Application of Bifidobacterium adolescentis and its metabolites in the preparation of drugs for improving liver damage

Abstract

The present invention belongs to the field of biomedical technology, and specifically relates to the use of Bifidobacterium adolescentis and its metabolites in the preparation of drugs for improving liver damage. The present invention provides the use of Bifidobacterium adolescentis and its metabolites in the preparation of drugs for improving liver damage. The classification and name of the Bifidobacterium adolescentis are: Bifidobacterium adolescentis, the deposit unit is: China General Microbiological Culture Collection Management Center, and the deposit number is: CGMCC 1.2190. The present invention has found that Bifidobacterium adolescentis CGMCC 1.2190 has a significant improvement effect on acetaminophen-induced liver damage, can significantly reduce ALT and AST levels, and reduce the levels of proinflammatory cytokines TNF-α, IL-6, MCP-1 and MCP-3. Its metabolite erythrine acts as an effective ingredient.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to the application of Bifidobacterium adolescentis and its metabolites in the preparation of a drug for improving liver damage. Background Art

[0002] The liver contains a special enzyme system with detoxification function, and is also an endocrine control station that can degrade many hormones that pass through the liver blood flow. During the entire life process of the human body, certain foreign substances (such as poisons, drugs, carcinogens) often enter the body, and some biologically active substances and metabolites (hormones, amines, etc.) are constantly produced in the body during the metabolism process. Most of these foreign and endogenous substances are metabolized and transformed in the liver. Any process that destroys the stability of the liver's internal environment may cause liver cell damage, thereby affecting the normal physiological function of the liver.

[0003] Acute liver injury refers to abnormal liver function caused by various reasons. Liver injury is the basis of acute liver failure. Severe or persistent liver injury will eventually lead to liver failure. Acute liver injury is a special critical state between deterioration and recovery. If diagnosed and treated in time, it is expected to be cured; otherwise, it may deteriorate or even cause liver failure, causing serious damage to the body. Therefore, the diagnosis and treatment of acute liver injury is extremely important. In recent years, the study of the mechanism of liver injury, prevention and treatment has become one of the hot topics in biological and medical research at home and abroad. Acetaminophen (APAP) is a common clinical drug for the treatment of fever and pain, and is considered safe at therapeutic doses. However, high doses of APAP can cause acute liver injury (ALI), and severe ALI can develop into acute liver failure (ALF), and even lead to death.

[0004] Chinese invention patent application CN114568703A discloses a new use of a strain of Bifidobacterium lactis, specifically a new use of Bifidobacterium lactis for preventing or alleviating acute alcoholic liver injury; the strain is Bifidobacterium lactis TY-S01 (Bifidobacterium lactis TY-S01), and the Bifidobacterium lactis TY-S01 provided by the invention with a deposit number of CGMCC No.21255 can prevent or alleviate acute alcoholic liver injury, has no side effects and has good effects.

[0005] Another Chinese invention patent application CN110354148A discloses the use of Bifidobacterium adolescentis CCFM1061 in the preparation of functional bacterial agents, foods and or medicines. The Bifidobacterium adolescentis CCFM1061 of the invention can quickly colonize in the intestines, significantly improve fasting blood sugar and oral glucose tolerance of type 2 diabetes induced by STZ and high-fat diet, and reduce the area under the curve of glucose tolerance; significantly improve the increase of total cholesterol and decrease of high-density lipoprotein cholesterol in serum caused by STZ and high-fat diet in type 2 diabetes; can significantly improve the insulin resistance of type 2 diabetes; can significantly improve the level of inflammation in liver tissue of type 2 diabetes; and significantly improve the pathological damage of pancreas, liver and other tissues of type 2 diabetes. In addition, Bifidobacterium adolescentis CCFM1061 has a strong adsorption capacity for perfluorooctanoic acid (PFOA) and has the ability to alleviate the toxicity of PFOA.

[0006] However, the prior art does not report the use of Bifidobacterium adolescentis and its metabolites in the preparation of drugs for improving liver damage. Summary of the invention

[0007] The present invention aims at the problems existing in the prior art and provides the use of Bifidobacterium adolescentis and its metabolites in the preparation of a drug for improving liver damage.

[0008] The use of Bifidobacterium adolescentis and its metabolites in the preparation of drugs for improving liver damage. The classification name of the Bifidobacterium adolescentis is: Bifidobacterium adolescentis, the preservation unit is: China General Microbiological Culture Collection Administration Center, and the preservation number is: CGMCC 1.2190.

[0009] Preferably, the liver injury comprises acute liver injury.

[0010] Preferably, the acute liver injury comprises drug-induced liver injury induced by acetaminophen.

[0011] Preferably, the bacterial content of Bifidobacterium adolescentis is 2×10 8 -3×10 8 CFU / mL.

[0012] Preferably, the metabolite comprises erythrine.

[0013] Preferably, the Bifidobacterium adolescentis and its metabolites are capable of reducing ALT and / or AST levels.

[0014] Preferably, the Bifidobacterium adolescentis and its metabolites can reduce the levels of pro-inflammatory cytokines TNF-α, IL-6, MCP-1 and / or MCP-3.

[0015] Preferably, the drug also includes other drugs and pharmaceutically acceptable carriers.

[0016] Preferably, the pharmaceutical carrier comprises at least one of lactose, glucose, sucrose, sorbitol, trehalose, starch, gum arabic, alginate, gelatin, calcium silicate, cellulose, polyvinyl pyrrolidone, methyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0017] Preferably, the drug is in the form of powder, capsule, tablet or oral solution.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The present invention found that Bifidobacterium adolescentis CGMCC 1.2190 significantly improved acetaminophen-induced liver damage and had a positive effect on inhibiting cell apoptosis and improving acute liver damage.

[0020] (2) The present invention found that Bifidobacterium adolescentis CGMCC 1.2190 can significantly reduce the levels of ALT and AST, and reduce the levels of proinflammatory cytokines TNF-α, IL-6, MCP-1 and MCP-3.

[0021] (3) The present invention also found that erythrine, a metabolite of Bifidobacterium adolescentis CGMCC 1.2190, plays a role as an active ingredient in acetaminophen-induced liver damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figure shows the difference between the food intake and body weight of mice (n=7-8).

[0023] Figure 2 ALT and AST levels in mouse serum (n=6, ***p<0.001).

[0024] Figure 3 H&E staining images of mouse liver tissue (n=6, dotted line indicates central lobular tissue necrosis, scale bar=100 μm).

[0025] Figure 4 The figure is a statistical diagram of the necrosis area of ​​mouse liver cells (n=6, ***p<0.001).

[0026] Figure 5 TUNEL staining images of mouse liver tissue (n=4-6, red indicates apoptotic cells, scale bar=100 μm).

[0027] Figure 6 The figure is a statistical diagram of the number of apoptotic hepatocytes in mice (n=4-6, **p<0.01, ***p<0.001).

[0028] Figure 7This is an IHC staining image of the inflammatory protein CD11b (n=5, scale bar=100 μm).

[0029] Figure 8 The statistical diagram of the positive expression area of ​​inflammatory protein CD11b (n=5, *p<0.05, ***p<0.001).

[0030] Fig. 9 This is an IHC staining image of the inflammatory protein TNF-α (n=5, scale bar=100 μm).

[0031] Fig.10 The figure is a statistical diagram of the positive expression area of ​​inflammatory protein TNF-α (n=5, **p<0.01).

[0032] Fig.11 The figure is a statistical graph of ELISA results in mouse serum (n=6, **p<0.01, ***p<0.001).

[0033] Fig.12 This is an α diversity analysis diagram (n=16, **p<0.01, ***p<0.001).

[0034] Fig.13 This is a β diversity analysis diagram (n=16).

[0035] Fig.14 This is the relative abundance diagram of bacterial communities at the phylum level. (n=16)

[0036] Fig.15 The relative abundance quantitative graph of Bifidobacterium adolescentis CGMCC 1.2190 (n=7-8, *p<0.05).

[0037] Fig.16 The PLS-DA analysis of mouse cecal contents based on UPLC-MS (n=7-8).

[0038] Fig.17 Volcano plot of differential metabolites (n=7-8).

[0039] Fig.18 Lollipop plots of fold differences and p-value changes of differential metabolites in mice (n=7-8).

[0040] Fig.19 The graph shows the erythrine content in the supernatant of Bifidobacterium adolescentis CGMCC 1.2190 (n=7-8, ***p<0.001).

[0041] Fig. 20 is the content of erythrine in the cecal contents of mice (n=6, *p<0.05).

[0042] Fig.21 The effect of Bifidobacterium adolescentis CGMCC 1.2190 on the content of erythrine in mouse plasma (n=6-7, *p<0.05).

[0043] Fig. 22 Flowchart of the in vivo experimental protocol for mice.

[0044] Fig.23 The graph shows the serum ALT and AST levels in mice (n=5-12, *p<0.05, **p<0.01, ***p<0.001).

[0045] Fig.24 H&E staining of mouse liver tissue and statistical diagram of hepatocyte necrosis area (scale bar = 100 μm, n = 5-7, **p < 0.01, ***p < 0.001).

[0046] Fig.25 TUNEL staining of mouse liver tissue and statistical diagram of hepatocyte apoptosis (scale bar = 100 μm, n = 5-7, **p < 0.01, ***p < 0.001).

[0047] Fig.26 Statistical graph of mouse serum ELISA test results (n=5-8, *p<0.05, **p<0.01, ***p<0.001).

[0048] Fig. 27 The relative viability of primary hepatocytes (n=6).

[0049] Fig.28 The graph shows the amount of LDH released from primary hepatocytes (n=5-6; *p<0.05; **p<0.01; ***p<0.001). DETAILED DESCRIPTION

[0050] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0051] It is worth noting that the raw materials used in the present invention are all common commercially available products, among which Bifidobacterium adolescentis CGMCC1.2190 was purchased from China General Microbiological Culture Collection Center; acetaminophen was purchased from Shanghai McLean Biochemical Technology Co., Ltd.; ALT or AST kit was purchased from Nanjing Jiancheng Bioengineering Institute; MRS broth liquid culture medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.; TUNEL detection kit was purchased from Jiangsu Keygen Biotechnology Co., Ltd.; DAB color developer was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; mouse fecal DNA extraction kit was purchased from Guangzhou Maibao Biotechnology Co., Ltd.

[0052] Example 1 Improvement effect of Bifidobacterium adolescentis CGMCC 1.2190 on acetaminophen-induced acute liver injury in mice

[0053] 1. Experimental Materials

[0054] 1.1 Mice: 8-week-old C57BL / 6 male mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd., animal license: SCXK (Liao) 2020-0001, body weight 22-24g, raised in the SPF-level experimental animal environment of Guangdong Pharmaceutical University, with an optimal temperature of 21±1.5℃ and an environmental relative humidity of 55±5%. Mice were given free access to water and mouse maintenance feed.

[0055] 1.2 Bifidobacterium adolescentis CGMCC 1.2190 (B.adolescentis) strain: The B.adolescentis strain was revived in MRS broth liquid culture medium, placed in anaerobic bags and anaerobic bags, and cultured in a 37°C incubator for 48-72 hours. When the OD value measured at a wavelength of 600nm reached about 0.8, the bacteria were counted. Subsequently, 1mL of 10% glycerol was added to the sample, and the sample was packaged and frozen at -80°C. Before use, the counted and packaged B.adolescentis bacterial solution was taken out for thawing, and the bacterial solution was placed in a centrifuge and centrifuged at 8000rpm for 5 minutes. The supernatant was discarded, and the bacterial precipitate was retained. 1mL of sterile PBS solution was added for elution, and finally sterile PBS solution (phosphate buffered saline) was added to dilute it to a bacterial content of 2×10 8 CFU / mL.

[0056] 2. Animal Grouping

[0057] Thirty-two C57BL / 6 mice were weighed and randomly divided into four groups: PBS control group, B. adolescentis group, APAP (acetaminophen) model group and B. adolescentis + APAP group.

[0058] 3. Method of administration

[0059] The B.adolescentis group and the B.adolescentis+APAP group were given 200 μL of 2×108 CFU / mouse Bifidobacterium adolescentis CGMCC 1.2190 (B.adolescentis) bacterial solution by gavage every day, and the other two groups were given the same volume of sterile PBS solution as a control every day for 7 days. At 8:00 pm on the 7th day of gavage with Bifidobacterium adolescentis CGMCC 1.2190, the APAP group and the B.adolescentis+APAP group were given APAP at a dose of 300 mg / kg by gavage.

[0060] 4. Body Weight and Food Intake Experiment

[0061] Experimental results and data analysis: The results are as follows Figure 1 As shown, compared with the PBS control group, the mice in the B. adolescentis group did not show significant differences in body weight and food intake, indicating that the intake of Bifidobacterium adolescentis CGMCC 1.2190 had no significant effect on the daily growth and energy metabolism of mice.

[0062] 5. Serum ALT and AST activity assay

[0063] 5.1 Experimental method: ALT and AST activities were detected in the sera of mice in the PBS control group, B.adolescentis group, APAP model group and B.adolescentis+APAP group. The ALT and AST detection methods were as follows:

[0064] Using the ALT or AST kit, add 200 μL R1 reagent, 50 μL R2 reagent and 10 μL serum sample to a 96-well plate, use an ELISA reader to read the absorbance at a wavelength of 340 nm at 1 min, 2 min and 3 min of incubation, and calculate the sample ALT and AST values ​​according to the formula in the instruction manual.

[0065] 5.2 Experimental results and data analysis: The results are as follows Figure 2 As shown in the data, compared with the PBS control group, the levels of ALT and AST in the serum of mice in the APAP model group were significantly increased; compared with the APAP model group, the levels of ALT and AST in the B. adolescentis + APAP group were significantly decreased (p < 0.001), indicating that liver damage was significantly alleviated, and Bifidobacterium adolescentis CGMCC 1.2190 had an improving effect on the liver function of mice with acute liver injury (AILI).

[0066] 6. H&E staining experiment

[0067] 6.1 Experimental methods: Hematoxylin-eosin (H&E) staining experiments were performed on the liver tissues of mice in the PBS control group, B. adolescentis group, APAP model group, and B. adolescentis + APAP group. The specific steps are as follows:

[0068] Fresh mouse livers were fixed with 4% paraformaldehyde for 36-48 hours, dehydrated, transparentized, paraffin-impregnated and embedded, and then prepared into 5 μm thick paraffin sections, which were dried at 60°C for 1 hour.

[0069] (1) Tissue dewaxing: xylene for 3 min × 3 times.

[0070] (2) Tissue rehydration: anhydrous ethanol for 2 min × 2 times, 95% ethanol for 1 min, 90% ethanol for 1 min, 80% ethanol for 1 min, and drain for 30 s.

[0071] (3) Staining cell nuclei: hematoxylin for 10 min, ultrapure water for 5 s, and tap water for 10 min.

[0072] (4) Differentiation: 1% hydrochloric acid alcohol for 10 seconds, rinse with tap water for 10 minutes.

[0073] (5) Cytoplasm staining: stain with eosin solution for 3 minutes.

[0074] (6) Dehydration: 80% ethanol for 10 s, 90% ethanol for 3 s, 95% ethanol for 2 min, and anhydrous ethanol for 3 min × 3 times.

[0075] (7) Permeabilization: xylene for 3 min × 3 times.

[0076] (8) Sealing: Add an appropriate amount of neutral resin to seal the slices, let them dry naturally, and then take a picture. For each slice, 6-8 fields of view were randomly photographed under an optical microscope, and the liver injury area was counted using Image J software.

[0077] 6.2 Experimental results and data analysis: H&E staining results are as follows Figure 3 As shown in the figure, the liver structure of mice in the PBS control group was clear and no obvious damage was observed. However, the liver of mice in the APAP model group showed extensive inflammatory cell infiltration and severe liver necrosis, with obvious central lobular tissue necrosis (indicated by dotted lines), blurred liver tissue contours, and loss of normal morphological structure. Figure 4 It can be seen that compared with the APAP model group, the liver damage area in the B. adolescentis + APAP group was significantly reduced (p < 0.001), indicating that Bifidobacterium adolescentis CGMCC 1.2190 has a significant reversal effect on AILI.

[0078] 7. TUNEL staining experiment

[0079] 7.1 Experimental method: TUNEL staining experiment was performed on the liver tissues of mice in the PBS control group, B.adolescentis group, APAP model group and B.adolescentis+APAP group using TUNEL detection kit. The specific steps are as follows:

[0080] (1) Dewax and rehydrate the paraffin sections using xylene and anhydrous ethanol.

[0081] (2) Add 100 μL of Proteinase K working solution to each sample, place in a wet box, and incubate at 37°C for 30 min. After incubation, wash with PBS three times, 5 min each time.

[0082] (3) Add 50 μL of TdT enzyme reaction solution to each sample and incubate in a humidified chamber at 37°C in the dark for 60 min. Wash with PBS three times, 5 min each time.

[0083] (4) Add 50 μL of Streptavidin-TRICT labeling solution to each sample and incubate in a humidified chamber at 37°C in the dark for 60 min. Wash with PBS three times, 5 min each time.

[0084] (5) Counterstain the cell nucleus with 50 μL of DAPI staining solution and react at room temperature in the dark for 10 min. Wash again with PBS three times and seal the slide with glycerol.

[0085] The apoptotic staining was observed under a fluorescence microscope (excitation wavelength 543 nm, emission wavelength 571 nm). 6-8 fields of view were randomly photographed for each slice, and the apoptotic cells in the tissue were statistically analyzed using Image J software.

[0086] 7.2 Experimental results and data analysis: TUNEL staining results are as follows Figure 5 As shown, in the PBS control group and the B. adolescentis group, the number of apoptotic cells was very small or almost nonexistent (red indicates TUNEL-positive cells). Figure 6 It can be seen that in the APAP model group mice, the number of apoptotic cells increased significantly, showing a significant apoptosis phenomenon. However, when the mice were treated with Bifidobacterium adolescentis CGMCC 1.2190, the number of apoptotic cells in the B. adolescentis + APAP group was significantly reduced (p < 0.01), proving that Bifidobacterium adolescentis CGMCC 1.2190 has a positive effect on inhibiting apoptosis and improving acute liver damage.

[0087] 8. Detection of positive expression of inflammatory proteins CD11b and TNF-α

[0088] 8.1 Experimental methods: The positive expression of inflammatory proteins CD11b and TNF-α was detected in the sera of mice in the PBS control group, B.adolescentis group, APAP model group and B.adolescentis+APAP group. The specific steps are as follows:

[0089] (1) Prepare in advance 10 mM sodium citrate containing 0.5% Tween 20 and a pH of 6.0.

[0090] (2) Dewax and rehydrate the tissue sections and soak them in PBS solution for 5 min.

[0091] (3) Soak the slices in sodium citrate, seal with plastic wrap and poke small holes in them.

[0092] (4) Antigen repair: Place the beaker in a microwave oven and heat until boiling. After boiling, take it out and let it cool naturally to room temperature.

[0093] (5) Take out the slide, circle the tissue with an immunohistochemistry pen, and wash twice with PBS solution, each time for 5 minutes.

[0094] (6) Endogenous blocking: Add 3% H2O2 to block for 20 min, and wash with PBS solution three times, each time for 5 min.

[0095] (7) Exogenous blocking: Add 5% BSA blocking solution and incubate for 10 min.

[0096] (8) Add primary antibody (CD11b, TNF-α antibody) and incubate at 4°C overnight.

[0097] (9) Recover the primary antibody, add the secondary antibody (HRP-labeled goat anti-rabbit IgG), incubate at 37°C for 20 min, and wash three times with PBS solution.

[0098] (10) Color development: Add DAB color developer to each slide, keep track of time, and protect from light. Determine the staining time based on the color change observed under a microscope.

[0099] (11) Finally, the slides were restained, dehydrated, and sealed.

[0100] 8.2 Experimental results and data analysis: Compared with the APAP model group, the positive expression area of ​​CD11b in the liver of mice in the B. adolescentis + APAP group treated with Bifidobacterium adolescentis CGMCC 1.2190 was significantly reduced (p < 0.05) ( Figure 7-8 ), the positive expression area of ​​TNF-α was significantly reduced (p<0.01)( Figure 9-10 ), this result intuitively demonstrated the effective reduction of inflammatory response, indicating that Bifidobacterium adolescentis CGMCC 1.2190 has the effect of improving APAP-induced acute liver injury.

[0101] 9. Experiments to detect the levels of inflammatory factors TNF-α, IL-6, MCP-1 and MCP-3

[0102] 9.1 Experimental method: ELISA kits were used to detect inflammatory cytokines in the peripheral blood serum of mice in the PBS control group, B.adolescentis group, APAP model group and B.adolescentis+APAP group. The kit catalog numbers are as follows: TNF-α (Xinbosheng Company, catalog number: EMC102a), IL-6 (Xinbosheng Company, catalog number: EMC004), MCP-1 (Xinbosheng Company, catalog number: EMC113) and MCP-3 (Huamei Company, catalog number: E07426m). The specific steps are as follows:

[0103] Use the universal diluent for standards and specimens to dilute the mouse serum 30 times, and dilute the lyophilized standard into different gradients of standard solution to prepare blank control wells, test sample wells, and standard wells. Add the sample diluent, test sample, and standard solution in order to ensure that the volume added to each well is consistent. After sealing the plate, place the plate in a 37°C incubator and incubate for 90 minutes in the dark. After the incubation, shake off the liquid in the well, wash the plate with washing solution, and repeat 5 times. Add 100μL of the pre-configured biotinylated antibody working solution to each well, place the plate in a 37°C incubator again, and incubate for 60 minutes in the dark. Shake off the biotinylated antibody working solution and wash the plate with washing solution, and repeat 5 times. Add 100μL of enzyme binding working solution to each well, except for the blank well. Place the plate in a 37°C incubator and incubate for 30 minutes in the dark. Wash the plate again with washing solution, and repeat 5 times. Add colorimetric reagent A and colorimetric reagent B to each well in turn, ensuring that they are fully mixed. Place the plate in a 37°C incubator and incubate for 15 minutes away from light. Finally, add 50 μL of the reaction stop solution to each well to terminate the reaction. Immediately use an ELISA reader to measure the OD value of each well at a wavelength of 450 nm. According to the standard curve, the concentrations of cytokines TNF-α, IL-6, MCP-1, and MCP-3 in mouse peripheral blood were calculated.

[0104] 9.2 Experimental results and data analysis: The results are as follows Fig.11 As shown in the figure, the expression level of inflammatory chemokines in the APAP model group increased significantly, which directly reflected the aggravation of liver inflammation. However, after the mice were treated with Bifidobacterium adolescentis CGMCC 1.2190, the expression level of inflammatory factors in the B. adolescentis + APAP group decreased significantly. Although the decrease in inflammatory factor IL-6 was not significant (p>0.05), the significant decrease in inflammatory factor TNF-α (p<0.01) indicated that Bifidobacterium adolescentis CGMCC1.2190 effectively inhibited the inflammatory response in mice to a certain extent, while the inflammatory factors MCP-1 and MCP-3 were significantly reduced (p<0.001), which further proved the positive role of Bifidobacterium adolescentis CGMCC 1.2190 in inhibiting monocyte infiltration and reducing liver inflammation.

[0105] Example 2 Bifidobacterium adolescentis CGMCC 1.2190 directly produces erythrine (HYP)

[0106] 1. Experimental Materials

[0107] 1.1 Mice: 8-week-old C57BL / 6 male mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd., animal license: SCXK (Liao) 2020-0001, body weight 22-24g, raised in the SPF-level experimental animal environment of Guangdong Pharmaceutical University, with an optimal temperature of 21±1.5℃ and an environmental relative humidity of 55±5%. Mice were given free access to water and mouse maintenance feed.

[0108] 1.2 Bifidobacterium adolescentis CGMCC 1.2190 (B.adolescentis) strain: The B.adolescentis strain was revived in MRS broth liquid culture medium, placed in anaerobic bags and anaerobic bags, and cultured in a 37°C incubator for 48-72 hours. When the OD value measured at a wavelength of 600nm reached about 0.8, the bacteria were counted. Subsequently, 1mL of 10% glycerol was added to the sample, and the sample was packaged and frozen at -80°C. Before use, the counted and packaged B.adolescentis bacterial solution was taken out for thawing, and the bacterial solution was placed in a centrifuge and centrifuged at 8000rpm for 5 minutes. The supernatant was discarded, and the bacterial precipitate was retained. 1mL of sterile PBS solution was added for elution, and finally sterile PBS solution (phosphate buffered saline) was added to dilute it to a bacterial content of 2×10 8 CFU / mL.

[0109] 2. Animal Grouping

[0110] Eight C57BL / 6 mice were weighed and randomly divided into two groups: a PBS control group and a B. adolescentis + APAP group.

[0111] 3. Method of administration

[0112] Mice in the B. adolescentis + APAP group were intragastrically administered with 200 μL of 2×10 8 CFU / mouse B.adolescentis liquid, the PBS control group was gavaged with 200 μL PBS every day for seven days. At 8:00 pm on the seventh day, the B.adolescentis+APAP group was gavaged with 300 mg / kg APA.

[0113] 4.16S rRNA sequencing technology to detect changes in rat intestinal flora

[0114] 4.1 Experimental methods: The cecal contents of mice in the PBS control group and the B. adolescentis + APAP group were sent to Tianjin Novogene Company for 250 bp fragment paired-end high-throughput sequencing using the Illumina Hiseq 2500 platform.

[0115] Statistical analysis: Data were processed by Tianjin Novogene using the QIIME1.9.1 platform.

[0116] 4.2 Experimental results and data analysis: The Shannon index is used to measure species diversity. The larger the Shannon index value, the higher the species diversity.

[0117] 4.2.1α Diversity Analysis: Results Fig.12 As shown, although there was no significant difference in the Shannon index (p>0.05), there were significant changes in the Faith's PD index (p<0.001) and the Observed Features index (p<0.01).

[0118] 4.2.2β diversity analysis: Results are as follows Fig.13 As shown, principal coordinates analysis (PCoA) based on the Bray Curtis distance matrix showed that there were significant differences in the composition of the intestinal microbiota between the PBS control group and the B. adolescentis group treated mice.

[0119] 4.2.3 Analysis of differences at the phylum level: Results Fig.14 As shown, the abundance of Bacteroidetes was higher in the B. adolescentis group of mice treated with B. adolescentis CGMCC 1.2190 compared with the PBS control group.

[0120] 4.2.4qRT-PCR analysis: Results Fig.15 As shown, the abundance of B. adolescentis in mice treated with Bifidobacterium adolescentis CGMCC 1.2190 was significantly increased (p < 0.05).

[0121] In summary, it was preliminarily inferred that Bifidobacterium adolescentis CGMCC 1.2190 had been successfully colonized in mice.

[0122] 5. Non-targeted metabolomics research

[0123] 5.1 Experimental methods: The cecal contents of mice in the PBS control group and B. adolescentis + APAP group were sent to BGI for non-targeted metabolomics testing.

[0124] 5.2 Experimental results and data analysis: The thresholds are set to: VIP ≥ 1, Fold Change ≥ 1.2 or ≤ 0.83, p < 0.05, and the results are as follows Fig.16 As shown, significant differences in the metabolite composition were found between the B. adolescentis group treated with B. adolescentis CGMCC 1.2190 and the PBS control group.

[0125] Depend on Fig.17 It can be seen that compared with the PBS control group, the contents of 52 metabolites in the B. adolescentis group were significantly increased, while the contents of 49 metabolites were significantly decreased.

[0126] Depend on Fig.18 It can be seen that among these differential metabolites, dethiobiotin, N-acetylleucylleucine and hypaphorine are the three most significantly upregulated metabolites.

[0127] In addition, a large number of studies have confirmed that erythrine has antioxidant and anti-inflammatory properties. Therefore, it is speculated that erythrine may serve as an important protective metabolite and play a key role in the protective effect of Bifidobacterium adolescentis CGMCC 1.2190 against APAP-induced liver injury.

[0128] 6. Evaluation of the Ability of Bifidobacterium adolescentis CGMCC 1.2190 to Produce Erythrine

[0129] 6.1 Experimental method: Collect Bifidobacterium adolescentis CGMCC 1.2190 in MRS medium, culture at 37°C for 48h, and then centrifuge at 8000rpm for 5min to obtain bacterial supernatant. Extract with bacterial supernatant: methanol at a volume ratio of 1:10, centrifuge at 13000rpm for 10min at 4°C, aspirate the supernatant, dry in a vacuum container, and reconstitute with methanol. The mobile phase is acetonitrile and 0.1% formic acid water at a volume ratio of 10:90, the flow rate is 1.0mL / min, and the isocratic elution is 15min.

[0130] 6.2 Experimental results and data analysis: Fig.19 It can be seen that the content of erythrine in the culture supernatant of Bifidobacterium adolescentis CGMCC 1.2190 treated B. adolescentis group was significantly increased compared with that in the culture medium MRS group (p < 0.001).

[0131] Depend on Figure 20-21 It can be seen that the levels of erythrine in the plasma and cecal contents of the mice in the B. adolescentis group also showed an upward trend compared with the culture medium MRS group (p < 0.05).

[0132] Based on the above experimental results, it was shown that erythrine metabolites may play a role as potential active ingredients in the protection of Bifidobacterium adolescentis CGMCC 1.2190 against APAP-induced acute liver injury.

[0133] Example 3 Effect of erythrine (HYP) on the improvement of acute liver injury induced by acetaminophen in mice

[0134] 1. In vivo experiments

[0135] 1.1 Experimental Materials

[0136] Mice: 8-week-old C57BL / 6 male mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd., animal license: SCXK (Liao) 2020-0001, body weight 22-24g, raised in the SPF-level experimental animal environment of Guangdong Pharmaceutical University, with an optimal temperature of 21±1.5℃ and an environmental relative humidity of 55±5%. Mice were free to drink water and eat mouse maintenance feed.

[0137] Erythrinaine (HYP): derived from Example 2.

[0138] 1.2 Animal grouping: Thirty-two C57BL / 6 mice were weighed and randomly divided into four groups: PBS control group, HYP group, APAP model group and HYP+APAP group.

[0139] 1.3 Administration method: The mice in the HYP group and the HYP+APAP group were intraperitoneally injected with HYP at a dose of 10 mg / kg at 18:00 on the same day. The HYP+APAP group and the APAP group were treated with 300 mg / kg APAP at 20:00 on the same day, and the other two groups were treated with the same volume of PBS solution (phosphate buffered saline).

[0140] 1.4 Serum ALT and AST activity assay

[0141] 1.4.1 Experimental method: ALT and AST activities were detected in the mouse sera of the PBS control group, HYP group, APAP model group and HYP+APAP group, respectively. The specific steps were the same as those in Example 1.

[0142] The experimental process is as follows Fig. 22 As shown, specifically: mice in the HYP group and the HYP+APAP group were intraperitoneally injected with HYP at a dose of 10 mg / kg at 18:00 on the same day. The HYP+APAP group and the APAP group were treated with 300 mg / kg APAP at 20:00 on the same day, and the other two groups were treated with the same volume of PBS solution (phosphate buffered saline). After 24 hours, the liver, cecum and serum of the mice were collected.

[0143] 1.4.2 Experimental results and data analysis: The results are as follows Fig.23 As shown, compared with the mice in the APAP model group, the ALT and AST levels in the serum of the mice pretreated with erythrinain (HYP) in the HYP+APAP group were significantly decreased (p < 0.01), indicating the protective effect of erythrinain on liver function.

[0144] 1.5 H&E staining experiment

[0145] 1.5.1 Experimental method: Hematoxylin-eosin (H&E) staining experiments were performed on the liver tissues of mice in the PBS control group, HYP group, APAP model group and HYP+APAP group. The specific steps were the same as those in Example 1.

[0146] 1.5.2 Experimental results and data analysis: The results are as follows Fig.24 As shown, erythrinain treatment significantly reduced the necrotic area around the central vein of the liver in the APAP-treated mice (p < 0.01), confirming the improvement effect of erythrinain on liver injury.

[0147] 1.6 TUNEL staining experiment

[0148] 1.6.1 Experimental method: TUNEL staining experiments were performed on the liver tissues of mice in the PBS control group, HYP group, APAP model group and HYP+APAP group. The specific steps were the same as those in Example 1.

[0149] 1.6.2 Experimental results and data analysis: The results are as follows Fig.25 As shown, erythrine significantly reduced APAP-induced hepatocyte apoptosis (p < 0.01), indicating that it plays a protective role in cell apoptosis.

[0150] 1.7 Detection of inflammatory factors TNF-α, IL-6, MCP-1 and MCP-3 expression

[0151] 1.7.1 Experimental method: The positive expression of inflammatory protein CD11b and TNF-α was detected in the mouse sera of the PBS control group, HYP group, APAP model group and HYP+APAP group. The specific steps were the same as those in Example 1.

[0152] 1.7.2 Experimental results and data analysis: The results are as follows Fig.26 As shown in the results, the levels of inflammatory factors such as IL-6, MCP-1 and MCP-3 in the serum of mice pretreated with erythrina alkali were significantly reduced (p < 0.05). Although the decrease in TNF-α was relatively small, it also indicated that erythrina alkali may alleviate liver damage by regulating the inflammatory response.

[0153] 2. In vitro experiments

[0154] 2.1 Experimental method: The cells in the 96-well plate were divided into 5 groups: PBS control group, APAP model group, 5μM HYP+APAP group, 2.5μM HYP+APAP group, and 1μM HYP+APAP group, where the APAP concentration was 5mM. The supernatant was discarded, and 100μL of the pre-prepared working solution was added to each well of the drug-treated groups with different concentrations. The PBS control group and the APAP model group were given 100μL of culture medium, respectively, and cultured in a 37℃ incubator for 2h, and then 100μL of the pre-prepared 5μM HYP+APAP, 2.5μM HYP+APAP, and 1μM HYP+APAP working solutions were added to each well and cultured in a 37℃ incubator for 24h.

[0155] (1) CCK-8 detection (Mei Lun Bio, catalog number: MA0218): After taking the 96-well plate out of the incubator, aspirate 50 μL of supernatant from each well and transfer it to an empty 96-well plate for subsequent LDH release detection. At the same time, discard the remaining supernatant, add 100 μL of culture medium containing 10% CCK-8 to each well, incubate in a 37°C incubator for 0.5-4 hours, observe the degree of color development, and place the 96-well plate in an ELISA instrument to measure the absorbance at 450 nm. The cell viability calculation formula is as follows:

[0156] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0157] Where As is the absorbance of the experimental well (culture medium containing cells, CCK-8, and the drug to be tested); Ac is the absorbance of the control well (culture medium containing cells, CCK-8, and no drug to be tested); Ab is the absorbance of the blank well (culture medium without cells and the drug to be tested, CCK-8).

[0158] (2) LDH assay: Detect the supernatant reserved in the previous step by adding 50 μL of LDH working solution to each well and incubating in a 37°C incubator for 15-30 min. After observing the color development of each well in the 96-well plate, place the plate in an ELISA reader and measure the absorbance of each well at a wavelength of 490 nm and calculate according to the formula in the instruction manual.

[0159] 2.2 Experimental results and data analysis: The relative activity results of primary hepatocytes are shown in the figure below: Fig. 27 As shown, the results of LDH release in primary hepatocytes are shown in Fig.28 As shown, it can be seen that different concentrations of erythrina alkaloids (HYP) had no toxicity to the primary hepatocytes in the APAP model group, but significantly improved the cell viability, confirming the positive effect of erythrina alkaloids in alleviating AILI.

[0160] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. Use of Bifidobacterium adolescentis and its metabolites in the preparation of a drug for improving liver damage, characterized in that: The classification name of the Bifidobacterium adolescentis is Bifidobacterium adolescentis, the depository unit is China General Microbiological Culture Collection Center, the deposit number is CGMCC 1.2190, The liver injury is drug-induced liver injury induced by acetaminophen, and the metabolites include erythrine.

2. The use according to claim 1, characterized in that: The bacterial content of Bifidobacterium adolescentis is 2×10 8 -3×10 8 CFU / mL.

3. The use according to any one of claims 1-2, characterized in that: The Bifidobacterium adolescentis and its metabolites can reduce the level of ALT and / or AST.

4. The use according to claim 1, characterized in that: The bifidobacterium adolescentis and its metabolites can reduce the levels of pro-inflammatory cytokines TNF-α, IL-6, MCP-1 and / or MCP-3.

5. The use according to claim 1, characterized in that: The medicine also includes other drugs and pharmaceutically acceptable pharmaceutical carriers.

6. The use according to claim 5, characterized in that: The pharmaceutical carrier includes at least one of lactose, glucose, sucrose, sorbitol, trehalose, starch, gum arabic, alginate, gelatin, calcium silicate, cellulose, polyvinyl pyrrolidone, methyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

7. The use according to claim 1, characterized in that: The dosage form of the medicine is powder, capsule, tablet or oral solution.

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