CN111437294A - Lactic acid bacteria formula for preventing acute and chronic alcoholic liver injury and application thereof - Google Patents

Abstract

The invention discloses a lactobacillus formula for preventing acute and chronic alcoholic liver injury and application thereof, wherein lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS1.0901 have good gastrointestinal fluid tolerance simulating capacity, have certain adhesion capacity to HT-29 cells and have basic probiotic characteristics, lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS1.0901 can prevent acute alcoholic liver injury, lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS1.0901 can prevent chronic alcoholic liver injury, and specifically, lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS1.0901 have the functions of reducing liver index, improving intestinal barrier, improving liver function, reducing inflammation level in liver, increasing short-chain fatty acid content in intestinal tract, reducing protein expression of CYP2E1 in liver and the like.

Description

Lactic acid bacteria formula for preventing acute and chronic alcoholic liver injury and application thereof

Technical Field

The invention relates to a lactobacillus formula for preventing acute and chronic alcoholic liver injury and application thereof, belonging to the technical field of medicines.

Background

However, acute and chronic alcoholic liver injury can be caused by a large amount of alcohol in a short term or excessive alcohol in a long term respectively, alcoholic liver disease (A L D) comprises the following histopathological changes of hepatic steatosis, alcoholic steatohepatitis, hepatic fibrosis, cirrhosis and liver cancer A L D becomes one of the diseases seriously threatening human health in the world, and along with the transformation of the public concept, people are more eager to use functional food or dietary supplements rather than medicines to prevent or relieve A L D, so that the demand for safe and healthy lactic acid bacteria capable of preventing acute and chronic alcoholic liver injury is urgent.

Disclosure of Invention

The invention aims to solve the technical problem of providing a lactobacillus formula for preventing acute and chronic alcoholic liver injury and application thereof.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a lactobacillus formula for preventing acute and chronic alcoholic liver injury comprises Lactobacillus plantarum K L DS1.0344 and Lactobacillus acidophilus K L DS 1.0901.

The volume ratio of the lactobacillus plantarum K L DS1.0344 to the lactobacillus acidophilus K L DS1.0901 is 1:1, and the thallus concentrations of the lactobacillus plantarum K L DS1.0344 and the lactobacillus acidophilus K L DS1.0901 are both 10⁸～10¹⁰CFU/mL。

An application of lactobacillus formula for preventing acute and chronic alcoholic liver injury in preventing acute and chronic alcoholic liver injury is provided.

Preferably, the use of a lactic acid bacteria formulation as described above for reducing the liver index.

Preferably, the use of a lactic acid bacteria formulation as described above for improving the intestinal barrier, inhibiting the reduction of the expression of the claudin-1, occludin and claudin-1 in the ileum.

Preferably, the lactic acid bacteria formula is applied to improving liver functions, inhibiting increase of A L T and AST levels in liver serum, reducing L PS content in liver serum and inhibiting liver inflammation.

Preferably, the use of the above lactic acid bacteria formulation for reducing the level of inflammation in the liver, reducing the levels of TNF- α and I L-6 in the liver and inhibiting an increase in MPO activity.

Preferably, the lactic acid bacteria formulation is used for reducing the level of inflammation in the liver, reducing the expression level of T L R4 protein on the cell membrane of the liver and p65 protein in the nucleus, and increasing the expression level of I к B α protein in the cytoplasm.

Preferably, the lactobacillus formula is applied to increase the content of short-chain fatty acids in intestinal tracts, and the short-chain fatty acids are used as signal molecules to participate in regulating Nrf-2 signal pathways closely related to oxidative stress, so that the reduction of protein expression levels of Nrf-2 in liver cell nuclei and HO-1, NQO1, SOD1, CAT and GPx-1 in cytoplasm is inhibited.

Preferably, the use of a lactic acid bacteria formulation as described above for reducing protein expression of CYP2E1 in the liver.

The invention has the following beneficial effects:

(1) the lactobacillus plantarum K L DS1.0344 and the lactobacillus acidophilus K L DS1.0901 have good capability of simulating gastrointestinal fluid tolerance, have certain adhesive capacity to HT-29 cells and have basic probiotic characteristics.

(2) The potential mechanism is that the interference of the lactobacillus plantarum K L DS1.0344 in combination with the lactobacillus acidophilus K L DS1.0901 can inhibit the intestinal permeability from increasing, so that L PS is prevented from reaching the liver through portal circulation, activation of T L R4 and a NF-kB signal channel downstream of the T L R4 is further inhibited, inflammation in the liver is prevented, and the effect of preventing acute alcoholic liver injury can be achieved by inhibiting lipid accumulation in the liver and improving the antioxidation capability of the liver.

(3) The potential mechanism is that the lactobacillus plantarum K L DS1.0344 and the lactobacillus acidophilus K L DS1.0901 are combined to regulate the intestinal flora so as to improve the level of short-chain fatty acids in the intestinal tract, the short-chain fatty acids further reach the liver through the intestinal hepatic axis and participate in regulating an AMPK signal path and an Nrf-2 signal path as signal molecules to inhibit lipid accumulation in the liver and improve the anti-oxidation capacity of the liver, and the lactobacillus plantarum K L DS1.0344 and the lactobacillus acidophilus K L DS1.0901 are combined to inhibit the growth of gram-negative bacteria in the intestinal tract and improve the intestinal barrier function so as to prevent L PS from entering portal circulation to reach the liver, so that the NF-kB signal path mediated by T L R4 is indirectly inhibited to prevent inflammation in the liver.

Drawings

FIG. 1 is a graph showing the effect of acute alcoholic liver injury lactic acid bacteria on the expression of ZO-1, occludin and claudin-1 in the present invention;

FIG. 2 is a graph showing the effect of acute alcoholic liver injury lactic acid bacteria on serum A L T, AST and L PS levels in accordance with the present invention;

FIG. 3 is a graph showing the effect of acute alcoholic liver injury lactic acid bacteria on liver histopathology in accordance with the present invention;

FIG. 4 is a graph showing the effect of acute alcoholic liver injury lactic acid bacteria on the level of liver inflammation in accordance with the present invention;

FIG. 5 is a graph showing the effect of acute alcoholic liver injury lactic acid bacteria on protein expression of related genes in T L R4 receptor and NF- κ B signaling pathway in the present invention;

FIG. 6 is a graph showing the effect of acute alcoholic liver injury lactic acid bacteria on liver lipid accumulation in accordance with the present invention;

FIG. 7 is a graph showing the effect of acute alcoholic liver injury lactic acid bacteria on the antioxidant level of the liver in accordance with the present invention;

FIG. 8 is a graph showing the effect of chronic alcoholic liver injury lactic acid bacteria on mouse body weight and liver index in accordance with the present invention;

FIG. 9 is a graph showing the effect of chronic alcoholic liver injury lactic acid bacteria on the expression of ZO-1, occludin and claudin-1 in the present invention;

FIG. 10 is a graph showing the effect of chronic alcoholic liver injury lactic acid bacteria on serum A L T, AST and L PS levels in accordance with the present invention;

FIG. 11 is a graph showing the effect of chronic alcoholic liver injury lactic acid bacteria on liver histopathology in accordance with the present invention;

FIG. 12 is a graph showing the effect of chronic alcoholic liver injury lactic acid bacteria on liver lipid accumulation in accordance with the present invention;

FIG. 13 is a graph showing the effect of chronic alcoholic liver injury lactic acid bacteria on the level of liver oxidation resistance in accordance with the present invention;

FIG. 14 is a graph showing the effect of chronic alcoholic liver injury lactic acid bacteria on the level of liver inflammation in accordance with the present invention;

FIG. 15 is a graph showing the effect of chronic alcoholic liver injury lactic acid bacteria on the content of short chain fatty acids in intestinal contents in accordance with the present invention;

FIG. 16 is a graph showing the effect of chronic alcoholic liver injury lactic acid bacteria on protein expression of related genes in the AMPK signaling pathway in accordance with the present invention;

FIG. 17 is a graph showing the effect of chronic alcoholic liver injury lactic acid bacteria on protein expression of T L R4 receptors and related genes in the NF- κ B signaling pathway in accordance with the present invention;

FIG. 18 is a graph showing the effect of chronic alcoholic liver injury lactic acid bacteria on CYP2E1 protein expression in accordance with the present invention.

Detailed Description

The present invention will be further described with reference to the accompanying drawings.

1 materials and methods

1.1 Experimental materials

1.1.1 test strains, Lactobacillus plantarum (L Acobacter plantium), K L DS1.0344 and Lactobacillus acidophilus (L Acobacter acidophilus), K L DS1.0901, were both provided by the Collection of Industrial microorganism strains (K L DS-DICC) of the department of Dairy science education, northeast university of agriculture, both strains were isolated from traditional fermented milk products of China.

1.1.2 Experimental cells: the cells used in the experiments were human colorectal adenocarcinoma cells HT-29, purchased from QINGTAI (Shanghai) Biotech development Co., Ltd.

1.1.3 Experimental animals the experimental animals were clean grade 6 week old male C57B L/6J mice, 90 in total, purchased from Beijing Wittingle laboratory animals technology, Inc., having a license number of SCXK (Jing) 2016-.

1.1.4 Medium

1.1.4.1 culture medium for lactic acid bacteria

MRS liquid culture medium, adding the components of the culture medium according to the proportion of (5.0 g of peptone, 5.0g of beef extract, 10.0g of tryptone, 5.0g of anhydrous sodium acetate, 2.0g of dipotassium phosphate, 5.0g of yeast extract powder, 0.25g of manganese sulfate, 0.58g of magnesium sulfate, 2.0g of diammonium hydrogen citrate, 2. 801.0g of tween-8932 and 20.0g of glucose), adding distilled water to a constant volume of 1L, fully and uniformly stirring, adjusting the pH to 5.8, subpackaging in 5m L or 10m L test tubes, sterilizing at 121 ℃ for 15min, and storing in a refrigerator at 4 ℃ for later use.

Adding 16g of agar powder into each component of the MRS solid culture medium after adding the components of the MRS liquid culture medium, metering the volume to 1L by using distilled water, fully and uniformly stirring, adjusting the pH to 5.8, sterilizing for 15min at the temperature of 121 ℃, then respectively pouring the mixture into culture dishes (each 20-25 m L) in a super clean bench, and turning over the culture dishes for later use after the culture medium is solidified.

High-sugar DMEM medium, namely 10% of heat-inactivated fetal bovine serum and 1% of antibiotic (penicillin concentration 100U/m L and streptomycin 100 mu g/m L) are added into the high-sugar DMEM medium, and the mixture is filtered and sterilized by a 0.22 mu m filter membrane and stored in a refrigerator at 4 ℃ for later use.

1.2 Experimental methods

1.2.1 culture and preservation of the Strain

Respectively inoculating two strains of bacteria in an MRS liquid culture medium according to the inoculation amount of 2% (v/v), culturing in a constant-temperature incubator at 37 ℃ after marking, carrying out passage 1 time every 24h, carrying out three-region lineation on an MRS solid culture medium by using an inoculating loop when the 3 rd generation culture reaches 18h, culturing in the constant-temperature incubator at 37 ℃ for 48h, picking out a single colony for gram staining, carrying out passage 2 times in the MRS liquid culture medium after observation and identification, and carrying out passage 2 times when the 2 nd generation culture reaches 18h, mixing a culture solution with 50% of glycerol, and culturing in a constant-temperature incubator at a temperature of 3: 2, and storing the mixture in a refrigerator at the temperature of minus 80 ℃ for later use.

Before the experiment, two frozen strains are taken out from a refrigerator at the temperature of-80 ℃, activated for 3 generations by the inoculation amount of 2% (v/v), subcultured for 1 time every 24h, and cultured for the 3 rd generation till 18h later to be used for the experiment.

1.2.2 HT-29 cell culture and preservation

1.2.2.1 recovery of HT-29 cells

From liquid nitrogen biological containerTaking out frozen HT-29 cells, quickly placing into preheated 37 deg.C water bath to melt (within 1 min), immediately transferring into 15m L sterile centrifuge tube in ultra clean bench, adding 3m L complete high sugar DMEM culture medium, centrifuging for 5min at 1000 × g, discarding supernatant, adding 5m L complete high sugar DMEM culture medium, gently blowing with pipette to mix, transferring into 50m L cell culture bottle, and culturing at 37 deg.C with 5% CO₂The culture was carried out in an incubator, and the medium was replaced with fresh medium every 2 days.

1.2.2 passage of HT-29 cells

When the cells grow to be a monolayer in an adherent manner and reach the coverage rate of the bottom of a cell bottle of about 80%, washing the cells for 3 times by using sterile PBS of 1m L, adding 0.25% trypsin-EDTA of 1m L for digestion, after the cells are digested for 2min, adding a complete high-sugar DMEM culture medium of 1m L to stop digestion, sucking liquid by using a liquid-transferring gun to repeatedly and lightly blow and beat the cells to completely shed to obtain a cell suspension, transferring the cell suspension to a sterile centrifuge tube of 15m L, centrifuging for 5min at 1000 × g, discarding supernatant, adding the complete high-sugar DMEM culture medium of 2-3 m L again, lightly blowing and uniformly mixing by using the liquid-transferring gun, subpackaging the mixture into a new cell culture bottle (2-3 bottles), adding the complete high-sugar DMEM culture medium of 4m L into each bottle, and then adding the complete high-sugar DMEM culture medium of 5% CO at 37 ℃ and 5 ℃₂Culturing in an incubator to complete passage.

1.2.2.3 cryopreservation of HT-29 cells

Taking out the cell culture bottle from the incubator, washing the cells for 3 times by using sterile PBS (1m L), adding 0.25% trypsin-EDTA (ethylene diamine tetraacetic acid) with 1m L for digestion, after the cells are digested for 2min, adding complete high-sugar DMEM (DMEM) medium with 1m L to stop the digestion, sucking liquid by using a pipette gun to repeatedly and lightly blow and beat the cells to completely shed the cells to obtain cell suspension, transferring the cell suspension to a sterile centrifuge tube with 15m L, centrifuging for 5min at 1000 × g, removing supernatant, adding cell freezing solution (10% dimethyl sulfoxide (DMSO) and 90% serum) of 1m L to mix uniformly), lightly blowing and uniformly dispersing the cells, transferring the cell suspension to a freezing tube, then placing the cell suspension in a refrigerator with the temperature of 4 ℃ for 30min, then transferring to a refrigerator with the temperature of-20 ℃ for 2h, then transferring to a refrigerator with the temperature of-80 ℃ for overnight, and then placing the cell suspension in a liquid nitrogen biological container for later use.

1.2.3 Strain tolerance simulation gastrointestinal fluid experiment

The simulated gastric fluid and simulated intestinal fluid are prepared by dissolving pepsin (1:10000) in sterile 0.5% NaCl solution (w/v) to give a concentration of 3 g/L, adjusting pH to 3.0 with sterile HCl solution, filtering with 0.22 μm filter to remove bacteria to give simulated gastric fluid, dissolving trypsin (1:250) in sterile 0.5% NaCl solution (w/v) to give a concentration of 1 g/L, adding 0.3% bile salt, adjusting pH to 8.0 with sterile NaOH solution, and filtering with 0.22 μm filter to remove bacteria to give simulated intestinal fluid.

Taking out Lactobacillus plantarum K L DS1.0344 and Lactobacillus acidophilus K L DS1.0901 cultured for 18h in the 3 rd generation from the incubator, centrifuging for 10min at 8000 × g, discarding supernatant, washing thallus for 3 times with sterile PBS, re-suspending the thallus in simulated gastric juice, mixing well, adjusting thallus concentration to 10⁹CFU/m L, after 3h incubation at 37 ℃ in an incubator, viable cell count was performed using dilution-spread plate method, followed by transfer to simulated intestinal fluid followed by 8h incubation at 37 ℃ in an incubator, viable cell count was also performed using dilution-spread plate method.

Survival (%) > 1g CFU N₁/1g CFU N₀× 100% of formula (I), wherein N is₁: the number of treated viable bacteria; n is a radical of₀: viable count before treatment.

1.2.4 Strain adhesion HT-29 cell assay

(1) Preparation of lactic acid bacteria suspension

Taking out Lactobacillus plantarum K L DS1.0344 and Lactobacillus acidophilus K L DS1.0901 cultured for 18h from the 3 rd generation from the incubator, centrifuging for 10min at 8000 × g, discarding supernatant, washing thallus for 3 times with sterile PBS, suspending thallus in high-sugar DMEM medium containing 10% fetal calf serum, mixing, and adjusting the suspension concentration to 10⁸CFU/mL。

(2) Preparation of cells

HT-29 cells were seeded from cell culture flasks into 12-well cell culture plates (10)⁵One cell/well), replacing the culture medium every 2d, and paving after the cells are completely attached to the wallAfter the bottom surface of the well plate was filled, the medium was changed to a high-glucose DMEM medium without diabody overnight, and the adhesion experiment was performed the next day.

(3) Adhesion test of lactic acid bacteria to HT-29 cells

The adhesion test of lactic acid bacteria to HT-29 cells was carried out by washing a monolayer of HT-29 cells twice in each well of a 12-well cell culture plate with sterile PBS, and then adding 500. mu. L to the wells at a concentration of 10⁸Lactic acid bacterium (V) of CFU/m L₀＝5×10⁷CFU). The 12-well cell culture plates were then transferred to 37 ℃ with 5% CO₂Incubating for 2h in an incubator, washing the monolayer cells of each well of a 12-well cell culture plate with sterile PBS 3-5 times to elute the non-adhered bacteria, adding 250 mu L of 0.25% trypsin-EDTA into each well for digestion for 10min, adding 250 mu L of serum to stop digestion, collecting the liquid in each well, and counting the adhered viable bacteria by a dilution coating plate method (V)₁). The adhesion rate of the strain is calculated by the following formula: adhesion rate (%) - (V)₁/V₀)×100％。

1.2.5 study of lactic acid bacteria to prevent acute alcoholic liver injury

1.2.5.1 preparation of lactic acid bacteria

Taking out Lactobacillus plantarum K L DS1.0344 and Lactobacillus acidophilus K L DS1.0901 cultured for 18h from the 3 rd generation from the incubator, centrifuging for 10min at 8000 × g, discarding supernatant, washing thallus for 3 times with sterile PBS, suspending thallus in sterile PBS, adjusting thallus concentration to 10¹⁰CFU/m L, and mixing Lactobacillus plantarum K L DS1.0344 suspension and Lactobacillus acidophilus K L DS1.0901 suspension at a volume ratio of 1:1 to form mixed lactobacillus suspension (10)¹⁰CFU/m L), spare.

1.2.5.2 Experimental groups of mice and design of experiment

A clean 6-week-old male C57B L/6J mouse is bred in an animal house, the temperature is controlled to be 22 +/-2 ℃, the humidity is controlled to be 55% +/-5%, the light is 12 h/12 h dark circulation, the mouse is freely drunk and fed, after the mouse is adaptively fed with standard feed for 1 week, the mouse is randomly divided into 3 groups, and each group is 10 mice, namely a Control group (Control) and an alcohol group (A)lcohol) and lactobacillus group (L AB) mice of control and alcohol groups were gavaged daily in the morning with 0.2m L of sterile PBS and lactobacillus group mice were gavaged with 2 × 10⁹CFU mixed lactic acid bacteria, in the afternoon, the mice of the control group were still gavaged with 0.2m L sterile PBS, while the mice of the alcohol and lactic acid bacteria groups were gavaged with 40% (w/v) wine (12m L/kg body weight; prepared from 56 degrees red star Erguotou plus distilled water) for 10 d.

TABLE 1 mouse grouping and daily treatment comparison Table

1.2.5.3 sample Collection

After the experiment is finished, all mice are fasted for 12h, anesthetized, blood is taken out through eyeballs and then killed, the blood is stood still for 1h at room temperature, after 1500 × g is centrifuged for 10min at 4 ℃, upper serum is absorbed and placed in a refrigerator at-80 ℃ for biochemical analysis, the mice are dissected, ileum at the same position of each mouse is taken out and placed in the refrigerator at-80 ℃ for immunofluorescence detection, the liver of the mouse is taken out, one part of the liver of the mouse is placed in the refrigerator at-80 ℃ for Western blot detection and biochemical analysis, and the other part of the liver of the mouse is placed in 4% paraformaldehyde for pathological analysis.

1.2.5.4 determination of liver index

After the mouse was dissected, the liver was removed, blood on the surface was washed with pre-cooled physiological saline, blotted with filter paper and quickly weighed, and then the liver index (%) was calculated as × 100% of the weight of liver (g)/weight of body (g) using the following formula.

1.2.5.5 Biochemical analysis

The detection of A L T, AST and L PS in serum was performed according to the protocol of the kit.

Weighing a proper amount of liver, measuring physiological saline 9 times the weight of the tissue block, putting the liver homogenate and the physiological saline together into a manual homogenizer for grinding under an ice bath condition to form homogenate, centrifuging the liver homogenate for 10min at 2500 × g under the condition of 4 ℃, taking out supernatant fluid to detect lipid, inflammation indexes and oxidation indexes in the liver, and detecting TG, TNF- α, I L-6, MPO, SOD, Gpx, CAT and MDA in the liver of a mouse according to the operation on the instruction manual of the kit.

1.2.5.6 immunofluorescence detection

(1) Embedding, namely 4% paraformaldehyde fixed ileum sample, washing with running water for 30min, performing tissue repair, putting into a pathological embedding plastic basket for dehydration, transparentizing, dipping wax for 3h, finally embedding the tissue block into paraffin, (2) slicing, namely, cutting the tissue into slices with the thickness of 4 mu m by using a slicer, flattening the slices in warm water, putting on an anti-falling glass slide, baking the slices at 60 ℃ for at least 2h, (3) staining, (a) dewaxing and hydrating, namely, putting the paraffin slices into fresh xylene, soaking for 10min × times, removing excessive liquid, putting into absolute ethyl alcohol, soaking for 3min × times, removing the excessive liquid, putting into 95% ethyl alcohol, soaking for 3min × times, removing the excessive liquid, putting into 75% ethyl alcohol, soaking for 3min × times, washing with distilled water for 1min, putting into PBS, incubating, adding a proper amount of PBS (pH 6.0) for antigen repair, (c) blocking PBS, (b) adding a proper amount of PBS, incubating, adding a proper amount of PBS, (3 min, soaking for 3min, rinsing for 10min, adding a proper amount of a normal serum (3 h), incubating, adding a proper amount of a PBS, adding a normal temperature blocking agent, (3 h, incubating, and rinsing for 24 h), and rinsing for 100 mu) and rinsing (3 h), and rinsing for 100 mu) and rinsing (3 h), and rinsing, incubating (3 h), and rinsing for 100 h), and rinsing (3 h), and rinsing for 100 h), and rinsing

And observing the target fluorescence expression condition of the ileum tissue under a fluorescence microscope, photographing and recording by adopting a fluorescence microscopy imaging system, and detecting the average fluorescence optical density value of the positive fluorescence result by adopting Image ProPlus6.0 software.

1.2.5.7 liver Pathology analysis

(1) Embedding: a liver tissue sample fixed by 4% paraformaldehyde is washed for 30min by running water, subjected to tissue block repairing, put into a pathological embedding plastic basket for dehydration, transparent, and subjected to paraffin soaking for 3h, and the tissue block is embedded in paraffin. (2) Slicing: the tissue was cut into 5 μm thick sections with a microtome, the sections were flattened in warm water and placed on glass slides and the sections were baked at 60 ℃ for at least 2 h. (3) Dyeing: dewaxing the slices with xylene, washing with flowing water for 20min, staining with hematoxylin for 30min, washing with flowing water for 20min, differentiating with hydrochloric acid and ethanol, staining with eosin for 5min, dehydrating with gradient ethanol, clearing xylene, and sealing with resin adhesive. (4) Reading the film: and (3) observing the pathological change of the liver tissue under a microscope, completely browsing the whole section, and taking a picture and recording the normal part or the part with obvious pathological change by using a microscopic imaging system.

1.2.5.8 Westernblot detection

(1) Protein production

Putting a proper amount of liver tissue and a proper amount of precooled RIPA lysis buffer (a small tissue block of about 3mm × 3mm is put into RIPA lysis buffer of 0.5-1 m L) into a manual homogenizer together, grinding the mixture into homogenate under the ice bath condition, centrifuging the homogenate for 15min at 12000 × g at the temperature of 4 ℃, sucking out supernatant to obtain total protein extract, separating cell nuclear protein and cytoplasmic protein by using a MinuteTM cytoplasmic nuclear separation kit, and separating cell membrane protein by using a MinuteTM plasma membrane protein and cell component separation kit.

(2) Protein quantification

Protein concentration was determined using the BCA protein concentration assay kit.

(3) Polyacrylamide gel electrophoresis

Sucking a proper amount of sample supernatant, adding the sample supernatant into a sample hole, adding a pre-stained protein Marker into a hole beside the sample, adding l × SDS loading buffer solution into a hole without the sample supernatant to keep the gel surface balance, turning on a power supply, setting the voltage to be 60V, increasing the voltage to 90V after the protein sample enters the separation gel, referring to the position of the pre-stained Marker, and stopping electrophoresis when a target strip enters the optimal separation area (about 2/3 of the gel).

(4) Rotary film

Precooling the membrane transferring liquid at 4 ℃, and soaking the fiber pad, the filter paper and the NC/PVDF membrane soaked with methanol in advance by the membrane transferring liquid. Carefully prying off a glass plate, placing the gel in a tray containing a membrane transferring solution, cutting off a separation gel containing a target strip, manufacturing a transfer printing interlayer (the sizes of the membrane, the filter paper and the gel are approximately the same) of a fiber mat-filter paper-gel-NC/PVDF membrane-filter paper-fiber mat, placing the transfer printing interlayer into a transfer groove filled with the membrane transferring solution, turning on a power supply, and stabilizing the current for 200mA for 60-120 min.

(5) Sealing of

And (3) after the membrane is transferred, taking out the NC/PVDF membrane and marking, putting the NC/PVDF membrane into a plate, adding a confining liquid containing 5% of skimmed milk powder, and oscillating for 1.5-2h by a shaking table.

(6) Antigen antibody reaction

And after the sealing is finished, washing the membrane for 5min × 3 times by using TBST, putting the membrane into a dish containing primary antibody (diluted by Western primary antibody diluent according to the instruction of the antibody), shaking and incubating overnight by using a shaking table at 4 ℃, taking out the membrane after the second day, discarding the primary antibody, washing the membrane for 5min × 3 times by using TBST, putting the membrane into secondary antibody solution diluted by using 5% skimmed milk powder sealing solution, carrying out shaking and reacting for 1-2h at room temperature by using the shaking table, and after the secondary antibody reaction is finished, washing the membrane for 5-10min × 3 times by using TBST.

(7) Color development

Mixing A, B two liquids in the EC L chemiluminescence kit according to l: l equal volume, preparing a working solution for standby (in-situ preparation), taking an NC/PVDF film out of TBST, throwing off redundant liquid, putting the film with the front side facing upwards on a preservative film, dripping a proper amount of working solution, covering with the preservative film, and imaging by using a gel imager.

(8) Image analysis

Results were grey scale analyzed using Gel-Pro Analyzer 4 software.

1.2.6 study of lactic acid bacteria to prevent chronic alcoholic liver injury

1.2.6.1 intragastric lavage sample preparation

Taking out Lactobacillus plantarum K L DS1.0344 and Lactobacillus acidophilus K L DS1.0901 cultured for 18h from the 3 rd generation from the incubator, centrifuging for 10min at 8000 × g, discarding supernatant, washing thallus for 3 times with sterile PBS, suspending the thallus in sterile PBS, adjusting thallus concentration to 10⁸CFU/m L and 10¹⁰CFU/m L, followed by two concentrations of Lactobacillus plantarum K L DS1.0344 suspension and Lactobacillus acidophilus K L DS1.0901 suspensionAre respectively expressed by 1:1 to form a low-concentration mixed lactic acid bacteria suspension (10)⁸CFU/m L) and high concentration mixed lactic acid bacteria suspension (10)¹⁰CFU/m L), and mixing silymarin in sterile PBS.

1.2.6.2 grouping of experimental mice and design of experiment

A clean-grade 6-week-old male C57B L/6J mouse is raised in an animal house at 22 + -2 deg.C and humidity 55% + -5% with 12H light/12H dark cycle, the mouse is freely drunk and fed with standard feed during adaptive feeding, after 1 week of adaptive feeding, the mouse is randomly divided into 5 groups of 12 mice each comprising a control group (PF), an alcohol group (AF), a low-dose lactic acid bacteria group (LL), a lactic acid bacteria high-dose group (L H) and a silymarin group (PC), wherein the AF group of mice is adaptively fed with an alcohol-containing L ieber-Decali liquid feed for 1 week (the alcohol content is from 0 to 4% (w/v)), and the mice fed with an alcohol-containing L ieber-Decali liquid feed for 6 weeks, and are fed with a daily gavage of 0.2m L sterile PBS for 6 weeks, the LL group, L H group and PC group are fed with the same method as the AF group, and the low-dose lactic acid bacteria (10) are respectively fed in 6 weeks (10 g) each day¹⁰CFU/m L CFU), high dose of lactic acid bacteria (2 × 10)⁹CFU) and silymarin (100mg/kg body weight), PF group mice were fed in L ieber-DeCarli liquid diet pair containing maltodextrin with calories such as alcohol for 6 weeks with gavage of 0.2m L sterile PBS per day (1 week with L ieber-DeCarli liquid diet containing maltodextrin with calories such as alcohol prior to the start of the experiment), AF group, LL group, L H group and PC group mice were fed ad libitum, while each mouse in PF group was given daily feed in the amount of average food intake of mice in the previous day AF group, LL group, L H group and PC group, the feed formulation of mice is shown in Table 2. the specific operating conditions of each group of mice are shown in Table 3. mice in the experimental period, mice in 2 days were replaced with litter and mice were weighed weekly.

TABLE 2 mouse feed formulation

TABLE 3 mouse grouping and daily treatment condition comparison table

1.2.6.3 sample Collection

After the experiment is finished, all mice are fasted for 12 hours, anesthetized, blood is taken out through eyeballs and then killed, the blood is stood still for 1 hour at room temperature, after 1500 × g is centrifuged for 10 minutes at 4 ℃, upper serum is absorbed and placed in a refrigerator at-80 ℃ for biochemical analysis, the mice are dissected, the caecum content of the mice is collected in a sterile freezing tube on ice under the sterile condition and placed in the refrigerator at-80 ℃ for detecting the intestinal flora composition and short chain fatty acids, the ileum at the same position of each mouse is taken out and placed in the refrigerator at-80 ℃ for immunofluorescence detection, the livers of the mice are taken out, one part of the livers are placed in the refrigerator at-80 ℃ for Western blot detection and biochemical analysis, and the other part of the livers are placed in 4% paraformaldehyde for pathological analysis.

1.2.6.4 intestinal flora analysis

Total microbial DNA in caecum contents of control group, alcohol group and lactobacillus high-dose group mice according to the following formula

The bacterial 16S rDNA V3-V4 region was amplified using Polymerase Chain Reaction (PCR) with forward 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and reverse 806R (5 '-GGACTACHVGGGTWTCTAAT-3') primers, PCR conditions were pre-denaturation at 95 ℃ for 3min, 30 cycles of 95 ℃ for 30S (denaturation), 55 ℃ for 30S (annealing), 72 ℃ for 30S (extension), and extension at 72 ℃ for 5min PCR with 20. mu. L reaction system, 4. mu. L FastFU buffer (5 ×), 2. mu. L dNTPs (2.5mM), 0.8. mu. L forward primer (5. mu.M), 0.8. mu. L reverse primer (5. mu.M), 0. L. mu. FasFasFU 4 and 10. mu. ang PCR products were detected by electrophoresis using 1% agarose gel electrophoresis, and the DNA concentration was determined using 10% agarose gel electrophoresis with 10 PCRThe gel extraction kit was purified and quantified using a fluorescence photometer Qubit 2.0. Then, Miseq libraries were constructed and sequenced on the Illumina Miseq platform.

The generated raw data were pooled using F L ASH software (V1.2.11) and mass filtered using QIIME software (V1.7.0) chimaera sequences were identified and removed using UCHIME algorithm to collect high quality sequence tags (cleartags), sequence tags with 97% sequence homology were clustered using UPARSE software to get operational classification units (OTUs), these OTUs were aligned to Greengenes database using PyNAST software and labeling classification information, α -diversity and β -diversity were analyzed using QIIME software (V1.7.0) and R software (V3.4.1), primary coordinate analysis (PCoA) was performed using R software (V3.4.1), the magnitude of the effect of each species abundance on the difference effect was estimated using L efSe online analysis platform to determine the species with significant differences between the groups.

1.2.6.5 determination of short-chain fatty acids

Placing 50mg of cecum content into a 2m L centrifuge tube, adding 0.5m L pure water, whirling for 10s, adding steel balls, adjusting to 35Hz by a grinder for 4min, performing ultrasonic treatment for 5min (ice water bath), centrifuging 5000 × g of the sample for 20min at 4 ℃, taking out 0.4m L supernatant (placing the supernatant into a new 2m L centrifuge tube), adding 0.5m L pure water, whirling for 10s, adjusting to 35Hz by a grinder for 4min, performing ultrasonic treatment for 5min (ice water bath), centrifuging 5000 × g of the sample for 20min at 4 ℃, taking out 0.4m L supernatant, uniformly mixing the 0.4m L supernatant with the original supernatant (0.4m L), and adding 0.1m L50% of H₂SO₄And 0.8m L internal standard solution (2-methyl valeric acid dissolved in methyl tert-butyl ether to a concentration of 25 mg/L), vortexing for 10s, shaking for 5min, centrifuging the sample at 4 ℃ for 15min at 10000 × g, standing at-20 ℃ for 30min, taking out the supernatant, placing the supernatant in a sample bottle (silica-based), and detecting by gas chromatography-mass spectrometry (GC-MS).

An Agilent7890 gas chromatography-mass spectrometer equipped with an Agilent HP-FFAP capillary column (30m × 250μm × 0.25.25μm) is used for GC-MS detection, and the detection conditions are that the sample feeding amount is 1μm L, the flow splitting Mode is a Splitmode (5:1), the spacer purge flow rate is 3m L/min, the carrier gas is Helium, the chromatographic column is HP-FFAP (30m × 250μm × 0.25.25μm), the column flow rate is 1m L/min, the column box temperature raising program lasts for 1min at 80 ℃, is increased to 150 ℃ at 5 ℃/min, is increased to 230 ℃ at 40 ℃/min, lasts for 12min at 230 ℃, the sample inlet temperature is 240 ℃, the ion source temperature is 230 ℃, the quadrupole rod temperature is 150 ℃, the ionization voltage is-70 eV, the mass range is m/z 33-200, and the solvent delay is 5 min.

1.2.7 data analysis

All experimental data are in mean ± sd

To show that each experiment is repeated for more than 3 times, the experimental data is statistically analyzed by SPSS 22.0 software, and the comparison between multiple groups of data and the comparison between two groups of data are respectively carried out by adopting one-factor analysis of variance and paired sample T test, p<0.05 indicates significant difference, p<0.01 indicates that the difference is extremely significant, p>0.05 indicated no significant difference. The graph was drawn using GraphPad Prism 8.0, Excel 2019 and the R language.

2 results and analysis

2.1 tolerance of the strains to simulated gastrointestinal fluids

The survival rates of lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS1.0901 in simulated gastric fluid (pH 3) after 3h can reach 94.37 +/-1.31% and 93.52 +/-0.86% respectively, and the survival rates of lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS1.0901 in simulated intestinal fluid (pH 8) after 8h can reach 91.61 +/-0.95% and 92.40 +/-1.58% respectively, so that the survival rates of lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS1.0901 in the simulated gastric fluid and the simulated intestinal fluid are both more than 90%, indicating that the two have good antagonistic properties to the gastrointestinal simulated fluid.

2.2 adhesion Capacity of strains to HT-29 cells

The adhesion rates of the lactobacillus plantarum K L DS1.0344 and the lactobacillus acidophilus K L DS1.0901 to HT-29 cells are respectively 10.13 +/-0.73% and 13.25 +/-1.29%, and both are more than 10%, which indicates that both have good adhesion capability to HT-29 cells.

2.3 analysis of the Effect of lactic acid bacteria on preventing acute alcoholic liver injury

2.3.1 body weight and liver index analysis

The body weights of the mice were weighed at the beginning and end of the official experiment, respectively, and after dissecting the mice, the mass of the liver was weighed for calculating the liver index, as shown in table 4, the initial body weights and the final body weights of the mice of the alcohol group and the lactic acid bacteria group were not significantly different (p >0.05), the body weight gain of the mice of the alcohol group was significantly lower than that of the control group (p <0.05), while the body weight gain of the mice of the lactic acid bacteria group was higher than that of the alcohol group but was not significant (p >0.05), the liver index of the mice of the alcohol group was significantly higher than that of the control group (p <0.01), and the liver index of the mice of the lactic acid bacteria group was significantly lower than that of the alcohol group (p <0.01), it was shown that the liver index was effectively improved by lactobacillus plantarum K L DS1.0344 in combination with lactobacillus K L DS 1.0901.

TABLE 4 Effect of lactic acid bacteria on mouse body weight and liver index

Note: represents a significant difference compared to the alcohol group mice (p <0.05) and represents a very significant difference compared to the alcohol group mice (p < 0.01).

2.3.2 analysis of the expression level of claudin in the ileum

In order to determine the effect of lactobacillus plantarum K L DS1.0344 in combination with lactobacillus acidophilus K L DS1.0901 on the intestinal barrier, the expression of claudin ZO-1, occludin and claudin-1 in the ileum was examined using immunofluorescence techniques the results are shown in fig. 1, where (a) is the fluorescence intensity of ZO-1, (B) is the fluorescence intensity of occludin, (C) is the fluorescence intensity of claudin-1, magnification × represents a significant difference (p <0.05) compared to the alcoholic mice, a significant difference (p <0.01) compared to the alcoholic mice, the fluorescence intensities of occludin ZO-1, occludin and claudin-1 in the alcoholic mice are significantly reduced (p <0.05, p <0.01 and p <0.01) compared to the control group, and lactobacillus significantly inhibits the fluorescence intensity of claudin-1, occludin and claudin-1 in the ileum (p <0.05, p <0.01) and lactobacillus strains significantly inhibit the decrease of lactobacillus claudin-1 in combination with lactobacillus acidophilus K3875, thereby improving the intestinal barrier expression of lactobacillus claudin K1.0344.

2.3.3 serum A L T, AST and L PS level analysis

In order to determine the effect of lactobacillus plantarum K L DS1.0344 in combination with lactobacillus acidophilus K L DS1.0901 on liver function, a L T and AST levels in serum were determined as shown in fig. 2 a and B, the a L T and AST levels in serum of the alcohol group mice were significantly increased (p <0.01) compared to the control group, indicating impaired liver function, whereas the lactic acid bacteria significantly inhibited the increase of both (p <0.01), indicating that lactobacillus plantarum K L DS1.0344 in combination with lactobacillus acidophilus K L DS1.0901 was able to improve liver function.

As shown in C in fig. 2, L PS content in serum of mice in the alcohol group was significantly increased (p <0.01) compared to the control group, L PS content in serum of mice in the lactic acid bacteria group was significantly decreased (p <0.01) compared to the alcohol group, (a) was serum a L T level, (B) was serum AST level, and (C) was serum L PS level, which represents a very significant difference (p <0.01) compared to the mice in the alcohol group.

2.3.4 histopathological analysis of liver

As shown in figure 3 (H & E staining, magnification × 200), the liver tissue structure of the control group mouse is normal, no obvious histopathological injury is seen, the liver of the alcohol group mouse has focal necrosis, inflammatory cell infiltration and slight blood stasis in liver sinuses, the liver injury of the lactobacillus group mouse is obviously improved, only slight granular degeneration exists, and the combination of the lactobacillus plantarum K L DS1.0344 and the lactobacillus acidophilus K L DS1.0901 can effectively prevent the histopathological injury.

2.3.5 analysis of inflammation levels in the liver

L PS induces pro-inflammatory cytokine production in the liver, and excessive secretion of TNF- α and I α 0-6 results in inflammation production, and thus levels of TNF- α and I α -6 in mouse liver were examined as shown in a and B in fig. 4, levels of TNF- α and I L-6 in liver of mice in the alcohol group were significantly increased (p <0.01) compared to the control group, and levels of TNF- α and I L-6 in liver of mice in the lactobacillus group were significantly decreased (p <0.01) compared to the alcohol group, MPO activity was an important indicator for quantifying the degree of inflammation, as shown in C in fig. 4, the activity of MPO in liver of mice in the alcohol group was significantly higher than that of control group (p <0.01), while lactobacillus significantly inhibited the increase of MPO activity (p <0.01) compared to the liver of mice in the lactobacillus group, it was shown that lactobacillus K L DS1.0344 in combination with lactobacillus K L was significantly decreased (p 3) (p <0.01) and that the level of liver was significantly decreased as shown in liver B42 (p < 0.42) compared to the liver).

2.3.6 protein expression analysis of T L R4 and NF-kB signal channel related gene in liver

To further investigate whether lactobacillus plantarum K L DS1.0344 in combination with lactobacillus acidophilus K L DS1.0901 reduces the level of inflammation in the liver by modulating T1.0901 2R 1.0901 receptors and their downstream NF- κ B signaling pathways, we examined the significant increase in the expression levels of T1.0901R 1.0901 on the cell membrane, p 1.0901 in the cell nucleus, and I1.0901B 1.0901 in the cytoplasm as shown in fig. 5, the expression levels of T1.0901R 1.0901 on the cell membrane and p 1.0901 protein in the cell nucleus of mice in the alcohol group (p <0.01), the significant decrease in the expression levels of I1.0901B 1.0901 in the cytoplasm (p <0.01) compared to the control group, while the expression levels of T1.0901R 1.0901 protein in the cell membrane and p 1.0901 protein in the cell nucleus of the lactobacillus group mice (p <0.01), the significant increase in the expression levels of I1.0901B in the cytoplasm (p <0.01) compared to the expression levels of T1.0901B 1.0901 in the cell membrane and its downstream NF-1.0901B 1.0901 (p 1.0901B 3B) showed that the p < 0.3672B 3B signal was significantly decreased as shown by the p < 0.01).

2.3.7 analysis of lipid levels in liver

As shown in fig. 6, TG content in liver of mice in the alcohol group was significantly increased (p <0.01) compared to the control group, while intervention of lactic acid bacteria significantly suppressed its increase (p <0.01), indicating that pretreatment of lactobacillus plantarum K L DS1.0344 in combination with lactobacillus acidophilus K L DS1.0901 could prevent lipid accumulation in liver of mice in fig. 6, representing significant difference (p <0.05) compared to mice in the alcohol group, and representing significant difference (p <0.01) compared to mice in the alcohol group.

2.3.8 analysis of antioxidant levels in liver

As shown in fig. 7, the activities of SOD, GPx and CAT in liver of mice in alcohol group were significantly decreased (p <0.01), the MDA content was significantly increased (p <0.01), compared to the control group, the activities of SOD, GPx and CAT in liver of mice in lactic acid bacteria group were significantly increased (p <0.01 or p <0.05), and the MDA content was significantly decreased (p <0.01), compared to the alcohol group, the above results showed that the intervention of lactobacillus plantarum K L DS1.0344 in combination with lactobacillus acidophilus K L DS1.0901 could increase the antioxidant level in liver of mice, in fig. 7, (a) liver SOD activity, (B) liver GPx activity, (C) liver activity, (D) liver MDA content represented a significant difference (p <0.05) compared to mice in alcohol group, and (p <0.01) compared to mice in liver group.

2.4 analysis of the action of lactic acid bacteria in preventing chronic alcoholic liver injury

The method comprises the steps of intragastrically feeding mice fed with L ieber-Decalli liquid feed with alcohol by using mixed bacterial suspensions of different doses of lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS1.0901 every day, taking silymarin for treating alcoholic liver injury as a positive treatment control, researching the effect of lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS1.0901 on preventing chronic alcoholic liver injury in the mice fed with L ieber-Decalli liquid feed with alcohol, selecting mixed bacterial suspensions of lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS1.0901 with obvious prevention effect, and further excavating the action mechanism of preventing the chronic alcoholic liver injury.

2.4.1 body weight and liver index analysis

After the start of the official experiment, the body weight of the mice was measured once a week, and after dissecting the mice, the livers were weighed for calculation of the liver index. As shown in a in fig. 8, before the start of the actual experiment, the body weight difference of each group of mice was insignificant (p >0.05), and by week 6, the body weight of the alcohol group of mice was significantly reduced (p <0.05) compared to the control group, while the body weight of the low-dose lactic acid bacteria group and the lactic acid bacteria high-dose group of mice was significantly increased (p <0.01) compared to the alcohol group, wherein the body weight of the lactic acid bacteria high-dose group of mice was closer to the silymarin group.

The liver index of the alcohol group mice was significantly higher than that of the control group (p <0.01), while the liver index of the low dose lactic acid bacteria group, the lactic acid bacteria high dose group and the silymarin group mice was significantly lower than that of the alcohol group (p <0.05, p <0.01 and p <0.01), the lactic acid bacteria high dose group was closer to the silymarin group. In FIG. 8, (A) mouse body weight and (B) liver index. Represents a significant difference compared to the alcohol group mice (p <0.05) and represents a very significant difference compared to the alcohol group mice (p < 0.01).

2.4.2 analysis of the expression level of claudin in the ileum

As shown in FIG. 9, the expression of Claudin ZO-1, occludin and claudin-1 was significantly decreased in the ileum of mice in the alcohol group compared to the control group (p < 0.01). The expression of Claudin ZO-1, occludin and claudin-1 was significantly increased in the ileum of mice in the low dose group of lactic acid bacteria and the high dose group of lactic acid bacteria compared to the alcohol group (p <0.01 or p <0.05), wherein the expression of Claudin ZO-1, occludin and claudin-1 in the ileum of mice in the high dose group of lactic acid bacteria was equivalent to that of the silymarin group.9, (A, D) the fluorescence intensity of ZO-1, (B, E) the fluorescence intensity of occludin, (C, F) the fluorescence intensity of claudin-1, and the magnification × 200 represents a significant difference compared to mice in the alcohol group (p <0.05) and the mouse represents a significant difference to alcohol group (p < 0.01).

2.4.3 serum A L T, AST and L PS level analysis

A L T and AST water in serum are commonly used as indices for evaluating liver function, as shown in A and B in FIG. 10, chronic alcohol exposure significantly increased the A L T and AST levels (p <0.01) in mouse serum compared to the control group, indicating impaired liver function, A L T and AST levels (p <0.05) in mouse serum of the low-dose lactobacillus group were significantly reduced compared to the alcohol group, A L T and AST levels (p <0.01) in mouse serum of the high-dose lactobacillus group were extremely significantly reduced, having the same tendency as that of the silymarin group, showing that the mixed bacterial suspension of Lactobacillus plantarum K L DS1.0344 and Lactobacillus acidophilus K L DS1.0901 at high dose could more effectively prevent the impaired liver function of mice.

To determine the effect of lactobacillus plantarum K L DS1.0344 in combination with lactobacillus acidophilus K L DS1.0901 on L0 PS content in serum, L1 PS content in mouse serum was examined as shown in C in fig. 10, L PS content in mouse serum was significantly higher in the alcohol group than in the control group (p <0.01), L PS content in mouse serum was significantly lower in the low dose lactobacillus group than in the alcohol group (p <0.05), L PS content in mouse serum was significantly lower in the high dose lactobacillus group than in the alcohol group (p <0.01), and was closer to the silymarin group, indicating that mixed bacterial suspensions of lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS1.0901 at high doses were able to more effectively reduce L PS content in mouse serum, in fig. 10, (a) serum a L T level, (B) AST level, (C) L PS level represents a significant difference compared to the mouse serum (p <0.05) and the alcohol group (p < 0.01).

2.4.4 histopathological analysis of liver

As shown in FIG. 11 (H & E staining, magnification × 200), the liver tissue structure of the control group mice was normal, and no significant histopathological damage was observed, and a large number of lipid droplets appeared in the liver of the alcohol group mice accompanied by the degeneration of the vacuoles, the liver damage of the low dose lactobacillus group mice was significantly improved, including the reduction of lipid droplets and the transformation of the degeneration of the vacuoles into the degeneration of the granules, the improvement effect of the high dose group of lactobacillus was more significant, and only very few lipid droplets existed, similar to the results observed for the silymarin group, and the above results showed that the mixed bacterial suspension of the high dose lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS1.0901 could more effectively prevent the pathological damage of the liver tissue, and in FIG. 11, A-E were respectively the control group, the alcohol group, the low dose group of lactobacillus, the high dose group of lactobacillus, and the silymarin group.

2.4.5 analysis of lipid levels in liver

To further investigate the effect of lactobacillus plantarum K L DS1.0344 in combination with lactobacillus acidophilus K L DS1.0901 on lipid levels in mouse livers, the content of TG in mouse livers was determined as shown in fig. 12, chronic alcohol intake significantly increased the TG content in mouse livers (p <0.01) compared to the control group, TG content in mouse livers was significantly decreased in low dose lactobacillus group (p <0.05) compared to the alcohol group, TG content in mouse livers was significantly decreased in both lactobacillus high dose group and silymarin group (p <0.01), with the same trend, indicating that mixed bacterial suspensions of lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS1.0901 at high doses had a stronger ability to inhibit lipid accumulation in mice in alcohol group (p <0.05), and mice in alcohol group (p < 0.01).

2.4.6 analysis of antioxidant levels in liver

The above antioxidant indices of mice in the alcohol group were significantly decreased in liver (p <0.01), and the MDA content was significantly increased (p <0.01), compared to the control group, the low dose intervention of lactic acid bacteria and the high dose intervention of lactic acid bacteria significantly improved the above antioxidant indices (p <0.05 and p <0.01), compared to the alcohol group, wherein the effect after the high dose intervention of lactic acid bacteria was close to silymarin, these results indicate that the mixed suspensions of lactobacillus plantarum K L DS1.0344 and lactobacillus K L DS1.0901 in the high dose were more effective in increasing the antioxidant level of liver of body, (a) liver GPx activity, (B) liver gx activity, (C) liver MDA activity, (D) represents significant difference in liver content from the liver of mice (p <0.01), compared to the alcohol group (p < 0.01).

2.4.7 analysis of inflammation level in liver

As shown in fig. 14, TNF- α, I L-6 and MPO levels in mouse liver were significantly increased (p <0.01), MPO activity was significantly increased (p <0.01), supplementation with low-dose lactic acid bacteria significantly decreased TNF- α and I L-6 levels (p <0.05), MPO activity was significantly decreased (p <0.05), supplementation with high-dose lactic acid bacteria significantly decreased TNF- α and I L-6 levels and MPO activity in mouse liver (p <0.01), comparable to silymarin intervention, indicating that mixed suspensions of high-dose lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS1.0901 were more effective in inhibiting inflammation in mouse liver, inflammation production in mouse liver, (a) was induced by (a) t 62, and (I) t 2d, (p <0.05) representing a significant difference in mouse liver activity, after chronic alcohol intake, i.1-6 and I466 levels (p < 0.05).

2.4.8 analysis of intestinal flora

In addition, the abundance of enterobacteria L, acetobacter asiatica, bifidus, atobia, ak L, eosonococcaceae and erysiperioides in the intestine of mice in the alcohol group was decreased compared to the control group at the family level, while the dry prognosis of mixed suspensions of lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS1.0901 was improved in the intestine of mice in the alcohol group compared to the control group, while the abundance of enterobacteria DS 3623, lactobacillus acidophilus 3, lactobacillus acidophilus, pseudomonas aeruginosa, lactobacillus acidophilus, desuccineae and lactobacillus acidophilus L DS1.0901 was increased in the intestine of mice in the alcohol group compared to the control group, while the abundance of lactobacillus acidophilus DS 3623 and lactobacillus acidophilus 4934 was increased in the mixed suspensions of lactobacillus acidophilus DS.

At the genus level, L Acobacillus, Bifidobacterium, Erysipellucidium, Faecalibacillus, Enterobacter and Marvinbryantia were found to be less abundant in the intestines of the alcoholic mice than the control group, while the abundance of these genera was improved after dry-treatment of the mixed suspension of high doses of Lactobacillus plantarum K L DS1.0344 and Lactobacillus acidophilus K L DS 1.0901.

2.4.9 analysis of content of short-chain fatty acid in intestinal tract

Changes in the intestinal flora lead to changes in their metabolites, which are followed by the determination of short chain fatty acids in intestinal microbial metabolites using GC-MS. As shown in fig. 15, the content of acetic acid, propionic acid and butyric acid in the intestinal tract of the alcohol group mice was significantly reduced compared to the control group (p <0.05, p <0.05 and p < 0.01). Compared with the alcohol group, the content of acetic acid, propionic acid and butyric acid in intestinal tracts of the lactobacillus high-dose mice is remarkably increased (p <0.01, p <0.05 and p < 0.01). This is consistent with the changes in intestinal flora described above.

2.4.10 analysis of protein expression of genes related to AMPK Signal pathway in liver

The protein expression levels of AMPK signaling pathway-related genes in mouse liver were determined using Western blot method as shown in fig. 16, and the protein expression levels of AMPK, PPAR- α and CPT-1 in mouse liver were significantly reduced in the alcohol group compared to the control group (p <0.01), while the protein expression levels of ACC, SREBP-1 and FAS were significantly increased in the alcohol group (p <0.01), the protein expression of AMPK, PPAR- α and CPT-1 in mouse liver was significantly increased in the mixed suspension of lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS1.0901 compared to the alcohol group (p <0.01, p <0.01 and p <0.05), the protein expression of ACC, SREBP-1 and FAS (p <0.01), and the protein expression of PPAR-351 was significantly reduced in mouse liver (p <0.01), and the protein expression of AMPK-csa, PPAR-358 and CPT-1 was significantly decreased in mouse liver, (p <0.01, p < 0.493) and the protein expression level of AMPK, PPAR-csa protein expression level of protein in mouse liver was significantly decreased in mouse liver (p <0.01, p < 28, csa) compared to the mixed protein expression of mouse liver, and protein expression of PPAR-csa 3, a protein expression of mouse liver was significantly decreased in mouse liver, a < 3, a 3, and fab < 3, and p < 3, respectively, (p < 3, and p < 3, showing that protein expression of mouse liver, and fab.

2.4.11 protein expression analysis of T L R4 and NF-kB signal channel related gene in liver

As shown in fig. 17, the protein expression levels of T L R4 receptor on the cell membrane of liver and p65 in the cell nucleus were significantly increased (p <0.01), the protein expression level of I κ B α in the cytoplasm was significantly decreased (p <0.01) in the alcohol group mice compared to the control group, while the protein expression levels of T L R4 receptor on the cell membrane of liver and p65 in the cell nucleus were significantly decreased (p <0.01) and the protein expression level of I κ B α in the cytoplasm was significantly increased (p <0.01) in the lactobacillus high dose group mice compared to the alcohol group, which indicates that the intervention of the mixed bacterial suspension of lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS1.0901 prevented the occurrence of inflammation by inhibiting the NF- κ B signal pathway in fig. 17, (a) T L R4 protein expression level, (B) p65 protein expression level, (C) I к B α protein expression level represents a significant difference from the alcohol group (p < 0.01).

2.4.12 analysis of CYP2E1 protein expression in liver

The level of protein expression of CYP2E 3875 in the liver of mice was significantly increased by alcohol intake (p <0.01) compared to the control group as shown in figure 18, the intervention of a mixed suspension of lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS1.0901 at high doses significantly decreased the protein expression of CYP2E1 in the liver of mice compared to the alcohol group (p <0.01) in figure 18, a marked difference (p <0.01) compared to the alcohol group mice is represented by CYP2E1, which is generated by CYP2E1 when oxidizing alcohol to acetaldehyde.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Claims (10)

1. The lactobacillus formula for preventing acute and chronic alcoholic liver injury is characterized by comprising lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS 1.0901.

2. The lactic acid bacteria formula for preventing acute and chronic alcoholic liver injury according to claim 1, wherein the volume ratio of lactobacillus plantarum K L DS1.0344 to lactobacillus acidophilus K L DS1.0901 is 1:1, and the cell concentration of lactobacillus plantarum K L DS1.0344 and lactobacillus acidophilus K L DS1.0901 is 10⁸~10¹⁰CFU/mL。

3. Use of a formulation according to any one of claims 1 to 2 for the prevention of acute and chronic alcoholic liver injury.

4. Use according to claim 3, for reducing the liver index.

5. Use according to claim 3, for improving the intestinal barrier, inhibiting the reduction of the expression of the claudin-1, occludin and claudin-1 tight junction proteins in the ileum.

6. The use according to claim 3 for improving liver function, inhibiting increased levels of A L T and AST in liver serum, reducing L PS levels in liver serum, and inhibiting liver inflammation.

7. The use according to claim 3, for reducing the level of inflammation in the liver, for reducing the levels of TNF- α and I L-6 in the liver, and for inhibiting an increase in MPO activity.

8. The use according to claim 3, characterized by the use for reducing the level of inflammation in the liver, reducing the expression of T L R4 on the cell membrane of the liver and p65 protein in the nucleus and increasing the expression of I к B α protein in the cytoplasm.

9. Use according to claim 3, for increasing the content of short chain fatty acids in the gut, the short chain fatty acids being involved as signalling molecules in the regulation of the Nrf-2 signalling pathway closely associated with oxidative stress, thereby inhibiting Nrf-2 in the nucleus of the liver and the reduction of protein expression levels of HO-1, NQO1, SOD1, CAT and GPx-1 in the cytoplasm.

10. Use according to claim 3, for reducing the protein expression of CYP2E1 in the liver.

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