A drug for treating ulcerative colitis

Abstract

The present invention discloses a drug for treating ulcerative colitis, belonging to the field of medical technology. The preparation method of the drug is as follows: take 3-7mg of BSA, add 6-10mg of BBR and 1-3mg of DHCL, prepare 4-6ml with pure water and DMSO, the ratio of pure water to DMSO is 3-5:0.5-1.5, heat treat at 55-65℃ and stir for 20-28h, centrifuge at 28-32℃ to separate the drug-loaded protein aggregates BD / PA, add 0.5-1.5ml of 1-3% concentration of SA after lyophilization, mix evenly, and then use a saturated CaCl2 solution at room temperature as a coagulation bath to prepare the drug BD / PA@SA for treating ulcerative colitis. The present invention is based on the guidance of the traditional Chinese medicine "monarch-minister-assistant-envoy" medication theory, constructs a carrier through drug delivery technology, and improves the bioavailability of BBR and DHCL, the main active ingredients of Xianglian Pills.

Description

Technical Field

[0001] The present 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, listed by the World Health Organization as one of the modern intractable diseases. Modern medicine treats UC primarily through oral administration, due to its convenience, cost-effectiveness, and good patient compliance. Currently, 5-aminosalicylic acid (5-ASA), corticosteroids, immunosuppressants, and biologics 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, the continued search and development of new drugs for the treatment of UC remains worthy of attention.

[0003] In China, Traditional Chinese Medicine (TCM) commonly uses oral medication to treat ulcerative colitis (UC). By exploring the "Compendium of Traditional Chinese Medicine Formulas," researchers identified Xianglian Pills, a century-old classic formula for treating UC-related symptoms. Xianglian Pills consist of a 4:1 ratio of Coptis chinensis and Aucklandia lappa. Modern pharmacological research and clinical application have confirmed the efficacy of Xianglian Pills in treating UC. However, the traditional Xianglian Pills formulation suffers from numerous limitations, including complex ingredients, low active ingredient content, poor utilization, and high dosage, which limit its widespread application. Utilizing the primary natural bioactive compounds (BBR and DHCL) in Xianglian Pills to improve their therapeutic formulation is a promising approach. Summary of the Invention

[0004] One of the objectives 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, and prepare 4-6 ml with pure water and DMSO, with the ratio of pure water to DMSO being 3-5:0.5-1.5. Heat treat at 55-65°C and stir for 20-28 hours. After the end, centrifuge at 28-32°C to separate the drug-loaded protein aggregates to precipitate BD / PA. After freeze-drying, add 0.5-1.5 ml of 1-3% concentration of SA, 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. 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 amount 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 complexation, and processing plasticity 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 UC ulcer lesions to improve drug utilization. In addition, it is necessary to add a layer of shell that is protected from digestion by gastric and small intestinal proteases to protect the drug-loaded protein core so that it can reach the colon lesions directly to exert its effect. It is planned to use natural polysaccharides that are degradable by colon bacteria, 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 "monarch-minister-adjuvant-envoy" medication theory of traditional Chinese medicine, with BBR (berberine, the main component of Coptis chinensis) as the monarch drug and DHCL (dehydrocostus lactone, the main component of costus root) as the minister drug. Carriers (PA and SA microspheres) are constructed through drug delivery technology to serve as the adjuvant and minister drugs in the treatment system, so as to overcome the shortcomings of Xianglian Pills in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1Characterization results of protein aggregates (PAs) in Example 1. (A) Schematic diagram of BSA forming PAs. (B) Transmission electron microscopy (TEM) and atomic force microscopy (AFM) images of BSA before and after heat treatment. Scale bars: 10 μm (TEM) and 2 μm (AFM). (CD) Zeta potential (C) and fluorescence emission wavelength changes (D) of BSA before and after heat treatment. (EF) Fluorescence emission wavelength changes after mixing the 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 Observation results of the PA formation kinetics in Example 1. (A) Absorbance of BSA solution at 630 nm at different heat treatment times. (B) BSA size distribution. (C) PA size distribution. (D) Optical microscopy and AFM observations of PA formation during heat treatment. Scale bars: 100 μm (optical microscopy, BF mode) and 4 μm (AFM).

[0018] Figure 3 Synthesis and characterization 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, 10 μm. (B) Size distribution of BD / PA. (C) Shift in the fluorescence emission spectrum of the drug after binding to PA. (D) Localization of the fluorescent dye DiD on FITC-labeled PA. (E) Drug loading and encapsulation efficiency of BD / PA. (F) Light microscopy observation of empty microspheres (@SA) and drug-loaded protein aggregate microspheres (BD / PA@SA). Scale bars, 500 μm (40x magnification) and 100 μm (100x magnification). (G) Size distribution of @SA and BD / PA@SA. (H) Total drug release profiles of BD / PA and BD / PA@SA formulations. (I) Percentage of total drug released from BD / PA and BD / PA@SA formulations after exposure to different digestive fluids.

[0019] Figure 4 Figure 1 shows the morphology of BD / PA and the changes in PA after heat treatment of BSA with different components. (A) Light microscopy of a solution mixed with BSA and drug BDs (BBR and DHCL) before and after heat treatment. Scale bar, 100 μm. (B) AFM observation of PA morphology and roughness calculation after heat treatment with and without BSA. Scale bars, 2 μm (Oligomer) and 4 μm (Fibril). (C) Zeta potential measurements of BD, BD / PA, and BD+BSA. (D) Light microscopy of BSA after heat treatment with different components for 12 hours. Scale bar, 100 μm. (E) Yields of PA and BD / PA after 24 hours of heat treatment.

[0020] Figure 5 Fluorescence spectra of the BD / PA-related components 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 from Example 1 show the morphology of SA microspheres and the release behavior of the two drugs individually. (A) Scanning electron microscopy (SEM) images of freeze-dried @SA and BD / PA@SA. (B) Drug loading and encapsulation efficiency of BD / PA@SA. (C) Drug release profiles of BBR (C) and DHCL (D) and the percentage of drug released after exposure to different digestive fluids.

[0022] Figure 7 Figure 1 shows the targeting and retention of BD / PA@SA in the mouse colon. (A) In vitro organotypic imaging at 24, 48, and 168 hours demonstrates the colon targeting and retention of BD / PA@SA. (B) Quantification of the Cy5.5 fluorescence signal in the colon at 24, 48, and 168 hours. (C) Deposition of Cy5.5-labeled BD / PA in colonic ulcers at 24 hours. Scale bar, 200 μm.

[0023] Figure 8 Figure 1 shows the deposition of Cy5.5-labeled BD / PA in other major organs. In vitro organ imaging at 24 h, 48 h, and 168 h 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] Figure 9 Results of the efficacy of BD / PA@SA in alleviating UC in mice in Example 1. (A) Schematic diagram of mouse UC model construction and treatment. (B) Body weight change. (C) Photograph of colon length. (D) Colon length statistics. (E) Colon HE staining. Scale bars: 50 μm (lower row) and 100 μm (upper row). (F) Colonic MPO levels. (G) Colonic inflammatory cytokine levels. (HI) Colonic CD86 and CD206 immunohistochemical staining (H) and quantification (I).

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

[0026] Figure 11The efficacy of BD / PA@SA in the delayed UC treatment model described in Example 1. (A) Schematic diagram of the delayed UC treatment model in mice. (B) Body weight change. (C) Colon length photograph. (D) Colon length statistics. (E) Colon HE staining. Scale bars: 50 μm (lower row) and 100 μm (upper row).

[0027] Figure 12 Figure 1 shows histological sections of the colonic mucosa of UC mice repaired with BD / PA@SA in Example 1. (A) Immunofluorescence staining of the colonic tight junction proteins occludin-1 and ZO-1. Scale bar, 100 μm. (B) PAS staining of the colon (B), immunohistochemical staining of Muc-2 protein (C), and immunohistochemical staining of Ki67 (D). Scale bar, 300 μm.

[0028] Figure 13 Figures showing the regulation of intestinal microbiota by BD / PA@SA in Example 1. (A-C) Sobs (A), Ace (B), and Shannon (C) indices at the OTU level. (D) PLS-DA analysis at the OTU level. (E) Histogram of relative abundance of intestinal microbiota in different treatment groups at the phylum level. (F) Heat map showing the relative abundance of the 50 most important bacteria in different treatment groups at the genus level. (G) Schematic diagram of the construction of the intestinal microbiota pre-clearance model and treatment. (H) Colon length photograph. (I) Colon length statistics. (J) Colon HE staining. Scale bars: 50 μm (lower row) and 100 μm (upper row). (K) Colon MPO levels.

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

[0030] Figure 15 Figure 1 shows the changes in SCFA-producing bacteria abundance and intestinal SCFA levels after BD / PA@SA treatment. (A) Relative abundance of species that differ between groups at the genus level. (B) LDA discriminant analysis bar chart. (C) SCFA levels in mouse intestinal contents. (D) Correlation analysis of specific bacteria with UC-related efficacy indicators at the species level.

[0031] Figure 16 Results of the efficacy of the various components of BD / PA@SA in treating UC mice in Example 1. (A) Schematic diagram of mouse UC model establishment and treatment. (B) Body weight change. (C) Photograph of colon length. (D) Colon length statistics. (E) Colon HE staining. Scale bars: 50 μm (lower row) and 100 μm (upper row). (F) Colonic MPO and inflammatory cytokine levels.

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

[0033] Figure 18 Supplementary illustration 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] Figure 19 Figures showing the biosafety analysis of BD / PA@SA in Example 1. (A) Cell viability of mouse macrophage RAW264.7 cells after incubation with different concentrations of PA. (B) Cell viability of mouse colon cells CT26.WT cells after incubation with different concentrations of PA. (C) Body weight changes of mice orally administered with BD / PA@SA. (D) Major organ indices. (E) HE staining of major organs. Scale bar, 100 μm. (F) Liver and kidney function and blood routine test results. DETAILED DESCRIPTION

[0035] Example 1

[0036] 1. Materials

[0037] Berberine (BBR), 5-aminosalicylic acid (5-ASA), 8-Anilino-1-naphthalenesulfonic acid (ANS), Thioflavin T (ThT), 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 420,000, mannuronic acid:guluronic acid ratio 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 36,000–50,000) 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. The cell membrane far-infrared fluorescent probe DiD was purchased from White Shark Biotechnology. Cellulose dialysis bags (molecular weight cutoff 50,000), artificial gastric fluid, artificial small intestinal fluid, and artificial colonic fluid 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 aid dissolution of the hydrophobic drug. The mixed solution was stirred at 300 rpm at 60°C for 24 hours. Protein aggregates (PA) or drug-loaded protein aggregates (BD / PA) were then centrifuged at 4000 rpm at 30°C for 5 minutes and lyophilized. To investigate or exclude the effects of drug and DMSO on protein aggregation, separate experiments were performed with different combinations of BSA and heat-treated for 12 hours under the aforementioned conditions: BSA (with or without DMSO); BSA supplemented with DHCL (without DMSO); and BSA supplemented with DHCL, BBR, or DHCL and BBR (with DMSO).

[0041] 2.2 Characterization of aggregates

[0042] The absorbance at 630 nm during the heat treatment of the BSA solution was measured using a microplate reader (BioTek, USA) to monitor the occurrence of aggregation. The morphology of the protein aggregates PA and BD / PA was initially observed under an optical microscope (Olympus, Japan). The micromorphology 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 of BSA during heat treatment were analyzed using a circular dichroism (CD) spectrometer (Applied Photophysics, UK). The fluorescence spectra of the drug, BSA, PA, or a mixture of these components were analyzed using a fluorescence spectrophotometer (Edinburgh, UK). The zeta potential and size distribution were measured using a Zetasizer Nano ZS and a 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 the protein aggregates, and the proportions of particles and fibers were estimated respectively. The difference in protein concentration in the supernatant of the PA solution before and after centrifugation was measured using a micro-UV-visible spectrophotometer (Thermo, USA) to estimate the PA yield. For BD / PA, knowing the drug loading allows estimation of protein content and conversion of yield based on this information. The fluorescent dye DiD, used to simulate the hydrophobic drug, was heat-treated with FITC-labeled BSA. After washing with DMSO, the localization of PA and DiD was observed under a confocal microscope (Olympus, Japan). Image J was used to measure the Pearson correlation coefficient (R), which reflects the degree of DiD localization on the FITC-labeled PA.

[0043] 2.3 Synthesis and characterization of microspheres

[0044] Sodium alginate (SA) was prepared at a 2% (w / v) concentration using pure water alone. A 1ml aliquot was added with or without the protein aggregates (prepared according to the weight of each component in Section 2.1) or the drug component, prepared and lyophilized as described in Section 2.1. Mixing was followed by ultrasonic dispersion. Microspheres (including bare microspheres@SA, protein aggregate microspheres PA@SA, traditional Chinese medicine monomer microspheres BD@SA (traditional Chinese medicine monomers were directly mixed with SA and then prepared using electrostatic droplet technology), and drug-loaded protein aggregate microspheres BD / PA@SA) were then prepared using a saturated CaCl2 solution at room temperature as a coagulation bath. The electrostatic droplet experiment involves drawing SA (with or without added ingredients) through a syringe (e.g., 1 ml) and placing it on a microsyringe pump. The syringe pump speed is set to 0.1 ml / min, and the syringe needle is clamped with a high-voltage (6 kV) iron clamp. Pump thrust, gravity, and the electric field force cause tiny droplets to flow from the needle and drip into a coagulation bath, forming microspheres or drug-loaded microspheres. Finally, the microspheres are separated and freeze-dried. The morphology of microspheres containing or without BD / PA (BD / PA@SA, @SA) was observed using brightfield and scanning electron microscopy (SEM, Zeiss, Germany). Size distribution was measured using a 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, respectively. For BD / PA@SA, 1 mL of 0.5 mol / L EDTA-2Na was added to lyse the microspheres. After shaking 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), with a flow rate of 0.2mL / min. The proportion of the gradient elution mobile phase changed with time: 0-3min, 70% A; 3-7min, 70%-35% A; 7-10min, 35% A; 10-11min, 35%-70% A; 11-12min, 70% A. Standard samples of different concentrations were set in advance to obtain a standard curve of chromatographic peak area and sample concentration, and then the drug amount in the unknown sample was converted. Finally, the drug loading (LC) and encapsulation efficiency (EE) were 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 lyophilized) and BD / PA@SA prepared according to Section 2.3 (1 mL of sodium alginate (SA) was prepared in pure water at a 2% (w / v) concentration and added or not with the protein aggregates or drug component prepared and lyophilized according to Section 2.1, mixed, and ultrasonically dispersed uniformly. Then, microspheres were prepared by electrostatic droplet technology using a saturated CaCl2 solution at room temperature as a coagulation bath, in which BD / PA and SA were mixed to form microspheres BD / PA@SA) were prepared) were analyzed in artificial gastric fluid, artificial small intestinal fluid, and artificial colonic fluid. First, a control sample BD+BSA was prepared (which contained the same mass of drug and protein as the BD / PA sample (the drug loading and encapsulation efficiency of BD / PA were determined in Section 2.4: approximately 0.916 mg BBR, 0.173 mg DHCL, and 4.08 mg BSA). They were thoroughly mixed and lyophilized to obtain BD+BSA). It contained the same mass of drug and protein as the BD / PA sample (the drug loading and encapsulation efficiency of BD / PA were determined in Section 2.4: approximately 0.916 mg BBR, 0.173 mg DHCL, and 4.08 mg BSA. After thorough mixing, they were lyophilized to obtain BD+BSA. Each lyophilized sample was then placed in a dialysis bag, immersed in 30 mL of artificial gastric fluid, and shaken at 37°C and 100 rpm. After 2 hours, the solution was replaced with 30 mL of artificial small intestinal fluid and shaken for another 4 hours at 37°C and 100 rpm. Finally, the solution was replaced with 30 mL of artificial colonic fluid and shaken for another 42 hours at 37°C and 100 rpm. At specific time points (0.5 h, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, and 48 h), 3 mL of digestive fluid was aspirated (and replenished promptly to maintain a constant volume), and an equal volume of acetonitrile was added to extract the drug. The samples were analyzed using the above-mentioned UPLC program, and the drug release at different time points was calculated to observe the drug release behavior of the BD / PA and BD / PA@SA formulations.

[0050] 2.6 Animal Source and Rearing

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

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

[0053] Cyanine 5.5 (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. After stirring in dark-protected water at 4°C for 6 hours, BD / PA-Cy5.5 was collected using ultrafiltration tubes (molecular weight cutoff: 3000) and encapsulated in SA (BD / PA-Cy5.5@SA). Subsequently, mice with 2.5% (w / v) DSS-induced UC were administered only once with free Cy5.5, BD / PA-Cy5.5, or BD / PA-Cy5.5@SA (containing 0.365 mg / kg of Cy5.5). Healthy mice received only a single dose of BD / PA-Cy5.5@SA. Mice were euthanized 24, 48, and 168 hours after oral administration, and major organs were harvested. Fluorescence images were captured using an IVIS Lumina Series III (PerkinElmer, USA) to analyze the Cy5.5 signal distribution and thus to assess the in vivo biodistribution of BD / PA. Additionally, colonic tissues were collected 24 hours after oral administration and snap-frozen in liquid nitrogen. Frozen sections were prepared and stained with DAPI. Cy5.5-labeled BD / PA deposition in colonic lesions was then observed using an Olympus VS200 Research Slide Scanner.

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

[0055] After a one-week acclimation period, 8-week-old male C57BL / 6J mice were provided with 2.5% (w / v) DSS-containing water for 7 consecutive days, followed by normal water for 3 days. Healthy mice were provided with normal water at all times. Different animal experiments may require different dosing requirements. Generally, during the experimental period following the acclimation period, mice were orally administered 100 mg / kg of drug (5-ASA, the Chinese herbal 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 of SA, ultrasonically dispersed, and then electrostatically droplet-formed)) or saline once daily for a total of 10 times. For experiments investigating antibiotic disruption of the intestinal microbiota, mice were pretreated with a cocktail of antibiotics (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 initiating DSS modeling and drug administration. The mice's body weight was monitored throughout the experiment. On the final day of the experiment, the mice were euthanized and their entire colons were removed. After colon length was measured, tissue sections were prepared from the distal colon. The remaining colon tissue was used for ELISA and transcriptome analysis. In addition, colonic feces were collected for microbial analysis and short-chain fatty acid analysis.

[0056] 2.9 Histological evaluation and immunohistochemical staining analysis

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

[0058] 2.10 Immunofluorescence imaging

[0059] Distal colon tissue sections were blocked with 10% goat serum and incubated with primary antibodies against ZO-1 (1:200) and occludin-1 (1:200). After overnight incubation at 4°C, the sections were incubated with corresponding secondary antibodies for 1 hour at 37°C. Cell nuclei were counterstained with DAPI and observed using an Olympus SpinSR10 confocal microscope, and fluorescence signals were analyzed using 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 on dry ice. The fecal intestinal microbiota was analyzed using the 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] The SCFAs content was detected by Wuhan Maitwell 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 ultrasonicated 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, ultrasonicated for 5 minutes, and then centrifuged at 12000 rpm for 10 minutes (4 ° C). Finally, the supernatant was analyzed using an Agilent7890B-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] Colonic samples were prepared and sequenced using RNA-seq libraries by Wuhan Matwell Biotechnology Co., Ltd. Gene alignments were calculated using HISAT2 software, and FPKM values ​​were calculated to quantify gene expression levels. Differentially expressed genes (DEGs) between groups were identified using DESeq2 software, with an FDR threshold of <0.05 and |log2 FC| ≥1. Gene classification and pathway function annotation were performed using the GO and KEGG databases. Protein-protein interactions within DEGs were analyzed using the STRING database.

[0068] 2.15 In vivo toxicity evaluation

[0069] After a week of acclimation, 8-week-old male C57BL / 6J mice were orally administered with saline or BD / PA@SA (100 mg / kg) once daily for a total of 10 doses. The mice were observed for changes in behavior or weight during the experiment. On day 10, the mice were euthanized, and their blood and major organs (heart, liver, kidney, lung, spleen, and colon) were collected. Major organ indices were assessed, and liver and kidney function and blood routine tests were performed. Finally, H&E staining was used to observe the presence of organ lesions.

[0070] 2.16 Cell culture and CCK-8 assay

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

[0072] 2.17 Statistical Analysis

[0073] Statistical analysis and graphs were performed using GraphPad Prism 10.0. Results are presented as mean ± SEM. Differences between groups were assessed by t-test, one-way or two-way analysis of variance. 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 drug addition was analyzed ( Figure 1 A). BSA changes from dissolved to 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 the lesion areas of different shapes and sizes, while preparing a mixture of two aggregates is more likely to achieve efficient retention. Nano-scale BSA is induced to form nano-micron-scale PA ( Figure 2BC), in which 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 gradual unfolding and exposure of the hydrophobic structure of BSA. ThT fluorescence and circular dichroism spectroscopy further revealed the changes in the secondary structure of the protein ( Figure 1 FH and Table 1). These results indicate that compared to pristine BSA, the β-sheet ratio of PA slightly increased, while the α-helix ratio decreased. Heat-treated BSA not only exhibited molecular aggregation but also altered its peptide chain conformation.

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

[0078]

[0079] Then, 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 due to changes in the solution ionic strength. Previous studies have shown that higher ionic strength can form shorter and more flexible fibers. However, 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), which formed more aggregates ( Figure 4 D), the yield in 24 hours can reach 86% ( Figure 4 E). One 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 support 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 580 nm to 604 nm ( Figure 3C). At the same time, BD also showed similar changes after being mixed with PA. This may be because the energy transfer originally caused by π-π stacking of BBR was reduced after binding to the exposed hydrophobic area of ​​PA, ultimately resulting in higher fluorescence. In addition, the experiment found that the hydrophobic fluorescent dye DiD (simulating hydrophobic drugs) had a good co-localization relationship with FITC-PA ( Figure 3 D). This not only demonstrates the feasibility of PA-based drug loading but also supports the existence of hydrophobic interactions. It can be speculated that these characteristics facilitate the further adaptation of BD / PA to colonic ulcers and drug loading.

[0080] Subsequently, we determined the drug loading and encapsulation efficiency of BD / PA ( Figure 3 E), and based on electrostatic droplet technology, SA was used to encapsulate the 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 contents@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 administering 2.5% (w / v) DSS in drinking water for 7 days, followed by ordinary drinking water for 3 days. The normal group was given ordinary water all the time. The drug was orally administered 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 biodistribution of the drug carrier in the body. The following four groups were specifically set up:

[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 fluorescence signal in the colon of mice orally administered with Cy5.5 and BD / PA-Cy5.5 was weaker and the fluorescence duration was shorter ( Figure 7 AB). This is because the fluorescent molecules are affected by the physiological barriers in the gastrointestinal tract due to the lack of delivery of the SA polysaccharide shell, making it difficult for them to concentrate and remain in the colon. When BD / PA@SA releases its contents BD / PA in the colon, the latter, relying on its nano-micron size, matches different types of ulcer surfaces, and remains in the colon for a long time and releases the drug ( 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 has excellent efficacy in treating DSS-induced acute colitis ( Figure 9 A). Similarly, C57BL / 6J mice were given 2.5% (w / v) DSS in their drinking water for 7 days to establish an acute colitis model. They were then given regular drinking water for 3 days. A control group was given regular water throughout the experiment. Mice were orally administered 100 mg / kg of either drug (5-ASA, the traditional Chinese medicine monomer BD [BBR / DHCL mass ratio = 4:1], BD / PA, or BD / PA@SA) or saline once daily 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 improved weight loss and shortened colon length, and inhibited colon tissue damage, achieving an effect similar to that of the clinical first-line drug 5-ASA ( Figure 9 BE and Figure 10 Regarding the important inflammatory pathological state in UC, we also found that even with a very small amount of drug loading, 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) ( Figure 9FG). However, the therapeutic effects of BD or BD / PA were relatively poor. 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 ( Figure 9 HI). These studies have shown that BD / PA@SA also has excellent anti-inflammatory effects.

[0091] In the traditional formulation of Xianglian Wan, BBR and DHCL are the main synergistic herbal ingredients, with BBR playing the primary anti-inflammatory role. Previous studies have shown that BBR exerts anti-inflammatory effects by inhibiting the NLRP3 inflammasome, arachidonic acid metabolism, and the AKT signaling pathway, while DHCL modulates the MAPK and NF-κB pathways to improve inflammation. Therefore, following the traditional Chinese medicine theory of "monarch, minister, assistant, and envoy," we proposed designing BBR as the "monarch" herb and DHCL as the "minister" herb, aiming for their synergistic effect in UC inflammation. However, limited by the poor bioavailability of BBR and DHCL, these studies typically require high oral doses (e.g., 100 mg / kg and above) to achieve efficacy. Guided by this theory, we designed both "assistant" and "envoy" herbs using modern drug delivery technologies. By leveraging the properties of protein aggregates (PAs) (to aid retention and serve as the "adjuvant" herb) and polysaccharides (SAs) (to target and deliver the "envoy" herb), we achieved superior therapeutic efficacy with reduced doses of the individual herbal drugs (3.73 mg / kg for BBR and 0.67 mg / kg for DHCL). This not only greatly improves the bioavailability of the hydrophobic active ingredient but also reduces the risk of drug accumulation and toxic side effects. It is important to note that the lack of the SA component in the BD / PA group may have a significant impact on the reduced efficacy, and the lack of colon targeting may be a key factor.

[0092] In addition, UC patients often seek treatment after disease onset, and we also investigated the efficacy of BD / PA@SA in a delayed intervention model ( Figure 11 A). An acute model of colitis was established by first administering 2.5% (w / v) DSS to the drinking water of C57BL / 6J mice for 7 days, followed by 11 days of normal drinking water. 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 drugs (5-ASA or BD / PA@SA) or normal saline once a day for a total of 10 doses. Compared with natural recovery (DSS group) and clinical first-line drug treatment (5-ASA group), the mice under BD / PA@SA intervention recovered their weight more quickly, and their weight was significantly improved at the end of treatment ( Figure 11 B), and maintained colon length and intestinal epithelial health ( Figure 11 CE). This suggests 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 investigated 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 ( Figure 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 ( Figure 12 BC), also showed the ability of BD / PA@SA to repair the colonic 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 colonic 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 was decreased ( Figure 12 D) This result also indirectly reflects the restoration of epithelial health after BD / PA@SA treatment. Furthermore, bacteria are part of the colonic environment, and some pathogenic bacteria contribute to the pathogenesis of UC. In addition to anti-inflammatory and colonic epithelial repair, BD / PA@SA may also alleviate UC by regulating the intestinal microbiome.

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

[0096] There is increasing evidence that UC is accompanied by intestinal microbial 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 ( Figure 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 ( Figure 13 AC), the OTU level Venn diagram also supports this point ( Figure 14 B). PLS-DA 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 ( Figure 13 D and Figure 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 ( Figure 13 E), and the bacterial abundances differed between groups at the genus level ( Figure 13 F). Clearly, these results indicate that BD / PA@SA modulates the intestinal flora of colitis mice. To further understand the role of the intestinal microbiota in the therapeutic effects of BD / PA@SA, we conducted an experiment to disrupt the intestinal flora with antibiotics. We first pretreated mice with a broad-spectrum oral antibiotic (abbreviated as ABX) for 5 days ( Figure 13 G), and then an acute colitis model was established by administering 2.5% (w / v) DSS in drinking water for 7 days. Mice were then orally administered 100 mg / kg of the drug (BD / PA@SA) or saline once daily for a total of 10 doses. The results showed that the efficacy of the ABX-BD / PA@SA group on DSS-induced colitis was significantly reduced compared to the BD / PA@SA group without antibiotic pretreatment ( Figure 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] To explore the microbial communities and their potential roles in mediating the therapeutic effects of BD / PA@SA, we conducted species difference analysis ( Figure 15 A). At the genus level, on the one hand, BD / PA@SA significantly reduced the abundance of Desulfovibrio, whose H2S production 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. At the same time, linear discriminant analysis specifically pointed out that Lactobacillus and Lactobacillus_johnsonii were the dominant genera / species in the BD / PA@SA group ( Figure 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 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 ( Figure 15 C). We believe that the PA and SA microspheres that deliver drugs to the colon are the main reason for this result. Studies have reported that SA, as a dietary fiber, becomes a nutrient for intestinal bacteria in vitro and increases 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 are not only "adjuvant" drugs that assist in retention and "enabler" drugs for targeted delivery in our treatment system, but their ordinary properties are transformed into SCFAs after intestinal microbial metabolism. Their therapeutic role has also been transformed into the direction of "minister" drugs, and they have cooperated with "lord" drugs to fight the disease. In addition, in order to further clarify the relationship between the dominant flora and UC indicators, we carried out Spearman correlation analysis ( Figure 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, Lactobacillus johnsonii is considered a key bacterial species regulated by BD / PA@SA, contributing to its therapeutic efficacy to a certain extent.

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

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

[0101] Normal control group CON;

[0102] Disease model DSS group;

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

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

[0105] PA@SA group of disease models treated with SA microspheres encapsulated with PA;

[0106] SA microspheres encapsulating the traditional Chinese medicine monomer BD are used to treat disease models in the BD@SA group;

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

[0108] Similarly, an acute colitis model was established by administering 2.5% (w / v) DSS to C57BL / 6J mice in their drinking water for 7 days, followed by 3 days of regular water. The CON group was given regular 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 daily for a total of 10 doses. BD / PA@SA, which had intact ingredients, still protected mice from DSS-induced weight loss and shortened colon length, while suppressing colon tissue damage and inflammatory state ( Figure 16 In contrast, PA@SA without BBR and DHCL was insufficient to alleviate UC, despite their metabolization into beneficial short-chain fatty acids. Furthermore, BD@SA failed to protect animals from DSS-induced colitis, primarily because the absence of PA prevented the hydrophobic drug from being retained in the ulcer for long enough to exert its effect. In summary, the components of BD / PA@SA are inseparable. While PA (the "adjuvant" drug) and SA (the "enabling" drug) possess certain therapeutic benefits, they must both retain and target the drug. The absence of any of these components compromises the therapeutic efficacy of BD / PA@SA.

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

[0110] To reveal the underlying molecular mechanism by which BD / PA@SA alleviates DSS-induced colitis, we performed RNA-Seq analysis on the colonic 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 ( Figure 18 AB). Next, by counting the significantly differentially expressed genes (DEGs, p-adjust < 0.05, FC > 2) between the different groups, we found that compared with the DSS group, only 5 DEGs were found after BD treatment. 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. The changes in 327 genes were unique to BD / PA@SA treatment ( Figure 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 immune system ( Figure 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 ( Figure 17 D). The connections between these DEGs were further reflected in the protein interaction (PPI) network ( Figure 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. The mechanism is that plasma cells transform from IgA secretory to IgG secretory. 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 ( Figure 17D). Existing reports indicate that SCFAs can participate in the activation and differentiation of intestinal B cells, enhancing the expression of genes related to IgA production. They can also promote B cells to provide the energy and raw materials required for IgA synthesis, effectively driving the conversion of intestinal B cell antibody secretion to the IgA class. BD / PA@SA treatment increased SCFA levels, which may be one of its mechanisms for intervening in intestinal mucosal immunity and treating UC. Furthermore, linoleic acid metabolism, arachidonic acid metabolism, and α-linolenic acid metabolism were also the main pathways enriched for 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 pathogenesis of UC. AA is generally believed to play a key 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, the metabolic pathways of α-linolenic acid (ALA), an ω-3 polyunsaturated fatty acid, may alleviate inflammation in UC, exerting anti-inflammatory effects by inhibiting M1 macrophages and affecting AA metabolism. In this study, alterations in these metabolic pathways may also contribute to the reduced inflammation observed after BD / PA@SA treatment. For example, BBR in BD / PA@SA has been reported to downregulate AA metabolic pathways and improve UC. As a natural active ingredient that inhibits the AA pathway, it has been shown to reduce the expression of COX-2, PGE2, and their corresponding receptors. Furthermore, previous molecular docking experiments have shown that BBR can bind to PLA2 and 5-LOX, potentially affecting AA metabolism by inhibiting enzyme activity. Notably, the upregulated Lactobacillus species after BD / PA@SA treatment can also regulate LA metabolism, alleviating colitis by converting LA into pre-conjugated linoleic acid (CLA), which is beneficial for gut health. In summary, RNA-Seq analysis suggests a potential molecular mechanism by which BD / PA@SA alleviates 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 ( Figure 19 In an in vivo experiment, healthy mice were orally administered 100 mg / kg of BD / PA@SA once daily for 10 doses. Following the study, the mice were euthanized and their blood and major organ samples were analyzed. The results showed that the BD / PA@SA group showed a gradual increase in body weight. Compared to the control group (CON group) that received normal saline, the indices of major organs remained stable, and H&E staining of the tissues showed no abnormalities. Figure 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 ( Figure 19F). This shows that BD / PA@SA has good biosafety.

[0114] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined 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 with 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, and then centrifuged at 28-32°C to separate the drug-loaded protein aggregates to precipitate BD / PA. After freeze-drying, 0.5-1.5ml of 1-3% concentration SA was added, and the mixture was evenly mixed under ultrasound. A saturated CaCl2 solution at room temperature was used as a coagulation bath, and the drug microspheres BD / PA@SA were prepared by electrostatic droplet technology. BD / PA@SA is a drug used to treat 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 amount of DHCL used 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.

Images