Use of indole-3-propionic acid for the preparation of a medicament for the prevention or treatment of blood-borne infections

Abstract

The application discloses application of indole-3-propionic acid in preparation of a medicine for preventing or treating blood-borne infection and belongs to the technical field of biological medicine. The application discloses, for the first time, the use of indole-3-propionic acid in resisting blood-borne infection. The use of indole-3-propionic acid can significantly enhance the ability of liver Kupffer cells to capture blood-borne pathogenic bacteria and reduce the pathogenic bacterial load in circulating blood. The application provides a new idea for the treatment of blood-borne infection, especially the anti-infection treatment of susceptible populations such as severe hepatitis or cirrhosis. Indole-3-propionic acid is cheap and easy to obtain, can be produced in large scale, is beneficial to development into a medicine for treating blood-borne infection and has good application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, and particularly relates to application of indole-3-propionic acid in preparation of a drug for preventing or treating blood-borne infection. BACKGROUND

[0002] Bloodstream infection (BSI) is a major cause of morbidity and mortality of infectious diseases worldwide. It can be a primary infection caused by surgical intervention, intravenous catheter use, etc., or a secondary complication caused by other diseases such as cirrhosis, etc. Excessive growth and spread of invasive pathogens in the blood circulation can exacerbate damage to important organs at a distance, often leading to more serious diseases such as infective endocarditis, meningitis, sepsis or septic shock. The mortality risk of BSI is 7%-35%, accounting for one-third of bacterial infection deaths. Although great progress has been made in understanding the pathophysiological mechanisms of BSI and clinical intensive care treatment in recent years, how to quickly and effectively clear bacteria in the circulation is still crucial for effective prevention and treatment of fatal consequences caused by BSI.

[0003] Currently, the prevention and treatment of blood-borne infections in the clinic mainly relies on the use of broad-spectrum antibiotics based on experience, but long-term use of antibiotics has the problem of drug resistance, so it is necessary to develop alternative therapies to traditional antibiotics.

[0004] Kupffer cells, as resident macrophages in the liver, form a unique immune defense barrier in the vascular space of the liver sinusoid. Kupffer cells not only can capture and kill invading bacteria themselves, but also can synergize with other immune cells to resist pathogenic bacterial infection, thereby playing a crucial role in clearing blood-borne bacteria and preventing pathogens from spreading to the whole body.

[0005] Hepcidin is an acute phase protein produced mainly by hepatocytes to regulate body iron metabolism. Hepcidin regulates the concentration of iron in the extracellular fluid by binding to the iron transporter on the cell membrane (the only known cellular iron transporter) and inducing its degradation. Hepcidin plays a crucial role in host defense against bacterial infection. It not only has direct bactericidal activity at non-physiological high concentrations in vitro, but also controls and even kills bacteria by regulating intracellular / extracellular iron status to form hypoferritinemia, depriving the iron element necessary for the growth of pathogenic microorganisms. Low levels of hepcidin are common in patients with severe hepatitis and cirrhosis, and these patients are more likely to develop serious blood-borne infections, which are life-threatening. At the same time, animal experiments found that in a mouse model of peritonitis, inhibition of liver hepcidin led to systemic spread of bacteria. These studies suggest the importance of hepcidin in preventing systemic bacterial infection. However, the mechanism by which hepcidn protects the body from systemic bacterial spread is still unclear.

[0006] In the research of the research group, Hamp1 - / - ) mice and wildtype (WT) mice were used to study blood-borne bacterial infection. It was found that compared with WT mice, Hamp1 - / - mice had significantly decreased ability of liver Kupffer cells to capture cells, significantly increased bacterial load in circulating blood, and significantly increased mortality. No study has reported that hepcidin is related to the ability of Kupffer cells to capture bacteria. SUMMARY

[0007] The purpose of the present application is to provide an active substance that can enhance the ability of Kupffer cells to capture pathogenic bacteria, for the development of drugs for the prevention or treatment of blood-borne infections, especially anti-infection treatment for susceptible populations such as severe hepatitis or cirrhosis.

[0008] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] The present application provides the use of indole-3-propionic acid (IPA) in the preparation of a drug for preventing or treating blood-borne infections.

[0010] In the present application, the hepcidin gene knockout animal model was used to study the blood-borne pathogenic bacteria infection. Compared with the wild type animals, the ability of liver Kupffer cells to capture pathogenic bacteria in the hepcidin gene knockout animal model was significantly decreased, the bacterial load in the circulating blood was significantly increased, and the mortality was significantly increased. By analyzing the portal vein blood metabolomics of the animal model, it was found that compared with the wild type, the concentration of indole-3-propionic acid, a tryptophan metabolite in the portal vein blood, was significantly reduced after the hepcidin gene knockout, suggesting that indole-3-propionic acid may be a potential therapeutic target for resisting blood-borne infection.

[0011] In the present application, the hepcidin gene knockout animal model was used to study the blood-borne pathogenic bacteria infection. Compared with the wild type animals, the ability of liver Kupffer cells to capture pathogenic bacteria in the hepcidin gene knockout animal model was significantly decreased, the bacterial load in the circulating blood was significantly increased, and the mortality was significantly increased. By analyzing the portal vein blood metabolomics of the animal model, it was found that compared with the wild type, the concentration of indole-3-propionic acid, a tryptophan metabolite in the portal vein blood, was significantly reduced after the hepcidin gene knockout, suggesting that indole-3-propionic acid may be a potential therapeutic target for resisting blood-borne infection.

[0012] Indole-3-propionic acid is a tryptophan metabolite, which naturally exists in animals and has high biological safety. Therefore, indole-3-propionic acid can be used for the development and preparation of drugs for resisting blood-borne infection.

[0013] Further, the blood-borne infection includes hepcidin gene function loss or decrease accompanied by blood-borne infection. Patients with hepcidin gene loss or low level of hepcidin are more likely to have severe blood-borne infection, and indole-3-propionic acid can effectively resist blood-borne infection.

[0014] Further, the blood-borne infection includes severe hepatitis or liver cirrhosis accompanied by blood-borne infection. The manifestations of the severe hepatitis or liver cirrhosis include low level of hepcidin.

[0015] Further, the blood-borne infection includes decreased ability of liver Kupffer cells to capture pathogenic bacteria accompanied by blood-borne infection. Patients with decreased ability of liver Kupffer cells to capture pathogenic bacteria are more likely to have blood-borne infection, and indole-3-propionic acid can effectively resist blood-borne infection.

[0016] The mechanism of the present application shows that indole-3-propionic acid can prevent or treat blood-borne infection by enhancing the ability of liver Kupffer cells to capture pathogenic bacteria.

[0017] Further, the pathogen of the blood-borne infection can be but is not limited to bacteria, and the bacteria include Escherichia coli.

[0018] In the present application, the drug uses indole-3-propionic acid as the main active ingredient, adds a pharmaceutically acceptable carrier to form, and can be prepared according to the preparation method recorded in pharmacy. The specific dosage is adjusted according to the type of disease, the degree of disease, age and the purpose of administration.

[0019] Further, the preparation form of the medicine can be, but is not limited to, oral preparation.

[0020] In a mouse model, the effective dose of indole-3-propionic acid is 50 mg / kg.bw / d, and there is no obvious toxic side effect.

[0021] The present application also provides a medicine composition for resisting blood-borne infection, which comprises an effective dose of indole-3-propionic acid.

[0022] The present application uses indole-3-propionic acid as the main active ingredient to prepare a medicine for preventing or treating blood-borne infection. Further, the medicine composition also comprises a pharmaceutically acceptable carrier.

[0023] Term explanation:

[0024] The term "comprising" is an open-ended term, i.e. it includes what the specification states but also anything else.

[0025] The term "treatment", in some embodiments, means improving a disease or condition (i.e. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In other embodiments, it means alleviating or relieving at least one physical parameter, including a parameter that can not be perceived by the subject. In other embodiments, it means modulating a disease or condition physically (e.g. stabilizing a perceptible symptom) or physiologically (e.g. stabilizing a parameter of the body) or both. In other embodiments, it means preventing or delaying the onset, onset or worsening of a disease or condition.

[0026] The term "effective dose" means the amount of a compound that is sufficient to have an effect on the treatment of a disease when administered to a subject. The effective dose can vary depending on the severity of the disease, and the physical condition, age, weight, gender, etc. of the subject to be treated.

[0027] The term "pharmaceutically acceptable carrier" means any formulation or carrier medium capable of delivering an effective dose of active substance of the present application, not interfering with the biological activity of the active substance and having no toxic side effects on the host or subject.

[0028] The present application has the following beneficial effects:

[0029] (1) The present application discloses the use of indole-3-propionic acid in resisting blood-borne infection for the first time. Animal experiments show that the administration of indole-3-propionic acid can significantly enhance the ability of liver Kupffer cells to capture blood-borne pathogenic bacteria, and reduce the pathogenic bacterial load in circulating blood. The present application provides a new idea for the treatment of blood-borne infection, especially the anti-infection treatment of susceptible populations such as severe hepatitis or cirrhosis.

[0030] (2) Indole-3-propionic acid is cheap and easy to obtain, and can be produced on a large scale, which is beneficial to the development of drugs for treating blood-borne infections and has good application value. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Hamp1 - / - Results of live imaging of the liver of mice after blood-borne E. coli infection to detect the ability of Kupffer cells to capture bacteria, wherein A is a bacteria capture image under a live microscope; and B is a quantitative analysis of the bacteria capture ability of KC. * indicates P < 0.05.

[0032] Figure 2 Hamp1 - / - Results of detection of bacterial load in the circulating blood of mice after blood-borne E. coli infection and statistics of mortality, wherein A is the bacterial load; and B is the mortality rate. * indicates P < 0.05.

[0033] Figure 3 Hamp1 - / - Results of morphological detection of Kupffer cells in the liver of mice, wherein A is the morphology under a microscope; B is the morphology after three-dimensional reconstruction; C is the volume of KC cells; and D is the surface area of KC cells. **** indicates P < 0.0001.

[0034] Figure 4 Hamp1 - / - Results of detection of the concentration of IPA in the portal vein blood of mice. ** indicates P < 0.01.

[0035] Figure 5 Results of morphological detection of Kupffer cells after IPA treatment, wherein A is the morphology under a microscope; B is the morphology after three-dimensional reconstruction; C is the volume of KC cells; and D is the surface area of KC cells. **** indicates P < 0.0001.

[0036] Figure 6 Hamp1 - / - Results of detection of the level of IPA in the portal vein blood of mice 7 days after IPA gavage. * indicates P < 0.05.

[0037] Figure 7 Results of live imaging of the liver of mice after IPA gavage to detect the ability of Kupffer cells to capture bacteria, wherein A is a bacteria capture image under a live microscope; and B is a quantitative analysis of the bacteria capture ability of KC. * indicates P < 0.05.

[0038] Figure 8 Results of morphological detection of Kupffer cells in the liver of mice after IPA gavage, wherein A is the morphology under a microscope; B is the morphology after three-dimensional reconstruction; C is the volume of KC cells; and D is the surface area of KC cells. **** indicates P < 0.0001.

[0039] Figure 9 The results of liver and circulating blood bacterial load detection after 2 hours and 24 hours of blood-borne infection after intragastric administration of IPA, wherein A and B are liver and circulating blood bacterial load after 2 hours of blood-borne infection, respectively; C and D are liver and circulating blood bacterial load after 24 hours of blood-borne infection, respectively. * indicates P < 0.05. DETAILED DESCRIPTION

[0040] The application will be further described below in conjunction with specific examples. The following examples are only used to illustrate the application and are not used to limit the application. Modifications or replacements of the method, steps or conditions of the application, without departing from the spirit and essence of the application, all belong to the scope of the application.

[0041] The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0042] Hamp1 - / - The mice are hepcidin gene knockout mice, see Yang Y, Zeng C, Yang S, Zhang Y, Song S, Liu S, Shu Q, Fang X, Chen Q. Airway Epithelial Hepcidin Coordinates Lung Macrophages and Immunity Against Bacterial Pneumonia. Shock. 2020 Sep; 54(3): 402-412.; the WT mice are Hamp1 - / - The mice are littermates. E. coli carrying green fluorescent protein GFP, E. coli are purchased from ATCC, USA. Indole-3-propionic acid (IPA) is purchased from Aladdin, China.

[0043] Example 1: In vivo imaging experiment of liver Kupffer cells capturing bacteria after blood-borne infection

[0044] 1. Preparation of E. coli blood-borne infection model

[0045] For the in vivo imaging experiment of liver Kupffer cells capturing bacteria, Hamp1 - / - The mice and WT mice were intravenously injected with 5.5 x 10 7 The E. coli carrying green fluorescent protein GFP was prepared into a blood-borne infection model at a CFU dose.

[0046] 2. Liver in vivo imaging detection

[0047] At 2 hours post blood-borne infection model, in vivo imaging of the liver was performed as follows:

[0048] Kupffer cells (KCs) were stained in vivo by intravenous injection of Brilliant Violet 421-conjugated anti-F4 / 80 antibody 10 minutes before surgery. For in vivo imaging of the liver, a midline incision from the pubic bone to the xiphoid process of the abdomen was made in mice, and the skin and abdominal muscle tissue were excised to expose the liver. The xiphoid process of the mouse was fixed with a tied suture to release the falciform ligament. The experimental mice were placed in a right lateral position, and the right lobe of the liver was gently exposed on a coverslip. A small piece of a pre-wetted wipe with 1x PBS was then gently placed over the liver lobe to eliminate liver movement caused by respiration and to obtain a relatively flat area for imaging. The liver was then imaged on a Nikon A1R confocal microscope using a 20x objective. Image analysis was performed using ImageJ (NIH) and Imaris (Olympus) software. The bacterial capture amount of KCs after E. coli GFP infection was quantified as the number of bacterial particles captured by individual KCs per field of view (FOV).

[0049] 3. Results analysis

[0050] As shown in Figure 1 compared with WT mice, Hamp1 - / - mice showed a significant decrease in the ability of liver Kupffer cells to capture cells.

[0051] Example 2: Bacterial load detection after blood-borne infection

[0052] 1. Preparation of E. coli blood-borne infection model

[0053] For bacterial load and mortality after infection, Hamp1 - / - mice and WT mice were prepared for blood-borne infection model by intravenous injection of 4.5x10 7 CFU of E. coli.

[0054] 2. Bacterial load detection and mortality statistics of mice

[0055] Bacterial load detection: Mice were sacrificed at 2 hours after blood-borne infection model. Peripheral blood was collected in an EDTA anticoagulant tube, and liver tissue was weighed and homogenized in pre-cooled sterile phosphate buffered saline (PBS). After serial dilution of tissue homogenate and blood samples by 10-fold gradient, inoculate Luria-Bertani agar plates, and count individual colonies after 12 to 16 hours of culture at 37°C. The number of colonies was calculated as CFU per milliliter of blood or CFU per gram of tissue.

[0056] Mortality statistics: The mice were continuously observed for death after the mouse model, and a total of 7 days were observed. The mortality of mice was statistically analyzed by Kaplan-Meier log-rank test.

[0057] 3. Result analysis

[0058] As shown in Figure 2 , compared with WT mice, Hamp1 - / - mice had significantly increased bacterial load in circulating blood (A) and significantly increased mortality (B). Figure 2 Figure 2

[0059] Example 3: Morphological detection of liver Kupffer cells and IPA detection of portal vein blood of Hamp1

[0060] 1. Morphological detection of liver Kupffer cells

[0061] Hamp1 - / - mice and WT mice were intravenously injected with Brilliant Violet 421 conjugated anti-F4 / 80 antibody to stain Kupffer cells (KC) in vivo. Then the liver was detected by in vivo imaging, and the method was the same as in Example 1. The cell volume and surface area of KC were measured by three-dimensional measurement using Imaris software for three-dimensional reconstruction.

[0062] 2. IPA detection of portal vein blood

[0063] The portal vein blood of Hamp1 - / - mice and WT mice was taken respectively for IPA concentration detection. The ELISA kit of Enzyme-Linked Biotechnology Co., Ltd. was used, and the operation was carried out according to the instructions provided by the kit.

[0064] 3. Result analysis

[0065] As shown in Figure 3 , compared with WT mice, Hamp1 - / - mice had significantly reduced liver Kupffer cell volume and surface area.

[0066] As shown in Figure 4 , compared with WT mice, Hamp1 - / - mice had significantly reduced concentration of tryptophan metabolite indole-3-propionic acid (IPA) in portal vein blood.

[0067] The above results suggest that IPA may be closely related to the occurrence and development of blood-borne infection in Hamp1 - / - mice, and is a potential therapeutic target for Hamp1 - / - mice to resist blood-borne infection.

[0068] ​​Example 3: Effect of indole-3-propionic acid (IPA) on KC morphology

[0069] 1. Isolation and culture of Kupffer cells (KC)

[0070] From Hamp1 - / - Non-parenchymal cells of liver were isolated from Hamp1 - / - mice, and flow cytometry sorting was performed to sort KCs among live CD45+F4 / 80+ cells using Beckman MoFlo-Astrios EQ flow cytometer. The sorted KCs were washed with sterile PBS and resuspended in DMEM containing 10% FBS and 1% penicillin, streptomycin.

[0071] 2. Indole-3-propionic acid (IPA) treatment

[0072] To evaluate the effect of indole-3-propionic acid (IPA) on KC morphology, KCs were stimulated with 100 mM IPA or sterile PBS. After 24 hours of IPA treatment, cells were stained with Brilliant Violet 421-conjugated anti-F4 / 80 antibody, and then cell morphology was photographed under an Olympus FV3000 microscope, and three-dimensional reconstruction of KC volume and surface area was performed using Imaris software.

[0073] 3. Results analysis

[0074] As shown in Figure 5 , compared with the PBS control group, the volume and surface area of KCs were significantly increased after IPA treatment.

[0075] Example 4: Detection of IPA concentration in portal vein blood after IPA gavage treatment

[0076] 1. Preparation and gavage of IPA

[0077] The purified IPA stock solution was diluted in sterile PBS to a final concentration of 5 mg / mL. The IPA was gavaged to Hamp1 - / - mice at a dose of 50 mg / kg once a day for 7 consecutive days. The control group was gavaged with an equal volume of sterile PBS. After 7 days, the portal vein blood of the mice was collected. The concentration of IPA in the portal vein blood was detected by ELISA.

[0078] 2. Results analysis

[0079] As shown in Figure 6 , the IPA level in the portal vein blood of mice was significantly increased after 7 days of IPA gavage.

[0080] Example 5: Effect of IPA gavage treatment on the ability of liver Kupffer cells to capture bacteria

[0081] 1. Hamp1 - / - Mice were pretreated with IPA gavage, as in Example 4.

[0082] 2. Hamp1 - / - Mice were injected intravenously with E. coli to simulate blood-borne infection, as in Example 1.

[0083] 3. Liver bioluminescence imaging was performed at 0.5 hour after blood-borne infection, as in Example 1.

[0084] 4. Results analysis

[0085] As shown in Figure 7 , the bacteria-trapping ability of KCs in mice was significantly enhanced after IPA gavage.

[0086] Example 6: Effect of IPA gavage treatment on KC morphology

[0087] 1. Hamp1 - / - Mice were pretreated with IPA gavage, as in Example 4.

[0088] 2. Hamp1 - / - Mice were injected intravenously with E. coli to simulate blood-borne infection, as in Example 1.

[0089] 3. At 0.5 hour after blood-borne infection, the KCs were stained in vivo by intravenous injection of Brilliant Violet 421-conjugated anti-F4 / 80 antibody, and liver bioluminescence imaging was performed, as in Example 1. The volume and surface area of KCs were measured by three-dimensional measurement using Imaris software for three-dimensional reconstruction.

[0090] 4. Results analysis

[0091] As shown in Figure 8 , the volume and surface area of KCs in the livers of Hamp1 - / - mice were significantly increased after IPA gavage.

[0092] Example 7: Effect of IPA gavage treatment on bacterial load in the livers and circulating blood of infected mice

[0093] 1. Hamp1 - / - Mice were pretreated with IPA gavage, as in Example 4.

[0094] 2. Hamp1 - / - Mice were injected intravenously with E. coli to simulate blood-borne infection, as in Example 2.

[0095] 3. Mice were sacrificed 2 hours and 24 hours after the bloodstream infection model was established. Peripheral blood was collected in EDTA anticoagulant tubes, and liver tissue was weighed and homogenized in pre-cooled sterile phosphate-buffered saline (PBS). Tissue homogenates and blood samples were serially diluted 10-fold and inoculated onto Luria–Bertani agar plates. After incubation at 37°C for 12 to 16 hours, individual colonies were counted. Colony counts were calculated as CFU / mL blood or CFU / g tissue.

[0096] 4. Results Analysis

[0097] like Figure 9 As shown, Hamp1 after IPA gavage - / - Two hours after bloodborne infection in mice, the bacterial load in the liver increases. Figure 9 A) Reduced bacterial load in circulating blood ( Figure 9 B); Liver bacterial load 24 hours later ( Figure 9 C) and circulating blood bacterial load ( Figure 9 D) All decreased.

Claims

1. Use of indole-3-propionic acid for the preparation of a medicament for the prevention or treatment of a blood-borne infection, characterized in that, The blood-borne infection is severe hepatitis or cirrhosis with blood-borne infection, the manifestations of which include low levels of hepcidin.

2. Use according to claim 1, wherein The indole-3-propionic acid prevents or treats blood-borne infection by enhancing the ability of liver Kupffer cells to capture pathogenic bacteria.

3. Use according to claim 1 or 2, characterized in that, The pathogen of the blood-borne infection includes Escherichia coli.

4. Use according to claim 1 or 2, characterized in that, The preparation form of the drug is oral preparation.

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