Combination drug for treating colitis and use thereof

Abstract

The present application relates to the technical field of biological pharmacy, in particular to a combined drug for treating colitis and application thereof, wherein the volume ratio of Coptis exosome and Salmonella outer membrane vesicle in the combined drug is 1-2:1-3; the concentration of the Coptis exosome and the Salmonella outer membrane vesicle is 9-11 mg / mL. The present application overcomes the limitation of single treatment strategy by innovatively combining the Coptis exosome and the Salmonella outer membrane vesicle, significantly improves the treatment effect, and avoids the side effects of existing drugs. The present application has lower treatment cost, is safer and has no drug resistance problem, and can provide a more effective, safe and economical treatment method for inflammatory diseases such as colitis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biopharmaceuticals, in particular to a combined drug for treating colitis and application thereof. BACKGROUND

[0002] Colitis is a common intestinal disease, mainly including ulcerative colitis (UC) and Crohn's disease (CD), which belongs to inflammatory bowel disease (IBD). Its characteristic is long-term inflammation of the colon and rectum, which is manifested as symptoms such as abdominal pain, diarrhea, weight loss, etc. Long-term ineffective treatment may lead to complications such as loss of intestinal function and colon cancer. At present, the treatment methods for colitis mainly include drug treatment (such as glucocorticoids, immunosuppressants and biological agents) and surgical treatment. However, the existing single drug treatment is often accompanied by significant side effects (such as infection, liver damage, osteoporosis, etc.), and patients are prone to drug resistance, resulting in unstable treatment effect.

[0003] Therefore, it is urgent to develop a new strategy for treating colitis more effectively and safely. SUMMARY

[0004] To solve the above problems, the present application provides a combined drug for treating colitis and application thereof.

[0005] The present application is realized by the following technical solutions:

[0006] A combined drug for treating colitis, wherein the volume ratio of Coptis exosome and Salmonella outer membrane vesicle in the combined drug is 1-2:1-3; the concentration of Coptis exosome and Salmonella outer membrane vesicle is 9mg / mL-11mg / mL.

[0007] Preferably, the volume ratio of Coptis exosome and Salmonella outer membrane vesicle in the combined drug is 1:2; the mass concentration of Coptis exosome and Salmonella outer membrane vesicle is 10mg / mL.

[0008] Preferably, the Coptis exosome is obtained by the following steps:

[0009] Wash Coptis and soak in water for 19-21min;

[0010] Juice the soaked Coptis and collect the Coptis juice;

[0011] Centrifuge the Coptis juice at 5000r / min at low speed under the condition of 3-5℃ for 19-21min, collect supernatant I; centrifuge supernatant I at 10000r / min at high speed for 29-31min, collect supernatant II; filter supernatant II and retain the filtrate;

[0012] centrifuging at 10000 r / min for 29-31 min, collecting supernatant III; filtering supernatant III with filters with pore sizes of 0.44-0.46 μm and 0.21-0.23 μm, respectively, and retaining the final filtrate; and separating and purifying the final filtrate to obtain the Coptis exosome.

[0013] Preferably, the Coptis exosome is obtained by separating and purifying the final filtrate using an exosome extraction and purification kit.

[0014] Preferably, the Salmonella outer membrane vesicle is obtained by the following steps:

[0015] The Salmonella is amplified and cultured, and the bacterial cells are collected by centrifugation, resuspended in a PBS solution containing deoxycholic acid sodium, and incubated to obtain a mixed solution.

[0016] The mixed solution is centrifuged, and the supernatant is collected, concentrated to a volume of 1 / 10 of the original solution, and filtered through a sterile filter membrane to obtain a filtrate.

[0017] The filtrate is added to the upper layer of a sucrose density gradient solution, and ultracentrifuged at 80000-120000 g at 3-5°C to collect the Salmonella outer membrane vesicle layer at the interface of 20-30% w / v sucrose to obtain a Salmonella outer membrane vesicle suspension; the sucrose density gradient is 20-50% w / v.

[0018] The Salmonella outer membrane vesicle suspension is dialyzed in a PBS buffer at 3-5°C, and the dialysis solution is replaced during the dialysis to obtain the Salmonella outer membrane vesicle.

[0019] Preferably, the content of deoxycholic acid sodium in the PBS solution is 0.3-0.7% w / v.

[0020] Preferably, the molecular weight cut-off of the dialysis is 100 kDa.

[0021] Preferably, the combination drug can be formulated into a clinically acceptable preparation form together with or separately from a pharmaceutically acceptable excipient.

[0022] Preferably, the pharmaceutically acceptable excipient is one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickening agent, an emulsifying agent, a preservative, and a stabilizer.

[0023] Preferably, the dosage form of the combination drug is an oral preparation, an infusion, or an injection.

[0024] The combination drug for use in the treatment of colitis.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] The present application provides a combined drug for treating colitis, wherein the volume ratio of Coptis exosome and Salmonella outer membrane vesicle is 1-2:1-3; and the concentration of the Coptis exosome and the Salmonella outer membrane vesicle is 9-11 mg / mL. (1) The present application first uses Coptis exosome and Salmonella outer membrane vesicle in combination to treat colitis. Experimental studies show that the combination of exosome and outer membrane vesicle can produce a synergistic effect in multiple mechanisms, making the treatment of colitis more effective. The anti-inflammatory and antioxidant effects of Coptis exosome complement the immune regulation of Salmonella outer membrane vesicle, which can improve the pathological state of colitis in multiple dimensions and provide a new treatment strategy. (2) The combination of Coptis exosome and Salmonella outer membrane vesicle is safe. The Coptis exosome in the present application is derived from natural plants, and the Salmonella outer membrane vesicle is a microbial derivative. Compared with chemical drugs or immunosuppressants, the combination has a lower risk of side effects. Coptis exosome has natural anti-inflammatory and antioxidant effects and does not depend on immune system suppression, thereby reducing the side effects and health risks caused by immune suppression. (3) The combination of Coptis exosome and Salmonella outer membrane vesicle has no drug resistance problem. Unlike some drug treatments that may cause drug resistance, the use of natural products and microbial derivatives is less likely to cause drug resistance, and is expected to provide long-term effective treatment. (4) Reduce treatment cost: Coptis exosome and Salmonella outer membrane vesicle are widely available, and the extraction method is relatively mature and low in cost. Compared with some expensive biological agents and immunosuppressants, the present application provides a low-cost alternative, with a production cost reduced by more than 40%, which can be more widely used in clinical practice. (5) Simplify the treatment plan: Coptis exosome and Salmonella outer membrane vesicle do not require special equipment for extraction and application, and thus the treatment plan can be applied to more medical institutions, especially primary medical units, so that the treatment of colitis can be popularized to a wider patient group.

[0027] In summary, compared with the shortcomings of the prior art, the present application overcomes the limitations of single treatment strategy by innovatively combining Coptis exosome and Salmonella outer membrane vesicle, significantly improves the treatment effect, and avoids the side effects of existing drugs. The present application has a lower treatment cost, is safer and has no drug resistance problem, and can provide a more effective, safe and economical treatment method for inflammatory diseases such as colitis. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0029] Figure 1 Identification and basic information chart of the exosomes of Coptis chinensis in the present application; Figure 1 In the figure, A is a cup-shaped morphology chart of the exosomes of Coptis chinensis under a transmission electron microscope; B is a particle size chart of the exosomes of Coptis chinensis; and C is an extraction information chart of the exosomes of Coptis chinensis.

[0030] Figure 2 Identification and basic information chart of the outer membrane vesicles of Salmonella in the present application; A is a cup-shaped morphology chart of the outer membrane vesicles of Salmonella under a transmission electron microscope; B is a particle size distribution chart of the outer membrane vesicles of Salmonella; and C is a concentration and diameter chart of the outer membrane vesicles of Salmonella.

[0031] Figure 3 Pathological score chart of mice in different treatment groups in the present application.

[0032] Figure 4 Body weight loss rate chart of mice in different treatment groups in the present application.

[0033] Figure 5 Colon length and HE staining chart of mice in different treatment groups in the present application; Figure 5 In the figure, A is a colon length chart of mice; and B is an HE staining chart of the colon of mice.

[0034] Figure 6 Relative expression amount chart of extracted genes of the colon of mice in different treatment groups in the present application; Figure 6 In the figure, A is a relative expression amount chart of occludin genes; and B is a relative expression amount chart of claudin genes.

[0035] Figure 7 Inflammation factor detection chart of serum of mice in different treatment groups in the present application; Figure 7 In the figure, A is a relative expression amount chart of tumor necrosis factor alpha genes; B is a relative expression amount chart of interleukin 6 genes; C is a relative expression amount chart of interleukin 1 beta; D is a tumor necrosis factor alpha protein content chart; E is an interleukin 6 protein content chart; and F is an interleukin 1 beta protein content chart.

[0036] Figure 8 Antioxidant component detection chart of supernatant of cells in different treatment groups in the present application; Figure 8 In the figure, A is a superoxide dismutase content chart; B is a malondialdehyde content chart; and C is a glutathione content chart.

[0037] Figure 9Figure of anti-oxidation detection of different treatment groups of the cell model of the present application; Figure 9 In the figure, A is a protein print of superoxide dismutase, malondialdehyde and internal reference; B is a relative expression amount figure of superoxide dismutase protein; C is a relative expression amount figure of malondialdehyde protein. DETAILED DESCRIPTION

[0038] In order to facilitate the understanding of the present application, the present application will be described more fully below, and preferred embodiments of the present application will be given. However, the present application can be realized in many different forms and is not limited to the embodiments described in the present application. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0039] Unless otherwise defined, all technical and scientific terms used in the present application have the same meanings as commonly understood by those skilled in the art to which the present application belongs. The terms used in the present application in the specification of the present application are only for the purpose of describing the specific embodiments and are not intended to limit the present application.

[0040] The beneficial effects of the present application will be described below through specific embodiments.

[0041] In the present application, the rhizomes of Coptis chinensis Franch. are gifted by Coptis chinensis Franch. industry system in Zhuxi County, Shiyan City, Hubei Province.

[0042] Salmonella is preserved by the Basic Medical College of Hubei Medical College.

[0043] Example 1

[0044] A combined drug for treating colitis, wherein the combined drug is composed of Coptis chinensis Franch. exosomes and Salmonella outer membrane vesicles in a volume ratio of 1:2; the concentration of Coptis chinensis Franch. exosomes and Salmonella outer membrane vesicles is both 10 mg / mL.

[0045] The extraction method of Coptis chinensis Franch. exosomes is as follows:

[0046] Extraction raw materials: mature Coptis chinensis Franch. rhizomes are selected, washed and dried.

[0047] Exosome extraction steps:

[0048] Step 1: Pretreatment of raw materials

[0049] (1) Wash the Coptis chinensis Franch. raw materials: wash the Coptis chinensis Franch. with 28 kHz ultrasonic wave for 5 min, rinse with tap water for 3 times, rinse with ultrapure water for 3 times, and then soak in ultrapure water for 20 min.

[0050] (2) Cutting treatment: cut the washed Coptis chinensis Franch. into 1.5 cm long fragments using a ceramic knife.

[0051] Step 2: Juice the raw materials

[0052] The Coptis fragment was placed in the Mijia blender N1, and juice was extracted at a speed of 35000 r / min for 5 min, and the Coptis juice was collected.

[0053] Step 3: Two-step centrifugal impurity removal

[0054] (1) Low-speed centrifugation: The extracted liquid was placed in a 4℃ low-temperature high-speed centrifuge, and centrifuged at 5000 r / min for 20 min, and the supernatant 1 was collected.

[0055] (2) High-speed centrifugation: The supernatant obtained in step 3(1) was centrifuged at 10000 r / min for 30 min at 4℃, and the supernatant 2 was collected again.

[0056] Step 4: Primary filtration

[0057] The supernatant 2 obtained in step 3(2) was filtered through a needle filter with a pore size of 0.45 μm, and the filtrate was retained.

[0058] Step 5: pH adjustment and secondary centrifugation

[0059] (1) pH adjustment: NaHCO3 solution was slowly added to the filtrate to adjust the pH to 7.0, and the solution color change was observed and a small amount of flocculent precipitate was generated.

[0060] (2) Centrifugal purification: The adjusted solution was centrifuged at 10000 r / min for 30 min at 4℃, and the supernatant 3 was collected.

[0061] Step 6: Secondary filtration

[0062] The supernatant obtained in step 5(2) was sequentially filtered using needle filters with pore sizes of 0.45 μm and 0.22 μm, and the final filtrate was retained. The final filtrate was treated with an exosome extraction and purification kit to obtain Coptis exosomes.

[0063] Step 7: Characterization

[0064] The morphology and integrity of Coptis exosomes were observed by transmission electron microscopy (TEM). As shown in Figure 1 , the extract showed a cup-shaped morphology with typical exosome characteristics, and had a clear double-layer phospholipid membrane structure.

[0065] The suspension was analyzed by NTA using a Malvern nanoparticle tracking analyzer (NanoSight NS300) to determine the particle size of the exosomes. As shown in Figure 3 , the average particle size was 128.8 nm; the concentration range was 5.6E+7 particles / mL.

[0066] The extraction method of Salmonella outer membrane vesicles is as follows:

[0067] Extracted raw material: Salmonella enterica (S. enterica) was selected and cultured in LB liquid medium. Typhimurium

[0068] Outer membrane vesicle extraction procedure:

[0069] 1. Bacterial cell amplification culture:

[0070] Salmonella enterica was inoculated into LB liquid medium (pH 7.4) and cultured at 37°C with 200 rpm shaking until the logarithmic growth phase (OD 600 = 0.8). The bacterial cells were collected by centrifugation (4°C, 6000 x g, 15 min) and washed with phosphate buffer solution (PBS, pH 7.4) for 3 times.

[0071] 2. Induced release of Salmonella enterica outer membrane vesicles (OMVs):

[0072] The washed bacterial cells were resuspended in PBS solution (pH 8.0) containing 0.5% (w / v) deoxycholic acid sodium (DOC) and incubated at 37°C with 150 rpm shaking for 40 min to induce the release of OMVs.

[0073] 3. Crude extract preparation:

[0074] The mixture was centrifuged (4°C, 10000 x g, 20 min) to remove intact bacterial cells and large particle impurities, and the supernatant was collected.

[0075] 4. Ultrafiltration concentration:

[0076] The supernatant was concentrated using a 100 kDa tangential flow ultrafiltration system until the volume was reduced to 1 / 10 of the original solution, and then filtered through a 0.22 μm sterile filter to remove residual impurities.

[0077] 5. Gradient centrifugation purification:

[0078] The filtrate was loaded into a pre-prepared sucrose density gradient solution with a sucrose density gradient of 20% w / v ~ 50% w / v, the upper layer, and ultracentrifuged at 4°C, 100000 x g, for 2 hours. The milky white OMVs layer at the 20% ~ 30% sucrose interface was collected.

[0079] 6. Detergent removal:

[0080] The OMVs suspension was placed in a dialysis bag with a molecular weight cutoff of 100 kDa and dialyzed in PBS buffer (pH 7.4) at 4°C for 24 hours, with the dialysis solution being replaced every 6 hours to remove residual sucrose and deoxycholic acid sodium, to obtain Salmonella enterica outer membrane vesicles (OMVs).

[0081] 7. Final product verification:

[0082] ​The morphology integrity of OMVs was observed by transmission electron microscopy (TEM); the results are shown in Figure 4 Figure 1, the extract has a shape like a tea tray, has typical outer membrane vesicle characteristics, and has a clear lipid bilayer membrane (derived from the outer membrane of bacteria).

[0083] The particle size distribution was 50nm-100nm detected by dynamic light scattering (DLS); the results are shown in Figure 2 Figure 2, the particle size distribution is 124.4nm, and the concentration range is 1.9E+7 particles / mL.

[0084] The protein concentration was determined by BCA method, and the content of lipopolysaccharide (LPS) was controlled by limulus reagent method ≤1EU / μg.

[0085] Example 2

[0086] A combined drug for treating colitis, wherein the combined drug is composed of Coptis exosome and Salmonella outer membrane vesicle according to a volume ratio of 1:2; the concentration of Coptis exosome and Salmonella outer membrane vesicle is 9mg / mL.

[0087] The extraction method of Coptis exosome is as follows:

[0088] The Coptis was cleaned by 28kHz ultrasonic wave for 4min, washed with ultrapure water for 2 times, and soaked in water for 19min;

[0089] The soaked Coptis was cut into Coptis fragments with a length of 1.4cm;

[0090] The Coptis fragments were squeezed for 4min at a speed of 35000r / min, and the Coptis juice was collected;

[0091] The Coptis juice was centrifuged at a low speed of 5000r / min at 3℃ for 19min, and the supernatant I was collected; the supernatant I was centrifuged at a high speed of 10000r / min for 29min, and the supernatant II was collected; the supernatant II was filtered through a filter with a pore size of 0.44μm, and the filtrate was retained;

[0092] After adjusting the pH of the filtrate to 6.9, the filtrate was centrifuged at 10000r / min for 29min at 3℃, and the supernatant III was collected; the supernatant III was sequentially filtered through filters with pore sizes of 0.44μm and 0.21μm, and the final filtrate was retained. The remaining steps are exactly the same as the extraction method of Coptis exosome in Example 1.

[0093] The extraction method of Salmonella outer membrane vesicle is as follows:

[0094] The content of sodium deoxycholate in PBS solution is 0.3%w / v.

[0095] The filtrate was added to the upper layer of sucrose density gradient solution and ultracentrifuged at 80000xg for 2 hours at 3°C;

[0096] At 3°C, the Salmonella outer membrane vesicle suspension was dialyzed in PBS buffer for 24 hours. The remaining steps were exactly the same as the extraction method of Salmonella outer membrane vesicles in Example 1.

[0097] Example 3

[0098] A combined drug for treating colitis, wherein the combined drug is composed of Coptis exosomes and Salmonella outer membrane vesicles in a volume ratio of 1:3; the concentration of Coptis exosomes and Salmonella outer membrane vesicles is both 11 mg / mL.

[0099] The Coptis was cleaned with 28 kHz ultrasonic wave for 6 min, washed with ultrapure water for 4 times, and soaked in water for 21 min;

[0100] The soaked Coptis was cut into Coptis fragments with a length of 1.6 cm;

[0101] The Coptis fragments were squeezed for 6 min at a speed of 35000 r / min, and the Coptis juice was collected;

[0102] The Coptis juice was centrifuged at a low speed of 5000 r / min for 21 min at 5°C, and the supernatant I was collected; the supernatant I was centrifuged at a high speed of 10000 r / min for 31 min, and the supernatant II was collected; the supernatant II was filtered through a filter with a pore size of 0.46 μm, and the filtrate was retained;

[0103] After adjusting the pH of the filtrate to 7.1 at 5°C, the filtrate was centrifuged at 10000 r / min for 31 min, and the supernatant III was collected; the supernatant III was fractionally filtered through filters with pore sizes of 0.46 μm and 0.23 μm in turn, and the final filtrate was retained. The remaining steps were exactly the same as in Example 1.

[0104] The extraction method of Salmonella outer membrane vesicles is as follows:

[0105] The content of sodium deoxycholate in the PBS solution was 0.7% w / v.

[0106] The filtrate was added to the upper layer of sucrose density gradient solution and ultracentrifuged at 120000xg for 2 hours at 5°C.

[0107] At 5°C, the Salmonella outer membrane vesicle suspension was dialyzed in PBS buffer for 24 hours. The remaining steps were exactly the same as in Example 1.

[0108] The yield of OMVs extracted by the above method was 15 mg-20 mg protein / L of culture solution, the vesicle integrity was ≥90%, and the particle size distribution was concentrated (120±25 nm), which was suitable for the development of vaccine adjuvant or drug delivery system.

[0109] Experimental Example 1: Effect verification of combined use of Coptis exosome and Salmonella outer membrane vesicle in the treatment of colitis

[0110] The following experiments were performed using the combined medication of Example 1 as an example.

[0111] The standardized experimental procedure of the colitis model was induced by Dextran Sulfate Sodium (DSS) using Balb / c mice.

[0112] I. Preparation of experimental materials

[0113] 1. Experimental animals: 6-week-old Balb / c female mice were adaptively fed in a SPF-level animal laboratory for 7 days, free to eat and drink, and the environmental temperature was controlled at 22°C, with a light cycle of 12 hours light / 12 hours dark.

[0114] 2. Preparation of DSS solution

[0115] Dextran Sulfate Sodium (DSS) powder was dissolved in sterile drinking water, stirred until completely dissolved, and a 3% (w / v) DSS aqueous solution was prepared, 3g diluted in 100mL water, and used immediately after preparation.

[0116] 3. Experimental grouping and administration

[0117] The mice were randomly divided into a control group, a DSS model group, a Coptis exosome treatment group, a Salmonella outer membrane vesicle treatment group, and a combined treatment group, 6 mice in each group:

[0118] Control group: continuously given normal drinking water.

[0119] DSS model group: each mouse was given 200μL PBS by gavage.

[0120] Coptis exosome treatment group: each mouse was given 200μL Coptis exosome by gavage, with a concentration of 10mg / mL.

[0121] Salmonella outer membrane vesicle treatment group: each mouse was given 200μL Salmonella outer membrane vesicle by gavage, with a concentration of 10mg / mL.

[0122] Combined treatment group: each mouse was given 66.7μL Coptis exosome + 133.3μL Salmonella outer membrane vesicle (volume ratio 1:2) by gavage, both with a concentration of 10mg / mL;

[0123] All drugs were diluted with PBS, and the gavage time was unified at 9:00 am.

[0124] II. Experimental procedure

[0125] 1. Adaptation period: Mice were purchased and adapted in SPF environment for 1 week, free access to food and water.

[0126] 2. DSS induction phase

[0127] DSS preparation: DSS powder was dissolved in sterile drinking water, stirred to complete dissolution under light protection (3g diluted in 100mL water, prepared fresh).

[0128] Dosing period: 3% DSS solution (mass / volume) was administered continuously for 7 days.

[0129] Monitoring indicators: Body weight was recorded daily, and the experiment was terminated if body weight loss was ≥20% (ethical requirement).

[0130] Disease activity index (DAI): Scored based on body weight loss, stool consistency (diarrhea / bloody stool), and activity status (0-4 points).

[0131] Food and water intake: Ensure stable DSS intake.

[0132] 3. Sample collection and processing

[0133] Euthanasia: Cervical dislocation.

[0134] Colonic collection: The colon was dissected out, and its length was measured (shortened in inflammation). The colon was opened longitudinally, and the contents were flushed with saline.

[0135] Pathological section: Paraffin-embedded after 24 hours of fixation with 4% paraformaldehyde, and H&E staining was used to evaluate inflammation and pathological damage.

[0136] Molecular biology: Frozen in liquid nitrogen and stored at -80°C (for qPCR, ELISA, etc. to detect inflammatory factors).

[0137] (DAI scoring criteria (0-4 points, comprehensive evaluation of the following indicators):

[0138] Body weight loss: 0 (none), 1 (1-5%), 2 (5-10%), 3 (10-15%), 4 (>15%).

[0139] Stool consistency: 0 (normal), 1 (soft stool), 2 (diarrhea), 3 (mucus stool), 4 (bloody stool).

[0140] Degree of blood in stool: 0 (none), 1 (occult blood), 2 (gross blood), 3 (severe bleeding)).

[0141] 4. Inflammatory factor gene and intestinal protein gene detection:

[0142] qPCR detection of mRNA expression:

[0143] The colon tissue was ground in liquid nitrogen, total RNA was extracted by TRIzol method, and reverse transcribed into cDNA.

[0144] Primer design:

[0145] The upstream primer sequence (F) of tumor necrosis factor alpha (TNF-α) is shown in SEQ ID NO. 1, which is 5'-CAGAGGGAAGAGTTCCCCAG-3'; the downstream primer sequence (R) is shown in SEQ ID NO. 2, which is 5'-CCTTGGTCTGGTAGGAGACG-3'.

[0146] The upstream primer sequence (F) of interleukin 6 (IL-6) is shown in SEQ ID NO. 3, which is F: 5'-ACTCACCTCTTCAGAACGAATTG-3'; the downstream primer sequence (R) is shown in SEQ ID NO. 4, which is R: 5'-CCATCTTTGGAAGGTTCAGGTTG-3'.

[0147] The upstream primer sequence (F) of interleukin 1 beta (IL-1β) is shown in SEQ ID NO. 5, which is F: 5'-TCCAGGATGAGGACCCAAGC-3'; the downstream primer sequence (R) is shown in SEQ ID NO. 6, which is R: 5'-GAACGTCACACACCAGCAGGTTA-3'.

[0148] The upstream primer sequence (F) of the reference (β-actin) is shown in SEQ ID NO. 7, which is F: 5'-CATCCGTAAAGACCTCTATGCCAAC-3'; the downstream primer sequence (R) is shown in SEQ ID NO. 8, which is R: 5'-ATGGAGCCACCGATCCACA-3'.

[0149] The upstream primer sequence (F) of the closed connection protein (Claudin-1) is shown in SEQ ID NO. 9, which is F: 5'-CCTGGGACTTCATCGCTGTG-3'; the downstream primer sequence (R) is shown in SEQ ID NO. 10, which is R: 5'-TGGACAGCCCATTCACATCC-3'.

[0150] The upstream primer sequence (F) of the closed connection protein (Occludin) is shown in SEQ ID NO. 11, which is F: 5'-TTCTGCTGCCTGGAGACGAT-3'; the downstream primer sequence (R) is shown in SEQ ID NO. 12, which is R: 5'-GCTGCTGGTACCGTTGTCTT-3'.

[0151] By 2 −ΔΔCt Method to calculate the relative expression of genes.

[0152] 5. Inflammatory factor protein detection:

[0153] ELISA detection of protein expression: Take serum, use ELISA method to detect the level of IL-6, IL-1β and TNF-α cytokines in mouse serum, and the specific steps are operated according to the kit instructions. Use the enzyme label instrument to read the absorbance value at wavelength of 450 nm.

[0154] 1. Sample addition: Set standard holes, sample holes and blank holes. Set 7 standard holes, add 100 μL of different concentrations of standard samples in turn. Add 100 μL of standard sample diluent to the blank hole, and add 100 μL of sample to be tested to the remaining holes. Cover the enzyme label plate with film and incubate at 37°C for 1 hour.

[0155] 2. Discard the liquid and spin dry without washing.

[0156] 3. Add 100 μL of detection solution A working solution to each hole, cover the enzyme label plate with film, and incubate at 37°C for 1 hour.

[0157] 4. Discard the liquid in the hole, wash each hole with 350 μL of washing solution, soak for 2 minutes, and gently pat the enzyme label plate on the absorbent paper to remove all the liquid in the hole. Repeat the plate washing 3 times. After the last washing, absorb or pour out the remaining washing buffer, and invert the enzyme label plate on the absorbent paper to completely absorb the liquid remaining in the hole.

[0158] 5. Add 100 μL of detection solution B working solution to each hole, cover the enzyme label plate with film, and incubate at 37°C for 30 minutes.

[0159] 6. Discard the liquid in the hole, spin dry, and wash the plate 5 times as in step 4.

[0160] 7. Add 90 μL of TMB substrate solution to each hole, cover the enzyme label plate with film, and develop color at 37°C in the dark (reaction time 20 minutes).

[0161] 8. Add 50 μL of termination solution to each hole to terminate the reaction.

[0162] 9. Immediately measure the optical density value (OD value) of each hole with the enzyme label instrument at 450 nm wavelength.

[0163] 2. Experimental results

[0164] 1. Body weight change and pathological score as shown in Figure 3 and Figure 4 .

[0165] The model group mice showed significant weight loss from the 2nd day, and the trend was downward all the time, which indicated that the acute colitis model was successfully established. The total decrease was more than 18% until the end of the experimentp <0.01), and the pathological score reached 2±0.1 ( p <0.01).

[0166] The body weight of the Coptis exosome treatment group decreased by 9% ( p <0.01), and the pathological score decreased to 0.9±0.1 ( p <0.01), indicating that it has certain anti-inflammatory and metabolic regulation effects, which may be related to the rich active ingredients of berberine.

[0167] The body weight of the Salmonella outer membrane vesicle treatment group decreased by 11% ( p <0.01), and the pathological score was 0.6±0.1 ( p <0.01), indicating its immunomodulatory potential, or related to the outer membrane protein of Salmonella outer membrane vesicles regulating innate immune tolerance.

[0168] The combined treatment group had the most significant effect, with a body weight loss rate of only 4% ( p <0.01), which was significantly lower than other groups, and the pathological score was only 0.2±0.1 ( p <0.01), significantly better than the single treatment group, verifying the synergistic therapeutic effect of the combination of the two.

[0169] 2. Colon length and histopathological analysis

[0170] The colon of the model group was significantly shortened to 6±0.5 cm ( p <0.01), the crypt structure was severely damaged, accompanied by a large number of neutrophil infiltration, and the pathological score was the highest at the end, reaching 2±0.1 ( p <0.01), which was consistent with the characteristics of ulcerative colitis.

[0171] The colon length of the Coptis exosome treatment group recovered to 7±0.2 cm ( p <0.01), and the pathological score decreased to 0.9±0.1 ( p <0.01), and the number of goblet cells recovered to 65% of the control group ( p <0.01).

[0172] The colon length of the Salmonella outer membrane vesicle treatment group recovered to 7±0.1 cm ( p <0.01), which was similar to the colon length of the Coptis exosome treatment group, and the pathological score was 0.6±0.1 ( p <0.01). Histology showed reduced inflammatory cell infiltration and partial regeneration of crypt structure.

[0173] The colon length of the combined treatment group reached 7.8±0.1 cm ( p <0.01), and the pathological score decreased to 0.2±0.1 ( p<0.01), crypt structure was basically restored to normal, and the number of goblet cells was restored to 85% of the control group p <0.01). It is shown that a complementary mechanism between exosomes and vesicles may be formed to promote intestinal tissue repair. As Figure 5 shown.

[0174] 3. Combined evaluation of body weight and pathological changes

[0175] The experimental data shown in Tables 1 and 2 clearly support that the combined treatment group is superior to the single treatment group in terms of body weight maintenance, inflammation suppression, tissue repair, etc., showing a synergistic mechanism between Coptis exosomes and Salmonella outer membrane vesicles, which may include the following aspects: inhibition of the expression of TNF-a, IL-6 and other pro-inflammatory factors; exosomes inhibit apoptosis and enhance intestinal barrier; Salmonella outer membrane vesicles activate moderate immune tolerance and promote mucosal regeneration; and combined promotion of goblet cell function and MUC2 synthesis.

[0176] Table 1 Body weight change and pathological score

[0177]

[0178] Table 2 Colon length and histopathological analysis

[0179]

[0180] 4. Combined treatment group improves intestinal barrier integrity

[0181] The qPCR detection results show that the expression of claudin gene and occludin gene in the colonitis group is significantly lower than that in the control group; the expression of claudin gene and occludin gene in the Coptis exosome treatment group and the Salmonella outer membrane vesicle treatment group is significantly improved; and the expression of claudin gene and occludin gene in the combined treatment group is significantly higher than that in the colonitis model group, the Coptis exosome treatment group and the Salmonella outer membrane vesicle treatment group. The above results show that the combined treatment group has the best effect, and improves the intestinal barrier integrity by improving the expression of claudin gene and occludin gene. As Figure 6 shown.

[0182] 5. Combined treatment group reduces the level of inflammatory factors

[0183] (1) qPCR detection results showed that compared with the control group, the tumor necrosis factor alpha gene, interleukin 6 protein gene and interleukin 1 beta protein gene of the colonitis group were significantly increased; the tumor necrosis factor alpha gene, interleukin 6 protein gene and interleukin 1 beta protein gene of the Huanglian exosome treatment group and the Salmonella outer membrane vesicle treatment group were significantly reduced; the tumor necrosis factor alpha gene, interleukin 6 protein gene and interleukin 1 beta protein gene of the combined treatment group were significantly lower than those of the colonitis model group, the Huanglian exosome treatment group and the Salmonella outer membrane vesicle treatment group. The above results show that the combined treatment group has the best effect, and reduces the intestinal inflammatory response by reducing the tumor necrosis factor alpha gene, interleukin 6 protein gene and interleukin 1 beta protein gene. Figure 7

[0184] In summary, the expression of inflammation-related factors in the colonitis model group of mice was significantly increased, disrupting the homeostasis of inflammation regulation; the Huanglian exosome treatment group and the Salmonella outer membrane vesicle treatment group can significantly reduce the inflammatory factors; and the combined treatment group has the best effect, which is significantly better than the effect of single treatment.

[0185] Experimental Example 2, Verification of the Synergistic Mechanism of Huanglian Exosomes and Salmonella Outer Membrane Vesicles at the Cell Level

[0186] Establishment and treatment of cell model (cell culture, Western blot, Elisa, qPCR).

[0187] 3. Experimental method

[0188] Caco-2 cells were cultured in DMEM medium containing 20% fetal bovine serum (FBS) at 37°C, 5% CO2. 22 in a cell incubator.

[0189] Caco-2 cells were seeded in a 6-well plate at a density of 2x10 5 cells per well, and cultured at 37°C, 5% CO2 until adherent (about 48 hours).

[0190] ​Discard the old culture medium, add LPS (Biyun Tian) to the fresh complete culture medium to make its final concentration 50 μg / mL, set 3 replicate wells for each group, continue to culture for 24 hours. Then add different solutions according to the following grouping for 24 hours, collect the supernatant and cell protein for subsequent detection.

[0191] Control group: add 50 μL PBS solution to each well (without adding LPS).

[0192] Inflammation model group: add 50 μL LPS with a concentration of 2 mg / mL to each well to make its final concentration 50 μg / mL;

[0193] Coptis exosome treatment group: add 50 μL Coptis exosome with a concentration of 0.4 mg / mL to each well to make its final concentration 10 μg / mL;

[0194] Salmonella outer membrane vesicle treatment group: add 50 μL Salmonella outer membrane vesicle with a concentration of 0.4 mg / mL to each well to make its final concentration 10 mg / mL;

[0195] Combined treatment group: add 16.7 μL Coptis exosome with a concentration of 0.4 mg / mL and 33.3 μL Salmonella outer membrane vesicle with a concentration of 0.4 mg / mL to each well, with a volume ratio of 1:2.

[0196] 1. Antioxidant detection (kit method)

[0197] Three detection kits (Bi Yun Tian) of Bi Yun Tian were used, and the information is as follows: total SOD activity detection kit (WST-8 method), lipid oxidation (MDA) detection kit, GSH and GSSG detection kit, and detection was carried out according to the instruction steps.

[0198] 2. Antioxidant detection (Western blot method)

[0199] Collect cells, use protein extraction kit (protein extraction steps), Western blot test steps

[0200] Cell treatment:

[0201] Aspirate the culture solution, gently wash the cells with pre-cooled PBS for 2 times, and aspirate the residual liquid completely.

[0202] Add an appropriate amount of lysis solution, such as 200 μL per well of a 6-well plate, and incubate on ice for 20 minutes.

[0203] Scrape the cells with a cell scraper, and transfer the lysis solution to a pre-cooled centrifuge tube.

[0204] Centrifuge at 12000 rpm for 15 minutes at 4°C. Aspirate the supernatant (containing total protein) to a new centrifuge tube, avoiding aspiration of the precipitate.

[0205] Immediately proceed with BCA quantitative detection, standard preparation: BSA standard was diluted according to the gradient of 0 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL.

[0206] Mix according to the volume ratio of reagent A: reagent B = 50:1 (such as 5 mL of reagent A + 100 μL of reagent B).

[0207] Add 25 μL of standard or sample to be tested to each well (dilute appropriately to the linear range).

[0208] Add 200 μL of BCA working solution to each well, and incubate at 37°C for 30 minutes.

[0209] Determine the absorbance (OD value) at 562 nm using an enzyme-labeled instrument.

[0210] Draw a standard curve and calculate the protein concentration of the sample (formula: concentration = standard curve corresponding value x dilution multiple).

[0211] The whole operation is carried out on ice to prevent protein degradation.

[0212] Mix the sample with SDS-PAGE loading buffer and boil. Perform electrophoretic separation and transfer to membrane. Block the PVDF membrane in Tris buffer saline containing 0.5% Tween-20 (TBST) and 10% skim milk powder by mass fraction at room temperature for 2 hours, then incubate in blocking solution containing anti-SOD (1:2000) he and MDA (1:2000) at 4°C overnight. Wash the membrane with TBST 4 times, each for 8 min, incubate with the corresponding HRP-conjugated secondary antibody (1:5000) for 2 hours, and repeat the membrane washing. Visualize the signal using an ECL chemiluminescence kit, and image by a gel imaging system, and finally analyze the gray value.

[0213] II. Experimental results

[0214] 1. The combination therapy group reduces oxidative stress

[0215] (1) The combination therapy group reduces superoxide dismutase (SOD) activity: SOD is a cell that scavenges superoxide anion free radicals (O2 -SOD is a key antioxidant enzyme that mainly maintains cellular redox homeostasis. The experimental results show that the SOD activity of the inflammation model group is significantly decreased, indicating that its antioxidant defense system is inhibited, leading to the continuous accumulation of reactive oxygen species (ROS) in cells, making the cells in an oxidative stress level. The SOD activity of the Huanglian exosome treatment group and the Salmonella outer membrane vesicle treatment group can be significantly reduced; the SOD activity of the combined treatment group is significantly lower than that of the inflammation model group, the Huanglian exosome treatment group and the Salmonella outer membrane vesicle treatment group. The above results show that the combined treatment group has the best effect, which can alleviate the oxidative stress level of cells by reducing the SOD activity. As shown in Figure 8 and Figure 9 .

[0216] (2) The combined treatment group increases the content of malondialdehyde (MDA): MDA is one of the end products of lipid peroxidation and is widely used to reflect the degree of oxidative damage to the lipid components of the cell membrane. In this experiment, the MDA level of the inflammation model group is significantly increased, indicating that the lipid peroxidation reaction is enhanced, the stability of the cell membrane structure is threatened, and membrane permeability changes and cell dysfunction may be induced. The MDA activity of the Huanglian exosome treatment group and the Salmonella outer membrane vesicle treatment group can be significantly increased; the MDA activity of the combined treatment group is significantly higher than that of the inflammation model group, the Huanglian exosome treatment group and the Salmonella outer membrane vesicle treatment group. The above results show that the combined treatment group has the best effect, which can alleviate the oxidative stress level of cells by increasing the MDA activity. As shown in Figure 8 and Figure 9 .

[0217] (3) The combined treatment group reduces the glutathione (GSH / GSSG) ratio: glutathione is one of the most important reduced antioxidants in cells, which can remove free radicals directly or participate in various redox reactions as a reducing agent. The results show that the GSH / GSSG ratio of the inflammation model group is significantly reduced, reflecting that a large amount of GSH in the cell is oxidized to oxidized glutathione, indicating that the cell is in a significant oxidative stress state. The GSH / GSSG ratio of the Huanglian exosome treatment group and the Salmonella outer membrane vesicle treatment group can be significantly reduced; the glutathione activity of the combined treatment group is significantly lower than that of the inflammation model group, the Huanglian exosome treatment group and the Salmonella outer membrane vesicle treatment group. The above results show that the combined treatment group has the best effect, which can alleviate the oxidative stress level of cells by reducing the GSH / GSSG ratio. As shown in Figure 8 .

[0218] In summary, the combined treatment group has the best effect and can alleviate the oxidative stress level and reduce the inflammatory response.

[0219] Any combination of the technical features in the above-described embodiments can be made, and for the sake of brevity, not all possible combinations are described, however, as long as the combination of the technical features does not exist in contradiction, it shall be considered within the scope of the present disclosure.

[0220] The above-described embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but it shall not be understood as a limitation on the patent scope of the present application. For ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and these shall all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

Claims

1. A combination drug for treating colitis, characterized in that, The volume ratio of the Huanglian exosome and the Salmonella outer membrane vesicle in the combined medicament is 1-2:1-3; the concentration of the Huanglian exosome and the Salmonella outer membrane vesicle is 9-11 mg / mL.

2. The combination of claim 1, wherein The volume ratio of the Huanglian exosome and the Salmonella outer membrane vesicle in the combined medicament is 1:2; the mass concentration of the Huanglian exosome and the Salmonella outer membrane vesicle is 10 mg / mL.

3. The combination of claim 1 or 2, wherein The Huanglian exosome is obtained by the following steps: The Huanglian is washed and soaked in water for 19-21 min; The soaked Huanglian is squeezed to obtain Huanglian juice; The Huanglian juice is centrifuged at 5000 r / min at 3-5 ℃ for 19-21 min to obtain supernatant I; the supernatant I is centrifuged at 10000 r / min for 29-31 min to obtain supernatant II; the supernatant II is filtered to obtain a filtrate; The filtrate is adjusted to pH 6.9-7.1 and centrifuged at 10000 r / min at 3-5 ℃ for 29-31 min to obtain supernatant III; the supernatant III is filtered by filters with pore sizes of 0.44-0.46 μm and 0.21-0.23 μm in sequence to obtain a final filtrate; the final filtrate is separated and purified to obtain the Huanglian exosome.

4. The combination of claim 1 or 2, wherein The Salmonella outer membrane vesicle is obtained by the following steps: The Salmonella is expanded and cultured, and the bacterial cells are collected by centrifugation; the bacterial cells are resuspended in a PBS solution containing sodium deoxycholate to obtain a mixed solution; The mixed solution is centrifuged to obtain supernatant; the supernatant is concentrated to a volume of 1 / 10 of the original volume and filtered by a sterile filter to obtain a filtrate; The filtrate is added to the upper layer of a sucrose density gradient solution, and ultracentrifuged at 80000-120000 g at 3-5 ℃ to obtain a Salmonella outer membrane vesicle layer at the interface of 20-30% w / v sucrose; the sucrose density gradient is 20-50% w / v; The Salmonella outer membrane vesicle suspension is dialyzed in a PBS buffer at 3-5 ℃, and the dialysis solution is replaced during the dialysis to obtain the Salmonella outer membrane vesicle.

5. The combination of claim 4, wherein The content of the sodium deoxycholate in the PBS solution is 0.3-0.7% w / v.

6. The combination of claim 4, wherein The molecular weight cut-off of the dialysis is 100 kDa.

7. The combination of any one of claims 1 to 6, wherein The combined medicament can be prepared into a clinically acceptable preparation form together with or separately from a pharmaceutically acceptable excipient.

8. The combination of claim 7, wherein the combination is administered simultaneously. The pharmaceutically acceptable excipient is one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickening agent, an emulsifying agent, a preservative and a stabilizer.

9. The combination of claim 7, wherein The dosage form of the combined medicament is an oral preparation, an infusion or an injection.

10. Use of the combined medicament according to any one of claims 1-9 in the preparation of a preparation for treating colitis.

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