Application of biliverdin molecule in preparation of medicine for treating clostridium difficile infection

Abstract

The invention discloses an application of a biliverdin molecule in preparation of a medicine for treating clostridium difficile infection. By constructing a meriones unguiculatus clostridium difficile infection model, it is proved that biliverdin can significantly improve the survival rate of the meriones unguiculatus infected with clostridium difficile and reduce diarrhea symptoms; the biliverdin is combined with a TcdB-DRBD region, so that the TcdB activity is reduced, and a host is protected. Therefore, biliverdin can be used as a potential therapeutic drug for clostridium difficile infection. The biliverdin is a non-toxic green linear water-insoluble pyrrole pigment and is a byproduct of heme oxygenase-1 (HO-1), so that the biliverdin is used for treating the CDI and has no toxic or side effect on the body, and the biliverdin is a pigment micromolecule, has no bacteriostatic action and cannot cause disorder of the flora environment of the body.

Description

(1) Technical Field

[0001] The present invention belongs to the technical field of new applications of drugs, and particularly relates to the application of biliverdin molecules in the preparation of drugs for treating Clostridium difficile infection. (2) Background Art

[0002] Clostridium difficile is an obligate anaerobic Gram-positive spore-forming bacterium, which is widely distributed in natural environments, animal and human feces. Its spores have strong resistance and can survive in the external environment for several weeks to several months. Clostridium difficile infection (CDI) is an important factor leading to antibiotic-associated diarrhea (ADD), and about 30% of ADD cases are caused by Clostridium difficile, so it is also called Clostridium difficile associated diarrhea (CDAD). The related diseases caused by CDI mainly include abdominal pain and diarrhea, pseudomembranous colitis, toxic megacolon, renal failure, sepsis, etc., and even cause death of the body. In recent years, due to the emergence of the highly virulent strain 027 / NAP1 / BI (ribosomal typing: 027, pulsed-field gel electrophoresis typing: NAP1, restriction endonuclease typing: BI) of Clostridium difficile, the incidence and severity of CDI have been continuously increasing globally, and the number of affected regions has been increasing. Highly virulent strains of Clostridium difficile and outbreaks have occurred in countries such as North America, Australia, and Europe. According to the statistical data of the US CDC, about 500,000 Americans are infected with Clostridium difficile every year, and 29,000 of them die from the infection. 13‰ of inpatients in the United States are infected with Clostridium difficile (11‰ in Europe), bringing a huge economic and social burden.

[0003] Currently, the treatment method for Clostridium difficile infection is anti-infection treatment with vancomycin plus metronidazole. However, current data show that the clinical positive rate of Clostridium difficile in China is about 3%-23% and varies, and there is a high resistance rate to various antibiotics. Research shows that up to 93.1% of strains in the Asia-Pacific region show multidrug resistance, the proportion of clinical multidrug-resistant strains in China exceeds 70%, and up to 30.3% of strains show resistance to MLSB (macrolide-lincosamide-streptogramin B) antibiotics. At the same time, there have been severe CDI cases caused by a large amount of toxins secreted by multidrug-resistant strains in clinical practice in China, bringing unprecedented difficulties and challenges to clinical anti-infection treatment.

[0004] Biliverdin (BV) is a non-toxic green linear water-insoluble pyrrole pigment and is a by-product of heme oxygenase-1 (HO-1). In recent years, biliverdin has been applied to the protection of various diseases, such as vascular injury, organ transplantation, protection against acute lung injury in rats, and inhibition of the deterioration and development of adenotoxemia. However, its role in Clostridium difficile has not been reported. (III) Summary of the Invention

[0005] The object of the present invention is to provide an application of biliverdin molecules in the preparation of drugs for treating Clostridium difficile infection. By constructing a Clostridium difficile infection (CDI) model in Mongolian gerbils, the present invention proves that biliverdin can significantly improve the survival rate of CDI-infected gerbils, reduce diarrhea symptoms, lower the concentration of Clostridium difficile toxin B (TcdB) in intestinal tissues, and inhibit the damage of Clostridium difficile to intestinal epithelial tissues, thus confirming that biliverdin is a potential therapeutic drug for CDI and solving the problem of drug resistance in the existing anti-infection treatment using vancomycin plus metronidazole.

[0006] The technical solution adopted by the present invention is as follows:

[0007] The present invention provides an application of biliverdin molecules in the preparation of drugs for treating Clostridium difficile infection.

[0008] Preferably, the drug can treat Clostridium difficile infection induced by the highly virulent strain 027 / NAP1 / BI of Clostridium difficile.

[0009] Preferably, the drug can reduce the concentration of TcdB in intestinal tissues and inhibit the damage of Clostridium difficile to intestinal epithelial tissues.

[0010] Preferably, the administration method of the drug is gavage, and the administration dose is 35 mg / kg.

[0011] Preferably, the biliverdin has the characteristic of extremely low water solubility. It is first dissolved in dimethylformamide (DMSO) and then diluted with physiological saline for gavage. The volume ratio of DMSO to physiological saline is 1:20.

[0012] The present invention also provides an application of the said biliverdin in the preparation of drugs for treating diarrhea caused by Clostridium difficile infection.

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

[0014] By constructing a Clostridium difficile infection model in Mongolian gerbils, the present invention proves that biliverdin can significantly improve the survival rate of gerbils infected with Clostridium difficile and reduce diarrhea symptoms; biliverdin binds to the TcdB-DRBD region to reduce the activity of TcdB and protect the host. Therefore, biliverdin can be used as a potential therapeutic drug for Clostridium difficile infection.

[0015] Since biliverdin is a non-toxic green linear water-insoluble pyrrole pigment and a by-product of heme oxygenase-1 (HO-1), the present invention uses biliverdin to treat CDI, which has no toxic side effects on the organism. Moreover, biliverdin is a small molecule of pigment class and has no antibacterial effect, so it will not cause the imbalance of the body's flora environment. (IV) BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It shows the effect of biliverdin on the survival rate of gerbils with CDI induced by the highly virulent strain 027 / NAP1 / BI. Ctrl group, n = 5; CDI group, n = 7; CDI + BV group, n = 7.

[0017] Figure 2 It is a bar graph of the TcdB levels in gerbils with CDI induced by the highly virulent strain 027 / NAP1 / BI in different treatment groups. Ctrl group, n = 5; CDI group, n = 3; CDI + BV group, n = 4.

[0018] Figure 3 It is an H&E staining image of paraffin sections of intestinal tissues of gerbils with CDI induced by the highly virulent strain 027 / NAP1 / BI in different treatment groups. Ctrl group, n = 5; CDI group, n = 3; CDI + BV group, n = 4.

[0019] Figure 4 It is the toxicity test of biliverdin on Caco-2 cells. A represents a bar graph of the effect of different concentrations of biliverdin on the concentration of Caco-2 cells; B represents a bar graph of the effect of different concentrations of biliverdin on the survival rate of Caco-2 cells; C represents a microscopic photograph (40× magnification) of the effect of biliverdin on the activity of Caco-2 cells induced by tcdB. n = 3 for each group.

[0020] Figure 5 It is a photograph of Clostridium difficile cultured with different concentrations of biliverdin and vancomycin.

[0021] Figure 6 It is the molecular docking diagram of biliverdin and TcdB-4. A: Scatter plot of the binding energy of small molecule compounds, with the X-axis being the number and the Y-axis being the affinity (kcal / mol). B. Structure of the TcdB4-biliverdin complex. TcdB4 and biliverdin are shown in green and red respectively. The enlarged view shows the optimal configuration simulated by AutoDock Vina. C. Weak interaction between BV and TcdB4 residues. D. Affinity of TcdB binding to biliverdin calculated using the MM / PBSA method.

[0022] Figure 7 It is the schematic flow diagram of the examples of the present invention. (V) DETAILED IMPLEMENTATION MANNER

[0023] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0024] The reagents / consumables used in the embodiments of the present invention are as shown in Table 1 below:

[0025] Table 1 Reagents / Consumables

[0026]

[0027]

[0028] The instruments / manufacturers involved in the embodiments of the present invention are as shown in Table 2 below:

[0029] Table 2 Instruments / Manufacturers

[0030] Instrument type Manufacturer Microscope Olympus Ultrasonic cell disruptor SCIENTZ Microplate reader Molecular Devices Multi-point inoculator HengAo Technology McFarland turbidimeter BioMérieux

[0031] This study involving animal experiments complied with relevant ethical regulations and was carried out in accordance with the protocol approved by the Laboratory Animal Management and Use Committee of Zhejiang Institute for Food and Drug Control. Mongolian gerbils are an ideal animal model recognized in this experiment, and this experimental protocol uses the minimum number of animals to obtain reliable experimental results. To reduce the pain of experimental animals, oral administration and cervical dislocation were used in this experiment to quickly euthanize the gerbils, and the experimental process complied with the humanitarian standard for ending the life of animals.

[0032] Example 1. Therapeutic effect of biliverdin on CDI gerbils

[0033] 1. Experimental protocol

[0034] 1.1 Experimental subjects and reagents:

[0035] Experimental subjects: Male Mongolian gerbils aged 5 - 8 weeks, weighing 60 - 70 g. The gerbils were housed separately in cages, fed and watered ad libitum, and raised in an environment with 12 hours of light and 12 hours of darkness.

[0036] Biliverdin stock solution: Weigh 100 mg of biliverdin, add an appropriate amount of DMSO to prepare a 100 mM stock solution, and store it in a -80 °C freezer.

[0037] Preparation of biliverdin working solution: Take 0.3 μL of biliverdin stock solution, add an appropriate amount of DMSO to prepare 30 μM; take 0.5 μL of biliverdin stock solution, add an appropriate amount of DMSO to prepare 50 μM; take 1 μL of biliverdin stock solution, add an appropriate amount of DMSO to prepare 100 μM. Use it after thawing at room temperature.

[0038] Composition of spore medium: Tryptone 10 g / L, Yeast extract 10 g / L, (NH 4 ) 2 SO 40.6 g / L, MgSO 4 ·7H 2 O 0.12 g / L, CaCl 2 ·2H 2 O 0.08 g / L, K 2 CO 3 3.48 g / L, KH 2 PO 4 0.6 g / L, and the solvent is water.

[0039] Composition of BHIYT medium: 37 g / L of BHI medium, 5 g / L of yeast powder, 1 g / L of L-cysteine, 1 g / L of taurocholate, 15 g / L of agar powder, and the solvent is water.

[0040] 1.2 Preparation of Clostridium difficile spore suspension

[0041] (1) Broth enrichment: The highly virulent strain 027 / NAP1 / BI of Clostridium difficile (standard strain ATCC BAA-1870, genotype ST1 / RT027) was inoculated into a 15 mL centrifuge tube containing 5 mL of BHI medium and anaerobically cultured at 37 °C for 24 h.

[0042] (2) Spore culture: 1 mL of the bacterial solution from step (1) (if there is precipitation in the bacterial solution, vortex and mix well first) was added to a 50 mL centrifuge tube containing 20 mL of spore medium and cultured at 37 °C for 72 h.

[0043] (3) Spore counting: Take out the cultured bacterial solution from step (2), vortex to mix well. Take 1 mL of the mixed bacterial solution into a 1.5 mL EP tube and incubate at 65 °C for 20 min. The incubated bacterial solution was serially diluted five-fold with PBS (pH = 7.4) at a 10-fold gradient. Take 5 EP tubes, add 900 μL of PBS (pH = 7.4) and 100 μL of the diluted bacterial solution to each tube, and mix well. Take 100 μL and evenly spread it on the BHIYT medium plate (3 parallels for each gradient), and anaerobically culture at 37 °C for 2 days, then take out and count.

[0044] (4) Clostridium difficile spore suspension: The spores from step (3) were prepared into a Clostridium difficile spore suspension with a concentration of 5x10^4 CFU / mL using PBS (pH = 7.4) for gavage.

[0045] 1.3 Establish a CDI model induced by the highly virulent strain 027 / NAP1 / BI: Randomly divide the experimental gerbils into three groups: blank control group (Ctrl, n = 5, where n represents the number of mice in each group), CDI experimental group (CDI, n = 7), and biliverdin treatment group (CDI + BV, n = 7). The mice in each group drank tap water containing an antibiotic mixture, and the final concentration composition of the antibiotic mixture was: 0.4 mg / mL kanamycin, 0.035 mg / mL gentamicin, 850 U / mL polymyxin, 0.215 mg / mL metronidazole, and 0.045 mg / mL vancomycin. After 3 days of treatment, they drank tap water. On the 6th day, 10 mg / kg clindamycin was injected intraperitoneally. The CDI group and the CDI + BV group were respectively gavaged with a suspension of Clostridium difficile spores, and the gavage volume was 100 μL. The blank control group orally administered an equal volume of PBS. After gavage, the tails of the gerbils became wet and significant diarrhea occurred, indicating successful construction of the CDI model.

[0046] 1.4 Administration: After successful construction of the CDI model, the CDI + BV group orally administered 400 μL of the biliverdin working solution, and the dosage was 35 mg / kg body weight. The CDI group and the blank control group orally administered an equal volume of PBS (pH = 7.4). Three days after administration, observe the status and survival rate of the gerbils, and detect the TcdB level and intestinal histopathology.

[0047] 1.5 Effects of biliverdin on the status and survival rate of gerbils in the CDI model

[0048] Observe whether diarrhea occurs in the mice in each group (Ctrl group, n = 5; CDI group, n = 7; CDI + BV group, n = 7, where n represents the number of mice in each group), measure the body weight, and count the survival rate.

[0049] 1.6 Effects of biliverdin on the TcdB level and intestinal histopathology of gerbils in the CDI model

[0050] Three to five gerbils were sacrificed in each group (Ctrl group, n = 5; CDI group, n = 3; CDI + BV group, n = 4). The intestinal contents and intestinal tissues of the gerbils were collected and stored in a -80 °C refrigerator. The intestinal contents were used to detect the TcdB content by ELISA method. The intestinal tissues were sectioned and stained with H&E to observe the morphological and pathological changes of the intestinal tissues.

[0051] ELISA method:

[0052] The extracted intestinal contents were soaked overnight in PBS. 100 μL of the soaking supernatant and 100 μL of purified TcdB antigen at 200 ng / mL (prepared by the method of Example 2) were added to a 96-well plate and incubated overnight at 4°C. Then, the soaking solution was discarded, and the plate was rinsed 3 times with PBS, incubated with the blocking solution (PBST + 0.5% BSA) for 2 hours, rinsed 3 times with PBST, incubated with the primary antibody E3 (1:1000) for 2 hours, and rinsed 4 times with PBST. The secondary antibody Myc (1:5000) was incubated for 1 hour, and the washing method was the same as above. ELISA TMB chromogenic solution was added, and the plate was incubated in the dark for 15 minutes. Finally, the TMB substrate stop solution was added, and the OD value was measured with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm to calculate the TcdB content.

[0053] 1.7 Statistical analysis: Data are presented as mean ± standard error. Statistical differences were analyzed using GraphPad Prism 10 medical drawing analysis software by one-way analysis of variance and Tukey's multiple comparison test or Dunnett's multiple comparison test. A P value < 0.05 was considered statistically significant, and the smaller the P value, the more significant the difference. In the figures, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0054] 2. Experimental results

[0055] 2.1 Effects of biliverdin on the status and survival rate of gerbils in the CDI model:

[0056] After induction with the highly virulent strain 027 / NAP1 / BI spores, the tails of the gerbils in the CDI group and the CDI + BV group became wet, showed significant diarrhea, and their body weights decreased significantly.

[0057] Figure 1 Shown in the figure is the survival curve of mice in each group. It can be seen from the figure that the survival rate of gerbils in the CDI group was 42%. The survival rate of gerbils treated with biliverdin in the CDI + BV group increased to 57%.

[0058] 2.2 Effects of biliverdin on the TcdB level in gerbils in the CDI model:

[0059] Figure 2 Shown in the figure is the TcdB level in gerbils in each group 3 days after administration of biliverdin. Compared with the Ctrl group, the TcdB level in the CDI group was significantly increased (p < 0.0001), and the TcdB level in the CDI + BV group was significantly lower than that in the CDI group (p < 0.0001).

[0060] 2.3 Effects of biliverdin on the intestinal histopathology of gerbils in the CDI model:

[0061] From Figure 3It was clearly seen that in the gerbil intestinal tissues of the CDI group, epithelial damage occurred, myofibroblasts were irregularly arranged, the cell arrangement was loose, the number of goblet cells in the crypts was significantly reduced, and there was infiltration of neutrophils; in the pathological examination of the gerbil intestinal tissues of the CDI+BV group after treatment with biliverdin, only irregularly arranged myofibroblasts and loose cell arrangement were observed.

[0062] In summary, the above results suggest that the CDI Mongolian gerbil model induced by the highly virulent strain 027 / NAP1 / BI was successfully constructed. Biliverdin can alleviate the CDI symptoms and pathological phenomena of gerbils, improve the survival rate, and has an obvious protective effect.

[0063] Example 2. Effect of biliverdin on human colorectal adenocarcinoma cell line Caco-2

[0064] 1 Experimental protocol

[0065] 1.1 Culture media

[0066] Complete culture medium: MEM medium (purchased from Gibco, catalog number 11095500) supplemented with 20% (v / v) fetal bovine serum (FBS), 100 units / mL penicillin, 100 μg / mL streptomycin, and 1% (v / v) non-essential amino acids (NEAA).

[0067] 2×YT liquid medium: 16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride, 1 mM chloramphenicol, and the solvent is water.

[0068] 1.2 Cell culture:

[0069] Human colorectal adenocarcinoma cell line Caco-2 was inoculated into the complete culture medium and incubated in a 37 °C constant temperature and humidity incubator containing 5% CO 2 for 24 h.

[0070] Cell seeding: After carefully pipetting, digesting, and centrifuging the above-mentioned Caco-2 cells with ice-cold PBS, the cell concentration was detected using a Neubauer counting chamber. According to the cell concentration, Caco-2 was diluted with the complete culture medium and seeded into a 96-well plate, with 5*10 4 cells per well.

[0071] 1.3 Effect of biliverdin on the proliferation activity of human colorectal adenocarcinoma cell line Caco-2

[0072] The 96-well plates in the above 1.2 were divided into a blank group (complete medium, no cells), a control group (Ctrl, complete medium + cells), and a biliverdin group (10 μM, 30 μM, 50 μM, and 100 μM biliverdin + complete medium + cells), with 3 wells in each group. After culturing for 24 hours, the original medium was discarded. The biliverdin group was added with 100 μL of complete medium containing the corresponding biliverdin concentration, and the blank group and the control group were added with 100 μL of complete medium. They were placed in a 37 °C constant temperature and humidified incubator containing 5% CO 2 After culturing for 24, 48, and 72 hours respectively, 10 μL of CCK8 working solution was added to each well of each group. After continuing to culture for 1 hour, the OD value at 450 nm absorbance was measured on an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate was calculated.

[0073] Cell survival rate = (OD of drug-treated cells - OD of blank cells) / (OD of control cells - OD of blank cells) × 100%

[0074] Preparation of 1.4 TcdB

[0075] The Bacillus subtilis WB800N expressing TcdB was inoculated in 2×YT liquid medium and cultured at 37 °C until OD600 = 0.6 - 1.0. 1 mM IPTG was added, and induction was carried out overnight at 25 °C. Then, it was centrifuged at 5000×g for 5 min, and the cell pellet was resuspended with cell lysis buffer (50 ml of PBS + 500 μl of 100×(24.83 mg / ml) PMSF + 500 μl of 50 mg / ml lysozyme). The bacterial solution was sonicated using an ultrasonic cell disruptor with a power of 200 W, 5 s pulse / 3 s pause. After 5 cycles, the mixture was cooled on ice for 2 min until the bacterial solution became clear and transparent. It was centrifuged at 10000×g for 10 min to separate the supernatant, and 10 mM imidazole was added to the final concentration as the loading solution.

[0076] Fresh nickel beads were mixed evenly and added to an empty affinity chromatography column, which was then washed successively with ultrapure water and PBS (pH 7.4, 30 mM). It was equilibrated with PBS containing 10 mM imidazole for 5 min, and the liquid was filtered out. 30 mL of the loading solution was added and allowed to stand for 5 min. Then, the outflowing liquid was collected and loaded again. This step was repeated three times. PBS containing 20, 40, 60, and 80 mM imidazole at the final concentration was added successively, 30 mL each time, for elution, and the liquid was filtered out. Subsequently, 30 mL of PBS containing 500 mM imidazole at the final concentration was added to equilibrate for 5 min, and the outflowing liquid was collected and repeated three times to obtain the purified protein liquid. The above 10 mL of protein liquid was added to a concentrator tube and centrifuged at 6000×g for 5 min until 500 μL of protein solution remained in the concentrator tube. 4 mL of PBS was added, and it was centrifuged at 4000×g for 5 min until 500 μL of protein solution remained in the concentrator tube. This step was repeated three times. Finally, concentrated pure protein TcdB was obtained and stored at -80 °C.

[0077] 1.5 Effect of biliverdin on the proliferative activity of human colorectal adenocarcinoma Caco-2 cells induced by TcdB:

[0078] The 96-well plates in the above 1.2 were divided into a Ctrl group, a TcdB group, and a TcdB + BV group, with 3 wells in each group.

[0079] After seeding the plates for 24 hours, the culture medium was discarded. 100 μL of complete medium was added to each well in the Ctrl group. 100 μL of complete medium containing 200 ng / mL of TcdB was added to each well in the TcdB group. 100 μL of complete medium containing 30 μM biliverdin and 200 ng / mL of TcdB was added to each well in the TcdB + BV group. They were co-cultured for 24 hours under the conditions in the above 1.1, and photographed under a microscope to observe the cell rounding reaction.

[0080] 1.6 Statistical analysis: The statistical analysis was the same as in Example 1. * represents p < 0.05, ** represents p < 0.01, **** represents p < 0.0001. * represents p < 0.05, ** represents p < 0.01.

[0081] 2. Experimental results

[0082] 2.1 Effect of biliverdin on the proliferative activity of human colorectal adenocarcinoma Caco-2 cells

[0083] The results are shown in Figure 4 A and B below. Biliverdin at 10 - 100 μM has no obvious toxicity to Caco-2 cells and does not significantly inhibit cell growth.

[0084] 2.2 Effect of biliverdin on the activity of Caco-2 cells induced by TcdB

[0085] The results are shown in Figure 4 C below. The cell rounding experiment showed that compared with the cells not given TcdB, TcdB could induce cell rounding and atrophy, and 30 μM biliverdin significantly inhibited the cell rounding reaction of TcdB.

[0086] Example 3. Effect of biliverdin on the activity of Clostridium difficile

[0087] 1. Experimental protocol

[0088] 1.1 Preparation of culture medium:

[0089] Quality composition of brain heart infusion broth medium (BHIS): 3.7% brain heart infusion broth (BHI), 0.5% yeast powder, 0.1% L-cysteine.

[0090] Bruce agar plate: Weigh 5.0 g of tryptone, 5.0 g of peptone, 0.5 g of D-(+)-glucose, 2.5 g of yeast extract, 2.5 g of sodium chloride, 0.05 g of sodium bisulfite, and 7.5 g of agar powder. Add 500 mL of ultrapure water and shake well. Adjust the pH to 7.2 - 7.4 with 8.0 M NaOH solution and glacial acetic acid. After sealing, autoclave at 121 °C for 15 min. When cooled to 45 °C, add 25 mL of fresh frozen sheep blood, 500 μL of hemin, and 500 μL of 1 mg / mL vitamin K1 working solution, and shake well.

[0091] 1.2 Clostridium difficile culture:

[0092] The highly virulent strain of Clostridium difficile 027 / NAP1 / BI (ATCC BAA-1870, ST1 / RT027) was inoculated in BHIS and cultured in an anaerobic condition (10% H 2 , 10% CO 2 , 80% N 2 ) in a 37 °C incubator for 24 hours. Adjust the culture to a McFarland turbidity of 0.5 (measured with a McFarland nephelometer) with BHIS to obtain a Clostridium difficile bacterial solution.

[0093] 1.3 In vitro antibacterial experiment:

[0094] Take 1 μL of the Clostridium difficile bacterial solution prepared in 1.2 above and inoculate it into Bruce agar plates supplemented with 1 mL of biliverdin at corresponding concentrations (30, 50, and 100 μM) and Bruce agar plates supplemented with 1 mL of vancomycin (4, 8, 16 μg / mL) using a multi-point inoculator. The control group (Ctrl) was not supplemented with any drugs. Incubate in an anaerobic condition (10% H 2 , 10% CO 2 , 80% N 2 ) in a 37 °C incubator for 48 hours.

[0095] 1.4 Statistical analysis: The statistical analysis was the same as in Example 1. * represents p < 0.05, ** represents p < 0.01, **** represents p < 0.0001. * represents p < 0.05, ** represents p < 0.01.

[0096] 2. Experimental results

[0097] The results are shown in Figure 5, The in vitro antibacterial experiment results showed that the Clostridium difficile strain (ATCC BAA-1870, ST1 / RT027) could grow normally in the medium with biliverdin at 30 - 100 μM, but could not grow in vancomycin at 4 - 16 μg / mL. There was no significant difference between the biliverdin group and the control group (Ctrl), and there was a significant difference between the biliverdin group and the vancomycin group. This indicated that, different from vancomycin, BV had no obvious inhibitory effect on the growth of Clostridium difficile.

[0098] Example 4, Molecular Docking of Biliverdin and TcdB-4

[0099] 1. Experimental Scheme

[0100] 1.1 Small Molecule Design and Processing

[0101] Download the 3D structure of biliverdin in PDB format from the PubChem database, import the structure into ChemBio3D Ultra 14.0 for energy minimization, set the Minimum RMS Gradient to: 0.001, and save the small molecule as mol2 format. Import the optimized small molecule into AutodockTools - 1.5.6 for hydrogenation, calculation of charges, assignment of charges, setting of rotatable bonds, and then save it as "pdbqt" format.

[0102] 1.2 Protein Preparation and Processing

[0103] Download Clostridium difficile TcdB-4 (PDB ID: 7V1N) from the RCSB database, use Pymol 2.3.0 to remove the protein crystal water, original ligands, etc., import the protein structure into AutoDockTools for hydrogenation, calculation of charges, assignment of charges, specifying atom types, and save it as "pdbqt" format.

[0104] 1.3 Preparation of Parameter Files

[0105] Use POCASA 1.1 to predict the protein binding site, and perform docking using AutoDockVina 1.1.2. The relevant parameters of STAT3 are set as: center_x = 221.8, center_y = 157.5, center_z = 138.5; search space: size_x: 20, size_y: 20, size_z: 20 (the spacing of each grid point is set to 0.375 Å), exhaustiveness: 10, and the remaining parameters are default settings.

[0106] 1.4 Result Analysis

[0107] Use PyMOL 2.3.0 and LigPlot v2.2.5 to analyze the interaction mode of the docking results.

[0108] 2. Experimental results

[0109] The binding energy of biliverdin and TcdB-4 is -9.5 kcal / mol, demonstrating good binding. Biliverdin interacts with TcdB-4 mainly through hydrogen bonding and hydrophobic forces, forming hydrogen bonds with TYR-1510 and SER-1598. At the same time, the research results show that biliverdin can also bind to TcdBs of other lineages at different amino acid sites, and the binding affinity is also very high. The binding affinity between BV and TcdB was quantitatively evaluated using the molecular mechanics / Poisson-Boltzmann surface area (MM / PBSA) method. The calculated binding energy is approximately -40 kcal / mol( Figure 6 ).

[0110] In summary, biliverdin can effectively improve the survival rate of gerbils with CDI induced by Clostridium difficile 027 / NAP1 / BI and reduce diarrhea symptoms; this may be related to biliverdin reducing the concentration of TcdB by binding to TcdB-DRBD, thereby alleviating CDI symptoms. The application of biliverdin in CDI has great potential and is a potential therapeutic drug for CDI.

Claims

1. Application of a biliverdin molecule in the preparation of a drug for treating Clostridium difficile infection.

2. The use according to claim 1, characterized in that The drug can treat Clostridium difficile infection induced by the highly virulent Clostridium difficile strain 027 / NAP1 / BI.

3. The use according to claim 1, characterized in that The drug can reduce the concentration of TcdB in intestinal tissue and inhibit the destruction of intestinal epithelial tissue by Clostridium difficile.

4. The use according to claim 1, characterized in that The drug is administered by intragastric administration, and the dosage is 35 mg / kg.

5. The use according to claim 1, characterized in that The biliverdin molecules were first dissolved in dimethylformamide, and then diluted with physiological saline before oral administration.

6. Use of the biliverdin molecule according to claim 1 in the preparation of a drug for treating diarrhea caused by Clostridium difficile infection.

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