CN111388499A

CN111388499A - Application of miR-31 in preparation of colon-targeted nano-drug for preventing and treating ulcerative colitis

Info

Publication number: CN111388499A
Application number: CN201811642767.9A
Authority: CN
Inventors: 于政权; 杜素娟; 徐久智
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2018-12-29
Filing date: 2018-12-29
Publication date: 2020-07-10

Abstract

The invention relates to the technical field of biological medicines, in particular to application of miR-31 in preparation of a colon-targeted nano-medicament for preventing and treating ulcerative colitis. The invention provides application of miR-31 or miR-31 analogue in preparation of a medicament for preventing or treating digestive tract diseases, in particular colitis, wherein the sequence of miR-31 is shown in SEQ ID NO.1 or SEQ ID NO. 2. The miR-31 is found to be an ideal active ingredient for preventing and treating ulcerative colitis, and can effectively relieve the occurrence and development of ulcerative colitis; by utilizing the protein nano-particle-konjac glucomannan gel microsphere carrier to deliver miR-31 molecules and matching with an enema administration mode, colon intestinal tracts can be targeted, the drug slow-release effect can be exerted, the bioavailability of nano-drugs is remarkably improved, and the prevention and treatment effects of ulcerative colitis are further effectively improved.

Description

Application of miR-31 in preparation of colon-targeted nano-drug for preventing and treating ulcerative colitis

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of miR-31 or an analogue thereof in preparation of a protein particle-konjak microsphere nano-medicament for preventing or treating colitis.

Background

Ulcerative colitis is an inflammatory bowel disease. Along with the increasing living standard of people, the incidence of the disease is higher and higher, and the attention of people is gradually aroused. The pathogenesis of ulcerative colitis is currently unknown, and the lack of effective therapeutic agents is an incurable disease that severely threatens human health (Colombel J F, Mahadevan U, Gastroenterology,2016,152(2): 309.). Therefore, exploring the pathological mechanisms of ulcerative colitis and finding effective therapeutic strategies is of great importance to prevent the development of ulcerative colitis. Currently, research on the treatment of ulcerative colitis is focused mainly on the regulation of the immune system. The treatment strategy of ulcerative colitis is mainly immunosuppression, and although the treatment strategy has a certain curative effect, the ulcerative colitis is easy to relapse and cannot be cured radically. Therefore, there is a great need to find effective treatment methods for ulcerative colitis.

α -lactalbumin can be processed into a nano carrier which is easy to modify and has good histocompatibility, and the carrier can carry the drug for treating diseases (L i Y, L i W, Bao W, et al, Nanoscale,2017 and 9), however, the drug taking α -lactalbumin nanoparticles as the carrier is difficult to safely and efficiently reach the colon no matter in an intravenous injection mode or an oral administration mode, so that the drug is difficult to be used for treating colitis.

The oxidized konjak polysaccharide derived from konjak can be processed into various forms such as gel, microsphere and the like, is expected to be a novel drug carrier for targeting colon due to the pH response characteristic, particularly the konjak microsphere has the advantages of uniform particle size, certain drug loading capacity, good biocompatibility and the like, and has the potential of embedding nanoparticles (Chen X, Wang S, L u M, et al, Biomacromolecules,2014,15(6): 2166).

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide application of miR-31 or an analogue thereof in preparation of a protein particle-konjak microsphere nano-medicament for preventing or treating colitis.

In order to achieve the purpose, the technical scheme of the invention is as follows: firstly, miR-31 is found to have the function of relieving gastrointestinal diseases such as colorectal diseases and the like, particularly the occurrence and development of ulcerative colitis; on the basis, miR-31 is tried to develop a medicament for preventing and treating intestinal diseases, however, miR-31 has the problems of easy degradation, lack of targeting property, low utilization rate and difficult administration, aiming at the problems, the inventor screens a large number of medicament carriers, but the medicament carrier system in the prior art can not simultaneously solve the problems of miR-31 degradation and targeted colon transportation, so the inventor develops a composite carrier of protein nano-particles and konjac polysaccharide gel, in the carrier, the protein nano-particles have a high-efficiency RNA adsorption function, the konjac polysaccharide gel has a function of targeted colon, a miR-31 medicament delivery system prepared on the basis of the carrier not only can be administered to the colon in a targeted manner, but also has a good slow release effect, the degradation of miR-31 is effectively avoided, and the colon-targeted slow release of miR-31 is realized, the utilization rate of miR-31 is improved.

The mammalian miRNA-31 has high conservation, wherein the GenBank accession numbers of human miR-31 genes and mouse miR-31 genes are NR _029505.1 and NR _029747.1 respectively, the gene sequences of the human miR-31 genes and the mouse miR-31 genes are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively, the difference between the gene sequences of the human miR-31 genes and the gene sequences of the mouse miR-31 genes is 1 base at the 3' end, and the functional region sequences are completely conserved.

Firstly, the invention provides application of miR-31 or miR-31 analogue in preparation of a medicament for preventing or treating digestive tract diseases.

In the invention, the active ingredient of the medicament for preventing or treating the digestive tract diseases comprises miR-31 or miR-31 analogue.

In the invention, the miR-31 analogue is a miRNA molecule with the same function as miR-31, and comprises a miR-31 agonist or a chemically modified miR-31.

Preferably, the sequence of the miR-31 is shown in SEQ ID NO.1 or SEQ ID NO. 2.

In the present invention, the disease of the digestive tract is an intestinal disease.

Preferably, the intestinal disease is a disease of the colon or rectum.

More preferably, the disease of the colon or rectum is ulcerative colitis.

On the basis, the invention provides a pharmaceutical composition for preventing or treating digestive tract diseases, which contains miR-31 or miR-31 analogue.

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

As a preferred embodiment of the present invention, the pharmaceutically acceptable carrier is a protein nanoparticle-konjac glucomannan gel microsphere carrier system.

Further, the present invention provides a drug delivery system targeting colon, which comprises protein nanoparticles, a drug adsorbed on the surface of the protein nanoparticles, and konjac glucomannan gel microspheres coated outside the drug-adsorbed protein nanoparticles.

In the above drug delivery system for targeting the colon, the drug is a negatively charged molecule; the protein nanoparticles are albumin nanoparticles; the konjac glucomannan gel microspheres are oxidized konjac glucomannan gel microspheres.

Such negatively charged molecules include, but are not limited to, nucleic acids, negatively charged protein molecules. Preferably, the drug is a nucleic acid.

More preferably, the drug is an RNA molecule.

The RNA molecule can be any RNA molecule with a large molecular weight, and can be coding RNA or non-coding RNA, and the non-coding RNA includes but is not limited to miRNA, siRNA, lncRNA, circRNA, and the like.

The miRNA includes, but is not limited to miR-31, miR-29 and miR-155.

As an example of the invention, the drug is miR-31 or an analogue thereof.

When the drug is miR-31 or an analogue thereof, the nanoparticle prepared from lactalbumin can be better combined with a miR-31 molecule, so that the miR-31 drug delivery system comprises the lactalbumin nanoparticle, the miR-31 or the analogue thereof adsorbed on the surface of the lactalbumin nanoparticle and oxidized konjac glucomannan gel microspheres coated outside the lactalbumin nanoparticle adsorbed with the miR-31 or the analogue thereof.

In order to realize better protein particle adsorption effect and konjak microsphere embedding effect and prepare uniform and stable nano-medicament, in the miR-31 medicament delivery system, the mass ratio of the lactalbumin nanoparticles to the miR-31 or the like is 60:1-80:1

The mass ratio of the lactalbumin nanoparticles to the oxidized konjac glucomannan gel microspheres is 3: 25-6: 25.

Preferably, the particle size of the drug targeting system is 20-30 μm.

The invention also provides a preparation method of the drug delivery system, which comprises the following steps:

preparing protein nanoparticles adsorbing drugs: preparing protein nanoparticles by treating protein with protease and ultrasound; activating the protein nanoparticles, and adding a medicament to perform an adsorption reaction; the particle size of the protein nanoparticles is 35-40nm, and the potential of the protein nanoparticles is 11-13 mV;

(II) embedding and adsorbing protein nanoparticles of the medicine by konjac glucomannan gel microspheres: respectively preparing an oil phase containing a surfactant and a water phase containing konjac glucomannan gel, a cross-linking agent and protein nanoparticles for adsorbing drugs, and embedding by adopting a W/O emulsification method.

Preferably, the step (ii) includes the steps of:

(1) preparing an oil phase: mixing span-80 and liquid paraffin according to the mass ratio of 1: 20;

(2) preparation of an aqueous phase: mixing konjac glucomannan gel, ferrous sulfate heptahydrate and protein nanoparticles for adsorbing drugs according to the mass ratio of 25:6:3, uniformly mixing;

(3) dropwise adding the water phase into the oil phase, carrying out ultrasonic shearing, stirring the sheared mixed solution at 35 ℃ for 4-6 h, and carrying out crosslinking and embedding reaction;

(4) centrifuging and discarding the supernatant, and washing the embedded microspheres by respectively adopting normal hexane and anhydrous methanol.

The invention also provides application of the drug delivery system in preparing a drug for preventing or treating colon diseases.

Preferably, the colonic disease is ulcerative colitis.

The invention has the beneficial effects that:

(1) the miR-31 is found to be an ideal active ingredient for preventing and treating ulcerative colitis, and provides a new medicinal active ingredient for treating ulcerative colitis.

(2) The invention discovers that the konjac polysaccharide gel microspheres can embed protein nanoparticles and can efficiently target colon to transport drugs, and the konjac polysaccharide gel microspheres, which are protein nanoparticles provided by the invention, can be used as carriers of a drug delivery system to realize effective colon-targeted sustained release effect, so that the colon utilization rate of the drugs is effectively improved.

(3) According to the invention, miR-31 molecules are delivered by utilizing a protein nanoparticle-konjac glucomannan gel microsphere carrier, and the problems that the traditional nano-drug cannot target colon, the drug utilization rate is low and the like are solved by matching with an enema administration mode; the carrier can directly reach the affected part in an enema administration mode, can be adhered to the surface of the colon through the carrier to play a drug slow release effect, obviously improves the bioavailability of the nano-drug, further effectively improves the prevention and treatment effects of ulcerative colitis, provides a new method for preparing the microRNA nano-drug and treating the ulcerative colitis by utilizing the microRNA nano-drug, and has great application potential.

Drawings

Fig. 1 is a schematic diagram of a preparation process of the miR-31 lactalbumin particle-konjac microsphere nano-drug in embodiment 1 of the invention, wherein PS is an lactalbumin nanoparticle, PS/miR-31 is an lactalbumin nanoparticle adsorbing miR-31, and OKGM is oxidized konjac glucomannan.

FIG. 2 is a graph showing the results of fluorescent staining analysis of konjak microspheres successfully embedding lactalbumin nanoparticles in example 1 of the present invention.

FIG. 3 is a graph showing the potential and particle size stability analysis of the lactalbumin particles of miR-31 lactalbumin particle-konjak microsphere nano-drug in example 1 of the invention, wherein A is the particle size stability analysis; b is stability analysis of potential.

FIG. 4 is a colon targeting analysis of miR-31 lactalbumin particles-konjac microsphere nano-drugs in example 2 of the invention, wherein A is a fluorescence microscope observation result image of a frozen section of a mouse colon to which a fluorescently-labeled miR-31 nano-drug is administered; b is miR-31 expression quantity analysis of colon cells.

Fig. 5 shows HE staining and immunohistochemical results of small intestine and colon of mice administered with nano-drug empty carrier OKGM-PS in example 2 of the present invention, wherein Ctrl is a control of enema physiological saline, OKGM-PS is administered with nano-drug empty carrier, H & E is a graph of HE staining results, and P65 and P-STAT are immunohistochemical results of P65 and P-STAT, respectively.

Fig. 6 is a HE staining result of lung, kidney, liver and spleen tissues of a mouse after administration of the nano-drug empty carrier OKGM-PS in example 2 of the present invention, wherein Ctrl is a control of enema physiological saline, and OKGM-PS is an administration nano-drug empty carrier.

FIG. 7 is a body weight analysis of mice after administration of the nano-drug in example 2 of the present invention.

Fig. 8 is a colon length analysis of a mouse after the administration of the nano-drug in example 2 of the present invention, wherein a is a colon picture of the mouse, and B is a statistical result of the colon length of the mouse.

FIG. 9 shows the HE staining of the colon of mice after administration of OKGM-PS-miR-31 in example 2 of the present invention.

FIG. 10 is a graph showing the effect of administration of OKGM-PS-miR-31 on the expression level of mouse Ki67 in immunofluorescence staining analysis in example 2 of the present invention.

FIG. 11 is a graph showing the expression levels of P-Stat3 and P65 in mice after administration of immunohistochemical staining in example 2 of the present invention.

FIG. 12 shows the expression levels of Gp130, Gsk3 β, L ats2 and Dlg1 in mice after administration of immunohistochemical staining analysis in example 2 of the present invention.

FIG. 13 shows Western blot analysis of the expression levels of mouse I L-17, Gp130, Axin1, Gsk3 β and Dlg1 after administration in example 2 of the present invention.

FIG. 14 is a schematic view showing the administration of the drug in example 3 of the present invention, after 5 days of DSS treatment, the withdrawal of DSS and the administration of enema are carried out for seven days, once a day.

FIG. 15 is a body weight analysis of mice administered with the nano-drug in example 3 of the present invention.

FIG. 16 is a colon length analysis of a mouse after administration of the nano-drug in example 3 of the present invention.

FIG. 17 shows the HE staining of the colon of mice after administration of OKGM-PS-miR-31 in example 3 of the present invention.

FIG. 18 is a body weight profile analysis of miR-31 knockout mice after administration of the nano-drug in example 4 of the present invention.

FIG. 19 is a colon length analysis of miR-31 knockout mice after administration of the nano-drug in example 4 of the present invention.

FIG. 20 shows the expression level of Ki67 in miR-31 knockout mice after administration according to the immunofluorescence staining analysis in example 4 of the present invention.

FIG. 21 is a schematic diagram of gene splicing and miR-31 knockout sequences for constructing miR-31 knockout mice by using a CRISPR-Cas9 system in example 4 of the invention.

Fig. 22 is a PCR identification result of miR-31 knockout mice constructed by using CRISPR-Cas9 system in example 4 of the present invention, wherein M is DNA marker,

lanes

1, 4, 9, 10, and 11 are mice in which miR-31 gene is successfully knocked out,

lanes

2, 5, 6, 7, and 8 are heterozygous mice, and lane 3 is wild-type mice.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of miR-31 lactalbumin particles-konjak microsphere nano-drug

In this example, human miR-31 is taken as an example (the gene sequence of human miRNA is shown in SEQ ID No. 1), the lactalbumin particle-konjac microsphere nano-drug carrying miR-31 is prepared, and the embedding structure of the nano-drug and the storage stability of the particle size and potential of the nano-drug are analyzed.

1. Synthesis of miR-31

According to the sequence of the human miR-31 (shown as SEQ ID NO. 1), the miR-31 molecules are synthesized by a synthesis company, and the sequences of two chains of the synthesized miR-31 molecules are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.

2. Preparation of protein particles adsorbing miR-31 mimics

(1) Weighing α -lactalbumin 6mg, dissolving in Tris-HCl 6ml (pH 7.5), adding Protease 2.5 μ l, shaking, mixing, water bathing at 50 deg.C for 1h, cooling to room temperature, and subjecting to ultrasound for 30s to obtain lactalbumin nanospheres;

(2) putting the prepared protein nanospheres into deionized water by using a 3500d dialysis bag for dialysis for 2h, changing water every 0.5h, and completing dialysis when the measured potential is about-11 mv;

(3) adding 0.5mg of EDC into 6ml of the nanospheres obtained in the step (2), activating for 15min, adding 0.5mg of mal, performing rotary reaction for 2h, dialyzing in deionized water for 2h, changing water every 0.5h, and measuring the potential to be about-3 mv to finish the reaction;

(4) adding miR-31 into the 6ml reaction system obtained in the step (3) to continue rotating and reacting for 2h, dialyzing in deionized water for 2h, changing water every 0.5h, and measuring the potential about 12mv to finish the reaction;

(5) and (3) concentrating the 6ml nanosphere system prepared in the step (4) to 200 μ l, adding 2OD (about 66 μ g) synthesized miR-31 (dissolved in 100 μ l DEPC water) into the 200 μ l system, and shaking at room temperature for 10 s.

Through detection, the particle size of the prepared lactalbumin nanoparticles is 35-40nm, and the potential is 11-13 mV. The mass ratio of the miR-31 to the lactalbumin nanoparticles in the lactalbumin nanoparticles (nanospheres) adsorbing the miR-31 is 60:1-80: 1.

Replacing miR-31 with NC (miRNA control without miR-31 function, sequences of two strands of the miRNA control are respectively shown in SEQ ID NO.5 and SEQ ID NO. 6) and DEPC water to serve as a negative control and a blank control, wherein the preparation method of the miRNA control is only different from the preparation method of the lactalbumin particles adsorbing miR-31 in that equimolar NC or DEPC water is added in the step (4).

2. Opal protein particles with miR-31 adsorbed by konjac microspheres

(1) In a 50ml beaker, 1.5g span80 and 40ml liquid paraffin were added, sealed and stirred for use.

(2) 12mg of FeSO₄.7H₂Dissolving O in 100 mul deionized water for later use;

(3) taking 1ml of oxidized konjac glucomannan (OKGM, 50mg/m L), and adding the FeSO prepared in the step (2) into the oxidized konjac glucomannan₄.7H₂Mixing the O solution and the lactalbumin particles adsorbing miR-31 prepared in the step 1 uniformly;

(4) dropwise adding the water-phase mixed solution obtained in the step (3) into the oil-phase mixed solution obtained in the step (1) on an ultrasonic crusher, and shearing by adopting ultrasonic crushing under the ultrasonic condition of 3000r for 2 min;

(5) and (4) placing the sheared oil-water mixture obtained in the step (4) on a magnetic stirring instrument, and stirring for 4 hours at 35 ℃.

(6) Centrifuge at 3000rpm for 2min, and discard the supernatant.

(7) Adding 5ml of n-hexane, blowing and beating uniformly, centrifuging at 3000r for 2min, discarding the supernatant, and repeating for three times.

(8) Adding 5ml of anhydrous methanol, blowing and beating uniformly, centrifuging at 3000r for 2min, discarding the supernatant, and repeating for three times.

(9) The precipitate obtained after the above washing was dissolved in 4ml of deionized water (pH 3) and stored at 4 ℃.

The preparation method of the control group NC and DEPC is the same as that of miR-31.

The concentration of the miR-31 lactalbumin particle-konjak microsphere nano-drug prepared by the method is 21.425 mu g/ml, and the particle size is 20-30 mu m. The schematic diagram of the process for preparing the miR-31 lactalbumin particle-konjak microsphere nano-drug by adsorption and embedding is shown in figure 1.

3. Embedding structure for analyzing miR-31 lactalbumin particle-konjak microsphere nano-drug by fluorescent staining

The α -lactalbumin particles prepared above were stained with cy3SE dye and embedded in konjac microspheres, and observed using a fluorescence microscope, as shown in fig. 2, indicating that we successfully embedded the protein particles in konjac microspheres.

4. Stability analysis of lactalbumin nanoparticles

The protein particles are stored at 4 ℃ statically, the particle size and potential change of the protein particles are detected regularly, the result is shown in figure 3, the particle size potential of the lactoalbumin particles does not change obviously within one week of storage at 4 ℃, and the protein particle carrier is very stable.

Example 2 application of miR-31 lactalbumin particle-konjak microsphere nano-drug in prevention of DSS-induced colitis

In this embodiment, a mouse colitis model is constructed by means of Dextran Sulfate Sodium Salt (DSS) induction, the miR-31 lactalbumin particles-konjac microsphere nano-drug prepared in example 1 is used for prevention of DSS-induced colitis, the miR-31 lactalbumin particles-konjac microsphere nano-drug is enemata-administered before and during DSS treatment, phenotypic change is observed by means of HE staining and Ki67 immunofluorescence staining, and the action mechanism and the mechanism of phenotypic recovery of the drug are further analyzed by means of Western blot.

Preparation of (I) DSS (direct sequence-derived) induced colitis mouse model

Selecting 15C 57 male mice with the age of 8 weeks, punching ear tags for numbering, and randomly grouping into three groups of nano-drug empty carriers OKGM-PS (blank control group added with DEPC water for adsorption), OKGM-PS-NC (negative control group added with NC molecules) and OKGM-PS-miR-31 (experimental group), wherein the OKGM-PS and OKGM-PS-NC groups are control groups, and the OKGM-PS-miR-31 group is experimental group. By administering 3.5% DSS with water for 5 consecutive days, symptoms of acute colitis can appear.

(II) administration of miR-31 lactalbumin particle-konjak microsphere nano-drug

By means of enema administration, the mice in each group were subjected to enema treatment with the miR-31 lactalbumin particle-konjac microsphere nano-drug OKGM-PS-miR-31 prepared in example 1 and the control drugs OKGM-PS and OKGM-PS-NC without miR-31, 1 time per day with 100 mu L/mouse per enema, starting enema administration 2 days before DSS treatment, continuing administration for 5 days during DSS treatment, and sampling for subsequent analysis.

(III) sampling

After 5 days of DSS treatment, three groups of mice, namely OKGM-PS, OKGM-PS-NC and OKGM-PS-miR-31, are subjected to neck dislocation and killed, the colorectal is taken out by using scissors after medical alcohol disinfection, the intestinal contents are washed clean by PBS, a proper amount of the intestinal contents are reserved as a protein sample and an mRNA sample, the rest colon is fixed by 4% paraformaldehyde at normal temperature for 24 hours, the PBS is washed for 3 times, 10min is carried out each time, and then tissue dehydration and paraffin section preparation are carried out.

(IV) miR-31 lactalbumin particle-konjak microsphere nano-drug targeting colon condition analysis

The α -lactalbumin particles prepared by the method of example 1 are dyed by cy3SE dye, then miR-31 with a green fluorescent marker is adsorbed by the method of example 1, the mice are subjected to enema treatment by the prepared fluorescent-labeled nano-drug, and after 4 hours, the colon is taken for frozen sectioning, and the result is shown in figure 4, and the result shows that the miR-31 lactalbumin particle-konjak microsphere nano-drug successfully reaches the colon cells of the mice after 4 hours of administration, and the expression level of miR-31 in the colon cells of the mice in the experimental group is obviously improved compared with that in an NC control group.

(V) pathological section, immunohistochemistry and western blot analysis of mouse phenotype and tissues such as intestine, lung, kidney, liver and spleen

1. Body weight and colon length, integrity analysis

The body weight before DSS treatment was taken as the initial body weight, and the body weight of mice at each of the other time points was recorded. The change in body weight can be macroscopically indicative of the rate of enteritis lesion, the extent of lesion, and the rate of lesion recovery in the mouse. The degree of inflammation of the colon also varies in mice, and the body weight and the length of the colon also vary.

2. Preparation of Paraffin-Embedded sections

(1) Tissue dehydration

The dehydration procedure was as follows: 50% ethanol for 30 min; 70% ethanol for 4-7.5 h; 80% ethanol for 1 hour; 95% ethanol, 40 min; 100% ethanol I, 15 min; 100% ethanol II, 15 min; xylene: ethanol (volume ratio 1:1), 5 min; xylene I, 10 min; xylene II, 5 min; xylene: soft wax (volume 1:1), 30 min; softening wax for 30 min; hard wax, 3 h; the temperature of the last 3 steps is set to 60 ℃ for dissolving the wax, and the rest steps are carried out at normal temperature.

(2) Embedding in paraffin wax

Embedding normal tissue in paraffin, cutting into 3-5 μm thick slices, spreading in 39 deg.C water bath, observing under solid microscope whether the slices are completely spread, taking out the flattened slices with adhesive glass slide, air drying, and performing subsequent histopathological staining and immunostaining.

3. Mouse tissue phenotype analysis (HE staining)

Hematoxylin-eosin staining (HE staining for short) is the most common method used in cytology, histology, embryology and pathology, and can visually observe abnormal changes of tissue cells, and the specific steps are as follows:

(1) baking slices: 63 ℃ for 1 h; (2) dewaxing and hydrating; (3) treating with xylene I for 15 min; (4) treating with xylene II for 15 min; (5) gradient ethanol treatment: treating with 100% ethanol I and II for 5min respectively; treating with 95% ethanol I and II for 5min respectively; treating with 80% ethanol for 5 min; treating with 70% ethanol for 5 min; (6) treating with distilled water for 5 min; (7) performing hematoxylin treatment for 10 min; (8) treating tap water for 5 min; (9) treating with 95% ethanol I and II for 3min respectively; (10) eosin treatment for 10 s; (11) treating with 95% ethanol I and II for 3min respectively; (12) treating with 100% ethanol I and II for 3min respectively; (13) treating with xylene I for 10 min; treating with xylene II for 10 min; (14) and sealing and taking a picture.

4. Immunohistochemical analysis of mouse tissue

The specific method comprises the following steps: (1) baking slices: 63 ℃ for 1 h; (2) dewaxing and hydrating; (3) xylene I:15 min; (4) xylene II for 15 min; (5) gradient ethanol treatment: 100% ethanol I, II: each for 5 min; 95% ethanol I, II: each for 5 min; 80% ethanol for 5 min; 5min with 70% ethanol; distilled water: 5 min; (6) antigen retrieval: boiling sodium citrate (pH 6.0) for 20 min; (7) and (3) natural cooling: about 1 h; (8) PBS treatment for 3 times, 5 min/time; (9) h₂O₂Keeping out of the sun for 20 min; (10) PBS treatment for 3 times, 5 min/time; (11) and (3) sealing: the sealing liquid acts for at least 1 h; (12) primary antibody hybridization: adding the prepared primary antibody according to a proper proportion, and standing overnight at 4 ℃; (13) rewarming: standing at room temperature for half an hour; (14) PBS treatment for 3 times, 5 min/time; (15) and B, liquid B: room temperature for 30 min; (16) PBS treatment for 3 times, 5 min/time; (17) and C, liquid C: room temperature for 30 min; (18) PBS treatment 3 times, 5 min/time. (19) Distilled water: the treatment is carried out for 5 min. (20) DAB, stopping developing under a proper developing time according to the characteristics of the antibody, and putting the DAB into distilled water; (21) and (3) hematoxylin: re-dyeing for 5 min; (22) tap water: treating for 5 min; (23) gradient ethanol treatment: 70% ethanol for 3 min; 80% ethanol for 3 min; 90% ethanol for 3 min; 3min with 100% ethanol; (24) xylene I:5 min; xylene II for 5 min; (25) and sealing and taking a picture.

Primary antibodies used for the immunohistochemical staining were P65(cell signaling technology, 1; 1000, #8242), P-Stat3(cell signaling technology, 1; 1000, #9145), Ki67(cell signaling technology, 1; 1000, #9449), β -catenin (Sigma, 1; 2000, # C7082), Gp130(Santa cruz, 1; 600, # sc-376280), Gsk3 β (cell signaling technology, 1; 1000, #9323), L ats2(Abcam, 1; 2000, # ab70565), Dlg1(Abcam, 1; 2000, # 3437).

The second antibody used for the immunohistochemical staining is a murine antibody and a rabbit antibody, the DAB color development kit is purchased from China fir gold bridge, the B liquid and the C liquid are both from China fir gold bridge kit, and the catalog numbers are as follows: sp9001, sp 9002.

5. Western blot detection of colon tissue gene expression

The specific method comprises the following steps:

(1) extraction of proteins

① cutting colon tissue of proper size, and cutting with scissors;

② adding 400 μ L protein lysate (PMSF: IP volume ratio 1:100) into the above sample, homogenizing by homogenizer, and lysing the tissue on ice for 30 min;

③ 4 deg.C, centrifuging at 12000rpm for 10 min;

④ sucking the supernatant, taking part of the protein sample to detect the concentration, using in Western blot experiment, and storing the rest samples at-80 deg.C.

(2) And (3) detecting the protein concentration: protein concentration was measured using the Byrun sky kit BCA, 50. mu.g of each lane.

(3) Electrophoresis: concentrating the glue: 60V, separation gel: 80V.

(4) Film transfer: and (5) wet-rotating, and rotating the film for 1h at 330 mA.

(5) And (3) sealing: sealing with 5% skimmed milk powder for 1 hr.

(6) Primary antibody hybridization: according to the molecular weight of protein, the membrane with proper size is cut, sealed and added with proper primary antibody, and the mixture is kept at 4 ℃ overnight.

(7) Rewarming: shaking on shaking bed for 30min to recover primary antibody.

(8) Washing the membrane: TBST 3X10 min/time

(9) And (3) hybridization of a second antibody: secondary antibodies (purchased from Biyuntian) were added and incubated for 1h at room temperature.

(10) Washing the membrane: TBST treatment was carried out 3 times at a rate of 10 min.

(11) Color development: and developing by adopting a DAB developing kit.

Primary antibodies used in the Western blot were I L-7 r (Santa cruz, 1; 1000, # sc-662), Gp130(Santa cruz, 1; 500, # sc-376280), Axin1(cell signaling technology, 1; 1000, #2074), Gsk3 β (cell signaling technology, 1; 1000, #9323), Dlg1(Abcam, 1; 600, # ab3437), β -butulin (Shanghai san Biotech, Ltd., dilution ratio 1; 4000,30101ES 50).

Western blot was performed using secondary antibodies (Biyunyan, dilution ratio 1: 10000, cat # A0208 (rabbit source), A0216 (mouse source).

6. Results of the experiment

(1) Analysis of toxic and side effects of the nano-drug on mice: the HE staining results of small intestine and colon of mice fed with nano-drug empty carrier OKGM-PS and the 3 immunohistochemical results of P65 and P-STAT are shown in figure 5, and the HE staining results of lung, kidney, liver and spleen tissues are shown in figure 6.

(2) Analysis of the influence of the nano-drug on the body weight and colon length of mice: the body weight of the mice after administration is shown in fig. 7, and the results show that the body weight of the mice in the experimental group administered with OKGM-PS-miR-31 is obviously higher than that in the control group.

The analysis result of the colon character of the mice after administration is shown in figure 8, and the result shows that the colon length of the experimental group administered with OKGM-PS-miR-31 is obviously longer than that of the control group, and the colon structure is more complete.

(3) Analysis on the influence of the nano-drug on the occurrence and development of mouse DSS-induced colitis, the results of HE staining of mouse colon are shown in FIG. 9, and show that the colon structure of mice of an experimental group administered with OKGM-PS-miR-31 is obviously superior to that of a control group (administered with OKGM-PS and OKGM-PS-NC), the results of immunofluorescence staining are shown in FIG. 10, and show that the Ki67 expression level of mice of the experimental group administered with OKGM-PS-miR-31 is obviously improved, and that the damage to colon inflammation of mice of the experimental group administered with OKGM-PS-miR-31 is obviously reduced compared with that of the control group, the immunohistochemistry results are shown in FIGS. 11 and 12, and the Western blot results are shown in FIG. 13, and show that the results of P-Stat3, P65, Gp130, Gsk3 β, L ats2, Dlg1, I L-17, in1, and the like of mice administered with OKGM-miR-31 show that the target genes of the mice are remarkably reduced in the inhibition of the targeted gene expression of the mouse DSS-induced colitis, and the target gene of the AOK-miR-31, and the target gene after administration of the mouse is significantly reduced by the target gene induced colitis induced by the target gene induced by the OKG-miR-31, and the target gene.

Example 3 application of miR-31 lactalbumin particle-konjak microsphere nano-drug in treatment of DSS-induced colitis

Preparation of (I) DSS (direct sequence-derived) induced colitis mouse model

Selecting 15C 57 male mice with the age of 8 weeks, punching ear tags for numbering, and randomly grouping into three groups of nano-drug empty carriers OKGM-PS, OKGM-PS-NC and OKGM-PS-miR-31, wherein the OKGM-PS and OKGM-PS-NC groups are control groups, and the OKGM-PS-miR-31 group is an experimental group. By administering 3.5% DSS with water for 5 consecutive days, symptoms of acute colitis can appear.

(II) administration of miR-31 mimetic protein particle-konjak microsphere nano-drug

By means of enema administration, the mice of each group were individually subjected to enema treatment 1 time per day with 100 μ L/mouse per enema, using the miR-31 lactalbumin particle-konjac microsphere nano-drug OKGM-PS-miR-31 prepared in example 1 and the control drugs OKGM-PS and OKGM-PS-NC without miR-31, stopping DSS treatment and starting nano-drug enema 5 days after DSS treatment, taking materials seven days after enema treatment, sampling for subsequent analysis, and the administration mode is schematically shown in fig. 14.

(III) sampling

Seven days after enema treatment, carrying out neck dislocation and sacrifice on three groups of mice, namely OKGM-PS, OKGM-PS-NC and OKGM-PS-miR-31, taking out the colorectal by using scissors after medical alcohol disinfection, washing the intestinal contents by PBS, reserving a proper amount of colon as a protein sample and an mRNA sample, fixing the rest colon by 4% paraformaldehyde for 24 hours at normal temperature, washing by PBS for 3 times, 10min each time, and then carrying out tissue dehydration.

(IV) pathological section, immunohistochemistry and western blot analysis of mouse phenotype and tissues such as intestine, lung, kidney, liver and spleen

The experimental procedure was as in example 2.

(V) results of the experiment

1. Analysis of the influence of the nano-drug on the body weight and colon length of mice: the body weight of the mice after administration is shown in fig. 15, and the results show that the body weight of the mice in the experimental group administered with OKGM-PS-miR-31 is obviously higher than that in the control group.

The colon character analysis result of the mice after administration is shown in fig. 16, and the result shows that the colon length of the experimental group administered with OKGM-PS-miR-31 is obviously longer than that of the control group, and the colon structure is more complete, which shows that the inflammation of the mice of the experimental group administered with OKGM-PS-miR-31 is obviously reduced compared with that of the control group.

2. Analysis of the influence of the nano-drug on the occurrence and development of mouse DSS-induced colitis: the result of HE staining is shown in FIG. 17, and the result shows that the colon structure of mice in the experimental group administered with OKGM-PS-miR-31 is obviously superior to that of the control group (administered with OKGM-PS and OKGM-PS-NC), which indicates that the mice in the experimental group administered with OKGM-PS-miR-31 have less damage and faster repair after being damaged by DSS, and indicates that the miR-31 nano-drug has a good treatment effect on colitis.

Example 4 application of miR-31 lactalbumin particle-konjak microsphere nano-drug in prevention of miR-31 knock-out of mouse DSS (DSS-induced colitis)

(I) construction of miR-31 knock-out mice

A schematic diagram of a knockout process of constructing a miR-31 gene knockout mouse by using a CRISPR/Cas9 technology is shown in FIG. 21, and the specific method comprises the following steps:

the K14rtta transgenic mice and TRE-Msi1 transgenic mice are mated (the construction method of two transgenic mice is described in Wang S, L i N, Yousefi M, Nakauka-Ddamba A, L i F, Parada K, et al (2015) Transformation of the endogenous plasmid by the MSI2RNA-binding protein. nat Commun 6:6517), generating offspring mice, and when the offspring mice grow to 6 weeks, tetracycline with the final concentration of 0.2 g/L is added into the drinking water of the mice, so that the mice freely drink water, enough drinking water is ensured, and the induction gene Musa shi1 is overexpressed, and after tetracycline is fed for 48 hours, mice with mammary gland external paget disease-like symptoms can be presented, namely, mice with mammary gland external paget disease (DTG mice).

Identification of miR-31 knockout mouse genotype:

extracting mouse genome DNA as a template, taking F1 and R1 as primers, carrying out PCR amplification (the PCR target is miR-31 gene), detecting an amplification product, and generating two characteristic bands of 300bp and 700bp, wherein a PCR reaction system comprises mix 6 mu L (Kangwei century, cargo number: 01037/30252), F10.6 mu L, R10.6 mu L, a genome DNA template 1 mu L and ddH₂O4.3 mu L, reaction program, 95 ℃ for 5min, 95 ℃ for 30s,30s at 58 ℃ and 30s at 72 ℃ for 35 cycles; 72 ℃ for 2 min.

The identification result of the miR-31 knockout mouse genotype is shown in FIG. 22, wherein the mouse with a 300bp band is a miR-31 knockout mouse, and the mouse with a 700bp band is a wild-type mouse.

The primer sequences used were as follows (SEQ ID NO.7 and SEQ ID NO. 8):

F1:5′-AGGTCACTGCACTCCTATGGAC-3′

R1:5′-GTGCCCAATGATTCTGACAAGTCAG-3′

preparation of (II) DSS (direct sequence-derived suppressor) colitis mouse model

Selecting 15 miR-31 knockout mice of 8 weeks old, numbering by ear tag, randomly grouping into OKGM-PS-NC and OKGM-PS-miR-31 groups, wherein the OKGM-PS-NC group is a control group, and the OKGM-PS-miR-31 group is an experimental group. By administering 3.5% DSS with water for 5 consecutive days, symptoms of acute colitis can appear.

(III) administration of miR-31 protein particle-konjak microsphere nano-drug

By means of enema administration, the mice in each group were subjected to enema treatment with the miR-31 lactalbumin particle-konjac microsphere nano-drug OKGM-PS-miR-31 prepared in example 1 and the control drug OKGM-PS-NC without miR-31, respectively, 1 time per day, 100 μ L/mouse per enema, administration of the nano-drug by enema starting 2 days before DSS treatment, administration of the nano-drug continuing during DSS treatment until 5 days after DSS treatment, and sampling for subsequent analysis.

(III) sampling

Two groups of mice of OKGM-PS-NC and OKGM-PS-miR-31 are dislocated at the neck and killed, the colorectal is taken out by scissors after medical alcohol disinfection, the intestinal contents are washed clean by PBS, a proper amount of colon is reserved to be used as a protein sample and an mRNA sample, the rest colon is fixed by 4 percent paraformaldehyde for 24 hours at normal temperature, the PBS is washed for 3 times, 10min is carried out each time, and then tissue dehydration is carried out.

The experimental procedure was as in example 2.

(V) results of the experiment

1. Analysis of the influence of the nano-drug on the body weight and colon length of mice: the body weight of the mice after administration is shown in FIG. 18, and the results show that the body weight of the mice in the experimental group administered with OKGM-PS-miR-31 is obviously higher than that of the mice in the control group administered with OKGM-PS-NC.

The colon character analysis result of the mice after administration is shown in fig. 19, and the result shows that the colon length of the experimental group administered with OKGM-PS-miR-31 is obviously longer than that of the control group, and the colon structure is more complete, which shows that the inflammation of the mice of the experimental group administered with OKGM-PS-miR-31 is obviously reduced compared with that of the control group.

2. Analysis of the influence of the nano-drug on the occurrence and development of mouse DSS-induced colitis: the immunofluorescence staining result is shown in fig. 20, and the result shows that the Ki67 expression level of mice in an experimental group administered with OKGM-PS-miR-31 is remarkably improved, the damage to colon inflammation of miR-31 knockout mice administered with OKGM-PS-miR-31 is remarkably reduced compared with a control group, and the miR-31 protein particle-konjak microsphere nano-drug has a better prevention effect on colitis.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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Claims

Application of miR-31 or miR-31 analogue in preparation of medicines for preventing or treating digestive tract diseases.
2. The use according to claim 1, wherein the active ingredient of the medicament comprises miR-31 or a miR-31 analogue; the miR-31 analogue comprises a miR-31 agonist or a chemically modified miR-31;

preferably, the sequence of the miR-31 is shown in SEQ ID NO.1 or SEQ ID NO. 2.
3. The use according to claim 1 or 2, wherein the disease of the digestive tract is a disease of the intestinal tract;

preferably, the intestinal disease is a disease of the colon or rectum;

more preferably, the disease of the colon or rectum is ulcerative colitis.
4. A pharmaceutical composition for preventing or treating digestive tract diseases, which contains miR-31 or a miR-31 analogue;

preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
5. A drug delivery system targeting colon, which is characterized in that the drug delivery system comprises protein nanoparticles, drugs adsorbed on the surfaces of the protein nanoparticles and konjac glucomannan gel microspheres coated outside the drug-adsorbed protein nanoparticles.
6. The drug delivery system of claim 5, wherein the drug is a negatively charged molecule; the protein nanoparticles are albumin nanoparticles; the konjac glucomannan gel microspheres are oxidized konjac glucomannan gel microspheres;

preferably, the drug is a nucleic acid;

more preferably, the drug is an RNA molecule.
7. The drug delivery system of claim 5 or 6, wherein the drug is miR-31 or an analog thereof; the drug delivery system comprises lactalbumin nanoparticles, miR-31 or an analogue thereof adsorbed on the surfaces of the lactalbumin nanoparticles, and oxidized konjac glucomannan gel microspheres coated outside the lactalbumin nanoparticles adsorbed with the miR-31 or the analogue thereof.
8. The drug delivery system of claim 7, wherein the mass ratio of the lactalbumin nanoparticles to the miR-31 or the similar thereof is 60:1-80: 1;

the mass ratio of the lactalbumin nanoparticles to the oxidized konjac glucomannan gel microspheres is 3: 25-6: 25;

preferably, the particle size of the drug targeting system is 20-30 μm.
9. A method for the preparation of a drug delivery system according to any of claims 5 to 8, comprising the steps of:

preparing protein nanoparticles adsorbing drugs: preparing protein nanoparticles by treating protein with protease and ultrasound; activating the protein nanoparticles, and adding a medicament to perform an adsorption reaction; the particle size of the protein nanoparticles is 35-40nm, and the potential of the protein nanoparticles is 11-13 mV;

(II) embedding and adsorbing protein nanoparticles of the medicine by konjac glucomannan gel microspheres: respectively preparing an oil phase containing a surfactant and a water phase containing konjac glucomannan gel, a cross-linking agent and protein nanoparticles for adsorbing drugs, and embedding by adopting a W/O emulsification method;

preferably, step (two) comprises the steps of:

(1) preparing an oil phase: mixing span-80 and liquid paraffin according to the mass ratio of 1: 20;

(2) preparation of an aqueous phase: uniformly mixing konjac glucomannan gel, ferrous sulfate heptahydrate and protein nanoparticles for adsorbing drugs according to the mass ratio of 25:6: 3;

(3) dropwise adding the water phase into the oil phase, carrying out ultrasonic shearing, stirring the sheared mixed solution at 35 ℃ for 4-6 h, and carrying out crosslinking and embedding reaction;

(4) centrifuging and discarding the supernatant, and washing the embedded microspheres by respectively adopting normal hexane and anhydrous methanol.
10. Use of a drug delivery system according to any one of claims 5 to 8 in the manufacture of a medicament for the prophylaxis or treatment of colonic disease;

preferably, the colonic disease is ulcerative colitis.