Application of indole-3-propionic acid in preparation of medicine for preventing or treating hematogenous infection

Abstract

The invention discloses application of indole-3-propionic acid in preparation of a medicine for preventing or treating hematogenous infection, and belongs to the technical field of biological medicine. The invention discloses the application of indole-3-propionic acid in resisting blood-borne infection for the first time, the capability of capturing blood-borne pathogenic bacteria by liver Kupu cells can be remarkably enhanced by giving indole-3-propionic acid, and the load of pathogenic bacteria in circulating blood is reduced. The invention provides a new thought for treatment of hematogenous infection, especially anti-infection treatment of susceptible people with severe hepatitis or liver cirrhosis and the like. Indole-3-propionic acid is low in price and easy to obtain, can be produced in a large scale, is favorable for being developed into a medicine for treating hematogenous infection, and has a good application value.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of indole-3-propionic acid in preparing medicines for preventing or treating blood-borne infections. Background Art

[0002] Bloodstream infection (BSI) is the leading cause of morbidity and mortality from infectious diseases worldwide. It can be a primary infection caused by surgical intervention, use of intravenous catheters, etc., or it can be a secondary complication caused by other diseases such as cirrhosis. The excessive growth and spread of invading pathogens in the blood circulation can aggravate damage to distal vital organs and often lead to more serious diseases such as infective endocarditis, meningitis, sepsis or septic shock. The risk of death from BSI is 7%-35%, accounting for one-third of deaths from bacterial infections. Although great progress has been made in the understanding of the pathophysiological mechanisms of BSI and clinical intensive care treatment in recent years, how to quickly and effectively eliminate bacteria in the circulation is still critical to effectively prevent and treat the fatal consequences of BSI.

[0003] Currently, the clinical prevention and treatment of blood-borne infections mainly relies on the empirical use of broad-spectrum antibiotics. However, long-term use of antibiotics leads to the problem of drug resistance, so there is a need to develop alternative therapies to traditional antibiotics.

[0004] Kupffer cells, as macrophages resident in the liver, form a unique immune defense barrier in the vascular space of the hepatic sinusoids. Kupffer cells can not only capture and kill invading bacteria themselves, but also work synergistically with other immune cells to resist pathogen infection, thus playing a vital role in clearing blood-borne bacteria and preventing pathogens from spreading throughout the body.

[0005] Hepcidin is an acute phase protein that regulates the body's iron metabolism, mainly produced by hepatocytes. Hepcidin regulates the iron concentration in the extracellular fluid by binding to the ferroportin (the only known cellular iron transport protein) on the cell membrane and inducing its degradation. Hepcidin plays a vital role in host defense against bacterial infection. It not only has direct bactericidal activity at non-physiological high concentrations in vitro, but also forms hypoferremia by regulating the intracellular / extracellular iron status, depriving pathogenic microorganisms of the iron necessary for growth, thereby controlling or even killing bacteria. Low levels of hepcidin are common in patients with severe hepatitis and cirrhosis, who are more susceptible to severe blood-borne infections that are life-threatening. At the same time, animal experiments have found that in a peritonitis mouse model, inhibition of liver hepcidin can lead to systemic dissemination of bacteria. These studies indicate the importance of hepcidin in preventing systemic bacterial infection. However, the mechanism by which hepcidn protects the body against systemic bacterial dissemination remains unclear.

[0006] The research group used hepcidin gene knockout (Hamp1 - / - ) mice and wild-type mice (WT) were infected with blood-borne bacteria and found that compared with WT mice, Hamp1 - / - The ability of Kupffer cells in mouse liver to capture cells was significantly reduced, the bacterial load in the circulating blood was significantly increased, and the mortality rate was significantly increased. So far, no research has reported that Hepcidin is related to the ability of Kupffer cells to capture bacteria. Summary of the invention

[0007] The purpose of the present invention is to provide an active substance that can enhance the ability of Kupffer cells to capture pathogens, which is used to develop drugs for preventing or treating blood-borne infections, especially for anti-infection treatment of susceptible populations such as severe hepatitis or cirrhosis.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] The present invention provides application of indole-3-propionic acid (IPA) in preparing medicine for preventing or treating blood-borne infection.

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

[0011] In the present study, the animals were treated with indole-3-propionic acid by oral gavage and then infected with blood-borne pathogens. It was found that compared with the sterile PBS control group, the ability of liver Kupffer cells in the hepcidin gene knockout animal model treated with indole-3-propionic acid to capture pathogens was significantly enhanced, the pathogen load in the circulating blood was reduced, and the prognosis was significantly improved.

[0012] Indole-3-propionic acid is a metabolite of tryptophan, exists naturally in animals, and has high biological safety. Therefore, indole-3-propionic acid can be used to develop and prepare drugs for preventing blood-borne infections.

[0013] Furthermore, the blood-borne infection includes hepcidin gene function deficiency or low level associated with blood-borne infection. Patients with hepcidin gene deficiency or low level of hepcidin are more likely to develop severe blood-borne infection, and administration of indole-3-propionic acid can effectively resist blood-borne infection.

[0014] Furthermore, the blood-borne infection includes severe hepatitis or liver cirrhosis with blood-borne infection. The manifestations of severe hepatitis or liver cirrhosis include low levels of hepcidin.

[0015] Furthermore, the blood-borne infection includes a decrease in the ability of liver Kupffer cells to capture pathogens accompanied by blood-borne infection. Patients with decreased ability of liver Kupffer cells to capture pathogens are more susceptible to blood-borne infection, and administration of indole-3-propionic acid can effectively resist blood-borne infection.

[0016] The research mechanism of the present invention shows that indole-3-propionic acid prevents or treats blood-borne infection by enhancing the ability of liver Kupffer cells to capture pathogens.

[0017] Furthermore, the pathogen of the blood-borne infection may be, but is not limited to, bacteria, including Escherichia coli.

[0018] The drug of the present invention is prepared by adding indole-3-propionic acid as the main active ingredient and a pharmaceutically acceptable carrier, 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, the age and the purpose of administration.

[0019] Furthermore, the drug may be in the form of an oral preparation but is not limited thereto.

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

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

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

[0023] Terminology Note:

[0024] The term "comprising" is an open expression, that is, it includes the contents specified in the present invention but does not exclude other contents.

[0025] The term "treating" refers in some embodiments to ameliorating a disease or condition (i.e., slowing or preventing or alleviating the development of a disease or at least one clinical symptom thereof). In other embodiments, it refers to alleviating or improving at least one physical parameter, including physical parameters that may not be perceived by the subject. In other embodiments, it refers to regulating a disease or condition physically (e.g., stabilizing a perceptible symptom) or physiologically (e.g., stabilizing a physical parameter), or both. In other embodiments, it refers to preventing or delaying the onset, onset, or exacerbation of a disease or condition.

[0026] The term "effective dose" refers to an amount of a compound that is sufficient to be effective in treating a disease when administered to a subject. The effective dose may vary with the severity of the disease, and the physical condition, age, weight, sex, etc. of the subject to be treated.

[0027] The term "pharmaceutically acceptable carrier" refers to any preparation or carrier medium that can deliver an effective dose of the active substance of the present invention without interfering with the biological activity of the active substance and having no toxic side effects on the host or subject.

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

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

[0030] (2) Indole-3-propionic acid is cheap and readily available and can be produced in large quantities, which is conducive to the development of a drug for the treatment of blood-borne infections and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0032] Figure 2 For Hamp1 - / - Results of bacterial load detection and mortality in circulating blood of mice infected with hematogenous E. coli, where A is bacterial load and B is mortality. * indicates P < 0.05.

[0033] Figure 3 For Hamp1 - / - Results of morphological detection of mouse liver KC, where A is the morphology under the microscope; B is the morphology after three-dimensional reconstruction; C is the volume of KC cells; D is the surface area of ​​KC cells. **** indicates P < 0.0001.

[0034] Figure 4 For Hamp1 - / - IPA concentration in mouse portal vein blood. ** indicates P < 0.01.

[0035] Figure 5 The morphological test results of KC after IPA treatment, where A is the morphology under the microscope; B is the morphology after three-dimensional reconstruction; C is the volume of KC cells; D is the surface area of ​​KC cells. **** indicates P < 0.0001.

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

[0037] Figure 7 The results of in vivo imaging of the liver of mice after IPA gavage to detect the bacterial capture ability of Kupffer cells, where A is the bacterial capture image under a living microscope; B is the quantitative analysis of the bacterial capture ability of KC. * indicates P < 0.05.

[0038] Figure 8 The results of morphological examination of KC in mouse liver after oral administration of IPA, where A is the morphology under the 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] Fig. 9 Results of bacterial load detection in the liver and circulating blood of mice 2 hours and 24 hours after hematogenous infection after oral administration of IPA, where A and B are the bacterial loads in the liver and circulating blood 2 hours after hematogenous infection, respectively; C and D are the bacterial loads in the liver and circulating blood 24 hours after hematogenous infection, respectively. * indicates P < 0.05. DETAILED DESCRIPTION

[0040] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0042] Hamp1 - / - The mice were hepcidin 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.; WT mice were Hamp1 - / - The mice were littermates of mice. Escherichia coli and Escherichia coli carrying green fluorescent protein GFP were purchased from ATCC, USA. Indole-3-propionic acid (IPA) was purchased from Aladdin Company, China.

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

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

[0045] For live imaging of bacteria captured by liver Kupffer cells, Hamp1 - / - Mice and WT mice were intravenously injected with 5.5×10 7 The blood-borne infection model was prepared by adding CFU dose of Escherichia coli carrying green fluorescent protein GFP.

[0046] 2. Liver in vivo imaging

[0047] Two hours after the blood-borne infection model, liver in vivo imaging was performed as follows:

[0048] Kupffer cells (KC) 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, the mouse abdomen was incised in the midline from the pubis to the xiphoid process, and the skin and abdominal musculature were removed to expose the liver. The xiphoid process was fixed with knotted sutures to release the falciform ligament. The experimental mouse was placed in the right lateral position, and the right lobe of the liver was gently exposed on a coverslip. A small piece of wipes pre-moistened with 1× PBS was then gently covered with the liver lobe to eliminate liver movement caused by breathing and to obtain a relatively flat area for examination. The liver was then imaged on a Nikon A1R confocal microscope using a 20× objective. Image analysis was performed using ImageJ (NIH) and Imaris (Olympus) software. Bacterial capture by KCs after E. coli GFP infection was quantified as bacterial particles captured by a single KC per field of view (FOV).

[0049] 3. Results Analysis

[0050] like Figure 1 As shown, compared with WT mice, Hamp1 - / - The ability of Kupffer cells in mouse liver to capture cells was significantly reduced.

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

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

[0053] For bacterial load and mortality after infection, Hamp1 - / - Mice and WT mice were intravenously injected with 4.5×10 7 The blood-borne infection model was prepared by using CFU of Escherichia coli.

[0054] 2. Mouse bacterial load detection and mortality statistics

[0055] Bacterial load detection: Mice were sacrificed 2 hours after the blood-borne infection model. Peripheral blood was collected in EDTA anticoagulation tubes, and liver tissue was weighed and homogenized in pre-cooled sterile phosphate buffered saline (PBS). Tissue homogenates and blood samples were serially diluted in 10-fold gradients and inoculated on Luria–Bertani agar plates. After incubation at 37°C for 12 to 16 hours, single colonies were counted. The number of colonies was calculated as CFU / ml blood or CFU / g tissue.

[0056] Mortality statistics: The mortality of mice was observed continuously for 7 days after the model was established. The mortality of mice was analyzed by Kaplan–Meier log-rank test.

[0057] 3. Results Analysis

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

[0059] Example 3: Morphological detection of liver Kupffer cells without blood-borne infection and IPA detection of portal vein blood

[0060] 1. Detection of liver Kupffer cell morphology

[0061] Hamp1 - / - Mice and WT mice were intravenously injected with Brilliant Violet 421-coupled anti-F4 / 80 antibody to perform in vivo staining of Kupffer cells (KC). The liver was then subjected to in vivo imaging detection using the same method as in Example 1. Imaris software was used to perform three-dimensional reconstruction by three-dimensionally measuring the cell volume and surface area of ​​KC.

[0062] 2. Portal vein blood IPA test

[0063] Take Hamp1 respectively - / - The IPA concentration in the portal vein blood of mice and WT mice was detected using the ELISA kit of ELISA Biological Company according to the instructions provided by the kit.

[0064] 3. Results Analysis

[0065] like Figure 3 As shown, compared with WT mice, Hamp1 - / - The volume and surface area of ​​Kupffer cells in mouse liver were significantly reduced.

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

[0067] The above results suggest that IPA may be related to Hamp1 - / - The occurrence and development of blood-borne infection in mice is closely related to Hamp1 - / - Potential therapeutic targets for resistance to blood-borne infections in mice.

[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 the liver were isolated from mice and sorted by flow cytometry. KCs were sorted from live CD45+F4 / 80+ cells using a Beckman MoFlo-Astrios EQ flow cytometer. The sorted KCs were washed with sterile PBS and resuspended in DMEM containing 10% FBS and 1% penicillin and 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 μM IPA or sterile PBS. After 24 h 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 3D reconstruction was performed by 3D measurement of KC volume and surface area using Imaris software.

[0073] 3. Results Analysis

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

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

[0076] 1. Preparation and intragastric administration of IPA

[0077] The purified IPA stock solution was diluted in sterile PBS to a final concentration of 5 mg / mL. IPA was administered to Hamp1 at a dose of 50 mg / kg. - / - The mice were gavaged 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] like Figure 6 As shown, the IPA level in the portal vein blood of mice was significantly increased 7 days after oral administration of IPA.

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

[0081] 1. Hamp1 - / - Mice were pretreated by intragastric administration of IPA in the same manner as in Example 4.

[0082] 2. Preprocessed Hamp1 - / - Mice were intravenously injected with Escherichia coli (E. coli) to simulate blood-borne infection, using the same method as in Example 1.

[0083] 3. 0.5 hours after blood-borne infection, liver in vivo imaging detection was performed using the same method as in Example 1.

[0084] 4. Results Analysis

[0085] like Figure 7 As shown, the bacteriostasis-capturing ability of Kupffer cells in mice was significantly enhanced after oral administration of IPA.

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

[0087] 1. Hamp1 - / - Mice were pretreated by intragastric administration of IPA in the same manner as in Example 4.

[0088] 2. Preprocessed Hamp1 - / - Mice were intravenously injected with Escherichia coli (E. coli) to simulate blood-borne infection, using the same method as in Example 1.

[0089] 3. 0.5 hours after hematogenous infection, Kupffer cells (KC) were stained in vivo by intravenous injection of Brilliant Violet 421-coupled anti-F4 / 80 antibody, and the liver was imaged in vivo using the same method as in Example 1. The volume and surface area of ​​KC were measured in three dimensions using Imaris software for three-dimensional reconstruction.

[0090] 4. Results Analysis

[0091] like Figure 8 As shown, Hamp1 - / - The volume and surface area of ​​KC cells in mouse liver increased significantly.

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

[0093] 1. Hamp1 - / - Mice were pretreated by intragastric administration of IPA in the same manner as in Example 4.

[0094] 2. Preprocessed Hamp1 - / - Mice were intravenously injected with Escherichia coli (E. coli) to simulate blood-borne infection, using the same method as in Example 2.

[0095] 3. Mice were killed at 2 hours and 24 hours after the blood-borne infection model. Peripheral blood was collected in EDTA anticoagulation tubes, and liver tissue was weighed and homogenized in pre-cooled sterile phosphate buffered saline (PBS). Tissue homogenates and blood samples were diluted in a 10-fold gradient and inoculated onto Luria–Bertani agar plates. After incubation at 37°C for 12 to 16 hours, single colonies were counted. The number of colonies was calculated as CFU / ml blood or CFU / g tissue.

[0096] 4. Results Analysis

[0097] like Fig. 9 As shown, Hamp1 - / - The bacterial load in the liver of mice increased 2 hours after hematogenous infection ( Fig. 9 A), decreased circulating blood bacterial load ( Fig. 9 B); 24 hours later, the bacterial load in the liver ( Fig. 9 C) and circulating blood bacterial load ( Fig. 9 D) both decreased.

Claims

1. Use of indole-3-propionic acid in the preparation of drugs for preventing or treating blood-borne infections.

2. The use according to claim 1, characterized in that The blood-borne infection includes hepcidin gene function deficiency or low level associated with blood-borne infection.

3. The use according to claim 2, characterized in that The blood-borne infection includes severe hepatitis or cirrhosis associated with blood-borne infection.

4. The use according to claim 1, characterized in that The blood-borne infection includes blood-borne infection accompanied by decreased ability of liver Kupffer cells to capture pathogens.

5. The use according to claim 1, characterized in that The indole-3-propionic acid prevents or treats blood-borne infection by enhancing the ability of liver Kupffer cells to capture pathogens.

6. The use according to any one of claims 1 to 5, characterized in that: The pathogens of the blood-borne infection include Escherichia coli.

7. The use according to any one of claims 1 to 5, characterized in that: The preparation form of the drug is an oral preparation.

8. A pharmaceutical composition for preventing blood-borne infection, characterized in that: The pharmaceutical composition comprises an effective amount of indole-3-propionic acid.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

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