Lactobacillus reuteri LR-XR23 and application thereof in treatment and / or improvement of NAFLD

Abstract

The invention provides lactobacillus reuteri LR-XR23 and application of the lactobacillus reuteri LR-XR23 in treatment and / or improvement of NAFLD, and relates to the technical field of microorganisms and the technical field of biological medicine. According to the invention, lactobacillus reuteri LR-XR23 is successfully separated from the colon of a NAFLD model mouse induced by a high-fat feed after being treated by traditional Chinese medicine coix seed essential oil, and the preservation number is GDMCC 65010. The strain can significantly reduce the weight of a high-fat feed induced NAFLD model mouse and inhibit the formation of fat; the hyperlipidemia symptom of a high-fat feed induced NAFLD model mouse is improved, and the function of regulating lipid metabolism is achieved; meanwhile, the lactobacillus reuteri LR-XR23 can be colonized in intestinal tracts, so that the diversity of intestinal flora is increased, and the ecological balance of the intestinal tracts is restored. The lactobacillus reuteri LR-XR23 provided by the invention provides a new thought and method for improving and / or treating the NAFLD.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms and biomedicine, and in particular to a Lactobacillus reuteri LR-XR23 and an application thereof in treating and / or improving NAFLD. Background Art

[0002] As people's living standards improve, the prevalence of non-alcoholic fatty liver disease (NAFLD) has increased. At the same time, the prevalence of diabetes and metabolic syndrome is also increasing worldwide. NAFLD is a continuous liver abnormality from non-alcoholic fatty liver to non-alcoholic steatohepatitis. The course of the disease is variable, often manifested as hepatic steatosis or more severe inflammation and liver cell damage (steatohepatitis). According to literature reports, the main drugs for the treatment of NAFLD are the following: insulin sensitizers: biguanides such as metformin, thiazolidinediones such as pioglitazone; lipid-regulating drugs: statins, fibrates, niacin; liver-protecting drugs: reduced glutathione, vitamin E, polyenylphosphatidylcholine, ursodeoxycholic acid, silymarin, etc. However, due to side effects, poor compliance, lack of efficacy or changes in diet and lifestyle, the treatment of NAFLD is usually not effective.

[0003] Probiotics are a type of microorganism that can have beneficial effects on animals. After colonizing the intestines of the body, they can improve microbial balance, help absorb vitamins, and so on. The organic acids, secondary bile acids, short-chain fatty acids and other factors produced after probiotics intervene in the body have the effects of improving intestinal ecological health, helping to restore the intestinal barrier, anti-oxidation, anti-inflammatory, lowering blood sugar and lipids, and anti-tumor. At present, NAFLD treatment methods based on the gut-liver axis and targeting intestinal flora are gradually emerging, and various new metabolic regulating drugs are also under clinical development. The present invention aims to provide a Lactobacillus reuteri LR-XR23 and to explain its effect in treating and / or improving NAFLD. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the deficiencies in the prior art, the present invention provides a Lactobacillus reuteri LR-XR23 and use thereof in treating and / or improving and / or preventing NAFLD.

[0006] (II) Technical solution

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0008] In the first aspect, the present invention provides a Lactobacillus reuteri subsp. reuteri LR-XR23, wherein the Lactobacillus reuteri LR-XR23 belongs to the genus Limosilactobacillus, has been deposited in the Guangdong Provincial Microbiological Collection Center, the preservation date is August 15, 2024, and the preservation number is GDMCC 65010.

[0009] Specifically, the Lactobacillus reuteri LR-XR23 is derived from the colon of NAFLD model mice induced by a high-fat diet after being treated with traditional Chinese medicine essential oils. The strain was identified and analyzed, and its 16S rDNA sequence is shown in SEQ ID NO.1; the strain is Gram-positive, the colonies are mostly milky white, with smooth edges, and can appear as long rods or short rods under a microscope, without spores.

[0010] In a second aspect, the present invention provides the use of Lactobacillus reuteri LR-XR23 in the preparation of a product for treating and / or improving and / or preventing NAFLD.

[0011] In a third aspect, the present invention provides the use of Lactobacillus reuteri LR-XR23 in the preparation of a product for inhibiting animal weight gain.

[0012] In a fourth aspect, the present invention provides the use of Lactobacillus reuteri LR-XR23 in the preparation of a product for inhibiting the deposition of subcutaneous and / or visceral fat in animals.

[0013] In a fifth aspect, the present invention provides the use of Lactobacillus reuteri LR-XR23 in the preparation of a product for inhibiting animal lipid synthesis genes FASN and / or PPAR-r and / or SCD-1.

[0014] In a sixth aspect, the present invention provides the use of Lactobacillus reuteri LR-XR23 in the preparation of a product for improving and / or alleviating and / or restoring the health of intestinal symbiotic flora.

[0015] In the above application, the product is any one of a probiotic preparation, a medicine, a health product, a feed additive, a common food, and a special food.

[0016] In the above application, in the above product, the viable count of Lactobacillus reuteri LR-XR23 is not less than 1×10 9 CFU / mL.

[0017] In the above application, the above product also contains medically or food acceptable oral adjuvants, which include excipients and / or additives and / or a mixture of other probiotics.

[0018] (III) Beneficial effects

[0019] The present invention successfully isolated Lactobacillus reuteri LR-XR23 from the colon of NAFLD model mice induced by high-fat diet after treatment with coix seed essential oil, and identified it as Limosilactobacillus by 16S rDNA sequence. It has been deposited in Guangdong Microbiological Collection Center (GDMCC for short, address: Guangdong Academy of Sciences Institute of Microbiology, Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou), with a deposit date of August 15, 2024 and a deposit number of GDMCC 65010. Lactobacillus reuteri LR-XR23 provided by the present invention has the following advantages:

[0020] 1. The Lactobacillus reuteri LR-XR23 provided by the invention can significantly reduce the body weight of NAFLD model mice induced by a high-fat diet, and inhibit fat production in the liver, subcutaneous fat, epididymal fat, and perirenal areas;

[0021] 2. The Lactobacillus reuteri LR-XR23 provided by the invention can effectively reduce the levels of triglycerides, cholesterol and HDL in NAFLD model mice induced by high-fat diet, increase the level of GSH, improve the hyperlipidemia symptoms of NAFLD model mice induced by high-fat diet, improve the liver's antioxidant capacity, transport phospholipids and cholesterol, and eliminate cholesterol in peripheral blood vessels, thereby achieving the function of regulating lipid metabolism;

[0022] 3. The Lactobacillus reuteri LR-XR23 provided by the present invention can colonize in the intestine, increase the diversity of intestinal flora, and can achieve the effect of repairing the intestinal flora disorder caused by a high-fat diet and restore the intestinal ecological balance.

[0023] 4. The Lactobacillus reuteri LR-XR23 provided by the present invention has a significant preventive and / or alleviating and / or improving and / or therapeutic effect on NAFLD. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Results of body weight and fat weight of two groups of mice; a, body weight of two groups of mice; b, body weight growth rate of two groups of mice; c, epididymal fat weight; d, inguinal fat weight; e, perirenal fat weight; compared with HFD group, *p<0.05, **p<0.01.

[0025] Figure 2 The results of HE sections and Oil red staining of liver fat counts of the two groups of mice.

[0026] Figure 3Results of fat metabolism-related factors in serum and liver of two groups of mice; a, TG content in serum; b, TC content in serum; c, HDL-c content in serum; d, LDL-c content in serum; e, GSH content in liver tissue; f, TG content in liver tissue; g, TC content in liver tissue; compared with the HFD group, *p<0.05, **p<0.01.

[0027] Figure 4 The expression results of genes related to liver fat metabolism in the two groups of mice; compared with the HFD group, *p<0.05, **p<0.01.

[0028] Figure 5 The structural changes of intestinal flora in two groups of mice. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1

[0031] Isolation, cultivation and identification of Lactobacillus reuteri LR-XR23

[0032] Lactobacillus reuteri LR-XR23 was isolated from the colon of NAFLD model mice induced by high-fat diet after treatment with coix seed essential oil. It was identified as Limosilactobacillus by 16S rDNA sequence and has been deposited in Guangdong Microbiological Collection Center (GDMCC, address: Institute of Microbiology, Guangdong Academy of Sciences, Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou). The deposit date is August 15, 2024, and the deposit number is GDMCC 65010.

[0033] Lactobacillus reuteri LR-XR23 was inoculated into MRS solid culture medium and cultured at 37°C for 48 hours. The colonies were observed and Gram staining was positive. The colonies were mostly milky white in color with smooth edges. Under a microscope, they appeared to be long rods or short rods without spores.

[0034] 16S rDNA sequence of strain LR-XR23 of the present invention 16S rDNA identification: using bacterial 16SrDNA universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'), using the genome of strain LR-XR23 as a template, PCR amplification was performed, and the amplified product was sequenced. As shown in SEQ ID No.1; the sequencing results were sequenced on the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The homology with Limosilactobacillus reuteri subsp.reuteri, CICC_6132 was 99.93%.

[0035] SEQ ID No.1:

[0036]

[0037] Example 2

[0038] Effects of Lactobacillus reuteri LR-XR23 on NAFLD model mice induced by high-fat diet

[0039] 1 Experimental animals

[0040] C57 / Bl6J mice were of standard strain, 7 weeks old, weighing (20±1) g, and were housed in a room with appropriate temperature and humidity control, with a 12-h light / dark cycle, free access to food and water, and fed with standard laboratory diet (Cat. No. 1010001, a product of Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.). The experiments were conducted after one week of adaptation.

[0041] 2 Experimental strains

[0042] The Lactobacillus reuteri subsp. reuteri LR-XR23 isolated in Example 1 was inoculated into MRS liquid culture medium at a 2% inoculation amount, and after anaerobic culture at 37°C for 24 h, it was transferred to fresh MRS liquid culture medium and cultured under the same conditions for 24 h. The bacterial sludge was obtained by suction filtration in an anaerobic workstation, and then washed and resuspended with 0.8.1M PBS (pH 7.2, containing 0.05% cysteine) to obtain a bacterial concentration of 1×10 9 CFU / mL bacterial suspension was stored in a well-sealed threaded anaerobic tube with a stopper and used on the same day.

[0043] 3 Animal grouping and treatment

[0044] Ten mice were randomly selected after one week of adaptation and continued to be fed with a 60% high-fat diet (D12492, Research diets). After 10 weeks, NAFLD model mice induced by high-fat diet were obtained. The NAFLD model mice were randomly divided into two groups, 5 mice in each group, namely the high-fat diet group (HFD group) and the high-fat diet + LR-XR23 gavage group (LR-XR23 group). Among them, each mouse in the HFD group was gavaged with 0.2 mL of normal saline per day, and each mouse in the LR-XR23 group was gavaged with 0.2 mL of bacterial concentration of 1×10 9 CFU / mL of LR-XR23 bacterial suspension once a day; both groups continued to be fed a high-fat diet and had free access to food and water for 4 weeks.

[0045] At the end of the experiment, the mice were killed by cervical dislocation. The heart, liver, spleen, lung, kidney, blood, ileum, cecum, and colon were removed and immediately placed in liquid nitrogen and stored at -80°C for further analysis. The subcutaneous and visceral fat deposits were precisely dissected and weighed.

[0046] 4. Detection indicators

[0047] 4.1 Determination of serum and liver AST, ALT, GSH, HDL, etc.

[0048] Whole blood was centrifuged at 2500 rpm / min for 10 min to collect serum, and the test was performed according to the manufacturer's kit (Nanjing Jiancheng Bioengineering Institute).

[0049] 4.2 Pathological examination of mouse liver and adipose tissue

[0050] Mouse liver and epididymal tissues were fixed in formalin for 24 hours, dehydrated, embedded, stained, and finally imaged under a microscope.

[0051] 4.3 Detection of gene expression levels related to fat metabolism in mouse liver

[0052] The liver was taken out from the -80℃ ultra-low temperature refrigerator, and 20 mg of tissue was weighed to extract total RNA. The RNA was reverse transcribed into cDNA using a reverse transcription kit, and cDNA was used as a template for fluorescence quantitative PCR amplification. β-actin was used as an internal reference and 2 －ΔΔCT The mNRA expression of related genes was calculated by this method.

[0053] The primer sequences are as follows

[0054]

[0055]

[0056] Note: PPAR-α: peroxisome proliferator-activated receptor α; PPAR-γ: peroxisome proliferator-activated receptor gamma; CYP7A1: cholesterol 7-alpha hydroxylase; CPT-1: carnitine palmitoyltransferase 1; FAS: fatty acid synthase; SCD-1: stearoyl-CoA desaturase-1; GAPDH: 3-phosphate dehydrogenase.

[0057] 5 Statistical methods

[0058] Prism software was used to measure the data and analyze them statistically. Wilcoxon test was used for significance analysis. P<0.05 indicated statistical significance, and P<0.01 indicated significant statistical difference. For quantitative experiments, three replicates were performed unless otherwise specified, and the results were averaged.

[0059] 6 Results

[0060] 6.1 Effect of Lactobacillus reuteri LR-XR23 on the body weight of NAFLD model mice induced by high-fat diet

[0061] Compared with the HFD group, the body weight of mice treated with LR-XR23 for 4 weeks decreased significantly (p<0.05), with a body weight loss rate of 8.31% ( Figure 1 a, b). Compared with the HFD group, the inguinal subcutaneous fat, epididymal fat, and perirenal fat of the LR-XR23 group mice were significantly reduced (p<0.01) ( Figure 1 ce). HE and oil red staining of the liver revealed that fatty degeneration in the liver of mice in the LR-XR23 group was reduced and the diameter of fat droplets was reduced ( Figure 2 The above results show that LR-XR23 can reduce the weight gain of NAFLD model mice induced by high-fat diet and inhibit the lipogenesis in the liver, subcutaneous fat, epididymal fat and perirenal fat.

[0062] 6.2 Effects of Lactobacillus reuteri LR-XR23 on fat metabolism-related factors in NAFLD model mice induced by high-fat diet

[0063] Figure 3 The results of fat metabolism-related factors in serum and liver of two groups of mice after the experiment. The analysis showed that after LR-XR23 treatment, TC and TG in serum and liver of NAFLD model mice decreased significantly (p<0.01), HDL in serum increased significantly (p<0.01), while LDL showed a downward trend but not significantly (p>0.05). The GSH level in liver tissue increased significantly (p<0.01). This shows that LR-XR23 can improve the hyperlipidemia symptoms of NAFLD model mice after oral administration, and improve the antioxidant capacity of the liver of NAFLD model mice and the ability to transport phospholipids and cholesterol and eliminate cholesterol in peripheral blood vessels.

[0064] Figure 4 The results of QPCR detection of gene expression. The results of QPCR detection of gene expression showed that there was no significant change in the lipolysis genes PPAR-α, CPT-1 and CYP7A1 in the liver of NAFLD model mice after LR-XR23 treatment, but the three fat synthesis genes PPAR-γ, SCD-1 and FAS were significantly downregulated (p<0.01). This suggests that LR-XR23 may mainly inhibit the expression of the three fat synthesis genes PPAR-γ, SCD-1 and FAS in NAFLD model mice, thereby inhibiting liver fat synthesis and ultimately achieving the purpose of inhibiting the formation of fatty liver.

[0065] 6.3 Effect of Lactobacillus reuteri LR-XR23 on the intestinal flora structure of NAFLD model mice induced by high-fat diet

[0066] Figure 5The results of changes in the intestinal flora structure of the two groups of mice are shown. Diversity analysis ( Figure 5 a) and PCOA( Figure 5 b) It was found that the fecal flora of mice in the LR-XR23 group was significantly different from that in the HFD group, and the diversity was increased. This result showed that LR-XR23 could restore the intestinal flora structure disorder of NAFLD model mice induced by high-fat diet to normal. LEfSc analysis ( Figure 5 ce) The results showed that the abundance of Faecalibaculum rodentium, which is negatively correlated with obesity, was significantly reduced in the LR-XR23 group, while the abundance of LR-XR23 was significantly increased. The above results show that the LR-XR23 provided by the present invention can colonize in the intestines of NAFLD model mice induced by high-fat diet, and can repair the flora disorder caused by high-fat diet and restore the intestinal ecological balance.

[0067] 7 Conclusion

[0068] The present invention successfully isolated Lactobacillus reuteri LR-XR23 from the colon of NAFLD model mice induced by a high-fat diet after treatment with coix seed essential oil. The strain can significantly reduce the body weight of NAFLD model mice induced by a high-fat diet and inhibit the formation of fat; it can effectively reduce the levels of triglycerides, cholesterol and HDL in NAFLD model mice induced by a high-fat diet, increase the level of GSH, improve the hyperlipidemia symptoms of NAFLD model mice induced by a high-fat diet, improve the liver's ability to resist oxidation, transport phospholipids and cholesterol, and eliminate cholesterol in peripheral blood vessels, thereby regulating lipid metabolism; at the same time, Lactobacillus reuteri LR-XR23 can colonize in the intestine, increase the diversity of intestinal flora, and achieve the effect of repairing the disorder of intestinal flora caused by a high-fat diet, and restore the ecological balance of the intestine. The Lactobacillus reuteri provided by the present invention can be effectively used to prepare foods, medicines, microbial preparations or feed additives for preventing and / or alleviating and / or improving and / or treating NAFLD, and has broad application value and practical significance.

[0069] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Lactobacillus reuteri subsp. reuteri LR-XR23, wherein the Lactobacillus reuteri LR-XR23 belongs to the genus Limosilactobacillus, has been deposited in Guangdong Microbiological Collection Center, the deposit date is August 15, 2024, and the deposit number is GDMCC 65010.

2. Use of Lactobacillus reuteri LR-XR23 as claimed in claim 1 in the preparation of a product for treating and / or improving and / or preventing NAFLD.

3. Use of the Lactobacillus reuteri LR-XR23 as claimed in claim 1 in preparing a product for inhibiting animal weight gain.

4. Use of Lactobacillus reuteri LR-XR23 as claimed in claim 1 in the preparation of a product for inhibiting subcutaneous and / or visceral fat deposition in animals.

5. Use of Lactobacillus reuteri LR-XR23 as claimed in claim 1 in preparing products for regulating animal lipid metabolism.

6. Use of Lactobacillus reuteri LR-XR23 as claimed in claim 1 in the preparation of products for inhibiting animal lipid synthesis genes FASN and / or PPAR-r and / or SCD-1.

7. Use of Lactobacillus reuteri LR-XR23 as claimed in claim 1 in the preparation of a product for improving and / or alleviating and / or restoring the health of intestinal symbiotic flora.

8. The use according to any one of claims 2 to 6, characterized in that: The product is any one of probiotic preparations, medicines, health products, feed additives, ordinary foods, and special foods.

9. The use according to any one of claims 2 to 6, characterized in that: In the above products, the viable count of Lactobacillus reuteri LR-XR23 is not less than 1×10 9 CFU / mL.

10. The use according to any one of claims 2 to 6, characterized in that The above product also contains medically or food acceptable oral adjuvants, which include excipients and / or additives and / or a mixture of other probiotics.

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