Application of miR-601 in the preparation of drugs for preventing or treating ulcerative colitis and regulating blood sugar

Abstract

The present invention relates to the field of biomedicine, and more specifically, to the use of miR-601 in the preparation of drugs for preventing or treating ulcerative colitis and regulating blood sugar. Experimental results in the present invention demonstrate that miR-601 can alleviate colitis mucosal damage caused by ulcerative colitis, while also restoring intestinal microecological balance and improving glucose metabolism.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of miR-601 in preparing drugs for preventing or treating ulcerative colitis and regulating blood sugar. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Ulcerative colitis (UC) is a chronic immune-mediated disease characterized by diffuse mucosal inflammation and clinically characterized by abdominal pain, diarrhea, bloody stools containing mucus, pus, and tenesmus. In recent years, the incidence and prevalence of UC have increased, making it a significant global healthcare burden. Furthermore, type 2 diabetes mellitus (T2DM) is the most common metabolic disease in the world. Its etiology is complex, causing irreversible damage to the body and leading to various chronic complications. Its incidence continues to rise worldwide. Effectively preventing and treating diabetes has become a pressing global health issue.

[0004] Studies have shown that there is a clear clinical correlation between type 2 diabetes and ulcerative colitis. Mechanistically, patients with ulcerative colitis and type 2 diabetes have similar unhealthy lifestyles and common pathogenic factors, including systemic chronic inflammatory states and intestinal flora imbalance. Currently, whether it is the treatment of ulcerative colitis or the treatment of type 2 diabetes, complications will arise and worsen during the actual treatment process. When taking drugs to treat ulcerative colitis, it may lead to the aggravation of diabetes; and when taking drugs to treat type 2 diabetes, it may cause gastrointestinal discomfort. Therefore, the development of new small molecule drug preparations with low toxicity and side effects that take into account both intestinal microecological protection and systemic metabolic improvement is crucial for the treatment of ulcerative colitis and type 2 diabetes.

[0005] MicroRNA (miRNA) is a class of small, non-coding, single-stranded RNA molecules approximately 22 nucleotides in length. Studies have shown that miRNAs can regulate gene expression and play a crucial role in cell growth, development, proliferation, apoptosis, and differentiation. Due to their high conservation, multi-target regulatory capabilities, and close association with disease pathways, miRNAs are promising candidates for the treatment of ulcerative colitis and type 2 diabetes. Summary of the Invention

[0006] In order to overcome the above problems, the present invention provides the use of miR-601 in the preparation of drugs for preventing or treating ulcerative colitis and regulating blood sugar.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides use of miR-601 in preparing a drug for preventing or treating ulcerative colitis. The sequence of miR-601 is shown in SEQ ID NO.1.

[0009] In one or more embodiments, the application may be any of the following:

[0010] A1) miR-601 promotes Akkermansia in the intestine ( Akkermansia ) Application in proliferation;

[0011] A2) miR-601 promotes Akkermansia in the intestine ( Akkermansia ) application in proliferating products;

[0012] A3) Application of miR-601 in regulating the interaction network of intestinal flora, strengthening the intestinal barrier, and reducing inflammatory response;

[0013] A4) Application of miR-601 in the preparation of products that regulate the interaction network of intestinal flora, strengthen the intestinal barrier, and reduce inflammatory response;

[0014] A5) Application of miR-601 in reducing colon shortening, weight loss, and intestinal histopathological damage caused by ulcerative colitis;

[0015] A6) Use of miR-601 in the manufacture of products that reduce colon shortening, weight loss, and intestinal histopathological damage caused by ulcerative colitis;

[0016] A7) Application of miR-601 in reducing the number of inducible nitric oxide synthase (iNOS)-positive cells and caspase-3-positive cells in ulcerative colitis;

[0017] A8) Use of miR-601 in the preparation of a product for reducing the number of inducible nitric oxide synthase (iNOS)-positive cells and caspase-3-positive cells caused by ulcerative colitis.

[0018] Preferably, the interactions regulating intestinal flora in A3) and A4) include Akkermansia ( Akkermansia ) and the interactions between beneficial bacteria that promote intestinal mucosal healing and SCFA-producing flora.

[0019] Further preferably, the beneficial bacteria that promote intestinal mucosal healing are Mycospirillum ( Mucispirillum ) and Dunaliella ( Dubosiella ).

[0020] Further preferably, the SCFA-producing bacterial flora is an unclassified Lachnospira ( unidentified_ Lachnospiraceae ) and Bacillus subtilis ( Odoribacter ) 、Lachnoclostridium .

[0021] Preferably, the pathological damage of the intestinal tissue in A5) and A6) includes epithelial erosion, leukocyte infiltration, ulceration, etc.

[0022] Preferably, the ulcerative colitis in A5) to A8) is induced by dextran sulfate sodium salt (DSS).

[0023] In a second aspect, the present invention provides use of an agent for promoting miR-601 expression or enhancing miR-601 function in the preparation of a drug for preventing or treating ulcerative colitis.

[0024] In one or more embodiments, the reagent includes: a miR-601 mimic, a miR-601 precursor, a miR-601 agonist, and a vector carrying miR-601.

[0025] In a third aspect, the present invention provides use of miR-601 in the preparation of a drug for regulating blood sugar, wherein the sequence of miR-601 is shown in SEQ ID NO.1.

[0026] In one or more embodiments, the application may be any of the following:

[0027] B1) Application of miR-601 in lowering fasting blood glucose;

[0028] B2) Application of miR-601 in the preparation of products for lowering fasting blood glucose;

[0029] B3) Application of miR-601 in improving insulin sensitivity;

[0030] B4) Application of miR-601 in the preparation of products for improving insulin sensitivity.

[0031] A fourth aspect of the present invention provides use of a reagent for promoting miR-601 expression or enhancing miR-601 function in the preparation of a drug for regulating blood sugar.

[0032] In one or more embodiments, the reagent includes: a miR-601 mimic, a miR-601 precursor, a miR-601 agonist, and a vector carrying miR-601.

[0033] A fifth aspect of the present invention provides a pharmaceutical preparation comprising an effective amount of miR-601 or the agent for promoting miR-601 expression or enhancing miR-601 function as described in the second aspect.

[0034] In one or more embodiments, the drug further comprises a pharmaceutically acceptable carrier.

[0035] Preferably, the carrier includes cationic lipid carriers and nanoparticles and other carriers.

[0036] Preferably, the pharmaceutical preparation further comprises supplementary additives, which are selected from one or more of diluents, buffers, encapsulating agents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, colorants, flavoring agents and adsorption carriers.

[0037] Preferably, the dosage form of the drug is a suspension, emulsion, granule, spray, injection, transdermal absorbent, dosage form suitable for transfection, tablet, powder, granule or capsule.

[0038] A sixth aspect of the present invention provides a pharmaceutical composition comprising an effective amount of miR-601 or the agent for promoting miR-601 expression or enhancing miR-601 function as described in the second aspect.

[0039] The beneficial effects of the present invention are:

[0040] The experimental results of the present invention show that miR-601 can improve colitis mucosal damage caused by ulcerative colitis and simultaneously restore the intestinal microecological balance and improve the body's glucose metabolism. Specifically, first, compared with wild-type mice, the intestinal Akkermansia ( Akkermansia ) increased in abundance; while Akkermansia ( Akkermansia ) degrades host mucin (MUC2) by secreting mucins, while stimulating goblet cells to secrete new mucins, maintaining the dynamic balance of the mucus layer, promoting the expression of tight junction proteins ZO-1 and Occudin, and reducing intestinal permeability and inflammatory response. Second, in the intestinal flora of miR-601 overexpressing transgenic mice, Akkermansia ( Akkermansia ) and beneficial bacteria that promote intestinal mucosal healing (Mycospirillum ( Mucispirillum ) and Dunaliella ( Dubosiella ), SCFA-producing species (unclassified Lachnospira ( unidentified_Lachnospiraceae ) and Bacillus subtilis ( Odoribacter )、 Lachnoclostridium The results showed a strong mutual promotion effect (correlation coefficient > 0.7); miR-601 can strengthen the intestinal barrier and reduce inflammatory responses by regulating the microbial interaction network. Third, in a mouse model of ulcerative colitis induced by dextran sulfate sodium salt (DSS), miR-601 overexpression alleviated DSS-induced colitis-induced symptoms of colon shortening, weight loss, DAI score, and histopathological damage such as epithelial erosion, leukocyte infiltration, and ulceration. MiR-601 overexpression reduced the number of inducible nitric oxide synthase (iNOS)-positive cells and caspase-3-positive cells after DSS treatment and alleviated DSS-induced damage to the mucus layer. Fourth, in miR-601-overexpressing transgenic male mice, miR-601 overexpression reduced fasting blood glucose and improved insulin sensitivity. In miR-601-overexpressing transgenic female mice, miR-601 overexpression also reduced fasting blood glucose.

[0041] (2) The treatment of ulcerative colitis mainly relies on salicylic acid and glucocorticoids. The long-term use of these drugs will have significant toxic side effects and disrupt the intestinal microecological environment, causing intestinal flora disorder. The reduction of intestinal core bacteria and the increase of potential pathogenic bacteria will further damage the intestinal barrier, aggravate mucosal damage, and trigger a systemic inflammatory response. Compared with traditional chemical drugs, miR-601 can target and regulate bacterial gene expression without interfering with the normal function of cells, reducing toxicity and side effects on normal cells. Compared with traditional treatment methods, miR-601 has the advantages of simultaneously alleviating colitis and restoring the balance of intestinal microecology, providing an efficient treatment basis for the clinical transformation of ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0043] Figure 1 The process of screening and obtaining miR-601, where A is the Venn diagram of miRNAs expression in different parts; B is the abundance of co-expressed miRNAs in the three parts;

[0044] Figure 2 is the map of the recombinant plasmid;

[0045] Figure 3Comparison of intestinal microbiota diversity between wild-type mice (male WT-Male, female WT-Female) and miR-601 overexpressing transgenic mice (male TG-Male, female TG-Female). A is the Shannon index, representing the evenness in alpha diversity (higher diversity means more even distribution of species in the intestinal microbiota); B is the beta diversity based on the Bray-Curtis distance, representing the similarity between microbial communities, where R 2 The larger ( P <0.05), indicating a greater difference in species composition;

[0046] Figure 4 Figure 2 shows the differential bacterial analysis between wild-type and miR-601 overexpressing transgenic mice. A is the analysis using DeSEq2 (log2 fold change absolute value>1, P <0.05) (log10 transformation); B is a Venn diagram of sex differences in differential bacteria between male and female mice. The left circle in B is the differential bacteria between male mice with miR-601 overexpression transgenic (TG) and wild type (WT), and the small semicircles in the middle are the bacteria that are up-regulated and down-regulated in male TG, respectively, with the bacterial names marked. The right circle is the differential bacteria between female mice with TG and WT.

[0047] Figure 5 Figure 2 is a co-occurrence network analysis of intestinal flora, where A is the co-occurrence network interaction diagram of wild-type male mice; B is the co-occurrence network interaction diagram of miR-601 overexpressing transgenic male mice; C is the co-occurrence network interaction diagram of wild-type female mice; D is the co-occurrence network interaction diagram of miR-601 overexpressing transgenic female mice.

[0048] Figure 6 The differential metabolic pathways predicted by Tax4Fun (using one-way ANOVA analysis, P <0.05) and correlation analysis with the above differential species;

[0049] Figure 7 Overexpression of miR-601 does not affect the colon morphology, cell apoptosis, and mucus layer of mice. A is the colon length; B is the statistical result of the colon length in A; C is the disease activity index (DAI) of miR-601 overexpressing transgenic (TG) and wild-type (WT) mice measured daily; D is the colon tissue stained with HE; E is the histological score;

[0050] Figure 8Overexpression of miR-601 does not affect the caspase-3-positive cells and mucus layer thickness in mice; A is the immunohistochemical staining of caspase-3 in the colon tissues of miR-601-overexpressing transgenic (TG) and wild-type (WT) mice; B is the statistical result in A; C is the AB-PAS staining of the colon tissues of miR-601-overexpressing transgenic (TG) and wild-type (WT) mice; D is the statistical result in C; values ​​are expressed as mean ± SEM;

[0051] Figure 9 miR-601 alleviates the shortened colon length and weight loss caused by DSS-induced colitis 7 days after induction. A shows the colon length and statistical results of miR-601-overexpressing transgenic (TG) and wild-type (WT) mice; B shows the weight loss of the two groups of mice; C shows the disease activity index (DAI) of the two groups of mice.

[0052] Figure 10 miR-601 alleviates the tissue damage and inflammatory response caused by DSS-induced colitis 7 days later; A is the HE staining result of colon tissue; B is the histological score; C is the immunohistochemical staining of anti-CD-45 in colon tissue of WT+DSS and miR-601+DSS groups; D is the statistical result in C, and the values ​​are expressed as mean ± SEM, * P <0.05 vs WT + DSS group;** P <0.01 vs WT + DSS group;

[0053] Figure 11 miR-601 inhibits iNOS levels after DSS treatment; A is immunofluorescence staining of iNOS expression in wild-type (WT) colon after DSS induction; B is immunofluorescence staining of iNOS expression in the colon of miR-601 overexpressing transgenic (TG) mice after DSS induction;

[0054] Figure 12 Figure 3. MiR-601 inhibits epithelial cell apoptosis and mucus barrier damage after DSS treatment. A is the immunohistochemical staining of capase-3 in colon tissues of the WT+DSS and miR-601+DSS groups; B is the statistical result in A; C is the AB-PAS staining of colon tissues of the WT+DSS and miR-601+DSS groups; D is the statistical result in C; values ​​are expressed as mean ± SEM (n = 4 per group); * P <0.05 compared with the WT+DSS group;

[0055] Figure 13Overexpression of miR-601 reduces fasting blood glucose and improves insulin sensitivity in male mice; A is the body weight of wild-type male mice (WT-Male) and miR-601-overexpressing transgenic male mice (TG-Male); B is the blood glucose level of the two groups after a 6-h fast; C is the blood glucose level of the two groups after a 16-h fast; D is the insulin level of the two groups after a 6-h fast; E is the blood glucose level curve of the two groups 30, 60, 90, and 120 minutes after glucose (2 g / kg*bw) injection (GTT test); F is the area under the curve of the blood glucose level curve in the GTT test in E; G is the blood glucose level curve of the two groups 30, 60, 90, and 120 minutes after insulin (0.75 IU / kg*bw) injection (ITT test); F is the area under the curve of the blood glucose level curve in the ITT test in G;

[0056] Figure 14 Figure 3 Overexpression of miR-601 reduces fasting blood glucose in female mice. A is the body weight of wild-type female mice (WT-Female) and miR-601-overexpressing transgenic female mice (TG-Female). B is the blood glucose level of the two groups after a 6-h fast. C is the blood glucose level of the two groups after a 16-h fast. D is the insulin level of the two groups after a 6-h fast. E is the blood glucose level curve of the two groups 30, 60, 90, and 120 min after glucose (2 g / kg*bw) injection (GTT test). F is the area under the curve of the blood glucose level curve in the GTT test in E. G is the blood glucose level curve of the two groups 30, 60, 90, and 120 min after insulin (0.75 IU / kg*bw) injection (ITT test). F is the area under the curve of the blood glucose level curve in the ITT test in G. DETAILED DESCRIPTION

[0057] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0058] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0059] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0060] Example 1

[0061] Method for screening and obtaining miR-601:

[0062] Through comprehensive analysis of human-specific expression, human blood exosomes, colon cell exosomes and fecal expression miRNA profiles, miRNAs with potential research value were screened out. Among them, the miRNA profiles expressed in human blood exosomes and colon cell exosomes were obtained from the EVmiRNA database (https: / / guolab.wchscu.cn / EVmiRNA / #! / ), and the fecal miRNA profile has been published in Cell Host & Microbe (Liu, S., et al., The Host Shapes the Gut Microbiota via Fecal MicroRNA. Cell Host & Microbe, 2016. 19(1): p. 32-43.). Figure 1 As shown in Figure A, the intersection method was used to cross-compare the miRNA expression data in the three databases. It was found that among the co-expressed miRNA sets, miR-601 ranked second in fecal miRNA abundance ( Figure 1 Therefore, it highlights its stable expression characteristics and potential biological importance, and will be used as the object for subsequent research.

[0063] Example 2

[0064] (1) Construction of a miR-601 overexpression transgenic mouse model:

[0065] A miR-601 overexpression transgenic mouse model was constructed using a high-efficiency expression vector recombinant plasmid of the piggyBac transposon system. The vector used in the recombinant plasmid was VB161206-1091wre (Saiye Biotechnology Co., Ltd., TGMB161202CAQ01). The recombinant plasmid contains a CAG composite promoter, an EGFP reporter gene, and a human miR-601 precursor sequence. The map of the recombinant plasmid is shown in Figure 2. Figure 2As shown, stable genome integration was achieved through piggyBac transposon-mediated transposon; the recombinant plasmid was used to prepare transgenic mice by pronuclear microinjection, and the purified recombinant plasmid was injected into fertilized eggs and then transplanted into the oviduct of surrogate female mice (C57BL / 6). Tail tip tissue was harvested 21 days after birth, and genomic DNA was extracted. Exogenous gene integration in transgenic mice was verified using a duplex PCR system. The duplex PCR system included primers F1 (SEQ ID NO. 2) and R1 (SEQ ID NO. 3) (product size, 450 bp) and F2 (SEQ ID NO. 4) and R2 (SEQ ID NO. 5) (product size, 260 bp). The PCR primer system, as described in Table 1, also included piggyBac Helper plasmid primers (piggyBac Helper-F (SEQ ID NO. 6) and piggyBac HelperR (SEQ ID NO. 7)). False positives were excluded by setting up water controls (no DNA template added), positive controls (injected transgenic DNA), and wild-type (wild-type mouse genomic DNA) controls. No Helper plasmid integration occurred, and PCR-positive F0 heterozygous mice were obtained.

[0066] Table 1 PCR primer system

[0067]

[0068] (2) Transgenic mouse breeding:

[0069] After reaching sexual maturity (8 weeks), F0 mice were mated with wild-type mice to produce F1 mice. Genotyping of these F1 mice was performed using the same PCR system as in Table 1. Positive F1 heterozygous mice from the same F0 were sibimited to produce F2 mice. Genotyping of these F2 mice was also performed. Transgenic mice are generally bred to heterozygous mice. In the subsequent examples, transgenic mice overexpressing miR-601 were all F2 mice. All animals were housed in a specific pathogen-free (SPF) environment with a daily 12-h light / 12-h dark cycle and constant temperature and humidity. Mice were weaned at 21 days of age. If mice develop slowly and are thin, weaning may be extended to 28 days. After weaning, males and females should be housed separately.

[0070] Example 3

[0071] miR-601 improves the intestinal microecological environment in enteritis:

[0072] (1) Collection and sequencing of mouse intestinal flora: Colon contents of mice were collected, including 8 miR-601 overexpressing transgenic male mice, 8 miR-601 overexpressing transgenic female mice, 8 wild-type male mice, and 8 wild-type female mice. Genomic DNA was extracted using the CTAB method, and the quality of DNA extraction was tested by agarose gel electrophoresis. Then, 16S rRNA V4 region primers 515F (SEQ ID NO.8: 5'-GTGCCAGCMGCCGCGGTAA-3') and 806R (SEQ ID NO.9: 5'-GGACTACHVGGGTWTCTAAT-3') were used for amplification. PCR products were detected by 2% agarose gel electrophoresis. PCR products were recovered using a Qiagen recovery kit and library construction was performed using the TruSeq® DNA PCR-Free Sample Preparation Kit. After the library was qualified, it was sequenced on an Illumina NovaSeq6000.

[0073] (2) Intestinal flora data analysis

[0074] Sequencing data processing: The paired-end sequence data obtained by sequencing were split according to the sample barcode sequence and merged through FLASH. QIIME was used for quality control. The generated sequences were further de-redundant and chimeras. The quality-controlled sequences were clustered into OTUs (Operational Taxonomic Units) at 97% consistency using Uparse software. The Mothur algorithm was further used to align the sequences with the SILVA database (SSUrRNA database) and species annotation was performed on representative ASV sequences.

[0075] (3) Analysis of bacterial flora differences:

[0076] The alpha diversity between groups was compared by Wilcoxon test based on Shannon index (Shannon index) and the difference in community structure between the two groups was compared by multivariate permutation analysis of variance (PERMANOVA) based on Bray-Curtis distance. Figure 3 As shown in Figure 2, the intestinal flora uniformity index (Shannon index) of miR-601 overexpressing transgenic male mice tended to be higher than that of wild-type male mice, while the intestinal flora uniformity index of transgenic female mice was lower than that of wild-type female mice ( Figure 3 Middle A), Bray-Curtis distance-based beta diversity comparison showed that the confidence ellipses of the intestinal microbial communities of miR-601 overexpressing transgenic mice and wild-type mice were significantly different ( Figure 3Figure B), indicating that the intestinal microbiota composition of miR-601 overexpressing transgenic mice and wild-type mice is different (WT-M vs. TG-M: R 2 = 0.215, P <0.001; WT-F vs. TG-F: R 2 = 0.218, P = 0.002).

[0077] By DESeq2 differential analysis (log2 fold change absolute value>1, P <0.05) to identify the types of microorganisms that were significantly different at the genus level between miR-601 overexpressing transgenic mice and wild-type mice. Figure 4 A in the figure shows the relative abundance of these different bacterial genera. The results show that Akkermansia ( Akkermansia ) was the species with the most significant difference in abundance between miR-601 overexpressing transgenic mice and wild-type mice. Further comparison of gender differences revealed that compared with wild-type mice, the intestinal tract of miR-601 overexpressing transgenic mice was higher in both male and female mice than in wild-type mice. Akkermansia ) increased in relative abundance ( Figure 4 Middle B). Studies have reported that Akkermansia ( Akkermansia ) is associated with the occurrence and progression of ulcerative colitis. Mechanistically, Akkermansia ( Akkermansia ) degrades host mucin (MUC2) by secreting mucins, while stimulating goblet cells to secrete new mucins, maintaining the dynamic balance of the mucus layer, promoting the expression of tight junction proteins ZO-1 and Occudin, and reducing intestinal permeability and inflammatory response. Akkermansia ) has become a star potential probiotic for the treatment of intestinal inflammation by regulating the "mucus layer-immunity-metabolism" axis through multiple targets. miR-601 promotes Akkermansia ( Akkermansia ) proliferation, and is expected to be used as a potential therapeutic target for the clinical treatment of ulcerative colitis in the future.

[0078] (4) Microbial network analysis:

[0079] The correlation between species in each group was calculated using SparCC, and the intestinal flora co-occurrence network was constructed using Cytoscape software to reflect the stability of the intestinal microbial community and the potential interactions between species. The co-occurrence network analysis results showed that the intestinal flora network structure of both miR-601 overexpressing transgenic male and female mice was simpler than that of wild-type mice, and the mutual inhibition relationship between species was weaker ( Figure 5 ). Among them, in the intestinal flora interaction network of wild-type mice ( Figure 5A, C), Akkermansia ( Akkermansia, Akk in the figure) and species producing short-chain fatty acids (SCFA) ( Eubacterium_siraeum_group In the figure, it is abbreviated as Sir; Eubacterium_xylanophilum_group In the figure, it is abbreviated as Xyl; Rikenella In the figure, it is abbreviated as Rik; Lachnospiraceae_A2 In the figure, it is abbreviated as A2; GCA- 900066575 In the figure, it is abbreviated as GCA; Flavonifractor In the figure, abbreviated as Fla) there is a strong mutual inhibition (correlation coefficient <-0.7), while in miR-601 overexpressing transgenic male mice ( Figure 5 In the intestinal flora interaction network of (B), Akkermansia ( Akkermansia ) and the beneficial bacteria Mycospirillum ( Mucispirillum, Muc) and Dunaliella ( Dubosiella, Dub in the figure), SCFA-producing species (unclassified Lachnospira ( unidentified_ Lachnospiraceae, Abbreviated as U_La in the figure) and Bacillus odoratus ( Odoribacter, (Abbreviated as Odo in the figure) 、 Lachnoclostridium, In the figure, abbreviated as Lacl) showed a strong mutual promotion effect (correlation coefficient>0.7). Figure 5 In the interaction network of (D), the microbial interactions are simpler, and Akkermansia ( Akkermansia ) Strong interaction relationships are also simpler. Short-chain fatty acids mainly include acetic acid, propionic acid, butyric acid and their derivatives, which are mainly produced by intestinal flora fermenting dietary fiber ingested by the host, and have a profound impact on intestinal health. Among them, butyric acid is the main energy source for colonic epithelial cells. It promotes the expression of tight junction proteins by activating the AMPK / mTOR pathway and reduces intestinal permeability. It can also inhibit histone deacetylase (HDAC) activity, regulate MUC2 gene transcription, and increase the thickness of the intestinal mucus layer. In addition, propionic acid mediates the secretion of IL-10 from dendritic cells through the GPR43 receptor, promotes the differentiation of regulatory T cells (Treg), and inhibits the overactivation of Th17 cells. Therefore, miR-601 can strengthen the intestinal barrier and reduce inflammatory responses by regulating the microbial interaction network, and can be used as a target for future UC treatment.

[0080] (4) Functional prediction of intestinal flora:

[0081] Tax4Fun was used to predict the functional pathways of KEGG pathways of microorganisms, and one-way ANOVA analysis was used to compare the differential functional pathways between female and male miR-601 overexpressing transgenic mice and wild-type mice. P <0.05), Figure 6The heat map in the middle left shows the differential pathways (KEGG L2 level and KEGG L3 level) analyzed by ANOVA. The differential metabolic pathways mainly involve carbohydrate absorption, cell proliferation and differentiation, and steroid synthesis. The differential functional pathways and their correlation with the above differential species were further compared ( Figure 6 Bubble plot in the middle right half), upregulated pathways were similar to those in Akkermansia ( Akkermansia ) was positively correlated with Faecalibacterium ( Faecalibaculum ) and Rombutsiella ( Romboutsia ) were negatively correlated with the downregulated RIG-I-like receptor signaling pathway and showed an inverse correlation with these species. These results indicate that the altered intestinal microbiota function in miR-601 overexpressing transgenic mice is mainly caused by Akkermansia ( Akkermansia ), Faecalibacterium ( Faecalibaculum ) and Rombutsiella ( Romboutsia ) are driven by changes in their relative abundance.

[0082] Example 4

[0083] miR-601 improves ulcerative colitis:

[0084] (1) Preparation of dextran sulfate sodium salt (DSS)-induced colitis mouse model:

[0085] Ulcerative colitis was induced in miR-601-overexpressing transgenic mice and wild-type mice (3 months old) by adding 3% DSS to their drinking water for 7 days. The mice were randomly divided into four groups: (1) WT: wild-type mice drinking water; (2) miR-601: miR-601-overexpressing transgenic mice drinking water; (3) WT+DSS: wild-type mice drinking DSS; and (4) miR-601+DSS: miR-601-overexpressing transgenic mice drinking DSS. At the end of the experiment, all mice were euthanized under anesthesia, and colon tissues were collected for further analysis.

[0086] (2) Identification of intestinal morphology and function:

[0087] Disease Activity Index (DAI): During DSS induction, body weight, stool consistency, and occult blood were monitored daily. The DAI was calculated as (weight loss score + diarrhea score + rectal bleeding score) / 3.

[0088] The weight loss performance score is: 0 = no weight loss, 1 = 5-10%, 2 = 10-15%, 3 = 15-20%, 4 ≥ 20%; the diarrhea performance score is: 0 = none, 2 = mild, 4 = gross diarrhea; the rectal bleeding performance score is: 0 = no blood, 2 = positive, 4 = gross blood.

[0089] Histopathological analysis: Distal colon samples were collected and fixed with 4% paraformaldehyde. Tissue specimens were sectioned (4 μm) and stained with hematoxylin and eosin (HE). Histological scoring was performed based on the severity of inflammation, extent of ulceration, crypt damage, and percentage of affected area. The severity of inflammation was scored as follows: 0 = none, 1 = mild, 2 = moderate, and 3 = severe; the extent of ulceration was scored as follows: 0 = none, 1 = mild ulceration, 2 = moderate ulceration, and 3 = extensive ulceration; the crypt damage was scored as follows: 0 = none, 1 = submucosa, 2 = loss of one-third of the basal layer, 3 = loss of two-thirds of the basal layer, 4 = only the surface epithelium intact, and 5 = all crypts and epithelium destroyed; the percentage of affected area was scored as follows: 0 = none; 1 = 1-25%; 2 = 26-50%; 3 = 51-75%; and 4 = 76-100%.

[0090] Frozen sections: Mouse distal colons were fixed with 4% paraformaldehyde for 24 hours and then dehydrated using a gradient of sucrose solution (15% to 30%). Dehydrated tissue was embedded in OCT. Colonic tissue sections were cut into 6 μm sections using a freezing microtome and fixed with acetone.

[0091] Immunohistochemistry and Immunofluorescence: Paraffin-embedded colon tissue sections (4 μm) were used for immunohistochemistry or immunofluorescence staining. Paraffin sections were deparaffinized and antigen-retrieved, then blocked with blocking buffer (10% (volume fraction) goat serum, 2.5% (mass fraction) BSA, and 0.1% (volume fraction) Triton-X-100) for 1 hour at room temperature. Sections were incubated overnight at 4°C with primary antibodies against CD45 (1:300), cleaved caspase-3 (1:300), and iNOS (1:300). Alexa Fluor 594 secondary antibody (1:1000) was used for immunofluorescence staining, and Broad Spectrum Zymed Poly HRP secondary antibody (1:50) was used for immunohistochemistry.

[0092] AB-PAS staining: For the detection of acidic mucins, an AB-PAS kit was used according to the manufacturer's instructions. Paraffin-embedded sections were deparaffinized, rehydrated through graded ethanol solutions, and stained with Alcian blue (pH 2.5) for 15 minutes, followed by oxidation with periodate for 10 minutes, and then stained with Schiff's reagent for 15 minutes, followed by staining with hematoxylin for 3 minutes.

[0093] result:

[0094] Overexpression of miR-601 does not change colon morphology and function: Histopathological analysis showed that overexpression of miR-601 did not cause significant changes in colon morphology, including colon length ( Figure 7 A, B), disease activity index DAI ( Figure 7Middle C), colonic folds or overall colonic structure ( Figure 7 Immunohistochemical staining of cleaved caspase-3-positive cells showed no significant difference between miR-601-overexpressing transgenic mice and wild-type mice ( Figure 8 A, B), indicating that miR-601 overexpression itself does not affect the apoptosis of colon epithelial cells. Akkermansia ) degrades mucin and enhances intestinal barrier integrity. AB-PAS staining was further used to evaluate the intestinal mucin barrier. The results showed that miR-601 overexpression did not affect the thickness of the mucus layer ( Figure 8 (C, D).

[0095] miR-601 ameliorates DSS-induced ulcerative colitis: miR-601-overexpressing transgenic mice and wild-type mice were given 3% DSS for seven days to induce acute ulcerative colitis. The effects of miR-601 on colon morphology and function in DSS-induced mice were examined using the same method described above. Overexpression of miR-601 attenuated the colon shortening caused by DSS-induced colitis ( Figure 9 Middle A), weight loss ( Figure 9 Medium B), DAI score ( Figure 9 Middle C), epithelial erosion and ulcer and other histopathological damage manifestations are alleviated ( Figure 10 A, B) and reduced inflammatory infiltration ( Figure 10 Further experiments confirmed that miR-601 overexpression reduced the inflammatory response, apoptosis, and mucus layer thickness after DSS treatment. Immunofluorescence analysis of inducible nitric oxide synthase (iNOS) was performed on paraffin sections of mouse colon. iNOS is one of the most important pro-inflammatory biomarkers. Overexpression of miR-601 significantly reduced the number of iNOS-positive cells in the colon ( Figure 11 Compared with WT mice, the number of caspase-3-positive cells in miR-601-overexpressing transgenic mice decreased after DSS administration ( Figure 12 A, B), indicating that miR-601 overexpression can alleviate the apoptosis of colonic epithelial cells induced by DSS. AB-PAS staining results showed that the reduction of mucin in the miR-601 group mice was alleviated compared with the WT group after DSS administration ( Figure 12 (C and D) indicate that miR-601 overexpression alleviated DSS-induced damage to the mucus layer.

[0096] Example 5

[0097] miR-601 regulates the body's glucose metabolism:

[0098] (1) Experimental process:

[0099] Fasting blood glucose and fasting blood insulin determination: Tail tip blood glucose was measured in miR-601 overexpressing transgenic mice and wild-type mice (10 months old, male and female) after fasting for 6 hours and 16 hours. Eyeball blood was collected to obtain serum, and fasting blood insulin was measured using the ALPCO 80-INSMSU-E01, E10 rat and mouse ultrasensitive insulin ELISA detection kit.

[0100] Glucose tolerance test (GTT): Fasting blood glucose was measured by tail tip blood after mice fasted overnight for 16 hours. The glucose injection dose (2 g / kg*bw) was calculated based on the mice's fasting body weight. Blood glucose was measured by tail tip blood 15, 30, 60, and 120 minutes after intraperitoneal glucose injection.

[0101] Insulin tolerance test (ITT): The fasting blood glucose of mice was measured after fasting for 6 hours. The insulin dosage (0.75 IU / kg*bw) was calculated based on the mouse body weight. After intraperitoneal injection of insulin, the blood glucose of mice was measured using tail tip blood at 15 minutes, 30 minutes, 60 minutes, and 120 minutes.

[0102] (2) Results:

[0103] In male mice, miR-601 overexpression reduced blood glucose levels after 16 h of fasting ( Figure 13 C), 0 h blood glucose level in GTT test ( Figure 13 E), and can improve insulin sensitivity ( Figure 13 However, miR-601 did not change the body weight of mice ( Figure 13 Middle A), blood glucose level after 6 h fasting ( Figure 13 Middle B), insulin levels after 6 hours of fasting ( Figure 13 In female mice, miR-601 overexpression reduced fasting blood glucose ( Figure 14 B) and blood glucose level at 30 min in GTT test ( Figure 14 E), without changing the body weight of mice ( Figure 14 Middle A), blood glucose level after 16 h fasting ( Figure 14 Middle C), insulin levels after 6 hours of fasting ( Figure 14 D) and insulin sensitivity ( Figure 14 (G, H).

[0104] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Use of miR-601 in the preparation of a drug for preventing or treating ulcerative colitis, wherein the sequence of miR-601 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The application may be any of the following: A1) miR-601 promotes Akkermansia in the intestine ( Akkermansia ) Application in proliferation; A2) Application of miR-601 in regulating the interaction network of intestinal flora, strengthening the intestinal barrier, and reducing inflammatory response; A3) Application of miR-601 in reducing colon shortening, weight loss, and intestinal histopathological damage caused by ulcerative colitis; A4) Application of miR-601 in reducing the number of inducible nitric oxide synthase-positive cells and caspase-3-positive cells in ulcerative colitis.

3. The use according to claim 1, wherein A2) in the gut microbiota, including Akkermansia ( Akkermansia ) and the interaction between the beneficial bacteria that promote intestinal mucosal healing and the SCFA-producing flora; the beneficial bacteria that promote intestinal mucosal healing are Mycospirillum ( Mucispirillum ) and Dunaliella ( Dubosiella ); The SCFA-producing bacteria were unclassified Lachnospiraceae ( unidentified_Lachnospiraceae ) and Bacillus subtilis ( Odoribacter ) 、 Lachnoclostridium ; A3) Histopathological lesions in the midgut include epithelial erosions, leukocyte infiltration, and ulceration; In A3)~A4), ulcerative colitis is induced by dextran sulfate sodium salt.

4. Use of vectors carrying miR-601 in the preparation of drugs for preventing or treating ulcerative colitis.

5. Application of miR-601 in the preparation of drugs for regulating blood sugar, wherein the sequence of miR-601 is shown in SEQ ID NO.

1.

6. The use according to claim 5, characterized in that The application may be any of the following: B1) Application of miR-601 in lowering fasting blood glucose; B2) Application of miR-601 in improving insulin sensitivity.

7. Application of vectors carrying miR-601 in the preparation of drugs for regulating blood sugar.

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