Figure 8 The results of mRNA expression of genes related to liver cholesterol, bile acid and fatty acid synthesis;

[0030] Fig. 9 This is the gene expression result after A21094 strain intervened in NAFLD;

[0031] Fig.10 The results of KEGG enrichment and GO analysis of significantly up-regulated / down-regulated genes after A21094 strain intervention in NAFLD;

[0032] Fig.11 The results of the analysis of Alpha diversity and Beta diversity of colonic flora after A21094 strain intervention in NAFLD;

[0033] Fig.12 The results of the analysis of colonic flora Phylum abundance after A21094 strain intervention in NAFLD;

[0034] Fig.13 The results of analysis of the abundance of colonic flora Genus after A21094 strain intervention in NAFLD;

[0035] Fig.14 These are the results of LEfSe analysis of colonic flora after A21094 strain intervention in NAFLD.

[0036] Description of the preservation and survival of biological materials

[0037] Lactobacillus plantarum A21094, the deposit date is November 11, 2022, the depository is Guangdong Microbiological Culture Collection Center, the abbreviation of which is GDMCC, the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, the deposit number is GDMCC No: 62961. DETAILED DESCRIPTION

[0038] The invention provides application of a Lactobacillus plantarum strain A21094 in preparing a product for preventing and / or treating non-alcoholic fatty liver disease. The deposit number of the Lactobacillus plantarum strain A21094 is GDMCC No: 62961.

[0039] In the present invention, plant lactobacillus A21094 is derived from the intestinal microbial flora of centenarians, is a natural and safe beneficial bacteria, and animal experiments have shown that plant lactobacillus A21094 can significantly reduce blood lipid levels. At the same time, it can significantly reduce AST and ALT levels and reduce liver damage; it also has the ability to enhance oral glucose tolerance and improve insulin resistance, is better than the commercial strains of related products currently sold on the market, and is a strain with potential superior probiotic function. According to phylogenetic tree analysis, the plant lactobacillus A21094 is far away from the existing plant lactobacillus of related functions on the evolutionary tree, indicating that it is a new plant lactobacillus.

[0040] In the present invention, the product is preferably a drug. The present invention has no particular limitation on the preparation method of the drug, and any drug preparation method known in the art can be used.

[0041] In the embodiment of the present invention, the method of first establishing a non-alcoholic fatty liver disease model and then administering bacteria for treatment is a real treatment method.

[0042] In the embodiment of the present invention, Lactobacillus plantarum A21094 improves the liver tissue morphology in non-alcoholic fatty liver disease, and the effect is better than that of commercial strains. Experiments show that Lactobacillus plantarum A21094 treats non-alcoholic fatty liver disease by inhibiting cholesterol synthesis, promoting bile acid pathways, regulating fatty acid synthesis pathways, regulating IL-17 inflammatory signaling pathways, and regulating biological circadian rhythms.

[0043] The invention provides application of a Lactobacillus plantarum strain A21094 in preparing a product for inhibiting cholesterol synthesis in non-alcoholic fatty liver disease. The preservation number of the Lactobacillus plantarum strain A21094 is GDMCC No: 62961.

[0044] In the present invention, high levels of HMGR expression can promote the synthesis of cholesterol and steroid hormones, thereby increasing the risk of NAFLD; SCD1 (monounsaturated fatty acid synthase 1) expression also changes in the occurrence and development of non-alcoholic fatty liver disease (NAFLD). High expression of DGAT2 can promote the synthesis and deposition of fatty acids, leading to excessive fat deposition in the liver. In the embodiment of the present invention, A21094 can significantly reduce the expression levels of HMGR, SCD1 and DGAT2 mRNA, indicating that Lactobacillus plantarum A21094 has the function of regulating fatty acid synthesis and cholesterol metabolism disorders.

[0045] The invention provides application of a Lactobacillus plantarum strain A21094 in preparing a product for promoting bile acid synthesis in non-alcoholic fatty liver disease. The deposit number of the Lactobacillus plantarum strain A21094 is GDMCC No: 62961.

[0046] In the present invention, cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme of the classical pathway of bile acid anabolism, and its expression level reflects the speed of bile acid synthesis and the body's ability to remove cholesterol. In addition, cholesterol 12α-hydroxylase (CYP8B1) is involved in the synthesis of bile acids, which is very important for the digestion and absorption of fat. Mutations in the CYP8B1 gene may lead to various metabolic diseases, such as liver disease. The experimental results showed that compared with the normal group, the mRNA expression of CYP7A1 and CYP8B1 in the liver tissue of mice in the NAFLD group was significantly reduced; compared with the NAFLD group, the expression of CYP7A1 and CYP8B1 in the A21094 group was significantly increased, and CYP8B1 had significant differences; therefore, A21094 has the function of promoting bile acid synthesis and preventing cholesterol accumulation in the liver.

[0047] The invention provides application of a Lactobacillus plantarum strain A21094 in preparing a product for regulating fatty acid synthesis in non-alcoholic fatty liver disease. The deposit number of the Lactobacillus plantarum strain A21094 is GDMCC No:62961.

[0048] In the embodiment of the present invention, the indicators related to cholesterol synthesis in serum were detected, and the results showed that compared with the normal group, the total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C) contents of the NAFLD group were significantly increased, the total triglyceride (TG) content was increased, and the high-density lipoprotein-cholesterol (HDL-C) content was decreased, but there was no significant difference. Compared with the NAFLD group, the TC and TG of the probiotic group were significantly decreased, and compared with the commercial strain, the LDL-C of plant lactobacillus A21094 was significantly decreased, and the HDL-C content was significantly increased, and there was a significant difference, indicating that A21094 has a comprehensive effect of reducing cholesterol and blood lipid levels in non-alcoholic fatty liver.

[0049] In the embodiment of the present invention, Lactobacillus plantarum A21094 can significantly reduce blood lipid levels, reduce cholesterol by 70% in vivo, and reduce triglycerides by 62% in vivo. It can reduce aspartate aminotransferase (AST) and alanine aminotransferase (ALT) by 73% and 66% in vivo, respectively, which is better than the current related patented strains and commercial strains. It can effectively improve liver damage caused by fat accumulation.

[0050] The invention provides application of Lactobacillus plantarum strain A21094 in preparing a product for enhancing oral glucose tolerance in non-alcoholic fatty liver disease and / or improving insulin resistance in non-alcoholic fatty liver disease. The deposit number of the Lactobacillus plantarum strain A21094 is GDMCC No: 62961.

[0051] In the embodiment of the present invention, compared with the normal group, the blood sugar in the NAFLD group rose rapidly and by a large margin, and the blood sugar dropped relatively slowly within 120 minutes, and could not return to normal blood sugar levels, indicating that glucose tolerance had decreased. After insulin injection, the blood sugar in the NAFLD group dropped very slowly and could not return to normal blood sugar levels, indicating that insulin resistance had occurred, but compared with the NAFLD group and the commercial group, Lactobacillus plantarum A21094 could significantly enhance the glucose tolerance of mice and reduce insulin resistance, and improve the metabolic dysfunction caused by non-alcoholic fatty liver disease.

[0052] The invention provides application of a Lactobacillus plantarum strain A21094 in preparing a product for inhibiting the expression of inflammatory factors in non-alcoholic fatty liver disease. The deposit number of the Lactobacillus plantarum strain A21094 is GDMCC No: 62961.

[0053] In the present invention, the product is a product that inhibits the expression of inflammatory factors such as interleukin 6 (IL-6), interleukin 1β (IL-1β), interleukin 17 (IL-17), tumor necrosis factor (TNF-α) and reduces the level of lipopolysaccharide (LPS) in serum. The results of the embodiments of the present invention show that compared with the NAFLD group, Lactobacillus plantarum A21094 can reduce the levels of IL-6 and IL-17 inflammatory factors, with no significant difference. However, it can significantly reduce the levels of LPS, TNF-ɑ and IL-1β inflammatory factors, which were reduced by 66%, 59% and 79%, respectively, and there are significant differences. It shows that Lactobacillus plantarum A21094 regulates the inflammation of mice with non-alcoholic fatty liver disease through multiple inflammatory factor signaling pathways.

[0054] The invention provides application of a Lactobacillus plantarum strain A21094 in preparing a medicine for regulating the intestinal flora structure in non-alcoholic fatty liver disease. The deposit number of the Lactobacillus plantarum strain A21094 is GDMCC No:62961.

[0055] In the present invention, the regulation of the intestinal flora structure in non-alcoholic fatty liver disease is preferably to increase the abundance of Lactobacillales and Akkermansia in the intestine of patients with non-alcoholic fatty liver disease, and reduce the abundance of Turicibacter and Allobaculum.

[0056] In the present invention, the plant lactobacillus strain A21094 preferably includes a freeze-dried powder of the plant lactobacillus strain A21094. The plant lactobacillus strain A21094 freeze-dried powder comprises inoculating the activated plant lactobacillus strain A21094 into an MRS liquid culture medium for anaerobic fermentation to obtain a seed solution; amplifying the seed solution to obtain a large amount of bacterial solution; and freeze-drying the large amount of bacterial solution from the isolated bacterial body to obtain the freeze-dried powder.

[0057] In the present invention, the amplified culture includes multiple anaerobic fermentation cultures. The temperature of the anaerobic fermentation culture is preferably 36-38°C, more preferably 37°C. The time of the anaerobic fermentation culture is preferably 12-24h, more preferably 20h. The method for separating the bacteria is preferably centrifugation. The speed of the centrifugation is preferably 4800-5200rpm, more preferably 5000rpm. The time of the centrifugation is preferably 4-6min, more preferably 6min. The bacteria are preferably mixed with an equal mass of a lyophilizer before freeze-drying. The freeze-drying method is preferably treated at -20°C for 30min, then treated at -80°C for 1h, and then transferred to a freeze-drying bottle for freeze-drying for 24h.

[0058] In the present invention, the drug is preferably an oral dosage form. The effective live bacterial concentration of Lactobacillus plantarum strain A21094 in the drug is preferably (1-100)×10 9 CFU / ml, more preferably 1×10 9 CFU / ml.

[0059] The application of the Lactobacillus plantarum strain A21094 provided by the present invention in preparing products for preventing and / or treating non-alcoholic fatty liver disease is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0060] Example 1

[0061] The present invention provides the screening and identification of Lactobacillus plantarum A21094 for treating non-alcoholic fatty liver disease.

[0062] The strain has been identified in patent number: CN116144541A, and the purified strain was molecularly identified by 16S rRNA method. 16S rRNA amplification and sequencing were completed by Sangon Biotech (Shanghai) Co., Ltd. The 16S rRNA sequence of strain A21094 was compared in the NCBI (National Center for Biotechnology Information) database after sequencing. The result showed that the gene sequence of strain A21094 had a homology of 99.86% with Lactobacillus plantarum among all similar sequences. Therefore, combined with physiological and biochemical characteristics, strain A21094 was identified as Lactobacillus plantarum.

[0063] At the same time, phylogenetic tree analysis was performed with existing patented strains of Lactobacillus plantarum ( Figure 1), it was found that the plant lactobacillus A21094 involved in the present invention has a certain distance from the existing plant lactobacillus related patent strains on the evolutionary tree, indicating that the plant lactobacillus A21094 involved in the invention is a new subspecies. The plant lactobacillus (Lactiplantibacillus plantarum) isolated from the intestines of centenarians has been deposited in the Guangdong Microbiological Culture Collection Center on November 11, 2022, and its deposit number is GDMCC No: 62961.

[0064] Example 2

[0065] Preparation of freeze-dried powder of Lactobacillus plantarum A21094 and its animal experiment in treating non-alcoholic fatty liver disease

[0066] 1. Preparation of freeze-dried powder of Lactobacillus plantarum A21094

[0067] (1) Strain fermentation culture

[0068] Primary fermentation: pick a single colony of the experimental strain after activation twice to add it to 15 ml of MRS liquid culture medium, and culture it statically under anaerobic conditions at 37°C for 12 hours to obtain seed solution 1.

[0069] Secondary fermentation: Inoculate seed solution 1 into 150 ml of MRS liquid culture medium at an inoculation rate of 3%, and culture at 37° C. under anaerobic conditions for 20 to 22 hours to obtain seed solution 2.

[0070] Amplification culture: inoculate the seed solution 2 into 300 ml of MRS liquid culture medium at an inoculation rate of 3%, and culture it statically at 37° C. under anaerobic conditions for 20 to 24 hours to obtain the bacterial solution after amplification culture.

[0071] (2) Fermentation broth treatment: Collect the bacterial broth and centrifuge it in a refrigerated centrifuge at 4°C, 5000 rpm for 5 min, and discard the supernatant. Wash the bacteria twice with physiological saline, centrifuge it at 4°C, 5000 rpm for 5 min, and weigh the wet weight of the bacteria.

[0072] (3) Freeze drying

[0073] Freeze-dried protective agent: wet weight of bacteria = 1:1 volume ratio for resuspending, EP tube packaging, placed at -20℃ for 30min, and then placed at -80℃ for 1h. The freeze-dried protective agent is a skim milk powder solution with a mass concentration of 12%. The preparation method is as follows: weigh 12g skim milk powder, make up to 100ml deionized water, and sterilize with high-pressure steam at 115℃ for 15min. Put the packaged EP tube into the freeze-dried bottle, put the freeze-dried bottle into the freeze dryer for freeze drying, and continue freeze drying for 24h to finally obtain freeze-dried bacterial powder. Use the plate count method for technology and store the bacterial powder at 4℃ for standby use, and resuspend it with sterile saline when using.

[0074] 2. Animal Experiments

[0075] 24 SPF 6-8 week old female C57BL / 6J mice were selected as experimental mice and fed adaptively for 1 week. During the adaptation period, the light cycle was maintained at 12h, the temperature was 20-26℃, and the humidity was 40%-60%. The mice were allowed to eat and drink freely. After the adaptation period, they were randomly divided into 4 groups, 6 mice in each group, as follows:

[0076] Normal group: basic feed and 0.2 ml skim milk;

[0077] Model group (NAFLD group): high-fat (20%) high-fructose high-cholesterol diet and 0.2 ml 12% skim milk;

[0078] Lactobacillus casei Zhang group (casei Zhang group): high-fat (20%) high-fructose high-cholesterol feed and 0.2 ml bacterial powder (1×10 9 CFU / ml);

[0079] A21094 group: high-fat (20%) high-fructose high-cholesterol diet and 0.2 ml bacterial powder (1×10 9 CFU / ml).

[0080] The NAFLD group, Lactobacillus casei Zhang group, and A21094 group were fed a high-fat, high-fructose, high-cholesterol diet (customized by Jiangsu Collaborative Biological) for 4 consecutive weeks. After abnormal blood lipids and fatty degeneration of the liver were detected, the probiotic group began to be gavaged with 0.2 ml of bacterial powder (1×10 9 CFU / ml), and the mice were gavaged for 4 weeks. During the experiment, the mice were free to drink water and eat. In the 7th week of the experiment, an oral glucose tolerance test was performed, and in the 8th week, an insulin tolerance test was performed. The mice were dissected, blood was collected from the heart, and liver, colon tissue, colon content and feces were collected.

[0081] Example 3

[0082] Detection of mouse serum TC, TG, HDL-C and LDL-C

[0083] In Example 2, blood was collected from the heart before the mice were dissected, and the mice were fasted for 12 hours before blood collection. The collected blood was coagulated at 4°C for 2 hours, centrifuged at 4°C, 2600 rpm for 20 minutes, and serum was collected. The serum was stored at -20°C for future use; serum TC, TG, HDL-C and LDL-C detection kits (Prilai) were used for detection according to the instructions.

[0084] Figure 2The results showed that compared with the normal group, the TC and LDL-C levels in the NAFLD group increased significantly, by 68% and 30%, respectively, the TG content increased by 70%, and the HDL-C content decreased by 62%, and there were significant differences. Compared with the NAFLD group, the TC and TG in the A21094 group decreased significantly, by 70% and 62%, respectively, and there were significant differences; at the same time, compared with the commercial strain, the TC, TG, and LDL-C of Lactobacillus plantarum A21094 decreased significantly, and the HDL-C content increased significantly, and there were significant differences, indicating that A21094 has a good comprehensive effect in lowering blood lipid levels.

[0085] Example 4

[0086] H&E staining of mouse liver

[0087] In Example 2, part of the mouse liver tissue was fixed with 4% paraformaldehyde fixative for 48 hours during dissection, embedded in paraffin, sliced ​​with 4-5 μm using a paraffin slicer (Kodi KD2260), and then stained with H&E. The staining results were scored for non-alcoholic fatty liver disease using the NAS scoring system, and histopathological evaluation was performed.

[0088] Figure 3 The results of H&E staining of mouse liver tissue showed that compared with the normal group, the model group had narrowed sinusoids, marginalized nuclei, swollen cells, balloon-like degeneration, intracellular lipid droplets of varying sizes, and a large number of neutrophil inflammatory cells infiltrating. Compared with the model group, the vacuoles in the Zhang casei group became smaller and the fatty degeneration became less, while the contours of the liver lobules in the Lactobacillus plantarum A21094 group were slightly clearer, the nuclear morphology was basically regular, and the number of cells increased, which was almost the same as that of the normal group, indicating that A21094 can significantly improve non-alcoholic liver disease more than the commercial strain Zhang casei. Figure 4 The evaluation results of the non-alcoholic fatty liver disease NAS scoring system further demonstrated that A21094 is more effective in treating non-alcoholic fatty liver disease, can reverse fatty degeneration, and has significant differences.

[0089] Example 5

[0090] Analysis of liver injury and inflammation in mice

[0091] Liver damage occurs through direct liver cell damage and inflammation. Serum ALT, AST and total bile acid levels are important indicators for evaluating excessive liver damage. The serum collected after the implementation of Example 2 was tested according to the instructions of the ALT and AST detection kit (Nanjing Jiancheng Biological Research Institute). ELISA detection (Jianglai Biological ELISA detection kit) of inflammatory factors such as LPS, IL-6 and IL-17. Detection of IL-1β and TNF-ɑmRNA expression levels in liver tissue.

[0092] Figure 5 Results The test results showed that compared with the normal group, the AST, ALT and total bile acid levels in the NAFLD group were significantly increased, increasing by 60%, 86%, and 57%, respectively. Compared with the NAFLD group, the probiotic group could reduce the levels of these indicators, but compared with the commercial strain group Zhang group, Lactobacillus plantarum A21094 could significantly reduce the AST, ALT and total bile acid levels in serum, reducing by 76%, 66% and 70%, respectively, with significant differences, indicating that Lactobacillus plantarum A21094 can reduce liver damage and cholestasis.

[0093] Figure 6 The results of related inflammatory indicators showed that Lactobacillus plantarum A21094 could reduce the levels of IL-6 and IL-17 inflammatory factors, with no significant difference. However, it could significantly reduce the levels of LPS, TNF-ɑ and IL-1β inflammatory factors, by 66%, 59% and 79% respectively, with significant differences. This indicates that Lactobacillus plantarum A21094 regulates inflammation in mice with non-alcoholic fatty liver disease through multiple inflammatory factor signaling pathways.

[0094] Example 6

[0095] Lactobacillus plantarum A21094 enhances oral glucose tolerance and improves insulin resistance in mice

[0096] Oral glucose tolerance test (OGTT) was performed in the 7th week in Example 2. Fasting overnight the day before the test, then 2.5 g / kg of glucose was given, and the blood glucose levels at 0 min, 30 min, 60 min and 120 min were detected using test strips and a blood glucose meter (Roche blood glucose meter). Insulin tolerance test (ITT) was performed in the 8th week, fasting for 2 h before the test, 0.75 U / kg of insulin was injected intraperitoneally, and the blood glucose levels at 0 min, 30 min, 60 min and 120 min were detected using test strips and a blood glucose meter (Roche blood glucose meter).

[0097] Figure 6 The results showed that compared with the normal group, the blood sugar level in the NAFLD group rose rapidly and to a greater extent, and the blood sugar level dropped relatively slowly within 120 minutes, and could not return to normal blood sugar levels, indicating that glucose tolerance was impaired. After the injection of insulin, the blood sugar level in the NAFLD group dropped very slowly and could not return to normal blood sugar levels, indicating that insulin resistance occurred. However, compared with the NAFLD group and the commercial group, Lactobacillus plantarum A21094 could significantly enhance the glucose tolerance of mice and reduce insulin resistance, and improve the metabolic dysfunction caused by non-alcoholic fatty liver disease.

[0098] Example 7

[0099] Lactobacillus plantarum A21094 treats NAFLD by inhibiting cholesterol synthesis and promoting bile acid pathway

[0100] In Example 2, mouse liver tissue samples were collected and frozen at -80°C. 30 mg of liver tissue was weighed, and the total RNA of mouse liver tissue was extracted according to the instructions of the tissue total RNA kit (Suzhou Youyi Landi Biotechnology Co., Ltd.). cDNA synthesis was performed according to the instructions of the reverse transcription kit (Tiangen). Reference (doi:10.3389 / fnut.2022.1071284.) qPCR primers (Table 1). The comparative CT (△△CT) method was used to detect the expression of the target gene. The qPCR reaction was carried out in the QuantStudioTM System, and the fluorescence quantification program was: amplification curve: 94°C 30s, 94°C 15s, 58°C 30s, for 40 cycles; melting curve: 95°C 15s, 60°C 1min, 95°C 15s. The housekeeping gene 18S was used as the internal reference. The target gene was quantitatively analyzed.

[0101] Table 1 Fluorescence quantitative primers

[0102]

[0103]

[0104] Cholesterol 7-alpha hydroxylase (CYP7A1) belongs to the liver-specific microsomal cytochrome P450 enzyme system and is the rate-limiting enzyme in the classical pathway of bile acid synthesis metabolism. Nearly 50% of cholesterol in the body is converted into bile acid and excreted through the catalysis of CYP7AI. Its expression level reflects the speed of bile acid synthesis and the body's ability to eliminate cholesterol. CYP8B1 is involved in the synthesis of bile acid and is very important for the digestion and absorption of fat. Mutations in the CYP8B1 gene may lead to various metabolic diseases, including gallstones and liver disease. Figure 7 The results showed that compared with the normal group, the mRNA expression of CYP7A1 and CYP8B1 in the liver tissue of mice in the NAFLD group was significantly decreased; compared with the NAFLD group, the expression of CYP7A1 and CYP8B1 in the A21094 group was significantly increased, by 51% and 87%, respectively, and CYP8B1 had a significant difference; therefore, A21094 has the function of promoting bile acid synthesis and preventing cholesterol accumulation in the liver.

[0105] High levels of 3-hydroxy, 3-methylglutaryl-CoA reductase (HMGR) expression can promote the synthesis of cholesterol and steroid hormones, thereby increasing the risk of NAFLD. The expression level of HMGR increases with the severity of the disease. At the same time, the expression level of monounsaturated fatty acid synthase 1 (SCD1), another key enzyme in the fatty acid metabolism and synthesis pathway, also changes in the occurrence and development of non-alcoholic fatty liver disease (NAFLD). High expression of diacylglycerol acyltransferase 2 (DGAT2) can promote the synthesis and deposition of fatty acids, leading to excessive fat deposition in the liver. In addition, high expression of DGAT2 is also closely related to the pathological process of NAFLD and may accelerate the occurrence of liver inflammation and fibrosis. In this experiment, compared with the normal group, the expression levels of HMGR, SCD1 and DGAT2 mRNA in the liver tissue of mice in the NAFLD group were significantly increased, by 73%, 80% and 79%, respectively. However, compared with the NAFLD group, A21094 could significantly reduce the expression levels of HMGR, SCD1 and DGAT2 mRNA by 80%, 82% and 82%, respectively, indicating that Lactobacillus plantarum A21094 has the function of regulating fatty acid synthesis and cholesterol metabolism disorders.

[0106] Example 8

[0107] Transcriptome analysis of Lactobacillus plantarum A21094 in the treatment of non-alcoholic fatty liver disease

[0108] In Example 2, mouse liver tissue samples were collected and frozen in liquid nitrogen and then quickly frozen at -80°C. 100 mg of liver tissue was weighed and sent for transcriptome sequencing (Beijing Novogene Technology Co., Ltd.). The sequencing depth was 6G. The original count was normalized using TPM (the number of transcripts per kilobase per million mapped reads), and the differential expression threshold was: Log2 (FC) ≥ 1, P (FDR) < 0.05. The transcriptome results found that compared with the normal group, the NAFLD group had a total of 142 DEGs upregulated and 288 DEGs downregulated. In the A21094 group, a total of 11 DEGs upregulated and 34 DEGs downregulated ( Fig. 9 ). After A21094 strain intervention, the differentially expressed genes were used for KEGG enrichment analysis, and the main enriched pathways were glycerophospholipid metabolism, cholesterol metabolism, PPAR signaling pathway, adipose cytokine signaling pathway, AMPK signaling pathway, unsaturated fatty acid biosynthesis, IL-17 signaling pathway, etc. ( Fig.10 ). It is speculated that Lactobacillus plantarum A21094 may improve the symptoms of non-alcoholic fatty liver disease through multiple mechanisms such as regulating fatty acid metabolism, alleviating inflammatory response, regulating circadian rhythm and promoting cholesterol metabolism.

[0109] Example 9

[0110] Analysis of intestinal flora in the treatment of non-alcoholic fatty liver disease with Lactobacillus plantarum A21094

[0111] In Example 2, the effect of Lactobacillus plantarum A21094 on the diversity of intestinal flora in colon contents before and after treatment of non-alcoholic fatty liver disease was analyzed by Miseq sequencing of 16S rRNA. Fig.11 ), Chao1 index (P<0.05), Pielou's index (P<0.05), Shannon index (P<0.05) and Simpson index (P<0.05) of A21094 group were significantly lower than those of NAFLD group, and there was no significant difference compared with the normal group, indicating that Lactobacillus plantarum A21094 intervention reduced the diversity of intestinal microbial flora and had the function of regulating intestinal flora. PCoA analysis of Bray-curitis distance showed that ( Fig.11 ), the A21094 group and the NAFLD group were obviously separated in distribution, indicating that the intervention of Lactobacillus plantarum A21094 changed the intestinal microbial flora structure of NAFLD mice.

[0112] Phylum abundance differences showed ( Fig.12 ), compared with the NAFLD group, the abundance of Firmicutes in the A21094 group decreased, and the abundance of Verrucomicrobia increased significantly. Compared with the normal group, the Bacteroidetes and B / F ratio of A21094 in the NAFLD group were significantly reduced. The results of the difference in Genus abundance showed that ( Fig.13 ), in the colon, compared with the model group, the abundance of Akkermansia and Enterorhabdus in A21094 increased significantly, and Romboutsia decreased significantly. Compared with the normal group, the abundance of Lactobacillus in A21094 increased significantly. LEfSe analysis showed ( Fig.14 ), compared with the normal group, the abundance of Faeclibaculum in the model group increased, and the abundance of Clostridia decreased; compared with the model group, the abundance of Lactobacillales and Akkermansia in the A21094 group increased, and the abundance of Turicibacter and Allobaculum decreased.

[0113] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of Lactobacillus plantarum strain A21094 in the preparation of a medicament for preventing and / or treating non-alcoholic fatty liver disease, wherein the deposit number of the Lactobacillus plantarum strain A21094 is GDMCC No: 62961.

2. The use according to claim 1, characterized in that: The prevention and / or treatment of non-alcoholic fatty liver disease is to promote bile acid synthesis in non-alcoholic fatty liver disease, inhibit fatty acid synthesis in non-alcoholic fatty liver disease, enhance oral glucose tolerance in non-alcoholic fatty liver disease, improve insulin resistance in non-alcoholic fatty liver disease, inhibit inflammatory response in non-alcoholic fatty liver disease and regulate the intestinal flora structure in non-alcoholic fatty liver disease.

3. The application according to claim 2, characterized in that: The inhibition of inflammatory response in non-alcoholic fatty liver disease is to inhibit the expression of inflammatory factors such as interleukin 6, interleukin 1β, interleukin 17, and tumor necrosis factor and reduce the level of lipopolysaccharide in serum.

4. The application according to claim 2, characterized in that: The method for regulating the intestinal flora structure in non-alcoholic fatty liver disease is to increase the abundance of Lactobacillales and Akkermansia and reduce the abundance of Turicibacter and Allobaculum in the intestine of patients with non-alcoholic fatty liver disease.

5. The use according to claim 1, characterized in that: The Lactobacillus plantarum strain A21094 includes freeze-dried powder of the Lactobacillus plantarum strain A21094; The freeze-dried powder of the Lactobacillus plantarum strain A21094 comprises inoculating the activated Lactobacillus plantarum strain A21094 into an MRS liquid culture medium for anaerobic fermentation to obtain a seed liquid; amplifying and culturing the seed solution to obtain a large amount of bacterial solution; The large amount of bacterial liquid is freeze-dried from the separated bacterial cells to obtain a freeze-dried powder.

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