CN114806979A

CN114806979A - Probiotic compound for treating non-alcoholic fatty liver disease and application thereof

Info

Publication number: CN114806979A
Application number: CN202210745871.0A
Authority: CN
Inventors: 张彬彬; 付金龙; 施军平
Original assignee: Affiliated Hospital Of Hangzhou Normal University (hangzhou Second People's Hospital)
Current assignee: Affiliated Hospital Of Hangzhou Normal University (hangzhou Second People's Hospital)
Priority date: 2022-06-29
Filing date: 2022-06-29
Publication date: 2022-07-29

Abstract

The invention discloses a probiotic compound for treating non-alcoholic fatty liver disease and application thereof, wherein the probiotic compound comprises probiotics of Bradychis, Roseberry and Saccharomyces cerevisiae, and the volume ratio of the probiotics of Bradychis, Roseberry and Saccharomyces cerevisiae is 0.1-10: 0.1-7: 0.2-5; the invention regulates and controls the key link of the non-alcoholic fatty liver disease, not only can effectively control the occurrence and development of the disease course of the non-alcoholic fatty liver disease, but also can improve insulin resistance and adjust lipid metabolism abnormity; meanwhile, the medicine makes up the vacancy that no medicine on the market treats NAFLD, and is convenient to use, safe and effective.

Description

Probiotic compound for treating non-alcoholic fatty liver disease and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a probiotic compound for treating non-alcoholic fatty liver disease and application thereof.

Background

Nonalcoholic fatty liver disease (NAFLD) is a complex metabolic-related disease that affects approximately 1/4 worldwide population and constitutes a significant health and economic burden on society. NAFLD is closely related to T2DM and abdominal obesity, and its global prevalence is dramatically increasing with changes in lifestyle and dietary habits. NAFLD is common in all continents with the highest incidence in south america (31%) and middle east (32%), followed by asia (27%), the united states (24%) and europe (23%), and the least incidence in africa (14%). In particular, its histological phenotype, NASH, may progress to advanced liver disease, cirrhosis and hepatocellular carcinoma (HCC). The prevalence of NAFLD continues to increase (15% in 2005 and 25% in 2010), and as such, the incidence of NASH almost doubles (5.91% to 33%) over the same period of time. Now, NASH is considered the second most common liver transplantation indication in the united states following chronic viral hepatitis, and is also continuing to increase.

The primary goals for NAFLD, as indicated in the "guidelines for the prevention and treatment of non-alcoholic fatty liver disease" (2018 edition), are to reduce weight and improve IR, to prevent metabolic syndrome (MetS), T2DM and its associated complications, and the secondary goals are to reduce liver fat deposition, inhibit liver fibrosis and cirrhosis progression. The current treatment of NAFLD has no specific drug application and clinical application related to lipid deposition, and the current drug therapy is still oriented to adjuvant therapy.

The causes of NAFLD, although due to stress caused by an excessive supply of nutrients by the liver, in particular fructose, glucose and fatty acids, have now been investigated to indicate that intestinal micro-ecological disorders are an important pathological mechanism for the development of NAFLD, which acts mainly through the "gut-liver" axis. The gut and liver communicate extensively through the biliary tract, portal vein and systemic circulation, and this bidirectional crosstalk is called the "gut-liver" axis.

It has also been found that intestinal flora can influence the progression of NAFLD disease. Gut microbiota and bacterially derived metabolites may contribute to the development and progression of NAFLD through a variety of mechanisms including increased lipid absorption, energy absorption, increased gut permeability and induction of chronic inflammation. The feces of a metabolic synthesis model mouse fed with HFD diet are transplanted to a sterile recipient mouse, and the hepatic lipid synthesis of the recipient mouse is induced, so that the triglyceride content in the liver of the recipient mouse is increased by 3 times, and the expression of lipogenic genes in the liver is increased. In addition, gut flora microorganisms can regulate the progression of NAFLD-HCC, whose gut microbial structure is more disordered than healthy people, while bacterial extracts from NAFLD-HCC microorganisms can induce expansion of T cells and reduction of CD8+ T cells.

Currently, no medicine capable of improving hepatic fibrosis is on the market, and a new clinical bacterial therapy strategy is to use specific prebiotics, probiotics, synbiotics and excrement of healthy volunteers for transplantation to restore the balance of intestinal microflora, so that the prevention and treatment of metabolic diseases such as NASH are attracting attention. The study of the scientific research team of Lianjuan academy of China at the institute of Engineering, Engineering, 2021 at 5 th written text states that the most concerned probiotics in the treatment of nonalcoholic fatty liver disease mainly include the traditional probiotics represented by the genus Bifidobacterium and the genus Lactobacillus and the next generation of emerging probiotics, such as Ackermanomyces muciniphila and Clostridium pralatum.

In a human intestinal bacteria study, it was shown that the control of the weight of obese patients by transplantation of the stools of healthy lean donors to obese patients could be associated with a significant increase in the proportion of Roseburia intestinalis bacteria of butyrate producers. In a weight loss study, it was found that the abundance of Blautia wexlerae (MGS0575) and Bacteroides dorei (MGS0187) bacteria was significantly positively correlated with weight loss in patients, suggesting that these two bacteria are potential probiotics for weight loss.

Disclosure of Invention

The invention provides a probiotic compound for treating non-alcoholic fatty liver disease and application thereof, aiming at solving the problems in the prior art.

The scheme of the invention is as follows:

a probiotic compound for treating non-alcoholic fatty liver disease comprises probiotics Blauett bacteria, Roseburia bacteria and Saccharomyces cerevisiae, wherein the volume ratio of the probiotics Blauett bacteria, Roseburia bacteria and Saccharomyces cerevisiae is 0.1-10: 0.1-7: 0.2-5. Blauettia (Blautia wexlerae), Raosbai (Roseburia intestinalis) and Saccharomyces cerevisiae (Saccharomyces cerevisiae Hansen) were purchased from the Japanese type culture Collection (JCM).

As a preferred technical scheme, the volume ratio of the probiotics of the blautia genus, the Roseburia formula and the saccharomyces cerevisiae is 1:1: 1.

As a preferred technical scheme, the preparation method comprises the steps of respectively preparing suspensions of probiotics of blautia, Roseburia and saccharomyces cerevisiae according to the density of 1 x 10^8cfu/ml, and then mixing the suspensions according to the volume ratio.

The invention also discloses a method for preparing the probiotic compound for treating the non-alcoholic fatty liver disease, which is characterized by comprising the following steps:

1) carrying out metagenome detection on the feces of the NO-NASH and NASH patients, and screening probiotics obviously enriched in the NO-NASH to obtain obviously enriched probiotics, namely Brauteria bacteria;

2) culturing the probiotics of the blautia, the Roseburia and the saccharomyces cerevisiae in an anaerobic box according to an official net culture medium, and inoculating according to the density of 1 x 10^8 cfu/ml;

3) mixing probiotics of Bradychii, Roseburia bacteria and saccharomyces cerevisiae according to the volume ratio of 0.1-10: 0.1-7: 0.1-5 to obtain suspension, and storing the suspension in a refrigerator at the temperature of-80 ℃ for later use.

As a preferable technical scheme, in the step 3), the probiotics of the blautia genus and the Roseburia formula are mixed with the saccharomyces cerevisiae according to the volume of 1:1: 1.

As a preferable technical scheme, the probiotic blautia is cultured by using a culture medium 104 at 37 ℃; roseburia strain was cultured in 1611 medium at 37 ℃; saccharomyces cerevisiae is cultured in YM medium at 25-28 deg.C.

The invention also discloses application of the probiotic compound for treating the non-alcoholic fatty liver disease, and application of the probiotic compound in treating the non-alcoholic fatty liver disease, improving insulin resistance and adjusting lipid metabolism disorder.

Due to the adoption of the technical scheme, the probiotic compound for treating the non-alcoholic fatty liver disease and the application thereof comprise probiotics of blautia, Roseberry and saccharomyces cerevisiae, wherein the volume ratio of the probiotics of blautia, Roseberry and saccharomyces cerevisiae is 0.1-10: 0.1-7: 0.2-5.

The invention has the advantages that:

the invention carries out metagenomic analysis on feces collected from NO-NASH and NASH patients, screens out probiotics obviously enriched in NO-NASH for combination, and provides a probiotic compound for treating non-alcoholic fatty liver disease; meanwhile, the medicine makes up the vacancy that no medicine on the market treats NAFLD, and is convenient to use, safe and effective.

Drawings

FIG. 1 is a graph showing the comparison of the body weight, liver weight and fat weight of a mouse in an experiment of the present invention; wherein (A) the weight of the mouse (B) the weight of the mouse liver (C) the weight of the mouse fat; ctrl is normal diet group, HFD is high fat diet group, HFD + prebiotic is high fat diet + Probiotic permanent planting group; n =6, P <0.05, P < 0.01; the # is P <0.05, and the # is P < 0.01;

FIG. 2 is a graph showing the comparison of ALT, AST, TC and TG levels in serum in the experiment of the present invention; wherein (A) serum ALT content (B) serum AST content (C) serum TC content (D) serum TG content; ctrl is normal diet group, HFD is high fat diet group, HFD + prebiotic is high fat diet + Probiotic permanent planting group; n =6, P <0.05, P < 0.01;

FIG. 3 is a graph of a mouse insulin tolerance and glucose tolerance test in an experiment of the present invention; wherein (A) the insulin tolerance of the mice (B) the glucose tolerance of the mice, Ctrl is a normal diet group, HFD is a high fat diet group, and HFD + Probiotic is a high fat diet + Probiotic permanent planting group; n =6, P <0.05, P < 0.01;

FIG. 4 is a graph showing the hematoxylin-eosin staining and the oil red O staining of the liver of a mouse in an experiment of the present invention; wherein (A) the liver tissue of the mouse is stained by hematoxylin and eosin (B) the liver of the mouse is stained by oil red O, Ctrl is a normal diet group, HFD is a high-fat diet group, and HFD + Probiotic is a high-fat diet + Probiotic permanent planting group; n =6, P <0.05, P < 0.01.

Detailed Description

In order to make up for the above deficiencies, the present invention provides a probiotic composition for treating non-alcoholic fatty liver disease and its application to solve the problems of the background art.

The volume ratio of the probiotics of the blautia bacteria, the Roseburia bacteria and the saccharomyces cerevisiae is 1:1: 1.

Comprises probiotic bacteria of the genus Brabender, the genus Roseburia and Saccharomyces cerevisiae, which are respectively prepared into suspension according to the density of 1 x 10^8cfu/ml, and then are mixed according to the volume ratio.

1) metagenomic detection is carried out on the feces of patients with NO-NASH and NASH, and probiotics which are obviously enriched in NO-NASH are screened;

The probiotics of the Brabender genus, the Roseburia genus and the saccharomyces cerevisiae in the step 3) are mixed according to the volume of 1:1: 1.

The probiotic blautia is cultured by using a culture medium of 104 at 37 ℃; roseburia strain was cultured in 1611 medium at 37 ℃; saccharomyces cerevisiae is cultured in YM medium at 25-28 deg.C.

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

Example 1:

1) carrying out metagenome detection on the feces of patients with NO-NASH and NASH, and screening probiotics which are obviously enriched in the NO-NASH;

3) mixing probiotic bacteria of Blauteria, Roseburia bacteria and Saccharomyces cerevisiae at a volume ratio of 1:1:1 to obtain suspension, and storing in a refrigerator at-80 deg.C for use.

Example 2:

3) mixing probiotic bacteria of Blauteria, Roseburia bacteria and Saccharomyces cerevisiae at a volume ratio of 0.1:0.1:0.2 to obtain suspension, and storing in a refrigerator at-80 deg.C for use.

Example 3:

3) mixing probiotic bacteria of Blauteria, Roseburia bacteria and Saccharomyces cerevisiae at a volume ratio of 10:7:5 to obtain suspension, and storing in a refrigerator at-80 deg.C for use.

Experiment:

1. probiotic ratio

According to previous work, feces of patients with NO-NASH and NASH are collected for metagenomic detection, Probiotics which are obviously enriched in NO-NASH are screened, the obviously enriched Probiotics of Blautia (Blautia wexlerae), Roseburia instrinis (Roseburia intestinalis) and Saccharomyces cerevisiae (Saccharomyces cerevisiae Hansen) are purchased from a Japan typical strain preservation center (JCM), culture is carried out in an anaerobic box according to an official culture medium, suspension is manufactured according to the density of 1: 10^8cfu/ml, combination is carried out according to the proportion of example 1, wherein the effect of treating NAFLD mice by mixing three Probiotics according to the proportion of 1:1:1 is found to be most obvious, the three Probiotics are inoculated according to the density of 1: 10^8cfu/ml, suspension is manufactured according to the volume ratio of 1:1: 1:1 (Probiotics), and a refrigerator is kept at 80 ℃ for standby.

NAFLD mouse establishment

C57BL/6J Male mice, 6-8 weeks old, SPF grade, were purchased from Shanghai Slek laboratory animal center. After 1 week of adaptive feeding, the patients were divided into normal diet group (Ctrl, 10% calorie derived from fat, 12450B, Research Diets) and high fat diet group (HFD, 60% calorie derived from fat, D12492, Research Diets) according to the random number table, and NAFLD model was established for 8 weeks

3. Pseudo-germ free mouse establishment

According to the internationally accepted guidelines for antibiotic use, ABX, where mice were treated with the combination of the broad-spectrum antibiotics Ampicillin (Ampicillin, 1 g/L), Neomycin (Neomycin, 1 g/L), Vancomycin (Vancomycin, 0.5 g/L) and drinking water (ABX) for 2 weeks, a pseudo-sterile mouse model was established in which intestinal flora was extensively suppressed.

4. Grouping and administration of mice

After 8 weeks of HFD diet induction, 2 weeks after ABX-depleted enterobacteria administration, probiotic transplantation was given, and the experiment was terminated after 6 weeks, specifically grouped as follows:

1) normal diet group + physiological saline (Ctrl)

2) High fat diet group + ABX + physiological saline (HFD)

3) High fat diet group + ABX + probiotic colonization (HFD + Probiotics)

And finishing the experiment after 6 weeks, detecting the body weight, the liver weight, the glucose tolerance, the insulin tolerance and the serum biochemical indexes of the mouse, observing the liver steatosis degree of the mouse through histopathology, and observing the influence of the probiotics on the NAFLD mouse model.

The animal experiment process of the invention is as follows:

experiments prove the efficacy of the probiotic compound on the high-fat diet-induced non-alcoholic fatty liver disease of C57 mice. The invention has the following beneficial effects: the probiotic compound is given for field planting, the weight, the liver weight and the epididymal fat weight of the high-fat diet-induced NAFLD mouse can be obviously inhibited, serological indexes show that the probiotic transplantation improves the insulin tolerance and the glucose tolerance of the high-fat diet-induced NAFLD mouse, the activity of serum hepatoma, the content of serum cholesterol and liver triglyceride are reduced, and further, the hematoxylin-eosin and oil red O staining of the liver can show that the probiotic can inhibit the liver steatosis and the fat drop deposition.

The probiotic supplement can obviously improve the progress of the NAFLD mouse, and is probably related to the recovery of the high-fat diet induced NAFLD mouse intestinal flora disorder through probiotic colonization, the inhibition of lipid absorption in the intestinal tract, the correction of the metabolic disorder of intestinal microorganisms, the recovery of the damage of the intestinal tract barrier, and the inhibition of harmful substances from entering the liver through the intestinal barrier and the portal vein to cause the liver inflammatory reaction.

The body weight results of the mice show that the body weight of the mice in the high-fat diet group is obviously increased (P < 0.01) compared with the normal group, and the body weight of the mice in the probiotic group is obviously reduced (P < 0.01) compared with the high-fat diet group (A in figure 1); the results of the liver weights of the mice showed that the weight of the mice on the high fat diet group was significantly increased (P < 0.01) compared to the normal group, and the weight of the mice on the probiotic group was significantly decreased (P < 0.05) compared to the high fat diet group (B in fig. 1); the results of the fat weight of the mice showed a significant increase in body weight (P < 0.01) in the high-fat diet group compared to the normal group and a significant decrease in body weight (P < 0.05) in the probiotic group compared to the high-fat diet group (C in fig. 1).

TABLE 1 weight of mice, liver weight and fat weight Table

Group of	N	Body weight (g)	Liver weight (g)	Weight of fat (g)
					Ctrl	6	22.77±0.7342##	0.7571±0.0546***	0.2289±0.0584***
HFD	6	33.76±2.570	1.501±0.3207	1.440±0.4535
					HFD+Probiotic	6	30.01±2.402**	1.145±0.2623*	0.9630±0.3081*

Note: ctrl is normal diet group, HFD is high fat diet group, HFHS + prebiotic is high fat diet + Probiotic permanent planting group; n =6, isP<0.05P<0.01, compared to the HFD group.

Biochemical index results of mice showed that serum ALT was significantly increased in mice on the high-fat diet (P < 0.01) compared to the normal group and significantly decreased in mice on the probiotic group (P < 0.01) compared to the high-fat diet group (a in fig. 2); serum AST was significantly increased in mice of high fat diet group compared to normal group (P < 0.01), and significantly decreased in mice of probiotic group compared to high fat diet group (P < 0.05) (B in fig. 2); serum TC was significantly increased in mice on the high-fat diet (P < 0.01) compared to the normal group and significantly decreased in mice on the probiotic group (P < 0.01) compared to the high-fat diet group (C in fig. 2); the liver TG was significantly increased in mice on the high-fat diet group compared to the normal group (P < 0.01) and significantly decreased in mice on the probiotic group compared to the high-fat diet group (P < 0.05) (D in fig. 2).

TABLE 2 serum ALT, AST, TC and TG content table

Group of	N	ALT（IU/L）	AST（IU/L）	TC（mmoL/L）	TG（mmoL/L）
						Ctrl	6	12.50±2.197***	11.38±1.923***	2.651±0.4362***	5.525±1.259**
HFD	6	51.98±13.89	49.98±17.33	6.787±0.7999	13.55±3.282
						HFD+Probiotic	6	22.64±7.354**	29.43±16.49*	4.881±0.3391**	7.815±4.701*

The results of insulin tolerance and glucose tolerance of mice show that the area under the insulin tolerance curve of the mice in the high-fat diet group is obviously increased (P < 0.01) compared with the normal group, and the area under the insulin tolerance curve of the mice in the probiotic group is obviously reduced (P < 0.05) compared with the mice in the high-fat diet group, thereby prompting that the probiotics improves the insulin tolerance injury induced by the high-fat diet (A in figure 3); the area under the glucose tolerance curve of mice on the high fat diet was significantly increased (P < 0.01) compared to the normal group, and significantly decreased (P < 0.05) compared to the high fat diet group, suggesting that the probiotics improved the high fat diet-induced glucose tolerance impairment (B in fig. 3).

TABLE 3 Experimental Table for insulin tolerance and glucose tolerance in mice

Group of	N	Insulin resistance AUC area (g)	AUC area of glucose tolerance
				Ctrl	6	11.19±1.492***	6.506±1.147**
HFD	6	15.45±0.9945	11.75±3.753
				HFD+Probiotic	6	13.07±1.653*	7.579±0.3350*

To further observe the effect of probiotics on high fat diet induced fatty degeneration of liver tissue in NAFLD mice, hematoxylin-eosin staining was performed on liver tissue of each group of mice, and H & E staining (200 fold) of paraffin sections of liver tissue of each group showed: the normal group of mice has the advantages of compact liver tissue structure, normal arrangement, cytoplasmic red staining, nucleus centering, no abnormal liver cell morphology, no liver cord broadening, and no inflammatory cell infiltration in central venous and manifold areas: the liver tissue structure of mice in a high-fat diet group is loose, the liver cell swelling is obvious, the cytoplasm is lightly stained, the nucleus is centered or is deviated to one side, a large amount of lipid droplets are contained, the diffuse steatosis of the liver cells can be seen, and the central vein and the convergent region are scattered in inflammatory cell infiltration; the probiotic group had compact hepatocyte structure, red cytoplasm, no significant broadening of the liver plate, and the hepatocytes were radially arranged around the liver plate with occasional small amount of lipid droplets distributed (a in fig. 4).

Observing mouse liver cell oil under a red-dyeing mirror, wherein only a small amount of oil drops in a normal group are uniformly scattered in cytoplasm; compared with the normal diet group, the high fat diet group mice have loose hepatocyte structure, obvious swelling, diffuse steatosis of liver parenchyma, and a large amount of bright red lipid drops can be seen in the hepatocyte; compared with the high fat diet group, the probiotic group had smaller lipid droplets in the liver cells, decreased in number, and dispersed in the cytoplasm (fig. 4, B).

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A probiotic compound for the treatment of non-alcoholic fatty liver disease, characterized by: the wine brewing yeast strain comprises probiotics of blautia, Roseberry and wine brewing yeast, wherein the volume ratio of the probiotics of blautia, Roseberry and wine brewing yeast is 0.1-10: 0.1-7: 0.2-5.

2. The probiotic compound for treating non-alcoholic fatty liver disease of claim 1, wherein: the volume ratio of the probiotics of the blautia bacteria, the Roseburia bacteria and the saccharomyces cerevisiae is 1:1: 1.

3. The probiotic compound for treating non-alcoholic fatty liver disease according to claim 1 or 2, characterized in that: comprises probiotic bacteria of the genus Brabender, the genus Roseburia and Saccharomyces cerevisiae, which are respectively prepared into suspension according to the density of 1 x 10^8cfu/ml, and then are mixed according to the volume ratio.

4. A method of preparing a probiotic composition for the treatment of non-alcoholic fatty liver disease as claimed in claim 1, comprising the steps of:

carrying out metagenome detection on the feces of the NO-NASH and NASH patients, and screening probiotics obviously enriched in the NO-NASH to obtain obviously enriched probiotics, namely Brauteria bacteria;

respectively culturing the probiotics of the blautia, the Roseburia bacteria and the saccharomyces cerevisiae in an anaerobic box through culture media, and inoculating according to the density of 1 x 10^8 cfu/ml;

5. The method of claim 4, wherein the probiotic composition is administered to the patient in need of treatment for non-alcoholic fatty liver disease, wherein: the probiotics of the Brabender genus, the Roseburia genus and the saccharomyces cerevisiae in the step 3) are mixed according to the volume of 1:1: 1.

6. The method of claim 4, wherein the probiotic composition is administered to the patient in need of treatment for non-alcoholic fatty liver disease, wherein said probiotic composition comprises: the probiotic blautia is cultured by using a culture medium of 104 at 37 ℃; roseburia strain was cultured in 1611 medium at 37 ℃; saccharomyces cerevisiae is cultured in YM medium at 25-28 deg.C.

7. Use of a probiotic compound according to claim 1 for the treatment of non-alcoholic fatty liver disease, characterized in that: the probiotic compound is used for treating non-alcoholic fatty liver disease, improving insulin resistance and adjusting lipid metabolism disorder.