CN114288388B - Application of omelanin in preparation of medicines for treating inflammatory bowel disease - Google Patents
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Abstract
The invention belongs to the field of inflammatory bowel disease research, and particularly discloses application of omelanin in preparation of a medicament for treating inflammatory bowel disease. Application of omelanin in preparing medicine for preventing and treating inflammatory bowel disease is provided. Application of omelanin in preparation of preparation for inhibiting colitis intestinal inflammatory factor secretion is provided. Application of omelanin in preparation of preparation for inhibiting colitis intestinal inflammatory factor secretion is provided. Use of an intestinal inflammatory cytokine in the preparation of a formulation for inhibiting the expression of omelanin. The omelanin reduces secretion of inflammatory factors in intestinal tracts of colonitis induced by Dextran Sodium Sulfate (DSS), and effectively improves the intestinal tract inflammation; meanwhile, a corresponding scheme is provided for the adjustment of the omelanin.
Description
Technical Field
The invention belongs to the field of inflammatory bowel disease research, and particularly relates to application of omelanin in preparation of medicines for treating inflammatory bowel disease.
Background
Inflammatory bowel disease is a group of intestinal idiopathic diseases, and the incidence of IBD is continuously increasing in emerging industrialized countries including china, which brings great burden to society and countries. The treatment for IBD is mainly anti-inflammatory, hormonal, immunosuppressive, biological agents etc., but even with active treatment, it is difficult for about 50% of moderately severe IBD patients to achieve the goal of "complete mucosal healing". There is an urgent need to find new therapeutic targets that overcome the current bottleneck in IBD therapy.
Omelanin (Omentin) is a newly discovered fat factor, is expressed in visceral fat, mesothelial cells, vascular cells, airway goblet cells, ovaries, blood plasma and the like, and has the regulation effects on resisting atherosclerosis, improving insulin sensitivity, protecting cardiovascular and the like. The amino acid sequence of omeningin (Omentin) is: MRGSHHHHHH GMASTDEANT YFKEWTCSSS PSLPRSCKEI KDECPSAFDG LYFLRTENGV IYQTFCDMTS GGGGWTLVAS VHENDMRGKC TVGDRWSSQQ GSKAVYPEGD GNWANYNTFG SAEAATSDDY KNPGYYDIQA KDLGIWHVPN KSPMQHWRNS SLLRYRTDTG FLQTLGHNLF GIYQKYPVKY GEGKCWTDNG PVIPVVYDFG DAQKTASYYS PYGQREFTAG FVQFRVFNNE RAANALCAGM RVTGCNTEHH CIGGGGYFPE ASPQQCGDFS GFDWSGYGTH VGYS (source e.coli).
The omelanin was found to be significantly reduced in the serum of patients with inflammatory bowel disease. The role of Omentin in inflammatory bowel disease may play an anti-inflammatory role, so specific effects were observed using the Omentin intervention in the acute DSS mouse model.
There is no application of Omentin in preparing medicines for treating inflammatory bowel disease.
Disclosure of Invention
Aiming at the problems, the invention provides the application of the omeningin in preparing the medicines for treating the inflammatory bowel disease, mainly solves some research gaps of the traditional omeningin, and makes up some gaps of intestinal inflammation treatment.
In order to solve the problems, the invention adopts the following technical scheme:
application of omelanin in preparing medicine for preventing and treating inflammatory bowel disease is provided.
In some embodiments, the inflammatory bowel disease includes ulcerative colitis and crohn's disease.
In some aspects, the inflammatory bowel disease is DSS colitis.
Application of omelanin in preparation of preparation for inhibiting colitis intestinal inflammatory factor secretion is provided.
In some embodiments, the colitis intestinal inflammatory factor is at least one of IL-1β, IL-1α, TNF- α, IL-6, IL-17, GM-CSF mRNA.
In some aspects, the colitis intestinal inflammatory factor secretion is dextran sodium sulfate induced colitis intestinal inflammatory factor secretion.
Application of omelanin in preparation of preparation for inhibiting colitis intestinal inflammatory factor secretion is provided.
Use of an intestinal inflammatory cytokine in the preparation of a formulation for inhibiting the expression of omelanin.
In some embodiments, the intestinal inflammatory cytokine comprises L-1β, IL-1α, TNF- α, IL-6, IL-17, GM-CSF mRNA.
Application of omelanin detection agent in preparation of inflammatory bowel disease detection preparation is provided.
The beneficial effects of the invention are as follows:
omelanin reduces secretion of colonitis intestinal inflammatory factors induced by Dextran Sodium Sulfate (DSS), and effectively improves intestinal inflammation; a new detection mode is also provided. Meanwhile, a corresponding scheme is provided for the adjustment of the omelanin.
Drawings
FIG. 1 is a graph showing the intestinal tissue and serum expression of Omentin in mice with acute DSS colitis;
FIG. 2 shows the degree of variation in body weight and colon length of mice with Omentin improved acute DSS colitis;
FIG. 3 shows the reduction of intestinal inflammatory cytokine secretion by mice with acute DSS colitis by Omentin.
Detailed Description
The first aspect of this section describes the use of omelanin：
One of them, omelanin, is used in preparing medicine for preventing and treating inflammatory bowel disease.
In some embodiments, the inflammatory bowel disease includes ulcerative colitis and crohn's disease.
In some embodiments, the inflammatory bowel disease is DSS colitis (dextran sodium sulfate induced colitis).
And secondly, the application of omelanin in preparation of preparations for inhibiting the secretion of inflammatory factors in colonitis intestinal canal.
In some embodiments, the colitis intestinal inflammatory factor is at least one of IL-1β, IL-1α, TNF- α, IL-6, IL-17, GM-CSF mRNA.
In some aspects, the colitis intestinal inflammatory factor secretion is dextran sodium sulfate induced colitis intestinal inflammatory factor secretion.
And thirdly, the application of the omeningin in preparing the preparation for inhibiting the secretion of inflammatory factors in colonitis intestinal canal.Part I Two aspects describe the regulation of omeningin：
Use of an intestinal inflammatory cytokine in the preparation of a formulation for inhibiting the expression of omelanin.
The expression behavior of the omeningin can be reduced through the intestinal inflammatory cytokines, so that the regulation effect of the omeningin is realized in some scenes. Such as: in commercial omelanin property research experiments, in order to investigate conditions affecting omelanin expression, modulation was performed at this time using intestinal inflammatory cytokines.
In some embodiments, the intestinal inflammatory cytokine comprises L-1β, IL-1α, TNF- α, IL-6, IL-17, GM-CSF mRNA. Of course, other cytokines capable of affecting omestatin expression are also within the scope of the present invention.
Application of omelanin detection agent in preparation of inflammatory bowel disease detection preparation is provided. Inflammatory bowel disease can be characterized to some extent by detecting the reticulin level in a suspected inflammatory bowel disease test.
The third aspect of this section is described in connection with a specific study：
1. Material
1.1 animals: male 6-8 week C57BL/6J mice (purchased from Beijing vitamin Toril Lihua Co.) were bred in SPF environment;
1.2 reagent omeningin (Omentin Human E.coli), purchased from BioVender Co., europe, 294 amino acids, 32.7kDa, purity: 90%, endotoxin <1.0 EU/. Mu.g; mouse ITLN1 (intellect 1/Omentin) ELISAkit, purchased from the company Wuhan Elabscience.
2. Method of
2.1 construction of DSS-induced acute colitis model and Omentin intervention
Grouping: 18 male C57BL/6J mice are randomly divided into a control group, a DSS group and a DSS+omelanin group, 6 mice in each group are subjected to subsequent experiments after being adaptively fed by an SPF-class animal experiment center;
and (3) intervention:
control group: normal water was freely drunk for 7 days, and 200ul of PBS solution was injected per mouse per day intraperitoneally;
DSS group: mice were free to drink 3% dss solution for 7 days, each mouse being intraperitoneally injected with 200ul PBS solution per day;
dss+omentin group: mice were free to drink 3% dss solution for 7 days, each mouse was injected intraperitoneally with 0.5ug of omelanin per day;
general case record: 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.
Mice were sacrificed and specimens were collected: the mice were sacrificed on day 7 of molding, and were subjected to intraperitoneal anesthesia with sodium pentobarbital, and then were subjected to blood collection from the eyeballs, centrifuged, and the collected serum was stored in a-80 ℃ refrigerator for use. The abdominal cavity was opened along the midline of the abdomen, the length of the complete straight colon was measured and the spleen weight was weighed. The colon stool was cleaned and the colon tissue was left in a-80 ℃ freezer for subsequent experiments.
2.2RT-qPCR
(1) Extraction of tissue RNA
Taking and autoclaving 2ml EP tubes, numbering, adding 2 magnetic beads and 1ml Trizol solution into each EP tube, shearing 10mg colon tissue into the corresponding EP tube, and homogenizing on a grinder for 65HZ, 120s and 2 times;
adding 200ul chloroform into the tissue homogenate, vigorously shaking an EP tube for 15s, standing at room temperature for 5min after the solution turns pink;
and (3) centrifuging: the homogenate is divided into three layers at 4 ℃ and 12000rpm for 15min, wherein the upper colorless solution is RNA phase;
sucking the upper colorless solution of 300ul to a new EP tube, adding 300ul of isopropanol, slightly inverting the EP tube upside down, fully mixing, standing for 5min at room temperature, centrifuging at 4 ℃ at 12000rpm for 15min, and observing RNA precipitation at the bottom of the EP tube;
after carefully discarding the supernatant, slowly adding 1ml of pre-chilled 75% ethanol along the EP tube wall, gently washing the EP tube wall upside down, and centrifuging at 12000rpm for 5min at 4 ℃;
after carefully discarding the supernatant, the EP tube was inverted on filter paper and the RNA was dried for 5min;
adding a proper amount of RNase-free water to dissolve the precipitate according to the RNA precipitation amount, and lightly blowing the precipitate by a pipetting gun to fully dissolve the precipitate;
and (3) RNA concentration detection: RNA concentration was determined using a spectrophotometer. ddH 2 O washes the smooth surface of the cuvette multiple times, and adds 100ul of ddH2O to zero. Add 2ul of test sample and 98ul of ddH2O to cuvette, record reading and A260/A280, final RNA concentration (ug/ml) =reading×100.
(2) Reverse transcription
Calculating the required volumes of RNA and DEPC water according to the amplification reaction system and the concentration of RNA;
such as a 10ul system: RNA-Primermix (5X) 2ul+RNA test sample (1/concentration 1000) ul+DEPC water (10-2-VRNA) (ul);
preparing an RNA-Primer Mix, an RNA sample to be detected and DEPC water, and performing instantaneous separation; according to the system, calculating the volume of each component, and respectively adding 0.5ml of EP pipe and instantaneous separation;
reverse transcription: reverse transcription was performed in a reverse transcription apparatus at 37℃for 15min and at 85℃for 15sec to give the corresponding cDNA, which was stored in a-20℃refrigerator.
③RT-qPCR
Preparing an amplification reaction system: primer system SYBR Green 5 ul+primer 1ul, target gene system cDNA 1ul+ddH2O 3ul;
sample adding: according to experimental requirements, designing a sample adding form, making corresponding marks on a 96-well plate, adding 6ul of primer system and 4ul of target gene system on the side wall of each well, sealing the 96-well plate by using a sealing plate membrane after sample adding is finished, and centrifuging.
Amplification: putting the 96-well plate into a PCR instrument, setting a program for amplification, and repeating 40 cycles for amplification at 95 ℃ for 10 min-95 ℃ for 30 s-60 ℃ for 30 s-72 ℃ for 1 min;
analysis of results: the beta-actin is used as an internal reference gene, and a 2-delta CT method is adopted to calculate and analyze the expression condition of target genes of each group compared with a control group.
Primer sequence:
Mouseβ-actin:Forward——AGTGTGACGTTGACATCCGTA，Reverse——GCCAGAGCAGTAATCTCCTTCT；
Mouse omentin:Forward——AGTGCAGCTGAAGAGAACCT，Reverse——ACTTCCCACGCATGTTGTTC；
Mouse IL-1α:Forward——CGAAGACTACAGTTCTGCCATT，Reverse——GACGTTTCAGAGGTTCTCAGAG；
Mouse IL-1β:Forward——CCTCGTGCTGTCGGACCCATA，Reverse——CAGGCTTGTGCTCTGCTTGTGA；
Mouse TNFα:Forward——TACTGAACTTCGGGGTGATTGGTCC，Reverse——CAGCCTTGTCCCTTGAAGAGAACC；
Mouse IL-6:Forward——TAGTCCTTCCTACCCCAATTTCC，Reverse——TTGGTCCTTAGCCACTCCTTC；
Mouse IL-17:Forward——CAGCAGCGATCATCCCTCAAAG，Reverse——CAGGACCAGGATCTCTTGCTG；
Mouse GM-CSF:Forward——GCCATCAAAGAAGCCCTGAA，Reverse——GTGAAATTGCCCCGTAGACC。
2.3ELISA
(1) the plate was removed 20 minutes in advance and standards and samples were prepared.
(2) 100uL of standard working solution or sample is added into the corresponding plate hole, and the plate hole is incubated for 90 minutes at 37 ℃;
(3) immediately after removing the liquid in the plate, 100uL of biotinylated antibody working solution was added and incubated at 37℃for 60 minutes;
(4) discarding the liquid in the plate, and washing the plate for 3 times;
(5) adding 100uL of HRP enzyme conjugate working solution into each hole, incubating for 30 minutes at 37 ℃, discarding the liquid in the plate, and washing the plate for 5 times;
(6) adding 90uL of substrate solution into each hole, and incubating at 37 ℃ for about 15 minutes;
(7) adding 50uL of stop solution into each hole;
(8) immediately reading at the wavelength of 450nm, processing the data, establishing a standard curve, and calculating the content of the serum Omentin of each sample.
3. Statistical analysis
Experimental data analysis and mapping was performed using GraphPad Prism 8.0, and the results are expressed as mean ± standard error. Based on the type of variables and the number of groups, the inter-group variance analysis uses One-way analysis of variance (One-way anova) or t-test. The differences between the groups were considered statistically significant at P < 0.05.
4. Results and analysis
(1) Intestinal tissue and serum expression of Omentin in mice with acute DSS colitis
A mouse model of acute DSS colitis (dextran sodium sulfate induced colitis) was constructed, see in the methods section. RT-qPCR detects the levels of Omentin mRNA in intestinal tissues of two groups of mice, and ELISA detects the levels of Omentin in serum of two groups of mice. As shown in FIG. 1, the levels of Omentin in intestinal tissues and serum of mice in the DSS group were significantly reduced compared with those in the control group, and the difference was statistically significant, indicating that the expression of Omentin in intestinal tissues and serum of mice in acute DSS colitis was reduced.
(2) Omentin improves the degree of variation in body weight and colon length in mice with acute DSS colitis
Acute DSS colitis mice were intervened with Omentin, specific grouping and procedures section. The ratio of the daily body weight of each mouse to the body weight of the mouse on day 0 was measured and calculated, and the average value and standard deviation of each group were calculated. As shown in fig. 2, the average body weight of mice in the group that had been interfered with by Omentin was heavier than that of the DSS group, and the difference was statistically significant. In addition, the average colon length of the mice in the Omentin intervention group is longer than that of the mice in the DSS group, and the mice have statistical significance, which indicates that the Omentin can reduce the intestinal inflammation of the mice in the acute DSS colitis, and the mice can reduce the weight reduction degree and the colon shortening degree of the mice in the acute DSS colitis.
(3) Omentin reduces acute DSS colitis mouse intestinal inflammatory cytokine secretion
The inflammatory cytokines IL-1β, IL-1α, TNF- α, IL-6, IL-17, GM-CSF mRNA levels were measured in the intestinal tissue of each group of mice using RT-qPCR. The column height represents the average value of the ratio of mRNA level to beta-actin of the internal reference gene, and the distance between the horizontal line above the column and the column represents the standard deviation. As shown in FIG. 3, the mRNA levels of IL 1 beta, IL 1 alpha, TNF alpha, IL-6, IL 17 and GM-CSF were decreased in the Omentin-interfered group compared with the DSS group, and the difference was statistically significant, and it was further demonstrated from the gene expression point of view that Omentin could alleviate intestinal inflammation in mice with acute DSS colitis.
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.
Claims (1)
1. Application of omelanin in preparing medicines for preventing and treating inflammatory bowel disease; wherein the inflammatory bowel disease is any one of ulcerative colitis and Crohn's disease.
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| CN202210085098.XA CN114288388B (en) | 2022-01-25 | 2022-01-25 | Application of omelanin in preparation of medicines for treating inflammatory bowel disease |
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| CN202210085098.XA CN114288388B (en) | 2022-01-25 | 2022-01-25 | Application of omelanin in preparation of medicines for treating inflammatory bowel disease |
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