CN111265653B

CN111265653B - Application of atrial natriuretic peptide in preparation of medicines for treating inflammatory bowel disease

Info

Publication number: CN111265653B
Application number: CN202010083380.5A
Authority: CN
Inventors: 付妤; 陈超越
Original assignee: Tongji Medical College of Huazhong University of Science and Technology
Current assignee: Tongji Medical College of Huazhong University of Science and Technology
Priority date: 2020-02-09
Filing date: 2020-02-09
Publication date: 2023-05-09
Anticipated expiration: 2040-02-09
Also published as: CN111265653A

Abstract

The invention relates to application of an atrial Li Na peptide in preparing a medicament for treating inflammatory bowel disease. Use of atrial natriuretic peptide in the manufacture of a medicament for the treatment of inflammatory bowel disease, wherein the inflammatory bowel disease comprises ulcerative colitis, crohn's disease, indeterminate colitis; the beneficial effects are that: has no adverse effect on normal cells and no toxic or side effect, can effectively relieve colonic inflammation of colonitis and reduce secretion of inflammatory cytokines of colonic tissues.

Description

Application of atrial natriuretic peptide in preparation of medicines for treating inflammatory bowel disease

Technical Field

The invention belongs to the biomedical field, in particular to application of atrial natriuretic peptide in preparing anti-inflammatory drugs.

Background

Inflammatory bowel disease (inflammatory bowel disease, IBD) mainly includes ulcerative colitis (ulcerative colitis, UC) and Crohn's Disease (CD), and its breakthrough in treatment is not isolated from the development of new drugs.

The primary therapeutic agents for IBD include 5 aminosalicylic acid, glucocorticoids, immunosuppressants and biological agents, inducing and maintaining disease remission through immunomodulation. In recent years, drug development is mainly focused on biological agents and small molecule targeted drugs, but research on hormone drugs is relatively lacking. Although glucocorticoid can induce and relieve rapidly and has low cost, it has many side effects after long-term use due to the physiological functions of regulating growth, development, metabolism, etc., and is liable to cause gastrointestinal hemorrhage, osteoporosis, metabolic disorder, etc., and cannot be used for IBD maintenance therapy.

However, the powerful anti-inflammatory effect exhibited by hormonal drugs suggests that we can search for new break-through from the point of endocrine-immune regulation and develop effective and inexpensive drugs. Use of a chlamydia murine as disclosed in CN108815199a in the preparation of a medicament for the treatment of ulcerative colitis.

Atrial Natriuretic Peptide (ANP), also known as atrial natriuretic peptide (atrial natriuretic peptide), is a peptide hormone initially extracted from atrial myocytes. At present, the research field of ANP is also wider, and the ANP has the physiological functions of promoting urination, promoting natrium, dilating vascular smooth muscle, regulating and controlling water-salt balance and maintaining blood pressure.

Disclosure of Invention

Aiming at the problems, the invention provides application of atrial Li Na peptide in preparing medicines for treating inflammatory bowel disease, and mainly solves the problem that the application of atrial natriuretic peptide in anti-inflammatory medicines, especially medicines for treating inflammatory bowel disease, is blank in the prior art.

In order to solve the problems, the invention adopts the following technical scheme:

application of atrial natriuretic peptide in preparing antiinflammatory medicine is provided.

Application of atrial natriuretic peptide in preparing medicine for treating inflammatory bowel disease is provided.

One mode, wherein the atrial natriuretic peptide has an amino acid sequence of: ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr.

One way, the atrial natriuretic peptide has a chemical formula of C ₁₂₇ H ₂₀₅ N ₄₅ O ₃₉ S ₃ 。

In one form, the inflammatory bowel disease includes ulcerative colitis, crohn's disease.

In one mode, the inflammatory bowel disease comprises indeterminate colitis.

The beneficial effects of the invention are as follows:

has no adverse effect on normal cells and no toxic or side effect, can effectively relieve colonic inflammation of colonitis and reduce secretion of inflammatory cytokines of colonic tissues.

Drawings

FIG. 1 is a graph showing the expression of ANP and its receptor in the intestinal tract and related lymphoid organs of mice;

FIG. 2 is a graph of ANP in alleviating intestinal inflammation in mice;

FIG. 3 is a graph showing the expression of the enteroinflammatory factor and autophagy-related genes in mice with reduced ANP.

Detailed Description

EXAMPLE 1 therapeutic Effect on murine ulcerative colitis

Materials:

A. mice: c57BL/6J mice (purchased from Beijing vitamin Torilhua Co., ltd.) were kept under SPF conditions to 20-22g and about 8 weeks old.

B. Human atrial natriuretic peptide ANP (purchased from UK Tocris Bioscience company), amino acid sequence Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr, molecular weight of 3080.46Da; the purity was 95.8%.

Method and results:

1. constructing a Dextran Sodium Sulfate (DSS) induced colitis mouse model and interfering with ANP recombinant proteins.

(1) Grouping of experimental mice:

grouping of experimental mice: male C57BL/6 mice were randomly divided into a control group, a DSS group, a DSS+2ugANP group and a DSS+20ugANP group, 6 mice/group, and were adaptively fed for 7 days, and their diet, activity, and urination and defecation conditions were observed and the body weight was weighed.

(2) Intervention of mice:

control group: normal water was freely drunk for 7 days, and 400ul of PBS solution was injected intraperitoneally on days 0, 1, 3, 5, 7;

DSS group: mice were freely drinkable with 2.5% dss solution for 7 days, and were intraperitoneally injected with 200ul PBS solution on days 0, 1, 3, 5, 7;

ANP intervention group: mice were free to drink 2.5% dss solution for 7 days, and 200ul of PBS solution containing 2ug and 20ug recombinant ANP was injected intraperitoneally on days 0, 1, 3, 5, 7.

(3) Record of the general status of mice:

after the beginning of the molding, the weight, the behavior of the feces (normal, pasty or watery feces) and the blood-carrying condition (no, occult or obvious macroscopic feces) and the activity condition (active or listlessness) of the mice were recorded every day.

(4) Mice were sacrificed and specimens were collected:

on day 7, the mice are sacrificed, after the mice are anesthetized by pentobarbital sodium, the eyeballs are subjected to blood sampling, the abdominal cavity is opened along the midline of the abdomen, a complete straight colon is left from above the rectum to the ileocecum, and the colon length is measured and recorded; while spleen was left and weighed. The colon feces were washed and distal colon tissue was preserved at-80℃for RT-qPCR detection.

2. Real-time quantitative RT-qPCR

(1) Extraction of Total RNA and concentration determination

About 10mg of the tissue was cut out and placed in a 2ml EP tube, numbered, and 1ml Trizol solution and 2 magnetic beads were added;

homogenizing in a grinder for 2 times at 65Hz and 120 sec; adding 200ul (1/5 volume of trizol solution) of chloroform into the homogenized lysate, covering the tube cover, shaking vigorously for 15sec, and standing at room temperature for 5min after the solution is fully mixed until no phase separation phenomenon exists;

and (3) centrifuging: 4 ℃, 12000rpm, 15min; carefully remove the EP tube from the centrifuge and divide the homogenate into three layers: colorless supernatant, middle white layer, colored lower organic phase;

taking another new 1.5ml EP tube and numbering, sucking 300ul of supernatant, transferring to the new EP tube, adding 300ul of isopropyl alcohol with equal volume into the supernatant, gently inverting the EP tube upside down to fully mix, standing for 10min at room temperature, centrifuging: 4 ℃, 12000rpm, 10min; taking out the EP tube from the centrifuge, and observing that RNA sediment exists at the bottom of the test tube, carefully discarding the supernatant, and avoiding discarding the RNA sediment;

slowly add 1ml of pre-chilled 75% ethanol along the tube wall (note not touching the pellet), gently invert the tube upside down, centrifuge: 4 ℃, 12000rpm, 5min; carefully discarding the supernatant, inverting the EP tube on the filter paper, drying the precipitate at room temperature for about 5min, adding a proper amount of high-pressure double distilled water after drying the precipitate, fully dissolving the precipitate, and adjusting the water quantity according to the amount of the precipitate;

after the RNA precipitate was completely dissolved, the centrifuge was transiently centrifuged.

(2) RNA concentration determination

Washing the cuvette, opening the spectrophotometer, and adding double distilled water into the cuvette for zeroing.

Adding 2ul of sample to be tested and 98ul of double distilled water into a cuvette, measuring and recording A260 and A280 values,

to calculate the purity and concentration of RNA.

(3) Reverse transcription

Taking out the reagent required by reverse transcription, thawing at room temperature, and vortex oscillating and mixing uniformly, and then instantaneously separating; preparing a reaction system: calculating the dosage of each component required by proper volume according to the following reverse transcription reaction system proportion, respectively adding 0.5ml of EP tube, and carrying out instantaneous separation after shaking and mixing by a vortex instrument; reverse transcription: after the preparation of the reverse transcription reaction system, reverse transcription was performed in a reverse transcription apparatus at 37℃for 15min, at 85℃for 15sec. After which PCR was performed or stored at-20 ℃.

10ul reaction system: RNA-Primermix (5X) 2ul+RNA test sample (1/concentration 1000) ul+DEPC water (10-2-VRNA) (ul).

④RT-qPCR

Taking out the reagent required by PCR, thawing at room temperature, and performing vortex oscillation and mixing uniformly and then performing instantaneous separation;

preparing a reaction system: according to the quantity of the sample to be tested and the index to be tested, preparing the actual required dosage according to the following proportion of a PCR reaction system; the vortex instrument oscillates and mixes evenly and then instantaneously separates;

sample adding: taking a 96-well PCR plate, designing a sample adding sequence, marking, adding a mixture of 4ul cDNA and water on one side wall, rotating the 96-well plate, adding a mixture of 6ul SYBR Green and primers on the other side wall, sealing the 96-well plate by using a sealing plate film after sample adding, and centrifuging;

amplification: putting the 96-well plate into a PCR instrument, opening a software system, and setting a program for amplification;

amplification conditions: pre-denaturation at 95℃for 10min, denaturation at 95℃for 30sec, annealing at 60℃for 1min, extension at 72℃for 30sec per cycle, and amplification was performed by repeating 40 cycles;

analysis of results: GAPDH or beta-actin is used as reference gene, and is used

The expression of the target genes of each group relative to the control group is analyzed.

10ul reaction system: 5ul SYBR Green dye+0.5 ul, upper primer+0.5 ul, lower primer+1 ul, target gene+3 ul dd H ₂ O。

Primer sequences for RT-qPCR

3. Results

(1) As shown in FIG. 1, three wild type untreated 8-week-old male C57BL/6 mice were selected, and the mRNA levels of the gene NPPA encoding ANP and its two major receptor NPR-A, NPR-C genes in the mice were examined by RT-qPCR in the jejunum, ileum, colon and liver, spleen, kidney, appendix, small intestine PP-knot, mesenteric lymph node, thymus, and the mean and standard deviation thereof were represented by bar graphs.

The higher the column, the greater the relative expression of NPPA and NPR-A, NPR-C mRNA at that site, and the vertical axis value indicates the ratio of the expression of NPPAmRNA at that site to that of NPPAmRNA from the atrium of the same mouse.

(2) As in fig. 2, the specific grouping of mice is shown in the methods section. The ratio of the daily body weight of each mouse to the body weight of the mouse on day 0 is measured and calculated, and the average value and standard deviation of each group are calculated; the results show that the average body weight of the original ANP group mice was heavier than the DSS group for each mouse and had statistical significance, with no statistical differences in body weight for different doses of ANP treatment. The average spleen weight of the ANP mice is lighter than that of the DSS mice, and the average colon length of the ANP mice is longer than that of the DSS mice, and the ANP mice have statistical significance; there was no statistical difference in spleen weight and colon length between the two groups at different doses of ANP.

ANP-alleviating mice were characterized by reduced intestinal inflammation, which reduced the body weight loss, spleen enlargement and colon shortening in DSS model mice, and this effect was dose-independent.

(3) As shown in FIG. 3, the mRNA levels of inflammatory cytokines IL-1 beta, TNF-alpha, IL-6, IFN-gamma and autophagy related genes ATG5 and ATG12 of colon tissue of the tail end of the four groups of mice are detected by using an RT-qPCR method, the column heights represent average values of ratios of the mRNA levels to the internal reference gene beta-actin, and the distances between the horizontal lines above the columns represent standard deviations. The results showed that IL-1. Beta., TNF-. Alpha., IL-6, IFN-. Gamma., ATG5, ATG12 were decreased in mRNA expression to a different extent in the ANP group compared to the DSS group, and that the decrease in TNF-. Alpha., IFN-. Gamma., ATG5, ATG12 was independent of the dose of ANP, and the decrease in IL-1. Beta. And IL-6 were correlated with the dose of ANP, further demonstrating that ANP can reduce intestinal inflammation in mice from the viewpoint of gene expression.

It will be apparent to those skilled in the art that various modifications to the above embodiments may be made without departing from the general spirit and concepts of the invention. Which fall within the scope of the present invention. The protection scheme of the invention is subject to the appended claims.

Sequence listing

<110> affiliated synergetic Hospital of the university of science and technology, shangji medical college

Application of <120> atrial natriuretic peptide in preparation of inflammatory bowel disease treatment drug

<140> 202010083380.5

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 28

<212> PRT

<213> artificial sequence

<400> 1

Ser Leu Ala Ala Ser Ser Cys Pro Gly Gly Ala Met Ala Ala Ile Gly

1 5 10 15

Ala Gly Ser Gly Leu Gly Cys Ala Ser Pro Ala Thr

20 25

Claims

1. Application of atrial natriuretic peptide in preparing ulcerative colitis treatment medicament, wherein the amino acid sequence of the atrial natriuretic peptide is as follows:

Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr。

2. the use according to claim 1, wherein the atrial natriuretic peptide has the chemical formula C ₁₂₇ H ₂₀₅ N ₄₅ O ₃₉ S ₃ 。