CN114561320B - Application of lactobacillus probiotics CGMCC No.1.13855 in preparing medicines for treating liver diseases - Google Patents
Application of lactobacillus probiotics CGMCC No.1.13855 in preparing medicines for treating liver diseases Download PDFInfo
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Abstract
The invention discloses an application of a lactobacillus probiotic strain in preparing a liver disease treatment drug, wherein the preservation number of the strain is CGMCC No.1.13855, the preservation date is 2021, 12 and 16 days, the strain is classified and named as Xu Jianguo lactobacillus (Lactobacillus xujianguonis), and the preservation unit is China general microbiological culture Collection center. The strain is harmless to animals and has the effect of regulating liver function.
Description
Technical Field
The invention relates to probiotics and application thereof, and belongs to the field of microorganisms.
Background
Lactobacillus belongs to lactic acid bacteria, and is named because the lactobacillus ferments carbohydrate to generate lactic acid product, and the lactobacillus takes the shape of a short rod. Lactobacillus is widely distributed in nature, and can be separated from plants, animals such as pigs, foods such as beer and pickled Chinese cabbage. Members of the genus lactobacillus are gram-positive bacteria, do not produce spores, have low tolerance to oxygen, are mostly anaerobic and unpowered, and are partly microaerophilic and powered. Under the condition of strict fermentation, lactobacillus can ferment from carbohydrate to produce lactic acid as main final product, so that the strain has better acid tolerance and partial strain has acidophilic property. Because of this property of lactobacillus, they are often used in the food fermentation industry, such as in the manufacture of cheese, yogurt, and the like. In addition, lactobacillus is considered to be a non-pathogenic and food-grade safe genus, and many strains play an important role in food microbiology and human nutrition, and are used as probiotics.
Non-alcoholic fatty liver disease (NAFLD) is a global health problem due to the prevalence of obesity, and is also a major cause of chronic liver damage, whereas intestinal flora disorders may be one of the causes of NAFLD pathogenesis. In addition to the damage of intestinal mucosal barrier, intestinal epithelial permeability changes and increased intestinal endotoxemia caused by intestinal flora disorders, metabolites of intestinal bacteria play an important role. This has led to a great interest in probiotic supplements as potential treatments for NAFLD, as probiotic supplements can delay or even reverse intestinal dysbacteriosis, restoring normal intestinal flora. The probiotics have better tolerance and safety in treating NAFLD, and the probiotic therapy taking intestinal flora as a target point is likely to be an important measure for treating NAFLD in the future.
The invention aims at separating and screening new lactobacillus species from the excrement of Qinghai wild woodchuck which only ingests plants, and the application of the new lactobacillus species in preparing medicaments for treating liver diseases.
Disclosure of Invention
Based on the above object, the present invention provides an application of lactobacillus probiotic bacterial strain in preparing liver disease therapeutic drugs, wherein the probiotic bacterial strain has a preservation number of CGMCC No.1.13855, a preservation date of 2021, 12 months and 16 days, and is classified and named Xu Jianguo lactobacillus (Lactobacillus xujianguonis), the preservation unit is China general microbiological culture center, and the address is the national institute of microbiology, national academy of sciences, 3, of North Chen West road 1, beijing, chao, and the post code is 100101.
In a preferred embodiment, the 16S rDNA of the strain has the sequence shown in SEQ ID NO: 1.
In a more preferred embodiment, the liver disease is liver dysfunction due to a lipid metabolism disorder.
In another more preferred embodiment, the liver disease is hepatitis and/or liver fibrosis caused by inflammatory factors.
More preferably, the inflammatory factor is interleukin 1-beta or serum monocyte chemotactic protein-1.
In yet a more preferred embodiment, the liver disease is serum endotoxin induced liver injury.
In a preferred embodiment, the liver disease therapeutic agent is also used as both a lipid-lowering agent and a diabetes therapeutic agent.
In a more preferred embodiment, the diabetes treatment agent is an agent that reduces the glucose level in human serum, an agent that reduces human serum insulin, or an agent that reduces inflammatory factors associated with insulin resistance in human serum.
In a preferred embodiment, the lactobacillus probiotic strain is prepared as a capsule, a lyophilized powder or a liquid formulation.
The invention separates and purifies lactobacillus with probiotic property from wild woodchuck faeces, and experiments prove that the separated lactobacillus is harmless to animals, and the lactobacillus has the effect of protecting liver function through animal experiments. The CGMCC No.1.13855 strain can obviously reduce serum glutamic pyruvic transaminase, serum glutamic pyruvic transaminase and liver malondialdehyde, raise the functions of liver superoxide dismutase and liver glutathione peroxidase, and has similar or even stronger liver protection effect with simvastatin. The physical sign of the rat which ingests the lactobacillus is normal, and the gastrointestinal liver and spleen are taken as pathology after the experiment is finished after five weeks of ingestion, so that the abnormality is not seen. The bacteria detection of the aseptic tissue does not see the ectopic field planting of the lactobacillus.
Drawings
FIG. 1. Phylogenetic tree analysis (maximum likelihood method) of CGMCC No.1.13855 strain based on 16S rDNA gene;
FIG. 2 effect of CGMCC No.1.13855 strain on serum Total Cholesterol (TC) of high-fat model rats;
FIG. 3 effect of CGMCC No.1.13855 strain on serum Triglyceride (TG) of high-fat model rats;
FIG. 4 effects of CGMCC No.1.13855 strain on serum Low Density Lipoprotein (LDL) of high fat model rats;
FIG. 5 effects of CGMCC No.1.13855 strain on serum High Density Lipoprotein (HDL) of a high fat model rat;
FIG. 6 effect of CGMCC 1.13855 strain on liver fat droplet content of high fat model rats (oil red O staining);
FIG. 7 effects of CGMCC 1.13855 strain on serum glutamic pyruvic transaminase (ALT) of a high-fat model rat;
FIG. 8 effects of CGMCC 1.13855 strain on serum glutamic-oxaloacetic transaminase (AST) of a high-fat model rat;
FIG. 9 effect of CGMCC 1.13855 strain on hepatic Malondialdehyde (MDA) of high-fat model rats;
FIG. 10. Influence of CGMCC 1.13855 strain on liver superoxide dismutase (SOD) of high-fat model rat;
FIG. 11 effect of CGMCC 1.13855 strain on liver glutathione peroxidase (GSH-Px) of high fat model rats;
FIG. 12 effects of CGMCC 1.13855 strain on serum interleukin 1-beta (IL-1 beta) of high-fat model rats;
FIG. 13 effect of CGMCC 1.13855 strain on serum monocyte chemotactic protein-1 (MCP-1) of high-fat model rats;
FIG. 14 effect of CGMCC 1.13855 strain on serum Endotoxin (ET) of high fat model rats;
FIG. 15. Influence of CGMCC 1.13855 strain on fasting blood glucose of a high-fat model rat;
FIG. 16 effect of CGMCC 1.13855 strain on fasting serum insulin in a high-fat model rat;
FIG. 17 effect of CGMCC 1.13855 strain on serum tumor necrosis factor-alpha (TNF-alpha) of high-fat model rats;
FIG. 18 effect of CGMCC 1.13855 strain on serum interleukin 6 (IL-6) of high-fat model rats.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way.
EXAMPLE 1 isolation, screening and identification of lactic acid bacteria
1. Isolation of lactic acid bacteria
1) From the bacteria-retaining tubeTaking out 100 μl of sample, adding into EP tube preloaded with 900 μl of sterile PBS, sequentially performing gradient dilution on the sample, and diluting woodchuck faeces sample to 10 -6 Doubling;
2) 100 mu L of samples with different dilutions are coated on MRS culture medium and put into an incubator;
3) At 37℃0.5% CO 2 Culturing in the environment for 48h;
4) Taking out the culture dish, picking colonies with different forms of characteristics by using a sterile inoculating loop, transferring to a new MRS solid culture medium for purification, performing anaerobic culture at 37 ℃ for 48 hours, continuously transferring for 3 times, culturing the purified strain in liquid MRS with pH of=3.5, and screening strains with excellent acid resistance growth for experiment or freezing preservation.
2. Preservation of bacterial species
The laboratory uses MRS culture medium containing 25% glycerol as bacteria-preserving liquid to carry out the freezing preservation of strains, and the method is as follows:
1) Sterilizing 2mL of bacteria-retaining tube at 121deg.C for 15 min;
2) After lactobacillus is continuously transferred on MRS solid culture medium for 3 times, 1.5ml of aseptic bacteria-preserving liquid is added on a culture dish;
3) Scraping and coating the culture dish by using an L rod to fully integrate bacterial colonies into the bacteria-retaining liquid;
4) Transferring the bacterial liquid into a bacteria-preserving tube, uniformly mixing and preserving at-80 ℃.
3. Colony appearance and bacterial morphology observations
Lactobacillus HT111-2 with the preservation number of CGMCC No.1.13855 belongs to anaerobic bacteria, grows well under anaerobic conditions, forms light yellow colonies with round shape, irregular edge, raised middle and rough surface on a Columbia blood culture medium, and has the diameter of about 1-1.2 mm; does not grow under aerobic conditions. Under an optical microscope, the thalli are light purple after gram staining, and can be arranged end to end. The thallus is rod-shaped under a transmission electron microscope, has no spores, no flagella, no bacteria and no power, and has a diameter of about 0.7-1 multiplied by 2.5-4.28 mu m.
4. Extraction of total DNA from bacteria
Single colonies were inoculated on MRS medium, cultured overnight at 37℃and DNA was extracted according to the bacterial genomic DNA extraction kit (TIANGEN) protocol.
5. Biochemical identification method of strain
The present study uses the "BioMerieux" bacterial System Biochemical identification cards API 50CH and API ZYM, manufactured by Merieux, france, to perform carbohydrate glycolysis and enzymatic biochemical identification of strain HT111-2 as well as of the reference strain.
Results: according to biochemical identification, a strain with biochemical characteristics of lactobacillus is obtained, the preservation number of the strain is CGMCC No.1.13855, the preservation date is 2021, 12 months and 16 days, the strain classification is Xu Jianguo lactobacillus (Lactobacillus xujianguonis), the preservation unit is China general microbiological culture Collection center, and the address is the institute of microbiological culture, the national academy of sciences, national institute of sciences, no. 3, north Chenxi, university, and the Beijing, korean, and the post code is 100101.
6. Bacterial universal primer 16S rRNA PCR amplification
Identification of bacterial 16S rRNA: extracting bacterial genome DNA, amplifying a lactobacillus universal primer 16S rDNA PCR product, sequencing, and performing BLAST comparison on the sequence on NCBI for preliminary identification.
The primers for PCR amplification of bacterial 16S rRNA and the PCR reaction conditions used in this experiment were as follows, and 50. Mu.L of PCR system was used for all experiments. And (3) carrying out BLAST comparison on the sequencing result of the lactobacillus universal primer 16S rRNA PCR product to be tested on NCBI, and identifying. The result suggests that the CGMCC No.1.13855 strain is a suspected new species of lactobacillus.
General primer 16S rRNA PCR amplification conditions:
primer sequence:
27F， 5'-AGAGTTTGATCMTGGCTCAG-3'；
1492R 5'-TACGGYTACCTTGTTACGACTT-3'；
reaction system (50 μl):
；
amplification conditions:
。
7. phylogenetic analysis
According to the 16S rDNA gene of the strain with the CGMCC No.1.13855 16S rDNA GenBank 31 mode as a reference sequence, the 16S rDNA gene sequence of the Enterococcus ATCC 19434 is used as an outer group, the MEGA 6.0 software is utilized, the CLUSTAL_W is adopted for carrying out multi-sequence comparison, and a system evolutionary tree is constructed based on a maximum likelihood method MaximumLikelihood (ML). The phylogenetic tree constructed based on the 16S rDNA gene sequence shows that the strain CGMCC No.1.13855 is clustered with adjacent lactobacillus, and an independent branch is formed on the phylogenetic tree, which is most similar to the evolutionary relationship of L.amylolyticus DSM 11664 and L.armteri DSM 5661, and is shown in the figure 1 (maximum likelihood method). The full genome frame map sequence of the strain CGMCC No.1.13855 is obtained by second generation sequencing, and hybridized with the genome of the reference strain L.amylolyticus DSM 11664 and L.hamsteri DSM 5661 on line, the DDH values are respectively 20.6 percent and 21.1 percent, and the DDH values are respectively hybridized with the genome sequences of 175 mode strains in the lactobacillus genus recorded in a GenBank database on line, and the result shows that the DDH value range is 17.2-42.5 percent and is lower than 70 percent, thereby meeting the judging standard of new species.
In conclusion, genome and phylogenetic analysis suggest that the strain CGMCC No.1.13855 is a novel species of Lactobacillus.
Example 2 hypolipidemic function of Lactobacillus CGMCC No.1.13855 on rat serum
32 female SD rats of about 200 g body weight were randomly divided into four groups, one group was given normal maintenance feed, and three groups were given high fat feed (cholesterol 1%, lard 10%,0.2 cholate, 10% yolk powder). Three groups of high fat diet groups were orally administered 10 a week after one week on alternate days 9 CFU CGMCC No.1.13855 lactobacillus liquid, physiological saline is administered every other day, simvastatin with the weight of 20mg/kg is administered every other day three weeks before the experiment is terminated, the experiment is terminated for five weeks, and the blood lipid level of serum is detected to evaluate the lactobacillus CGMPreventive intervention effect of CC No.1.13855 on lipid metabolism disorder model.
FIG. 2 shows CGMCC No.1.13855 T Effects on serum Total Cholesterol (TC) in high-fat model rats. The average content of serum TC in CGMCC No.1.13855 intervention group (namely HT111-2 intervention group) and simvastatin intervention group is 1.79+/-0.18 mmol/L and 1.77+/-0.08 mmol/L which are obviously lower than that in high-fat model group (p)<0.001 A) is provided; the average content of serum TC in the high-fat model group is 2.48+/-0.23 mmol/L, which is obviously higher than that in the normal control group by 1.40+/-0.10 mmol/L (p)<0.001）。
FIG. 3 shows the effect of CGMCC No.1.13855 on serum Triglyceride (TG) of a high-fat model rat. The average content of serum TG in the CGMCC No.1.13855 intervention group (namely HT111-2 intervention group) and the simvastatin intervention group is 2.55+/-0.17 mmol/L and 2.55+/-0.08 mmol/L, which are obviously lower than that in the high-fat model group (p < 0.001); the average content of serum TG in the high-fat model group is 3.07+/-0.15 mmol/L, which is obviously higher than that in the normal control group by 1.87+/-0.09 mmol/L (p < 0.001).
FIG. 4 shows the effect of CGMCC No.1.13855 on serum Low Density Lipoprotein (LDL) of high fat model rats. The average content of serum LDL in the CGMCC No.1.13855 intervention group (namely HT111-2 intervention group) and the simvastatin intervention group is 2.60+/-0.15 mmol/L and 2.17+/-0.32 mmol/L, which are obviously lower than that of the high-fat model group (p <0.01 and p < 0.001); the average content of serum LDL in the high-fat model group is 3.15+/-0.24 mmol/L, which is obviously higher than that in the normal control group by 1.79+/-0.26 mmol/L (p < 0.001).
FIG. 5 shows the effect of CGMCC No.1.13855 on serum High Density Lipoprotein (HDL) of a high lipid model rat. The average serum HDL content of the CGMCC No.1.13855 intervention group (namely HT111-2 intervention group) and the simvastatin intervention group is 2.10 plus or minus 0.22mmol/L and 2.33 plus or minus 0.17mmol/L, which are obviously higher than that of the high-fat model group by 1.51 plus or minus 0.08mmol/L (p <0.01 and p < 0.001); the average serum HDL content of the high-fat model group is 1.51+/-0.08 mmol/L, which is obviously lower than that of the normal control group by 3.10+/-0.41 mmol/L (p < 0.001).
FIG. 6 shows the effect of CGMCC No.1.13855 strain on liver fat droplet content (oil red O staining) of high fat model rats. Normal control oil red O staining showed no lipid droplets in the cytoplasm. The high-fat model group oil red O staining showed a large number of lipid droplets within the cytoplasm. The CGMCC No.1.13855 intervention group (i.e. HT111-2 intervention group) and simvastatin intervention group were stained with oil red O showing lipid droplets in the cytoplasm, but significantly less than the high-lipid model group.
The results show that the CGMCC No.1.13855 lactobacillus strain has the functions of obviously reducing serum total cholesterol, triglyceride, low density lipoprotein and increasing high density lipoprotein, can reduce the fat content of liver, and has similar blood fat reducing effect as simvastatin.
Rats ingested with CGMCC No.1.13855 lactobacillus appeared normally, and no abnormality was seen in taking gastrointestinal liver and spleen as pathology after five weeks of ingestion ended the experiment. The bacteria detection of the aseptic tissue does not see the ectopic field planting of the lactobacillus.
EXAMPLE 3 protective Effect of Lactobacillus CGMCC No.1.13855 on liver function of rat
32 female SD rats of about 200 g body weight were randomly divided into four groups, one group was given normal maintenance feed, and three groups were given high fat feed (cholesterol 1%, lard 10%,0.2 cholate, 10% yolk powder). Three groups of high fat diet groups were orally administered 10 a week after one week on alternate days 9 The method comprises the steps of (1) CFU CGMCC No.1.13855 lactobacillus bacterial liquid, administering physiological saline every other day, administering 20mg/kg body weight of simvastatin every other day three weeks before the experiment is terminated, terminating the experiment for five weeks, and detecting the levels of glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase and inflammatory factors of serum to evaluate the preventive intervention effect of the lactobacillus CGMCC No.1.13855 on liver function injury caused by lipid metabolism disorder models.
FIG. 7 shows the effect of CGMCC No.1.13855 on serum glutamic pyruvic transaminase (ALT) of a high-fat model rat. The average serum ALT content of the CGMCC No.1.13855 intervention group (namely HT111-2 intervention group) and the simvastatin intervention group is 1.05+/-0.09 ng/mL and 1.34+/-0.04 ng/mL, which are obviously lower than that of the high-fat model group (p < 0.001); serum ALT average content of the high-fat model group is 1.66+/-0.09 ng/mL, which is obviously higher than that of the normal control group by 0.94+/-0.05 ng/mL (p < 0.001).
FIG. 8 shows the effect of CGMCC No.1.13855 on serum glutamic-oxaloacetic transaminase (AST) of a high-fat model rat. The average serum AST content of the CGMCC No.1.13855 intervention group (namely HT111-2 intervention group) and the simvastatin intervention group is 1.67+/-0.26 ng/mL and 1.91+/-0.34 ng/mL, which are obviously lower than that of the high-fat model group (p < 0.001); serum AST average content of the high-fat model group is 2.70+/-0.12 ng/mL, which is obviously higher than that of the normal control group by 1.51+/-0.27 ng/mL (p < 0.001).
Figure 9 shows the effect of CGMCC No.1.13855 on hepatic Malondialdehyde (MDA) in high lipid model rats. The average content of liver MDA in CGMCC No.1.13855 intervention group (namely HT111-2 intervention group) and simvastatin intervention group is 3.12 plus or minus 0.38nmol/mL and 3.45 plus or minus 0.49nmol/mL, which are obviously lower than that of high-fat model group 4.54 plus or minus 0.20nmol/mL (p <0.01 and p < 0.001); the average content of liver MDA in the high-fat model group is 4.54+/-0.20 nmol/mL, which is obviously higher than that in the normal control group by 2.63+/-0.49 nmol/mL (p < 0.001).
FIG. 10 shows the effect of CGMCC No.1.13855 on liver superoxide dismutase (SOD) of a high-lipid model rat. The average content of liver SOD in CGMCC No.1.13855 intervention group (namely HT111-2 intervention group) and simvastatin intervention group is 1.28+/-0.07 ng/mL and 1.18+/-0.14 ng/mL, which are both obviously higher than that in high-fat model group by 0.90+/-0.05 ng/mL (p < 0.001); the average liver SOD content of the high-fat model group is 0.90+/-0.05 ng/mL, which is obviously lower than that of the normal control group by 1.32+/-0.06 ng/mL (p < 0.001).
FIG. 11 shows the effect of CGMCC No.1.13855 on liver glutathione peroxidase (GSH-Px) of a high fat model rat. The average content of liver GSH-Px of CGMCC No.1.13855 intervention group (namely HT111-2 intervention group) and simvastatin intervention group is 7.71+/-1.08 ng/mL and 7.80+/-0.63 ng/mL, which are obviously higher than that of high-fat model group 4.31+/-0.41 ng/mL (p < 0.001); the average liver GSH-Px content of the high fat model group is 4.31+/-0.41 ng/mL, which is obviously lower than that of the normal control group by 8.90+/-0.48 ng/mL (p < 0.001).
The results show that the CGMCC No.1.13855 lactobacillus strain has the effects of obviously reducing serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase and liver malondialdehyde, and simultaneously increases the effects of liver superoxide dismutase and liver glutathione peroxidase, and has the similar effect of protecting liver function with simvastatin.
EXAMPLE 4 liver inflammation and liver fibrosis-related inflammatory factor reduction function of Lactobacillus CGMCC No.1.13855 on rat serum
FIG. 12 shows the effect of CGMCC No.1.13855 on serum interleukin 1-beta (IL-1 beta) of a high-fat model rat. The average content of serum IL-1 beta in CGMCC No.1.13855 intervention group (namely HT111-2 intervention group) and simvastatin intervention group is 20.29+/-1.88 pg/mL and 17.98+/-1.16 pg/mL, which are obviously lower than that in a high-fat model group (p < 0.001); the average content of serum IL-1 beta in the high-fat model group is 28.40+/-1.96 pg/mL, which is obviously higher than that in the normal control group, namely 20.06+/-3.81 pg/mL (p < 0.001).
FIG. 13 shows the effect of CGMCC No.1.13855 on serum monocyte chemotactic protein-1 (MCP-1) of a high-fat model rat. The average content of serum MCP-1 in the CGMCC No.1.13855 intervention group (namely HT111-2 intervention group) is 536.98 +/-104.31 pg/mL, which is obviously lower than 733.69 +/-40.18 pg/mL (p < 0.001) of the high-fat model group; the average content of serum MCP-1 in the simvastatin intervention group is 628.89 +/-52.98 pg/mL, and has no obvious difference (p is more than 0.05) with 733.69 +/-40.18 pg/mL of the high-fat model group; the average serum MCP-1 content of the high-fat model group is 733.69 +/-40.18 pg/mL, which is obviously higher than that of the normal control group 527.28 +/-72.42 pg/mL (p < 0.001).
The results show that the CGMCC No.1.13855 lactobacillus strain has the effect of obviously reducing serum IL-1 beta and MCP-1 inflammatory factors and has stronger effect of reducing serum inflammatory factors than simvastatin. Liver fibrosis is the final outcome of the continued development of liver injury from a variety of causes, and MCP-l is an important macrophage chemokine in tissue injury repair, which plays an important role in liver injury and repair. IL-1β is another important inflammatory cell that causes fibrosis, is the primary inducer of the pro-inflammatory response, and can enhance the extent of the inflammatory response by coordinating and promoting the expression of other inflammatory factors, such as TNF- α. In summary, MCP-l and IL-1β are important inflammatory factors in liver fibrosis. The CGMCC No.1.13855 lactobacillus obviously reduces the content of serum IL-1 beta and MCP-1 inflammatory factors, thereby reducing the occurrence of liver inflammation and liver fibrosis.
EXAMPLE 5 endotoxin-reducing function of Lactobacillus CGMCC No.1.13855 on rat serum
Figure 14 shows the effect of CGMCC 1.13855 on serum Endotoxin (ET) of high fat model rats. The average content of serum ET in the CGMCC 1.13855 intervention group (namely HT111-2 intervention group) is 36.14 +/-5.59 pg/mL, which is obviously lower than 53.95+/-6.28 pg/mL (p < 0.001) of the high-fat model group; the average serum ET content of the simvastatin intervention group is 44.89+/-4.90 pg/mL, and has no obvious difference (p is more than 0.05) with 53.95+/-6.28 pg/mL of the high-fat model group; the average serum ET content of the high fat model group is 53.95+/-6.28 pg/mL, which is obviously higher than that of the normal control group which is 25.25+/-6.26 pg/mL (p < 0.001).
The results show that the CGMCC No.1.13855 lactobacillus strain has the effect of obviously reducing serum Endotoxin (ET), thereby reducing the liver injury degree and having stronger effect of reducing the serum Endotoxin (ET) than simvastatin.
Rats ingested with CGMCC No.1.13855 lactobacillus appeared normally, and no abnormality was seen in taking gastrointestinal liver and spleen as pathology after five weeks of ingestion ended the experiment. The bacteria detection of the aseptic tissue does not see the ectopic field planting of the lactobacillus.
EXAMPLE 6 in vivo evaluation of hypoglycemic Functions of Lactobacillus CGMCC No.1.13855
32 female SD rats of about 200 g body weight were randomly divided into four groups, one group was given normal maintenance feed, and three groups were given high fat feed (cholesterol 1%, lard 10%,0.2 cholate, 10% yolk powder). Three groups of high fat diet groups were orally administered 10 a week after one week on alternate days 9 The CFU CGMCC No.1.13855 lactobacillus liquid is administered with physiological saline every other day, and with simvastatin with the weight of 20mg/kg every other day three weeks before the termination of the experiment, and the blood sugar and inflammatory factor level of the fasting serum of the rat are detected to evaluate the preventive intervention effect of the lactobacillus CGMCC No.1.13855 on the metabolic disorder model.
1. The lactobacillus CGMCC No.1.13855 has the function of reducing fasting blood glucose and blood insulin of rats
Figure 15 shows the effect of CGMCC No.1.13855 on fasting blood glucose in a high-fat model rat. The average blood sugar content of the CGMCC No.1.13855 intervention group (namely HT111-2 intervention group) and the simvastatin intervention group is 3.30+/-0.78 mmol/L and 2.91+/-0.72 mmol/L, which are obviously lower than that of the high-fat model group (p < 0.001); the average blood sugar content of the high-fat model group is 5.09+/-0.29 mmol/L, which is obviously higher than that of the normal control group by 2.67+/-0.41 mmol/L (p < 0.001).
Figure 16 shows the effect of CGMCC No.1.13855 on fasting serum insulin in a high-fat model rat. The average serum insulin content of CGMCC No.1.13855 intervention group (namely HT111-2 intervention group) and simvastatin intervention group is 31.45 + -1.77 mU/L and 36.81+ -2.23 mU/L, which are obviously lower than that of high-fat model group 44.84+ -2.23 mU/L (p < 0.001); the average serum insulin content of the high-fat model group is 44.84+/-2.23 mU/L, which is obviously higher than that of the normal control group by 25.33+/-3.04 mU/L (p < 0.001).
The results show that the CGMCC No.1.13855 lactobacillus strain has the effect of obviously reducing blood sugar and serum insulin and has similar blood sugar reducing effect as simvastatin. Lipid metabolism disorders such as hypertriglyceridemia and hyperfree fatty acidemia are closely related to insulin resistance. Insulin resistance reduces the efficiency of insulin in promoting glucose uptake and utilization, and the body's compensatory hypersecretion of insulin produces hyperinsulinemia to maintain blood glucose stability. Insulin resistance is prone to metabolic syndrome and type 2 diabetes. The lactobacillus strain CGMCC No.1.13855 has the function of reducing blood fat, thereby reducing insulin resistance and leading to the reduction of blood sugar and serum insulin.
EXAMPLE 7 insulin resistance action-related inflammatory factor reducing function of Lactobacillus CGMCC No.1.13855 on rat serum
FIG. 17 shows the effect of CGMCC No.1.13855 on serum tumor necrosis factor-alpha (TNF-alpha) of a high-fat model rat. The average content of serum TNF-alpha in the CGMCC No.1.13855 intervention group (namely HT111-2 intervention group) and the simvastatin intervention group is 112.54 +/-20.53 pg/mL and 159.75 +/-7.95 pg/mL, which are obviously lower than 198.5+/-13.94 pg/mL (p < 0.001) of the high-fat model group; the average content of the serum TNF-alpha in the high-fat model group is 198.5+/-13.94 pg/mL, which is obviously higher than that in the normal control group 105.44 +/-25.22 pg/mL (p < 0.001).
FIG. 18 shows the effect of CGMCC No.1.13855 on serum interleukin 6 (IL-6) of a high-fat model rat. The average serum IL-6 content of the CGMCC No.1.13855 intervention group (namely HT111-2 intervention group) and the simvastatin intervention group is 115.43 +/-9.20 pg/mL and 132.24 +/-9.94 pg/mL, which are obviously lower than 155.93 +/-7.55 pg/mL (p <0.001 and p < 0.01) of the high-fat model group; the average content of serum IL-6 in the high-fat model group is 155.93 +/-7.55 pg/mL, which is obviously higher than 114.42 +/-8.90 pg/mL (p < 0.001) of the normal control group.
The results show that the CGMCC No.1.13855 lactobacillus strain has the effects of obviously reducing serum tumor necrosis factor-alpha and IL-6 inflammatory factors and reducing serum inflammatory factors more than simvastatin. Because the production of insulin resistance is closely related to serum inflammatory factor levels, long-term over-secretion of serum inflammatory factors may be a major factor in impaired insulin secretion by islet beta cells and in the development of insulin resistance. TNF- α and IL-6, as the most representative cytokines, play a central regulatory role in inflammation and immune responses. Elevated serum levels of TNF- α and IL-6 can induce insulin resistance by interfering with insulin signaling. The CGMCC No.1.13855 lactobacillus strain obviously reduces the content of inflammatory factors of TNF-alpha and IL-6 in serum, thereby reducing the insulin resistance effect and causing the blood sugar to be reduced.
Rats ingested with CGMCC No.1.13855 lactobacillus appeared normally, and no abnormality was seen in taking gastrointestinal liver and spleen as pathology after five weeks of ingestion ended the experiment. The bacteria detection of the aseptic tissue does not see the ectopic field planting of the lactobacillus.
Sequence listing
<110> infectious disease prevention and control institute of China center for disease prevention and control
Application of <120> lactobacillus probiotics CGMCC No.1.13855 in preparing liver disease treatment medicine
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1527
<212> DNA
<213> Lactobacillus xujianguonis
<400> 1
agagtttgat catggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60
gagcagaact aacagattta cttcggtaat gacgtttcgg acgcgagcgg cggatgggtg 120
agtaacacgt gggtaacctg cccttaagtc tgggatacca cttggaaaca ggtgctaata 180
ccggataaca actagtgctg catggcacta gcttaaaagg cggcgtaagc tgtcgctaaa 240
ggatggaccc gcggtgcatt agctagttgg taaggtaacg gcttaccaag gcgacgatgc 300
atagccgagt tgagagactg atcggccaca ttgggactga gacacggccc aaactcctac 360
gggaggcagc agtagggaat cttccacaat gggcgaaagc ctgatggagc aacgccgcgt 420
gagtgaagaa ggttttcgga tcgtaaagct ctgttgttgg tgaagaagga tagaggtagt 480
aactggcctt tatttgacgg taatcaacca gaaagtcacg gctaactacg tgccagcagc 540
cgcggtaata cgtaggtggc aagcgttgtc cggatttatt gggcgtaaag cgagcgcagg 600
cggagaaata agtctgatgt gaaagccctc ggcttaaccg aggaagtgca tcagaactgt 660
ttttcttgag tcagaagagg agagtgaact ccatgtgtag cggtggaatg cgtagatata 720
tggaagaaca ccagtggcga aggcggctct ctggtctgta actgacgctg aggctcgaaa 780
gcatgggtag cgaacaggat tagataccct ggtagtccat gccgtaaacg atgagtgcta 840
agtgttggga ggtttccgcc tctcagtgct gcagctaacg cattaagcac tccgcctggg 900
gagtacgacc gcaaggttga aactcaaagg aattgacggg ggcccgcaca agcggtggag 960
catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat ctggtgcaaa 1020
cctaagagat taggcgttcc cttcggggac accaagacag gtggtgcatg gctgtcgtca 1080
gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg ttattagttg 1140
ccagcattaa gttgggcact ctaatgagac tgccggtgac aaaccggagg aaggtgggga 1200
cggcgtcaag tcatcatgcc ccttatgacc tgggctacac acgtgctaca atgggcagta 1260
caacgaggag cgaacctgtg aaggcaagcg aatctctgaa agctgttctc agttcggact 1320
gtaggctgca actcgcctac acgaagctgg aatcgctagt aatcgcggat cagcacgccg 1380
cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccatggaa gtctgcaatg 1440
cccaaagccg gtggcctaac cttcgggaag gagccgtcta aggcagggca gatgactggg 1500
gtgaagtcgt aacaaggtaa ccgtaaa 1527
Claims (4)
1. The application of lactobacillus probiotic strain in preparing liver disease treating medicine is characterized in that the probiotic strain has the preservation number of CGMCC No.1.13855, the preservation date of 2021, 12 and 16 days and the preservation classification of Xu Jianguo lactobacillusLactobacillus xujianguonis) The preservation unit is China general microbiological culture Collection center, and the liver disease is liver dysfunction caused by lipid metabolism disorder.
2. The use according to claim 1, wherein the liver disease treatment drug is also a lipid lowering drug and a diabetes treatment drug.
3. The use according to claim 2, wherein the diabetes treatment agent is an agent that reduces the glucose content in human serum, an agent that reduces human serum insulin, or an agent that reduces an inflammatory factor in human serum that is associated with insulin resistance, which is TNF- α and/or IL-6.
4. The use according to claim 1, wherein the lactobacillus probiotic strain is prepared as a capsule, a lyophilized powder or a liquid formulation.
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