Hyaluronic acid nano-coating bacteria with anti-inflammatory and tissue repair effects as well as preparation method and application of hyaluronic acid nano-coating bacteria

Abstract

The invention discloses hyaluronic acid nano-coating bacteria with anti-inflammatory and tissue repair effects, which comprise spores, cationic chitosan is deposited on the surface of the spores, and hyaluronic acid is correspondingly arranged on the surface of the cationic chitosan. Bacteria are cultured in a sporulation culture medium to obtain a mixture of bacteria and spores, the mixture of bacteria and spores is purified to obtain spores, cationic chitosan is deposited on the surfaces of the spores through electrostatic interaction to obtain CS deposition spores, the CS deposition spores with reverse surface charges are mixed with hyaluronic acid to be further coated, and the CS deposition spores with reverse surface charges are obtained. The hyaluronic acid nano-coating bacteria are obtained. The hyaluronic acid nano-coating bacterium is applied to preparation of drugs with anti-inflammatory and tissue repair effects at the same time. According to the hyaluronic acid nano-coating bacteria with the anti-inflammatory and tissue repair effects, the anti-inflammatory and tissue repair effects are generated in damaged intestinal tracts and organs outside the intestinal tracts by means of the hyaluronic acid nano-coating clostridium butyricum.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, in particular to a hyaluronic acid nano-coated bacterium with anti-inflammatory and tissue repair effects, and a preparation method and application thereof. Background Art

[0002] Local inflammation and tissue damage play a crucial role in the pathophysiology of various extraintestinal diseases. Various endogenous and exogenous danger signals can cause tissue damage, which can lead to a sudden loss of organ function. In turn, different factors released locally by damaged tissues trigger inflammatory cascades and recruitment of immune cells, leading to further tissue damage. Although the causal relationship between inflammation and tissue damage is still inconclusive, this mutual influence is a major factor in the development of extraintestinal diseases. Local inflammation occurring in extraintestinal organs can immediately spread to the intestine and induce intestinal inflammation. Conversely, the mutual influence between intestinal inflammation and extraintestinal inflammation accelerates systemic inflammation, and toxins and pathogens are continuously transported from the intestine to the systemic circulation, eventually reaching extraintestinal organs, causing tissue damage, thus forming a vicious cycle and exacerbating extraintestinal diseases. Therefore, strategies that can produce a dual effect of inflammation suppression and tissue restoration in the damaged intestine and extraintestinal organs are of great significance for reversing extraintestinal diseases.

[0003] Traditional drugs, such as nonsteroidal anti-inflammatory drugs, glucocorticoids, and monoclonal antibodies, are widely used to reduce inflammation. These therapies include direct elimination of pathogenic stimuli, reduction of proinflammatory cytokines, and non-phagocytic elimination of immune cells. However, the use of these therapies is highly associated with an increased risk of bacterial infections, such as Clostridium difficile, Salmonella, and Shigella. For example, a clinical cohort study for Crohn's disease showed that the proportion of serious infections after treatment with infliximab, a monoclonal antibody against tumor necrosis factor-α (TNF-α), was significantly higher than that of patients who did not receive treatment. On the other hand, many small molecules and compounds targeting transforming growth factor-β (TGF-β), receptor tyrosine kinases, and phosphatidylinositol 3-kinase have been applied in clinical trials to repair organ damage. For example, most TGF-β inhibitors may induce cardiotoxicity, manifested as degeneration, hemorrhage, and hyperplasia of valvular interstitial cells. More importantly, these approaches cannot achieve anti-inflammatory effects and tissue repair effects in both intestinal and extraintestinal organs at the same time.

[0004] Therefore, it is necessary to design a hyaluronic acid nano-coated bacteria with anti-inflammatory and tissue repair effects, and a preparation method and application thereof. Summary of the invention

[0005] In order to overcome the defects in the prior art, a hyaluronic acid nano-coated bacterium with anti-inflammatory and tissue repair effects and a preparation method and application thereof are provided.

[0006] The present invention is achieved through the following scheme:

[0007] A hyaluronic acid nano-coated bacterium with anti-inflammatory and tissue repair effects comprises spores, on the surface of which cationic chitosan is deposited, and on the surface of which hyaluronic acid is correspondingly arranged.

[0008] The spores are dormant spores produced by culturing butyric acid bacteria in a spore culture medium.

[0009] Cationic chitosan was deposited on the surface of collected spores by electrostatic action.

[0010] A method for preparing hyaluronic acid nano-coated bacteria with anti-inflammatory and tissue repair effects, the method comprising the following steps: culturing bacteria in a spore-producing medium to obtain a mixture of bacteria and spores, purifying the mixture of bacteria and spores to obtain spores, depositing cationic chitosan on the surface of spores through electrostatic action to obtain CS-deposited spores, and mixing these CS-deposited spores with reversed surface charges with hyaluronic acid for further coating to obtain hyaluronic acid nano-coated bacteria.

[0011] The specific steps of culturing the bacteria in the spore-forming medium are: culturing the bacteria in the spore-forming medium in an anaerobic environment maintained at a temperature of 37° C.;

[0012] Caco-2 and HK-2 cell lines were obtained from the American Type Culture Collection and cultured in DMEM / F-12 and DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin, respectively, at 37°C in a 5% CO2 incubator.

[0013] The bacterium is Clostridium butyricum.

[0014] The sporulation medium contains 0.3% lactose, 0.035% MgSO4, 1% yeast extract, 0.2% NaHCO3, 0.3% K2HPO4, and 0.1% KH2PO4.

[0015] The specific steps of purifying the mixture of bacteria and spores are as follows: the bacteria are cultured in a spore-producing medium for 10-14 days to obtain a mixture of bacteria and spores, the obtained mixture of bacteria and spores is centrifuged at 5500×g for 10 minutes, boiled at 80°C for 20 minutes, and then the spores are further purified by density gradient centrifugation of a solution of 65% sucrose and 35% sucrose; the purified spores are then washed with phosphate buffered saline, and vacuum dried at room temperature to obtain the purified spores.

[0016] The invention discloses an application of hyaluronic acid nano-coated bacteria with anti-inflammatory and tissue repair effects, and an application of the hyaluronic acid nano-coated bacteria as a drug for preparing a drug with both anti-inflammatory and tissue repair effects.

[0017] Application of the hyaluronic acid nano-coated bacteria as a drug for reducing intestinal mucosal inflammation and restoring intestinal barrier integrity;

[0018] The hyaluronic acid nano-coated bacteria can also be used to prepare drugs for remodeling microbial metabolites and reducing microbial transport, which can reduce interstitial inflammation and pathological damage of extra-intestinal organs.

[0019] The beneficial effects of the present invention are:

[0020] The present application is a kind of hyaluronic acid nano-coated bacteria with anti-inflammatory and tissue repair effects, which uses hyaluronic acid nano-coated Clostridium butyricum to produce anti-inflammatory and tissue repair effects in damaged intestinal and extra-intestinal organs. With the help of cationic chitosan surface charge reversal, negatively charged butyric acid bacteria can be electrostatically combined with anionic HA nano-coated under cell-compatible conditions, and the formed HA nano-coating can protect butyric acid bacteria from gastrointestinal (GI) damage and improve its accumulation at the site of intestinal inflammation and damage. Due to the combination of the innate immunosuppressive properties of HA and the butyric acid production behavior of butyric acid bacteria, nano-coated bacteria can effectively alleviate intestinal mucosal inflammation and restore the integrity of the intestinal barrier. By reshaping microbial metabolites and reducing microbial transport, nano-coated bacteria can also reduce interstitial inflammation and pathological damage in extra-intestinal organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the efficacy of hyaluronic acid nano-coated bacteria in this application;

[0022] Figure 2 Schematic diagram of the characteristics of Spore@CH;

[0023] Figure 3 Schematic diagram of the retention of spores@CH in the pathological digestive tract;

[0024] Figure 4 Schematic diagram of the intestinal inflammation inhibition and barrier repair effects of spores@CH;

[0025] Figure 5 Schematic diagram for regulating the balance of intestinal microorganisms;

[0026] Figure 6 Schematic diagram of the extraintestinal inflammation inhibition and organ repair effects of spores@CH;

[0027] Figure 7 Schematic diagram of the in vivo experimental results of the therapeutic effect of Spore@CH in acute kidney injury;

[0028] Figure 8 Schematic diagram of the potential of Spore@CH to treat chronic kidney disease;

[0029] Fig. 9 Relative abundance of Clostridium A and Butyricum B in healthy stool samples and stool samples of patients with various diseases searched by GMrepo database. Data are mean ± SD. Statistical analysis was performed using Student's t-test. ***p < 0.001, **p < 0.01, *p < 0.05;

[0030] Fig.10 TEM images of spores, spores@C, and spores@CH. Scale bar is 500 nm;

[0031] Fig.11 LSCM images of spore@CH. Green and red indicate CS labeled with FITC and HA labeled with RhB. Scale bar is 2 μm;

[0032] Fig.12 Bacterial counts after 30 min incubation in SGF, 15 min incubation in bile salts, or 2 h incubation in SIF (n=3). Data are mean ± SD. Statistical analysis was performed using Student's t test.

[0033] *p<0.05;

[0034] Fig.13 Bacterial counts in the small intestine, cecum, and colon 6 hours after intragastric administration of equal amounts of spores or spores@CH (n=3). Data are mean ± SD. Statistical analysis was performed using Student's t test. **P<0.01, *P<0.05;

[0035] Fig.14 Retention rate of oral spore@CH in mice. A, B IVIS images of AKI mice 6 hours after administration and corresponding fluorescence intensity quantification (n=3). A and B represent spores labeled with Cy5.5 and HA labeled with Cy5, respectively. Data are mean ± SD. Statistical analysis was performed using one-way ANOVA or Student's t-test. *p<0.05;

[0036] Fig.15 A Flow cytometry scatter plot and B percentage of late apoptotic Caco-2 cells (Annexin V+PI+) stimulated by LPS after treatment with PBS, HA, Spore or Spore@CH (n=3). The control group represents unstimulated Caco-2 cells. Data are mean ± SD. One-way ANOVA was used for statistical analysis. ***P<0.001 and **P<0.01;

[0037] Fig.16A H&E staining images of colon sections of cisplatin- and B folic acid-induced mice after different treatments (n=3) and corresponding pathological score analysis. Blue and red arrows indicate edema and epithelial damage, respectively. Scale bar, 200 μm. Data are mean ± SD. One-way ANOVA was used for statistical analysis. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05;

[0038] Fig.17 Evaluation of intestinal barrier integrity in CKD mice after treatment with PBS, HA, spores, or spores@CH every other day for 14 days. A Immunofluorescence images of occludin (red) and ZO-1 (green) expression in the colon of treated mice. Cell nuclei were stained with DAPI (blue). Scale bar, 50 μm. B The average grayscale value of the red and green channels in the immunofluorescence images of colon sections was measured using ImageJ software (n=3). C PAS-AB staining images of the colon of treated mice and the average number of Altun Blue-positive cells per 20× field of view in colon sections (n=3). Scale bar, 100 μm. Data are mean ± SD. One-way ANOVA was used for statistical analysis. ***p<0.001, **p<0.01, *p<0.05;

[0039] Fig.18 Alterations in the intestinal microbiota of mice induced by folic acid after Spore@CH treatment. A Shannon index and Chao1 index of intestinal microbiota. B Species abundance of intestinal microbiota of mice in each group. C Relative abundance of Faecalibaculum in the intestinal microbiota (n=4 or 5). D IS, pCS, and IAA levels in feces of each group (n=4 or 5). Data are mean ± SD. One-way ANOVA was used for statistical analysis. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05;

[0040] Fig.19 FITC-dextran concentration in the culture medium on the basolateral side of the Transwell measured by a microwell fluorimeter after 6 hours of culture (n=3). Data are mean ± SD. One-way ANOVA was used for statistical analysis. **P<0.01 and *P<0.05;

[0041] Fig. 20 gating strategies to identify leukocytes, neutrophils, and macrophages;

[0042] Fig.21 A Flow cytometric dot plot of leukocytes (CD45+) in renal tissue of mice with acute renal injury after different treatments. B The percentage of leukocytes (CD45+) in the total number of renal cells. Data are mean ± SD. One-way ANOVA was used for statistical analysis. ***P < 0.001 and *P < 0.05;

[0043] Fig. 22 A Flow cytometry scatter plot and B percentage of late apoptotic HK-2 cells (Annexin V+PI+) stimulated by LPS after treatment with PBS, HA, Spore or Spore@CH (n=3). The control group represents unstimulated Caco-2 cells. Data are mean ± SD. One-way ANOVA was used for statistical analysis. *p<0.05;

[0044] Fig.23 A Flow cytometry scatter plot of leukocytes (CD45+), neutrophils (CD11b+Ly6G+), macrophages (F4 / 80+CD11b+), and CD4+T cells in the kidney tissue of mice with acute kidney injury after different treatments. B The percentage of leukocytes (CD45+) in the total number of kidney cells. Data are mean ± SD (n = 5);

[0045] Fig.24 Body weight changes of CKD mice after different treatments (n=5). Data are mean ± SD. Statistical analysis was performed using two-way ANOVA. *p<0.05;

[0046] Fig.25 Uncropped Western blot images of α-SMA, tubulin, FN-1, and β-actin;

[0047] Fig.26 A Flow cytometry scatter plot of leukocytes (CD45+), neutrophils (CD11b+Ly6G+), and CD4+T cells in renal tissue. B The percentage of leukocytes (CD45+), neutrophils (CD45+CD11b+Ly6G+), and CD4+T cells (CD45+) in the total number of renal cells in CKD mice after different treatments (n=5). Data are mean ± SD. One-way ANOVA was used for statistical analysis. ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05;

[0048] Fig. 27 H&E staining images of spleen, lung, heart, and liver of CKD mice after different treatments. Scale bar, 100 μm;

[0049] Fig.28 Folic acid-induced CKD mice were treated with PBS, Spoore@CH, or PD every other day for 14 days. PD was performed every other day with 5 ml of dialysate for 2 hours. Serum ABUN and B-creatinine levels of mice on day 14. Data are mean ± SD. One-way ANOVA was used for statistical analysis. ***P<0.001, **P<0.01, *P<0.05;

[0050] Fig.29 Images and quantification of AMasson and BSirus Red staining of kidney sections of CKD mice after different treatments. Scale bar, 100 μm. Data are mean ± SD. Statistical analysis was performed using one-way ANOVA. ***P < 0.001, **P < 0.01, *P < 0.05; DETAILED DESCRIPTION

[0051] The preferred embodiments of the present invention are further described below:

[0052] In this application, the Chinese meanings of some English words are explained as follows:

[0053] Clostridium butyricum

[0054] Cationic Chitosan: CS

[0055] Hyaluronic acid: HA

[0056] Spore

[0057] Spore@C: CS deposited spores

[0058] Spore@CH: HA coated Spore@C

[0059] The technical solution of this application includes:

[0060] A hyaluronic acid nano-coated bacterium with anti-inflammatory and tissue repair effects comprises spores, on the surface of which cationic chitosan is deposited, and on the surface of which hyaluronic acid is correspondingly arranged.

[0061] The spores are dormant spores produced by culturing butyric acid bacteria in a spore culture medium.

[0062] Cationic chitosan was deposited on the surface of collected spores by electrostatic action.

[0063] A method for preparing hyaluronic acid nano-coated bacteria with anti-inflammatory and tissue repair effects, the method comprising the following steps: culturing bacteria in a spore-producing medium to obtain a mixture of bacteria and spores, purifying the mixture of bacteria and spores to obtain spores, depositing cationic chitosan on the surface of spores through electrostatic action to obtain CS-deposited spores, and mixing these CS-deposited spores with reversed surface charges with hyaluronic acid for further coating to obtain hyaluronic acid nano-coated bacteria.

[0064] The specific steps of culturing the bacteria in the spore-forming medium are: culturing the bacteria in the spore-forming medium in an anaerobic environment maintained at a temperature of 37° C.;

[0065] Caco-2 and HK-2 cell lines were obtained from the American Type Culture Collection and cultured in DMEM / F-12 and DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin, respectively, at 37°C in a 5% CO2 incubator.

[0066] The bacterium is Clostridium butyricum.

[0067] The sporulation medium contains 0.3% lactose, 0.035% MgSO4, 1% yeast extract, 0.2% NaHCO3, 0.3% K2HPO4, and 0.1% KH2PO4.

[0068] The specific steps of purifying the mixture of bacteria and spores are as follows: the bacteria are cultured in a spore-producing medium for 10-14 days to obtain a mixture of bacteria and spores, the obtained mixture of bacteria and spores is centrifuged at 5500×g for 10 minutes, boiled at 80°C for 20 minutes, and then the spores are further purified by density gradient centrifugation of a solution of 65% sucrose and 35% sucrose; the purified spores are then washed with phosphate buffered saline, and vacuum dried at room temperature to obtain the purified spores.

[0069] The invention discloses an application of hyaluronic acid nano-coated bacteria with anti-inflammatory and tissue repair effects, and an application of the hyaluronic acid nano-coated bacteria as a drug for preparing a drug with both anti-inflammatory and tissue repair effects.

[0070] Application of the hyaluronic acid nano-coated bacteria as a drug for reducing intestinal mucosal inflammation and restoring intestinal barrier integrity;

[0071] The hyaluronic acid nano-coated bacteria can also be used to prepare drugs for remodeling microbial metabolites and reducing microbial transport, which can reduce interstitial inflammation and pathological damage of extra-intestinal organs.

[0072] Kidney diseases, including acute kidney injury (AKI) and chronic kidney disease (CKD), are common clinical diseases with high morbidity and mortality. It has been reported that intestinal microbiota can proteolyze aromatic amino acids in the diet to produce uremic toxin (UTx) precursors, which are an important cause of renal failure. On the contrary, kidney damage can lead to intestinal dysfunction, manifested as impaired intestinal barrier function and the occurrence of inflammatory response, which promotes the transport of bacteria and bacteria-derived UTx to the kidneys, thereby exacerbating the process of kidney damage. At the same time, the leakage of bacterial derivatives (such as lipopolysaccharide) can also aggravate local inflammation in the kidneys. Therefore, it is very ideal to be able to simultaneously produce a dual effect of inflammation suppression and tissue recovery in the damaged intestine and kidney. Unfortunately, to the best of our knowledge, such therapies have rarely been reported. The nano-coated bacteria of the present application have an attractive therapeutic potential in the treatment of kidney disease after oral administration. In cisplatin-induced AKI and folic acid-induced CKD mouse models, the nano-coated bacteria have the effects of restoring renal function and eliminating renal fibrosis, respectively. It can be expected that the dual-function and dual-localization intervention mediated by the nano-encapsulated bacteria of the present application provides a unique strategy for regulating the tandem between the intestine and distal organs, providing a feasible path for the treatment of extraintestinal diseases.

[0073] Figure 1 This is a schematic diagram of the efficacy of the hyaluronic acid nano-coated bacteria of this application; it can be seen from the figure that oral spores@CH can be preferentially retained in the diseased intestine, which can not only reduce intestinal inflammation and repair the intestinal barrier, but also reduce intestinal interstitial inflammation and pathological damage to extraintestinal organs by remodeling microbial metabolites and reducing microbial transport. Oral spores@CH can restore renal function and eliminate renal fibrosis, respectively, and is an alternative therapy for the treatment of AKI and CKD.

[0074] The present application is further described below in conjunction with specific embodiments:

[0075] method

[0076] Fetal bovine serum (FBS, 10099-141), DMEM F-12 (DMEM / F-12, 11320033), DMEM (11965092) and RPMI 1640 Medium (11875119) were purchased from Gibco. Penicillin-streptomycin (15140-122) was purchased from Hyclone. Cationic chitosan (CS, 850122-100g) and hyaluronic acid (HA, MB7238) were purchased from McLean Biotech and Meilun Biotech, respectively. Reinforced Clostridium medium (RCM, HB0316) was purchased from Qingdao Hope Biotech. Trypsin (A610681-0100) and bile salt (S30875) were purchased from Sangon Biotech and Shanghai Yuanye Biotech, respectively. Cy5.5 (D10013) and Cy5 (D10012) were purchased from Dofluoro. FITC-dextran (FD4-100MG), lipopolysaccharide (LPS, L4130), cisplatin (P4394), N-acetylcysteine ​​(NAC, PHR1098), collagenase IV (5138), folic acid (F7876) and DNase I (D4263) were purchased from Sigma-Aldrich. Annexin V-FITC apoptosis detection kit (AP101) was from MultiSciences Biotech. Urea detection kit (DIUR-100) and QuantiChrom creatinine detection kit (DICT-500) were purchased from BioAssay Systems. Peritoneal dialysis fluid (Lactate-G1.5%, H20133291) was purchased from Baxter Healthcare Corporation. Hemoglobin and eosin (H&E) staining kit (C0105), myeloperoxidase (MPO) staining kit (AF7494), persulfate Schiff (PAS) staining kit (C0142), and 4',6-diamidino-2-phenylindole (DAPI, C1002) were purchased from Beyotime Biotechnology. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) apoptosis detection kit (G1504) was purchased from Servicebio. TRIzol reagent (dp419) was purchased from Tiangen Biotechnology. PrimeScript RT Master Mix (R323) and SYBR green Master Mix (Q711) were purchased from Vazyme Biotec Co., LTD. Anti-tubulin (66031-1) and anti-β-actin (8457T) were purchased from Cell Signaling Technology. Anti-FN-1 (ab45688) and anti-α-SMA (ab124964) were purchased from Abcam.HRP goat anti-rabbit IgG (SA00001-2) and HRP horse anti-mouse IgG (SA00001-1) were purchased from Proteintech, antiglobulin (sc-133256) was purchased from Santa Cruz Biotechnology, and anti-ZO-1 (AF5145) was purchased from Affinity Biosciences. Anti-CD16 / 32 (101303, clone name: 93), anti-mouse CD45-APC / Fire 750 (103154, clone name: 30-F11), anti-mouse CD11b-FITC (101205, clone name: M1 / 70), anti-mouse CD45-APC / Fire 750 (103154, clone name: 30-F11), anti-mouse CD11b-FITC (101205, clone name: M1 / 70), anti-mouse Ly6G-PE (127607, clone name: 1A8), anti-mouse F4 / 80-APC (123115, clone name: BM8) and anti-mouse CD4-PE / Cyanine7 (100422, clone name: GK1.5) were purchased from BioLegend.

[0077] Bacteria and cell lines

[0078] C. butyricum purchased from China General Microbiological Culture Collection Center was cultured in a spore-forming medium containing 0.3% lactose, 0.035% MgSO4, 1% yeast extract, 0.2% NaHCO3, 0.3% K2HPO4, and 0.1% KH2PO4 in an anaerobic chamber at 37° C. Caco-2 and HK-2 cell lines were obtained from American Type Culture Collection and cultured in DMEM / F-12 and DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin, respectively, at 37° C. in a 5% CO2 incubator.

[0079] Purification of spores

[0080] After culturing Clostridium butyricum for 10-14 days, the obtained mixture of bacteria and spores was centrifuged at 5500×g for 10 min, boiled at 80°C for 20 min, and further purified by density gradient centrifugation of a solution of 65% sucrose and 35% sucrose. The purified spores were then washed with phosphate buffered saline (PBS) and vacuum dried at room temperature (RT) for future use.

[0081] Characterization of Spore@CH

[0082] The morphology of spores, Spore@C and Spore@CH was observed by transmission electron microscopy (TEM, H7700s, HITACH) and scanning electron microscopy (SEM, Sirion 200, USA). For transmission electron microscopy, 10 μl of sample was dropped on a 300-mesh Formvar / carbon screen and incubated for 1 h. After washing twice with 10 μl of double distilled water (ddH2O), the sample was completely dried at room temperature. For scanning electron microscopy, the sample was immediately fixed in 2.5% glutaraldehyde solution, fixed at room temperature for 1 h, and then dehydrated continuously with alcohol. Before observation, the sample needed to be dropped on a silicon wafer. Dynamic light scattering (DLS, Malvern Zetasizer nano ZS, UK) was used to measure the size and surface zeta potential of spores, Spore@C and Spore@CH. Spore@CH was prepared using FITC-labeled CS and Cy3-labeled HA for laser scanning confocal microscopy (LSCM, Leica TCS SP8, Germany) and flow cytometry (Beckman CytoFlex, USA).

[0083] Survival rate and growth curve of Spore@CH

[0084] To confirm the survival rate of Spore@CH, 1×10 spores or Spore@CH were added. 7 Colony forming units (cfu) were spread on RCM agar plates and incubated overnight at 37°C under anaerobic conditions. At the same time, spores or Spore@CH were resuspended in RCM and incubated at 37°C. The optical density (OD) value at 600 nm (OD600) was measured and recorded for 12 h with a microplate reader (BioTek, USA) at 2 h intervals.

[0085] Tolerance of Spore@CH to Simulated Gastrointestinal Fluid

[0086] Equal amounts of spores or spores @CH (5×10 7 CFUs) were suspended in 1 ml of simulated gastric fluid (SGF, 32 g / L sodium chloride solution containing 2 g / L pepsin, pH 2.0), simulated intestinal fluid (SIF, 6.8 g / L KH2PO4 solution containing 10 g / L trypsin, pH 6.8) or bile salt solution (0.3 mg / ml), and then gently shaken in an anaerobic chamber at 37°C. After 30 minutes, 15 minutes and 2 hours of incubation, the spores were centrifuged at 5500×g for 10 minutes and resuspended in PBS. Then, the spores were spread on RCM agar plates and incubated overnight at 37°C to determine the bacterial count.

[0087] Apoptosis detection

[0088] After exposure to 1 μg / ml LPS for 24 h, the cells were harvested and mixed with equal amounts of HA, spores, or spores@CH (1×10 6 CFUs) were co-cultured with Caco-2 cells. The apoptosis level of Caco-2 cells was then assessed using the Annexin V-FITC apoptosis detection kit according to the kit provided by the manufacturer. The results were detected using a FACSVerse flow cytometer (BD Biosciences, USA), and the data were analyzed using FlowJo (TreeStar). To further simulate the in vivo environment, a Caco-2 and HK-2 cell co-culture system was used as described previously [1]. Briefly, 5×10 5 Caco-2 cells were inoculated into the top insertion of a transwell (Corning, 1.12 cm2, pore size 0.4 μm) and cultured for 14 days, with the culture medium replaced every 2 days. 5 HK-2 cells were added to the basolateral chamber of the transwell and cultured for another 7 days. Equal amounts of HA, spores, or spores@CH (1×106 CFUs) were added to the apical insert, and LPS (1 μg / ml) was added to the basolateral side to simulate the cells. After overnight culture at 37°C, HK-2 cells were collected for apoptosis detection.

[0089] In vitro intestinal permeability assay

[0090] In order to establish biomimetic intestinal epithelial cells in vitro, 5×10 5 Caco-2 cells were seeded into the apical insert of the transwell and cultured for 14 days. LPS (1 μg / ml) was added to the basolateral side to induce intestinal barrier impairment. After 12 h of stimulation, 500 μl of 100 μg / ml FITC-dextran (4 kDa) was added to the apical insert and cultured for 6 h. The concentration of FITC-dextran was determined by a microplate fluorimeter (Infinite M200 PRO, Tecan) with an excitation wavelength of 490 nm and an emission wavelength of 530 nm.

[0091] Intestinal adhesion in vivo

[0092] Freshly sampled mouse intestinal tissue (including ileum and colon) was cut into small segments of 1 cm and then peeled to expose the intestinal lumen. The intestinal segments were then gently rinsed twice with PBS to remove unattached spores and imaged by a live imaging system (IVIS, LuminaII, Caliper). In order to quantify the retained spores, the intestinal segments were further homogenized and bacterial plate counts were performed.

[0093] Microbiome 16S ribosomal RNA gene sequencing

[0094] Folic acid-induced mice were gavaged with equal amounts of HA, spores, or spores@CH (1 × 107 CFUs per mouse) every other day for 14 consecutive days, and then intestinal samples were collected for 16S ribosomal RNA gene sequencing. 16S ribosomal RNA gene sequencing and analysis were performed at Biomarker Technologies. First, total bacterial DNA was extracted from colon samples of folate-induced mice after different treatments. The V3-V4 hypervariable region of the bacterial 16S ribosomal RNA gene was amplified with primers 27F-1492R, and the obtained sequences were detected on the Illumina HiSeq platform. Data analysis was performed on BMKCloud. The chao1 index was used to analyze alpha diversity to examine the richness and diversity of species in a single sample; the principal coordinate analysis (PCoA) was used to analyze beta diversity. In addition, species were classified according to the sequence composition of the operational taxonomic unit. The microbial composition was analyzed by family and genus, respectively.

[0095] Western blotting

[0096] Renal tissue protein was extracted and quantified using an enhanced BCA protein assay kit. Equal amounts of total protein (10–20 μg) from different groups were layered by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a 0.22 μm polyvinylidene difluoride membrane (Merk Millipore, Germany). After blocking with 5% skim milk in tris-buffered saline (TBST) containing 0.05% Tween-20 for 1 h, the membranes were incubated with primary antibodies overnight at 4 °C. The primary antibodies used were as follows: anti-tubulin (dilution 1:1000), anti-FN-1 (dilution 1:2000), anti-α-SMA (dilution 1:10000), and anti-β-actin (dilution 1:4000). The membranes were washed three times with TBST for 10 min each and then stained with secondary antibodies: HRP goat anti-rabbit IgG (dilution 1:3000) and HRP horse anti-mouse IgG (dilution 1:3000) for 2 h at RT. Wash three times with TBST for 10 minutes each time, and then use electrochemiluminescence (ECL) system (GE & Amersham Imager 680R) to visualize protein bands. Use ImagePro Plus software to semi-quantitatively analyze protein expression levels. Band density is normalized to the band density of internal control (β-actin or tubulin).

[0097] Histopathology and immunofluorescence analysis

[0098] The collected colon or kidney tissues were fixed with 4% paraformaldehyde and then embedded in paraffin. The colon samples were sliced ​​into 4-μm slides and then deparaffinized and subjected to H&E staining and MPO staining according to the manufacturer's instructions. To evaluate the pathological damage of the renal tubules after induction, PAS staining was performed on the kidney sections. The tubular injury score was determined according to the previous criteria [2]: 0 (no damage), 1 (damage <25%), 2 (damage 25-50%), 3 (damage 50-75%), and 4 (damage >75%). Masson staining and Sirius red staining were used to estimate the degree of renal fibrosis in CKD. The percentage of fibrosis area was analyzed using ImagePro Plus software.

[0099] For immunofluorescence analysis, sections were deparaffinized and antigen retrieval was performed with citrate buffer (pH 6.0). After blocking with 10% normal goat serum for 1 h, sections were incubated with primary antibodies (including occludin and ZO-1) at 4 °C overnight, followed by incubation with corresponding secondary antibodies at RT for 1 h. Kidney sections were stained with TUNEL following the same protocol as above. All samples were covered with antifading membranes containing DAPI. Finally, CLSM was used to observe and capture images. Quantitative analysis of colon staining areas was performed using ImagePro software (MediaCybernetics, USA).

[0100] Real-time quantitative polymerase chain reaction (RT-qPCR)

[0101] Total RNA was extracted from kidney tissue samples using TRIzol reagent, and cDNA was synthesized using PrimeScript RT MasterMix according to the manufacturer's protocol. RT-qPCR was performed using SYBRgreen Master Mix and Applied Biosystems 7500 system. The specificity of RT-qPCR was verified by melting curve analysis. The relative expression of target genes was determined by the 2-ΔΔCt method and normalized to the internal control β-actin. The primer sequences are listed in Table 1.

[0102] Table 1 RT-qPCR primer pairs

[0103]

[0104] Detection of kidney-infiltrating inflammatory cells

[0105] To detect kidney-infiltrating neutrophils, macrophages, and CD4+T cells, kidney tissue was cut into small pieces and digested in 1640 medium containing 1 mg / ml collagenase IV and 10 μg / ml DNaseI, shaking at 37°C for 45 minutes. The digested suspension was passed through a 70 μm cell strainer and rinsed twice with ice-cold 1× PBS. The resulting cells were blocked with anti-CD16 / 32. To detect neutrophils, kidney single-cell suspensions were further stained with anti-mouse CD45-APC / Fire 750, anti-mouse CD11b-FITC, and anti-mouse Ly6G-PE. To detect macrophages, kidney single-cell suspensions were further stained with anti-mouse CD45-APC / Fire 750, anti-mouse CD11b-FITC, and anti-mouse F4 / 80-APC. To detect CD4+T cells, kidney single cell suspensions were further stained with anti-mouse CD45-APC / Fire 750 and anti-mouse CD4-PE / Cyanine7. Antibodies for flow cytometry were purchased from BioLegend. Data were collected on a FACSVerse flow cytometer (BD Biosciences, USA) and analyzed using FlowJo software (TreeStar). The percentages of neutrophils, macrophages, and CD4+T cells in the total number of renal cells were calculated as the percentages of CD45+CD11b+Ly6G+, CD45+CD11b+F4 / 80+, and CD45+CD4+ cells in the total number of renal cells, respectively.

[0106] Quantification of metabolites

[0107] Fecal samples (0.2 g) were thoroughly mixed with 2 ml of ddH2O and then centrifuged at 19,000×g for 10 min at 4°C. The supernatant was collected and 10 μl of 50% sulfuric acid was added to the supernatant. Then, 2 μl of diethyl ether was added to the supernatant and mixed. Centrifuged at 4,000×g for 10 min, and the diethyl ether layer was discarded. The samples were filtered through a 0.22 μm filter and measured by high-performance liquid chromatography (Agilent Technologies, Dover, DE, USA) at a wavelength of 206 nm. The operating conditions were set as follows: flow rate 0.6 ml / min, column temperature 35°C, and injection volume 10 μl. The analysis time for each sample was 30 min. For the qualitative and quantitative analysis of short-chain fatty acids (SCFA), SCFA standard solutions of different concentrations were prepared. The SCFA content in serum was evaluated according to the same protocol as above.

[0108] Quantitative analysis of uremic toxins (UTx)

[0109] UTx concentrations were measured by fluorescence. Briefly, 10 μl of 35% perchloric acid was added to 50 μl of serum or stool samples, and the mixture was vortexed for 30 s. After centrifugation at 16,000 × g for 10 min, the supernatant was diluted with ddH2O. A volume of 2 μl of the sample was then injected into an HPLC system (Agilent Technologies, Dover, TX, USA). A reversed-phase C18 column (Kromasil 100-3.5, 150 mm × 2.1 mm, 3 μm, Akzo Nobel, Sweden) and a C18 guard column were used to separate all target compounds. The mobile phase consisted of 85% solvent A (containing 17 mmol / l NaH2PO4 and 0.1% H3PO4 as ion pairing agent in water) and 15% methanol (as solvent B) at a flow rate of 0.16 ml / min and a temperature of 35 °C during isocratic elution of ninhydrin sulfate (IS). The mobile phase of p-cresol sulfate (pCS) contained 75% solvent A and 25% methanol as solvent B, while the mobile phase of indole-3-acetic acid (IAA) contained 58% solvent A and 42% methanol as solvent B. Quantitative detection was performed using a fluorescence detector (RF-20 model, Shimadzu Corporation, Kyoto, Japan) with excitation / emission wavelengths of 278 / 348 nm for IS and IAA and 265 / 290 nm for pCS. The retention times of IS, pCS, and IAA were approximately 10.2 ± 0.2, 11.0 ± 0.2, and 9.1 ± 0.2 min, respectively. Calibration curves of these UTx were established using the external standard method, showing a strong linear relationship between peak area and concentration (R2>0.999).

[0110] Experimental Section

[0111] Animals BALB / c mice (male, 6–8 weeks old) and C57BL / 6 mice (male, 6–8 weeks old) were purchased from Spief Biotechnology Co., Ltd. (Beijing, China) and housed in an SPF environment with an average temperature of 22°C and a standard light–dark cycle of 12 h. Animal experiments were performed in accordance with relevant guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of the Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine (SH9H-2023-A822-1).

[0112] Preparation of Spore@CH: 10 mg of spores were rinsed twice with 1 ml of 0.5 M NaCl solution (pH 6) and suspended. To prepare Spore@C, 600 μl of 2 mg / ml CS dissolved in NaCl solution was added to the spore solution. The resulting mixture was shaken at 55×g for 30 min at room temperature and then centrifuged at 1000×g for 10 min. After being rinsed twice with NaCl solution, Spore@C was dispersed in 1 ml of NaCl solution. To further prepare Spore@CH, 800 μl of 4 mg / ml HA dissolved in NaCl solution was added to the Spore@C solution, and the resulting mixture was shaken at 55×g for 30 min at room temperature. After being washed twice with NaCl solution, Spore@CH was collected.

[0113] In vivo oral bioavailability: BALB / c mice were orally administered with 1×108 CFUs of spores or spores@CH. Four hours after administration, the mice were euthanized and the digestive tract tissues were collected. Tissues including the small intestine, cecum, and colon, as well as the luminal contents, were collected separately. Equal amounts of the samples were diluted with PBS, and the volume of each dilution (50 μl) was spread on solid RCM agar plates and incubated overnight in an anaerobic chamber at 37°C.

[0114] Accumulation of Spore@CH in the inflamed intestine: To investigate the retention capacity of Spore@CH in the inflamed intestine, spores or HA were labeled with Cy5.5. AKI mice were gavaged with 1×108 CFU of fluorescently labeled spores or Spore@CH (spores were labeled with Cy5.5). Similarly, AKI mice were orally administered with fluorescently labeled HA or 1×108 CFU of Spore@CH (HA was labeled with Cy5). Six hours after administration, treated mice were imaged using IVIS. Then, mice were euthanized and digestive tract tissues were collected for IVIS imaging. Images of mice and tissues were recorded and analyzed by Living Image 4.2.

[0115] Efficacy of Spore@CH in the Treatment of AKI and CKD: To establish the mouse AKI model, cisplatin was dissolved in sterile PBS at a concentration of 1 mg / ml, and C57BL / 6 mice (approximately 20-25 g) were intraperitoneally injected with cisplatin solution (20 mg / kg body weight) once. Then, the mice were randomly divided into six groups and orally administered 200 μl of PBS, HA, spores, spore@C, spore@CH (1×107 CFUs) or NAC (400 mg / kg) daily for 3 consecutive days. At the end of the experiment, the mice were euthanized for efficacy evaluation. Serum was collected to detect BUN and creatinine levels. BUN was detected using a urea detection kit and creatinine was detected using a QuantiChrom creatinine detection kit according to the manufacturer's instructions.

[0116] To induce the mouse CKD model, male C57BL / 6 mice were intraperitoneally injected with 300 mg / kg of folic acid dissolved in 0.3 M sodium bicarbonate. Then, the mice were randomly divided into five groups. On the third day after the induction of CKD, the mice were orally administered 200 μl of PBS containing HA, spores, spores@C or spores@CH (1×107 CFUs per mouse) at intervals of 2 days for a total of 14 days. Healthy mice that did not take folic acid served as the control group. To evaluate the efficacy of spores@CH, a PD group was further applied. CKD mice were intraperitoneally injected with 3 ml of low-calcium PD solution (Lactate-G1.5%) every two days. The body weight of the mice was monitored every day. At the end of the experiment, the mice were euthanized for efficacy evaluation.

[0117] Statistical analysis Data are presented as mean ± SD. Unpaired two-tailed Student's t-test was used to compare statistical differences between the two groups. One-way ANOVA with Tukey's multiple comparisons or two-way ANOVA with Fisher's LSD multiple comparisons were used for multiple comparisons. Statistical analysis was performed using GraphPad Prism 8.0. In all cases, statistical significance was based on p < 0.05, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0118] In order to search for bacteria with the potential to treat extra-intestinal injuries, we evaluated the profile of microbial composition in fecal samples from patients with different diseases by analyzing metagenomic sequence data extracted from public databases. In particular, the number of Clostridium butyricum was significantly reduced in diseases of the kidney, liver, nervous system, metabolism, and digestive system. It is well known that Butyricum is a strictly anaerobic spore-forming bacillus that is commonly used to maintain the integrity of the intestinal barrier due to its ability to produce butyric acid. On the other hand, HA is a natural glycosaminoglycan biopolymer approved by the US Food and Drug Administration, and its efficacy in inhibiting inflammatory responses has been widely recognized. Given this inherent property, we chose to combine HA with Butyricum to form a therapeutic platform with dual anti-inflammatory and tissue repair effects.

[0119] Since Butyricum is susceptible to oxygen, Butyricum is cultured in spore medium to produce dormant spores (referred to as "spores"). Considering the tolerance of Butyricum to oxygen in the environment, the use of spores instead of live bacteria can facilitate preparation, purification and characterization. It should be noted that in nutrient media (such as intestinal fluid), spores can germinate immediately and enter normally growing bacteriophages. Through electrostatic effects, CS is easily deposited on the surface of collected spores. We call the resulting CS-deposited spores Spores@C. These Spores@C with reversed surface charge can be further coated by simply mixing with HA ( Figure 2A). The obtained HA-coated spore@C (referred to as spore@CH) was first observed by scanning electron microscopy (SEM). Compared with the wrinkled edges of spores and spore@C, the surface of spore@C was smooth ( Figure 2 B). Transmission electron microscopy (TEM) images show that Spore@CH is surrounded by an additional nanocoating layer ( Fig.10 Dynamic light scattering (DLS) measurements showed that the average size of spores@CH increased from 907.6 ± 31.1 μm to 1249 ± 45.6 μm compared with spores ( Figure 2 C). As expected, after the deposition of cationic CS, the zeta potential of the spore increased from -30.2 ± 0.6 mV to 45.9 ± 0.2 mV, while the subsequent coating of anionic HA decreased the zeta potential to -13.1 ± 0.5 mV ( Figure 2 D). To confirm that both CS and HA were decorated on the spore surface, CS labeled with fluorescein isothiocyanate (FITC) and HA labeled with rhodamine B (RhB) were used. Laser scanning confocal microscopy (LSCM) images of spore@CH showed confocal FITC and RhB signals, and a clear fluorescent shell appeared around each spore ( Figure 2 E and Fig.11 ). Flow cytometry analysis showed that the fluorescence intensity of FITC and RhB signals of Spore@CH was significantly increased ( Figure 2 F, G), further verifying the successful decoration of CS and HA. Quantitative analysis of the proportion of CS+HA+ double-positive spores showed that the coating efficiency exceeded 92% ( Figure 2 H). By fluorescence detection, the loading amounts of CS and HA were calculated to be 4.8% and 6.8%, respectively ( Figure 2 I). The loading efficiencies of CS and HA were calculated to be 40.0% and 21.3%, respectively. Specifically, quantification of the loading in Spore@CH revealed that 1×107 colony forming units (CFU) of spores were modified by 113μg CS and 160μg HA, respectively. Comparable numbers of viable bacteria were observed after culturing the spores and spore@CH groups in reinforced clostridial medium (RCM) plates under anaerobic conditions ( Figure 2 J), indicating that the coating and preparation process have satisfactory cell compatibility. At the same time, there was no significant difference in bacterial growth between the spores@CH group and the uncoated spore group ( Figure 2 K). In conclusion, these results validate the successful nanocoating of Butyricum spores with CS and HA under cytocompatible conditions.

[0120] Figure 2The characteristics of Spore@CH were analyzed in detail. A is the preparation of Spore@CH by electrostatic interaction. B is the scanning electron microscope image of spores, spore@C and spore@CH. The scale bar is 500 nm. C is the particle size distribution and Dzeta potential of Spore, Spore@C and Spore@CH measured by DLS (n=3). E is the LSCM image of Spore@CH. Green and red represent FITC-labeled CS and RhB-labeled HA, respectively. The scale bar is 2 μm. F is the flow cytometric histogram and G mean fluorescence intensity (MFI) of CS labeled with FITC and HA labeled with RhB on Spore@CH (n=3). H is the representative flow cytometric scatter plot and the percentage of RhB+FITC+ spores in the total number of spores after coating (n=3). I is the loading capacity and encapsulation efficiency of CS and HA on Spore@CH (n=3). J is the count of spores and spores@CH after 24 h of culture on RCM agar plates at 37°C (n=3). K is the growth curve of spores and spores@CH cultured in RCM medium at 37°C (n=3). Data are mean ± standard deviation (SD). Statistical analysis was performed using Student's t test. ***p<0.001

[0121] Improve the survival rate and preferential colonization rate of Spore@CH in the intestine

[0122] Figure 3 Schematic diagram of the retention of spores@CH in the pathological digestive tract. A is an IVIS image of the ileum and colon after culture of equal amounts of spores or spores@CH (n=3). B is the relative fluorescence intensity of spores or spores@CH retained in the ileum and colon (n=3). C is the count of spores or spores@CH retained in the ileum and colon (n=3). D and E are IVIS images of digestive tract sections 6 hours after administration and the corresponding fluorescence intensity quantification. D and E represent spores labeled with Cy5.5 and HA labeled with Cy5, respectively. Data are mean ± SD. Statistical analysis was performed using one-way analysis of variance or Student's t-test. ***P<0.001, **P<0.01, *P<0.05.

[0123] One of the major challenges facing bacterial therapeutics is the low oral bioavailability in the digestive tract, primarily due to the harsh digestive tract environment and limited intestinal retention. While dormant spores are more resistant to environmental insults than live bacteria, the nanocoating provides additional protection from the digestive tract environment, as evidenced by a significant increase in spore survival after incubation in simulated gastric fluid, bile salts, and simulated intestinal fluid for a period of time ( Fig.12To determine whether the nanocoating could enhance the survival of Butyricum in the intestine, FITC-conjugated spores and spore@CH were dropped onto intestinal segments sampled from mice, which were then rinsed with phosphate-buffered saline (PBS) and measured using an in vivo imaging system (IVIS) after 0.5 h of incubation. Figure 3 As shown in A and B, the relative fluorescence intensity of the spore@CH treatment group was significantly higher than that of the spore group, indicating that the nanocoating has the effect of enhancing the spore retention ability. In addition, quantitative analysis of bacterial plate counts further confirmed that the number of bacteria retained in the colon increased by 1.4 times ( Figure 3 C). Since the colon is covered with a large amount of mucus, we speculated that the hydrogen bonding interaction between the hydrophilic HA surface and mucin promoted the retention of spores in the intestine. Next, the in vivo retention of spores@CH was further evaluated by oral administration of 5×107 CFU of spores and spores@CH, and the number of butyric acid bacteria in the small intestine, cecum, and colon of mice was calculated by plate counting. Fig.13 As shown, 6 h after oral administration of spores@CH, the survival rate of all these intestinal segments was significantly increased. Interestingly, the colon had the highest survival rate among all the intestinal segments tested. These results indicate that the nanocoating is able to improve the oral bioavailability of Butyricum spp.

[0124] Given that a pathological intestine impedes the retention of oral probiotics and that HA facilitates the delivery of various oral formulations to the inflamed colon, we next investigated whether the nanocoating could enhance the accumulation of Butyricum in the pathological intestine. We treated mice intraperitoneally with cisplatin, which induces intestinal pathological changes such as an inflammatory environment and extensive epithelial cell death. With the help of Cy5.5 labeling, IVIS analysis showed that Spores@CH had a higher fluorescence intensity in the abdomen of mice 6 hours after gavage compared with Spores and Spores@C ( Fig.14 A), indicating an increase in the number of Butyricum in the intestine. In particular, digestive tract imaging collected from treated mice showed improved retention of Butyricum in the pathological intestine, as reflected by the highest fluorescence intensity in the Spore@CH group ( Figure 3 D). In addition, by labeling HA with Cy5, IVIS images of mice and sampled digestive tracts showed that the enrichment of HA in the pathological intestine of the spore@CH group was enhanced ( Figure 3 E and Fig.14 B). The above results confirm that Spore@CH can preferentially localize to the pathological digestive tract, reflecting the stability of the nanocoating in protecting the spores during oral administration.

[0125] Spore@CH inhibits intestinal inflammation and repairs intestinal barrier

[0126] Figure 4Schematic diagram of the intestinal inflammation inhibition and barrier repair effects of spores@CH. Evaluation of inflammatory response and intestinal barrier integrity in mice induced by cisplatin after treatment with PBS, spores, HA or spores@CH for 3 days. A is the image of colonic MPO staining and the corresponding quantification of BMPO-positive cells (n=3). Red arrows represent MPO-positive cells. Scale bar, 50 μm. Real-time quantitative PCR (RT-qPCR) analysis of C Tnfa, D Il1β and E Il6 mRNA levels in colon tissues (n=5). mRNA level data were normalized to the mean value of the healthy group. F Immunofluorescence images of colonic obliterans (red) and ZO-1 (green) expression in treated mice. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 50 μm. The average gray value of the red channel (G) and the green channel (H) in the immunofluorescence images of colon sections was measured using ImageJ software (n=3). I Average number of alginate blue-positive cells per 20× field of view in colon sections, and J PAS-AB staining images of colons of treated mice (n=3). Scale bar, 100 μm. Data are mean ± SD. Statistical analysis was performed using one-way ANOVA. ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05

[0127] Given that HA itself has the ability to inhibit inflammation, we speculated that enriching Spore@CH at the lesion site can effectively reduce the inflammatory response. To evaluate the efficacy of Spore@CH in inhibiting intestinal inflammation, we intraperitoneally injected mice with cisplatin and then orally administered 1×107 CFUs of Spore@CH daily for 3 consecutive days. Myeloperoxidase (MPO) is a signature enzyme expressed by neutrophils and is a typical feature of neutrophil infiltration and intestinal inflammation. Figure 4 As shown in A and B, under cisplatin conditions, neutrophils influxed into the epithelial cells. Notably, the inflammatory response was significantly attenuated after treatment with Spore@CH and HA, as evidenced by the reduction of MPO-positive cell infiltration in the colon tissue. In addition, the mRNA levels of multiple proinflammatory cytokines were increased in the colon of cisplatin-treated mice, including TNF-α, interleukin (IL)-1β, and IL-6 ( Figure 4 CE). Spore@CH significantly reduced cytokine levels, which were comparable to those in the healthy group. Of note, similar to PBS, the anti-inflammatory effect of spores was minimal. It can be concluded that the ability of spore@CH to inhibit intestinal inflammation is attributed to the action of released HA. Given that inflammatory responses are often accompanied by apoptosis, we next evaluated the effect of spore@CH on apoptosis in Caco-2 enterocytes. Notably, spore@CH had significant anti-apoptotic ability, as evidenced by a reduction in the number of apoptotic Caco-2 cells induced by lipopolysaccharide (LPS) ( Fig.15). In short, oral administration of Spore@CH can effectively inhibit intestinal inflammation.

[0128] Given that HA and C. butyricum can restore intestinal barrier function, we turned to explore whether spore@CH can maintain the structure and function of the intestinal barrier. Hemoglobin and eosin (H&E) staining was first performed, and representative images showed obvious pathological damage in the colon induced by cisplatin, manifested by massive epithelial cell death, severe submucosal edema, and disappearance of intestinal glands ( Fig.16 A). Compared with HA and spores, spores@CH can significantly reduce the pathological symptoms of the colon. The deterioration of the intestinal physical barrier is closely related to the reduction of tight junction complex expression. After intraperitoneal injection of cisplatin, the expression of occludin and ZO-1 on colon epithelial cells decreased and stopped ( Figure 4 FH). Compared with the other groups, the ZO-1 levels in the HA group and the Spore@CH group were increased, and the enhancing effect of Spore@CH on occludin expression was the most significant among all groups. Since the enhancement of intestinal inflammatory signals can lead to increased barrier permeability, we speculate that the inhibition of inflammation by HA is related to its beneficial effects on the damaged intestinal barrier. Considering the effectiveness of Spore@CH in strengthening the intestinal barrier in acute pathological stimulation, we further evaluated its efficacy in the case of chronic stimulation, that is, intraperitoneal injection of high-dose (300 mg / mouse) folic acid. After stimulation, 1×107 CFUs of Spore@CH were orally administered every 2 days for 14 days. Fig.16 B , 17A , B , after folate-induced mice were treated with spore@CH, intestinal epithelial damage was also significantly reduced, intestinal damage was not visible, and colonic expression of occludin and ZO-1 was increased.

[0129] Thrush cells are specialized epithelial cells that secrete mucin to form the intestinal mucus layer and play an important role in maintaining the intestinal barrier. According to PAS and AB staining, these nephrotoxic drugs caused severe destruction of thrush cells in the colon, which was manifested by pathological cavitation and decreased thrush cell density ( Figure 4 I, J and Fig.17 C). Spore@CH can significantly improve the number and function of thrush cells compared with other groups. In summary, Spore@CH has a dual effect of inhibiting inflammation and restoring the intestinal barrier during pathological damage to the intestine.

[0130] Regulates intestinal microbial balance

[0131] Figure 5To regulate the balance of intestinal microorganisms. Folic acid-induced mice were gavaged with PBS, HA, Spore, or Spore@CH every other day for 14 days, and intestinal samples were collected for 16S ribosomal RNA gene sequencing. A PCoA results at the species level of intestinal microbiota (n = 4 or 5). B Classification data based on the comparison of LDA values ​​between the healthy group (blue) and the CKD group (orange). Only LDA scores (log10) > 4.0 are shown. C Taxonomic cladogram from LEfSe shows the differences between the healthy group (blue) and the CKD group (orange). D Abundance of intestinal microbiota at the family level in each group. E Relative abundance of Erysipelotrichaceae and F Ruminococcaceae in the intestinal microbiota (n = 4 or 5). G Relative abundance of Roseburia in the intestinal microbiota (n = 4 or 5). H Counts of anaerobic bacteria, I aerobic bacteria, and J total bacteria in kidney tissue after treatment (n = 5). K Acetate, propionate, and butyrate levels in feces of each group (n = 5). N IS, OpCS and PIAA levels in serum of each group (n=5). Q Urea and creatinine levels in feces of each group (n=5). Data are mean ± SD. One-way ANOVA was used for statistical analysis. ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05

[0132] After confirming that Spore@CH can alleviate inflammation and restore intestinal barrier dysfunction, we further explored its potential effects on extra-intestinal organs. Considering that the role of intestinal microbiota in regulating distant organs by producing metabolites in various diseases has been confirmed, we analyzed the microbial structure of folic acid-treated mice after Spore@CH treatment by 16S ribosomal RNA gene sequencing. Shannon and Chao1 indices and principal coordinate analysis (PCoA) showed that the composition and diversity of the intestinal microbiota in the folic acid group were significantly altered compared with healthy mice ( Figure 5 A and Fig.18 A). Unlike spores and HA, the spores@CH treated group was more similar to the healthy group. To characterize the gut microbiota, a linear discriminant analysis (LDA) effect size (LEfSe) was performed and the results showed that folate-induced mice were enriched in Erysipelotrichaceae, while the healthy group was enriched in Oscillospiraceae and Ruminococcaceae ( Figure 5B, C, score > 4). In addition, Oscillospiraceae and Ruminococcaceae are well-known producers of short-chain fatty acids (SCFAs) and can improve acute renal dysfunction. In folic acid-induced mice, Spore@CH treatment significantly reduced the relative abundance of Erysipelotrichaceae, but increased the relative abundance of Ruminococcaceae ( Figure 5 DF). We further analyzed the major bacterial genera and found that the abundance of Faecalibaculum, which is reported to be associated with uremic toxins (UTx), was decreased in Spore@CH-treated mice ( Fig.18 B, C). Meanwhile, the number of SCFA-producing bacteria (such as Roseburia) was significantly higher than that in the spore@CH treatment group ( Figure 5 G).

[0133] To evaluate the effect of Spore@CH on intestinal permeability, spores, HA, and Spore@CH were added to Transwell inserts pre-seeded with Caco-2 cell monolayers. After the addition of FITC-dextran to the inserts, it was observed that the fluorescence intensity of the FITC channel of the basolateral chamber medium increased upon LPS stimulation, indicating that the permeability of the monolayer was greatly increased ( Fig.19 ). It is noteworthy that after treatment with Spore@CH, the permeability of FITC-dextran was reduced to normal levels, comparable to the unstimulated control group. Figure 5 As shown in HJ, after folic acid induction, the transport of aerobic and anaerobic bacteria in the kidney increased significantly, triggering tubulointerstitial damage. Impressively, the number of transported bacteria in the Spore@CH group and the healthy group was comparable and almost negligible, indicating that Spore@CH almost eliminated the transport of bacteria through the intestine. These data indicate that oral administration of Spore@CH can effectively reverse the imbalance of intestinal microbial flora, specifically manifested by an increase in the number of SCFA-producing bacteria, a decrease in the number of toxin-producing bacteria, and a decrease in bacterial transport.

[0134] Given the changes in gut microbial composition, we set out to determine the associated changes in microbial metabolism. Encouraged by the increase in SCFA-producing bacteria, we investigated the levels of SCFA in the gut after spore@CH treatment. Figure 5As shown by KM, Spore@CH reversed the folic acid-induced decrease in the levels of primary SCFA (including acetate, propionate, and butyrate) in the feces of mice. Notably, mice treated with Spore@CH had a stronger ability to increase butyrate content than other mice. Given the importance of butyrate in maintaining the integrity of the intestinal barrier, we inferred that the increase in butyrate levels after Spore@CH treatment may be the main factor in strengthening the intestinal barrier. In addition, the reduction in the number of UTx-associated bacteria also prompted us to evaluate the effect of Spore@CH on UTx levels. Multiple evidences indicate that UTx composed of indole sulfate (IS), p-cresyl sulfate (pCS), and indole-3-acetic acid (IAA) produced by intestinal microbial metabolism can have harmful effects on the kidneys. As Figure 5 NP and Fig.18 As shown in Figure 2D, oral administration of Spores@CH reduced UTx levels in both fecal and serum samples. In particular, the Spores@CH group showed a significant advantage in reducing IS levels, which is considered to be one of the most critical protein-bound UTx, leading to intestinal barrier destruction and stimulating glomerular sclerosis. In addition, the reduction in IS content in serum was more significant than that in feces, which means that the reduction in UTx content in serum may be due to the combined reduction of harmful intestinal microbiota and intestinal barrier permeability. In addition, it is generally believed that circulating nitrogenous waste produced in renal failure can enter the intestinal lumen and promote the growth of UTx-related bacteria. In all groups, Spores@CH showed excellent efficacy in clearing urea and creatinine ( Figure 5 Q, R), which may be due to the enhanced metabolism of urea and creatinine by microorganisms. These results indicate the good efficacy of spore@CH in improving microbial and host metabolites.

[0135] Extraintestinal inflammation inhibition and organ repair effects of spores@CH

[0136] Figure 6Figure 4 Inhibitory and organ-repairing effects of Spore@CH on extraintestinal inflammation. Evaluation of inflammatory responses in the kidneys of mice induced by cisplatin after 3 days of treatment with PBS, spores, HA, or Spore@CH. A Distribution of neutrophils (CD11b+Ly6G+) and macrophages (CD11b+F4 / 80+) among leukocytes (CD45+) in renal tissues. B Percentages of neutrophils (CD45+CD11b+Ly6G+) and macrophages (CD45+CD11b+F4 / 80+) among the total number of renal cells in mice induced by cisplatin after different treatments (n=5). C Heat map depicts the expression pattern of six inflammatory cytokines in the kidneys after treatment. RT-qPCR analysis of mRNA levels of D Tnfa, E Il1β, FIl6, G Cxcl1, H Cxcl2, and ICcl2 in renal tissues (n=5). mRNA level data were normalized to the mean values ​​of the healthy group. Evaluation of tubulointerstitial injury in mice induced by folate after 14 days of treatment with HA, spores, or spores@CH every other day. J PAS-stained images of renal sections and K corresponding analysis of tubular injury scores (n = 5). Red, blue, and green arrows represent tubular cell degeneration, tubular cell flattening, and tubular dilatation, respectively. Scale bar: 100 μm. Data are mean ± SD. One-way ANOVA was used for statistical analysis. ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05.

[0137] Since metabolites in both the gut and systemic circulation were remodeled and microbial transport from the gut to the kidney was reduced, we hypothesized that Spore@CH could ameliorate extraintestinal disease. Since tubulointerstitial inflammation is thought to initiate a cascade of deleterious events leading to tubular damage, we evaluated whether oral administration of Spore@CH could alter the infiltration of inflammatory cells and the inflammatory cytokine profile that injures the kidney. Figure 6 A, B and Fig. 20 , 21 As shown, cisplatin-induced infiltration of neutrophils and macrophages in mice was significantly increased, which was significantly reversed by spore@CH treatment. In contrast, HA showed only a slight reduction in inflammatory cell infiltration in the kidney. In addition, treatment with spore@CH or HA significantly reduced the expression of proinflammatory cytokines in kidney tissue compared with the PBS group and spore group ( Figure 6CI). In addition, the expression of chemokines in the renal tissue of mice induced by cisplatin was upgraded, including CXC motif chemokine ligand 1 (CXCL1), CXCL2, and CC motif ligand 2 (CCL2), which can guide leukocytes into the kidney and aggravate inflammation. Spore@CH can significantly reduce the levels of chemokines, especially CXCL2, which is an important chemokine for recruiting neutrophils to the site of inflammation. Subsequently, we evaluated the repair effect of Spore@CH on renal injury. PAS staining of kidney sections showed that pathological damage occurred in the kidneys of mice induced by folic acid, manifested as patchy or diffuse degeneration of tubular cells, disappearance of brush border, flattening of tubular cells, and dilation of tubules ( Figure 6 J, K). The group treated with Spore@CH showed less renal lesions compared with the other groups, as confirmed by the tubular injury score. We also investigated whether Spore@CH could reduce apoptosis in tubular epithelial cells, as apoptosis is a key cellular event in the progression of renal injury. LPS-stimulated HK-2 cells, a renal epithelial cell line, showed a significantly increased percentage of late apoptotic cells, which was reversed by Spore@CH treatment ( Fig. 22 ). In conclusion, Spore@CH demonstrated its remarkable ability to alleviate extra-intestinal inflammation and the potential to mitigate extra-intestinal organ damage.

[0138] The value of Spore@CH in the intervention of AKI

[0139] Figure 7 The results of in vivo experiments on the therapeutic effect of Spore@CH in acute kidney injury. A is a schematic diagram of the in vivo animal experiment. Mice with acute kidney injury induced by cisplatin were treated with PBS, HA, Spore, Spore@C, Spore@CH or NAC three times a day. They were euthanized on the fourth day after cisplatin injection and samples were collected for subsequent experiments (n=5). B urea nitrogen and C creatinine levels in the serum of mice after different treatments. D PAS staining of renal sections and tubular injury score results. Scale bar, 100 μm. Red, blue and green arrows indicate the exfoliation of tubular cells, flattening of tubular cells and dilation of renal tubules, respectively. Representative images of renal sections stained with TUNEL (green) and F correspond to the quantification of TUNEL-positive cells. Cell nuclei were stained with DAPI (blue). Scale bar, 50 μm. Flow cytometry was used to detect the percentage of renal infiltrating neutrophils (G), macrophages (H) and CD4+T cells (I) in the total renal cells. Data are mean ± SD. One-way ANOVA was used for statistical analysis. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

[0140] AKI is a common and critical clinical condition characterized by a dramatic decline in renal function. Having demonstrated the in vivo effectiveness of Spore@CH in maintaining intestinal homeostasis and regulating the gut-kidney tandem, we next sought to evaluate the therapeutic potential of Spore@CH in intervening in cisplatin-induced renal injury, a representative model of drug-induced AKI. HA (0.16 mg / mouse) and the same doses of spores and Spore@C served as control groups. Cisplatin-induced renal injury resulted in a dramatic increase in serum urea nitrogen (BUN) and creatinine levels (primary indicators of impaired renal function), while Spore@CH-treated mice showed a significant decrease in serum urea nitrogen (BUN) and creatinine levels ( Figure 7 B, C). PAS staining of kidney sections confirmed the damage of renal structure in AKI mice, showing obvious pathological damage, including tubular necrosis, tubular dilatation, intraluminal casting formation, and disappearance of brush border. ( Figure 7 D). Spore@CH treatment significantly reduced renal tubular damage compared with all other groups. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay showed that the number of apoptotic cells in the spore@CH group was the lowest among all groups ( Figure 7 E, F). It is noteworthy that the efficacy of spore@CH treatment in AKI mice was superior to that of NAC treatment. At the same time, cisplatin induction greatly increased the infiltration of macrophages, neutrophils, and CD4+ T cells into the renal cortex, thereby exacerbating cell apoptosis. Figure 7 GI and Fig.23 As shown, Spoore@CH can more effectively reduce the infiltration of these immune cells in the kidney compared with NAC. Taken together, these findings illustrate that Spoore@CH can effectively reduce renal injury and thus alleviate cisplatin-induced AKI.

[0141] Potential of Spore@CH for the treatment of chronic kidney disease

[0142] Figure 8Potential of Spore@CH for the treatment of chronic kidney disease. A Schematic timeline of in vivo studies. Folic acid-induced CKD mice were treated with PBS, HA, spores, Spore@C, or Spore@CH every other day for 14 days. Blood and kidney samples of each group were collected and examined at the end of the experiment (n = 5). Serum BUN and creatinine levels of BE mice on days B, C 3 and D, E 14 after different treatments. F Digital photos of left kidneys of each group after different treatments. G Masson-stained images of kidney sections and corresponding analysis of Masson-positive areas. Scale bar, 100 μm. H Sirius red-stained images of kidney sections. Scale bar, 100 μm. Corresponding quantification of Sirius red staining is shown. I Western blot analysis of α-SMA and FN-1 expression. J, K Quantitative analysis of FN-1 and α-SMA protein expression normalized to the expression of internal genes (tubulin or β-actin). Data are mean ± SD. One-way ANOVA was used for statistical analysis. ****p<0.0001,***p<0.001,**p<0.01,and*p<0.05

[0143] Given that increased UTx and decreased butyrate can lead to CKD progression, and that Spores@CH can regulate intestinal microbial balance, we also evaluated its value in alleviating CKD. CKD mice were intraperitoneally injected with folic acid (300 mg / mouse) to establish a CKD model characterized by renal fibrosis. CKD mice were gavaged with HA (0.16 mg / mouse) or an equivalent dose (1×107 CFUs / mouse) of Spores, Spores@C, or Spores@CH every other day for 14 consecutive days ( Figure 8 A). We monitored the weight of mice every day, and the results showed that the weight of mice showed a pattern of first decreasing and then recovering ( Fig.24 ). Notably, spore@CH had the strongest protective effect, as reflected by stable body weight and the lowest serum BUN and creatinine levels in mice ( Figure 8 BE). Renal morphology and Masson staining of sampled renal tissues showed that renal interstitial fibrosis was obvious in CKD mice, while Spore@CH reversed this phenomenon to a large extent ( Figure 8 BE).

[0144] Spore@CH largely reversed this phenomenon ( Figure 8 F, G). In addition, Sirius red staining confirmed the excellent efficacy of Spore@CH in improving excessive deposition of renal collagen fibers ( Figure 8 H). Western blot analysis showed that the expression levels of pro-fibrotic markers such as fibronectin 1 (FN-1) and α-smooth muscle actin (α-SMA) were significantly reduced in the kidneys of mice treated with spore@CH ( Figure 8 IK and Fig.25 ).like Fig.26As shown in Figure 2, compared with the other groups, the number of neutrophils and CD4+T cells infiltrating the kidneys of CKD mice decreased dramatically after spore@CH treatment. In addition, no pathological abnormalities were found in the main organs after treatment ( Fig. 27 ), indicating that Spore@CH has good biosafety. Next, we selected peritoneal dialysis (PD) as a benchmark to evaluate the efficacy of Spore@CH, which is a clinical method for removing nitrogenous wastes from the body in patients with chronic kidney disease. Notably, mice treated with Spore@CH and PD showed comparable efficacy in reducing serum urea nitrogen and creatinine and alleviating fibrosis ( Fig.28 and Fig.29 ). In contrast, oral administration is a convenient, non-invasive method with high patient compliance and better safety.

[0145] The present application has a dual-function, dual-position intervention strategy to effectively regulate the series connection between the intestine and distal organs to treat extraintestinal diseases. Under simple but cell-compatible conditions, cationic CS can generate a layer on the negatively charged surface of butyric acid bacteria, and further decorate the anionic HA layer on the positively charged surface through electrostatic action. We observed that the oral bioavailability of nano-coated butyric acid bacteria was improved, including enhanced resistance to gastrointestinal damage and preferential localization in the pathological intestine. After oral administration of nano-coated butyric acid bacteria, the inflammatory response can be effectively reduced and intestinal barrier damage can be restored, which is mainly due to the combination of the anti-inflammatory inherent properties of HA and the butyrate-generating ability of butyric acid bacteria. At the same time, nano-coated butyric acid bacteria can reshape microbial metabolites, reduce microbial transport, and then affect extraintestinal organs, with the characteristics of reducing inflammation and repairing tissue damage. In in vivo therapeutic evaluation, nano-coated butyric acid bacteria greatly restored renal function and eliminated renal fibrosis in cisplatin-induced AKI and folic acid-induced CKD mouse models, respectively. Given that surface modification can flexibly introduce different functional motifs on bacteria, the integration of dual-functional and dual-localization intervention modes opens a window for the treatment of extraintestinal diseases.

[0146] Although the technical solutions of the present invention have been described and listed in detail, it should be understood that it is obvious to those skilled in the art to make modifications to the above embodiments or adopt equivalent alternatives. These modifications or improvements made without departing from the spirit of the present invention are within the scope of protection required by the present invention.

Claims

1. A hyaluronic acid nano-coated bacteria with anti-inflammatory and tissue repair effects, characterized in that: The hyaluronic acid nano-coated bacteria comprises spores, on the surface of which cationic chitosan is deposited, and on the surface of which hyaluronic acid is correspondingly arranged.

2. The hyaluronic acid nano-coated bacteria with anti-inflammatory and tissue repair effects according to claim 1, characterized in that: The spores are dormant spores produced by culturing butyric acid bacteria in a spore culture medium.

3. The hyaluronic acid nano-coated bacteria with anti-inflammatory and tissue repair effects according to claim 1, characterized in that: Cationic chitosan was deposited on the surface of collected spores by electrostatic action.

4. A method for preparing hyaluronic acid nano-coated bacteria with anti-inflammatory and tissue repair effects as claimed in any one of claims 1 to 3, characterized in that: The method comprises the following steps: culturing bacteria in a spore-producing medium to obtain a mixture of bacteria and spores, purifying the mixture of bacteria and spores to obtain spores, depositing cationic chitosan on the surface of the spores through electrostatic action to obtain CS-deposited spores, and mixing these CS-deposited spores with reversed surface charges with hyaluronic acid for further coating to obtain hyaluronic acid nano-coated bacteria.

5. The method for preparing a hyaluronic acid nano-coated bacteria having anti-inflammatory and tissue repair effects according to claim 4, characterized in that: The specific steps of culturing the bacteria in the spore-forming medium are: culturing the bacteria in the spore-forming medium in an anaerobic environment maintained at a temperature of 37° C.; Caco-2 and HK-2 cell lines were obtained from the American Type Culture Collection and cultured in DMEM / F-12 and DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin, respectively, at 37°C in a 5% CO2 incubator.

6. The method for preparing a hyaluronic acid nano-coated bacteria having anti-inflammatory and tissue repair effects according to claim 5, characterized in that: The bacterium is Clostridium butyricum.

7. The method for preparing a hyaluronic acid nano-coated bacteria having anti-inflammatory and tissue repair effects according to claim 5, characterized in that: The spore production medium contains 0.3% lactose, 0.035% MgSO4, 1% yeast extract, 0.2% NaHCO3, 0.3% K2HPO4, and 0.1% KH2PO4.

8. The method for preparing a hyaluronic acid nano-coated bacteria having anti-inflammatory and tissue repair effects according to claim 4, characterized in that: The specific steps of purifying the mixture of bacteria and spores are as follows: the bacteria are cultured in a spore-producing medium for 10-14 days to obtain a mixture of bacteria and spores, the obtained mixture of bacteria and spores is centrifuged at 5500×g for 10 minutes, boiled at 80°C for 20 minutes, and then the spores are further purified by density gradient centrifugation of a solution of 65% sucrose and 35% sucrose; the purified spores are then washed with phosphate buffered saline, and vacuum dried at room temperature to obtain the purified spores.

9. A use of hyaluronic acid nano-coated bacteria having anti-inflammatory and tissue repair effects as claimed in any one of claims 1 to 3, characterized in that: The hyaluronic acid nano-coated bacteria are used to prepare drugs with both anti-inflammatory and tissue repair effects.

10. The use of a hyaluronic acid nano-coated bacteria having anti-inflammatory and tissue repair effects according to claim 9, characterized in that: Application of the hyaluronic acid nano-coated bacteria as a drug for reducing intestinal mucosal inflammation and restoring intestinal barrier integrity; The hyaluronic acid nano-coated bacteria can also be used to prepare drugs for remodeling microbial metabolites and reducing microbial transport, which can reduce interstitial inflammation and pathological damage of extra-intestinal organs.

Images