CN111135197A

CN111135197A - Application of animal bifidobacterium A6 in preparing health-care products

Info

Publication number: CN111135197A
Application number: CN201811299227.5A
Authority: CN
Inventors: 任发政; 张筱琳; 赵亮; 张明
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2018-11-02
Filing date: 2018-11-02
Publication date: 2020-05-12

Abstract

The invention provides application of animal bifidobacterium A6 in preparing a health-care product for relieving chronic kidney disease. The inventor finds that the bifidobacterium animalis A6 can achieve the effects of inhibiting the generation of enterogenic uremic toxin and protecting the residual kidney function of 5/6 nephrectomized rats by regulating intestinal microenvironment and improving intestinal metabolism, and the health-care product prepared from the bifidobacterium animalis A6 can be effectively used for relieving chronic kidney diseases.

Description

Application of animal bifidobacterium A6 in preparing health-care products

Technical Field

The invention relates to the field of biological pharmacy, in particular to application of bifidobacterium animalis A6 in preparing health care products, and more particularly relates to application of bifidobacterium animalis A6 in preparing health care products, a method for screening medicines, a medicinal composition for relieving chronic kidney diseases and a medicinal combination.

Background

Chronic Kidney Disease (CKD) causes toxins such as urea in blood to be unable to be metabolized by the kidney and to be retained in the body, accumulated toxins can flow through the whole body by means of blood circulation, and various organs of the body such as the kidney, the liver, the brain, the heart, the intestinal tract and the like are damaged, so that the incidence rate of anemia, fracture, urinary tract infection, pneumonia, septicemia and cardiovascular and cerebrovascular diseases is remarkably increased. As the renal function of CKD patients decreases, the overall mortality rate from various complications and CKD increases, and numerous studies indicate that the most common cause of death in CKD patients is cardiovascular disease, not CKD itself, and is not related to the course of CKD. In addition, studies show that the risk of concurrent cancer in CKD stage 5 patients is significantly increased, and the high or low risk is related to the age, and the risk of concurrent cancer in young patients is highest and gradually decreases with the age.

Kidney transplantation is the most effective means for treating chronic kidney disease, but it is difficult to find a matched kidney source, which is one of the main reasons for limiting kidney transplantation for patients with kidney disease. Dialysis is currently the main treatment in hospitals, mainly for unbound small molecule water-soluble substances, and the kinetic index for assessing dialysis efficiency is assessed based on the content of water-soluble components urea and creatinine. The dialysis membrane used in hemodialysis is substantially hydrophilic or semi-hydrophilic, the dialysate is also a hydrophilic environment, but the small molecules bound to the proteins are at least partially hydrophobic, and these components are only removed from the proteins and possibly filtered by the dialysis membrane. Therefore, a portion of the protein-bound toxins remains difficult to remove from the patient after dialysis, and these substances continue to have toxic effects on the body. Because current dialysis procedures are not effective in removing protein-bound uremic toxins from the blood, new treatment strategies may be required if sustained reduction of such substances is desired.

Thus, the treatment and prevention of chronic kidney disease remains to be developed and improved.

Disclosure of Invention

The prior art discloses that 30 metabolites are undetectable or very low in blood of a colectomy dialysis patient compared with a common dialysis patient, wherein the 30 metabolites comprise 5 enterotoxuria toxins such as indoxyl sulfate, p-cresol sulfate, indoxyl glucuronide, 5-oxindole, α -phenylacetyl-I-glutamine, which indicate that the toxins are related to colonic metabolism, and wherein the indoxyl sulfate, the p-cresol sulfate and the cardiovascular and cerebrovascular death rate of a CKD patient have strong correlation.

Based on the findings of the facts and the problems, the inventor successfully constructs a rat CKD model through 5/6nephrectomy, researches the influence of bifidobacterium animalis A6 on the intestinal flora structure of 5/6 nephrectomized rats through a 16S rRNA high-throughput sequencing and bioinformatics method, and determines that clostridium difficile is an intestinal flora target of bifidobacterium animalis A6 for inhibiting renal injury for the first time, which is shown in figure 1. Therefore, the inventor firstly proposes that the animal bifidobacterium A6 can effectively maintain the residual kidney function of 5/6 nephrectomized rats, and simultaneously can effectively improve dysbacteriosis caused by CKD and down-regulate clostridium difficile.

In a first aspect of the invention, the invention proposes the use of bifidobacterium animalis a6 in the manufacture of a health product for use in the alleviation of chronic kidney disease. The inventor finds that the bifidobacterium animalis A6 can achieve the effects of inhibiting the generation of enterogenic uremic toxin and protecting the residual kidney function of 5/6 nephrectomized rats by regulating intestinal microenvironment and improving intestinal metabolism, and the health-care product prepared from the bifidobacterium animalis A6 can be effectively used for relieving chronic kidney diseases.

According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:

according to an embodiment of the invention, the chronic kidney disease comprises at least one selected from the group consisting of kidney injury, kidney fibrosis, compensatory glomerular hypertrophy, glomerulosclerosis, tubulointerstitial fibrosis, kidney failure and uremia. The inventor finds through experiments that the bifidobacterium animalis A6 can improve the glomerular filtration rate, improve the glomerular sclerosis and the kidney fibrosis state, further protect the kidney function, and meanwhile, the bifidobacterium animalis A6 can relieve the tubulointerstitial fibrosis, reduce the compensatory glomerular hypertrophy, reduce the kidney injury, relieve the kidney failure, treat uremia and the like. Further, the health product prepared from bifidobacterium animalis a6 can be effectively used for relieving at least one disease of renal injury, renal fibrosis, compensatory glomerular hypertrophy, glomerulosclerosis, tubulointerstitial fibrosis, renal failure and uremia.

According to an embodiment of the invention, the nutraceutical is for use in at least one of: regulating intestinal microecology, improving intestinal metabolism, inhibiting enterogenous uremia toxin, and inhibiting growth of Clostridium difficile. The inventor discovers for the first time that clostridium difficile can aggravate kidney injury, and simultaneously discovers for the first time that clostridium difficile is an intestinal flora target of bifidobacterium animalis A6 for inhibiting kidney injury, inhibits the growth of clostridium difficile and can effectively inhibit kidney injury. The inventor also finds that the animal bifidobacterium A6 mainly inhibits the generation of enterogenic uremic urea, reduces the content of uremic toxin in blood and further prevents the oxidative stress damage of uremic toxin to kidney by inhibiting the proliferation of bacteria related to protein metabolism such as clostridium difficile in intestinal tracts. Furthermore, the health product prepared from animal bifidobacterium A6 can relieve chronic kidney disease by at least one of regulating intestinal microecology, improving intestinal metabolism, inhibiting the generation of enterogenic uremic toxin, inhibiting the growth of clostridium difficile and the like.

According to an embodiment of the invention, the enterogenic uremic toxin is indoxyl sulfate, p-cresol sulfate. The inventor finds that bifidobacterium animalis A6 can inhibit the generation of indoxyl sulfate and p-cresol sulfate, and furthermore, the health-care product prepared from bifidobacterium animalis A6 has good effect of relieving kidney injury caused by indoxyl sulfate and p-cresol sulfate.

In a second aspect of the invention, the invention proposes the use of bifidobacterium animalis a6 in the manufacture of a medicament for use in modulating the content of clostridium difficile. As described above, the inventor firstly discovers the relationship between the bifidobacterium animalis A6 and clostridium difficile, and further, the medicament prepared by using the bifidobacterium animalis A6 can be used for regulating the content of the clostridium difficile. According to an embodiment of the invention, the use may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, said modulating the content of clostridium difficile is a down-regulating the content of clostridium difficile. As described above, the inventors found for the first time that bifidobacterium animalis a6 can inhibit the growth of clostridium difficile, and clostridium difficile can cause kidney injury, and further that the medicament prepared by using bifidobacterium animalis a6 can reduce the content of clostridium difficile, relieve kidney injury and protect residual kidney function.

In a third aspect of the invention, a method of screening for a drug for the treatment or prevention of chronic kidney disease is presented. According to an embodiment of the invention, the method comprises administering a candidate drug to an animal model, wherein the animal model is a model of chronic kidney disease, and comparing the levels of bifidobacterium animalis a6 in the model before and after administration to determine whether the drug is a candidate drug. The inventor finds that the animal bifidobacterium A6 can achieve the effects of inhibiting the generation of enterogenic uremic toxin and protecting the residual kidney function of 5/6 nephrectomized rats by regulating the intestinal microenvironment, improving the intestinal metabolism and down-regulating clostridium difficile. The chronic kidney disease can be effectively relieved by using the medicament screened by the method according to the embodiment of the invention.

According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:

according to an embodiment of the invention, the method further comprises: an increased content of bifidobacterium animalis a6 in the model after administration compared to before administration is an indication that the drug is the target drug. As mentioned above, the animal bifidobacterium A6 can achieve the effects of inhibiting the generation of enterogenic uremic toxin and protecting the residual kidney function of 5/6 nephrectomized rats by regulating the intestinal microenvironment, improving the intestinal metabolism and down-regulating clostridium difficile. After administration, the content of animal bifidobacterium A6 in the model is increased, so that the chronic kidney disease can be relieved.

According to an embodiment of the invention, the method further comprises: a decrease in the clostridium difficile content of the gut compared to that of the model after administration compared to that before administration is an indication that the drug is the drug of interest. The inventor discovers for the first time that clostridium difficile can aggravate kidney injury, inhibit the growth of clostridium difficile and effectively inhibit kidney injury. A decrease in clostridium difficile content can further be an effective indicator that the drug of interest can be used to alleviate chronic kidney disease.

According to an embodiment of the invention, the method further comprises: comparing changes in the model before and after dosing of at least one of: a change in enterogenic uremic toxin, blood creatinine, blood urea nitrogen, wherein a decrease in at least one of the above in the model following administration is indicative that the drug candidate is the drug of interest. A reduction in at least one of the enterogenic uremic toxins, blood creatinine, and blood urea nitrogen is further effective to indicate that the drug of interest can be used to alleviate chronic kidney disease.

According to an embodiment of the invention, the enterogenic uremic toxin is indoxyl sulfate, p-cresol sulfate. The reduction of indoxyl sulfate and p-cresol sulfate is further effective in indicating that the target drug can be used for relieving chronic kidney disease.

According to an embodiment of the invention, the chronic kidney disease comprises at least one selected from the group consisting of kidney injury, kidney fibrosis, compensatory glomerular hypertrophy, glomerulosclerosis, tubulointerstitial fibrosis, kidney failure and uremia.

In a fourth aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition for alleviating chronic kidney disease or modulating the content of clostridium difficile comprises bifidobacterium animalis a 6. As mentioned above, the bifidobacterium animalis A6 can achieve the effects of inhibiting the generation of enterogenic uremic toxin and protecting the residual kidney function of 5/6 nephrectomized rats by regulating the intestinal microenvironment and improving the intestinal metabolism, and further the bifidobacterium animalis A6 can be effectively used for relieving chronic kidney diseases; meanwhile, the inventor also discovers the relationship between clostridium difficile and bifidobacterium animalis A6 for the first time, and the clostridium difficile can cause kidney injury. The pharmaceutical composition comprising bifidobacterium animalis a6 according to embodiments of the invention may be effective in alleviating chronic kidney disease.

According to an embodiment of the present invention, the above pharmaceutical composition may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the pharmaceutical composition further comprises one or more therapeutic agents. According to an embodiment of the invention, the therapeutic agent is for the treatment of chronic kidney disease. Thus, the pharmaceutical composition according to the embodiment of the present invention can be more effectively used for treating or alleviating chronic kidney disease.

According to an embodiment of the invention, the therapeutic agent comprises AST-120 or fidaxomicin. Thus, the pharmaceutical composition according to the embodiment of the present invention can be further effectively used for treating or alleviating chronic kidney disease.

According to an embodiment of the invention, said modulating the content of clostridium difficile is a down-regulating the content of clostridium difficile. As described above, the inventors found for the first time that bifidobacterium animalis a6 can inhibit the growth of clostridium difficile, and clostridium difficile can cause kidney injury, and further that the pharmaceutical composition comprising bifidobacterium animalis a6 can down-regulate the content of clostridium difficile, relieve kidney injury and protect residual kidney function.

In a fifth aspect of the invention, a pharmaceutical combination is presented. According to an embodiment of the present invention, bifidobacterium animalis a6 and other drugs useful for treating or preventing chronic kidney disease include: AST-120 or fidaxomicin. The bifidobacterium animalis A6 is combined with AST-120 or fidaxomicin, and the composition is used simultaneously or in time-sharing mode, so that the treatment effect on the chronic kidney disease is more remarkable.

According to an embodiment of the present invention, the above-mentioned pharmaceutical combination may further comprise at least one of the following additional technical features:

According to some embodiments of the present invention, the pharmaceutical composition for alleviating chronic kidney disease of the present invention may further include a pharmaceutically acceptable carrier, and the dosage form of the pharmaceutical composition is not particularly limited. For oral administration, the pharmaceutically acceptable carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants and flavoring agents. For formulations for topical administration, pharmaceutically acceptable carriers may include bases, excipients, lubricants and preservatives. The pharmaceutical composition of the present invention may be prepared in various dosage forms in combination with the above pharmaceutically acceptable carrier. For example, for oral administration, the pharmaceutical compositions may be prepared as tablets, troches, capsules, elixirs, suspensions, syrups or wafers. The pharmaceutical compositions may also be formulated as solutions, suspensions, tablets, pills, capsules and depot preparations.

Among the carriers suitable for pharmaceutical formulations, according to some specific examples of the present invention, are excipients and diluents that may include: lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

According to other embodiments of the present invention, fillers, anticoagulants, lubricants, moisturizers, fragrances, and preservatives may also be included in the pharmaceutical compositions of the present invention.

According to the embodiment of the invention, the pharmaceutical composition can be used for regulating intestinal microecology, improving intestinal metabolism, inhibiting the generation of enterogenic uremic toxins and inhibiting the growth of clostridium difficile, so as to relieve or improve disease states such as renal injury, renal fibrosis, compensatory glomerular hypertrophy, glomerulosclerosis, tubulointerstitial fibrosis, renal failure and uremia. Thus, the pharmaceutical composition of the present invention may be administered in the alleviation of chronic kidney disease.

The term "administering" as used herein means introducing a predetermined amount of a substance into a patient by some suitable means. The drug of the present invention can be administered by a usual route as long as it can reach the desired tissue. The most proper use mode of the pharmaceutical composition is oral administration, and A6 has the characteristics of acid resistance and bile salt resistance and can survive to the intestinal tract to play a role. Injection means cannot be used. In addition, the pharmaceutical compositions of the present invention may be administered using a specific device that delivers the active ingredient to the target cells.

The administration frequency and dose of the pharmaceutical composition of the present invention can be determined by a number of relevant factors, including the type of disease to be treated, the administration route, the age, sex, body weight and severity of the disease of the patient and the type of drug as an active ingredient. According to some embodiments of the invention, the daily dose may be divided into 1, 2 or more doses in a suitable form for

administration

1, 2 or more times over the entire period, as long as a therapeutically effective amount is achieved.

The term "therapeutically effective amount" refers to an amount of a compound sufficient to significantly ameliorate some of the symptoms associated with a disease or condition, i.e., to provide a therapeutic effect for a given condition and administration regimen. For example, a drug or compound that reduces, prevents, retards, inhibits, or arrests any symptoms of a disease or disorder in chronic kidney disease should be therapeutically effective. A therapeutically effective amount of a drug or compound need not cure a disease or condition, but will provide treatment for a disease or condition such that the onset of the disease or condition in an individual is delayed, prevented or prevented, or the symptoms of the disease or condition are alleviated, or the duration of the disease or condition is altered, or the disease or condition becomes less severe, or recovery is accelerated, for example.

The term "treatment" is used to refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or adverse effects resulting from the disease. As used herein, "treatment" encompasses the treatment of disease (primarily chronic kidney disease) in mammals, particularly humans, including: (a) preventing disease (e.g., preventing chronic kidney disease) or the occurrence of a condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting a disease, e.g., arresting disease progression; or (c) alleviating the disease, e.g., alleviating symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administration of a drug containing bifidobacterium animalis a6 as described herein to an individual in need thereof.

According to embodiments of the present invention, the medicament or pharmaceutical composition of the present invention may be used in combination with conventional treatment methods and/or therapies, or may be used separately from conventional treatment methods and/or therapies. When the drugs or pharmaceutical compositions of the present invention are administered in combination therapy with other drugs, they may be administered to the individual sequentially or simultaneously. Alternatively, the pharmaceutical composition of the invention may comprise a combination of bifidobacterium animalis a6 of the invention, a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient and other therapeutic or prophylactic agents known in the art.

Drawings

FIG. 1 is an overall technical roadmap according to an embodiment of the present invention;

fig. 2 is a graph of creatinine and urea nitrogen content in serum of rats in each group after 14 days of a nephrectomy operation according to an embodiment of the present invention, in which the ratio of left: the change of the serum creatinine content of each group of rats after nephrectomy, right: the blood urea nitrogen content of rats in each group is changed after nephrectomy;

FIG. 3 is a graph of serum creatinine and urea nitrogen content changes in various groups of rats according to an embodiment of the present invention, wherein: the blood urea nitrogen content of rats in each group was varied as follows: the change of the serum creatinine content of rats in each group;

FIG. 4 shows trichrome staining of the kidney of rats in groups after 10 weeks of intragastric administration of Bifidobacterium animalis A6 according to an embodiment of the present invention;

FIG. 5 shows the kidney fibrosis ratio of rats in each group after 10 weeks of intragastric administration of Bifidobacterium animalis A6 according to an embodiment of the present invention;

fig. 6 shows PAS staining of rats in each group after 10 weeks of gastric gavage with bifidobacterium animalis a6 according to an embodiment of the invention;

FIG. 7 is a glomerular sclerosis index of groups of rats after 10 weeks of intragastric gavage with Bifidobacterium animalis A6 according to an embodiment of the invention;

fig. 8 shows the serum contents of indoxyl sulfate and P-cresol sulfate in rats of each group 10 weeks after the administration of bifidobacterium animalis a6, wherein the difference between groups is marked by different lower case letters in the same index (P < 0.05);

FIG. 9 is a principal coordinate analysis at the level of the rat intestinal flora according to an embodiment of the present invention;

FIG. 10 is a comparison of differences between groups (family level) according to an embodiment of the invention;

FIG. 11 shows the serum content changes of indoxyl sulfate and p-cresol sulfate in rats of different treatment groups after the gavage of Clostridium difficile according to an embodiment of the present invention;

FIG. 12 is a graph of the change in serum creatinine and serum urea nitrogen levels in rats of different treatment groups following gavage with Clostridium difficile according to an embodiment of the present invention;

FIG. 13 is a trichrome stain of rats in different treatment groups after lavage with Clostridium difficile according to an embodiment of the invention;

fig. 14 is a graph of kidney fibrosis ratios for rats in different treatment groups after clostridium difficile gavage in accordance with an embodiment of the present invention, wherein the different lower case letters are marked to indicate significant differences between groups (P < 0.05).

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

The bifidobacterium animalis A6 used in the invention is a strain separated from intestinal tracts of longevity old people of Guangxi Bama by the inventor.

Abbreviations referred to in the present invention are as follows:

full English-name and full Chinese-name abbreviations

Chronic Kidney Disease of CKD Chronic Kidney Disease

Infection of CDI Clostridium difficile with Clostridium difficile

CFU Colony-Forming Units

Nx 5/6nephrectomy 5/6nephrectomy

MRM Multiple Reaction Monitoring

BUN Blood Urea Nitrogen from Urea Nitrogen

Creatine Cre

Indoxyl Sulfate indophenol of IS

PCS rho-cresol Sulfate p-cresol

OTU Operational Taxonomic Units colony-forming Units

PCR Polymerase Chain Reaction (PCR) method

PCoA Principal Coordinates Analysis

SDS Sodium Dodeccyl Sulfate Sodium Dodecyl Sulfate

Tris Trishydroxymethyl amino methane

PAS periodate Schiff periodic Acid Schiff

The following describes embodiments of the present invention in detail.

Example 1 Effect of Bifidobacterium animalis A6 on the residual renal function in 5/6 nephrectomized rats

1. Materials and methods

1.1 Experimental animals

SPF grade Sprague Dawley (SD) rats 39 male, 6 week old, body weight 120-: SCXK (Jing) 2012 and 0001. The transgenic organism food is bred in an SPF animal house of the transgenic organism food safety supervision and inspection test center of China university of agriculture, the room temperature is 22 +/-2 ℃, the illumination/darkness cycle is 12 hours, and the transgenic organism food is free to drink water and eat food.

1.2 Main reagents and formulations

The main reagents are shown in table 1:

table 1:

note: the other reagents which are not indicated are domestic biochemical or analytical pure reagents.

(1) Chloroacetaldehyde hydrate solution: before use, dissolving chloroacetaldehyde hydrate with sterile water to prepare a chloroacetaldehyde hydrate solution of 40mg/mL, and storing the solution in a refrigerator at 4 ℃ in a dark place after preparation;

(2) MRS culture medium: dissolving MRS meat soup powder in 1L culture medium, and sterilizing at 121 deg.C for 15 min.

(3)PBS：1.44g/L Na₂HPO₄，0.24g/L KH₂PO₄0.2g/L KCl, 8g/L NaCl. Sterilizing with high pressure steam at 121 deg.C for 15 min.

(4) Physiological saline: 8.5g/L NaCl, and sterilizing at 121 ℃ for 15min for later use.

1.3 Main instruments

1.4 Experimental strains and treatments

The test strains are: bifidobacterium animalis A6(Bifidobacterium animalis A6) deposited in the functional dairy laboratory of the department of education under the accession number: CGMCC 9273.

Strain treatment: inoculating Bifidobacterium animalis A6 in MRS liquid culture medium, and anaerobically culturing at 37 deg.C for 12 hr to reach bacteria concentration of 10⁹CFU/mL, the bacterial solution was centrifuged at 2770 Xg for 15min at room temperature, and the cells were collected. The harvested thallus is washed twice with normal saline and then resuspended in normal saline, and when in use, the thallus suspension is diluted to 10 times¹⁰CFU/mL concentration.

1.55/6 establishment of nephrectomized rat model

Rats marked with picric acid were acclimatized for 7 days, followed by 5/6nephrectomy, as follows:

5/6nephrectomy is mainly divided into two steps, first, 6-week-old rats are intraperitoneally injected with anesthetic chloracetal hydrate at a dose of 30mg chloracetal hydrate/100 g body weight. After the rats were fully anesthetized, a wound of appropriate size was cut from the left back of the rat with surgical scissors, and after finding the left kidney at the wound, the kidney capsule was first peeled off, and 1/3 of the left kidney was then excised from each of the upper and lower ends of the left kidney, leaving a central portion 1/3. After the excision is finished, a stitching worker stitches the wound of the rat, the rat is placed in a warm environment to wake up, and after the rat recovers consciousness and can freely eat and drink water, the rat is placed back into the cage to recover; after the left kidney is removed for one week, the second step of operation is continued, the rat is firstly anesthetized, the anesthetic dose and the method are unchanged, the operation is carried out from the right back of the rat, after the envelope of the right kidney is stripped, the right kidney blood vessel is ligated by using a suture line, the whole right kidney is removed, the wound is sutured after the removal is finished, and the rat is placed in a warm environment until the rat is completely awake. To eliminate the effect of the entire operation and the peeling of the kidney capsule on the rats, this example also specifically designed a group of rats to undergo a sham operation, which was identical to the 5/6 nephrectomized rats except that the left and right kidneys were not resected. After the whole 5/6nephrectomy and sham surgery were completed, rats were observed daily for postoperative recovery and recorded for abnormalities. After 14 days of operation, blood of rats is collected, after serum is obtained by centrifugation, the Cre and BUN content in the serum is measured by using a full-automatic biochemical analyzer. And (3) standard of successful molding: the Cre and BUN content in the serum of the nephrectomized rats is remarkably increased (P <0.05) compared with that of the sham operated rats, namely 5/6 nephrectomized rats are considered to be successfully modeled.

1.6 Experimental groups and dose settings

The 5/6 rats successfully modeled after nephrectomy were divided into 2 groups (model group and probiotic group) by weight, and each group was fed with 13 animals and 5 animals in one cage. Wherein the probiotic group rats are gavaged with 2mL A6 bacterial liquid (3X 10) per day¹⁰CFU/mL); the model and control (sham operated, 13) rats were gavaged daily with an equal volume of saline for a fixed period of time for 10 weeks.

1.7 sample Collection

Rat feces and blood samples were collected every two weeks. Collecting 1-1.5ml of rat blood by using the mode of inner canthus vein of eye socket, standing the blood for 2h, centrifuging at 600 Xg for 10min to obtain serum, reserving a part of the serum at-20 ℃ for Cre and BUN detection in the blood, reserving a part of the serum at-80 ℃ for uremia toxin detection in the blood. The fecal samples were frozen at-80 ℃ for subsequent analysis.

The stomach was perfused for 10 weeks, and after anesthetizing the rats with ether, the rats were sacrificed by cervical dislocation and subjected to subsequent dissection. The kidney of the rat is kept, one part of the kidney is placed in tissue fixing liquid for histological analysis, the other part of the kidney is frozen at the temperature of minus 80 ℃ for standby, and abnormal phenomena in the anatomical process are recorded by a camera.

1.8 measurement of creatinine and Urea Nitrogen in serum

The Cre and BUN content in the serum of each group of rats was determined using a Mindray BS-350E fully automatic biochemical analyzer.

1.9 Kidney morphological analysis

Kidneys were fixed with 10% formaldehyde and embedded in paraffin blocks. Glomerular sclerosis index and renal fibrosis were assessed by periodic acid-Schiff (PAS) staining and Masson-trichrome staining. The glomerular sclerosis index and the proportion of fibrotic regions in the kidney sections were quantitatively evaluated with the software Image-Pro Plus 3.0(Media Cybernetics, Silver Spring, MD, USA).

1.10 detection of indoxyl sulfate and p-cresol sulfate in blood

Preparation of a standard solution: dilute the indoxyl sulfate and p-cresol sulfate standards to 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, and 100ng/mL with methanol.

Pretreatment of a serum sample: mu.L of serum was added to 200. mu.L of an extractant, which was a methanol solution containing 20ng/mL indoxyl sulfate-d 3 and p-cresol sulfate-d 7. The samples were then vortexed for 30s, 14000g and centrifuged for 15 min. And then sucking 180 mu L of supernatant liquid, freeze-drying in a centrifuge tube for 36h, finally re-dissolving the freeze-dried powder in 50 mu L of 10% methanol solution, carrying out vortex oscillation for 30s, transferring the solution into a sample injection bottle, and preparing for on-machine analysis.

The content of IS and PCS in rat serum was determined using Bruker EVOQ triple quadrupole liquid chromatography system, and the monitoring conditions were as follows:

chromatographic conditions are as follows: waters HSST3 column (2.1 × 100mm 1.8 μm), 40 ℃ column box temperature, 250 μ L/min flow rate, and 10 μ L sample.

Mobile phase a was water containing 0.1% formic acid and mobile phase B was acetonitrile containing 0.1% formic acid. The liquid phase ramp procedure was as follows: 0% B for 0-2min, 0% B-15% B for 2-5min, 15% B for 5-11min, and 15% B-55% B for 11-15 min.

Mass spectrum conditions: the ion source is HESI, a positive and negative ion mode is adopted, the positive and negative ion voltages are 4000V and 3500V respectively, the temperature of a heating probe is 400 ℃, the pressure of heating gas is 40psi, the pressure of atomization gas is 50psi, a Multiple Reaction Monitoring (MRM) method is adopted to monitor the compound, and the optimized parameters of the MRM are as shown in the following table 2:

table 2:

compound (I)	Prime ratio (1)	Prime ratio (2)	Collision energy (eV)
				Indoxyl sulfate	212	80.2	13
Sulfuric acid p-cresol	187	107.2	16

1.11 data analysis

The experimental data were analyzed using SPSS 22 statistical software, with the results expressed as ` x ± s, and the significance of serum creatinine, serum urea nitrogen, renal fibrosis and indoxyl sulfate was analyzed using the Ducan test, and the significance of cresol and glomerulosclerosis index was analyzed using the Tamhane's T2 test for sulfuric acid. P <0.05 was considered significantly different, P <0.01 was considered very significantly different, with statistical significance.

1.12 Experimental results and analysis

1.12.15/6 nephrectomy rat model establishment

Urea (BUN) was one of the first substances used to evaluate glomerular filtration rate. Urea is the end product of protein metabolism in the human body and is mainly excreted out of the human body by glomerular filtration. BUN sensitivity is low, BUN will be maintained at normal levels in the early stages of kidney injury, but blood BUN concentrations will continue to rise as glomerular filtration function falls above normal 1/2.

Serum creatinine (Cre) is currently the most widely used indicator for indirect assessment of glomerular filtration rate. Serum creatinine is nontoxic, is a creatine metabolism end product in muscle tissues, is not combined with protein in blood circulation, is not metabolized by the kidney, and can freely pass through glomeruli and be excreted by the renal tubules. Serum Cre levels are generally dependent on glomerular filtration function in situations where muscle volume and activity are relatively stable, excretion of Cre by the tubules and renal excretion of Cre are constant, and diet is tightly controlled.

As can be seen from fig. 2, after 5/6nephrectomy for 14 days, the blood Cre and BUN concentrations of 5/6nephrectomy rats (operation group) were significantly increased (P <0.05) compared to the control group, indicating that the rat CKD model was successfully modeled, and the subsequent intervention experiments can be continued.

1.12.2A 6 Effect on blood urea Nitrogen and blood creatinine in 5/6 nephrectomized rats

As can be seen from fig. 3, during the gavage, the blood Cre and BUN contents of the model group and probiotic group rats were significantly increased (P <0.05) compared to the control group, and after oral administration of bifidobacterium a 68 for weeks and 10 weeks, the blood Cre and BUN contents of the probiotic group rats were significantly decreased (P <0.05) compared to the model group.

1.12.3A 6 Effect on kidney morphology in 5/6 nephrectomized rats

Renal fibrosis is the main pathological change in the development process of various primary or secondary renal diseases, is the main cause and pathological basis of end-stage renal failure, and is accompanied by progressive irreversible damage of renal function, and the pathological features of the renal fibrosis include extracellular matrix increase, renal tubular atrophy or expansion, apoptosis and the like. The degree and extent of renal interstitial fibrosis is highly correlated with the absence and decline of renal function. Therefore, the improvement of the renal fibrosis condition can effectively improve the renal function and slow down and reverse the chronic renal disease process.

Glomerulosclerosis is the ultimate result of glomerular injury caused by a variety of causes and is the major pathological basis of kidney failure. Studies have shown that massive accumulation of extracellular matrix within the glomeruli is the major pathological basis for glomerulosclerosis. The extracellular matrix is located around cells and mainly consists of three main substances, namely collagen, glycoprotein and proteoglycan, secreted by inherent cells of glomeruli. In addition, glomerulosclerosis and basement membrane injury are main pathological changes represented by the increase of Cre and BUN content in serum, and the glomerular sclerosis condition is improved, so that the reduction of blood Cre and blood BUN content of chronic kidney disease rats is facilitated.

The ratio of the area of renal fibrosis (blue area) to the total area of the visual field was calculated by Masson's trichrome stain assessment, with 25 non-overlapping areas/group randomly selected under 200 x magnification.

As can be seen from fig. 4 and 5, after 10 weeks of gavage, the rate of renal fibrosis in the model group was significantly increased (P <0.01) compared to the control group; after gavage, the kidney fibrosis rate was significantly reduced in the probiotic group compared to the model group (P < 0.05).

In histology, PAS staining is mainly used for glycogen staining, periodic acid can oxidize hydroxyl groups in intracellular polysaccharide glycol into aldehyde groups, and colorless fuchsin in Schiff's solution reacts with the aldehyde groups to make reaction sites show purplish red, so as to locate positive sites. Therefore, the PAS staining can locate the extracellular matrix in the glomerular basement membrane and mesangial stroma, and the distribution and content change of the extracellular matrix of each group of rats are observed, so that the glomerular sclerosis degree of each group of rats is judged.

By evaluation of PAS staining, 50-80 glomeruli were randomly selected per group, and the hardening index of each glomerulus was calculated and scored, 0 for no hardening, 1 for hardened area of 0-25%, 2 for 25-50%, 3 for 50-75%, and 4 for 75-100% of the area of the glomerulus.

The formula is score ═ Fi × i, Fi represents the ratio of the number of glomeruli scored as i to the total number of glomeruli.

As can be seen from fig. 6 and 7, after 10 weeks of gavage, the glomerulosclerosis index of the model group was very significantly increased (P <0.01) compared to the control group; after gavage, the glomerular sclerosis index of the probiotic group was significantly reduced compared to the model group (P < 0.05).

1.12.4A 6 Effect on 5/6 Renephrectomized rat serum indoxyl sulfate and sulfuric acid on cresol

Indoxyl Sulfate (IS) belongs to enterogenic uremia toxin, can inhibit the combination of drugs and protein, and accelerates glomerulosclerosis. Indoxyl sulfate is produced by intestinal bacteria metabolising in the colon, tryptophan being first broken down by intestinal bacteria to indole in the colon, followed by further hydroxylation of indole in the liver to produce 3-hydroxyindole, most of which is sulpholated to the indole sulfate. The increase in indoxyl sulfate is associated with the development of compensatory glomerular hypertrophy, glomerulosclerosis and tubulointerstitial fibrosis following renal insufficiency.

Para-cresol sulfate (PCS) is a phenol with high affinity for plasma albumin, and its precursor substance is para-cresol. The enteroanaerobes are able to convert phenylalanine and tyrosine into 4-hydroxyphenylacetic acid, which is then decarboxylated into p-cresol, most of which is able to convert into p-cresol sulphate in the intestinal cells. P-cresol sulfate is strongly bound to albumin, and is discharged from the kidney without being compensated for, and is easily accumulated in the body, and the rate of progression of renal failure is accelerated by the action of oxidative stress or the like. The common dialysis means is difficult to remove the uremic toxin combined with protein, such as indoxyl sulfate, p-cresol sulfate and the like.

As can be seen from FIG. 8, after 10 weeks of gavage, the serum IS and PCS in the rat model group were significantly increased (P <0.05) compared with the control group; after intragastric administration of A6, the content of IS and PCS in the serum of the rats in the probiotic group IS remarkably reduced compared with that in the model group (P < 0.05).

The embodiment of the invention shows that the bifidobacterium A6 can obviously reduce the Cre and BUN content in the serum of 5/6 nephrectomized rats, reduce the renal fibrosis and glomerular sclerosis degree of 5/6 nephrectomized rats, and indicate that the bifidobacterium A6 effectively protects the residual renal function of 5/6 nephrectomized rats and inhibits the further deterioration of CKD.

At least one of the following conclusions is demonstrated in example 1:

(1) this example successfully simulates CKD in rats using 5/6nephrectomy, with a significant rise in blood Cre and BUN (P <0.05) in rats two weeks after surgery;

(2) in terms of renal function, after b.gastri a6, blood Cre, BUN, renal fibrosis ratio and glomerular sclerosis index of 5/6 nephrectomized rats were significantly decreased (P <0.05), indicating that a6 effectively protected residual renal function of 5/6 nephrectomized rats.

(3) In the aspect of uremic toxin, after bifidobacterium gastricum A6 IS infused, the content of IS and PCS in blood of 5/6 nephrectomized rats IS reduced remarkably (P <0.05), which shows that A6 can effectively reduce IS and PCS in 5/6 nephrectomized rats.

Example 2 target analysis of Bifidobacterium animalis A6 on 5/6 intestinal flora in nephrectomized rats

2.1 Main reagents and formulations

The other reagents which are not indicated are domestic biochemical or analytical pure reagents.

(1)50 × TAE: 242g Tris, 57.1mL glacial acetic acid, 100mL EDTA 0.5mol/L, adding ultrapure water to the volume of 1L, and adjusting the pH value to 8.0.

(2) Tris-SDS (sodium dodecyl sulfate): 250mL (200mM Tris-HCl, 80mM EDTA; pH 9.0), 50mL 10% SDS, and mixing them well for use.

(3) Sodium acetate solution: adjusting pH to 5.2, diluting to 3mol/L, and sterilizing at 121 deg.C for 15 min.

(4) Tris-EDTA: adjusting pH of 10mmol/L Tris solution to 8.0 with 1mol/L HCl, adding 1mmol/LEDTA, diluting to 1L with ultrapure water, and sterilizing at 121 deg.C for 15 min.

(5) Chloroform/isoamyl alcohol (V/V24: 1): 96mL of chloroform and 4mL of isoamyl alcohol are mixed uniformly and placed in a brown bottle for later use.

2.2 Main Instrument

2.3 fecal bacteria Total DNA extraction

In the experiment, the phenol chloroform method is adopted to extract the total DNA in the rat feces, and the integrity of the DNA is detected by 1 percent agarose gel electrophoresis after the extraction is finished.

2.4 diversity analysis of intestinal flora in feces

PCR amplification

V3-V4 variable region was PCR amplified with primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5 '-GGACTACHVGGGTWTCTAAT-3'), using the following protocol: pre-denaturation at 95 ℃ for 3min, 27 cycles (denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s), and final extension at 72 ℃ for 10min (PCR apparatus: ABI)

Model 9700). The amplification system was 20uL, 4uL 5 × FastPfu buffer, 2uL 2.5mM dNTPs, 0.8uL primer (5uM), 0.4uL FastPfu polymerase; 10ng of DNA template.

Illumina Miseq sequencing

All samples were run under the formal experimental conditions, three replicates per sample, PCR products from the same sample were pooled, recovered using 2% agarose Gel, purified using AxyPrep DNA Gel Extraction Kit (Axygen biosciences, Union City, Calif., USA), eluted with Tris-HCl, and detected by 2% agarose electrophoresis. Referring to the preliminary quantitative result of electrophoresis, QuantiFluor is used^TMAssay quantification was performed by ST (Promega, USA). The purified amplified fragments were used to construct a library of PE 2 x 300 according to the standard protocol of the Illumina miseq platform (Illumina, San Diego, USA).

Constructing a library: (1) connecting a Y-shaped joint; (2) removing the adaptor self-connecting fragment by magnetic bead screening; (3) enriching the library template by utilizing PCR amplification; (4) sodium hydroxide denaturation can generate single-stranded DNA fragments.

Sequencing was performed using the Miseq PE300 platform from Illumina (Megi biomedical science and technology, Inc., Shanghai).

Sequence analysis

The original sequencing sequence is subjected to quality control by using Trimmomatic software, and spliced by using FLASH software:

(1) setting a 50bp window, if the average mass value in the window is lower than 20, cutting off a rear-end base from the window, and removing a sequence with the length lower than 50bp after quality control;

(2) the barcode needs to be matched accurately, the primer allows the mismatching of 2 bases, and fuzzy bases are removed;

(3) and splicing the sequences at two ends according to the overlapping base overlap, wherein the overlap needs to be more than 10 bp. And removing sequences which cannot be spliced.

The used UPARSE software (version 7.1http:// drive5.com/UPARSE /) carries out OTUs (operational taxonomic units) clustering on the sequence according to the similarity of 97 percent; and (3) removing chimeras by using UCHIME software to obtain a final effective sequence. Species class annotation was performed on each sequence using RDP classifier (http:// RDP. cme. msu. edu /), and the silvera database (SSU123) was aligned to obtain the basic analysis results of OTUs and classification pedigree for each sample, setting the alignment threshold at 70%.

The abundance, diversity index and the like of OTUs are analyzed by using an I-Sanger one-stop biological information cloud (http:// www.i-Sanger. com /), the colony structure IS statistically analyzed at each classification level by using cluster analysis and PCoA analysis for species annotation, and the correlation analysis IS performed on the relative abundance of floras and related indexes of renal function (Cre, BUN, IS and PCS) by using Pearson correlation coefficients.

2.5 fecal Clostridium difficile assay

And detecting the content of clostridium difficile in excrement by using real-time quantitative PCR (polymerase chain reaction), and judging the existence condition of clostridium difficile in the intestinal tract.

Determination of specific primers and quantitative PCR conditions

Specific primers of clostridium difficile are searched according to a reference document, and technologies such as agarose gel electrophoresis detection, fluorescence quantitative PCR and the like are used for verification, so that a primer sequence and quantitative PCR reaction conditions are established.

PCR amplification was performed with primers 5'-TTGAGCGATTTACTTCGGTAAAGA-3' and 5'-CCATCCTGTACTGGCTCACCT-3' and the amplification procedure was: pre-denaturation at 95 ℃ for 10 min; annealing at 95 ℃ for 20s, annealing at 58 ℃ for 60s, extending at 72 ℃ for 10s, detecting SYBR Green fluorescent signals at 80 ℃ for 10s, and performing 35 cycles. (real-time fluorescent quantitative PCR instrument: LightCycler 96). The amplification system is 20uL, 1uL template DNA, 10uL SYBR Premix Ex Taq Premix, 0.5uL upstream and downstream primers (10uM), 8uL sterile ultrapure water, and the size of the final product is 157 bp.

Acquisition of a Standard DNA template and determination of the copy number concentration

Extracting Clostridium difficile DNA by using a bacterial genome DNA extraction kit (DP302), carrying out real-time fluorescence quantitative PCR on the Clostridium difficile DNA by using a confirmed specific primer, loading a PCR product, carrying out electrophoresis on a 1% agarose gel, cutting a clear and single target DNA band in the gel by using a sterile scalpel, putting the DNA band into a sterile 1.5mL centrifuge tube, and carrying out DNA recovery by using a Tiangen agarose gel DNA recovery kit to finally obtain a standard DNA template of Clostridium difficile. The operation steps are as follows:

cutting the gel under ultraviolet irradiation to find a clear single target DNA gel electrophoresis strip, cutting the gel from the agarose gel by using a scalpel (cutting a blank gel part as much as possible), putting the cut gel into a clean centrifugal tube, weighing and recording;

adding sol solution with volume 3 times (gel density calculated by 1 kg/L) into the centrifuge tube (when the size of the recovered target fragment is less than 150bp or the agarose gel concentration is more than 2%, 6 times volume of sol solution is needed), and standing in a metal bath at 50 deg.C for 10min to fully dissolve the sol block.

Washing off impurities, carefully transferring the solution in the centrifugal tube to an adsorption column in a collecting tube, centrifuging for 1min at 17000 Xg, pouring off waste liquid in the collecting tube, and putting the adsorption column into the corresponding collecting tube; adding 200 μ L special rinsing liquid into the adsorption column, centrifuging at 17000 × g for 1min, pouring off waste liquid in the collection tube, and placing the adsorption column into the corresponding collection tube;

collecting a standard DNA template, taking out the adsorption column, putting the adsorption column into a sterile and clean 1.5mL centrifuge tube, hanging and dropwise adding 25 mu L of DNA elution buffer solution to the center of the adsorption membrane, standing at room temperature for 2min, centrifuging at 17000 Xg for 2min, and collecting a DNA product solution.

After obtaining the standard DNA template, the standard DNA concentration was determined using Qubit. The operation steps are as follows:

preparing a standard curve to prepare 0 mu g/mL and 5 mu g/mL standard DNA solutions, putting the standard DNA solutions into a sample tank, measuring the DNA concentration, and preparing the standard curve;

and (3) measuring the concentration of the standard DNA template, adding 1 mu L of the standard DNA template into 199 mu L of the Qubit working solution, mixing uniformly, putting into a sample tank, and reading the concentration of the DNA.

According to 1. mu.g of 1000bp double-stranded DNA 1.52pmol 9.1X 10¹¹And calculating the copy number concentration of the clostridium difficile standard DNA template by using the copy number and the size of the clostridium difficile standard DNA.

And simultaneously amplifying and detecting the rat fecal DNA sample and the standard DNA template of the clostridium difficile by using a fluorescent quantitative PCR instrument. And establishing a standard curve and a corresponding linear equation thereof according to the log value and the Ct value of the standard DNA template copy number concentration, and simultaneously calculating the amplification efficiency. And calculating the copy number concentration of the clostridium difficile in the fecal sample by a standard curve equation according to the Ct value of the fecal DNA sample.

2.6 data analysis

(1) Intestinal flora

The diversity of intestinal flora Alpha is analyzed by adopting a student T test method, and the difference bacteria among groups are screened by adopting a Wilcoxon rank sum test method in a pairwise comparison mode, wherein P <0.05 shows that the difference is obvious, P <0.01 shows that the difference is extremely obvious, and the statistical significance is achieved.

(2) Correlation analysis

The significance of the relative abundance of the differential bacteria and the correlation coefficient between the blood creatinine, the blood urea nitrogen, the indoxyl sulfate and the P-cresol sulfate is tested by adopting a Wilcoxon rank sum test method, wherein P <0.05 shows that the significant difference exists, and P <0.01 shows that the significant difference exists, so that the method has statistical significance.

2.7 Experimental results and analysis

2.7.1 Effect of A6 on Alpha diversity of intestinal flora in 5/6 nephrectomized rats

Alpha diversity reflects the species abundance and richness of the microflora in the gut of rats in each group, including Ace, Chao, Shannon, Simpson and Coverage indices. The Ace index and the Chao index reflect the abundance of gut microbes by different algorithms to estimate the number of OTUs in each group of samples. Both the Shannon index and Simpson index may be used to reflect the abundance of a microbial community, with a greater Shannon value indicating greater abundance of a community and a greater Simpson value indicating lesser abundance of a community.

The inventor utilizes an Illumina Miseq platform to carry out high-throughput sequencing, the Miseq platform carries out quality control filtration on the quality of reads and the effect of merge, 1413986 effective sequences are obtained after data are optimized, and the average length is 438.50 bp.

In this example, it can be seen from Table 3 that the Ace and Chao indices of the three groups of rats are lower than the number of OTUs in the group, indicating that as sequencing progresses, new microbial species are substantially not detected any more. The coverage (coverage) of the three groups of samples is more than 99.97 percent, which shows that the sequencing result well reflects the abundance and abundance of the microorganisms in each group of samples. The Shannon indexes of the intestinal flora of the control group, the model group and the probiotic group are respectively 4.04 +/-0.37, 3.86 +/-0.35 and 3.81 +/-0.30, and the Simpson indexes are respectively 0.048 +/-0.02, 0.064 +/-0.04 and 0.068 +/-0.04, which shows that the abundance of the intestinal flora of rats after the nephrectomy operation is reduced, and the A6 does not obviously increase the abundance of the intestinal flora of CKD rats.

Table 3:

remarking: the different lower case letters in the same index indicate significant differences between groups (P < 0.05).

2.7.2 Effect of A6 on Beta diversity in 5/6 nephrectomized rats

Beta diversity reflects species composition changes between different communities, i.e. "magnitude of community species changes or degree of community differentiation in relation to environmental gradients or environmental patterns" (Rosenzweig, 1995), and currently is widely used to determine changes in flora species composition in the temporal or spatial dimensions. Among the microbial diversity Analysis, commonly used Beta diversity Analysis methods include sample level cluster Analysis, Principal Component Analysis (PCA), Principal coordinate Analysis (PCoA), and non-metric multidimensional scaling Analysis.

PCoA is a non-restrictive data dimension reduction analysis method and can be used for researching the difference or similarity of sample community compositions. To investigate the effect of bifidobacterium a6 on the intestinal flora of 5/6 nephrectomized rats, pcoa (unweighted unifrac) analysis was performed on the intestinal flora (family level) of rats in the control, model and probiotic groups.

As shown in fig. 9, the Unweight Unifrac analysis showed differences in the intestinal flora structures (family levels) between the control and model groups, where PC 1-45.84% and PC 2-18.65; a6 has certain effect on intestinal flora (family level) of probiotic rats, wherein PC1 is 37.44%, and PC2 is 11.42%. On the X-axis, the intestinal flora structure of the control and probiotic rats was more dispersed than that of the model group.

2.7.3A6 Effect on the composition of intestinal flora in 5/6 nephrectomized rats

In order to observe the influence of probiotics on the relative abundance of intestinal flora of rats with chronic kidney diseases, the inventor performs species difference analysis on the intestinal flora of three groups of rats. The species difference analysis refers to analyzing by using a related analysis method according to community abundance data obtained by sequencing, and detecting abundance differences shown by different groups of microbial communities. The inventor conducts a significant difference test among groups on intestinal flora of three groups of rats by using a Wilcox rank-sum test method, analyzes to obtain strains with significant relative abundance difference among the three groups, and the result is shown in FIG. 10. As can be seen from fig. 10A, the relative abundance of 5 families was significantly increased in the intestinal flora of the model group rats compared to the control group (P <0.05), i.e., bacteroides _ S24-7_ group, Peptostreptococcaceae, Erysipelotrichaceae, rilaceae, and clostridium _1, respectively, and the cumulative total of all the altered flora accounted for about 38.81% of the total flora. As can be seen from fig. 10B, 3 of the intestinal flora of the probiotic rats were significantly reduced (P <0.01) compared to the model group, namely Peptostreptococcaceae, Erysipelotrichaceae and clostridium _1, and the cumulative total of the changed flora accounted for about 7.20% of the total flora.

The comprehensive analysis of two groups of differential bacteria with remarkably changed relative abundances shows that the relative abundances of Peptostreptococcus acaae, Erysipelotrichaceae and Clostridium _1 in the intestinal tracts of rats in the model group are remarkably up-regulated, and the relative abundances of the three families in the intestinal tracts of rats in the probiotic group are remarkably down-regulated after A6 is supplemented. Subsequently, the relative abundances of the three families of bacteria are subjected to Pearson correlation analysis with the blood creatinine, blood urea nitrogen, indoxyl sulfate and p-cresol sulfate content at the gastric perfusion end point of the rat, and the results are shown in Table 4, and as can be seen from the results in Table 4, the relative abundances of the three families of bacteria in the three groups of rat intestinal flora are all in obvious positive correlation with the Cre and BUN in rat serum and the indoxyl sulfate content, wherein the correlation of the Peptostreptococcaceae is highest. In addition, the relative abundance of two families, Peptostreptococcaceae and Erysipelotrichaceae, is also in a significant positive correlation with the content of p-cresol sulfate in rat serum.

Table 4:

according to the above correlation analysis results, the inventors considered that a certain bacterium in the family Peptostreptococcus may be an intestinal target for A6 to maintain 5/6 renal function in a nephrectomized rat. The Peptostreptococcaceae mainly comprises clostridium, Romboutsia, paracystidium, peptocristidium, Peptostreptococcus and Proteocatella. Difficile (Cd) is an important component strain of Peptostreptococcaceae, and clinical research evidence indicates that the recurrence rate of Clostridium difficile infection is significantly correlated with kidney disease progression. Thus the inventors hypothesized that clostridium difficile may aggravate kidney injury by increasing the p-cresol sulfate content in CKD rats. To confirm the above guess, the inventors tested 5/6 the number of clostridium difficile in the intestine of nephrectomized rats and analyzed the effect of a6 on the clostridium difficile content in the intestine of 5/6 nephrectomized rats. The inventors detected changes in clostridium difficile in various groups of stools by a fluorescent quantitative PCR method. And (3) taking a standard DNA template of clostridium difficile as an external standard, carrying out real-time quantitative PCR on the excrement DNA samples of different groups of rats, and calculating the copy number concentration of the samples according to the standard curve and the concentration of the standard DNA template. The results are shown in table 5, and it is clear from table 5 that the content of clostridium difficile in the feces of the model rats is significantly increased (P <0.05) compared with the control group, while the content of clostridium difficile in the feces of the 5/6 nephrectomized rats is significantly decreased (P <0.05) after the administration of bifidobacterium gastrum a 6. In combination with the results of the gut-to-flora analysis, c.difficile may be an important target for gut flora in a6 protected 5/6 nephrectomized rat kidney function.

Table 5:

example 2 at least one of the following conclusions was verified:

(1) in the aspect of flora structure, PCoA (unweighted Unifrac) and cluster analysis results show that the composition of the intestinal flora in the model group is obviously different from that in the control group. After supplementation with a6, the flora structure of 5/6 nephrectomized rats was closer to that of the control group.

(2) In terms of the analysis of intestinal flora targets, the relative abundance of intestinal bacteria of three groups of rats was compared two by two in the present example, and as a result, a6 was found to be capable of significantly down-regulating the relative abundance of Peptostreptococcaceae, clostridium _1 and Erysipelotrichaceae (P < 0.01).

(3) The relative abundance of Peptostreptococcaceae was found to be significantly and positively correlated with Cre, BUN, IS and PCS content in rat serum by Pearson correlation analysis (P < 0.01). Difficile belongs to an important constituent strain of Peptostreptococcaceae, and after intragastric gavage A6, the content of difficile in the intestinal tract of 5/6 nephrectomized rats is significantly reduced (P < 0.05).

Example 3 Clostridium difficile injury 5/6nephrectomy of residual renal function in rats

3.1 materials and methods

3.1.1 Experimental animals

SPF grade SD rats 24 male, 6 weeks old, 120-: SCXK (Jing) 2012 and 0001. The transgenic organism food is bred in an SPF animal house of the transgenic organism food safety supervision and inspection test center of China university of agriculture, the room temperature is 22 +/-2 ℃, the illumination/darkness cycle is 12 hours, and the transgenic organism food is free to drink water and eat food.

3.1.2 Main reagents and formulations

Reagent name reagent grade reagent manufacturer

Analytical pure Qingdao high-tech industrial garden Haibo Biotech limited by reinforced Clostridium medium

(1) Clostridium liquid medium: dissolving 38.0g of reinforced clostridium culture medium in 1L of culture medium, and sterilizing at 121 ℃ for 15min for later use.

Otherwise, as described in examples 1 and 2.

3.1.3 Experimental instruments

As described in examples 1 and 2.

3.1.4 Experimental strains and treatments

The test strains are: clostridium difficile (Clostridium difficile), strain No. ATCC 43593, purchased from the China center for Industrial collections of microorganisms.

Strain culture: inoculating Clostridium difficile into Clostridium difficile liquid culture medium, and anaerobically culturing at 37 deg.C for 12 hr to reach bacteria concentration of 10⁹CFU/mL, the bacterial solution was centrifuged at 2770 Xg for 15min at room temperature, and the cells were collected. The harvested thallus is washed twice with normal saline and then resuspended in normal saline, and when in use, the thallus suspension is diluted to 10 times⁹CFU/mL concentration.

3.1.5 Experimental groups and dose settings

Rats were labeled with picric acid and then acclimatized for 1 week, followed by 5/6nephrectomy, a specific procedure for which is described in example 1 for the establishment of the 5/6nephrectomy rat model.

After the model building is successful, the rats are divided into 3 groups according to the weight, and the 3 groups are respectively a model group (operation control, 2mL physiological saline per time for intragastric administration, 7 rats) and a bacterium infusion group (2mL 10 rats)⁹C, 8 clostridium difficile bacteria liquid/time with CFU/mL concentration) and an antibiotic group (2mL 2mg/mL fidaxomicin/time, 8) which is used for intragastric administration once every two days for 12 weeks, wherein the clostridium difficile content in the intestinal tract of the rat is increased by intragastric administration of clostridium difficile, and the growth of clostridium difficile in the intestinal tract of the rat is inhibited by the antibiotic group through intragastric administration of fidaxomicin.

3.2 sample Collection

Rat feces and blood samples were collected every four weeks. Collecting 1-1.5mL of rat blood by using a mode of inner canthus veins of an orbit, standing the blood for 2h, centrifuging 600 Xg for 10min to obtain serum, reserving a part of the serum at-20 ℃ for Cre and BUN detection in the blood, and reserving a part of the serum at-80 ℃ for uremia toxin detection in the blood. The fecal samples were frozen at-80 ℃ for subsequent analysis.

After 12 weeks of gavage, the rats were stunned with ether and then sacrificed by cervical dislocation for subsequent dissection. The left kidney of the rat is kept, one part of the left kidney is placed in tissue fixing liquid for tissue section analysis, all the remaining kidneys are frozen at-80 ℃ for standby, and abnormal phenomena occurring in the process of dissection are recorded by a camera.

3.3 detection of creatinine and Urea Nitrogen in serum, morphological analysis of Kidney, detection of indoxyl sulfate and p-cresol sulfate in serum

See example 1 for a specific method.

3.4 determination of Clostridium difficile content in feces

And detecting the content of clostridium difficile in excrement by real-time quantitative PCR (polymerase chain reaction), and judging the planting condition of the clostridium difficile in the intestinal tract. The specific method is shown in the content determination of clostridium difficile in the excrement in the example 2.

3.5 data analysis

The experimental data were analyzed using SPSS 22 statistical software, with the results expressed as ` x ± s, using the Ducan test for significance analysis of indoxyl sulfate, renal fibrosis and clostridium difficile content in feces, and using the Tamhane's T2 test for significance analysis of cresol, serum creatinine and serum urea nitrogen with sulfuric acid. P <0.05 was considered significantly different, P <0.01 was considered very significantly different, with statistical significance.

3.6 results and analysis of the experiment

3.6.1 Effect of Clostridium difficile on IS and PCS in serum of 5/6 nephrectomized rats

After 12 weeks of gastric lavage, the inventors detected the content of IS and PCS in the serum of rats of different treatment groups, and the results are shown in FIG. 11, and as can be seen from FIG. 11, after the clostridium difficile IS gavaged, the content of IS and PCS in the serum of rats of the bacteria lavage group IS remarkably increased (P <0.05) compared with that of the model group; after the gavage of the antibiotics, the content of IS and PCS in the serum of rats in the antibiotic group IS remarkably reduced compared with that in the model group (P < 0.05). Difficile IS proved to be capable of obviously increasing the IS and PCS content in the serum of CKD rats from the positive and negative aspects.

3.6.2 Effect of Clostridium difficile on blood creatinine and blood urea nitrogen in 5/6 nephrectomized rats

After the rats had completed 5/6nephrectomy, the rats were divided into three groups, one group being the model group (gavage saline), one group being the gavage group (clostridium difficile), the last group being the antibiotic group (gavage fidaxomicin), for a total of 12 weeks.

During the gavage process, the inventor continuously monitors the Cre and BUN content changes in the serum of rats of different treatment groups, the detection result is shown in figure 12, and as can be seen from figure 12, after the clostridium difficile is gavaged for 12 weeks, the Cre and BUN content of rats of the gavage group is remarkably increased (P <0.05) compared with that of the model group, and after the clostridium difficile is gavaged, the Cre and BUN content of rats of the antibiotic group is remarkably reduced (P <0.05) compared with that of the model group. Clostridium difficile was thus demonstrated to be indeed able to exacerbate kidney damage.

3.6.3 Effect of Clostridium difficile on kidney morphology in 5/6 nephrectomized rats

The kidney tissues of three groups of rats were subjected to Masson trichrome staining, and the fibrosis ratio of the rat kidney was counted, and the results are shown in FIGS. 13 and 14 (see the cases of renal fibrosis at the end of gastric perfusion in example 2 for specific statistical methods).

As can be seen from fig. 13 and 14, after clostridium difficile gavage, the kidney fibrosis ratio of the rats in the gavage group is significantly increased (P <0.05) compared with that in the model group; after the gavage of the antibiotics, the kidney fibrosis ratio of the rats in the antibiotic group is remarkably reduced (P <0.05) compared with that in the model group, which indicates that the clostridium difficile can aggravate the kidney fibrosis degree.

3.6.4 Experimental endpoint Clostridium difficile content in intestinal tract of rats in each group

The copy number concentration of clostridium difficile in rat fecal DNA samples of different treatment groups was determined using clostridium difficile standard DNA template as external standard, and the results are shown in table 6. As can be seen from Table 6, the content of Clostridium difficile in the feces of the rats in the perfusate group is significantly increased (P <0.05) compared with that in the model group, which indicates that the perfused Clostridium difficile successfully colonizes in the intestinal tract; after the antibiotic is infused into the stomach, the content of clostridium difficile in the feces of the rats in the antibiotic group is remarkably reduced (P <0.05) compared with that in the model group, which indicates that fidaxomicin can reduce the content of clostridium difficile in the intestinal tracts of the rats with chronic kidney diseases.

Table 6:

example 3 demonstrates at least one of the following conclusions:

(1) in terms of residual renal functions, 5/6 showed a significant increase in Cre and BUN contents and the rate of renal fibrosis in serum of nephrectomized rats (P <0.05) after supplementation with clostridium difficile, and a significant decrease in Cre, BUN and rate of renal fibrosis in serum (P <0.05) after supplementation with fidaxomicin, thereby demonstrating that clostridium difficile in the intestinal tract can aggravate renal injury.

(2) In the aspect of uremic toxins, the content of IS and PCS in serum of 5/6 nephrectomized rats IS remarkably increased (P <0.05) after the clostridium difficile IS perfused, and the content of two uremic toxins in serum IS remarkably reduced (P <0.05) after the fidaxomicin IS supplemented, thereby proving that the clostridium difficile in intestinal tracts can promote the generation of IS and PCS.

(3) After fidaxomicin supplementation, the content of clostridium difficile in the intestine of 5/6 nephrectomized rats was significantly reduced (P <0.05), even with 47.5% of 5/6 nephrectomized rat intestines in which clostridium difficile was not detected.

Example 3 demonstrates that c.difficile can exacerbate 5/6 kidney injury in nephrectomized rats, both positively and negatively. Difficile after gastric lavage can obviously increase 5/6 Cre and BUN contents in serum of a nephrectomized rat and the kidney fibrosis proportion, and the Cre and BUN contents in blood and the kidney fibrosis proportion are obviously reduced after gastric lavage by fidaxomicin.

The inventor finds that clostridium difficile in intestinal tracts can promote the generation of enterogenic uremic toxins such as IS and PCS and aggravate 5/6 renal failure injury of nephrectomized rats, and A6 can maintain 5/6 renal failure of nephrectomized rats by inhibiting the colonization of clostridium difficile in intestinal tracts of 5/6 nephrectomized rats.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. Use of bifidobacterium animalis a6 in the manufacture of a health product for the relief of chronic kidney disease.

2. The use of claim 1, wherein the chronic kidney disease comprises at least one member selected from the group consisting of renal injury, renal fibrosis, compensatory glomerular hypertrophy, glomerulosclerosis, tubulointerstitial fibrosis, renal failure, and uremia;

optionally, the nutraceutical is for use in at least one of:

regulating intestinal microecology, improving intestinal metabolism, inhibiting enterogenous uremia toxin, and inhibiting growth of Clostridium difficile;

optionally, the enterogenic uremic toxin is indoxyl sulfate, p-cresol sulfate.

3. Use of bifidobacterium animalis a6 in the manufacture of a medicament for use in modulating the content of clostridium difficile.

4. Use according to claim 3, wherein the modulation of the content of Clostridium difficile is a downregulation of the content of Clostridium difficile.

5. A method of screening for a drug for ameliorating chronic kidney disease,

and (3) administering the candidate drug to an animal model, wherein the animal model is a chronic kidney disease model, and comparing the content of the animal bifidobacterium A6 in the model before and after administration to determine whether the drug is the candidate drug.

6. The method of claim 5, wherein an increase in Bifidobacterium animalis A6 in the model after administration compared to before administration is indicative of the drug being the target drug.

7. The method of claim 6, further comprising a decrease in the Clostridium difficile content of the gut in the model after administration as compared to before administration is indicative of the drug being a target drug;

optionally, the method further comprises: comparing changes in the model before and after dosing of at least one of:

changes in enterogenic uremic toxins, blood creatinine, blood urea nitrogen,

wherein a decrease in at least one of the above after administration of the model is indicative that the drug candidate is a drug of interest;

optionally, the enterogenic uremic toxin is indoxyl sulfate, p-cresol sulfate;

optionally, the chronic kidney disease includes at least one selected from the group consisting of renal injury, renal fibrosis, compensatory glomerular hypertrophy, glomerulosclerosis, tubulointerstitial fibrosis, renal failure, and uremia.

8. A pharmaceutical composition for alleviating chronic kidney disease or regulating the content of Clostridium difficile, comprising Bifidobacterium animalis A6.

9. The pharmaceutical composition of claim 8, further comprising one or more therapeutic agents;

optionally, the therapeutic agent comprises AST-120 or fidaxomicin;

optionally, the modulating the content of clostridium difficile is down-regulating the content of clostridium difficile;

10. A pharmaceutical combination comprising Bifidobacterium animalis A6 and other agents useful in the treatment or prevention of chronic kidney disease, said other agents useful in the treatment of chronic kidney disease comprising: AST-120 or fidaxomicin.