Active therapeutic coating formed on mucous membrane interface in situ as well as preparation method and application of active therapeutic coating

Abstract

The invention provides an active therapeutic coating formed on a mucous membrane interface in situ and a preparation method and application thereof, and belongs to the technical field of biological medicine. The invention provides a coating composition for treating mucosal inflammatory diseases. The coating composition comprises probiotics, trivalent bismuth and ethyl gallate. The coating composition can be prepared into active therapeutic coatings (LSTC) formed on different mucous membrane surfaces in situ, and the LSTC has the effect of synergistically regulating a physicochemical barrier, a microbial barrier and an immune barrier. The LSTC disclosed by the invention has a relatively good treatment effect on mucosal inflammation related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to an in-situ formed active therapeutic coating at the mucosal interface, a preparation method thereof, and an application thereof. Background Art

[0002] The mucosal barrier is the first line of defense against infections and environmental stimuli. Its dynamic balance is mainly maintained by commensal microorganisms, physicochemical barriers, and mucosal immune responses. By physically blocking the entry of harmful foreign antigens, microorganisms, and their toxins into the host, the mucosal structure and homeostasis are maintained. The disruption of the mucosal barrier can lead to the occurrence of various diseases, including infectious, allergic, and inflammatory diseases. Restoring mucosal barrier function has become an effective strategy for protecting the host from exogenous stimuli. For example, immunomodulators, anti-tumor necrosis factor (TNF) antibody drugs, are currently popular treatment methods, which reverse the dysregulated mucosal barrier by blocking specific inflammatory pathways. However, suppressing immune function often leads to severe infections and even life-threatening situations. In addition, enzyme inhibitors that block myosin light chain kinase are used to treat inflammatory bowel disease (IBD) by reducing the permeability of tight junctions. Myosin light chain kinase is a key effector of vascular barrier dysfunction. However, these inhibitors can target myosin in different types of tissues, resulting in severe side effects, including hypotension, intestinal obstruction, and even death. More importantly, the single treatment mode makes these treatment methods ineffective clinically. Therefore, exploring innovative methods that can comprehensively restore mucosal barrier function and resist exogenous damaging factors is of great significance for the treatment of various diseases.

[0003] The mucosal surface exposed to the external environment is colonized by a large number of microorganisms. Microorganisms play an important role in regulating mucosal barrier function by communicating with epithelial cells and the mucosal immune system. For example, bacteria in mucus can degrade mucins produced by special epithelial cells and secrete metabolites, thereby affecting mucus production and epithelial cell integrity. At the same time, certain bacteria can affect the functions of the innate immune system and the adaptive immune system to maintain immune homeostasis, but immune dysregulation can trigger various immune disorders through the activation of macrophages, neutrophils, or T cells. The disruption of the epithelial barrier leads to the leakage of bacteria and bacterial derivatives, resulting in systemic deterioration and subsequently aggravating the local disruption of the mucosal barrier. In addition, the inflammatory environment is conducive to the massive reproduction of certain opportunistic pathogens. The specific toxins secreted by opportunistic pathogens can kill beneficial bacteria, thereby promoting epithelial cell necrosis. Therefore, methods for restoring mucosal barrier function may be beneficial for the treatment of mucosal diseases, but methods that can coordinately regulate mucosal barrier function in multiple aspects are rarely reported. Summary of the Invention

[0004] The present invention provides a living-synthetic therapeutic coating formed in situ at the mucosal interface, and a preparation method and application thereof. The living-synthetic therapeutic coating (LSTC) that can be synthesized in situ on the mucosal surface restores and remodels the mucosal barrier function by regulating the physical barrier, immune barrier, and microbial barrier.

[0005] The present invention provides a coating composition for treating mucosal diseases, comprising probiotics, trivalent bismuth, and ethyl gallate.

[0006] In a preferred embodiment of the present invention, the probiotics can act on the mucosal surface.

[0007] In a preferred embodiment of the present invention, the types of probiotics include probiotic bacilli, Clostridium butyricum, Lactobacillus, and Bifidobacterium.

[0008] In a preferred embodiment of the present invention, the trivalent bismuth includes soluble trivalent bismuth salts. When the trivalent bismuth is Bi(NO3)3·5H2O, the concentration ratio of Bi(NO3)3·5H2O to ethyl gallate is (0.05 - 0.8):(0.1 - 4.8).

[0009] In a preferred embodiment of the present invention, the mucosal inflammatory diseases include at least one of the following: oral mucosal diseases, nasal mucosal diseases, ocular mucosal diseases, vaginal mucosal diseases, gastric mucosal diseases, and intestinal mucosal diseases.

[0010] The present invention also provides an application of the above coating composition in the preparation of a living-synthetic therapeutic coating synthesized in situ on the mucosal surface.

[0011] The present invention also provides a living-synthetic therapeutic coating comprising the above coating composition and synthesized in situ on the mucosal surface.

[0012] The present invention also provides a preparation method of the above living-synthetic therapeutic coating, comprising the following steps: dissolving and stirring trivalent bismuth and ethyl gallate to obtain a nano-coating; adding probiotics to the nano-coating and incubating to obtain the living-synthetic therapeutic coating.

[0013] The present invention also provides an application of the above coating composition or the above living-synthetic therapeutic coating in the preparation of a drug for treating mucosal inflammatory diseases.

[0014] In a preferred embodiment of the present invention, the therapeutic effects of the drug include at least one of the following: improving mucosal physicochemical barrier damage, regulating the mucosal microbial barrier, and activating the mucosal immune barrier.

[0015] Beneficial effects: The present invention provides a method capable of in-situ synthesizing an active therapeutic coating (LSTC) on the mucosal surface, and remodeling the mucosal barrier function by synergistically regulating the physical and chemical barrier, microbial barrier and immune barrier of the mucosa to treat mucosal inflammatory diseases. The LSTC includes probiotics, trivalent bismuth and ethyl gallate. The probiotics are mainly derived from various mucosal surfaces and play a key role in the health of the host. Ethyl gallate (EG) has various biological properties, such as anti-inflammatory, antioxidant and mucosal repair. Trivalent bismuth (Bi III ) has a stable structure and can aggregate at the ulcer site to form a polymer coating, which has a unique pharmacological activity of protecting the mucosa. Based on polyphenol-metal complexation and π-π interaction, EG can chelate with Bi III to form a nano-coating in-situ on the mucosal surface through chelation, and has catechol groups in its structure, and the mediated adhesion ability can adhere probiotics to the surface of the nano-coating.

[0016] The present invention can in-situ synthesize the active therapeutic coating (LSTC) on the mucosal surface, and when the LSTC is used to treat different mucosal diseases, it can be prepared by adjusting the composition of probiotics and the coating thickness according to the properties of different mucosal surfaces. In the examples of the present invention, different in-vitro tissues and in-vivo experiments have proved that LSTC can be formed on different mucosal surfaces and has remarkable stability in vivo. Subsequently, the effects of LSTC on regulating the physical and chemical barrier, microbial barrier and immune barrier were evaluated in vitro and in vivo respectively.

[0017] The present invention evaluated the therapeutic effect of LSTC on mucosal inflammation-related diseases in vivo. For example, in the examples, the LSTC prepared with the probiotic Lactobacillus rhamnosus (LGG) was used to treat the mouse vaginitis model. After treatment, it could effectively inhibit the distribution of pathogenic bacteria in the vagina of mice, significantly restore the body weight of mice, reduce the redness and swelling at the vaginal orifice of mice, restore the exfoliation, keratinization and leukocyte infiltration of the vaginal mucosal stratum corneum caused by infection, reduce the infiltration of inflammatory cells, and reduce the expression of inflammatory factors. In the constructed mouse ulcerative colitis model, after treatment with LSTC, the body weight and colon length of mice were significantly increased, the spleen index was decreased, the infiltration of inflammatory cells in the colon was significantly reduced, and pathological changes such as mucosal edema and crypt structure damage were improved. In summary, LSTC has a good therapeutic effect on mucosal inflammation-related diseases. Description of the Drawings

[0018] Figure 1 Schematic diagram for the preparation of LSTC (EG-Bi-LGG) (A) and schematic diagram for restoring mucosal barrier function (B);

[0019] Figure 2The number of LGG adhered to the EG-Bi coatings formed by the reaction of different concentrations of Bi(NO3)3·5H2O (A) and EG (B) (n = 3). In the figure, A: the concentration of EG is 0.6 mg / mL, and the p-value indicates statistical significance compared with 0.2 mg / mL Bi(NO3)3·5H2O in each group; B: the concentration of Bi(NO3)3·5H2O is 0.2 mg / mL, and the p-value indicates statistical significance compared with 0.6 mg / mL EG in each group;

[0020] Figure 3 Cell viability of 293T cells (A) and J774A.1 cells (B) after incubation with EG and Bi(NO3)3·5H2O respectively (n = 3);

[0021] Figure 4 Growth curve of LGG in MRS medium containing 0.6 mg / mL EG and 0.2 mg / mL Bi(NO3)3·5H2O, with untreated LGG as the control (n = 6);

[0022] Figure 5 Characterization diagrams of the EG-Bi coating. In the figure, A: SEM; B: TEM; C: AFM; D: Contact angle of the EG-Bi coating formed on the silicon wafer; E: Fluorescence image of the BSA-FITC-labeled EG-Bi coating; F: 3D LSCM images of the low-concentration EG-Bi coating and the high-concentration EG-Bi coating;

[0023] Figure 6 Characterization diagrams of LSTC (LGG, EF, LD, EcN). In the figure, A: SEM; B: TEM; C: LSCM; D-G: Bacterial counts of LGG, EF, LD, and EcN on LSTC after culturing on MRS or LB solid medium for 24 h (n = 3);

[0024] Figure 7 Result diagrams of the preparation of LSTC in vitro tissues. In the figure, A-B: IVIS images (A) of mouse vaginal tissues after incubation with LGG or LSTC and the corresponding quantitative statistical charts of fluorescence intensity (B) (n = 5); C: Statistical count of the number of LGG adhered to mouse vaginal tissues (n = 5); D: ICP-MS detection of the content of Bi III ions on the vaginal surface (n = 4); E-F: IVIS images (E) of porcine vaginal tissues after incubation with LGG or LSTC (n = 3) and the corresponding quantitative statistical charts of fluorescence intensity (F); G: Statistical count of the number of LGG adhered to the porcine vaginal surface (n = 3); H: IVIS images (H) of the colon after incubation with LGG or LSTC solution and the corresponding quantitative statistical charts of fluorescence intensity (I) (n = 5); J: Statistical count of the number of LGG adhered to the colon surface (n = 5);

[0025] Figure 8 Figure showing the results of preparing LSTC in in vivo tissues. In the figure, A - B: IVIS images (A) and corresponding quantitative fluorescence intensity statistics (B) of the vagina of mice injected with LGG or LSTC intravaginally for 6 h (n = 5); C: Statistical count of the number of LGG attached to the vagina of mice (n = 5); D - E: IVIS images (D) and corresponding quantitative fluorescence intensity statistics (E) of the colon tissue of mice 6 h after colon administration; F: Statistical count of the number of LGG attached to the colon of mice (n = 5); G - H: IVIS images (G) and corresponding quantitative fluorescence intensity statistics (H) of the vagina of mice at different time points after intravaginal administration of LGG or LSTC (n = 5).

[0026] Figure 9 Figure showing the effect of LSTC on regulating the mucosal physical and chemical barrier in vivo and in vitro. In the figure, A: AF488 - WGA (red) staining images of the vagina tissue of mice infected with S. aureus and treated continuously with PBS, LGG, EG - Bi, or LSTC for 7 days; B: Statistical count of the average fluorescence intensity of AF488 - WGA in the vagina tissue of mice (n = 4); Blue indicates the nucleus stained with DAPI; C: Immunofluorescence images of the expression of occludin (green) and ZO - 1 (red) in the vagina tissue of mice infected with S. aureus after treatment; Statistical count of the average fluorescence intensity of (D) occludin and (E) ZO - 1 in the vagina tissue of mice (n = 3); Blue indicates the nucleus stained with DAPI.

[0027] Figure 10 Figure showing the effect of LSTC on regulating the mucosal immune barrier in vivo. After mice infected with S. aureus were continuously treated with PBS, LGG, EG - Bi, or LSTC for 7 days, vaginal samples of mice were collected for mRNA transcriptome sequencing results (n = 3). In the figure, A: Volcano plot of differentially expressed genes; B: KEGG pathway enrichment analysis results showing the KEGG pathway enrichment analysis of differentially expressed genes down - regulated in the LSTC group compared with the PBS group (p < 0.05), and the top 30 enriched KEGG pathways are shown in the figure; C: GO enrichment analysis of down - regulated genes in the PBS group and the LSTC group (p < 0.05); D: GSEA showing the enrichment of genes related to inflammatory responses; E: Heat map of the expression levels of genes related to inflammasome activation in all groups; F: GSEA analysis of the differences in immune infiltration of mice in different treatment groups.

[0028] Figure 11Figure showing the effect of LSTC on regulating the mucosal biological barrier in vitro and in vivo. After continuous treatment with PBS, LGG, EG-Bi, or LSTC for 7 days in S. aureus-infected mice, vaginal lavage fluid was collected for 16S rRNA microbiota sequencing. In the figure: A: Fluorescent image of the distribution of S. aureus in mouse vaginal tissue; B: ACE index (n = 5); C: Chao1 index (n = 5); D: PCoA results of vaginal microbiota at the OTU level (n = 5); E: Relative abundance of vaginal microbiota at the class level; F-G: Relative abundance of Lachnospiraceae NK4A136 group (F) and Clostridium sensu stricto 1 (G) at the genus level (n = 5);

[0029] Figure 12 Figure showing the results of the efficacy evaluation of LSTC in the treatment of vulvovaginal candidiasis. In the figure: A: Construction and treatment plan of the mouse vulvovaginal candidiasis model; B: Changes in mouse body weight during treatment (n = 6); C: PAS staining image of mouse vaginal secretions; D: Quantitative statistics of the number of C. albicans in mouse vaginal lavage on the 7th day after treatment (n = 6); E-F: Expression levels of IL-1β and TNF-α in mouse serum (n = 6); G: Quantitative statistics of leukocyte infiltration in mouse vaginal tissue (n = 3); H: H&E staining map of mouse vaginal tissue; I: MPO staining image of mouse vaginal tissue after treatment; J: Quantitative statistics of the number of MPO + cells per 10× field of view of vaginal sections (n = 3);

[0030] Figure 13 Figure showing the efficacy evaluation of LSTC in the treatment of aerobic vaginitis. In the figure: A: Construction and treatment plan of the mouse aerobic vaginitis model; B: Changes in mouse body weight during treatment (n = 5); C-D: Quantitative statistics of the number of S. aureus in mouse vaginal lavage (C) and the corresponding fluorescent image of the LB agar plate (D) on the 7th day after treatment (n = 5); E: H&E staining map of mouse vaginal tissue; F: Expression level of IL-6 in mouse serum (n = 5); G: Quantitative statistics of the number of MPO + cells per 10× field of view of vaginal sections (n = 3); H: MPO staining image of mouse vaginal tissue after treatment;

[0031] Figure 14 Figure showing the results of the efficacy evaluation of LSTC in the treatment of ulcerative colitis in mice. In the figure: A: Establishment and treatment plan of the mouse UC model; B: Body weight curve of mice during treatment (n = 5); C: Spleen index of mice (n = 5); D: Average length of the colon (n = 5); E: Photo of the colon; F: Representative H&E staining map of the colon. Detailed implementation methods

[0032] The present invention provides a coating composition for treating mucosal inflammatory diseases, comprising probiotics, trivalent bismuth, and ethyl gallate.

[0033] The mucosal diseases described in the present invention include at least one of the following: oral mucosal diseases, nasal mucosal diseases, ocular mucosal diseases, vaginal mucosal diseases, gastric mucosal diseases, and intestinal mucosal diseases, and there are no special limitations on the types and causes of various mucosal inflammatory diseases.

[0034] The probiotics described in the present invention can act on various mucosal surfaces, such as probiotics mainly present on the mucosal surface and playing a key role in the health of the host, including probiotic bacilli, Clostridium butyricum, Lactobacillus, and Bifidobacterium, etc. In one embodiment, the probiotic Lactobacillus rhamnosus (LGG) mainly present in the gastrointestinal tract, respiratory tract, and vagina is taken as an example for illustration, but it cannot be regarded as the entire protection scope of the present invention.

[0035] The ethyl gallate (EG) described in the present invention is an active ingredient derived from gallnuts and has various biological properties, such as anti-inflammatory, antioxidant, and mucosal repair effects. The Bi III is the most stable ionic form of bismuth element, which can aggregate at the ulcer site to form a polymer coating and has a unique pharmacological activity of protecting the mucosa. And based on polyphenol-metal complexation and π-π interaction, EG can chelate with Bi III to in-situ form a nano-coating on the mucosal surface ( Figure 1 in A), and has a catechol group in its structure, and the adhesion ability mediated by it can adhere LGG to the surface of the nano-coating.

[0036] In a preferred embodiment of the present invention, the Bi III can be a soluble ionic salt of trivalent bismuth known in the art, such as Bi(NO3)3·5H2O used in one embodiment, and it is confirmed in the embodiment that the optimal final concentrations of Bi(NO3)3·5H2O and EG are 0.2 mg / mL and 0.6 mg / mL, and at this time, the formed nano-coating (EG-Bi) has the strongest ability to adhere probiotics.

[0037] The present invention also provides the application of the above coating composition in the preparation of an active therapeutic coating synthesized in-situ on the mucosal surface.

[0038] In the embodiments of the present invention, animal models are respectively used to prepare at the ex-vivo tissue interface and in-vivo tissue interface, and it is found that the LSTC can be synthesized in-situ at the tissue interface.

[0039] The present invention also provides an active therapeutic coating comprising the above coating composition and synthesized in-situ on the mucosal surface.

[0040] In one embodiment of the present invention, the effects of LSTC on regulating the physical and chemical barrier, microbial barrier, and immune barrier were evaluated in vitro and in vivo, as follows: Figure 1 as shown in B below, the specific manifestations are as follows:

[0041] (1) The regulatory effect of LSTC on the mucosal physical and chemical barrier: By constructing a vaginal disorder model in mice, it was found that LSTC can repair the damaged goblet cells due to vaginal disorders in terms of quantity and function, and at the same time increase the expression of tight junction proteins, improving the damage of the mucosal physical and chemical barrier caused by vaginal disorders.

[0042] (2) The regulatory effect of LSTC on the mucosal microbial barrier: A mouse vaginal disorder model was established to evaluate the inhibitory effect of LSTC on pathogenic bacteria and the regulatory effect on microorganisms in vivo. It was found in the mouse vaginal tissue that LSTC can significantly improve the distribution of the pathogenic bacterium S. aureus in the tissue, and improve the richness and diversity of the mouse vaginal microbiota, and there was no significant difference in the microbial composition of the mice treated with LSTC and that of healthy mice.

[0043] (3) The regulatory effect of LSTC on the mucosal immune barrier: Verified by the mouse vaginitis disorder model, it was found that LSTC mainly alleviates mucosal inflammation by inhibiting the activation of inflammasomes, indicating that LSTC effectively down-regulates the inflammatory response and triggers anti-infection immunity in the vagina.

[0044] In one embodiment of the present invention, the therapeutic effect of LSTC on mucosal inflammation-related diseases was evaluated in vivo. First, an aerobic vaginitis model in mice was constructed with S. aureus. After treatment with LSTC, the distribution of the pathogenic bacterium S. aureus in the vagina of mice could be effectively inhibited, the body weight of the mice could be significantly restored, the exfoliation, keratinization, and leukocyte infiltration of the vaginal mucosal stratum corneum caused by the infection could be restored, the infiltration of inflammatory cells could be reduced, and the expression of inflammatory factors IL-6, TNF-α, and IL-1β could be decreased. Subsequently, a fungal vaginitis model in mice was constructed with C. albicans. After treatment with LSTC, the distribution of the pathogenic bacterium C. albicans in the mice could also be inhibited, while the pathological damage of the vaginal tissue was alleviated, the infiltration of inflammatory cells was reduced, and the expression of inflammatory factors IL-6, TNF-α, and IL-1β was decreased. In addition to vaginal mucosal inflammation, in another embodiment of the present invention, a mouse ulcerative colitis model was constructed with 4% DSS. After treatment with LSTC, the body weight and colon length of the mice could be significantly increased, the spleen index could be decreased, the infiltration of inflammatory cells in the colon could be significantly reduced, and pathological changes such as mucosal edema and crypt structure damage could be improved. In summary, LSTC has a good therapeutic effect on mucosal inflammation-related diseases.

[0045] The present invention also provides a method for preparing the above-mentioned active therapeutic coating, which includes the following steps: dissolving trivalent bismuth and ethyl gallate and then stirring and mixing them to obtain a nano-coating; adding probiotics to the nano-coating, mixing and incubating to obtain the active therapeutic coating of the coating composition.

[0046] In the present invention, Bi III and EG are dissolved in ddH2O and stirred at 300 rpm for 10 min at room temperature; the supernatant is discarded, and after adding probiotics, it is incubated at 200 rpm at room temperature for 5 min to prepare the LSTC.

[0047] The present invention also provides the application of the above-mentioned therapeutic coating in the preparation of a drug for treating mucosal diseases.

[0048] In a preferred embodiment of the present invention, the therapeutic effects of the drug include at least one of the following: improving mucosal physicochemical barrier damage, regulating mucosal microbial barrier, and regulating mucosal immune barrier.

[0049] To further illustrate the present invention, the following examples are used to describe in detail an active therapeutic coating formed in situ at the mucosal interface provided by the present invention, its preparation method and application, but they should not be construed as limiting the protection scope of the present invention.

[0050] In the embodiments of the present invention, unless otherwise specified, the solutions, reagents and methods used are all conventional in the art.

[0051] 1. Experimental strains and plasmids: Escherichia coli Nissle 1917 (EcN), Lactobacillus rhamnosus (LGG), Enterococcus faecalis (EF), Lactobacillus bulgaricus (LB), Staphylococcus aureus (S. aureus), and Candida albicans (C. albicans) were purchased from the China General Microbiological Culture Collection Center (CGMCC, China). Before each experiment, S. aureus and EF were stored on LB agar plates, and single colonies were picked and cultured overnight (200 rpm, 37 °C) in LB liquid medium. Single colonies of LGG and LB were picked on MRS agar plates and cultured overnight (200 rpm, 37 °C) in MRS liquid medium. C. albicans was stored on YPD agar plates, and single colonies were picked and recultured overnight (200 rpm, 37 °C) in YPD liquid medium. Plasmids RN4220T61-eGFP (erythromycin resistance) and pBBR1MCS2-Tac-mCherry (kanamycin resistance) were purchased from domestic suppliers and used according to the method.

[0052] 2. Cell culture: J774A.1 mouse macrophages, 293T human embryonic kidney cells, Caco-2 human colorectal adenocarcinoma cells, and HT29-MTX-E12 goblet cells were purchased from the American Type Culture Collection (ATCC). VK2 / E6E7 human vaginal epithelial cells were provided by the research group of Researcher Lizeng Gao from the Institute of Nanozymes, Chinese Academy of Medical Sciences (Metastable Iron Sulfides Gram-Dependently Counteract Resistant Gardnerella Vaginalis for Bacterial Vaginosis Treatment). All cells were cultured in DMEM complete medium (containing 10% serum and 1% penicillin-streptomycin solution) in a cell culture incubator (37 °C, 5% CO2).

[0053] 3. Animals: Mice (ICR, female, 6 - 8 weeks old) were purchased from Beijing Sino - Bio Biotechnology Co., Ltd. and raised in an SPF environment at an environmental temperature of 22°C with a standard 12 - h day - night cycle. All animal experiments were conducted in accordance with institutional guidelines and approved by the Animal Care and Use Committee of Shanghai Yishang Biotechnology Co., Ltd. (IACUC - 2023 - Mi - 214).

[0054] Example 1: Preparation and Property Study of EG - Bi Nanocoating

[0055] 1. Preparation of artificial mucosa

[0056] Take 1.5 g of agar, add 100 mL of distilled water, dissolve it by heating in a microwave oven, cool it to about 60°C, add 1 mg / mL mucin, and vortex - mix to form artificial mucosa.

[0057] 2. Preparation of EG - Bi nanocoating

[0058] Determine the optimal concentration ratio of EG and Bi to form the coating according to the ability to adhere LGG. Dissolve Bi(NO3)3·5H2O with a final concentration of 0.2 mg / mL and different final concentrations of EG (0.1, 0.3, 0.6, 1.2, 2.4, 4.8 mg / mL) in ddH2O, add it to the surface of the artificial mucosa, and stir at 300 rpm for 10 min at room temperature. Discard the supernatant, add 5.0×10 7 CFUs / mL of LGG, incubate at 200 rpm for 5 min at room temperature, discard the supernatant, wash 3 times with PBS to remove un - adhered LGG, homogenize, evenly coat on MRS solid medium, and perform colony counting after culturing in an incubator at 37°C for 48 h.

[0059] Similarly, add 0.6 mg / mL EG and different concentrations of Bi(NO3)3·5H2O (0.05, 0.1, 0.2, 0.4, 0.8 mg / mL) to the artificial mucosa layer, stir at 300 rpm for 10 min at room temperature. Discard the supernatant, add 5.0×10 7 CFUs / mL of LGG, incubate at 200 rpm for 5 min at room temperature, discard the supernatant, wash 3 times with PBS to remove un - adhered LGG, homogenize, evenly coat on MRS solid medium, and perform colony counting after culturing in an incubator at 37°C for 48 h.

[0060] The results are as Figure 2 shown. The nanocoating (EG - Bi) formed by 0.6 mg / mL EG and 0.2 mg / mL Bi(NO3)3·5H2O has the strongest ability to adhere LGG.

[0061] 3. Biocompatibility of EG - Bi nanocoating

[0062] (1) Cytotoxicity assay

[0063] The cytotoxicity of the EG-Bi nanocoating was evaluated using a cell counting kit-8 (CCK-8). 293T and J774A.1 cells in the logarithmic growth phase (1.0×10 4 / well, 100 μL) were seeded in 96-well plates and cultured overnight in a cell incubator at 37 °C. After the cells adhered, the medium in each well plate was replaced with 100 μL of serum-free DMEM medium, and a mixture of 0.6 mg / mL EG and 0.2 mg / mL Bi(NO3)3·5H2O was added for co-incubation. At 6, 12, and 24 h, 10 μL of CCK-8 solution was added to each well, and the absorbance value (OD) was measured at 450 nm using a microplate reader, and the cell viability was calculated.

[0064] The results are as Figure 3 shown. Even after 24 h of culture, the EG-Bi nanocoating had no cytotoxicity to 293T and J774A.1 cells.

[0065] (2) Bacterial viability assay

[0066] To evaluate the effect of the EG-Bi nanocoating on bacterial growth, Lactobacillus rhamnosus GG (LGG) in the logarithmic growth phase at 37 °C (1×10 5 CFUs / well, 200 μL) was resuspended in MRS medium in 96-well plates, and a mixture of 0.6 mg / mL EG and 0.2 mg / mL Bi(NO3)3·5H2O was added. At the same time, LGG was used as a control and incubated with gentle shaking at 37 °C. Then, the OD of LGG was recorded at 600 nm every 0.5 h using a microplate reader. 600 value.

[0067] The results are as Figure 4 shown. LGG still maintained the ability to grow and divide in the EG-Bi nanocoating, and its growth curve was similar to that of LGG without the nanocoating.

[0068] 4. Characterization of the EG-Bi nanocoating

[0069] (1) Transmission electron microscopy (TEM) characterization

[0070] 10 μL of an aqueous solution containing 0.6 mg / mL EG and 0.2 mg / mL Bi(NO3)3·5H2O was carefully dropped onto a carbon-coated copper grid, and after standing at room temperature for drying, the morphology of the sample was observed by TEM.

[0071] (2) Scanning electron microscopy (SEM) characterization

[0072] After the silicon wafer was treated with a surface plasma cleaner, 2 μL of an aqueous solution containing 0.6 mg / mL EG and 0.2 mg / mL Bi(NO3)3·5H2O was carefully dropped onto the silicon wafer. After drying at room temperature, gold was sputtered, and the morphology of the EG-Bi nanocoating was observed by SEM.

[0073] (3) Atomic force microscope (AFM) characterization

[0074] After the silicon wafer was treated with a surface plasma cleaner, 2 μL of an aqueous solution containing 0.6 mg / mL EG and 0.2 mg / mL Bi(NO3)3·5H2O was carefully dropped onto the silicon wafer. After drying at room temperature, its morphology was observed using AFM.

[0075] (4) Hydrophilicity and hydrophobicity detection

[0076] After the silicon wafer was treated with a surface plasma cleaner, the prepared artificial mucosa (1.5% agar containing 1 mg / mL mucin) was added. 2 μL of an aqueous solution containing 0.6 mg / mL EG and 0.2 mg / mL Bi(NO3)3·5H2O was carefully dropped onto the surface of the artificial mucosa. After drying at room temperature, the contact angle was measured using a contact angle analyzer.

[0077] (5) Confocal laser scanning microscope (CLSM) characterization

[0078] Take a 6-well plate and add the prepared artificial mucosa (1.5% agar containing 1 mg / mL mucin) to the bottom of the well. Add 500 μL of an aqueous solution containing 0.6 mg / mL EG and 0.2 mg / mL Bi(NO3)3·5H2O, stir at 300 rpm for 10 min, discard the supernatant, add 0.2 mg / mL BSA-FITC (just cover the coating), incubate at room temperature for 10 min, discard the supernatant, wash twice with ultrapure water. At the same time, use the sample without the EG-Bi nanocoating as a control, take images with a chemiluminescence imaging system. At the same time, cut it into about 1 cm 2 and place it upside down on a cover glass, and observe the coating through CLSM.

[0079] The results of SEM and TEM are as Figure 5 shown in A - B in the figure. Dense coatings were formed by EG-Bi nanoparticles on the surface. The AFM image is as Figure 5 shown in C in the figure. The thickness of the EG-Bi nanocoating is about 96.8 ± 1.1 nm. The hydrophilicity and hydrophobicity results are as Figure 5 shown in D in the figure. After the surface of the silicon wafer was modified with the EG-Bi nanocoating, the water contact angle decreased from 88.5 ± 2.0° to 13.9 ± 1.2°, indicating that the nanocoating has excellent hydrophilic surface properties and can better adhere to probiotics.

[0080] The mucosal barrier is usually a gel formed by the covering of mucus. Therefore, in this invention, an artificial mucosa was prepared in vitro. After adding an EG-Bi nanocoating to its surface, it was incubated with bovine serum albumin-fluorescein isothiocyanate (BSA-FITC) for 10 min, and fluorescence scanning was performed using a chemiluminescence gel imaging scanner, while the unmodified artificial mucosa was used as a control group. The results showed that there was a uniform FITC-labeled EG-Bi nanocoating on the surface of the artificial mucosa ( Figure 5 in E), indicating that EG-Bi was successfully modified on the mucosal surface. The EG-Bi nanocoatings formed at different concentrations were characterized by LSCM three-dimensional laser scanning confocal microscopy (3D-SCM). The results showed that the thickness of the EG-Bi nanocoating increased with the increase in the concentrations of EG and Bi(NO3)3·5H2O. When the concentrations of EG and Bi(NO3)3·5H2O were increased from 0.6 and 0.2 mg / mL to 1.2 and 0.4 mg / mL respectively, the thickness of the EG-Bi nanocoating increased by about 1.5 times ( Figure 5 in F). Therefore, the thickness of the coating formation can be regulated according to different application environments.

[0081] Example 2 Preparation of LSTC

[0082] On the basis of preparing the EG-Bi nanocoating in Example 1, 5.0×10 7 CFUs / mL of LGG, 2.0×10 8 CFUs / mL of EF, LD, and EcN were added to the surface of the EG-Bi nanocoating, and stirred at 300 rpm for 5 min at room temperature to form LSTC.

[0083] (1) TEM Characterization

[0084] 10 μL of an aqueous solution containing 0.6 mg / mL of EG and 0.2 mg / mL of Bi(NO3)3·5H2O was carefully dropped onto a carbon-coated copper grid. After standing at room temperature and drying, 10 μL of 5.0×10 7 CFUs / mL of LGG, 2.0×10 8 CFUs / mL of EF, LD, and EcN were respectively added. After drying at room temperature, it was washed 3 times with ultrapure water, and after standing at room temperature and drying, the sample morphology was observed by TEM, while the sample without the EG-Bi nanocoating was used as a control.

[0085] (2) SEM Characterization

[0086] After the silicon wafer was treated by a surface plasma cleaner, 2 μL of an aqueous solution containing 0.6 mg / mL of EG and 0.2 mg / mL of Bi(NO3)3·5H2O was carefully dropped onto the silicon wafer. After drying at room temperature, 10 μL of 5.0×107 CFUs / mL of LGG, 2.0×10 8 CFUs / mL of EF, LD, and EcN. After drying at room temperature, they were washed three times with ultrapure water, and their morphologies were observed using SEM, with the EG-Bi nanocoating-free sample used as a control.

[0087] (3) CLSM Characterization

[0088] Take a 6-well plate and add the prepared artificial mucosa (1.5% agar containing 1 mg / mL mucin) to the bottom of the wells. Add 500 μL of an aqueous solution containing 0.6 mg / mL of EG and 0.2 mg / mL of Bi(NO3)3·5H2O, stir at 300 rpm for 10 min, discard the supernatant, and take 1 mL of 5.0×10 7 CFUs / mL of LGG_cy5.5, LB_cy5.5, EF_cy5.5, EcN (carrying the pBBR1MCS2-Tac-mCherry plasmid, kanamycin resistance) (Encoding bacterial colonization and therapeuticmodality by wrapping with an adhesive drug-loadable nanocoa), incubate at room temperature for 5 min, discard the supernatant, wash three times with ultrapure water, and use the sample without the EG-Bi nanocoating as a control. Cut it into pieces about 1 cm 2 and place it upside down on a coverslip. Observe the coating through CLSM. Put the sample into a centrifuge tube, add 1 mL of sterile PBS for homogenization, and dilute it serially. Take 50 μL of the diluted sample containing LGG and LB and spread it on MRS solid medium, EF on LB solid medium, and EcN on LB solid medium containing 100 μg / mL kanamycin resistance. Culture in a 37°C bacterial incubator until single colonies grow and then perform colony counting.

[0089] After incubating LGG on the EG-Bi nanocoating for 10 min, both SEM images and TEM images showed that the number of LGG attached to the surface modified with the EG-Bi nanocoating was significantly increased compared to the unmodified mucosal surface ( Figure 6 A and B in). Subsequently, LGG was labeled with Cy5.5, and the ability of the EG-Bi nanocoating to adhere bacteria was further confirmed by LSCM, and the results showed obvious fluorescence signals in the EG-Bi nanocoating ( Figure 6 C in).

[0090] To quantify the number of bacteria adhered to the coating, 5.0×10 7 CFUs of LGG were added to 4.5 cm 2Incubate on the surface of the EG-Bi nanocoating for 5 min, wash with PBS to remove unattached bacteria, then homogenize, coat, and perform colony counting on the adhered LGG. The results showed that the number of LGG adhered to the EG-Bi coating surface increased by 9.8 times compared to the unmodified surface ( Figure 6 in D). After calculation, approximately 2.7×10 7 CFUs of LGG can adhere to the 4.5 cm 2 EG-Bi nanocoating surface.

[0091] Next, the present invention studied the universality of the EG-Bi nanocoating's adhesion ability to different probiotics. Gram-positive bacteria, including EF and LD, and Gram-negative bacteria EcN were selected. The results of SEM, TEM, and CLSM showed that the amount of EF, LD, and EcN adhered to the EG-Bi nanocoating surface increased significantly compared to the unmodified mucosal surface ( Figure 6 in A-C). Through quantitative statistics of the adhered bacteria, it was found that the adhesion amounts of EF, LD, and EcN on the EG-Bi nanocoating surface increased by 20.7 times, 30.1 times, and 37.2 times, respectively ( Figure 6 in E-G). In summary, these results indicate that LSTC can be formed on the in vitro mucosal surface and has high biocompatibility.

[0092] Example 3 Preparation of LSTC at the tissue interface

[0093] 1. Prepare LSTC on the vaginal tissues of mice and pigs

[0094] Cover the bottom of the wells in a 6-well plate with a layer of 1.5% agar. Take out the vaginal tissues of mice and pigs, longitudinally dissect them, fix them on the surface of the agar layer with the mucosal layer facing up, and then add an aqueous solution containing 0.6 mg / mL EG and 0.2 mg / mL Bi(NO3)3·5H2O to the surface of the vaginal mucosal tissue and stir at 300 rpm for 10 min. Take out the vaginal tissues with the adhered EG-Bi nanocoating, add them to 500 μL of 5×10 7 CFUs / mL LGG_Cy5.5 bacterial solution, and incubate at 300 rpm for 5 min. Wash 3 times with PBS to remove unattached bacteria. Then observe the formed LSTC in the vaginal tissues of mice using IVIS. Subsequently, put the vaginal tissues into PBS for tissue homogenization, perform gradient dilution with PBS, take 50 μL and evenly coat it on the MRS solid medium, and incubate at 37 °C for 48 h to count the LGG colonies. At the same time, use the vaginal tissues without the EG-Bi nanocoating as a control.

[0095] The results showed that LSTC was successfully prepared in the vaginal tissues of pigs and mice ( Figure 7in A-G). The IVIS results showed that after LSTC treatment, the fluorescence intensity of the vaginal tissue in mice increased significantly ( Figure 7 in A-B), indicating that LSTC was successfully formed at the mucosal interface of the vagina in mice. The vaginal tissue was collected, homogenized, and subjected to bacterial plate counting to quantitatively analyze the number of bacteria attached in LSTC. The results showed that the amount of LGG attached increased by 5.7 times after the formation of LSTC in the vaginal tissue of mice compared with the control group ( Figure 7 in C). In addition, the concentration of bismuth ions in the LSTC-modified tissue was detected by inductively coupled plasma mass spectrometry (ICP-MS). The content of bismuth ions in the LSTC group was significantly higher than that in the control group, demonstrating the presence of bismuth ions in the coating ( Figure 7 in D). In addition, we also prepared LSTC in the vaginal tissue of pigs, and the results were consistent with those of the vaginal tissue of mice ( Figure 7 E-G).

[0096] 2. Preparation of LSTC in mouse colon tissue

[0097] Six- to eight-week-old female ICR mice were selected, anesthetized and sacrificed, and the colon tissue of the mice was removed, divided into segments of 1-1.5 cm, the intestinal segments were everted to expose the mucosal layer, and the two everted ends were carefully ligated with surgical sutures. The prepared colon segments were randomly divided into two groups, with 5 segments in each group. One group of each segment was placed in an aqueous solution containing 0.6 mg / mL EG and 0.2 mg / mL Bi(NO3)3·5H2O and stirred at 300 rpm for 10 min. The intestinal segments with EG-Bi nanocoating attached were taken out and added to 500 μL of 5×10 7 CFUs / mL LGG_Cy5.5 bacterial solution and incubated at 300 rpm for 5 min. The intestinal segments were washed 3 times with PBS to remove unattached bacteria. Then, the LSTC formed in the mouse intestinal segments was observed using an in vivo imaging system (Invivoimage system, IVIS). Subsequently, the intestinal segments were placed in PBS for tissue homogenization, serially diluted with PBS, and 50 μL was evenly spread on MRS solid medium and cultured in a 37 °C bacterial incubator for 48 h to count the LGG colonies. At the same time, the intestinal segment tissue without EG-Bi nanocoating was used as a control.

[0098] Imaging was performed using the in vivo imaging system. The results were consistent with the data of the vaginal tissue. The fluorescence intensity of the colon tissue with LSTC formation increased significantly, and the number of bacteria attached in the colon tissue increased by 4.9 times compared with the unmodified colon tissue. The above results all indicated that LSTC could be successfully formed on the in vitro mucosal tissue ( Figure 7 in H-J).

[0099] 3. Preparation of LSTC at the in vivo tissue interface

[0100] Mice (ICR, female, 6 - 8 weeks old) were randomly divided into 2 groups, with 5 mice in each group. After anesthetizing the mice, 100 μL of a solution containing 0.6 mg / mL EG, 0.2 mg / mL Bi(NO3)3·5H2O, and 5×10 7 CFUs / mL of LGG_Cy5.5 solution was injected vaginally. The control group was given an equal amount of LGG_Cy5.5. After 6 h, the mice were anesthetized and imaged using IVIS. Then, the mice were euthanized, and the vaginal tissues were removed for IVIS imaging. The tissues were placed in 1 mL of PBS for homogenization, serially diluted with PBS, and 50 μL was evenly spread on MRS solid medium and incubated at 37 °C for 48 h to count the LGG colonies. The preparation process of LSTC in the mouse intestine was the same as above.

[0101] By injecting the LSTC solution into the vagina of mice, while using an equal amount of Cy5.5-labeled LGG solution as a control. Six hours after administering LSTC, the fluorescence intensity in the vaginal area of the mice increased significantly. Subsequently, collecting the vagina of the mice for ex vivo imaging further confirmed this result ( Figure 8 in A - B). The vaginal tissues of the mice were collected for homogenization, plating and counting to quantify the number of LGG adhered after LSTC formation. The results showed that the number of LGG adhered in the LSTC group increased by 4.5 times compared with the control ( Figure 8 in C). It indicates that LSTC can be successfully formed in the vagina of mice in vivo. In addition, using the same method, the LSTC solution was administered to the rectal part of the mice. After 6 h, the colon tissues were collected for imaging, and the results were the same as above. The fluorescence intensity in the colon tissues of the mice treated with LSTC increased significantly, and the number of LGG adhered also increased significantly, further verifying that LSTC can be formed in vivo ( Figure 8 in D - F).

[0102] 4. Evaluation of the in vivo stability of LSTC

[0103] Using the same method as in 3, LSTC was prepared in the vaginal tissues of mice. The mice were imaged every 3 h using IVIS to observe the in vivo stability of LSTC for 48 h continuously. At the same time, an equal amount of LGG_Cy5.5 was injected vaginally as a control.

[0104] The results are as Figure 8 shown in G - H. When only LGG was given, the fluorescence signal at the vagina of the mice decreased rapidly within 12 h. In contrast, after administering LSTC, even after 2 days, the fluorescence signal at the vagina of the mice was still higher than that of the LGG group, indicating that LSTC has significant stability in vivo. It shows that LSTC can be formed on multiple mucosal interfaces in vivo and has good stability in vivo.

[0105] Example 4 Regulation of mucosal physical and chemical barriers by LSTC in vitro and in vivo

[0106] 1. Effect of LSTC on mucin secretion in tissues

[0107] To evaluate the regulatory effect of LSTC on the physical and chemical barrier in vivo, a mouse vaginal disorder model was constructed by intravaginal injection of 4.0×10 9 CFUs S. aureus into mice, and then the mice were treated with intravaginal injection of LSTC solution for 7 consecutive days. The healthy group, PBS, and equal amounts of EG-Bi and LGG groups were used as controls. After the mice were anesthetized and euthanized, the vaginal tissues were taken out, frozen quickly in liquid nitrogen, fixed, sectioned to make frozen sections for mucus staining, so as to analyze the regulatory effect of LSTC on the chemical barrier of the mouse vagina. The mucus in the mouse vaginal tissue was stained with Alexa Fluor 488-labeled wheat germ agglutinin (AF488-WGA). The fluorescence images of the vaginal tissue showed that the vaginal epithelial cells of healthy mice were covered with a dense mucus layer. After S. aureus infection, the secretion of mucin protein decreased significantly and the mucosal barrier was damaged. Compared with the EG-Bi and LGG control groups, after treatment with LSTC, the level of mucus secretion could be significantly increased ( Figure 9 in A-B).

[0108] 2. Effect of LSTC on the expression of tight junction proteins in epithelial cells in tissues

[0109] After the mouse vaginal tissues were taken out, fixed, dehydrated, embedded, and sectioned, immunofluorescence staining was performed on the tight junction proteins occludin and ZO-1 in the tissues. The stained samples were observed and photographed with CLSM. The fluorescence values of the expression of occludin and ZO-1 in the vaginal tissue were quantitatively analyzed with ImageJ software.

[0110] S. aureus infection led to a significant decrease in the expression of the tight junction proteins occludin and ZO-1 in vaginal epithelial cells. After treatment with LSTC, the expression of these two proteins was significantly restored ( Figure 9 in C-E). C. albicans infection led to a significant decrease in the expression of the tight junction proteins occludin and ZO-1 in vaginal epithelial cells. After treatment with LSTC, the expression of these two proteins was significantly restored. It indicates that LSTC can effectively regulate the mucosal physical and chemical barrier in vivo.

[0111] Example 5 Regulation of mucosal immune barrier by LSTC in vitro and in vivo

[0112] 1. Transcriptome sequencing of mouse vaginal tissues

[0113] To evaluate the regulatory effect of LSTC on the immune barrier in vivo, by intravaginal injection of 4.0×109 CFUs S. aureus was used to establish a murine vaginal disorder model, and then the mice were treated with intravaginal injection of LSTC solution for 7 consecutive days. Healthy group, PBS group, and equal-dose EG-Bi and LGG groups were used as controls. The vaginal tissues of the mice were collected for transcriptome sequencing (mRNA-Seq).

[0114] Analysis of differentially expressed genes (DEGs) showed that compared with the healthy group, in the vaginal tissue samples of the PBS group, there were 134 downregulated and 349 upregulated DEGs. Notably, among the DEGs significantly upregulated in the PBS group, 15, 5, and 218 genes were significantly downregulated in the LGG, EG-Bi, and LSTC treatment groups, respectively ( Figure 10 in A), indicating that LSTC has a significant effect on alleviating inflammation activation caused by S. aureus. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology enrichment analysis (GO) results showed that compared with the PBS group, the downregulated genes in the LSTC group were enriched in immune-related pathways, such as cytokine-cytokine receptor signaling, NOD-like receptor-mediated signaling pathway, etc. ( Figure 10 in B-C). Gene Set Enrichment Analysis (GSEA) further confirmed the downregulated immune-related pathways, including inflammatory response, cytokine-cytokine interaction, Toll-like receptor signaling pathway, antigen processing and presentation, natural killer cell-mediated cytotoxicity, immune response activation, lymphocyte-mediated immunity, immunoglobulin-mediated immune response, and B cell-mediated immunity ( Figure 10 in D).

[0115] LSTC significantly decreased the expression of multiple genes related to inflammasomes, including Gbp2, Itga9, Il1r2, Irgm1, Itgam, and Tlr4 ( Figure 10 in E). Subsequently, enrichment analysis of cell types in different treatment groups was further performed to quantify the distribution of immune cells in the vagina. As Figure 10 shown in F, S. aureus infection led to significant enrichment of innate immune cells, such as granulocyte-monocyte precursors, neutrophils, macrophages, and myeloid dendritic cells. Compared with EG-Bi and LGG, LSTC treatment significantly reduced the levels of granulocyte-monocyte precursors, neutrophils, macrophages, and myeloid dendritic cells. And S. aureus infection triggered the activation of B cells but inhibited T cell immunity, including CD8 + T cells and T helper 1 (Th1) cells. After LSTC treatment, the activation of CD8 + T cells and Th1 cells was enhanced, and these cells are the main subsets mediating effective anti-infective immunity. It indicates that LSTC effectively downregulates the inflammatory response and triggers anti-infective immunity in the vagina.

[0116] Example 6: In vitro and in vivo regulation of mucosal microbial barrier by LSTC

[0117] 1. Inhibitory effect of LSTC on pathogenic bacteria in vivo

[0118] Female mice were intravaginally inoculated with 4.0×10 9 CFUs of S. aureus (carrying the RN4220 T61-eGFP plasmid and erythromycin resistance) to establish a vaginal dysbiosis model, and then treated with intravaginal injection of LSTC solution for 7 consecutive days. Healthy group, PBS, clindamycin, and equal amounts of EG-Bi and LGG groups were used as controls. On the 8th day, vaginal tissues of mice were collected, and the distribution of S. aureus in vaginal tissues was observed by confocal microscopy.

[0119] Fluorescence images of vaginal tissues showed that compared with S. aureus-infected mice, the distribution of S. aureus in vaginal tissues of LGG-treated mice decreased ( Figure 11 in A). Notably, among all treatment groups, the amount of S. aureus in the LSTC group was significantly reduced, indicating that LSTC can significantly inhibit the growth of S. aureus in vivo in vaginal dysbiosis mice.

[0120] 2. 16S rRNA gene sequencing of murine vaginal microbiota

[0121] To evaluate the inhibitory effect of LSTC on pathogenic bacteria and the regulatory effect on microbiota in vivo, female mice were intravaginally inoculated with 4.0×10 9 CFUs of S. aureus to establish a vaginal dysbiosis model, and then treated with intravaginal injection of LSTC solution for 7 consecutive days. Healthy group, PBS, clindamycin, and equal amounts of EG-Bi and LGG groups were used as controls. On the 8th day, vaginal lavage fluid of mice was collected for microbial composition analysis by 16S rRNA gene sequencing, and vaginal tissues were collected to detect the distribution of S. aureus in tissues.

[0122] Sequencing results showed that compared with healthy mice, the ACE index and Chao1 index in PBS-treated mice decreased significantly, indicating a significant reduction in the richness of their vaginal microbiota ( Figure 11 in B-C). Principal coordinate analysis (PCoA) at the operational taxonomic unit (OTU) level showed that the microbial composition of PBS-treated mice was significantly different from that of healthy mice. Compared with EG-Bi and clindamycin-treated groups, the microbial compositions of LGG and LSTC-treated groups were more similar to those of healthy mice, indicating that LGG and LSTC interventions reduced the structural changes in the vaginal microbiota caused by S. aureus infection ( Figure 11In D). It should be noted that at the phylum level, LSTC significantly increased the relative abundance of probiotics in S. aureus-infected mice ( Figure 11 In E). At the genus level, LSTC significantly increased the abundances of Lachnospiraceae NK4A136 group and Clostridium sensu stricto 1 in S. aureus-infected mice ( Figure 11 In F-G). It indicates that LSTC has a good regulatory effect on the vaginal microbiota of mice.

[0123] Example 7 Evaluation of the Efficacy of LSTC on Mucosal Inflammatory Diseases

[0124] (1) Evaluation of the Efficacy of LSTC on Candidal Vaginitis in Mice

[0125] Mice (ICR, female, 6 - 8 weeks old) were randomly divided into 6 groups: healthy group, PBS group, clotrimazole positive drug group, EG-Bi group, LGG group, and LSTC group. Except for the healthy group, each group of mice was subcutaneously injected with 0.1 mL estradiol benzoate injection (2 mg / mL) every 2 days for 3 consecutive injections. On the 7th day, 2.0×10 9 CFUs of C. albicans solution was inoculated into the vagina of mice for 3 consecutive days to establish candidal vaginitis in mice. 20 μL of PBS, clotrimazole (0.4 mg / kg), EG-Bi (12 μg EG, 4 μg Bi(NO3)3·5H2O), LGG (5×10 7 CFUs), and LSTC (12 μg EG, 4 μg Bi(NO3)3·5H2O, 5×10 7 CFUs of LGG) were injected into the vagina of mice for treatment for 7 consecutive days.

[0126] The body weights of mice were recorded every day during the treatment. After the treatment, the vaginal contents of mice were collected and evenly spread on YPD solid medium containing 100 μg / mL kanamycin and ampicillin, and incubated overnight at 37 °C for bacterial counting. After the mice were anesthetized, blood was taken from the eyeballs to measure the expression of inflammatory factors in the serum. At the same time, the vaginal tissues of mice were taken for MPO staining and H&E staining.

[0127] The model construction and treatment plan are as Figure 12 shown in A. C. albicans infection led to a significant decrease in the body weight of mice. After treatment with PBS, LGG, or EG-Bi, the weight loss caused by C. albicans infection could not be improved. In contrast, LSTC could significantly restore the body weight of mice ( Figure 12In B). On the 8th day after treatment, mouse vaginal secretions were collected for microscopic examination of C. albicans. Through periodic acid-Schiff (PAS) staining, the number of C. albicans in the vaginas of mice treated with LSTC was significantly reduced ( Figure 12 In C). Vaginal lavage fluid was collected for plate counting. The results showed that the clotrimazole and LSTC treatment groups could significantly reduce the number of C. albicans ( Figure 12 In D). It was also found that after treatment with LSTC, the expression of inflammatory factors IL-1β and TNF-α could be significantly reduced ( Figure 12 In E-F). In addition, the H&E staining results of mouse vaginal tissues showed that compared with the healthy group, mice infected with C. albicans showed severe leukocyte infiltration, and there was varying degrees of exfoliation and keratinization of the mucosal keratin layer. After LSTC treatment, the exfoliation, keratinization of the mucosal keratin layer and leukocyte infiltration caused by the infection were significantly reduced ( Figure 12 In G-H). Immunohistochemistry was used to stain MPO in vaginal tissues. The results showed that compared with PBS, LSTC significantly reduced the infiltration of MPO-positive cells in vaginal tissues ( Figure 12 In I-J). It is indicated that LSTC can effectively inhibit the colonization of C. albicans in the vagina and improve the mucosal damage caused by C. albicans.

[0128] (2) Evaluation of the efficacy of LSTC on aerobic vaginitis in mice

[0129] Mice (ICR, female, 6-8 weeks old) were randomly divided into 6 groups: healthy group, PBS group, clindamycin positive drug group, EG-Bi group, LGG group and LSTC group. Except for the healthy group, mice were subcutaneously injected with 0.1 mL estradiol benzoate injection (2 mg / mL) once a day for 3 consecutive days. Subsequently, mice were vaginally inoculated with S. aureus (4.0×10 9 CFUs) for 7 consecutive days to establish a mouse vaginal disorder model. Then, 20 μL of PBS, clindamycin (6.5 mg / kg), EG-Bi (12 μg EG, 4 μg Bi(NO3)3·5H2O), LGG (5×10 7 CFUs) and LSTC (12 μg EG, 4 μg Bi(NO3)3·5H2O, 5×10 7 CFUs LGG) were injected into the vaginas of mice for treatment for 7 consecutive days.

[0130] The model construction and treatment plan were as Figure 13 shown in A. S. aureus infection caused a significant decrease in the body weight of mice, while clindamycin and LSTC treatment could significantly restore the body weight of mice ( Figure 13In part B). On the 8th day after treatment, vaginal lavage fluid of mice was collected to quantify the number of S. aureus in each group after treatment. The results showed that clindamycin and LSTC could significantly reduce the number of S. aureus in mice( Figure 13 In parts C-D). The results of H&E staining showed that compared with the healthy group, the vaginal tissues of mice infected with S. aureus showed severe leukocyte infiltration, and different degrees of exfoliation and keratinization of the mucosal keratin layer( Figure 13 In part E). On the contrary, the integrity of the vaginal mucosal tissue and the infiltration of neutrophils in the LSTC group were restored to varying degrees, indicating that LSTC may improve local vaginal infection symptoms by inhibiting neutrophil infiltration and relieve the pathological damage of vaginal mucosal tissue. The expression levels of inflammatory factors in the serum of mice were measured. The expression level of IL-6 in the serum of the PBS group mice increased significantly. After treatment with LSTC, the expression of inflammatory factors could be significantly reduced( Figure 13 In part F). By immunohistochemical staining of MPO in vaginal tissues, it was found that compared with PBS, LSTC significantly reduced the infiltration of MPO-positive cells in vaginal tissues( Figure 13 In parts G-H). It indicates that LSTC can effectively relieve the mucosal pathological damage and inflammatory infiltration of aerobic vaginitis.

[0131] (3) Evaluation of the efficacy of LSTC on ulcerative colitis (UC) in mice

[0132] Mice (ICR, female, 6-8 weeks old) were randomly divided into 6 groups: healthy group, PBS group, 5-ASA positive drug group, EG-Bi group, LGG group and LSTC group. Mice were continuously free to drink 4% DSS aqueous solution for 7 days, and the fresh DSS solution was replaced regularly to induce the UC model in mice. Healthy mice drank distilled water. The DAI score was evaluated by observing the changes in mouse body weight and blood in the stool to evaluate whether the model was successfully constructed. 20 μL of PBS, 5-ASA (100 mg / kg), EG-Bi (12 μg EG, 4 μg Bi(NO3)3·5H2O), LGG (5×10 7 CFUs), LSTC (12 μg EG, 4 μg Bi(NO3)3·5H2O, 5×10 7 CFUs LGG) were given by enema (i.e., a 0.2 mm diameter enema tube was slowly inserted about 4 cm into the anus of the mouse) once a day for 7 consecutive days. The DSS group and the healthy group were used as controls and given normal saline in the same way. At the same time of administration, except for the healthy group, the remaining mice continued to drink the DSS solution.

[0133] The model construction and treatment plan are as Figure 14As shown in A, body weight and fecal characteristics were monitored during the period for DAI scoring. After the treatment ended on the 7th day, the colon tissues were taken out to measure the length, and H&E staining was performed to evaluate pathological damage. As shown in the figure, in the UC model, the body weight of the mice decreased significantly. In contrast, LSTC could significantly restore the body weight of the mice ( Figure 14 in B). Similarly, compared with the PBS group, the spleen index of the mice in the LSTC administration group decreased significantly ( Figure 14 in C), and the colon length increased significantly ( Figure 14 in D-E). In addition to the above clinical symptoms, the results of H&E staining of the colon tissues showed that in the PBS group, there was a large amount of inflammatory cell infiltration in the colon of the mice, showing severe pathological changes. Compared with the PBS group, after LSTC treatment, the infiltration of inflammatory cells in the colon could be significantly reduced, and pathological changes such as mucosal edema and crypt structure damage were improved ( Figure 14 in F). It is indicated that LSTC can significantly improve the symptoms and pathological manifestations of DSS-induced ulcerative colitis.

[0134] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments according to these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A coating composition for treating mucosal inflammatory diseases, characterized in that, It includes probiotics, trivalent bismuth, and ethyl gallate.

2. The coating composition according to claim 1, wherein The probiotics include probiotics that act on the mucosal surface.

3. The coating composition according to claim 1 or 2, characterized in that, The types of the probiotics include probiotic bacilli, Clostridium butyricum, Lactobacillus, and Bifidobacterium.

4. The coating composition according to claim 1, characterized in that, The trivalent bismuth includes soluble trivalent bismuth salts. When the trivalent bismuth is Bi(NO3)3·5H2O, the concentration ratio of Bi(NO3)3·5H2O to ethyl gallate is (0.05 - 0.8):(0.1 - 4.8).

5. The coating composition according to claim 1, characterized in that, The mucosal inflammatory diseases include at least one of the following: oral mucosal diseases, nasal mucosal diseases, ocular mucosal diseases, vaginal mucosal diseases, gastric mucosal diseases, and intestinal mucosal diseases.

6. Use of the coating composition according to any one of claims 1 - 5 in the preparation of an active therapeutic coating synthesized in situ on the mucosal surface.

7. An active therapeutic coating comprising the coating composition according to any one of claims 1 - 5 and synthesized in situ on the mucosal surface.

8. The method for preparing the active therapeutic coating according to claim 7, characterized in that, It includes the following steps: Dissolve and stir - mix trivalent bismuth and ethyl gallate to obtain a nano - coating; Add probiotics to the nano - coating and incubate to obtain the active therapeutic coating.

9. Use of the coating composition according to any one of claims 1 - 5 or the active therapeutic coating according to claim 7 in the preparation of a drug for treating mucosal inflammatory diseases.

10. The application according to claim 9, characterized in that The therapeutic effects of the drug include at least one of the following: improving mucosal physicochemical barrier damage, regulating mucosal microbial barrier, and regulating mucosal immune barrier.

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