Medicine for treating ulcerative colitis

Abstract

The invention discloses a medicine for treating ulcerative colitis, and belongs to the technical field of medicines. The preparation method of the medicine comprises the following steps: taking 3-7mg of BSA, adding 6-10mg of BBR and 1-3mg of DHCl, preparing 4-6ml of pure water and DMSO according to a ratio of (3-5): (0.5-1.5), carrying out heat treatment at 55-65 DEG C and stirring for 20-28h, carrying out centrifugal separation at 28-32 DEG C to precipitate BD / PA, carrying out freeze-drying, adding 0.5-1.5 ml of SA with the concentration of 1-3%, uniformly mixing, taking a CaCl2 solution saturated at room temperature as a coagulating bath, and carrying out freeze-drying to obtain the medicine. And preparing the medicine BD / PA-SA for treating ulcerative colitis. Based on the guidance of the'monarch-minister-assistant-guide 'medication theory of traditional Chinese medicine, a carrier is constructed through a drug delivery technology, and the bioavailability of the main effective components BBR and DHCl of the Xianglian pill is improved.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a medicine for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a chronic, relapsing inflammatory bowel disease, which is listed as one of the modern intractable diseases by the World Health Organization. Modern medicine mainly treats UC through oral administration because of its convenience, economy, and good patient compliance. Currently, 5-aminosalicylic acid (5-ASA), corticosteroids, immunosuppressants, and biological agents are commonly used drugs for the treatment of UC. However, on the one hand, these clinically approved drugs have many adverse reactions, such as allergies and gastrointestinal symptoms. On the other hand, they usually cannot directly address the key pathophysiological links in UC, namely, damage to the intestinal mucosal barrier and dysbiosis of intestinal symbiotic microorganisms. Therefore, it is still worthwhile to continue to search for and develop new drugs for the treatment of UC.

[0003] In China, Chinese medicine usually uses oral administration to treat UC. By exploring the Dictionary of Chinese Medicine Formulas, the century-old classic prescription Xianglian Pills for treating UC-related symptoms was screened out. It is composed of Coptis chinensis and Aucklandia lappa in a 4:1 ratio. Modern pharmacological research and clinical application have confirmed the efficacy of Xianglian Pills in treating UC. However, the traditional Xianglian Pills have many problems such as complex ingredients, low content of effective substances, poor utilization rate, and large dosage, which limits its widespread application. Making good use of the main natural bioactive compounds (BBR and DHCL) in Xianglian Pills to improve the therapeutic dosage form is a promising solution. Summary of the invention

[0004] One of the purposes of the present invention is to provide a drug for treating ulcerative colitis. The preparation method of the drug is as follows: take 3-7 mg of BSA, add 6-10 mg of BBR and 1-3 mg of DHCL, prepare 4-6 ml of pure water and DMSO, the ratio of pure water to DMSO is 3-5:0.5-1.5, heat treat at 55-65°C and stir for 20-28 hours, after the end, centrifuge and separate the drug-loaded protein aggregates at 28-32°C to precipitate BD / PA, add 0.5-1.5 ml of 1-3% concentration SA after freeze-drying, mix evenly under ultrasound, and then use a saturated CaCl2 solution at room temperature as a coagulation bath to prepare microspheres BD / PA@SA using electrostatic droplet technology, and BD / PA@SA is a drug for treating ulcerative colitis.

[0005] Preferably, the amount of BSA is 5 mg.

[0006] More preferably, the amount of BBR is 8 mg.

[0007] More preferably, the dosage of DHCL is 2 mg.

[0008] More preferably, the ratio of pure water to DMSO is 4:1.

[0009] More preferably, the total amount of pure water and DMSO is 5 ml.

[0010] More preferably, the heat treatment temperature is 60° C. and the stirring time is 24 h.

[0011] More preferably, the concentration of SA is 2% and the volume is 1 ml.

[0012] The second object of the present invention is to provide the use of the above-mentioned drug in the preparation of a product for treating ulcerative colitis.

[0013] The delivery carrier materials in the present invention are all derived from natural ingredients. Albumin in animals (such as bovine serum albumin BSA) has the advantages of good biocompatibility, hydrophobic drug complex, processing plasticity, etc. in drug delivery. In order to reduce the addition of non-food-grade foreign substances and use simple, efficient and low-cost preparation methods as much as possible, it is planned to prepare drug-loaded protein aggregates (PA) through the technology of protein drug co-assembly under heat treatment, relying on the nano-micron scale of PA to target the ulcer lesions of UC to improve drug utilization. In addition, it is necessary to add a layer of shell that is free from digestion by gastric and small intestinal proteases to protect the drug-loaded protein core from reaching the colon lesions directly to play a role. It is planned to use natural polysaccharides with colon bacteria degradability, that is, sodium alginate (SA), a pharmaceutical excipient that has passed FDA certification, to complete this treatment system on the basis of fully ensuring safety.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention is based on the guidance of the "monarch-minister-adjuvant-envoy" medication theory of traditional Chinese medicine, with BBR (berberine, the main component of Coptis chinensis) as the monarch drug, DHCL (dehydrocostus lactone, the main component of costus root) as the minister drug, and constructs carriers (PA and SA microspheres) through drug delivery technology as adjuvant and minister drugs in the treatment system to overcome the shortcomings of Xianglian Pills in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1The characterization results of protein aggregates (PA) in Example 1, (A) Schematic diagram of BSA forming PA. (B) Transmission electron microscopy (TEM) and atomic force microscopy (AFM) images of BSA before and after heat treatment, scale, 10μm (TEM) and 2μm (AFM). (CD) Zeta potential (C) and fluorescence emission wavelength change (D) of BSA before and after heat treatment. (EF) Changes in fluorescence emission wavelength after mixing fluorescent dyes ANS (E) and ThT (F) with BSA at different heat treatment time points. (GH) Circular dichroism spectra of BSA at different heat treatment time points (G) and changes in protein secondary structure ratio (H).

[0017] Figure 2 The formation kinetics of PA in Example 1 are observed. (A) Absorbance of BSA solution at 630 nm at different heat treatment time points. (B) Size distribution of BSA. (C) Size distribution of PA. (D) Optical microscopy and AFM observation of PA formation during heat treatment. Scale bars, 100 μm (optical microscopy BF mode) and 4 μm (AFM).

[0018] Figure 3 The synthesis and characterization results of BD / PA and BD / PA@SA in Example 1, (A) TEM observation of the morphology of PA and drug-loaded protein aggregates (BD / PA), scale bar, 10μm. (B) Size distribution of BD / PA. (C) Shift of the drug fluorescence emission spectrum after binding to PA. (D) Localization of fluorescent dye DiD on FITC-labeled PA. (E) Drug loading and encapsulation efficiency of BD / PA. (F) Light microscopy observation of content-free microspheres (@SA) and drug-loaded protein aggregate microspheres (BD / PA@SA), scale bar, 500μm (magnification 40x) and 100μm (magnification 100x). (G) Size distribution of @SA and BD / PA@SA. (H) Total drug release curves in BD / PA and BD / PA@SA preparations. (I) Release percentage of total drugs in BD / PA and BD / PA@SA preparations when they are subjected to different digestive fluids.

[0019] Figure 4 The morphology of BD / PA and the changes of PA after heat treatment of BSA and different components in Example 1. (A) Light microscopic observation of the solution mixed with BSA and drug BD (BBR and DHCL) before and after heat treatment, scale bar, 100μm. (B) AFM observation of the morphology of PA and calculation of roughness value after heat treatment with or without BSA, scale bar, 2μm (Oligomer) and 4μm (Fibril). (C) Zeta potential measurement of BD, BD / PA and BD+BSA. (D) Light microscopic observation of BSA after heat treatment of different components for 12h, scale bar, 100μm. (E) The yield of PA and BD / PA after heat treatment for 24h.

[0020] Figure 5 These are the fluorescence spectra of the components related to BD / PA in Example 1, including the fluorescence emission and excitation wavelengths of (A) BBR, (B) DHCL, (C) BSA, (D) PA, (E) BD, (F) BD / PA, (G) BD+PA, and (H) BD+BSA.

[0021] Figure 6 Results of the morphology of SA microspheres and the release behavior of two drugs in Example 1. (A) Scanning electron microscope (SEM) images of @SA and BD / PA@SA after freeze-drying. (B) Drug loading and encapsulation efficiency of BD / PA@SA. (CD) Drug release curves of BBR (C) and DHCL (D) and the release ratios after different digestive fluids.

[0022] Figure 7 The targeting and retention of BD / PA@SA in the mouse colon in Example 1. (A) In vitro organ imaging at 24h, 48h and 168h to test the colon targeting and retention of BD / PA@SA. (B) Quantification of colon Cy5.5 fluorescence signal at 24h, 48h and 168h. (C) Deposition of Cy5.5-labeled BD / PA in colon ulcers at 24h, scale bar, 200μm.

[0023] Figure 8 The deposition results of Cy5.5-labeled BD / PA in other major organs in Example 1. In vitro organ imaging at 24h, 48h and 168h confirmed that Cy5.5-labeled BD / PA did not appear in other major organs (a. heart, b. liver, c. kidney, d. lung, e. spleen, f. testis).

[0024] Fig. 9 The results of the efficacy of BD / PA@SA in relieving UC in mice in Example 1. (A) Schematic diagram of mouse UC model construction and treatment. (B) Body weight change. (C) Colon length photo. (D) Colon length statistics. (E) Colon HE staining, scale bar, 50 μm (lower row) and 100 μm (upper row). (F) Colon MPO level. (G) Colon inflammatory factor level. (HI) Colon CD86 and CD206 immunohistochemical staining (H) and quantification (I).

[0025] Fig.10 The colon injury scoring results of BD / PA@SA in alleviating UC in mice in Example 1.

[0026] Fig.11The results of the efficacy of BD / PA@SA in the delayed treatment model of UC in Example 1. (A) Construction of the delayed treatment model of UC in mice and a schematic diagram of the treatment. (B) Body weight change. (C) Photos of colon length. (D) Colon length statistics. (E) Colon HE staining, scale bar, 50 μm (lower row) and 100 μm (upper row).

[0027] Fig.12 The tissue sections of the colon mucosa of UC mice repaired by BD / PA@SA in Example 1, (A) Immunofluorescence staining of colon tight junction proteins Occludin-1 and ZO-1, scale bar, 100 μm. (BD) Colon PAS staining (B), Muc-2 protein immunohistochemical staining (C) and Ki67 immunohistochemical staining (D), scale bar, 300 μm.

[0028] Fig.13 Figure 1 shows the situation of BD / PA@SA regulating intestinal flora in Example 1. (AC) Sobs (A), Ace (B) and Shannon (C) indexes at the OTU level. (D) PLS-DA analysis at the OTU level. (E) Histogram of relative abundance of intestinal flora in different treatment groups at the phylum classification level. (F) Heat map showing the relative abundance of the 50 most important bacteria in different treatment groups at the genus classification level. (G) Schematic diagram of intestinal flora pre-clearance model construction and treatment. (H) Colon length photo. (I) Colon length statistics. (J) Colon HE staining, scale bar, 50μm (lower row) and 100μm (upper row). (K) Colon MPO level.

[0029] Fig.14 Supplementary explanation of the results of intestinal flora regulation in Example 1. (A) Species dilution curves of different treatment groups. (B) Venn diagram of shared and unique species at the OTU level. (C) PCoA analysis at the OTU level.

[0030] Fig.15 The abundance of SCFAs-producing bacteria and the changes in intestinal SCFAs levels after BD / PA@SA treatment in Example 1 are shown in Figure 1. (A) Relative abundance of species with differences between groups at the genus classification level. (B) LDA discriminant analysis bar graph. (C) SCFAs levels in mouse intestinal contents. (D) Correlation analysis of specific bacteria and UC-related efficacy indicators at the species classification level.

[0031] Fig.16 The results of the efficacy of each component of BD / PA@SA in treating UC mice in Example 1. (A) Schematic diagram of mouse UC model construction and treatment. (B) Body weight change. (C) Colon length photo. (D) Colon length statistics. (E) Colon HE staining, scale bar, 50μm (lower row) and 100μm (upper row). (F) Colon MPO and inflammatory factor levels.

[0032] Fig.17 Figure 1 shows the RNA-Seq analysis of mouse colon in Example 1. (A) Statistics of the number of differentially expressed genes between different groups. (B) Venn diagram of differentially expressed genes between different groups. (C) Volcano diagram of differentially expressed genes between BD / PA@SA group and DSS group. (D) KEGG pathway enrichment of differentially expressed genes between BD / PA@SA group and DSS group. (E) PPI network diagram of genes related to KEGG pathway enrichment.

[0033] Fig.18 Supplementary explanation of the RNA-Seq analysis results in Example 1. (A) PCA plot of sample distribution in each group. (B) Heat map of sample gene expression cluster analysis. (C) GO term analysis of differentially expressed genes between the BD / PA@SA group and the DSS group.

[0034] Fig.19 The biosafety analysis diagram of BD / PA@SA in Example 1, (A) Cell viability of mouse macrophage RAW264.7 after incubation with different concentrations of PA. (B) Cell viability of mouse colon cells CT26.WT after incubation with different concentrations of PA. (C) Body weight changes of mice orally administered with BD / PA@SA. (D) Index of major organs. (E) HE staining of major organs, scale bar, 100μm. (F) Liver and kidney function and blood routine. DETAILED DESCRIPTION

[0035] Example 1

[0036] 1. Materials

[0037] Berberine (BBR), 5-aminosalicylic acid (5-ASA), ANS (8-Anilino-1-naphthalenesulfonic acid), ThT (Thioflavin T), acetonitrile (HPLC grade), potassium dihydrogen phosphate (KH2PO4, HPLC grade) and phosphoric acid (HPLC grade) were purchased from MacLean Biochemical Technology Co., Ltd. Dehydrocostus lactone (DHCL) was purchased from Weikeqi Biotechnology Co., Ltd. Bovine serum albumin (BSA) and Tween 20 were purchased from BioFroxx. Sodium Alginate (SA, molecular weight 420000, mannuronic acid and guluronic acid ratio of 1:1) and dimethyl sulfoxide (DMSO) were purchased from Aladdin Reagent Co., Ltd. Anhydrous calcium chloride (CaCl2), sodium hydroxide (NaOH) and sodium chloride (NaCl) were purchased from Damao Chemical Reagent Factory. Dextran Sulfate Sodium Salt (DSS, molecular weight 36000-50000) was purchased from MP Biomedicals. 0.5 mol / L EDTA solution was purchased from Solebao Technology Co., Ltd. Cyanine 5.5 NHS ester was purchased from Xi'an Dianhua Biotechnology Co., Ltd. FITC-BSA was purchased from Jizhi Biochemical Technology Co., Ltd. Cell membrane far-infrared fluorescent probe DiD was purchased from White Shark Biotechnology. Cellulose dialysis bags (molecular weight cutoff 50000), artificial gastric juice, artificial small intestinal juice and artificial colonic juice were purchased from Yuanye Biotechnology Co., Ltd. Interleukin-1β (IL-1β), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α) and myeloperoxidase (MPO) ELISA kits were purchased from Jiangsu Enzyme Immunity Industry Co., Ltd. CCK-8 (Cell Counting Kit-8) was purchased from Xinsaimei Biotechnology Co., Ltd. RAW264.7 and CT26.WT cell lines and culture consumables were provided by Wuhan Saiweier Biotechnology Co., Ltd., China. Unless otherwise noted, all other chemical reagents used were of analytical grade.

[0038] 2. Methods

[0039] 2.1 Synthesis of protein aggregates PA and drug-loaded protein aggregates BD / PA

[0040] 5 mg of BSA was dissolved in 4 mL of water and 1 mL of DMSO, with or without the addition of 8 mg of BBR and 2 mg of DHCL. DMSO was added to help dissolve the hydrophobic drug. The mixed substance solution was stirred at 300 rpm for 24 hours at 60 ° C. Then it was centrifuged at 4000 rpm for 5 minutes at 30 ° C to obtain protein aggregates (PA) or drug-loaded protein aggregates (BD / PA), and finally lyophilized. In order to observe or exclude the effects of drugs and DMSO on protein aggregation, a separate experiment was conducted, that is, different combinations were set up and heat-treated for 12 hours according to the above conditions: BSA (with or without DMSO); BSA with DHCL added (without DMSO); BSA with DHCL, BBR, or DHCL and BBR added (with DMSO).

[0041] 2.2 Characterization of aggregates

[0042] The absorbance at 630 nm during the heat treatment of BSA solution was measured using a microplate reader (BioTek, USA) to monitor the occurrence of aggregation. The morphology of protein aggregates PA and BD / PA was preliminarily observed under an optical microscope (Olympus, Japan). The microscopic morphology of PA and BD / PA was observed using a transmission electron microscope (TEM, Thermo, USA). The surface structure and roughness values ​​of PA and BD / PA were understood using an atomic force microscope (Bruker, USA). The changes in the secondary structure during the heat treatment of BSA were analyzed using a circular dichroism (CD) spectrometer (Applied Photophysics, UK). The fluorescence spectra of drugs, BSA, PA or mixtures of these components were analyzed using a fluorescence spectrophotometer (Edinburgh, UK). Zeta potential and size distribution were measured using Zetasizer Nano ZS and Mastersizer 3000 (Malvern, UK). After examining all particles in the TEM and AFM images, a size threshold of 8 μm was selected to distinguish particles from fibers in protein aggregates, and the proportions of particles and fibers were estimated respectively. A micro-UV-visible spectrophotometer (Thermo, USA) was used to measure the difference in protein concentration in the supernatant of the PA solution before and after centrifugation to estimate the yield of PA. As for BD / PA, its protein content can be estimated after knowing its drug loading, and the yield can be converted accordingly. Fluorescent dye DiD was used to simulate hydrophobic drugs, and heat-treated together with FITC-labeled BSA. After washing with DMSO, the positions of PA and DiD were observed under a confocal microscope (Olympus, Japan). Image J was used to measure the Pearson correlation coefficient (R) to reflect the degree of localization of DiD on FITC-labeled PA.

[0043] 2.3 Synthesis and characterization of microspheres

[0044] Only pure water was used to prepare sodium alginate (SA) with a concentration of 2% (w / v), and 1 ml was taken, and the protein aggregates prepared and lyophilized in Section 2.1 (prepared according to the mass of each component in Section 2.1) or drug components were added or not, and the mixture was dispersed evenly by ultrasonication. Then, a saturated CaCl2 solution at room temperature was used as a coagulation bath, and microspheres (content-free microspheres @SA, protein aggregate microspheres PA@SA, Chinese medicine monomer microspheres BD@SA (Chinese medicine monomers were directly mixed with SA, and then microspheres were prepared by electrostatic droplet technology) and drug-loaded protein aggregate microspheres BD / PA@SA) were prepared by electrostatic droplet technology. The experimental steps of electrostatic droplets are as follows: SA with or without ingredients is sucked through a syringe (such as 1 ml), placed on a microinjection pump, the syringe pump speed (0.1 ml / min) is set, and the syringe needle is clamped with an iron clamp with high voltage (6 kV). Through the pump thrust, gravity and electric field force, tiny droplets flow out of the needle and drip into the coagulation bath, thereby forming microspheres or drug-loaded microspheres, etc., and finally the microspheres are separated and freeze-dried. The morphology of microspheres containing or not containing BD / PA (BD / PA@SA, @SA) was observed by bright field and scanning electron microscopy (SEM, Zeiss, Germany). The size distribution was measured by Mastersizer 3000.

[0045] 2.4 Analysis of drug loading and encapsulation efficiency

[0046] 1 mL of 0.1 mol / L NaOH solution was added to the freeze-dried BD / PA and BD / PA@SA, and 1 mL of 0.5 mol / L EDTA-2Na was added to BD / PA@SA to lyse the microspheres. After oscillation at 300 rpm for 6 hours at 4°C, ultrasonic treatment was performed for 10 minutes. Subsequently, 4 mL and 3 mL of acetonitrile were added to BD / PA and BD / PA@SA, respectively, and vortexed for 5 minutes. After standing at -20°C for 1 hour, the upper layer of acetonitrile was transferred to a new EP tube. Then, 0.3 mL of acetonitrile was added to 0.7 mL of 0.01 mol / L KH2PO4 (phosphoric acid adjusted to pH = 3). After filtration, the BBR and DHCL contents in the sample were analyzed by Waters UPLC chromatography system. The ACQUITY UPLC BEH C18 column ( 1.7μm, 2.1mm*50mm), column temperature was 35℃, injection volume was 5μL, and absorbance wavelength was 225nm. The mobile phase was eluent A (0.01mol / L KH2PO4, pH=3) and eluent B (acetonitrile), and the flow rate was 0.2mL / min. The proportion of the gradient elution mobile phase changes with time: 0-3min, 70% A; 3-7min, 70%-35% A; 7-10min, 35% A; 10-11min, 35%-70% A; 11-12min, 70% A. Set standard samples of different concentrations in advance to obtain the standard curve of chromatographic peak area and sample concentration, and then convert the amount of drug in the unknown sample. Finally, the drug loading (LC) and encapsulation efficiency (EE) are calculated using the following formula:

[0047]

[0048] 2.5 Analysis of drug release behavior in vitro

[0049] The drug release behaviors of BD / PA prepared according to Section 2.1 (5 mg BSA was dissolved in 4 mL water and 1 mL DMSO, 8 mg BBR and 2 mg DHCL were added to obtain drug-loaded protein aggregates (BD / PA), and finally freeze-dried) and BD / PA@SA prepared according to Section 2.3 (2% (w / v) sodium alginate (SA) was prepared with pure water, 1 ml was taken, and the protein aggregates or drug components prepared and freeze-dried in Section 2.1 or not were added, and the mixture was ultrasonically dispersed evenly. Then, a saturated CaCl2 solution at room temperature was used as a coagulation bath, and microspheres were prepared by electrostatic droplet technology, in which SA of BD / PA was mixed to form microspheres BD / PA@SA) were analyzed by artificial gastric juice, artificial small intestinal juice and artificial colonic juice. First, a control sample BD+BSA was prepared (it contained the same mass of drug and protein as the BD / PA sample (after determining the drug loading and encapsulation efficiency of BD / PA in Section 2.4, it was found that: about 0.916 mg BBR, 0.173 mg DHCL and 4.08 mg BSA. After fully mixing them, lyophilization was performed to obtain BD+BSA). It contained the same mass of drug and protein as the BD / PA sample (after determining the drug loading and encapsulation efficiency of BD / PA in Section 2.4, it was found that: about 0.916 mg BBR, 0.173 mg DHCL and 4.08 mg BSA. After they are fully mixed, freeze-dried to obtain BD+BSA). Then each freeze-dried sample is placed in a dialysis bag, immersed in 30mL of artificial gastric juice, and shaken at 37°C and 100rpm. After 2 hours, it is replaced with 30mL of artificial small intestinal fluid and shaken for another 4 hours at 37°C and 100rpm. Finally, it is replaced with 30ml of artificial colonic fluid and shaken for another 42 hours at 37°C and 100rpm. At specific time points (0.5h, 1h, 2h, 4h, 6h, 12h, 24h, 48h), after absorbing 3mL of digestive fluid (and replenishing it in time to keep the volume constant), an equal volume of acetonitrile is added to extract the drug. The samples are analyzed by the above-mentioned UPLC program, and the drug release at different time points is calculated, and then the drug release behavior of BD / PA and BD / PA@SA preparations is observed.

[0050] 2.6 Animal Source and Breeding

[0051] 7-week-old male C57BL / 6J mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. and housed in the Experimental Animal Center of Jinan University. All animal experimental protocols were approved by the Experimental Animal Welfare and Ethics Committee of Jinan University (approval number: IACUC-20230329-01). After the mice were housed together for one week, they were randomly assigned to reduce the heterogeneity of the intestinal microbiome.

[0052] 2.7 In vivo biodistribution evaluation of BD / PA

[0053] Cyanine5.5 (Cy5.5) was used as a fluorescent probe to examine the biodistribution of protein aggregates in major organs. Briefly, Cy5.5 NHS ester was mixed with BD / PA prepared according to Section 2.1 at a molar ratio of Cy5.5:PA = 2:1, and stirred in water at 4°C for 6 h in the dark. BD / PA-Cy5.5 was collected using an ultrafiltration tube (molecular weight cutoff: 3000) and encapsulated in SA (BD / PA-Cy5.5@SA). Then, free Cy5.5, BD / PA-Cy5.5, or BD / PA-Cy5.5@SA (containing 0.365 mg / kg of Cy5.5) was administered only once to mice with UC successfully induced by 2.5% (w / v) DSS, and BD / PA-Cy5.5@SA was administered only once to healthy mice. The mice were euthanized at 24 hours, 48 ​​hours, and 168 hours after oral administration, and their main organs were taken and fluorescent images were taken by IVIS Lumina Series III (PerkinElmer, USA) to understand the distribution of Cy5.5 signals and the in vivo biodistribution of BD / PA. In addition, the colon tissue of mice was taken at 24 hours and quickly frozen in liquid nitrogen, and frozen sections were made. After staining with DAPI, the deposition of Cy5.5-labeled BD / PA in colon lesions was observed using Olympus VS200 Research Slide Scanner.

[0054] 2.8DSS-induced colitis and drug intervention (w / v)

[0055] After a one-week adaptation period, 8-week-old male C57BL / 6J mice were provided with water containing 2.5% (w / v) DSS for 7 consecutive days and then with normal water for 3 days. Healthy mice were always provided with normal water. Different animal experiments may have different dosing requirements. Generally speaking, during the experimental period after the adaptation period, mice were administered 100 mg / kg of drugs (5-ASA, Chinese medicine monomer BD [BBR / DHCL mass ratio = 4:1], BD / PA, BD / PA@SA, PA, @SA, PA@SA, or BD@SA (the drugs described in Section 2.1 were mixed with 1 ml SA, ultrasonically dispersed evenly, and then electrostatic droplet formation)) or saline once a day by the oral route for a total of 10 times. In the experiment of antibiotic disruption of intestinal flora, we first pretreated mice with an antibiotic mixture (ampicillin 1 g / L, vancomycin 0.5 g / L, neomycin 1 g / L, and metronidazole 1 g / L) in drinking water for 5 days before starting DSS modeling and dosing. The body weight of mice was monitored throughout the experiment. On the last day of the experiment, mice were euthanized and the entire colon was removed. After measuring the colon length, the distal colon was taken to prepare tissue sections. The remaining colon tissue was used for ELISA and transcriptome analysis. In addition, intracolonic feces were collected for microbial analysis and short-chain fatty acid determination.

[0056] 2.9 Histological evaluation and immunohistochemical staining analysis

[0057] The collected organs were placed in 4% paraformaldehyde for 24 hours. Subsequently, the distal colon (5 μm) sections were prepared and stained with H&E (hematoxylin and eosin) and PAS (periodic acid-Schiff), and the colon injury score was evaluated with reference to previous studies. At the same time, the expression levels of Muc-2, Ki67, CD86 and CD206 in colon tissues were observed using immunohistochemical staining. These images were analyzed using Image J.

[0058] 2.10 Immunofluorescence imaging

[0059] The distal colon tissue sections were blocked with 10% goat serum and then incubated with primary antibodies against ZO-1 (1:200) and Occludin-1 (1:200). After incubation at 4°C overnight, the corresponding secondary antibodies were incubated at 37°C for 1 hour. The nuclei were then counterstained with DAPI and observed using an Olympus SpinSR10 confocal microscope, and the fluorescence signals were analyzed by Image J.

[0060] 2.11 Enzyme-linked immunosorbent assay (ELISA) analysis

[0061] The levels of MPO, IL-1β, IL-10, and TNF-α in the supernatant of colonic tissue were measured according to the protocol of the ELISA kit.

[0062] 2.12 Microbiological analysis

[0063] The collected feces were frozen and stored in dry ice. The intestinal microbiota in feces was analyzed by 16S rRNA gene sequencing service of Shanghai Meiji Biopharmaceutical Technology Co., Ltd. The data obtained from this sequencing were analyzed on the free I-Sanger cloud platform.

[0064] 2.13 Short-chain fatty acids (SCFAs) detection

[0065] SCFAs content was detected by Wuhan Mattwell Biotechnology Co., Ltd. In short, 1 mL of 0.5% (v / v) phosphoric acid and 1 small steel ball were added to 20 mg of feces, ground for 10 minutes, and then sonicated for 5 minutes. Subsequently, the sample was centrifuged at 12000 rpm for 10 minutes (4 ° C). After 100 μL of supernatant was drawn, 500 μL of methyl tert-butyl ether (containing internal standard) was added. The resulting mixture was vortexed for 3 minutes, sonicated for 5 minutes, and then centrifuged at 12000 rpm for 10 minutes (4 ° C). Finally, the supernatant was analyzed using an Agilent 7890B-7000D GC-MS / MS platform equipped with a DB-FFAP column (30m×0.25mm×0.25μm film thickness, J&W Scientific, USA).

[0066] 2.14 RNA-seq analysis

[0067] Colon samples were constructed and sequenced by Wuhan Mattwell Biotechnology Co., Ltd. HISAT2 software was used to calculate gene alignments, and FPKM values ​​were calculated to quantify gene expression levels. DESeq2 software was used to identify differentially expressed genes (DEGs) between groups, with FDR thresholds of <0.05 and |log2 FC|≥1. GO and KEGG databases were used for gene classification and pathway function annotation. The interaction relationships between proteins of DEGs were analyzed using the STRING database.

[0068] 2.15 In vivo toxicity evaluation

[0069] After 1 week of adaptation, 8-week-old male C57BL / 6J mice were orally administered with saline or BD / PA@SA (100 mg / kg) once a day for a total of 10 times. The behavior or weight changes of the mice were observed during the experiment. On the 10th day, the mice were euthanized, and their blood and major organs (heart, liver, kidney, lung, spleen and colon) were collected. The main organ indexes were evaluated, and liver and kidney function and blood routine analysis were performed. Finally, H&E staining was used to observe whether the organs had lesions.

[0070] 2.16 Cell culture and CCK-8 assay

[0071] RAW264.7 cells were cultured in DMEM medium. CT26.WT cells were cultured in RPMI-1640 medium. All culture media contained 10% (v / v) FBS and 1% (v / v) penicillin / streptomycin. All cells were cultured in a humidified environment at 37°C and 5% CO2. RAW264.7 cells (2×10 cells / well) were plated. 4 cells) and CT26.WT cells (1×10 4 Cells) were inoculated in 96-well plates and cultured for 24 hours. Then the protein aggregate PA was evenly dispersed in the culture medium (final concentration was 7.5, 15, 30, 60, 125, 250 and 500 μg / mL), and after replacing the cell culture medium, incubated for 24 hours. After the incubation, the cells were washed with fresh culture medium, 100 μL of fresh culture medium was added to each well, and then 10 μL of CCK-8 was added. After incubation at 37°C for 1 hour, the absorbance was measured at 450nm using an enzyme reader.

[0072] 2.17 Statistical analysis

[0073] Statistical analysis was performed and graphs were generated using GraphPad Prism 10.0. Results are presented as mean ± SEM. Differences between groups were assessed by t-test, one-way or two-way ANOVA. Statistical differences were defined as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and P > 0.05 (ns, no significant difference).

[0074] 3. Experimental results and discussion

[0075] (1) Preparation and characterization of PA, BD / PA, and BD / PA@SA

[0076] First, the aggregation behavior of the protein without the addition of drugs was analyzed ( Figure 1 A). BSA changes from a dissolved state to an aggregated state after heat treatment ( Figure 2 A), TEM and AFM images showed that BSA formed granular and fibrous protein aggregates (Protein aggregate, PA) ( Figure 1 B), and both aggregates were visible at the same time at 6 h ( Figure 2 D). In reality, the ulcer surface of UC is diverse. Regular, single-type aggregates are difficult to fully adapt to lesion areas of different shapes and sizes, and preparing a mixture of two aggregates is more likely to achieve efficient retention. Nano-scale BSA is induced by heat treatment to form nano-micron-scale PA ( Figure 2BC), where the proportions of particles and fibers were approximately 39.4% and 60.6%, respectively. At the same time, the Zeta potential of BSA after aggregation decreased from -5.5±1.0mV to -10.2±0.8mV ( Figure 1 C). Protein intrinsic fluorescence and ANS fluorescence decrease with the extension of heat treatment time ( Figure 1 DE), reflecting the behavior of BSA gradually unfolding and exposing its hydrophobic structure. ThT fluorescence and circular dichroism spectra further revealed the changes in the secondary structure of the protein ( Figure 1 FH and Table 1). They show that the β-sheet ratio of PA slightly increased and the α-helix ratio decreased compared with the original BSA. Heat-treated BSA not only had molecular aggregation, but also its peptide chain conformation changed.

[0077] Table 1. Quantification of changes in the secondary structure of BSA protein during heat treatment from 0 to 24 hours

[0078]

[0079] Next, hydrophobic drugs (BBR and DHCL, referred to as BD) were added and heat treated with BSA at the same time, and the drug-loaded protein aggregates BD / PA ( Figure 4 A) has some new features. On the one hand, compared with PA, its fibers and particles are irregular in shape and have a rougher surface ( Figure 3 A and Figure 4 B). At the same time, although BD / PA is also a nano-micron aggregate ( Figure 3 B), but the size of its fibers tended to decrease, which may be the result of changes in the solution ionic strength. Previous studies have shown that higher ionic strength can form shorter and more flexible fibers. BBR can ionize in water, increasing the solution ionic strength, which is likely to change the morphology and size of the aggregates. On the other hand, the Zeta potential of BD / PA did not decrease ( Figure 4 C), the number of aggregates formed is greater ( Figure 4 D), the yield in 24 hours can reach 86% ( Figure 4 E). A possible explanation is that the hydrophobic interaction brought about by the presence of hydrophobic drugs promotes the aggregation of BSA, thereby forming more PA. To prove the existence of this interaction, we measured the fluorescence wavelength of the BD / PA related components ( Figure 5 The optimal emission wavelength of drug BD in BD / PA shifted from 580nm to 604nm ( Figure 3C), and BD also showed similar changes after simple mixing with aggregate PA. This may be because the energy transfer of BBR originally caused by π-π stacking was reduced after binding to the exposed hydrophobic area of ​​PA, and finally produced higher fluorescence. In addition, the experiment found that the hydrophobic fluorescent dye DiD (simulating hydrophobic drugs) had a good co-localization relationship on FITC-PA ( Figure 3 D). This not only illustrates the feasibility of PA loading drugs, but also supports the existence of hydrophobic interactions. It can be speculated that the above characteristics are conducive to the further adaptation of BD / PA to colon ulcers and loading drugs.

[0080] Then, by measuring the drug loading and encapsulation efficiency, we learned the drug loading status of BD / PA ( Figure 3 E), and based on electrostatic droplet technology, SA was used to encapsulate to form drug-loaded microspheres BD / PA@SA ( Figure 3 F and Figure 6 A). The size of BD / PA@SA tends to be smaller than that of microspheres without content@SA ( Figure 3 G), which may be due to the role of protein as a surfactant, reducing the surface tension when the droplets are formed, resulting in a reduction in the surface coating layer and the formation of smaller microspheres. It is worth noting that BD / PA@SA is loaded with about 0.75 mg of BBR and 0.13 mg of DHCL ( Figure 6 B), and in vitro release experiments also showed reduced intragastric burst release and sustained release of drugs in the colon ( Figure 3 HI and Figure 6 CD). With such a small amount of drug loading, we need to further test whether it can effectively target the colon and treat UC.

[0081] (2) Drug carriers accumulate in the inflamed colon

[0082] Before conducting the efficacy evaluation experiment, we first explored the distribution of the drug carrier in C57BL / 6J mice after oral administration. An acute model of colitis was established by giving 2.5% (w / v) DSS in drinking water for 7 days, followed by ordinary drinking water for 3 days, and the normal group was given ordinary water all the time. The drug was taken orally only once on the last day of the experiment (all groups of drugs contained Cy5.50.365mg / kg), and then the mice were euthanized and their organs were taken for testing to observe the biological distribution of the drug carrier in the body. The following four groups were set up specifically:

[0083] Normal group, oral administration of BD / PA-Cy5.5@SA;

[0084] Model group, oral administration of Cy5.5;

[0085] Model group, oral administration of BD / PA-Cy5.5;

[0086] Model group, oral administration of BD / PA-Cy5.5@SA.

[0087] SA-encapsulated BD / PA labeled with Cy5.5 (BD / PA-Cy5.5@SA) significantly accumulated in the DSS-induced inflamed colon and persisted for up to 1 week. However, in comparison, the colon fluorescence signal of mice orally administered with Cy5.5 and BD / PA-Cy5.5 was weak and the fluorescence duration was also shorter ( Figure 7 AB). This is because the delivery of SA polysaccharide shell is lacking, and the fluorescent molecules are affected by the physiological barrier in the gastrointestinal tract, making it difficult to concentrate and retain in the colon. When BD / PA@SA releases its contents BD / PA in the colon, the latter relies on its nano-micron size to match different types of ulcer surfaces, and is retained in the colon for a long time and releases drugs ( Figure 7 C), healthy mice cannot retain BD / PA for a long time despite the delivery of SA polysaccharide shell due to the intact intestinal epithelium. In addition, no fluorescent signals were observed in other major organs of mice in each group ( Figure 8 ), which reduces the possibility of PA deposition in major organs and causing toxicity.

[0088] (3) BD / PA@SA is effective against colitis and is also effective with delayed intervention

[0089] BD / PA@SA not only has excellent colon targeting and retention capabilities, but we also found that it also has excellent efficacy in the treatment of DSS-induced acute colitis ( Fig. 9 A). Similarly, C57BL / 6J mice were given 2.5% (w / v) DSS in drinking water for 7 days to establish an acute model of colitis, followed by normal drinking water for 3 days. The normal group was given normal water all the time. During the experiment, mice were orally administered 100 mg / kg of drugs (5-ASA, Chinese medicine monomer BD [BBR / DHCL mass ratio = 4:1], BD / PA or BD / PA@SA) or saline once a day for a total of 10 doses.

[0090] Compared with the DSS group, the colons of mice treated with the incomplete system (BD group and BD / PA group) still showed obvious UC characteristics such as epithelial defects, scar repair and crypt disappearance. Oral administration of BD / PA@SA can improve weight loss and shortened colon length, and inhibit colon tissue damage, achieving an effect similar to that of the clinical first-line drug 5-ASA ( Fig. 9 BE and Fig.10 ). Regarding the important inflammatory pathological state in UC, we also found that loading a very small amount of BD / PA@SA can still significantly reduce the levels of MPO and inflammatory factors (IL-1β and TNF-α) in the colon, and restore the level of anti-inflammatory factors (IL-10) ( Fig. 9FG). However, the therapeutic effects of BD or BD / PA were relatively inferior. In addition, BD / PA@SA reduced the infiltration of pro-inflammatory M1 macrophages in the colon and restored the proportion of anti-inflammatory M2 macrophages ( Fig. 9 HI). These studies show that BD / PA@SA also has a very good anti-inflammatory effect.

[0091] In the traditional preparation of Xianglian Pills, BBR and DHCL are the main ingredients of two synergistic herbs, respectively, and BBR plays a major anti-inflammatory role. Previous studies have shown that BBR is anti-inflammatory by inhibiting NLRP3 inflammasomes, arachidonic acid metabolism, and AKT signaling pathways, while DHCL regulates MAPK and NF-κB pathways to improve inflammation. Therefore, according to the traditional Chinese medicine theory of "monarch, minister, assistant, and messenger", we intend to design BBR as the "monarch" drug and DHCL as the "minister" drug, so that they can synergistically intervene in the inflammation of UC. However, limited by the poor bioavailability of BBR and DHCL, the above studies usually require a larger oral dose to achieve efficacy (such as 100 mg / kg and above). Based on the guidance of the "monarch, minister, assistant, and messenger" theory, we designed the "assistant" drug and the "messenger" drug through modern drug delivery technology, that is, with the help of the properties of protein aggregates PA (assisted retention, "adjuvant" drug) and polysaccharide SA (targeted delivery, "messenger" drug), a smaller dose of Chinese medicine monomers (BBR: 3.73 mg / kg, DHCL: 0.67 mg / kg) can produce better therapeutic effects. This not only greatly improves the bioavailability of hydrophobic active ingredients, but also reduces the risk of drug accumulation and toxic side effects to a certain extent. It should be noted that the lack of SA component in the BD / PA group has reduced efficacy, and the lack of colon targeting ability may be an important reason.

[0092] In addition, UC patients usually seek treatment after disease onset, and we also investigated the efficacy of BD / PA@SA in a delayed intervention model ( Fig.11 A). C57BL / 6J mice were first given 2.5% (w / v) DSS in their drinking water for 7 days to construct an acute model of colitis, and then given normal drinking water for 11 days. The normal group was given normal water all the time. One day after the successful construction of the UC model (day 8), the mice were orally administered 100 mg / kg of the drug (5-ASA or BD / PA@SA) or saline once a day for a total of 10 doses. Compared with natural recovery (DSS group) and first-line clinical drug treatment (5-ASA group), the mice under BD / PA@SA intervention recovered their weight more quickly, and their body weight was significantly improved at the end of treatment ( Fig.11 B), and maintained colon length and intestinal epithelial health ( Fig.11 CE). This reflects that BD / PA@SA is also effective even with delayed intervention.

[0093] (4) BD / PA@SA repairs intestinal epithelium

[0094] Curious about the efficacy of BD / PA@SA, we further studied its ability to repair colonic epithelium. Immunofluorescence staining of tissue sections showed that the expression of Occludin-1 and ZO-1 in the intestine was restored ( Fig.12 A). These two proteins are tight junction proteins that act as a mechanical barrier to prevent intestinal bacteria, toxins and other contents from "leaking" into the body. Similarly, PAS staining and Muc-2 immunohistochemical staining ( Fig.12 BC), and also showed the ability of BD / PA@SA to repair the colon mucus barrier. However, these indicators were not significantly improved after treatment with 5-ASA and BD / PA, and the BD group only had an advantage in repairing the mucus barrier. Ki67 is an indicator for evaluating intestinal proliferation. Previous studies have shown that its expression increases with the aggravation of colon inflammation. We found through immunohistochemical staining that compared with other treatment groups, the expression of Ki67 in the colon of mice in the BD / PA@SA group decreased ( Fig.12 D). This result also indirectly reflects the restoration of epithelial health after BD / PA@SA treatment. In addition, bacteria are part of the colon environment, and some pathogenic bacteria are also involved in the pathogenesis of UC. In addition to anti-inflammatory and repairing colon epithelium, BD / PA@SA may also alleviate UC by regulating intestinal microecology.

[0095] (5) BD / PA@SA regulates intestinal flora

[0096] There is increasing evidence that UC is accompanied by intestinal microbiota imbalance, and restoring intestinal flora is one of the emerging therapeutic measures to alleviate UC. Given that BBR and DHCL have been reported to affect the composition of intestinal microbiota, we analyzed mouse fecal samples by 16S rRNA gene sequencing. The six groups were: normal control CON, disease model DSS, western medicine treatment disease model 5-ASA, Chinese medicine monomer treatment disease model BD, drug-loaded protein aggregate treatment disease model BD / PA, and drug-loaded protein aggregate microsphere treatment disease model BD / PA@SA. The species dilution curves of different treatment groups reflect that the amount of sequencing data is sufficient ( Fig.14 A). The α-diversity analysis index showed that after BD and BD / PA@SA treatment, the community diversity in the mouse intestine did not recover, but there may be some dominant bacterial groups with a higher proportion ( Fig.13 AC), and the OTU-level Venn diagram also supports this point ( Fig.14 B). PLS-DA plots and PCoA plots showed that BD and BD / PA@SA treated mice had unique gut microbiota profiles compared with the DSS group, whereas no such changes were observed in 5-ASA treated mice ( Fig.13 D and Fig.14C). Further analysis of bacterial abundance at the phylum / genus level showed that BD / PA@SA treatment reversed the change in the ratio of Firmicutes to Bacteroidetes in the DSS group ( Fig.13 E), and the bacterial abundances differed between groups at the genus level ( Fig.13 F). Obviously, these results indicate that BD / PA@SA modulates the intestinal flora of colitis mice. To further understand the role of intestinal microbiota in the efficacy of BD / PA@SA, we conducted an experiment to disrupt intestinal flora with antibiotics. We first pretreated mice with broad-spectrum oral antibiotics (abbreviated as ABX) for 5 days ( Fig.13 G), and then an acute colitis model was established by administering 2.5% (w / v) DSS in drinking water for 7 days, and mice were orally administered 100 mg / kg of the drug (BD / PA@SA) or saline once a day for a total of 10 doses. The results showed that compared with the BD / PA@SA group without antibiotic pretreatment, the efficacy of the ABX-BD / PA@SA group on DSS-induced colitis was significantly reduced ( Fig.13 HK). This suggests that the therapeutic effect of BD / PA@SA is partly due to the regulation of the intestinal microbiota.

[0097] (6) BD / PA@SA increases the abundance of intestinal short-chain fatty acid-producing bacteria and improves the level of short-chain fatty acids

[0098] In order to explore the microbiota and its potential role in mediating the therapeutic effects of BD / PA@SA, we conducted species difference analysis ( Fig.15 A). At the genus level, on the one hand, BD / PA@SA significantly reduced the abundance of Desulfovibrio, which produces H2S that is thought to damage the intestinal epithelium. On the other hand, BD / PA@SA treatment increased the abundance of Lactobacillus, Eubacterium_fissicatena_group, Papillibacter, and Muribaculum. Meanwhile, linear discriminant analysis specifically pointed out that Lactobacillus and Lactobacillus_johnsonii were the dominant genera / species in the BD / PA@SA group ( Fig.15B). Related reports indicate that these bacteria with increased abundance are closely related to the production of short-chain fatty acids (SCFAs). As a bacterial metabolite, SCFAs are currently believed to have beneficial effects such as anti-inflammatory, changing the intestinal pH environment to inhibit harmful bacteria, and providing energy for the intestinal epithelium. Based on the evidence of microbiota regulation, we speculate that BD / PA@SA may also increase the level of SCFAs in the intestine and promote intestinal repair. Not surprisingly, targeted metabolomics showed that the levels of six SCFAs, namely acetate, propionate, butyrate, 2-methylbutyrate, isovaleric acid, and isobutyric acid, in the intestines of mice in the BD / PA@SA group rebounded ( Fig.15 C). We believe that the PA and SA microspheres that deliver drugs to the colon are the main reason for this result. SA, as a dietary fiber, has been reported to become a nutrient for intestinal bacteria in vitro and increase the level of SCFAs. Usually, the fermentation of dietary fiber is the main source of SCFAs. At the same time, microorganisms also metabolize proteins or amino acids to produce branched short-chain fatty acids (such as 2-methylbutyric acid, isovaleric acid and isobutyric acid). This phenomenon shows that PA and SA microspheres in our treatment system are not only "adjuvant" drugs that assist retention and "enabler" drugs for targeted delivery, but also that they are ordinary drugs that are metabolized by intestinal microorganisms and transformed into SCFAs. Their therapeutic role has also been transformed into "minister" drugs, and they have cooperated with "king" drugs to fight diseases. In addition, in order to further clarify the relationship between the dominant flora and UC indicators, we conducted a Spearman correlation analysis ( Fig.15 D). The results showed that IL-10, CD206, PAS, and short-chain fatty acids were positively correlated with the abundance of Lactobacillus_johnsonii, while colon injury score, IL-1β, and MPO were negatively correlated with its abundance. In our study, it can be considered that Lactobacillus_johnsonii is the key bacterial species regulated by BD / PA@SA, which to some extent assists BD / PA@SA in exerting its therapeutic effect.

[0099] (7) The components of BD / PA@SA are inseparable

[0100] The concept of "monarch, minister, assistant and envoy" in medication not only requires each component to play its unique advantages, but also emphasizes the importance of mutual cooperation and indivisibility between components. We continued to verify the drug carrier BD / PA@SA has this property in animal experiments by setting up other combinations of drugs, PA and SA components ( Fig.16 A):

[0101] Normal control group CON;

[0102] Disease model DSS group;

[0103] PA group of protein aggregate therapy disease models;

[0104] @SA group of disease models treated with blank microspheres;

[0105] The PA@SA group of the disease model treated with SA microspheres encapsulated with PA;

[0106] SA microspheres encapsulating the Chinese medicine monomer BD for the treatment of disease models BD@SA group;

[0107] BD / PA@SA group of drug-loaded protein aggregate microspheres for the treatment of disease models.

[0108] Similarly, an acute model of colitis was established by giving C57BL / 6J mice 2.5% (w / v) DSS in their drinking water for 7 days, followed by normal water for 3 days, while the CON group was given normal water all the time. Mice were orally administered 100 mg / kg of drug (PA, @SA, PA@SA, BD@SA, or BD / PA@SA) or saline once a day for a total of 10 doses. The intact BD / PA@SA still protected mice from DSS-induced weight loss and shortened colon length, while inhibiting colon tissue damage and inflammatory state ( Fig.16 BF). In contrast, PA@SA without BBR and DHCL was not sufficient to alleviate UC, although they could be metabolized to produce beneficial short-chain fatty acids. In addition, BD@SA did not protect animals from DSS-induced colitis, mainly because the lack of PA prevented the hydrophobic drug from being retained in the ulcer for a long time to exert its effect. In summary, the components of BD / PA@SA are inseparable. Although PA ("adjuvant" drug) and SA ("enabler" drug) have certain therapeutic benefits, they still need to have both retention and targeting functions. The lack of any component will affect the therapeutic effect of BD / PA@SA.

[0109] (8) RNA-Seq analysis of the colon of UC mice after BD / PA@SA treatment

[0110] To reveal the deep molecular mechanism of BD / PA@SA in alleviating DSS-induced colitis, we performed RNA-Seq analysis on the colon tissues of mice. The PCA plot and cluster analysis heat map showed that there were differences in gene expression between the DSS group and the BD / PA@SA group ( Fig.18 AB). Then, by counting the significantly differentially expressed genes (DEGs, p-adjust<0.05, FC>2) between different groups, we found that there were only 5 DEGs after BD treatment compared with the DSS group. However, BD / PA@SA treatment led to significant changes in the expression levels of 332 genes, of which 200 genes were upregulated and 132 genes were downregulated, and the changes in 327 genes were unique to BD / PA@SA treatment ( Fig.17 AC). DEGs between the BD / PA@SA group and the DSS group were evaluated in detail using the GO database, and the results showed that these genes were involved in biological processes such as cell physiology, metabolism, and the immune system ( Fig.18 C). In addition, in KEGG enrichment analysis, they were mainly enriched in humoral immunity (B cell receptor signaling pathway and intestinal IgA production) and lipid metabolism (linoleic acid, arachidonic acid and α-linolenic acid) pathways ( Fig.17 D). The connections between these DEGs were further reflected in the protein-protein interaction (PPI) network ( Fig.17 E).

[0111] First, the role of humoral immunity in the immune pathogenesis of IBD has long been recognized. In the healthy intestine, plasma cells steadily produce IgA, which, as a key mediator of intestinal immunity, neutralizes and rejects pathogens in a non-inflammatory manner, reducing the pro-inflammatory interaction between the intestinal immune system and a large number of microbial-associated molecular patterns. However, recent studies have shown that B cell responses in UC are highly dysregulated, and intestinal plasma cells have become one of the contributors to UC inflammatory infiltration, and the mechanism is that plasma cells transform from IgA secretion to IgG secretion. Not only is the consumption and deficiency of IgA conducive to intestinal inflammation, but IgG can also mediate further activation of colonic proinflammatory macrophages. KEGG enrichment analysis showed that the therapeutic components of BD / PA@SA regulated the B cell receptor signaling pathway and the intestinal IgA production pathway ( Fig.17D). According to existing reports, SCFAs can participate in the activation and differentiation of intestinal B cells and enhance the expression of genes related to IgA production. On the other hand, they can also promote B cells to provide the required energy and raw materials for the synthesis of IgA, effectively promoting the conversion of antibody secretion of intestinal B cells to the IgA category. BD / PA@SA treatment increased the level of SCFAs, which may be one of its mechanisms for intervening intestinal mucosal immunity and treating UC. In addition, linoleic acid metabolism, arachidonic acid metabolism and α-linolenic acid metabolism are also the main enrichment pathways of these DEGs. Linoleic acid (LA) and its derivative arachidonic acid (AA) are both ω-6 polyunsaturated fatty acids, and changes in their metabolic pathways are often observed in the onset of UC. It is generally believed that AA plays an important role in the development of inflammatory diseases because it is metabolized by cyclooxygenase (COX) and lipoxygenase (LOX) to produce proinflammatory mediators such as prostaglandin E2 (PGE2) and leukotriene B4 (LTB4). In contrast, α-linolenic acid (ALA), which belongs to the ω-3 polyunsaturated fatty acid, has a metabolic pathway that may alleviate UC inflammation and exert anti-inflammatory effects by inhibiting M1 macrophages and affecting AA metabolism. In this study, changes in these metabolic pathways may also be related to the reduced inflammation observed after BD / PA@SA treatment. For example, it has been reported that BBR in BD / PA@SA can downregulate the AA metabolic pathway to improve UC. As a natural active ingredient that inhibits the AA pathway, it has the effects of reducing the expression of COX-2, PGE2 and corresponding receptors. At the same time, previous molecular docking experiments have shown that BBR can bind to PLA2 and 5-LOX, which may affect AA metabolism by inhibiting the activity of the enzyme. Notably, the upregulated Lactobacillus genus after BD / PA@SA treatment can also regulate LA metabolism and alleviate colitis by converting LA into pre-conjugated linoleic acid (CLA) that is beneficial to intestinal health. In summary, RNA-Seq analysis suggests the potential molecular mechanism of BD / PA@SA in alleviating UC.

[0112] (9) Biosafety Verification of BD / PA@SA

[0113] Finally, we verified the safety of the drug carrier. In vitro experiments showed that our PA had no obvious toxicity to mouse colon epithelial cells and macrophages ( Fig.19 AB). In the in vivo experiment, we orally administered 100 mg / kg BD / PA@SA to healthy mice once a day for 10 times. Afterwards, the mice were euthanized and their blood and major organs were analyzed. The results showed that the body weight of the BD / PA@SA group gradually increased. Compared with the control group (CON group) that took normal saline orally, the indexes of its major organs were stable and no abnormalities were found in the tissue H&E staining ( Fig.19 CE). In addition, although there were slight differences in blood glucose, urea nitrogen and creatinine, these indicators were still within the normal reference range ( Fig.19F). It can be seen that BD / PA@SA has good biosafety.

[0114] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A drug for treating ulcerative colitis, characterized in that: The preparation method of the drug is as follows: take 3-7 mg of BSA, add 6-10 mg of BBR and 1-3 mg of DHCL, and mix 4-6 ml of pure water and DMSO, with the ratio of pure water to DMSO being 3-5: 0.5-1.5, heat treated at 55-65°C and stirred for 20-28h, then centrifuged at 28-32°C to separate the drug-loaded protein aggregates to precipitate BD / PA, and after freeze-drying, add 0.5-1.5ml of 1-3% concentration SA, mix evenly under ultrasound, and then use saturated CaCl2 solution at room temperature as a coagulation bath to prepare drug microspheres BD / PA@SA using electrostatic droplet technology. BD / PA@SA is a drug for the treatment of ulcerative colitis.

2. The drug according to claim 1, characterized in that The amount of BSA used is 5 mg.

3. The drug according to claim 2, characterized in that The amount of BBR used is 8 mg.

4. The drug according to claim 3, characterized in that The dosage of DHCL is 2 mg.

5. The drug according to claim 4, characterized in that The ratio of pure water to DMSO is 4:

1.

6. The drug according to claim 5, characterized in that The total amount of the pure water and DMSO is 5 ml.

7. The drug according to claim 6, characterized in that The heat treatment temperature is 60° C. and the stirring time is 24 h.

8. The drug according to claim 7, characterized in that The concentration of SA was 2% and the volume was 1 ml.

9. Use of the drug according to any one of claims 1 to 8 in the preparation of a product for treating ulcerative colitis.

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