CN114288388A - Application of omentum extract in preparation of medicine for treating inflammatory bowel disease - Google Patents
Application of omentum extract in preparation of medicine for treating inflammatory bowel disease Download PDFInfo
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Abstract
The invention belongs to the field of research of inflammatory bowel diseases, and particularly discloses application of omentum extract in preparation of a medicament for treating inflammatory bowel diseases. Application of omentum extract in preparing medicine for preventing and treating inflammatory bowel disease is provided. Application of omentum extract in preparing preparation for inhibiting colitis intestinal inflammatory factor secretion is provided. Application of omentum extract in preparing preparation for inhibiting colitis intestinal inflammatory factor secretion is provided. Application of intestinal inflammatory cytokines in preparing a preparation for inhibiting expression of reticulin. The omentum extract reduces the secretion of colitis intestinal inflammatory factors induced by Dextran Sodium Sulfate (DSS), and effectively improves intestinal inflammation; meanwhile, a corresponding scheme is provided for the adjustment of the reticulin.
Description
Technical Field
The invention belongs to the field of research of inflammatory bowel diseases, and particularly relates to application of omentum extract in preparation of a medicament for treating inflammatory bowel diseases.
Background
Inflammatory bowel disease is a group of intestinal idiopathic diseases, and the incidence rate of IBD is continuously increased in emerging industrialized countries including China at present, so that enormous burden is brought to society and countries. The therapeutic measures for IBD are mainly anti-inflammatory, hormonal, immunosuppressive, biological agents, etc., but even with active treatment, the goal of "complete mucosal healing" is difficult to achieve in about 50% of moderately severe IBD patients. There is an urgent need to find new therapeutic targets to overcome the current therapeutic bottleneck in IBD.
The reticulin (Omentin) is a newly discovered fat factor, has expression in visceral fat, mesothelial cells, vascular cells, airway goblet cells, ovaries, blood plasma and the like, and has the regulation effects on the aspects of resisting atherosclerosis, improving insulin sensitivity, protecting heart blood vessels and the like. The amino acid sequence of the reticulin (Omentin) is as follows: 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 (origin e.
The research finds that the reticulin is obviously reduced in the serum of patients with inflammatory bowel diseases. The role of Omentin in inflammatory bowel disease may play an anti-inflammatory role, so the specific effect was observed using Omentin to intervene in the acute DSS mouse model.
At present, the application of Omentin in preparing a medicament for treating inflammatory bowel disease is unavailable.
Disclosure of Invention
Aiming at the problems, the invention provides the application of omentum in preparing the medicine for treating inflammatory bowel diseases, mainly solving some research blanks of the existing omentum and making up some blanks of the treatment of intestinal inflammation.
In order to solve the problems, the invention adopts the following technical scheme:
application of omentum extract in preparing medicine for preventing and treating inflammatory bowel disease is provided.
In some forms, the inflammatory bowel disease includes ulcerative colitis and crohn's disease.
In some forms, the inflammatory bowel disease is DSS colitis.
Application of omentum extract in preparing preparation for inhibiting colitis intestinal inflammatory factor secretion is provided.
In some embodiments, the colitis enteroinflammatory factor is at least one of IL-1 beta, IL-1 alpha, TNF-alpha, IL-6, IL-17, GM-CSF mRNA.
In some embodiments, the colitis enteroinflammatory factor secretion is dextran sodium sulfate-induced colitis enteroinflammatory factor secretion.
Application of omentum extract in preparing preparation for inhibiting colitis intestinal inflammatory factor secretion is provided.
Application of intestinal inflammatory cytokines in preparing a preparation for inhibiting expression of reticulin.
In some embodiments, the inflammatory cytokine comprises L-1 β, IL-1 α, TNF- α, IL-6, IL-17, GM-CSF mRNA.
Application of a reticulin detection agent in preparation of an inflammatory bowel disease detection agent.
The invention has the beneficial effects that:
the reticulin reduces the secretion of colitis 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 reticulin.
Drawings
FIG. 1 is a graph showing the expression of Omentin in intestinal tissues and serum of mice with acute DSS colitis;
FIG. 2 shows the degree of change in body weight and colon length of mice with acute DSS colitis improved by Omentin;
FIG. 3 shows that Omentin reduces intestinal inflammatory cytokine secretion in mice with acute DSS colitis.
Detailed Description
The first aspect of this section describes the use of dictyosin：
One of the applications of omentum extract in preparing medicine for preventing and treating inflammatory bowel disease.
In some forms, the inflammatory bowel disease includes ulcerative colitis and crohn's disease.
In some forms, the inflammatory bowel disease is DSS colitis (colitis induced by sodium dextran sulfate).
In the second place, the omentum extract is applied to preparing preparations for inhibiting colitis intestinal inflammatory factor secretion.
In some embodiments, the colitis enteroinflammatory factor is at least one of IL-1 beta, IL-1 alpha, TNF-alpha, IL-6, IL-17, GM-CSF mRNA.
In some embodiments, the colitis enteroinflammatory factor secretion is dextran sodium sulfate-induced colitis enteroinflammatory factor secretion.
Third, the use of omentum extract in preparing preparation for inhibiting colitis intestinal inflammatory factor secretion is provided.This section is given in the first place Two aspects illustrate the modulation of reticulin：
Application of intestinal inflammatory cytokines in preparing a preparation for inhibiting expression of reticulin.
The expression behavior of omentum can be reduced by the intestinal inflammatory cytokines, so that the regulation effect on omentum is realized in some scenes. Such as: in a commercial omentum property study experiment, to study conditions affecting omentum expression, regulation was performed using inflammatory cytokines in the gut.
In some embodiments, the inflammatory cytokine comprises L-1 β, IL-1 α, TNF- α, IL-6, IL-17, GM-CSF mRNA. Of course, other cytokines capable of affecting omentum expression are also within the scope of the present invention.
Application of a reticulin detection agent in preparation of an inflammatory bowel disease detection agent. The inflammatory bowel disease can be characterized to a certain extent by detecting the level of the reticuline in a suspected inflammatory bowel disease sample.
The third aspect of this section is described in connection with a particular study：
1. Material
1.1 animals: male 6-8 week C57BL/6J mice (purchased from Wintolite, Beijing) were bred in SPF environment;
reagent reticulin (Omentin Human E. coli), purchased from European BioVender, 294 amino acids, 32.7kDa, purity: > 90%, endotoxin <1.0EU/μ g; mouse ITLN1 (Intelection 1/Omentin) ELISAkit, available from Elapscience, Wuhan, Inc.
2. Method of producing a composite material
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 + omentum group, each group comprises 6 mice, and the mice are subjected to follow-up experiments after being adaptively fed in an SPF-level animal experiment center;
and (3) intervention:
control group: drinking normal water freely for 7 days, and injecting 200ul PBS solution into the abdominal cavity of each mouse every day;
and (4) DSS group: mice had 3% DSS solution freely for 7 days, and 200ul PBS solution was injected into the abdominal cavity of each mouse every day;
DSS + omnitin group: mice freely drink 3% DSS solution for 7 days, and each mouse is injected with 0.5ug of netoglobin every day in the abdominal cavity;
general case recording: after the start of modeling, the weight, stool characteristics (normal, pasty stool or watery stool), bloody condition (no, occult blood or obvious bloody stool), activity condition (active or burnout) and the like of the mice were recorded every day.
Mice sacrifice and specimen collection: sacrificed on day 7 of modeling, the pentobarbital sodium abdominal cavity anesthetized mice were subjected to eye blood sampling, centrifuged, and the serum was stored in a refrigerator at-80 ℃ for later use. The abdominal cavity was opened along the ventral midline, the length of the intact rectus colon was measured and the spleen was weighed. Colon feces were cleaned and colon tissue was left in a-80 ℃ freezer for subsequent experiments.
2.2RT-qPCR
Extraction of tissue RNA
Taking an autoclaved 2ml EP tube, numbering, adding 2 magnetic beads and 1ml Trizol solution into each EP tube, shearing 10mg colon tissues, putting into the corresponding EP tube, and homogenizing on a grinder for 2 times at 65HZ for 120 s;
adding 200ul chloroform into the tissue homogenate, vigorously and violently oscillating an EP tube for 15s, and standing at room temperature for 5min after the solution is pink;
centrifuging: 4 ℃, 12000rpm, 15min, dividing the homogenate into three layers, wherein the upper colorless solution is an RNA phase;
sucking 300ul of the colorless upper layer solution to a new EP tube, adding 300ul of isopropanol, slightly inverting the EP tube upside down, mixing well, standing at room temperature for 5min, centrifuging at 4 ℃, 12000rpm for 15min, and allowing RNA precipitation to be visible at the bottom of the EP tube;
carefully discarding the supernatant, slowly adding 1ml of precooled 75% ethanol along the wall of the EP tube, slightly reversing the direction of the EP tube to wash the wall of the EP tube, and centrifuging the EP tube at 4 ℃ and 12000rpm for 5 min;
carefully discarding the supernatant, inverting the EP tube on filter paper, and drying the RNA precipitate for 5 min;
adding a proper amount of RNase-free water to dissolve the precipitate according to the RNA precipitation amount, and gently blowing and beating the precipitate by using a pipette gun to fully dissolve the precipitate;
and (3) RNA concentration detection: RNA concentration was determined using a spectrophotometer. ddH2O cleaning the inside and outside of the smooth surface of the cuvette for a plurality of times, and adding 100ul ddH2O for zero setting. 2ul of test sample and 98ul of ddH2O were added to the cuvette and the reading and A260/A280 were recorded, the final RNA concentration (ug/ml) being read × 100.
② reverse transcription
Calculating the required RNA and DEPC water volumes according to the amplification reaction system and the RNA concentration;
for example, 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 carrying out instantaneous dissociation; according to the system, the volume of each component is calculated, and 0.5ml of EP tube is respectively added for instantaneous separation;
reverse transcription: reverse transcription was performed in a reverse transcription apparatus at 37 ℃ for 15min and 85 ℃ for 15sec to obtain the corresponding cDNA, which was stored in a freezer at-20 ℃.
③RT-qPCR
Preparing an amplification reaction system: a primer system SYBR Green 5ul + primer 1ul, and a target gene system cDNA 1ul + ddH2O 3 ul;
sample adding: designing a sample adding form according to experimental requirements, making corresponding marks on a 96-well plate, adding 6ul of a primer system and 4ul of a target gene system on the side wall of each well respectively, 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 10min to 95 ℃ for 30s to 60 ℃ for 30s to 72 ℃ for 1 min;
and (4) analyzing results: and (3) calculating and analyzing the expression condition of the target genes of each group compared with the control group by using beta-actin as an internal reference gene and adopting a 2-delta-CT method.
The primer sequence is as follows:
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
taking out the plate 20 minutes in advance, and preparing a standard substance and a sample.
Adding 100uL of standard substance working solution or sample into the corresponding plate hole, and incubating for 90 minutes at 37 ℃;
thirdly, after the liquid in the plate is discarded, 100uL of biotinylation antibody working solution is immediately added, and incubation is carried out for 60 minutes at 37 ℃;
fourthly, discarding the liquid in the plate, and washing the plate for 3 times;
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;
sixthly, adding 90uL of substrate solution into each hole, and incubating for about 15 minutes at 37 ℃;
adding 50uL of stop solution into each hole;
and reading immediately at the wavelength of 450nm, processing data, establishing a standard curve, and calculating the content of Omentin in serum of each sample.
3. Statistical analysis
Data analysis and plotting were performed using GraphPad Prism 8.0 and results are expressed as mean ± sem. Analysis of differences between groups was performed using One-way analysis of variance (One-way anova) or t-test, depending on the type of variable and the number of groups. Differences between groups were considered statistically significant when P < 0.05.
4. Results and analysis
Expression of Omentin in intestinal tissues and serum of acute DSS colitis mice
An acute DSS colitis (colitis induced by dextran sodium sulfate) mouse model is constructed, and the method is concretely described. RT-qPCR detects the Omentin mRNA level of intestinal tissues of two groups of mice, and ELISA detects the Omentin level of serum of the two groups of mice. As shown in figure 1, the levels of Omentin in intestinal tissues and serum of mice in the DSS group are significantly reduced compared with those of a control group, and the difference has statistical significance, which indicates that the expression of Omentin in intestinal tissues and serum of mice with acute DSS colitis is reduced.
② Omentin improves the change degree of the body weight and the colon length of the mice with acute DSS colitis
The Omentin is used for intervening acute DSS (Severe Skoog colitis) mice, and the specific components and the operation method are shown in the 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 mean and standard deviation of each group were calculated. As shown in fig. 2, the average body weight of the mice in the Omentin intervention group was statistically different from that in the DSS group. 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 statistical significance is achieved, so that the Omentin can reduce the intestinal inflammation of the mice with acute DSS colitis, and the intestinal inflammation can be specifically shown in the effect of reducing the weight reduction degree and the colon shortening degree of the mice model with acute DSS colitis.
③ Omentin reduces the intestinal inflammatory cytokine secretion of mice with acute DSS colitis
RT-qPCR was used to detect mRNA levels of inflammatory cytokines IL-1 beta, IL-1 alpha, TNF-alpha, IL-6, IL-17, GM-CSF in each group of mice in intestinal tissue. The height of the column indicates the average value of the ratio of the mRNA level to the reference gene beta-actin, and the distance between the horizontal line above the column and the column indicates the standard deviation. As shown in figure 3, compared with the DSS group, the mRNA levels of IL 1 beta, IL 1 alpha, TNF alpha, IL-6, IL 17 and GM-CSF in the mice in the Omentin intervention group are reduced, the difference is statistically significant, and the fact that Omentin can reduce and relieve intestinal inflammation in the mice with acute DSS colitis is further proved from the aspect of gene expression.
It will be apparent to those skilled in the art that various modifications may be made to the above embodiments without departing from the general spirit and concept of the invention. All falling within the scope of protection of the present invention. The protection scheme of the invention is subject to the appended claims.
Claims (9)
1. Application of omentum extract in preparing medicine for preventing and treating inflammatory bowel disease is provided.
2. The use according to claim 1, wherein the inflammatory bowel disease comprises ulcerative colitis and Crohn's disease.
3. The use of claim 1, wherein the inflammatory bowel disease is DSS colitis.
4. Application of omentum extract in preparing preparation for inhibiting colitis intestinal inflammatory factor secretion is provided.
5. The use of claim 4, wherein the colitis enteroinflammatory factor is at least one of IL-1 β, IL-1 α, TNF- α, IL-6, IL-17, GM-CSF mRNA.
6. The use of claim 4, wherein the colitis enteroinflammatory factor secretion is dextran sodium sulfate induced colitis enteroinflammatory factor secretion.
7. Application of intestinal inflammatory cytokines in preparing a preparation for inhibiting expression of reticulin.
8. The use of claim 7, wherein said inflammatory cytokine comprises L-1 β, IL-1 α, TNF- α, IL-6, IL-17, GM-CSF mRNA.
9. Application of a reticulin detection agent in preparation of an inflammatory bowel disease detection agent.
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