CN116531413A - Application of lactobacillus metagen in preparation of medicine for preventing and/or treating alcoholic liver disease - Google Patents

Abstract

The invention provides an application of a lactobacillus metazoan in preparing a medicament for preventing and/or treating alcoholic liver disease, belonging to the technical field of biological medicaments. The lactic acid bacteria metazoan prepared by the invention does not contain any living bacteria, does not have the risks of microbial infection and translocation, has higher stability and safety compared with the living bacteria, has obvious advantages compared with other products for relieving Alcoholic Liver Disease (ALD), and has high application potential in the aspect of preventing and treating ALD. Meanwhile, experiments prove that the lactic acid bacteria metaplasia can effectively protect the liver injury mediated by alcohol and simultaneously relieve the lipid accumulation of liver cells mediated by alcohol. Therefore, the effect of the lactic acid bacteria metaplasia on relieving the alcoholic fatty liver disease is comprehensively obtained by detecting the mouse liver injury markers and pathological changes. The lactobacillus metazoan has high application potential in the aspect of ALD prevention and treatment, and can provide a new scheme for ALD prevention and treatment.

Description

Application of lactobacillus metagen in preparation of medicine for preventing and/or treating alcoholic liver disease

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of a lactobacillus metazoan in preparation of a medicine for preventing and/or treating alcoholic liver disease.

Background

Alcoholic liver disease (Alcoholic liver diseases, ALD) is a type of liver disease characterized by different clinical pathological changes such as fatty liver, hepatitis, cirrhosis, liver cancer, etc., and is mainly caused by excessive alcohol consumption. ALD is one of the most important liver disease burden worldwide and has also become a new public health problem in China. The products for relieving alcoholic liver disease on the market at present are mainly liver-protecting and gallbladder-benefiting series products such as silymarin and the like, have the characteristic of small toxic and side effects, and can be widely used in liver disease treatment. However, silymarin is a large amount of flavonoid glycoside mixture, and therefore, cannot be industrially synthesized, but depends on the extraction process, and obviously cannot meet the medical requirements.

The metazoan is an inanimate microorganism and/or a component thereof beneficial to host health, and has anti-inflammatory, immunomodulating, antiproliferative and antioxidant activities. The lactobacillus metazoan is one kind of metazoan obtained through compounding, mixing, sterilizing, cooling, inoculating one or several kinds of lactobacillus probiotics, fermenting, inactivating fermented liquid or solid, breaking cell wall, solid-liquid separation, adding carrier and drying. Lactic acid bacteria probiotics have been shown to have effects of regulating intestinal flora balance, anti-inflammatory, maintaining intestinal barrier, obesity, immunoregulation, hypertension, cardiovascular diseases and cancer, and are widely used for preventing and treating gastrointestinal diseases. However, there is no application study of the metazoan of lactic acid bacteria in the prevention and treatment of ALD.

Disclosure of Invention

In view of the above, the invention aims to provide an application of the lactobacillus metazoan in preparing a medicament for preventing and/or treating alcoholic liver disease, and the lactobacillus metazoan does not contain any living bacteria, so that the lactobacillus metazoan has the advantages of higher stability and safety, and has greater application potential in preventing and treating alcoholic fatty liver disease compared with the living bacteria.

The invention provides application of a lactobacillus metagen in preparation of a medicament for preventing and/or treating alcoholic liver disease, wherein the lactobacillus metagen is prepared by mixed fermentation of lactobacillus reuteri (Lactobacillus reuteri), lactobacillus acidophilus (Lactobacillus acidophilus) and lactobacillus rhamnosus (Lactobacillus rhamnosus).

Preferably, the preparation method of the lactobacillus metazoan comprises inoculating lactobacillus reuteri seed solution, lactobacillus acidophilus seed solution and lactobacillus rhamnosus seed solution into a metazoan preparation culture medium for culture to obtain a culture solution;

inoculating the culture solution into the metazoan preparation culture medium for fermentation culture to obtain fermentation liquor;

and inactivating the fermentation liquor and crushing the thalli to obtain the metazoan.

Preferably, the inoculation volume ratio of the lactobacillus reuteri seed solution to the lactobacillus acidophilus seed solution to the lactobacillus rhamnosus seed solution is (0.8-1.2): 0.8-1.2.

Preferably, the total inoculation amount of the lactobacillus reuteri seed solution, the lactobacillus acidophilus seed solution and the lactobacillus rhamnosus seed solution during the culture is 1.5-2.5%;

the culture time is 10-14 h, and the culture temperature is 37+/-1 ℃.

Preferably, the inoculation amount of the culture solution is 4% -6% during the fermentation culture;

the fermentation culture time is 20-23 h, and the fermentation culture temperature is 36-38 ℃.

Preferably, the method for inactivating the thalli is heat treatment;

the temperature of the heat treatment is 60-70 ℃, and the time of the heat treatment is 25-35 min.

Preferably, the method for crushing the thalli is ultrasonic crushing;

the power of the ultrasonic crushing is 40W, the total crushing time of the ultrasonic crushing is 14-16 min, and the intermittent time of the ultrasonic crushing is 4-5 s.

Preferably, the metazoan preparation medium is an aqueous solution containing the following components: 20g/L glucose, 10g/L peptone, 5g/L sodium acetate, 4g/L yeast extract, 2g/L ammonium citrate, 2g/LK ₂ HPO ₄ ·3H ₂ O, 0.2g/L magnesium sulfate, 0.1g/LMnSO ₄ ·H ₂ O and 0.05g/L manganese sulfate.

Preferably, the lactobacillus reuteri comprises lactobacillus reuteri DSM17938 strain;

the lactobacillus acidophilus comprises lactobacillus acidophilus CICC6096 strain;

the lactobacillus rhamnosus (Lactobacillus rhamnosus) is lactobacillus rhamnosus CICC6137 strain.

Preferably, the medicament has the effect of protecting the alcohol-mediated liver injury and alleviating alcohol-mediated hepatocyte lipid accumulation.

The invention provides application of a lactobacillus metagen in preparation of a medicament for preventing and/or treating alcoholic liver disease, wherein the lactobacillus metagen is prepared by mixed fermentation of lactobacillus reuteri (Lactobacillus reuteri), lactobacillus acidophilus (Lactobacillus acidophilus) and lactobacillus rhamnosus (Lactobacillus rhamnosus). In view of the fact that the lactobacillus metazoan does not contain any living bacteria, is free from the risks of microbial infection and translocation, has higher stability and safety compared with lactobacillus probiotics such as lactobacillus reuteri (Lactobacillus reuteri), lactobacillus acidophilus (Lactobacillus acidophilus) and lactobacillus rhamnosus (Lactobacillus rhamnosus), has obvious advantages compared with other products for relieving alcoholic liver disease, and has high application potential in the aspect of ALD prevention and treatment. Meanwhile, experiments prove that the lactic acid bacteria metaplasia can effectively protect the liver injury mediated by alcohol and simultaneously relieve the lipid accumulation of liver cells mediated by alcohol. Therefore, the effect of the lactic acid bacteria metaplasia on relieving the alcoholic fatty liver disease is comprehensively obtained by detecting the mouse liver injury markers and pathological changes. The lactobacillus metazoan has high application potential in the aspect of ALD prevention and treatment, and can provide a new scheme for ALD prevention and treatment.

Drawings

FIG. 1 is a morphology of a post-Lactobacillus prebiotic and a post-Lactobacillus prebiotic control formulation prepared according to the present invention;

FIG. 2 shows the results of live bacteria detection of the post-Lactobacillus prebiotics and the post-Lactobacillus prebiotic control formulation;

FIG. 3 shows the results of reducing inflammatory factors in serum and lipid accumulation in liver of ALD mice with lactic acid bacteria metazoans, wherein A and B: the lactic acid bacteria metagen relieves liver injury and liver lipid accumulation of ALD mice; c and D: lactic acid bacteria metazoans improve deregulated aspartate aminotransferase and alanine aminotransferase in serum of ALD mice; and (3) injection: AST: aspartate aminotransferase, ALT: alanine aminotransferase; e: TG content levels in serum of mice of each group; f: quantitative determination of oil red staining positive areas; ^* P＜0.05， ^** P＜0.01， ^*** p < 0.001, compared with the Control group; ^# P＜0.05， ^## p < 0.01, compared to EtOH group; ^$ P＜0.05， ^$$ p < 0.01, compared to the Vehicle group; control, normal group; eoTH group: a model group; vehicle treatment group: a lactic acid bacteria metazoan control formulation group; postbiotics group: a lactic acid bacteria metagroup;

FIG. 4 shows that the lactic acid bacteria metazoan effectively improved lipid accumulation in human hepatocytes (L-02) in an in vitro ALD model, wherein A: oil red dyeing results; b: TG content levels in cell supernatants of each group; c: quantitative determination of oil red staining positive areas; ^* p < 0.01, compared with the Control group; ^# P＜0.05， ^## p < 0.01, compared to EtOH group; ^$ P＜0.05， ^$$ p < 0.01, compared to the Vehicle group; control, normal group; eoTH group: a model group; vehicle group: lactic acid bacteriaMetazoan control formulation group; postbiotics group: lactic acid bacteria metagenome.

Fig. 5 is the results of the lactic acid bacteria compounding probiotics and lactic acid bacteria metazoans to alleviate alcohol-mediated liver damage, wherein a and B: preparing probiotics group and lactobacillus metazoan by compounding acid bacteria, and relieving liver injury and liver lipid accumulation of ALD mice; c and D: the lactic acid bacteria compound probiotics and the lactic acid bacteria metagen improve the maladjusted aspartate aminotransferase and alanine aminotransferase in the serum of the ALD mice; and (3) injection: AST: aspartate aminotransferase, ALT: alanine aminotransferase; e: TG content levels in serum of mice of each group; f: quantitative determination of oil red staining positive areas; ^* P＜0.05， ^** P＜0.01， ^*** p < 0.001, compared with the Control group; ^# P＜0.05， ^## p < 0.01, compared to EtOH group; ^$ P＜0.05， ^$$ p < 0.01, compared with the progenitics group; control, normal group; eoTH group: a model group; vehicle treatment group: a lactic acid bacteria metazoan control formulation group; probiotics group: compounding probiotics with lactobacillus; postbiotics group: a lactic acid bacteria metagroup;

Detailed Description

The invention provides application of a lactobacillus metagen in preparation of a medicament for preventing and/or treating alcoholic liver disease, wherein the lactobacillus metagen is prepared by mixed fermentation of lactobacillus reuteri (Lactobacillus reuteri), lactobacillus acidophilus (Lactobacillus acidophilus) and lactobacillus rhamnosus (Lactobacillus rhamnosus).

In the preparation method of the lactobacillus metazoan, lactobacillus reuteri seed solution, lactobacillus acidophilus seed solution and lactobacillus rhamnosus seed solution are preferably inoculated into a metazoan preparation culture medium for culture to obtain a culture solution;

inoculating the culture solution into the metazoan preparation culture medium for fermentation culture to obtain fermentation liquor;

and inactivating the fermentation liquor and crushing the thalli to obtain the metazoan.

In the present invention, the lactobacillus reuteri preferably comprises strain l.reuteri DSM17938 (Lactobacillus reuteri), purchased from biogaria; the lactobacillus acidophilus preferably comprises lactobacillus acidophilus CICC6096 strain (Lactobacillus acidophilus), and is preferably purchased from China industry microbiological culture Collection center; the lactobacillus rhamnosus (Lactobacillus rhamnosus) is preferably lactobacillus rhamnosus CICC6137 strain, and is preferably purchased from China industry microbiological culture Collection center. The preparation method of the lactobacillus reuteri seed solution, the lactobacillus acidophilus seed solution and the lactobacillus rhamnosus seed solution comprises the steps of inoculating strains of corresponding strains into an MRS culture medium, activating to the third generation, and collecting culture solutions to obtain seed solutions of the three strains. The activation temperature is preferably 36 to 38 ℃, more preferably 37 ℃. The activation time is preferably 10 to 14 hours, more preferably 12 hours.

In the present invention, the total inoculum size of the lactobacillus reuteri seed solution, lactobacillus acidophilus seed solution and lactobacillus rhamnosus seed solution in the culture is preferably 1.5 to 2.5%, more preferably 2.0%. The inoculation volume ratio of the lactobacillus reuteri seed solution to the lactobacillus acidophilus seed solution to the lactobacillus rhamnosus seed solution is preferably (0.8-1.2): 0.8-1.2, and more preferably 1:1:1. The time of the culture is preferably 10 to 14 hours, more preferably 12 hours. The temperature of the culture is preferably 36 to 38℃and more preferably 37 ℃. The metazoan preparation medium is preferably an aqueous solution containing the following components: 20g/L glucose, 10g/L peptone, 5g/L sodium acetate, 4g/L yeast extract, 2g/L ammonium citrate, 2g/LK ₂ HPO ₄ ·3H ₂ O, 0.2g/L magnesium sulfate, 0.1g/LMnSO ₄ ·H ₂ O and 0.05g/L manganese sulfate. The culture is beneficial to expanding the total amount of the fermentation agent so as to meet the inoculation requirement of the fermentation culture.

In the present invention, the inoculation amount of the culture medium at the time of the fermentation culture is preferably 4% to 6%, more preferably 5%. The fermentation time is preferably 20 to 23 hours, more preferably 22 hours. The temperature of the fermentation culture is preferably 36 to 38℃and more preferably 37 ℃. The fermentation culture is used for making lactobacillus reuteri, lactobacillus acidophilus and lactobacillus rhamnosus enter a secondary metabolite production stage, so as to obtain a large amount of active ingredients for preventing and treating alcoholic liver disease.

In the present invention, the method for inactivating the bacterial cells is preferably a heat treatment. The temperature of the heat treatment is preferably 60 to 70 ℃, more preferably 65 ℃. The time of the heat treatment is preferably 25 to 35 minutes, more preferably 30 minutes. The method for disrupting the cells is preferably ultrasonic disruption. The power of the ultrasonic crushing is preferably 40W, and the total crushing time of the ultrasonic crushing is preferably 14 to 16min, more preferably 15min. The intermittent time of the ultrasonic disruption is preferably 4 to 5 seconds. After the treatment solution after ultrasonic disruption is obtained, living bacteria detection is preferably performed. In the detection method, the treatment solution is preferably inoculated into MRS agar culture medium (1L of the culture medium contains the following components of 10g of peptone, 5g of beef powder, 20g of glucose, 4g of yeast powder, 0.2g of sodium acetate, 2g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate, 2g of tri-ammonium citrate, 0.05g of manganese sulfate, 1ml of Tween 80 and 15g of agar powder), and the plate result shows that the prepared metazoan does not contain any living bacteria.

In the present invention, the drug preferably has the effects of protecting the alcohol-mediated liver injury and alleviating alcohol-mediated hepatocyte lipid accumulation. The alcoholic liver disease preferably includes at least one of fatty liver, alcoholic liver disease and alcoholic liver cirrhosis.

In the embodiment of the invention, an alcoholic liver disease mouse model (ALD mouse) is taken as an experimental object, and after the injection of stomach, the injection of stomach is carried out, compared with a control preparation intervention group of a lactobacillus metagen, the lactobacillus metagen can obviously relieve the increase of liver index of the mouse induced by alcohol, simultaneously reduce the accumulation of lipid in the liver of the ALD mouse, and can also obviously reduce the levels of AST, ALT and TG in serum of the ALD mouse, so that the lactobacillus metagen can protect alcohol-mediated liver injury; compared with the intervention group of the lactobacillus metazoan control preparation, the lipid accumulation in the metazoan group L-02 liver cells is obviously reduced. Meanwhile, the invention also sets the L-02 cells to carry out the administration culture of the lactobacillus metagen while inducing by alcohol, and the result shows that the lactobacillus metagen can obviously reduce the accumulation of lipid and the TG level in the L-02 cells of the in-vitro ALD model.

The application of the lactobacillus metazoan provided by the present invention in preparing a medicament for preventing and/or treating alcoholic liver disease is described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Preparation method of lactobacillus metazoan

1 description of the Experimental Material Source

Lactobacillus acidophilus CICC6096 strain 6096 and Lactobacillus rhamnosus CICC strain 6137 were purchased from the China center for type culture Collection of microorganisms, strain Lactobacillus reuteri DSM17938, and from BioGaia.

The metazoan preparation medium is purchased from Beijing Soy Bao biological company;

bacterial microorganism incubator 303-5B was purchased from Shanghai Shang Yi instruments limited;

ultrasonic disruptors were purchased from Shanghai Michelson Biotechnology Co., ltd;

the freeze vacuum dryer was purchased from Sichuan Hengchuan Co-created new materials technology Co., ltd.

2 Experimental methods

The Lactobacillus acidophilus CICC6096 strain, lactobacillus rhamnosus CICC6137 strain and Lactobacillus reuteriDSM17938 strain were activated to the third generation in MRS medium, respectively. The seed solutions of the activated Lactobacillus acidophilus CICC6096 strain, lactobacillus rhamnosus CICC6137 strain and Lactobacillus reuteri DSM17938 strain were mixed at a ratio of 1:1:1 (v/v/v), and the mixed seed solution was inoculated to metazoan preparation medium (containing 20g glucose, 10g peptone, 5g sodium acetate, 4g yeast extract, 2g ammonium citrate, 2g K per liter of water) at 2% ₂ HPO ₄ ·3H ₂ O, 0.2g of magnesium sulfate, 0.1g of Mn SO ₄ ·H ₂ O and 0.05g of manganese sulfate) was cultured at 37℃for 12 hours. Inoculating the obtained culture solution into a metazoan preparation culture medium according to 5%, and fermenting and culturing for 21-25 h at 37 ℃. Performing heat treatment (65deg.C, 30 min), ultrasonic crushing (crushing power 40%, total crushing time 15min, intermittent time 4 s), lyophilizing (pre-freezing at-40deg.C for 2-3 hr, vacuum pumping to vacuum degree 5-10Pa, vacuum lyophilizing for 24-48 hr) to obtain the final productUntil the lactic acid bacteria metaplasia.

Example 2

Method for detecting viable bacteria in lactic acid bacteria metaplasia

The lactic acid bacteria metazoan obtained in the example were inoculated into MRS agar medium (peptone 10g, beef powder 5g, glucose 20g, yeast powder 4g, sodium acetate 0.2g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.2g, triammonium citrate 2g, manganese sulfate 0.05g, tween 80 1ml, agar powder 15g medium, and cultured at 37℃for 24 hours to observe whether colonies were produced on the plates.

Sterility checks ensure that the lactobacillus prebiotics do not contain live bacteria such as Lactobacillus acidophilus CICC6096, lactobacillus rhamnosus CICC6137 and Lactobacillus reuteri DSM17938 strains.

Comparative example 1

Preparation of Lactobacillus metazoan control preparation

MRS medium was inoculated at 2% to metazoan preparation medium (20 g glucose, 10g peptone, 5g sodium acetate, 4g yeast extract, 2g ammonium citrate, 2g K per liter of water) ₂ HPO ₄ ·3H ₂ O, 0.2g of magnesium sulfate, 0.1g of Mn SO ₄ ·H ₂ O and 0.05g of manganese sulfate) was cultured for 12 hours. Inoculating the obtained fermentation broth into a metazoan preparation culture medium according to 5%, and culturing for 22h. The fermentation broth was subjected to heat treatment (65 ℃ C., 30 min), ultrasonic crushing (crushing power 40%, total crushing time 15min, intermittent time 4 s), and freeze-drying to obtain a lactic acid bacteria metazoan control preparation. The resulting lactobacillus prebiotic control formulation was subjected to aseptic inspection as in example 2 to ensure that the lactobacillus prebiotic control formulation was free of viable bacteria.

Example 3

Relief of liver damage in ALD mice livers from Lactobacillus metazoans

1 Experimental materials and methods

1.1 Experimental materials

95% of the edible alcohol was purchased from Henan Xin Yang He wine industry Co., ltd, lieber-DeCarli liquid alcohol feed and control liquid feed were purchased from Nantong Telofei feed technologies Co., ltd. The glutamic-oxaloacetic transaminase (AST) and glutamic-pyruvic transaminase (ALT) biochemical detection kit is purchased from Nanjing building Limited liability company. SPF-grade C57BL/6 mice, 8 weeks old, body weight (18+ -2 g), purchased from Chongqing Tengxin Biotechnology Co.

1.2 Experimental methods

Establishment of 1.2.1ALD mouse model and different intervention treatment groups thereof

C57BL/6 mice ALD model and given Lactobacillus metazoan treatment were established according to the methods described in previous studies (Bertola, A., mathews, S., ki, S., wang, H., & Gao, B.J.N.p. (2013) & Mousemodel ofchronic andbinge ethanol feeding (the NIAAA model) & Nature Protocols,8 (3), 627-637.Https:// doi.org/10.1038/nprot.2013.032). Briefly, all C57BL/6 mice were housed in groups in an environment with controlled relative humidity (45% -65%), temperature (22.+ -. 2 ℃). All C57BL/6 mice were randomly divided into 4 groups of 7 by body weight:

(A) Normal group (4 weeks on Lieber-decrli liquid control diet);

(B) Alcohol feed group (Lieber-DeCarli liquid control feed was first used for 5 days to adapt to liquid diet, then Lieber-DeCarli alcohol feed containing 5% alcohol was used to continue feeding for 23 days, mice were subjected to high concentration alcohol gavage once at a concentration of 5g/kg body weight 9 hours before collecting specimens);

(C) A lactic acid bacteria metaplasia intervention group (feeding mode same as the model group, and feeding lactic acid bacteria metaplasia daily, wherein the concentration is 0.6g/kg for 4 weeks);

(D) The lactic acid bacteria metazoan control preparation intervenes in the group (the same feeding mode as the model group is adopted, and the concentration of the gastric lactic acid bacteria metazoan control preparation is 0.6g/kg for 4 weeks).

And collecting liver tissue and serum samples of mice in a normal group, an alcohol group, a lactobacillus metazoan intervention group and a lactobacillus metazoan control preparation intervention group, and respectively detecting changes of AST and ALT liver injury markers in serum by using an AST and ALT detection kit. The liver tissue frozen by liquid nitrogen is embedded by frozen tissue embedding agent (OCT), sliced by a frozen microtome, stained by oil red and hematoxylin, and then glycerogelatin sealing sheets and microscopic examination are carried out to observe the lipid accumulation condition in the liver tissue of mice in different treatment groups. Formalin-fixed liver tissue was dehydrated by different concentrations of alcohol gradient, xylene transparent, paraffin embedded, microtome sectioned, eosin and hematoxylin stained, then neutral resin sealed and microscopic observed for pathological changes in liver tissue in mice of different treatment groups. The effect of the lactic acid bacteria metaplasia on relieving the alcoholic fatty liver disease is comprehensively evaluated by detecting liver injury markers and pathological changes of mice in different treatment groups.

1.2.2 detection of liver injury markers and pathological changes

Taking out liver tissue and serum samples of mice in a normal group, an alcohol group, a lactic acid bacteria metazoan intervention group and a lactic acid bacteria metazoan control preparation intervention group collected in the section 1.2.1, respectively calculating liver indexes of the mice in different treatment groups, and respectively detecting changes of AST, ALT and TG liver injury markers in serum by using AST, ALT and TG biochemical detection kits.

The liver tissue frozen by liquid nitrogen is embedded by frozen tissue embedding agent (OCT), sliced by a frozen microtome, stained by oil red and hematoxylin, and then glycerogelatin sealing sheets and microscopic examination are carried out to observe the accumulation of lipid in the liver tissue of mice in different treatment groups.

Formalin-fixed liver tissue was dehydrated by different concentrations of alcohol gradient, xylene transparent, paraffin embedded, microtome sectioned, eosin and hematoxylin stained, then neutral resin sealed and microscopic observed for pathological changes in liver tissue in mice of different treatment groups.

2 experimental results

2.1 post-lactic acid accumulation metaplasia to alleviate alcohol-mediated liver injury

After 4 weeks of feeding with the alcohol feed, the liver index of the mice is obviously increased, the lipid accumulation in the liver is obviously increased, and the liver cell damage is obviously aggravated; AST, ALT and TG levels were significantly elevated in the blood of mice. Mice were perfused daily with lactic acid bacteria metazoan (0.6 g/kg concentration) while being induced with alcohol feed, and liver pathology was examined after 4 weeks, as shown in the results: the lactobacillus metazoan significantly alleviated the alcohol-induced liver index increase in mice while reducing lipid accumulation in the liver of ALD mice, and also significantly reduced AST, ALT and TG levels in serum of ALD mice (fig. 3 a-F).

Example 4

Alcohol-mediated hepatocyte lipid accumulation test for relief of metaplasia of lactobacillus

1 description of the Experimental Material Source

Human normal hepatocyte line L-02 was purchased from the China academy of sciences typical culture Collection Committee (Shanghai, china);

dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum were purchased from Thermo Fisher Scientific company;

penicillin-streptomycin mixed solution is purchased from Beijing Soy Bao technology Co., ltd, and absolute ethanol is purchased from Thermo Fisher Scientific company;

t25 cell culture flasks and 6-well cell culture plates were purchased from eppendorf corporation.

2 Experimental methods

2.1 establishment of ALD human L-02 cell model and different intervention treatment groupings

According to the experimental foundation of Zhang et al, a human L-02 cell ALD model is established and is subjected to the post-natal intervention of lactobacillus. Specifically, the experiments were divided into 4 groups:

(A) Control group: l-02 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics for 5d;

(B) Alcohol group: after L-02 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics for 96 hours, absolute ethanol (Sigma-Aldrich) was added to the filled L-02 cells DMEM medium at a final concentration of 150mM, and cultured for 48 hours at a fixed time daily, 5 PM;

(C) Lactic acid bacteria metaplasia intervention group: adding the final concentration of the lactobacillus metazoan of 10mg/mL into an L-02 cell DMEM medium and culturing for 96 hours, then replacing the medium with 300mM absolute ethyl alcohol medium, and keeping for 48 hours at 5 pm for a fixed time every day;

(D) Control group of lactobacillus metazoan preparation: the final concentration of the 10mg/mL lactic acid bacteria metazoan control preparation was added to the L-02 cell DMEM medium and cultured for 96 hours, then replaced with 300mM absolute ethanol medium, fixed time per day, 5 pm, for 48 hours. At least three independent experiments were performed throughout the course of the study.

2.2 detection of lipid levels in L-02 cells

After L-02 cells cultured in 6 wells were treated by the different methods described in section 2.1, they were washed 3 times with PBS, stained according to the instructions of the oil red 0 staining solution test (Leagene, beijing, china) kit, and examined microscopically for the accumulation of lipid in L-02 cells in the different treatment groups. Cell culture supernatants from different treatment groups were collected simultaneously and tested for TG content using TG test kit.

3 results of experiments

3.1 post-natal primordial reduction of alcohol-mediated lipid accumulation in human hepatocytes (L-02)

After 96h incubation in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics, the accumulation of intracellular lipids increased significantly after 48h of intervention with 300mM absolute ethanol; lipid accumulation was significantly reduced in the metagenomic L-02 hepatocytes relative to the EtOH group.

While inducing with alcohol, the final concentration of 10mg/mL of the lactic acid bacteria metazoan was added to the L-02 cell DMEM medium and cultured for 96 hours, and then, after intervention with 300mM absolute ethanol for 48 hours, the L-02 intracellular lipid accumulation was detected by oil red staining, and the results showed that: the lactic acid bacteria metazoans significantly reduced lipid accumulation and TG levels in vitro in ALD model L-02 cells (a-C in fig. 4).

Example 5

Lactic acid bacteria compound probiotics for relieving liver injury of ALD mouse liver

1 Experimental materials and methods

1.1 Experimental materials

95% of the edible alcohol was purchased from Henan Xin Yang He wine industry Co., ltd, lieber-DeCarli liquid alcohol feed and control liquid feed were purchased from Nantong Telofei feed technologies Co., ltd. The glutamic-oxaloacetic transaminase (AST) and glutamic-pyruvic transaminase (ALT) biochemical detection kit is purchased from Nanjing building Limited liability company. SPF-grade C57BL/6 mice, 8 weeks old, body weight (18+ -2 g), purchased from Chongqing Tengxin Biotechnology Co.

1.2 Experimental methods

Establishment of 1.2.1ALD mouse model and different intervention treatment groups thereof

C57BL/6 mice ALD model and given lactobacillus metazoan treatment were established according to the methods described in previous studies (Bertola, A., mathews, S., ki, S., wang, H., & Gao, B.J.N.p. (2013) & Mousemodel ofchronic andbinge ethanol feeding (the NIAAA model) & Nature Protocols,8 (3), 627-637.Https:// doi.org/10.1038/nprot.2013.032). Briefly, all C57BL/6 mice were housed in groups in an environment with controlled relative humidity (45% -65%), temperature (22.+ -. 2 ℃). All C57BL/6 mice were randomly divided into 4 groups of 7 by body weight:

(A) Normal group (4 weeks on Lieber-decrli liquid control diet);

(B) Alcohol feed group (Lieber-DeCarli liquid control feed was first used for 5 days to adapt to liquid diet, then Lieber-DeCarli alcohol feed containing 5% alcohol was used to continue feeding for 23 days, mice were subjected to high concentration alcohol gavage once at a concentration of 5g/kg body weight 9 hours before collecting specimens);

(C) Lactobacillus compound probiotic group (feeding mode same as model group, and lactobacillus reuteri (Lactobacillus reuteri), lactobacillus acidophilus (Lactobacillus acidophilus) and lactobacillus rhamnosus (Lactobacillus rhamnosus) compound preparation are fed daily, the strain seed solution is mixed according to a ratio of 1:1:1 (v/v/v), the gastric lavage volume of the mice is about 10ul/g, and the bacterial load is about 2×10) ⁷ CFU, once a day for 4 weeks);

(D) A lactic acid bacteria metaplasia intervention group (feeding mode same as the model group, and feeding lactic acid bacteria metaplasia daily, wherein the concentration is 0.6g/kg for 4 weeks);

and collecting liver tissue and serum samples of mice with normal groups, alcohol groups, lactobacillus metazoans and lactobacillus compound probiotics groups, and respectively detecting changes of AST and ALT liver injury markers in serum by using an AST and ALT detection kit. The liver tissue frozen by liquid nitrogen is embedded by frozen tissue embedding agent (OCT), sliced by a frozen microtome, stained by oil red and hematoxylin, and then glycerogelatin sealing sheets and microscopic examination are carried out to observe the lipid accumulation condition in the liver tissue of mice in different treatment groups. Formalin-fixed liver tissue was dehydrated by different concentrations of alcohol gradient, xylene transparent, paraffin embedded, microtome sectioned, eosin and hematoxylin stained, then neutral resin sealed and microscopic observed for pathological changes in liver tissue in mice of different treatment groups. The effect of the lactic acid bacteria metaplasia on relieving the alcoholic fatty liver disease is comprehensively evaluated by detecting liver injury markers and pathological changes of mice in different treatment groups.

1.2.2 detection of liver injury markers and pathological changes

And taking out liver tissue and serum samples of mice in a normal group, an alcohol group, a lactobacillus metazoan group and a lactobacillus compound probiotic group collected in the section 1.2.1, respectively calculating liver indexes of the mice in different treatment groups, and respectively detecting changes of AST, ALT and TG liver injury markers in serum by using AST, ALT and TG biochemical detection kits.

The liver tissue frozen by liquid nitrogen is embedded by frozen tissue embedding agent (OCT), sliced by a frozen microtome, stained by oil red and hematoxylin, and then glycerogelatin sealing sheets and microscopic examination are carried out to observe the accumulation of lipid in the liver tissue of mice in different treatment groups.

Formalin-fixed liver tissue was dehydrated by different concentrations of alcohol gradient, xylene transparent, paraffin embedded, microtome sectioned, eosin and hematoxylin stained, then neutral resin sealed and microscopic observed for pathological changes in liver tissue in mice of different treatment groups.

2 experimental results

2.1 lactic acid bacteria Complex probiotic groups and lactic acid bacteria metazoans to alleviate alcohol-mediated liver injury

After 4 weeks of feeding with the alcohol feed, the liver index of the mice is obviously increased, the lipid accumulation in the liver is obviously increased, and the liver cell damage is obviously aggravated; AST, ALT and TG levels were elevated in the blood of mice. The probiotic bacteria are compounded by lactic acid bacteria while the mice are induced by alcohol feed (the volume of the mice for gastric lavage is about 10 mu l/g, and the bacterial count is about 2 multiplied by 10) ⁷ CFU) daily gavage mice, 4After week, the pathological changes of the liver are detected, and the result shows that: the lactobacillus compound probiotics and the lactobacillus metagens can relieve the liver index increase of the mice induced by alcohol, simultaneously reduce the accumulation of lipid in the livers of the ALD mice, and can also reduce the levels of AST, ALT and TG in the serum of the ALD mice, and the effect of the lactobacillus metagens on relieving the liver injury mediated by alcohol is obviously superior to that of the lactobacillus compound probiotics group (A-F in figure 5).

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. The application of lactobacillus metagen in preparing a medicament for preventing and/or treating alcoholic liver disease is provided, wherein the lactobacillus metagen is prepared by mixed fermentation of lactobacillus reuteri (Lactobacillus reuteri), lactobacillus acidophilus (Lactobacillus acidophilus) and lactobacillus rhamnosus (Lactobacillus rhamnosus).

2. The use according to claim 1, characterized in that the preparation method of the lactobacillus metazoan comprises inoculating lactobacillus reuteri seed solution, lactobacillus acidophilus seed solution and lactobacillus rhamnosus seed solution into metazoan preparation medium for culture, and obtaining culture solution;

inoculating the culture solution into the metazoan preparation culture medium for fermentation culture to obtain fermentation liquor;

and inactivating the fermentation liquor and crushing the thalli to obtain the metazoan.

3. The use according to claim 2, wherein the inoculation volume ratio of lactobacillus reuteri seed solution, lactobacillus acidophilus seed solution and lactobacillus rhamnosus seed solution is (0.8-1.2): 0.8-1.2: (0.8-1.2).

4. The use according to claim 2, wherein the total inoculum size of lactobacillus reuteri seed solution, lactobacillus acidophilus seed solution and lactobacillus rhamnosus seed solution during said cultivation is 1.5-2.5%;

the fermentation culture time is 10-14 h, and the fermentation culture temperature is 36-38 ℃.

5. The use according to claim 2, wherein the inoculum size of the culture solution during the fermentation culture is 4% to 6%;

the time of the culture is 20-23 h, and the temperature of the culture is 37 ℃.

6. The use according to claim 2, wherein the method of bacterial inactivation is heat treatment;

the temperature of the heat treatment is 60-70 ℃, and the time of the heat treatment is 25-35 min.

7. The use according to claim 2, wherein the method of cell disruption is ultrasonication;

the power of the ultrasonic crushing is 40W, the total crushing time of the ultrasonic crushing is 14-16 min, and the intermittent time of the ultrasonic crushing is 4-5 s.

8. The use according to claim 2, wherein the metazoan preparation medium is an aqueous solution comprising: 20g/L glucose, 10g/L peptone, 5g/L sodium acetate, 4g/L yeast extract, 2g/L ammonium citrate, 2g/LK ₂ HPO ₄ ·3H ₂ O, 0.2g/L magnesium sulfate, 0.1g/LMnSO ₄ ·H ₂ O and 0.05g/L manganese sulfate.

9. The use according to any one of claims 1 to 8, wherein the lactobacillus reuteri comprises strain lactobacillus reuteri DSM 17938;

the lactobacillus acidophilus comprises lactobacillus acidophilus CICC6096 strain;

the lactobacillus rhamnosus (Lactobacillus rhamnosus) is lactobacillus rhamnosus CICC6137 strain.

10. The use according to any one of claims 1 to 8, wherein the medicament has the effect of protecting against alcohol-mediated liver damage and alleviating alcohol-mediated accumulation of hepatocyte lipids.