Prussian blue nano-enzyme compound as well as preparation method and application thereof

Abstract

The invention discloses a Prussian blue nano-enzyme compound as well as a preparation method and application thereof. The Prussian blue nano-enzyme compound provided by the invention can inhibit the activation of DSS-induced intestinal epithelial cell inflammasome, and relieve pyroptosis of cells and oxidative stress injury of mitochondria; besides, the Prussian blue nano-enzyme compound can break pyroptosis-macrophage polarization crosstalk and prevent inflammation, has an anti-inflammatory effect on acute colitis, and can regulate intestinal flora.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a Prussian blue nanozyme complex, a preparation method thereof and an application thereof. Background Art

[0002] Inflammatory bowel disease (IBD) has become a global disease posing a significant risk to health due to its persistence and vulnerability. In IBD, the intestinal microbiota invades damaged intestinal epithelial cells (IECs), generating a large number of inflammatory mediators such as reactive oxygen species (ROS), which further overactivate resident immune cells. This excessive immune activation disrupts normal mucosal homeostasis, leading to a continuous "cascade" of inflammatory responses and tissue damage. Currently, inflammatory bowel disease is characterized by incurable, lifelong recurrence, and disability, and patients suffer great physical pain and mental burden. Therefore, there is an urgent need to explore new strategies and mechanisms for the over-immunotherapy of IBD.

[0003] In the intestinal immune microenvironment, macrophages are the main participants in intestinal immune homeostasis and intestinal inflammation. They can be polarized into classically activated pro-inflammatory phenotypes (M1) and alternatively activated anti-inflammatory phenotypes (M2), regulating the immune microenvironment at the colitis site. However, with the occurrence and development of colitis, the polarization imbalance of M1 / M2 macrophages in the intestine leads to the imbalance of intestinal inflammation regulation, resulting in excessive inflammatory responses and exacerbating the progression and recurrence of the disease. Although the current treatment of inflammatory bowel disease mainly relies on the development of various immunosuppressive drugs to inhibit the excessive inflammatory responses of immune cells, they may not completely address the underlying mechanisms driving chronic inflammation. In recent years, there has been increasing interest in the phenomenon of pyroptosis, especially at the inflammatory site. Pyroptosis is characterized by the release of inflammatory cytokines and cellular contents, which can undergo complex crosstalk with surrounding immune cells. Recent studies have emphasized the complex interaction between pyroptotic cells and macrophages, which is an important factor leading to irreversible IBD. Pyroptosis can affect macrophage polarization, thereby affecting their functional state and role in inflammation. Regulating macrophage polarization through pyroptosis signaling plays a crucial role in the exacerbation or alleviation of inflammation. Therefore, the regulation of macrophage homeostasis is closely related to the pyroptosis process. In summary, in the face of the complex immune environment during the occurrence of enteritis, how to regulate the functions of these immune cells and inhibit their inflammatory responses has become a key issue and solution idea for the effective treatment of IBD. The innovation of arming probiotics with nanozymes marks a significant progress in the treatment management of colitis and similar inflammatory diseases.

[0004] There are currently many strategies for regulating macrophages to treat colitis, but the strategies are relatively single. Since intestinal inflammation involves intestinal tissues, the microbiota, and the immune microenvironment, regulating only immune cells (such as macrophages, etc.) is not sufficient to address the chronic inflammatory mechanism. Moreover, more and more current studies have shown that intestinal permeability is regulated by the microbiota and immune cells, revealing that coordinating the crosstalk between intestinal epithelial cells, microorganisms, and immune cells can maintain intestinal permeability and stability in the body. Summary of the Invention

[0005] In this study, we synthesized a selenium-enriched Bifidobacterium longum modified with PBzyme to reshape the healthy communication between intestinal IECs and intestinal macrophages. The introduction of PBzymes can enable probiotics to establish long-term colonization in the intestine and relieve intestinal inflammation. More importantly, PBzymes can inhibit the pyroptosis of IECs, thereby reshaping the healthy crosstalk between IECs and macrophages and effectively reprogramming M1 macrophages into M2 macrophages. We used the Transwell cell model to measure the levels of pyroptosis polarization-related proteins, pro-inflammatory cytokines, and the functional responses between IECs and intestinal macrophages. And we used a dextran sulfate sodium (DSS)-induced colitis mouse model to detect inflammatory factors and related proteins. Finally, Se-BL@PB intervention reduced the pyroptosis-related microbiota structure, and the potential mechanism of action was determined through microbiota analysis. These findings together demonstrate that Se-BL@PB treatment disrupts the circuit of the pro-inflammatory microenvironment caused by abnormal cell crosstalk in IBD, which provides a new therapeutic concept for any other chronic inflammatory conditions.

[0006] In the first aspect of the present invention, there is provided a Prussian blue nanozyme complex, which comprises selenium-enriched Bifidobacterium longum.

[0007] In some embodiments, the selenium-enriched Bifidobacterium longum is prepared by the following method:

[0008] a. Inoculate Bifidobacterium longum in RCM medium and culture to obtain a seed solution;

[0009] b. Inoculate the seed solution into an RCM medium containing 5 - 25 μg / ml sodium selenite for enrichment Perform selenium culture, collect the selenium-enriched cultured bacterial solution to obtain selenium-enriched Bifidobacterium longum.

[0010] In some embodiments, the Prussian blue nanozyme complex is prepared by the following method:

[0011] (1) Dissolve potassium ferricyanide and polyvinylpyrrolidone in hydrochloric acid solution, stir to obtain a yellow solution, react the yellow solution at 70-100 °C for 20-30 h to obtain a blue solution, and obtain Prussian blue nanozyme after centrifugation;

[0012] (2) Dissolve the Prussian blue nanozyme obtained in (1) in hydrochloric acid solution, add polyvinylpyrrolidone, stir and mix evenly at room temperature, and react at 120 - 160 °C for 2 - 4 h. After the reaction, obtain hollow Prussian blue nanozyme through centrifugation, dialysis, and freeze-drying steps;

[0013] (3) Cultivate selenium-enriched Bifidobacterium longum until the stationary phase, centrifuge to collect the selenium-enriched Bifidobacterium longum cells, wash the cells twice with PBS buffer, and then resuspend the cells with PBS buffer to obtain a cell suspension; The selenium-enriched Bifidobacterium longum is prepared through the following steps:

[0014] a. Inoculate Bifidobacterium longum in RCM medium for cultivation to obtain a seed solution;

[0015] b. Inoculate the seed solution in RCM medium containing 5 - 25 μg / ml sodium selenite for selenium enrichment cultivation, collect the selenium-enriched culture broth, and obtain selenium-enriched Bifidobacterium longum;

[0016] (4) Add the hollow Prussian blue nanozyme to the cell suspension, mix evenly, react at room temperature for 25 - 35 min (for example, 30 min), then adjust the pH to neutral, continue to react for 25 - 35 min (for example, 30 min), centrifuge to obtain the precipitate, and wash the precipitate 3 times with PBS buffer to obtain the Prussian blue nanozyme complex.

[0017] In some embodiments, in step (1), the addition ratio of potassium ferricyanide, polyvinylpyrrolidone, and hydrochloric acid is 132 mg: 3 g: 30 ml; the molecular weight of the polyvinylpyrrolidone is K30; the concentration of the hydrochloric acid is 0.005 - 0.015 M; the reaction time is 24 h; the reaction temperature is 80 °C.

[0018] In some embodiments, in step (2), the addition ratio of Prussian blue nanozyme, hydrochloric acid, and polyvinylpyrrolidone is 1 mg: 1 ml: 10 mg; the concentration of the hydrochloric acid is 0.8 - 1.2 M; the reaction temperature is 140 °C; the reaction time is 3 h.

[0019] In some embodiments, the concentration of the hydrochloric acid is 1.0 M.

[0020] In some embodiments, in step (3), the cell concentration at the stationary phase is 0.8×10 8 ~1.2×10 8 CFU / mL; the concentration of the cell suspension is 1.0×10 8 CFU / mL;

[0021] In some embodiments, in step (3), the cell concentration at the stationary phase is 1.0×108 CFU / mL.

[0022] In some embodiments, the addition ratio of the hollow Prussian blue nanozyme and the bacterial cell suspension described in step (4) is 0.5 - 2.0 mg: 1 ml; the pH is 7.0 - 8.0.

[0023] In some embodiments, the pH described in step (4) is 7.0.

[0024] The second aspect of the present invention provides the use of a Prussian blue nanozyme complex as described in the first aspect of the present invention in the preparation of a medicament for treating inflammatory bowel disease.

[0025] In some embodiments, the inflammatory bowel disease is DSS-induced colitis.

[0026] The third aspect of the present invention provides a method for preparing selenium-enriched Bifidobacterium longum, the method comprising the following steps:

[0027] a. Inoculate Bifidobacterium longum in RCM medium and culture to obtain a seed solution;

[0028] b. Inoculate the seed solution in RCM medium containing 5 - 25 μg / ml sodium selenite for selenium-enriched culture, collect the selenium-enriched culture broth, and obtain selenium-enriched Bifidobacterium longum.

[0029] In some embodiments, the conditions for the culture in a are: under anaerobic conditions, inoculate at an inoculation amount of 1 - 3%, and culture overnight in a constant temperature incubator at 35 - 39 °C.

[0030] In some embodiments, the conditions for the selenium-enriched culture in b are: under anaerobic conditions, inoculate at an inoculation amount of 2%, culture at a constant temperature of 37 °C for 12 hours, and the concentration of sodium selenite is 10 μg / ml.

[0031] The fourth aspect of the present invention provides a selenium-enriched Bifidobacterium longum, which is prepared by the method described in the third aspect of the present invention.

[0032] The fifth aspect of the present invention provides a method for preparing a Prussian blue nanozyme complex, the method comprising the following steps:

[0033] (1) Dissolve potassium ferricyanide and polyvinylpyrrolidone in hydrochloric acid solution, stir to obtain a yellow solution, react the yellow solution at 70 - 100 °C for 20 - 30 h to obtain a blue solution, and obtain Prussian blue nanozyme after centrifugation;

[0034] (2) Dissolve the Prussian blue nanozyme obtained in (1) in hydrochloric acid solution, add polyvinylpyrrolidone, stir and mix well at room temperature, and react at 120 - 160 °C for 2 - 4 h. After the reaction, obtain hollow Prussian blue nanozyme through centrifugation, dialysis, and freeze-drying steps;

[0035] (3) Culture the selenium-rich Bifidobacterium longum as described in the fourth aspect of the present invention until the stationary phase, centrifuge to collect the selenium-rich Bifidobacterium longum cells, wash the cells twice with PBS buffer, and then resuspend the cells with PBS buffer to obtain a cell suspension;

[0036] (4) Add the hollow Prussian blue nanozyme to the cell suspension, mix well, react at room temperature for 25 - 35 min (for example, 30 min), then adjust the pH to neutral, continue to react for 25 - 35 min (for example, 30 min), centrifuge to obtain the precipitate, wash the precipitate 3 times with PBS buffer, and obtain the Prussian blue nanozyme complex.

[0037] In some embodiments, the addition ratio of potassium ferricyanide, polyvinylpyrrolidone, and hydrochloric acid in step (1) is 132 mg: 3 g: 30 ml; the molecular weight of the polyvinylpyrrolidone is K30; the concentration of the hydrochloric acid is 0.005 - 0.015 M; the reaction time is 24 h; the reaction temperature is 80 °C.

[0038] In some embodiments, the addition ratio of the Prussian blue nanozyme, hydrochloric acid, and polyvinylpyrrolidone in step (2) is 1 mg: 1 ml: 10 mg; the concentration of the hydrochloric acid is 0.8 - 1.2 M; the reaction temperature is 140 °C; the reaction time is 3 h.

[0039] In some preferred embodiments, the concentration of the hydrochloric acid is 1.0 M.

[0040] In some embodiments, the cell concentration at the stationary phase in step (3) is 0.8×10 8 ~1.2×10 8 CFU / mL; the concentration of the cell suspension is 1.0×10 8 CFU / mL.

[0041] In some preferred embodiments, the cell concentration at the stationary phase is 1.0×10 8 CFU / mL.

[0042] In some embodiments, the addition ratio of the hollow Prussian blue nanozyme and the cell suspension in step (4) is 0.5 - 2.0 mg: 1 ml; the pH is 7.0 - 8.0.

[0043] In some preferred embodiments, the pH is 7.0.

[0044] On the basis of conforming to the common knowledge in this field, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0045] The reagents and raw materials used in the present invention are all commercially available.

[0046] The positive and progressive effects of the present invention are as follows:

[0047] 1. PB nanozyme enhances the antioxidant effect of selenium-enriched Bifidobacterium longum;

[0048] 2. Se-BL@PB can inhibit the activation of inflammasomes in intestinal epithelial cells induced by DSS and alleviate pyroptosis and mitochondrial oxidative stress damage;

[0049] 3. Se-BL@PB prevents inflammation by disrupting the pyroptosis-macrophage polarization crosstalk;

[0050] 4. Anti-inflammatory effect of Se-BL@PB on acute colitis;

[0051] 5. Se-BL@PB regulates the intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 It is a transmission electron microscope image of the prepared hollow Prussian blue nanozyme.

[0053] Figure 2 It is an ultraviolet spectrum of the prepared hollow Prussian blue nanozyme.

[0054] Figure 3 It is a Fourier transform infrared spectrum of the prepared hollow Prussian blue nanozyme.

[0055] Figure 4 It is a dynamic light scattering image of the prepared hollow Prussian blue nanozyme.

[0056] Figure 5 It is an X-ray diffraction pattern of the prepared hollow Prussian blue nanozyme.

[0057] Figure 6 It is a selenium enrichment culture result image of Bifidobacterium longum.

[0058] Figure 7 It is a transmission electron microscope image of the prepared Prussian blue nanozyme-selenium-enriched Bifidobacterium longum complex.

[0059] Figure 8 It is the fluorescence of PB, Se-BL and Se-BL@PB analyzed by confocal laser scanning microscopy.

[0060] Figure 9 It is the growth curves of BL, Se-BL and Se-BL@PB.

[0061] Figure 10 It is the cell viability of NCM460 cells co-incubated with Se-BL@PB at different concentrations.

[0062] Figure 11 It is the graph of the protective ability of Se-BL@PB against ROS-mediated cytotoxicity.

[0063] Figure 12 It is the fluorescence staining graph of ROS scavenging activity in vitro.

[0064] Figure 13 It is the relative fluorescence statistical graph of ROS scavenging activity in vitro.

[0065] Figure 14 It is the immunofluorescence staining results of GSDMD and NLRP3 in the THP-1-NCM460 co-culture system.

[0066] Figure 15 It is the protein expression of GSDMD and NLRP3 in the THP-1-NCM460 co-culture system.

[0067] Figure 16 It is the disease activity index of each group in the DSS colitis mouse model.

[0068] Figure 17 It is the statistical result of the colon length of each group in the DSS colitis mouse model.

[0069] Figure 18 It is the H&E staining graph of the colon tissue samples of each group in the DSS colitis mouse model.

[0070] Figure 19 It is the statistical graph of the tissue damage score of each group in the DSS colitis mouse model.

[0071] Figure 20 It is the representative immunofluorescence microscopic photograph of NLRP3 and GSDMD intestinal tissue staining.

[0072] Figure 21 It is the level of pyroptosis-related cytokine IL-1β in serum measured by ELISA.

[0073] Figure 22 It is the level of pyroptosis-related cytokine TNF-α in serum measured by ELISA.

[0074] Figure 23 It is the heat map of the intestinal microbiota abundance of each group in the DSS colitis mouse model.

[0075] Figure 24 It is the abundance of beneficial bacteria and harmful bacteria of each group in the DSS colitis mouse model. Detailed implementation manners

[0076] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0077] Example 1: Synthesis and characterization of Prussian blue nanozyme

[0078] Dissolve 132 mg of potassium ferricyanide and 3 g of polyvinylpyrrolidone (PVP, molecular weight K30) in 30 ml of 0.01 M hydrochloric acid solution, and obtain a clear yellow solution through magnetic stirring. Then place the yellow solution in an oven and react at 80 °C for 24 hours to obtain a blue solution. Centrifuge the blue solution to obtain Prussian blue nanozyme (Pbzyme, PB). Then dissolve 30 mg of PB powder in 30 ml of 1 M hydrochloric acid solution, add 300 mg of PVP, stir at room temperature for 3 h, and then place it in an oven and react at 140 °C for 3 hours. After the reaction, centrifuge, dialyze, and freeze-dry to obtain hollow Prussian blue nanozyme. The hollow Prussian blue nanozyme is characterized by transmission electron microscopy ( Figure 1 ), ultraviolet spectroscopy ( Figure 2 ), Fourier transform infrared spectroscopy ( Figure 3 ), DLS ( Figure 4 ), XRD ( Figure 5 ), etc.

[0079] It can be seen from the transmission electron microscopy image of the hollow Prussian blue nanozyme that the nanozyme has a cylindrical hollow structure, and the diameter of the cavity is 70 - 80 nm.

[0080] The results of ultraviolet spectroscopy show that there is a strong absorbance in the first biological window, corresponding to the characteristic absorbance peak of Prussian blue nanozyme.

[0081] Fourier transform infrared (FTIR) spectroscopy shows the characteristic Fe-CN-Fe peak of the prepared PBzyme, indicating the successful connection between PB and Se-BL.

[0082] Dynamic light scattering (DLS) analysis shows that the hydrodynamic diameter of PBzyme is approximately 100.1 nm.

[0083] X-ray diffraction analysis only shows the characteristic peaks of Prussian blue nanozyme, without other abnormal peaks, indicating that the main component of the synthesized product is indeed Prussian blue nanozyme.

[0084] Example 2: Selenium-enriched culture of Bifidobacterium longum

[0085] Bifidobacterium longum (BL, accession number: ATCC 15707) was inoculated into fresh RCM medium (Meilun Biology, product number MB0039-1) at an inoculation amount of 2%, and anaerobically cultured at 37°C. After overnight incubation, the bacterial solution was respectively inoculated into fresh RCM medium containing 5 μg / ml, 10 μg / ml, 15 μg / ml, 20 μg / ml, and 25 μg / ml sodium selenite at an inoculation amount of 2%, and continuously anaerobically cultured at 37°C for 12 h. Then, viable cell counts were performed on each culture solution, and the bacterial solution was freeze-dried to obtain bacterial powder. The selenium content in the bacterial powder was detected by ICP-MS (ICP-Q). The experimental results are as Figure 6 shown. Selenium-enriched Bifidobacterium longum (Se-BL) with a selenium content of 10 μg / g was obtained.

[0086] Example 3: Synthesis and characterization of selenium-enriched Bifidobacterium longum Se-BL@PB with Prussian blue nanozyme engineering

[0087] (1) Synthesis of selenium-enriched Bifidobacterium longum Se-BL@PB with Prussian blue nanozyme engineering

[0088] Hollow PBzymes were added to the surface of selenium-enriched Bifidobacterium longum (Se-BL) to construct engineered probiotic Se-BL@PB. Specifically, Se-BL was inoculated into RCM medium at an inoculation amount of 2% and incubated in an anaerobic incubator at 37°C for 24 hours until the concentration reached 1×10 8 CFU / mL (the plateau phase of the Se-BL growth curve). After cultivation, Se-BL was collected by centrifugation, the supernatant was discarded, and Se-BL was washed twice with PBS buffer at pH 7.2 - 7.4 to remove residual medium. Then the washed bacteria were resuspended in 10 mL of PBS buffer. Subsequently, PBzymes were added to the Se-BL suspension, and 0.5 - 2.0 mg of Prussian blue nanozyme was added per milliliter of Se-BL. The mixture was gently shaken to promote electrostatic adsorption, and the reaction was carried out at room temperature for 30 minutes. After the reaction, the pH was adjusted to 7.0 - 8.0, and then the mixture was gently stirred for another 30 minutes to optimize the formation of hydrogen bonds. Finally, the complex was separated by centrifugation, the supernatant was discarded, and the precipitate was washed three times with PBS buffer to obtain engineered probiotic Se-BL@PB.

[0089] The final selenium-enriched Bifidobacterium longum Se-BL@PB with Prussian blue nanozyme engineering was resuspended in an appropriate volume of PBS buffer for subsequent experiments.

[0090] (2) Characterization of Se-BL@PB

[0091] The morphology of BL, Se-BL, and Se-BL@PB was observed by scanning electron microscopy (Hitachi-SU8010 / Zeiss sigma300, Japan). The transmission electron microscopy image of the Prussian blue nanozyme-engineered selenium-enriched Bifidobacterium longum Se-BL@PB is as Figure 7 shown, and the connection between the Prussian blue nanozyme and the selenium-enriched Bifidobacterium longum can be seen from the figure.

[0092] To confirm the integration of PB onto the surface of Se-BL, we labeled Se-BL and PB with cyanine 5 (Cy5) and fluorescein isothiocyanate (FITC), respectively. Confocal laser scanning microscopy (CLSM, Carl Zeiss LSM900) showed a clear Cy5 fluorescent shell ( Figure 8 ). The results of the confocal fluorescence images demonstrated the effectiveness of our surface modification, that is, PB had been integrated onto the surface of Se-BL.

[0093] BL, Se-BL, and Se-BL@PB were inoculated into fresh RCM medium and cultured anaerobically at 38°C. The inoculated bacterial solution was evenly divided into 8 portions and then continued to be cultured anaerobically. One portion of the sample was taken every 12 h and the viable cell count was measured by the above method until the culture reached 96 h. The growth curves of BL, Se-BL, and Se-BL@PB are as Figure 9 shown. From Figure 9 it can be seen that the side effects of this combined method on bacterial viability can be ignored because the optical density (600 nm) detection of Se-BL@PB showed similar growth curves between Se-BL coated with Prussian blue nanozyme and Se-BL@PB without coated Prussian blue nanozyme.

[0094] Example 4: CCK-8 assay for cytotoxicity

[0095] Human colon epithelial cell line NCM460 cells (Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) were inoculated into a 96-well plate containing 100 μL / well of NCM460 special medium (Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) at a density of 1×10 4 cells per well and cultured overnight. To study the cytotoxicity of Se-BL@PB, we prepared Se-BL@PB with concentrations of 40, 60, 80, 100, 200, 300, 400, and 500 μg / ml in DMEM. NCM460 cells were treated with 100 μl of Se-BL@PB solutions at different concentrations and incubated for 24 h. After incubation, 20 μL of CCK8 was added to each well and incubated in the incubator in the dark for 1 h. Finally, the absorbance at 490 nm was measured, and the results are as Figure 10 shown. From Figure 10 it can be seen that the cell survival rate of Se-BL@PB was higher than 90% in the concentration range of 40 - 500 μg / mL,Indicates that Se - BL@PB has good cell compatibility.

[0096] To study the protective ability of Se-BL@PB against reactive oxygen species (ROS)-mediated cytotoxicity, 100 μl of 40 mg / mL dextran sulfate sodium salt (DSS) and Se-BL@PB at concentrations of 20, 40, 80, 100, 200, and 400 μg / ml were added to each well and incubated for 24 h. After treatment, NCM460 cells were washed with fresh NCM460 special medium, 100 μL of fresh NCM460 special medium was added to each well, and then 10 μL of CCK-8 was added. After incubation at 37 °C, the absorbance of each well at 450 nm was measured using a microplate reader. The results are as Figure 11 shown, and it can be seen from Figure 11 that the antioxidant ability of Se-BL@PB increases with increasing concentration.

[0097] Example 5: In vitro ROS scavenging activity

[0098] For imaging detection, NCM460 cells were cultured with Se-BL@PB in the absence or presence of DSS (40 mg / mL). After 2 h, 20 μM 2,7-dichlorofluorescein diacetate (DCFH-DA) was added to detect the generation of intracellular ROS. After washing 3 times with PBS, cells were qualitatively observed under a fluorescence microscope. The control group and experimental groups were: (1) Control group (NCM460); (2) Model group (DSS + NCM460); (3) Se-BL group (DSS + NCM460 + Se-BL); (4) PB group (DSS + NCM460 + PB); (5) Se-BL@PB group (DSS + NCM460 + Se-BL@PB). The staining results are as Figure 12 shown, and the statistical results of relative fluorescence of each group are as Figure 13 shown; it can be seen from Figure 12 and Figure 13 that the ROS fluorescence intensity of the Control group was weak, and the Model group produced more ROS under the stimulation of DSS; compared with the Model group, pretreatment with Se-BL@PB led to a significant decrease in ROS-induced green fluorescence, thus showing its strong ROS scavenging ability.

[0099] Example 6: Establishment of a THP-1-NCM460 co-culture system

[0100] A co-culture system of THP-1 and NCM460 cells treated with lipopolysaccharide (LPS) was established to evaluate the crosstalk between them.

[0101] The THP-1 cells (icell / Saibakang, iCell-h213) were seeded into a 6-well plate containing 2 ml of THP-1 special medium (Seville, GZ10907) at an inoculation density of 50%. After culturing for 6 h, 100 ng / mL phorbol (PMA) was added, and the THP-1 cells were treated for 24 h. Then, the medium was discarded, and the cells were washed with PBS. After digestion, the THP-1 cells were seeded into the upper chamber of a transwell (Guangzhou Zhenxuan Biotechnology Co., Ltd., corning, cat. no.: 3401) containing 500 μl of RPMI-1640 medium (Gibco, cat. no.: c11875500BT) at an inoculation density of 50%. Next, 500 μl of LPS was added to polarize the THP-1 cells at a concentration of 100 ng / mL for 48 h, and then subsequent experiments were carried out.

[0102] The untreated NCM460 cells were resuspended in RPMI-1640 medium and seeded into the lower chamber of a transwell containing 2 ml of RPMI-1640 medium at an inoculation density of 50%. The density ratio of THP-1 and NCM460 cells was approximately 2:1. Control groups and experimental groups were set up, where the experimental groups were the model group, Se-BL group, PB group, and Se-BL@PB group. Then, the transwell co-culture system was incubated in an incubator at 37 °C with 5% carbon dioxide for 24 h to evaluate the pyroptosis of NCM460 cells induced by M1-polarized THP-1 cells. The NCM460 cells in the lower chamber were collected for subsequent experiments.

[0103] Example 7: Cellular immunofluorescence staining

[0104] The NCM460 cells collected in Example 5 were fixed with 4% paraformaldehyde solution (PFA) at room temperature for 10 minutes. After blocking with 4% BSA, the cells were incubated with primary antibodies targeting GSDMD and NLRP3 (GSDMD rabbit monoclonal antibody, Zhengneng Biologics, R24514; NLRP3 rabbit polyclonal antibody, ABclonal A21906), and then incubated with secondary antibodies labeled with Alexa Fluor 594 or Alexa Fluor 488 ( 488-conjugated goat anti-rabbit IgG (H+L), ABclonal AS053; 594-conjugated goat anti-rabbit IgG (H+L), ABclonal AS074) at room temperature. DAPI counterstaining was used to observe the cell nuclei. Immunofluorescence images were obtained using a Nikon microscope (Nikon TS2R-FL). NIS Elements F software and ImageJ software were used to analyze the acquired images. The results of immunofluorescence staining are shown in Figure 14As shown, immunofluorescence staining of pyroptosis markers NLRP3 and GSDMD in the model group showed a significant increase in fluorescence intensity, while that in the Se-BL@PB group was significantly lower than that in the control group. This indicates that Se-BL@PB effectively disrupted the crosstalk and reduced the inflammatory response.

[0105] Example 8: Immunoblotting detection method

[0106] Detect the protein expression levels of target genes in NCM460 cells in the lower chamber of transwell or THP-1 cells in the upper chamber of transwell. Proteins were extracted from cells in different treatment groups (control group, model group, Se-BL group, PB group, Se-BL@PB group) in Example 5 and quantified using a BCA protein concentration detection kit (Yaenzyme, ZJ101). The proteins were boiled in loading buffer, separated by 10% SDS-PAGE, and transferred to a PVDF membrane. The proteins were blocked with 5% bovine serum albumin at room temperature for 15 minutes, and primary antibodies (GSDMD, 1:2000; NLRP3, 1:2000) were added and incubated overnight at 4°C. After washing three times with TBST, the blots were incubated with an HRP goat anti-rabbit IgG (H+L) secondary antibody (ABclonal AS002) at room temperature for 1 hour. An immunofluorescence system was used to observe the immunoreactive blots. The immunoblotting detection results are as Figure 15 shown. The protein levels of NLRP3 and GSDMD in the model group were significantly increased, while Se-BL@PB effectively attenuated the upregulation of these proteins induced by ROS. Compared with the model group, the protein expression levels of GSDMD and NLRP3 in the Se-BL group, PB group, and Se-BL@PB group were decreased. These results confirmed that ROS mediated by M1 polarization in THP-1 cells could induce pyroptosis in NCM460 cells, while Se-BL@PB could break cell crosstalk and reduce continuous inflammatory responses.

[0107] Example 9: Efficacy observation of treating murine colitis

[0108] To verify the ability of Se-BL@PB obtained in this example to relieve colitis, a murine colitis model induced by dextran sulfate sodium (DSS) was used for the following tests:

[0109] Mice were housed under specific pathogen-free conditions with a 12-hour light / dark cycle and fed standard mouse food ad libitum.

[0110] In the preventive treatment of colitis model, C57BL / 6 mice (male, 6 weeks old) were randomly divided into five groups (n = 5 per group): ① healthy group (PBS), ② PBS group (DSS + PBS, i.e., Model group), ③ DSS + Se-BL, ④ DSS + PB, and ⑤ DSS + Se-BL@PB. Mice in the DSS group were fed 3% DSS in sterile drinking water for 6 days to induce a colitis model. The healthy group and the PBS group (i.e., the Model group) were treated with PBS, while the other groups were orally administered Se-BL, Se-BL@PB bacterial solution, and PB from day 7 to 12. The bacterial solution concentrations of Se-BL and Se-BL@PB were both 1×10 8 CFU / ml -1 , and the oral gavage dose was 1.25 mg / kg; on the scheduled days. The mice were weighed daily and the disease activity index (DAI) was recorded. The disease activity index (DAI) is used to evaluate inflammatory bowel disease (IBD) and other gastrointestinal Key indicators of disease activity. Evaluated according to the Disease Activity Index (DAI) ( Figure 16 ), Se-BL@ PB Treatment significantly alleviated colitis symptoms, including weight loss, fecal texture, and fecal blood. Fecal samples were collected on day 12, and then the mice were euthanized. The colon tissues and sera of the mice were taken, and the collected colon lengths were measured. The colon lengths of the mice in each group were as Figure 17 shown. It can be seen from the figure that Se-BL@PB treatment significantly alleviated the symptom of shortened colon length in colitis model mice. The colon tissues and mouse sera were used for HE histological staining and inflammatory cytokine evaluation respectively.

[0111] Example 9: H&E staining

[0112] The colon tissue samples obtained in Example 8 were fixed in 4% paraformaldehyde solution and embedded in paraffin. Then they were cut into 5-μm sections and subjected to H&E staining to evaluate the severity of colon damage. The results of H&E staining were as Figure 18 shown, and the statistical chart of tissue damage score was as Figure 19 shown. From Figure 18 and Figure 19 it can be seen that the H&E staining of DSS mice (Model group) showed severe histological damage. Colitis mice showed severe crypt destruction, histological collapse, massive infiltration of immune cells, and severe damage to the colonic epithelium in the inflamed colon. While the histological microstructure of the DSS + Se-BL@PB group was almost normal, with fewer inflammatory cells present, which was almost the same as the histological microstructure of the control group, indicating that Se-BL@PB can alleviate DSS-induced intestinal inflammation.

[0113] Example 10: Tissue immunofluorescence

[0114] To detect the expression of GSDMD and NLRP3, antigen retrieval and blocking were performed on the colon sections obtained in Example 8. The primary antibodies (1:1000, rabbit monoclonal antibody against GSDMD, Zhengneng Biotechnology, R24514; rabbit polyclonal antibody against NLRP3, ABclonal A21906) were incubated overnight in PBST, and then combined with the secondary CoraLite488 conjugated goat anti-rabbit immunoglobulin (H+L) for 1 hour in the dark. The colon sections were washed three times with PBS and stained with DAPI solution for 10 minutes at room temperature. Subsequently, the residual solution was slightly removed, and mounting medium was added under the coverslip. The tissue immunofluorescence staining images are as shown in Figure 20 shown. In the PBS+DSS group (Model group), the expression of NLRP3 and GSDMD proteins was significantly increased. While in the Se-BL@PB treatment group, the expression of NLRP3 and GSDMD proteins decreased significantly, indicating that this treatment effectively inhibited the activation of NLRP3 inflammasome and pyroptosis.

[0115] Example 11: ELISA assay for serum levels of pyroptosis-related cytokines

[0116] To further investigate the anti-pyroptosis activity of Se-BL@PB, we analyzed the expression of key inflammatory cytokines such as interleukin-1β (IL-1β) and TNF-α.

[0117] After collecting mouse serum, the level of IL-1β in the serum was measured using an ELISA kit (ABclonal, RK00176). According to the kit instructions, first, 40 μL of sample diluent was added to the wells of the coated plate for testing, and then 10 μL of the sample to be tested was added (the final dilution of the sample was 5-fold). Subsequently, 100 μL of enzyme-labeled reagent was added to each well, and the plate was sealed with a sealing film and incubated at 37 °C for 60 minutes. After incubation, the enzyme-labeled plate was washed with a 20-fold diluted washing solution 5 times. Then, 50 μL of chromogenic reagent A and B were added to each well, and the color was developed at 37 °C in the dark for 15 minutes. Finally, 50 μL of stop solution was added to each well to terminate the reaction, and the absorbance (OD value) of each well was measured at a wavelength of 450 nm. By plotting the standard curve, the corresponding concentration of IL-1β was calculated based on the OD value of the sample. The results are as shown in Figure 21 shown. The results showed that the level of the pro-inflammatory factor IL-1β related to cellular inflammation in the Se-BL@PB group was lower than that in the DSS group, indicating that Se-BL@PB has significant anti-pyroptosis activity.

[0118] After collecting mouse serum, an ELISA kit (ABclonal, RK00176, containing standards, biotinylated antibody working solution, and enzyme conjugate working solution) was used to measure the level of TNF-α in the serum. The washing solution was prepared according to the ELISA kit instructions. Subsequently, 100 μL of the serum sample to be tested and standards of different concentrations were added to the corresponding wells respectively. The microplate was placed in an incubator at 37 °C for 90 minutes. During the incubation, the microplate could be gently shaken appropriately to promote the reaction. After incubation, 4 washes were performed. 100 μL of the biotinylated antibody working solution was added to each well, and the reaction wells were sealed with sealing tape and placed in an incubator at 37 °C for 60 minutes. After incubation, the above washing steps were repeated 4 times. 100 μL of the enzyme conjugate working solution was added to each well. The reaction wells were sealed with sealing tape and placed in an incubator at 37 °C for 30 minutes. After incubation, the washing steps were repeated 4 times again. 100 μL of the chromogenic reagent was added to each well, and care was taken to avoid light during the operation. The microplate was placed in an incubator at 37 °C for 10 - 20 minutes in the dark until a gradient blue color was visible to the naked eye in the first 3 - 4 wells, the difference in the last 3 - 4 wells was not obvious, and there was no blue color in the zero well. 50 μL of the stop solution was added to each well, and the microplate was gently shaken to mix the liquid and stop the color reaction. Finally, the absorbance value (OD value) of each well was measured using a microplate reader at a wavelength of 450 nm. The measurement needed to be completed within 5 minutes after the reaction was terminated. The test results are as Figure 22 shown. It can be seen from the figure It shows that compared with the DSS + PBS group (Model group), Se - BL@PB treatment significantly reduced the colon group the TNF-α levels in the tissue and serum, highlighting its strong anti-inflammatory effect in alleviating colitis and systemic inflammation.

[0119] Example 12: Effect of Se-BL@PB on the gut microbiome

[0120] Heatmap of the relative abundances of gut microbiota at the family level ( Figure 23 ) and bar chart ( Figure 24)Eight significantly changed bacterial families were selected from [[]] for in-depth analysis. The results showed that compared with the PBS+DSS group, the abundances of beneficial bacterial families in the Se-BL@PB treatment group were significantly increased, including Marinifilaceae, Oscillospiraceae, and Bifidobacteriaceae, while a large number of harmful bacteria related to pyroptosis (such as Helicobacteraceae and Peptostreptococcaceae) were significantly decreased. The interventions of Se-BL, PB, and Se-BL@PB significantly inhibited the abundance of Helicobacteraceae, which has been associated with host cell pyroptosis in various studies. The abundances of Bifidobacterium in the PBS group and the Se-BL@PB group were significantly higher than those in other groups, indicating that the Se-BL@PB group has potential advantages in maintaining the balance of the gut microbiota and promoting health. Bifidobacteriaceae can reduce the level of oxidative stress and regulate the host immune response by producing metabolites such as short-chain fatty acids, thereby affecting the occurrence of pyroptosis. Our experimental results confirmed that Se-BL@PB can regulate the gut microbiota by disrupting the crosstalk between pyroptosis and polarization, while PB may inhibit pyroptosis by regulating the composition of specific pathogenic bacteria in the gut.

Claims

1. A Prussian blue nanozyme complex, characterized in that, The Prussian blue nanozyme complex contains selenium-enriched Bifidobacterium longum.

2. The Prussian blue nanozyme complex according to claim 1, wherein The selenium-enriched Bifidobacterium longum is prepared by the following method: a. Inoculate Bifidobacterium longum ( Bifidobacterium longum ) into RCM medium for cultivation to obtain a seed solution; b. Inoculate the seed liquid into an RCM medium containing 5 - 25 mg / ml sodium selenite for selenium enrichment culture, collect the selenium-enriched culture broth, and obtain selenium-enriched Bifidobacterium longum.

3. The Prussian blue nanozyme complex according to claim 1, characterized in that, The Prussian blue nanozyme complex is prepared by the following method: (1) Dissolve potassium ferricyanide and polyvinylpyrrolidone in a hydrochloric acid solution, stir to obtain a yellow solution, react the yellow solution at 70 - 100 °C for 20 - 30 h to obtain a blue solution, and obtain Prussian blue nanozyme after centrifugation; (2) Dissolve the Prussian blue nanozyme obtained in (1) in a hydrochloric acid solution, add polyvinylpyrrolidone, stir and mix evenly at room temperature, and react at 120 - 160 °C for 2 - 4 h. After the reaction, obtain hollow Prussian blue nanozyme through centrifugation, dialysis, and freeze-drying steps; (3) Cultivate selenium-enriched Bifidobacterium longum until the stationary phase, centrifuge to collect the selenium-enriched Bifidobacterium longum cells, wash the cells twice with PBS buffer and then resuspend the cells with PBS buffer to obtain a cell suspension; the selenium-enriched Bifidobacterium longum is prepared by the following steps: a. Inoculate Bifidobacterium longum into an RCM medium for culture to obtain a seed liquid; b. Inoculate the seed liquid into an RCM medium containing 5 - 25 μg / ml sodium selenite for selenium enrichment culture, collect the selenium-enriched culture broth, and obtain selenium-enriched Bifidobacterium longum; (4) Add the hollow Prussian blue nanozyme to the cell suspension, mix evenly, react at room temperature for 25 - 35 min (for example, 30 min), then adjust the pH to neutral, continue to react for 25 - 35 min (for example, 30 min), centrifuge to obtain the precipitate, and wash the precipitate 3 times with PBS buffer to obtain the Prussian blue nanozyme complex; Preferably, in step (1), the addition ratio of potassium ferricyanide, polyvinylpyrrolidone, and hydrochloric acid is 132 mg: 3 g: 30 ml; the molecular weight of the polyvinylpyrrolidone is K30; the concentration of the hydrochloric acid is 0.005 - 0.015 M, preferably 0.01 M; the reaction time is 24 h; the reaction temperature is 80 °C; In step (2), the addition ratio of Prussian blue nanozyme, hydrochloric acid, and polyvinylpyrrolidone is 1 mg: 1 ml: 10 mg; the concentration of the hydrochloric acid is 0.8 - 1.2 M, preferably 1.0 M; the reaction temperature is 140 °C; the reaction time is 3 h; The cell concentration during the plateau phase described in step (3) is 0.8×10 8 ~1.2×10 8 CFU / mL, preferably 1.0×10 8 CFU / mL; the concentration of the cell suspension is 1.0×10 8 CFU / mL; In step (4), the addition ratio of the hollow Prussian blue nanozyme and the cell suspension is 0.5 - 2.0 mg: 1 ml; the pH is 7.0 - 8.0, preferably 7.

0.

4. Use of the Prussian blue nanozyme complex according to claim 1 in the preparation of a drug for treating inflammatory bowel disease; Preferably, the inflammatory bowel disease is DSS-induced colitis.

5. A method for preparing Bifidobacterium longum with rich selenium, characterized in that, The method includes the following steps: a. Inoculate Bifidobacterium longum into an RCM medium for culture to obtain a seed liquid; b. Inoculate the seed liquid into an RCM medium containing 5 - 25 μg / ml sodium selenite for selenium-enriched culture, collect the selenium-enriched culture broth, and obtain selenium-enriched Bifidobacterium longum.

6. The method according to claim 5, characterized in that The culture conditions in a are as follows: under anaerobic conditions, inoculate at an inoculation amount of 1 - 3%, and culture overnight in a constant temperature incubator at 35 - 39 °C.

7. The method according to claim 5, characterized in that, The selenium-enriched culture conditions in b are as follows: under anaerobic conditions, inoculate at an inoculation amount of 2%, culture at 37 °C for 12 hours, and the concentration of sodium selenite is 10 mg / ml.

8. A Bifidobacterium longum rich in selenium, characterized in that, The selenium-enriched Bifidobacterium longum is prepared by the method described in any one of claims 5 - 7.

9. A method for preparing a Prussian blue nanozyme complex, characterized in that, The method comprises the following steps: (1) Dissolve potassium ferricyanide and polyvinylpyrrolidone in hydrochloric acid solution, stir to obtain a yellow solution, place the yellow solution in a reaction at 70 - 100 °C for 20 - 30 h to obtain a blue solution, and obtain Prussian blue nanozyme after centrifugation; (2) Dissolve the Prussian blue nanozyme obtained in (1) in hydrochloric acid solution, add polyvinylpyrrolidone, stir and mix evenly at room temperature, and react at 120 - 160 °C for 2 - 4 h. After the reaction, obtain hollow Prussian blue nanozyme through centrifugation, dialysis, and freeze-drying steps; (3) Culture the selenium-enriched Bifidobacterium longum described in claim 8 until the plateau phase, centrifuge to collect the selenium-enriched Bifidobacterium longum cells, wash the cells twice with PBS buffer, and then resuspend the cells with PBS buffer to obtain a cell suspension; (4) Add the hollow Prussian blue nanozyme to the cell suspension, mix evenly, react at room temperature for 25 - 35 min (for example, 30 min), then adjust the pH to neutral, continue to react for 25 - 35 min (for example, 30 min), centrifuge to collect the precipitate, and wash the precipitate 3 times with PBS buffer to obtain a Prussian blue nanozyme complex.

10. The method according to claim 9, characterized in that, In step (1), the addition ratio of potassium ferricyanide, polyvinylpyrrolidone, and hydrochloric acid is 132 mg: 3 g: 30 ml; the molecular weight of the polyvinylpyrrolidone is K30; the concentration of the hydrochloric acid is 0.005 - 0.015 M, preferably 0.01 M; the reaction time is 24 h; the reaction temperature is 80 °C.

11. The method according to claim 9, characterized in that, In step (2), the addition ratio of Prussian blue nanozyme, hydrochloric acid, and polyvinylpyrrolidone is 1 mg: 1 ml: 10 mg; the concentration of the hydrochloric acid is 0.8 - 1.2 M, preferably 1.0 M; the reaction temperature is 140 °C; the reaction time is 3 h.

12. The method according to claim 9, wherein The cell concentration during the plateau phase described in step (3) is 0.8×10 8 ~1.2×10 8 CFU / mL, preferably 1.0×10 8 CFU / mL; the concentration of the cell suspension is 1.0×10 8 CFU / mL.

13. The method according to claim 9, wherein In step (4), the addition ratio of the hollow Prussian blue nanozyme and the cell suspension is 0.5 - 2.0 mg: 1 ml; the pH is 7.0 - 8.0, preferably 7.0.

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