CN112138029A

CN112138029A - Application of bacteroides ovatus in preparation of anti-renal fibrosis medicine

Info

Publication number: CN112138029A
Application number: CN202011198368.5A
Authority: CN
Inventors: 周伟; 张之昊; 陈敏敏; 谭宁华; 吴文辉
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-10-31
Filing date: 2020-10-31
Publication date: 2020-12-29
Anticipated expiration: 2040-10-31
Also published as: CN112138029B

Abstract

The invention discloses application of bacteroides ovatus in preparing a renal fibrosis resistant medicament. Renal fibrosis is the terminal stage of various chronic renal diseases and is a pathological process characterized by leukocyte infiltration, renal tubular cell apoptosis and necrosis, proliferation of tubular interstitial fibroblasts, and extracellular matrix deposition. Drugs against renal fibrosis are of great importance for the treatment of renal fibrosis. The invention discovers that the bacteria ovatus has obvious activity of resisting renal fibrosis on a renal fibrosis model mouse, can obviously improve the pathological changes of renal tissues and plays a role in protecting the kidney. The prior art does not disclose the technical scheme of the invention.

Description

Application of bacteroides ovatus in preparation of anti-renal fibrosis medicine

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of bacteroides ovatus in preparation of a renal fibrosis resistant medicine.

Background

Renal fibrosis is the terminal stage of various chronic renal diseases and is a pathological process characterized by leukocyte infiltration, renal tubular cell apoptosis and necrosis, proliferation of tubular interstitial fibroblasts, and extracellular matrix deposition.

The method for inducing renal fibrosis mainly comprises drugs or poisons (mercuric chloride, cyclosporine A, nephrotoxic drugs, aminoglycoside drugs and the like), operations (ureter unilateral ligation, 5/6 nephrectomy model, renal ischemia-reperfusion and the like), unilateral nephrectomy combined Ang II and other combined factors, transgenic models and the like. Wherein, ureter unilateral ligation (UUO) model renal fibrosis is obvious in characteristic, low in mortality, and more suitable for research on renal interstitial fibrosis. The model simulates renal interstitial injury caused by clinical ureteral obstruction, and is an ideal renal interstitial fibrosis model for researching relatively mature.

Drugs against renal fibrosis are of great importance for the treatment of renal fibrosis.

Disclosure of Invention

The invention aims to provide application of bacteroides ovatus in preparing a renal fibrosis resistant medicament.

The above purpose of the invention is realized by the following technical scheme:

use of viable bacteria of bacteroides ovatus for preparing medicine for resisting renal fibrosis is provided.

A pharmaceutical preparation for resisting renal fibrosis contains viable bacteria and pharmaceutically acceptable adjuvants, and is prepared into pharmaceutically acceptable dosage forms.

Further, the auxiliary material is solid, liquid or semisolid auxiliary material.

Further, the dosage form is a tablet, a capsule, a pill, an oral liquid or a powder.

Has the advantages that:

the invention discovers that the bacteria ovatus has obvious activity of resisting renal fibrosis on a renal fibrosis model mouse, can obviously improve the pathological changes of renal tissues and plays a role in protecting the kidney. The prior art does not disclose the technical scheme of the invention.

Drawings

FIG. 1 is a histogram comparing the serum content of triglyceride, urea nitrogen, creatinine and uric acid in mice of each group;

FIG. 2 is a graph of H & E staining and Masson staining of kidney tissue sections of various groups of mice;

fig. 3 is a WB histogram of fibrin in kidney tissue and a gray-scale scan histogram of fibrin in kidney tissue of each group of mice.

Detailed Description

The following detailed description of the present invention is provided in connection with the accompanying drawings and examples, but not intended to limit the scope of the invention.

First, experimental material

The bacteria belonging to live bacteria genus, originated from ATCC (ATCC 8483), and cultured strictly according to the ATCC instructions, and administered by gavage with a live bacterial medium; SPF-grade ICR mice were purchased from the Qinglongshan animal breeding farm in Jiangning, Nanjing (quality certification number: No. 201926974).

Paraformaldehyde (Tianjin Corp. Fine chemical research institute), xylene (national group chemical reagent Co., Ltd.), H & E dye liquor (Beijing Soxhalabao bioscience), massson dye liquor set (Beijing Soxhalabiosome), neutral gum (national group chemical reagent Co., Ltd.), fibrinectin antibody (Abcam, ab45688), Collagen I antibody (Abcam, ab34710), Collagen III antibody (Proteintech, 22734-1-AP), glycine and Tris (Shanghai Biotech Co., Ltd.), SDS (Shengxing bioscience), and Tween 20 (Nanjing chemical reagent Co., Ltd.).

Second, Experimental methods

1. Animal grouping, modeling and administration

After one week of acclimatization, the ICR mice were randomly divided into three groups, 6 in each group, namely a control group, a model group and a bacteria irrigation group. Performing unilateral ureter ligation (UUO) operation on a model group and a bacterium perfusion group, and only causing no ligation (Sham) on an operation wound in a control group, wherein the bacterium perfusion group starts to pre-administer live bacteria 7 days before the operation, the stomach is continuously perfused and administered with the live bacteria 14d after ligation, the daily dose is 200 mu L of live bacteria-containing culture medium/bacterium, the CFU is 2 x 10⁸[bacteroides ovatus(BO)]The viable bacteria are taken out from the anaerobic box 2h before the bacteria are given every day (the anaerobic box comprises 10% CO)₂；10％H₂And 80% N₂) The control group and the model group were administered with the same amount of vehicle per day.

2. Animal sacrifice, sampling and sample handling

After 14 days of live bacteria administration after surgery, whole blood was collected from the eye and the mice were dissected to remove the ligated side kidney tissue. Centrifuging whole blood at 3000rpm and 4 deg.C for 10min, sucking supernatant clear serum liquid, transferring to a sterile centrifuge tube for biochemical index detection, and placing the rest in-80 deg.C refrigerator for use; rapidly picking kidney tissue, washing in normal saline, removing surface adipose tissue and mucosa, fixing 1/3 in 4% paraformaldehyde for histopathological examination; the rest part is marked and put into a refrigerator at the temperature of 80 ℃ below zero for detecting the related protein level.

3. Serum biochemical and histopathological detection

3.1 Biochemical detection of serum

The full-automatic biochemical analyzer is automatically detected by the instrument after sample loading, the sample concentration is automatically generated by the instrument according to an index result, and then the excel is exported; operating according to the kit specification according to biochemistry, converting the measured absorbance of the sample into concentration according to the formula in the specification, using GraphPad software to make a histogram, and performing statistical test.

3.2 tissue sections and H & E, Masson staining

And (4) dehydrating the fixed kidney tissues, and embedding and slicing the dehydrated kidney tissues by paraffin.

H & E staining: sequentially placing the slices into xylene I for 20min, xylene II for 20min, anhydrous ethanol I for 5min, anhydrous ethanol II for 5min, and 75% alcohol for 5min, and washing with tap water; staining the slices in hematoxylin staining solution for 3-5min, washing with tap water, differentiating the differentiation solution, washing with tap water, returning blue to the blue solution, and washing with running water; the slices are dehydrated for 5min respectively by adding 85 percent and 95 percent gradient alcohol in sequence, and are dyed for 5min in eosin dye solution; placing the slices in anhydrous alcohol I for 5min, anhydrous alcohol II for 5min, anhydrous alcohol III for 5min, xylene I for 5min, and xylene II for 5min, and sealing with neutral gum.

And (3) massson staining: sequentially placing the slices into xylene I for 20min, xylene II for 20min, anhydrous ethanol I for 5min, anhydrous ethanol II for 5min, and 75% alcohol for 5min, and washing with tap water; soaking the slices in potassium dichromate overnight, and washing with tap water; mixing the solution A and the solution B in equal ratio to obtain a hematoxylin staining solution, slicing the stained solution into hematoxylin for 3min, washing with tap water, differentiating the differentiation solution, washing with tap water, returning blue to the blue returning solution, and washing with running water; soaking the slices in ponceau acid fuchsin for 5-10min, and rinsing with tap water; dip-dyeing with phosphomolybdic acid water solution for 1-3 min; directly dyeing the phosphomolybdic acid in aniline blue dye solution for 3-6min without washing; the slices are differentiated by 1% glacial acetic acid, and dehydrated by two cylinders of absolute ethyl alcohol; placing the slices in a third jar with anhydrous ethanol for 5min, transparent xylene for 5min, and sealing with neutral gum.

3.3 microscopic examination and image acquisition and analysis

The pathological section is evaluated and scored after microscopic examination, and representative images are taken.

4. Expression of fibrosis-associated proteins in kidney tissue

The relative expression of fibrosis-related proteins Fibronectin, Collagen I, Collagen III and alpha-SMA in kidney tissues is detected by using a western blot method.

4.1 preparation of tissue protein samples

Weighing a proper amount of tissue according to the operation instruction of the tissue protein extraction kit, adding lysis solution containing a protease inhibitor, and homogenizing and lysing in ice bath; high-speed freezing and centrifuging (4 ℃, 12000rpm, 15min), and then sucking supernatant to obtain a protein solution; quantifying the obtained tissue protein according to the operation instruction of the BCA protein quantification kit; after adjusting the concentration, adding a loading buffer solution, boiling to obtain a tissue protein sample, and performing gel electrophoresis.

4.2 gel electrophoresis and transfer of membranes

Preparing polyacrylamide gel according to a formula, putting the polyacrylamide gel into an electrophoresis tank, then loading the polyacrylamide gel, filling electrophoresis liquid, flattening a protein sample at 60V, and then performing electrophoresis at 120V until a bromophenol blue indicator approaches the bottom of the gel; and (3) performing wet membrane transfer after electrophoresis, fixing the membrane in a membrane transfer tank according to the sequence of sponge-filter paper-glue-PVDF membrane-filter paper-sponge, fully adding membrane transfer liquid, and performing membrane transfer in ice bath (280mA, about 110 min).

4.3 blocking and antibody incubation

After the membrane is transferred, the membrane is placed in 5% skimmed milk, and is slowly shaken for 2 hours at room temperature for sealing; after the sealing is finished, cutting strips according to the molecular weight of the target protein, putting the strips into a corresponding primary antibody, and incubating overnight at 4 ℃; recovering the primary antibody and washing the membrane for three times, wherein each time lasts for 10 min; putting the strips into a secondary antibody solution, and incubating for 2h at room temperature; after completion, the membrane was washed three times for 10min each time.

4.4 development and results processing

The developing solution is prepared according to the following steps of A: b is 1: 1, the medicine is prepared and used at present; carrying out development operation according to the operation specification of a developing instrument to obtain a protein fluorescent strip; and repeating each group of data for three times, performing gray scanning by using Image J software, and making a histogram by using GraphPad software to obtain the relative expression result of each protein.

5. Data processing and statistical analysis

Student's test, () p <0.05, () p <0.01, () p <0.001 was used.

Third, experimental results

1. Effect of bacteroides ovatus on serum biochemistry of UUO model mice

The results are shown in fig. 1, and compared with the control group (Sham), the serum Triglyceride (TG), urea nitrogen (BUN), creatinine (Scr) and Total Cholesterol (TC) of the model group (UUO) mice are obviously increased, which indicates that the kidney function is abnormal and the modeling is successful; the four indexes are obviously reduced after the bacteroides ovatus is continuously administrated for 14 days, and the protective effect on the renal function is shown.

2. Protective effect of bacteroides ovatus on ligated lateral kidneys of UUO model mice

As can be seen from the H & E staining results in FIG. 2, the renal tubules in the kidney tissue of the model group (UUO) mice were moderately atrophied, the lumen was enlarged, the local inflammation was significant, and the epithelial cells were necrotic or denatured, as compared with the control group (Sham); moderate atrophy of glomeruli; the interstitium has obvious inflammatory cell infiltration. After being administrated, the bacterioides ovatus can obviously improve the pathological changes, the atrophy and the cavity expansion degree of renal tubules are obviously reduced compared with a model group, the epithelium is only slightly necrotized by cells, and the mild inflammatory cell infiltration is realized.

As can be seen from the Masson staining results in FIG. 2, the major medullary part of the renal pelvis submucosal tissue in the renal interstitium of the model group (UUO) mice showed a large amount of blue-stained collagen fiber deposition, and some of them fused into a thick cord; while the kidney tissues of mice in the given strain group (UUO + bacteroides ovatus) have obviously reduced collagen deposition, and a small amount of blue-stained collagen fiber deposition is observed in the sub-mucosal tissues of the renal pelvis in the main medullary part, and no obvious cord-like fusion is observed.

The result of tissue section staining shows that the bacteria ovatus can improve the kidney tissue lesion, and the kidney protection effect is achieved mainly by improving the damage of renal tubules and the generation and accumulation of collagen.

3. Effect of bacteria ovatus on the expression of fibrotic proteins in Kidney tissue

The results of western blot detection of fibrosis-associated proteins in kidney tissues of each group of mice are shown in fig. 3, and the expressions of fibronectin fibrinectin (fn), Collagen type I (Collagen I) and Collagen type III (Collagen III) in the model group (UUO) are significantly improved, which indicates that the kidney of the mice is obviously fibrotic and diseased through UUO surgery; the fibrotic protein decreased significantly after 21 days of continuous oral administration of bacteroides ovatus, indicating that bacteroides ovatus has anti-renal fibrotic activity.

The pharmacological tests show that the bacteroides ovatus has obvious renal fibrosis resisting activity and has an application prospect in developing renal fibrosis resisting medicines.

The above-described embodiments are intended to be illustrative of the nature of the invention, but those skilled in the art will recognize that the scope of the invention is not limited to the specific embodiments.

Claims

1. Use of viable bacteria of bacteroides ovatus for preparing medicine for resisting renal fibrosis is provided.

2. A pharmaceutical preparation for resisting renal fibrosis is characterized in that: the active ingredient is live bacteria of bacteroides ovatus, and also contains pharmaceutically acceptable auxiliary materials, and the preparation can be prepared into pharmaceutically acceptable dosage forms.

3. The pharmaceutical formulation of claim 2, wherein: the auxiliary material is solid, liquid or semisolid auxiliary material.

4. The pharmaceutical formulation of claim 2, wherein: the dosage form is tablet, capsule, pill, oral liquid or powder.