Application of beta-nicotinamide mononucleotide in preparation of medicine for inhibiting clostridium difficile infection

Abstract

The invention discloses application of beta-nicotinamide mononucleotide in preparation of a medicine for inhibiting clostridium difficile infection, and relates to the technical field of biomedicine. The invention discloses application of beta-nicotinamide mononucleotide in preparation of a medicine for inhibiting intestinal diseases caused by clostridium difficile infection. According to the invention, the beta-nicotinamide mononucleotide realizes systematic intervention on clostridium difficile infection through a dual core mechanism of down-regulating the expression of a proinflammatory factor and inhibiting a pyroptosis pathway mediated by metronidatin D; the anti-infection survival ability of a host can be remarkably enhanced, intestinal inflammation injury is relieved, the occurrence risk of complications such as pseudomembranous colitis is reduced, and the defect that dysbacteriosis is aggravated by traditional antibiotics is avoided; a brand new intervention strategy is provided for the public health emergency threat disease, namely, clostridium difficile infection, a foundation is laid for expanded application of beta-nicotinamide mononucleotide in the field of intestinal inflammatory diseases, and the beta-nicotinamide mononucleotide has prominent clinical transformation potential and academic research value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of β-nicotinamide mononucleotide in the preparation of drugs that inhibit Clostridium difficile infection. Background Technology

[0002] Clostridium difficile infection (C. difficile, CDI) is a common intestinal infectious disease that seriously threatens human health. Currently, traditional treatments for CDI mainly rely on antibiotics, but these have several limitations: Firstly, while antibiotics kill C. difficile, they easily disrupt the balance of normal intestinal flora, leading to a persistently high recurrence rate. Secondly, for the intestinal inflammation caused by C. difficile infection, especially the severe inflammatory response caused by pyroptosis mediated by Gasdermin D (GSDMD), existing drugs lack precise intervention targets, making it difficult to effectively alleviate inflammatory damage. Furthermore, patients often exhibit overexpression of pro-inflammatory cytokines and insufficient expression of anti-inflammatory factors, further exacerbating intestinal lesions. In addition, serious complications caused by C. difficile infection, such as pseudomembranous colitis and toxic megacolon, also lack safe and effective prevention and treatment strategies, posing a significant challenge to clinical treatment.

[0003] Therefore, developing a novel Clostridium difficile infection prevention and treatment drug that can address multiple mechanisms, has high safety, and is less likely to induce drug resistance has become an urgent technical problem to be solved in this field. Summary of the Invention

[0004] Based on this, the present invention aims to explore the application of β-nicotinamide mononucleotide (NMN) in the preparation of drugs that inhibit Clostridium difficile infection, and to provide a novel treatment for Clostridium difficile infection by revealing its regulatory mechanism on Clostridium difficile infection-related pyroptosis, inflammatory response and intestinal flora homeostasis.

[0005] This invention provides the use of β-nicotinamide mononucleotide in the preparation of drugs that inhibit Clostridium difficile infection-induced intestinal diseases.

[0006] Furthermore, the Clostridium difficile infection is an intestinal infectious disease.

[0007] Furthermore, the aforementioned Clostridium difficile infection includes diarrhea, abdominal pain, fever, dehydration, pseudomembranous colitis, intestinal perforation, and toxic megacolon.

[0008] Furthermore, the drug exerts its therapeutic effect by inhibiting Clostridium difficile-induced pyroptosis mediated by dermatophyte D.

[0009] Furthermore, the drug uses β-nicotinamide mononucleotide as its active ingredient and also includes a pharmaceutically acceptable carrier or excipient.

[0010] Furthermore, the drug dosage form is an oral preparation or an injectable preparation.

[0011] Furthermore, the oral preparation is a tablet, capsule, or granule; the injectable preparation is an injection solution or powder for injection.

[0012] The present invention also provides a drug for inhibiting Clostridium difficile infection, characterized in that the drug comprises a therapeutically effective amount of β-nicotinamide mononucleotide.

[0013] Compared with the prior art, the beneficial effects of the present invention are:

[0014] 1. At the mechanistic level, it achieves systemic intervention against Clostridium difficile infection through a dual core mechanism of downregulating the expression of pro-inflammatory factors and inhibiting the pyroptosis pathway mediated by dermosin D.

[0015] 2. From the perspective of therapeutic efficacy, it can significantly enhance the host's ability to fight infection and survive, alleviate intestinal inflammation and damage, and reduce the risk of disease complications (such as pseudomembranous colitis and toxic megacolon). At the same time, it avoids the drawback of traditional antibiotics exacerbating dysbiosis and has certain therapeutic value.

[0016] 3. From an application perspective, it provides a novel intervention strategy for public health "urgent threat" diseases such as Clostridium difficile infection, and also lays a theoretical and experimental foundation for the expanded application of β-nicotinamide mononucleotide in the field of intestinal inflammatory diseases, with outstanding clinical translation potential and academic research value. Attached Figure Description

[0017] Figure 1 A and B in the text represent nicotinamide adenine dinucleotide (NAD) in the colon. + ) and NAD + mRNA expression levels of synthesis-related enzymes; Figure 1 The middle C represents the comparison of survival rates of mice in each group; Figure 1 D represents the Clinical Sickness Score (CSS) and the number of Clostridium difficile in the feces of mice in each group. Figure 1 E represents the survival rate of mice in each treatment group; Figure 1 Clinical disease scores and fecal Clostridium difficile counts in mice of the F group; Figure 1 G represents the expression level of colonic inflammatory factors mRNA; * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001; ns indicates no significant difference, the same below;

[0018] Figure 2 In Figure A, the survival rate of Clostridium difficile-infected mice with macrophage deficiency induced by clodronate liposomes (Clo-lip) after treatment with or without β-nicotinamide mononucleotide (NMN) was observed. Figure 2 In the middle section, B represents the clinical disease score and fecal Clostridium difficile count in clophosphonate liposome-induced macrophage-deficient mice after administration of β-nicotinamide mononucleotide supplementation with or without the use of β-nicotinamide mononucleotide supplementation. Figure 2 C represents the levels of IL-6, IL-1β, and TNF-α in the colon; Figure 2 In the middle D, the phagocytic capacity of bone marrow-derived macrophages (BMDMs) in each group was assessed by the neutral red phagocytosis assay. Figure 2 E and F represent the number of Clostridium difficile bacteria in each group 4 hours after infection with bone marrow-derived macrophages, determined by macrophage phagocytosis assay.

[0019] Figure 3 Image A shows cell morphology under a microscope (400x magnification). Figure 3 In experiment B, lactate dehydrogenase release was performed. Figure 3 C represents the mRNA levels of Tnf-α, Il6, and Il10 in bone marrow-derived macrophages; Figure 3 In the middle, D represents the immunoblotting analysis of GSDMD protein expression; Figure 3 E represents the mRNA levels of Il6 and Il1b in BMDMs; Figure 3 F represents the IL-1β level;

[0020] Figure 4 In section A, the expression of GSDMD and Caspase-1 proteins was detected by Western blotting. Figure 4 In the middle B, the gene expression level of Gsdmd in colon tissue is represented. Figure 4 C represents the serum IL-1β level; Figure 4 D represents the gene expression levels of Tnf-α, Il6, Il1b, and Il10 in colon tissue; Figure 4 E represents an infection rate of 2.0 × 10⁻⁶. 8 Survival rates of mice in each group after CFU / mL Clostridium difficile VPI 10463 strain; Figure 4 F represents the clinical disease score of mice in each group; Figure 4 G in the diagram shows the colon length of mice in each group and the quantitative statistical bar chart. Figure 4 H represents the number of Clostridium difficile in the feces of mice in each group;

[0021] Figure 5In section A, the expression of GSDMD and Caspase-1 proteins was detected by Western blotting. Figure 5 In the middle B, the mRNA levels of Gsdmd and Il1b in colon tissue are represented. Figure 5 C represents the serum levels of TNF-α, IL-6, and IL-1β. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Experimental Example 1

[0024] 1. Materials and Methods

[0025] 1.1. Strains and Culture Conditions

[0026] Clostridium difficile VPI 10463 was cultured in Brain Heart Infusion (BHI) broth. The overnight culture was diluted 1:100 to 5 mL of fresh BHI broth and cultured until the logarithmic growth phase. The bacterial concentration was then adjusted to 0.5 McFarland turbidity standard, equivalent to 1.5 × 10⁻⁶. 8 CFU / mL.

[0027] 1.2. Mice and Reagents

[0028] Eight-week-old female wild-type C57BL / 6 mice were purchased from Chongqing Laboratory Animal Center (Chongqing, China). Eight-week-old Gsdmd genotype knockout (Gsdmd KO) female mice and age-matched wild-type female mice were purchased from Nanjing Kingfa Pharmaceutical Co., Ltd. (Nanjing, China). All mice were housed in a 12-hour light / dark cycle environment at a temperature of 22±2℃ and humidity of 50±5%. An acclimatization period was implemented for five days prior to the experiment, during which mice had free access to standard feed and drinking water. β-Nicotinamide mononucleotide (NMN, 97% purity, catalog number BD116593) and (E)-N-[4-(1-benzoylpiperidin-4-yl)butyl]-3-(pyridin-3-yl)acrylamide ((E)-N-[4-(1-benzoylpiperidin-4-yl)butyl]-3-(pyridin-3-yl)acrylamide, FK866) (98% purity, catalog number BD334954) were purchased from Shanghai Bide Pharmaceutical Co., Ltd., China. Enzyme-Linked Immunosorbent Assay (ELISA) for mouse inflammatory factors including three interleukins (IL-6, IL-1β, and IL-10) and tumor necrosis factor-α (TNF-α) detection kits (IL-6 catalog number IC50325-1; IL-1β catalog number ml106733; TNF-α catalog number mIC50536-1; IL-10 catalog number mIC50274-1; Shanghai Enzyme-Linked Biotechnology Co., Ltd.) was used to detect relevant indicators in colon tissue according to the instructions. NAD + Detection using NAD + The test kit (catalog number S0175) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0029] 1.3. Antibiotic administration

[0030] Mice received a five-day antibiotic treatment regimen using a mixed antibiotic solution containing kanamycin (0.4 mg / mL), gentamicin (0.035 mg / mL), polymyxin (850 U / mL), metronidazole (0.215 mg / mL), and vancomycin (0.045 mg / mL). All antibiotics were purchased from Sigma-Aldrich and dissolved in sterile water. The antibiotic solution was provided freely in the mouse cages. Dosage was determined based on average body weight (20–25 g) and estimated daily water intake (4–6 mL per mouse over four days). Twenty-four hours after completing the antibiotic course, mice received an intraperitoneal injection of clindamycin (10 mg / kg) dissolved in sterile water as follow-up treatment.

[0031] 1.4. Animal Model Construction and Experimental Design

[0032] This study investigated whether β-nicotinamide mononucleotide (NMN) pretreatment could effectively prevent Clostridium difficile infection. Specifically, NMN was administered to mice 24 hours prior to exposure to Clostridium difficile infection. Clodronate liposomes and control liposomes were purchased from Shanghai Yisheng Biotechnology Co., Ltd. After macrophage clearance, mice were administered 1×10⁻⁶ NMN via gastric tube. 8 Clostridium difficile VPI 10463 CFU / mL.

[0033] The experiment used 6-8 week old littermate wild-type female mice and Gsdmd gene knockout female mice (genotyped using tail DNA analysis with specific primers). All mice were confirmed healthy by a professional veterinarian and acclimatized to the experimental environment for at least 5 days before the experiment, but no pre-screening for Clostridium difficile colonization or infection was performed. After acclimatization, the researchers used a mixed antibiotic solution to purge the intestinal flora for 7 days, followed by an intraperitoneal injection of clindamycin one day later. 24 hours after clindamycin administration, the mice received an intraperitoneal injection of β-nicotinamide mononucleotide, and one hour later, they were administered Clostridium difficile VPI 10463 by gavage (inoculation dose: 1.5 × 10⁻⁶). 8 The infection concentration (CFU / mL) was adjusted according to experimental conditions. Throughout the experiment, six mice in each group received either β-nicotinamide mononucleotide, saline, or Clostridium difficile alone. Survival rate, weight changes, and disease-related clinical symptoms (including wet tail, diarrhea, arched back, dehydration, etc.) were continuously monitored. Clinical disease scores were calculated daily according to the criteria specified in Table 1. The quantity of Clostridium difficile in mouse feces was quantified by collecting fecal samples from mice on day 6 after infection with Clostridium difficile, immediately flash-freezing them in liquid nitrogen, and storing them long-term at -80°C.

[0034] Table 1. Clinical Sickness Score (CSS)

[0035]

[0036] 1.5. Quantitative detection of Clostridium difficile count in mouse feces

[0037] (1) Pretreatment: Accurately weigh 1 gram of fecal sample, resuspend it in an equal volume of sterile phosphate-buffered saline (PBS), vortex mix for 30 seconds, and let stand at room temperature for 15 minutes. Collect the supernatant as a stock solution.

[0038] (2) Selective culture: Take 100 μL of serially diluted solution and spread it evenly on cycloserine-cefoxitin-fructose agar (CCFA) selective medium plates. Anaerobic culture and colony-forming unit counting: Place the petri dishes in an anaerobic incubator at 37℃ and incubate for 48 hours. The incubator environment is 85% N2, 10% H2 and 5% CO2. Finally, count the number of typical yellow colonies, which is the Clostridium difficile colony-forming units.

[0039] (3) Experimental replication: Each treatment group contained three independent biological replicates, and the data were analyzed using the average of the three replicates.

[0040] 1.6. Induction of bone marrow-derived macrophages

[0041] Bone marrow was isolated from the femur and tibia after euthanasia of mice. The isolated cells were seeded in Durbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 IU / mL penicillin, 100 µg / mL streptomycin, and 25 ng / mL macrophage colony-stimulating factor (M-CSF). The cultures were incubated at 37°C and 5% CO2, and bone marrow-derived macrophages were induced on day 6 for experimental use.

[0042] 1.7. Determination of Neutral Red Phagocytic Activity and Macrophage-Mediated Bacterial Phagocytosis

[0043] The assay began with drug preparation and grouping. A 50 mM stock solution of β-nicotinamide mononucleotide (NMN) was prepared by dissolving it in phosphate buffer, sterilizing it through a 0.22 μm filter, and then aliquoting and storing at -20°C. The FK866 inhibitor was prepared as a 2.5 mM stock solution using dimethyl sulfoxide (DMSO) and stored at -20°C protected from light (final DMSO concentration <0.1%, confirmed to be non-cytotoxic in preliminary experiments). Experimental groups were configured with three replicates per group: the control group used complete medium containing 0.1% DMSO; the NMN group was treated with 500 μM β-nicotinamide mononucleotide (diluted from the stock solution to the working concentration using complete medium); and the FK866 group was treated with 25 nM FK866. The NMN+FK866 group was co-diluted with a mixture of 500 μM NMN and 25 nM FK866. Bone marrow-derived macrophages were cultured at 5 × 10⁻⁶ cells / well. 4Cells were seeded at a density of 100 μL of complete culture medium per well in 96-well plates and incubated at 37°C for 24 hours. After discarding the old culture medium, each group was replenished with 100 μL of drug-containing culture medium and incubated for another 24 hours. For neutral red staining, the drug-containing culture medium was removed first, and each well was gently washed twice with 150 μL of phosphate buffer. Then, 100 μL of 0.075% neutral red solution (prepared in serum-free α-minimum essential medium) was added, and the cells were incubated at 37°C for 1 hour. Unphagocytosed dye was washed three times with ice-bathed phosphate buffer (4°C), allowing the cells to stand for 1 minute after each wash to thoroughly remove residual dye from the well walls. For phagocytosis activity assay, 100 μL of cell lysis buffer was added to each well, and the cells were lysed at 150 rpm for 30 minutes at room temperature in the dark. The absorbance was measured at 540 nm using a microplate reader, with wells containing only lysis buffer serving as blank controls. Clostridium difficile VPI 10463 was used in the experiment, with a multiplicity of infection (MOI) of 100:1. After 4 hours of cell incubation, the cells were treated with the corresponding drugs for 1 hour according to the four experimental group protocols described above. The culture medium was then aspirated, and the cells were washed three times with phosphate-buffered saline (PBFS). Cells were then lysed with 0.3% (v / v) Triton X-100 for 5 minutes. The cell lysates were serially diluted with PBFS and then seeded onto CCFA plates for Clostridium difficile colony-forming unit (CDR) counting.

[0044] 1.8. Detection of Clostridium difficile infection in bone marrow-derived macrophages

[0045] Bone marrow-derived macrophages were infected with *Clostridium difficile* VPI 10463 cells at a multiplicity of infection (MIR) of 100:1. The experiment was divided into four groups, with three replicates per group: Control group (CDI): cells were infected with *Clostridium difficile* at a MMR of 0.1% in complete medium; *C. difficile* infection group (CDI): cells were infected with *Clostridium difficile* at a MMR of 100:1 (without drug treatment); β-nicotinamide mononucleotide (NMN) pretreatment + infection group (CDI+NMN): cells were pretreated with 500 μM β-nicotinamide mononucleotide (diluted to working concentration in complete medium) one hour before *Clostridium difficile* infection; β-nicotinamide mononucleotide (NMN): cells were treated with only 500 μM β-nicotinamide mononucleotide (diluted as above) without infection. Cell morphology was observed four hours after infection, and cells and their supernatant were collected.

[0046] 1.9. Clostridium difficile infection of bone marrow-derived macrophages lactate dehydrogenase (LDH) release assay

[0047] Bone marrow-derived macrophages were seeded in 96-well plates and divided into four groups (three replicates per group), receiving the following treatments: Control group (using complete medium containing 0.1% dimethyl sulfoxide); Clostridium difficile infection group (CDI): cells were infected with Clostridium difficile at a multiplicity of infection (MOI) of 100:1 (without drug treatment); β-nicotinamide mononucleotide pretreatment + infection group (CDI+NMN): cells were pretreated with 500 μM β-nicotinamide mononucleotide (diluted to working concentration) one hour before Clostridium difficile infection (MOI 100:1) and then placed in complete medium; β-nicotinamide mononucleotide group (NMN): cells treated with only 500 μM β-nicotinamide mononucleotide (uninfected). Four hours post-infection, cell supernatant was carefully collected into sterile microcentrifuge tubes (avoiding cell debris contamination) and stored at -80°C until lactate dehydrogenase detection. Reagent Preparation: Thaw the components of the lactate dehydrogenase assay kit (substrate mixture, catalyst, stop solution) to room temperature. Prepare the working solution according to the instructions (e.g., mix the substrate and catalyst in a 1:1 ratio). Transfer 50 μL of the supernatant from each sample to a new 96-well plate. Add 50 μL of lactate dehydrogenase working solution to each well, mix gently, and incubate at 37°C in the dark for 30 minutes. Add 50 μL of stop solution to each well to terminate the reaction. Absorbance Measurement: Within 10 minutes after the reaction is terminated, read the optical density (OD) at 490 nm using a microplate reader. The lactate dehydrogenase release rate (%) is calculated as follows: Lactate dehydrogenase release rate = (OD value of experimental group - OD value of control group) / (OD value of maximum lactate dehydrogenase release - OD value of control group) × 100%. To determine the maximum lactate dehydrogenase release, cells lysed with 1% polyethylene glycol p-tert-octylphenyl ether (Triton X-100) can be used as a positive control.

[0048] 1.10. Real-time reverse transcription polymerase chain reaction (RT-qPCR)

[0049] We used TRIzol reagents and extracted total RNA from the samples according to the manufacturer's instructions. Complementary DNA was then synthesized using the Takara cDNA synthesis kit following the operating instructions. Finally, we performed real-time quantitative PCR amplification on the Light Cycler 96 three-step system using SYBR Green (Takara). The primer sequences used are detailed in Table 2.

[0050] Table 2 Primer Sequences

[0051]

[0052] 1.11. Western blot of proteins

[0053] Colon tissue samples were homogenized in RIPA lysis buffer containing a mixture of protease inhibitors and phosphatase inhibitors. The tissue lysis buffer was denatured using sodium dodecyl sulfate lysis buffer and heated at 95°C for 10 minutes. After protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the samples were transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk and incubated overnight at 4°C with primary antibodies: anti-GSDMD antibody (abcam, ab209845, 1:1000 dilution), anti-Caspase-1 antibody (abcam, T510200, 1:1000 dilution), or anti-Calnexin antibody (abcam, TA5362S, 1:1000 dilution). After washing three times with Tris buffered saline containing Tween 20, the protein bands were incubated with goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific, A21109, 1:10000 dilution) at room temperature for 2 hours. Finally, the protein bands were visualized using an Odyssey imaging system.

[0054] 1.12. Statistical Analysis

[0055] All data were derived from at least three independent replicates. Results are expressed as mean ± standard deviation. Survival analysis was performed using the Kaplan-Meier estimation method, and survival curves were plotted accordingly. Unpaired t-tests were used for comparisons between two groups; one-way ANOVA and two-way ANOVA were used for comparisons among multiple groups, with a statistical significance level set at P < 0.05. Figures and tables were created using Adobe Illustrator 2021 or GraphPadPrism 9.

[0056] 2. Experimental Results

[0057] 2.1. β-Nicotinamide mononucleotide protects mice from Clostridium difficile infection.

[0058] The results showed that Clostridium difficile infection led to NAD+ levels in the colon of mice. + The concentration decreased significantly ( Figure 1 A). To assess NAD + The transcriptional regulatory mechanism of biosynthetic enzymes was investigated using reverse transcription quantitative polymerase chain reaction analysis of mouse colon tissue. Results showed that enzymes involved in the de novo synthesis of NAD+... + The expression of the key enzyme Tdo2 was significantly downregulated, while the expression of NAD2 was significantly downregulated. +The expression of nicotinamide phosphoribosyltransferase (Nampt) in the salvage pathway was significantly upregulated. Figure 1 B).

[0059] Notably, nicotinamide phosphoribosyltransferase (NAM) is a key enzyme catalyzing the conversion of nicotinamide (NAM) to β-nicotinamide mononucleotide (NMN). To investigate the functional role of NMN in Clostridium difficile infection, mice were administered NMN via intraperitoneal injection one hour before the onset of Clostridium difficile infection. NMN supplementation significantly improved the survival rate of infected mice. Figure 1 C), and significantly reduced clinical disease scores, but failed to significantly reduce the colonization of Clostridium difficile in the colon ( Figure 1 D).

[0060] To further verify the correlation between the pathogenic mechanism of Clostridium difficile infection and the NAMPT-NMN axis, mice were treated with the selective intracellular nicotinamide phosphoribosyltransferase inhibitor FK866 to block the β-nicotinamide mononucleotide biosynthesis pathway. Experiments showed that FK866 treatment significantly increased mouse mortality, while β-nicotinamide mononucleotide supplementation effectively reversed this lethal effect. Figure 1 E), confirming that the NAMPT-NMN axis plays a crucial role in the host's resistance to Clostridium difficile infection. Consistent with this, FK866 administration significantly increased clinical disease scores and the number of Clostridium difficile colony-forming units; however, prior supplementation with β-nicotinamide mononucleotide before FK866 treatment significantly reduced clinical disease scores and the number of Clostridium difficile colony-forming units. Figure 1 F).

[0061] To investigate the effects of β-nicotinamide mononucleotide (NMN) on colonic inflammation, the mRNA expression levels of inflammatory factors were analyzed. The results showed that NMN supplementation significantly downregulated the expression of pro-inflammatory factors Il6, Il1b, and Tnf-α, while upregulating the expression of the anti-inflammatory factor Il10. Figure 1 (G). These findings suggest that Clostridium difficile infection reduces NAD+ levels in the mouse colon and activates the NAD+ rescue pathway. Supplementation with β-nicotinamide mononucleotide (NMN) provides protection against Clostridium difficile infection by alleviating colonic inflammation and tissue damage, while inhibition of NMN biosynthesis exacerbates mortality in infected mice.

[0062] 2.2. The protective effect of β-nicotinamide mononucleotide in mouse survival against Clostridium difficile infection is macrophage-dependent.

[0063] To investigate whether macrophages are essential for the protective mechanism of β-nicotinamide mononucleotide against Clostridium difficile infection, macrophages were depleted in mice using clophosphate liposomes. Following macrophage depletion, supplementation with β-nicotinamide mononucleotide did not improve mouse survival rate. Figure 2 A), and it also failed to reduce clinical disease scores, let alone reduce the number of Clostridium difficile in the colon ( Figure 2 B). Furthermore, after macrophage removal, there were no significant differences in the levels of IL-6, IL-1β, and TNF-α in the colon of mice from each group. Figure 2 C). These results indicate that the protective effect of β-nicotinamide mononucleotide against Clostridium difficile infection is macrophage-dependent.

[0064] To further investigate the role of β-nicotinamide mononucleotide (NMN) in macrophage-mediated bacterial clearance, bone marrow-derived macrophages were treated with NMN and the nicotinamide phosphoribosyltransferase inhibitor FK866, and their phagocytic activity was subsequently assessed using a neutral red phagocytosis assay. The results showed that NMN pretreatment did not enhance the phagocytic capacity of bone marrow-derived macrophages; however, FK866 pretreatment significantly inhibited macrophage phagocytic function, and NMN supplementation partially reversed this inhibition. Figure 2 D).

[0065] Consistent with the above observations, in macrophage phagocytosis experiments using *Clostridium difficile*, FK866 treatment significantly reduced the number of *Clostridium difficile* colony-forming units (CDUs) within cells, while β-nicotinamide mononucleotide (NMN) supplementation eliminated this effect. Compared to the control group, the FK866 treatment group showed a reduction of 6 *Clostridium difficile* CDUs colony-forming units, while the β-nicotinamide mononucleotide combined with FK866 treatment group had a similar number of CDUs to the control group. Figure 2 E and F). Based on these data, it can be inferred that the NAD+ salvage pathway is involved in the macrophage immune response to Clostridium difficile infection.

[0066] 2.3. β-Nicotinamide mononucleotide inhibits pyroptosis and inflammatory response in macrophages infected with Clostridium difficile.

[0067] To investigate the regulatory role of β-nicotinamide mononucleotide (NMN) in pyroptosis and inflammatory responses in Clostridium difficile-infected macrophages, we infected bone marrow-derived macrophages with Clostridium difficile strain VPI 10463 (multiple infection ratio 100:1). The experiment included a control group and three experimental groups: the control group received no treatment; the CDI group received only Clostridium difficile VPI 10463 infection; the CDI+NMN group received β-nicotinamide mononucleotide pretreatment 1 hour before infection; and the NMN group received only β-nicotinamide mononucleotide treatment. Four hours after infection, microscopic observation revealed that macrophages in the CDI group exhibited typical pyroptosis characteristics, including significant cell swelling, membrane rupture, and vacuolation; while the CDI+NMN group, pretreated with β-nicotinamide mononucleotide, significantly improved these pathological changes. Figure 3 A).

[0068] To quantify cell death, a lactate dehydrogenase release assay was performed. The results showed that the lactate dehydrogenase release rate in the CDI group exceeded 60%, while the lactate dehydrogenase release rate in the CDI+NMN group was significantly reduced. Figure 3 B). Furthermore, RT-qPCR analysis showed that β-nicotinamide mononucleotide pretreatment of the CDI+NMN group significantly downregulated the mRNA expression levels of pro-inflammatory factors Tnf-α and Il6, while upregulating the expression of anti-inflammatory factor Il10. Figure 3 C).

[0069] To verify the induction of pyroptosis, the activation level of the GSDMD pathway in bone marrow-derived macrophages was detected by Western blotting. The results showed that the protein level of the cleaved GSDMD-N-terminal fragment (GSDMD-NT) was significantly increased in the CDI group, while this phenomenon was significantly weakened in the CDI+NMN group pretreated with β-nicotinamide mononucleotide. Figure 3 D). RT-qPCR analysis further confirmed that the CDI+NMN group pretreated with β-nicotinamide mononucleotide downregulated the mRNA expression levels of Gsdmd and Il1b. Figure 3 E). Enzyme-linked immunosorbent assay (ELISA) also showed that the CDI+NMN group significantly reduced the secretion of IL-1β.

[0070] In summary, these results indicate that Clostridium difficile infection induces GSDMD-mediated pyroptosis in BMDMs, while β-nicotinamide mononucleotide pretreatment inhibits the activation of the GSDMD pyroptosis pathway and the production of pro-inflammatory cytokines.

[0071] 2.4. β-Nicotinamide mononucleotide ameliorate reduced Clostridium difficile-induced pyroptosis in mouse colon

[0072] To further validate the role of GSDMD-mediated pyroptosis in the intestinal tract of *C. difficile* infection in mice, the expression of pyroptosis-related proteins in the colon tissues of control and infected mice was examined. The results showed that the expression levels of Caspase-1-p10 and GSDMD-NT fragments were significantly increased in the *C. difficile* infection group (CDI). Figure 4 A). RT-qPCR analysis showed that the expression of the Gsdmd gene was upregulated in the CDI group ( Figure 4 B), while ELISA showed that the serum interleukin-1β (IL-1β) level in the infected group mice was significantly higher than that in the control group ( Figure 4 C). Further testing of other inflammatory factors revealed that the expression levels of Tnf-α, Il6, and Il1b genes were significantly increased in the CDI group, while the expression of the Il10 gene was significantly downregulated. Figure 4 D).

[0073] Subsequently, parallel experiments were conducted using purchased wild-type mice and Gsdmd knockout mice. Both groups of mice received β-nicotinamide mononucleotide pretreatment before the experiment. The study found that Gsdmd knockout significantly improved the survival rate of mice induced by *Clostridium difficile*, reduced clinical disease scores, and alleviated colonic shortening, but had no significant effect on the colonic colonization of *Clostridium difficile*. Compared with Gsdmd knockout mice pretreated with β-nicotinamide mononucleotide, wild-type mice showed no significant changes in survival rate, clinical disease scores, colonic shortening, or *Clostridium difficile* colonization after β-nicotinamide mononucleotide pretreatment. Figure 4 These results indicate that, in animal models, Gsdmd deletion improves Clostridium difficile infection, while β-nicotinamide mononucleotide exacerbates Clostridium difficile infection in mice via the GSDMD-mediated pyroptosis pathway.

[0074] 2.5. β-Nicotinamide mononucleotide (NMN) alleviates Clostridium difficile infection in mice by inhibiting inflammation through the Caspase-1 / GSDMD-mediated pyroptosis pathway.

[0075] We also examined the expression of pyroptosis-related proteins in the colon tissues of six groups of mice. In the wild-type mouse group, β-nicotinamide mononucleotide (NMN) significantly inhibited the expression of Caspase-1-p10 and GSDMD-NT fragments, a finding consistent with RT-qPCR results. Similarly, NMN intervention also showed a similar inhibitory effect on the expression of Caspase-1-p10 and GSDMD-NT fragments in Gsdmd knockout mice, a conclusion further validated by RT-qPCR analysis. ELISA analysis of serum inflammatory factors (including TNF-α, IL-6, and IL-1β) showed consistent reductions in these factor levels in both wild-type and knockout mice treated with NMN. These findings further suggest that NMN alleviates Clostridium difficile infection in mice by inhibiting the Caspase-1 / GSDMD-mediated pyroptosis pathway.

[0076] 3. Conclusion

[0077] This study demonstrates that β-nicotinamide mononucleotide (NMN) significantly alleviates the progression of Clostridium difficile infection through two core mechanisms: (1) downregulating the expression of pro-inflammatory factors, thereby enhancing host survival through anti-inflammatory effects via macrophages; and (2) inhibiting the GSDMD-dependent pyroptosis pathway and Caspase-1 inflammasome activation, thereby reducing Caspase-1 activity and IL-1β secretion. This study lays a theoretical foundation for β-nicotinamide mononucleotide to inhibit the progression of Clostridium difficile infection-related intestinal inflammation and supports its potential as an intervention strategy for Clostridium difficile infection.

[0078] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. Application of β-nicotinamide mononucleotide in the preparation of drugs that inhibit Clostridium difficile infection-induced intestinal diseases.

2. The application according to claim 1, characterized in that, Clostridium difficile infection is an intestinal infectious disease.

3. The application according to claim 1, characterized in that, The aforementioned Clostridium difficile infection includes diarrhea, abdominal pain, fever, dehydration, pseudomembranous colitis, intestinal perforation, and toxic megacolon.

4. The application according to claim 1, characterized in that, The drug exerts its therapeutic effect by inhibiting pyroptosis induced by Clostridium difficile and mediated by pyrolysis D.

5. The application as described in any one of claims 1-4, characterized in that, The drug uses β-nicotinamide mononucleotide as its active ingredient and also includes pharmaceutically acceptable carriers or excipients.

6. The application as described in any one of claims 1-4, characterized in that, The drug dosage form is an oral preparation or an injectable preparation.

7. The application as described in claim 6, characterized in that, The oral preparation is a tablet, capsule, or granule; the injectable preparation is an injection solution or powder for injection.

8. A drug for inhibiting Clostridium difficile infection, characterized in that, The drug contains a therapeutically effective amount of β-nicotinamide mononucleotide.

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