Application of L-citrulline in preparation of medicine for preventing or treating tuberculosis
L-citrulline significantly reduces bacterial load and prolongs survival time in the treatment of tuberculosis, solving the drug resistance problem in existing technologies, providing an innovative strategy based on host metabolic regulation, enhancing host immune defense, and shortening the treatment course.
Patent Information
- Application Number
- CN202510966086.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-14
- Publication Date
- 2025-09-16
AI Technical Summary
Existing tuberculosis treatment regimens rely on antibiotics, which have drug resistance problems and lack innovative strategies based on host metabolic regulation. How to use endogenous metabolites to regulate host immune defense to reduce bacterial load has become the key to breaking through the drug resistance dilemma.
L-citrulline is used as an endogenous amino acid metabolite, and its significant effect in anti-tuberculosis treatment is verified through a zebrafish-marimolecular mycobacterium infection model, which significantly reduces bacterial load and prolongs survival time. The drug form includes solution, injection or oral preparation, which may contain a pharmaceutically acceptable carrier.
L-citrulline significantly reduces the bacterial load of Mycobacterium marinum, prolongs the survival time of zebrafish, exerts anti-inflammatory effects, and provides a new metabolite intervention strategy for anti-tuberculosis treatment, enhances host immune defense, reduces the survival and spread of Mycobacterium tuberculosis, and shortens the course of treatment.
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Figure CN120643547A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of L-citrulline in preparing a medicine for preventing or treating tuberculosis. Background Art
[0002] Tuberculosis, a chronic infectious disease caused by Mycobacterium tuberculosis, remains a major challenge in global public health. According to the World Health Organization (WHO), approximately 25% of the world's population is latently infected with Mycobacterium tuberculosis, with over 10 million new cases each year. The spread of multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains has further exacerbated the difficulty of prevention and treatment. Traditional anti-tuberculosis treatment relies on the combination of antibiotics such as isoniazid and rifampicin, but the 6-9 month course of treatment leads to low patient compliance. At the same time, pathogens develop drug resistance by evading immune surveillance and colonizing in macrophages, significantly reducing the bactericidal efficiency of existing drugs.
[0003] In recent years, the role of metabolic small molecules in host immune regulation and pathogen inhibition has gradually become a research hotspot. For example, vitamin D and its metabolites can enhance anti-tuberculosis immunity by regulating macrophage function, but research on endogenous amino acid metabolites is still relatively limited. Citrulline, as a key intermediate in the arginine-ornithine metabolic pathway, has been shown to have the potential to regulate immune cell function in inflammatory diseases, but its role in Mycobacterium tuberculosis infection has not been reported. In addition, the zebrafish-marimyocobacterium infection model has become an important tool for anti-tuberculosis drug screening because it has a high degree of homology with human tuberculosis in immune response and pathological characteristics (genomic homology is about 87%), and has the advantages of transparent early development and short experimental cycle. However, research on the development of new metabolite drugs based on this model is still in the exploratory stage.
[0004] Currently, treatment options for Mycobacterium tuberculosis infection are still primarily antibiotic-based, lacking innovative strategies based on host metabolic regulation. Utilizing endogenous metabolites to modulate host immune defenses and reduce bacterial loads has become a key approach to overcoming the drug resistance dilemma. Summary of the Invention
[0005] The present invention aims to provide the use of L-citrulline in the preparation of a drug for preventing or treating tuberculosis. The present invention found that L-citrulline can significantly reduce the bacterial load after Mycobacterium marinum infection and prolong the survival time of zebrafish, and has the prospect of being developed into a drug related to the prevention and treatment of tuberculosis.
[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:
[0007] Application of L-citrulline (Citrulline) in preparing medicine for preventing or treating tuberculosis.
[0008] Preferably, the medicament comprises an effective dose of L-citrulline.
[0009] Preferably, the drug further comprises a pharmaceutically acceptable carrier.
[0010] Preferably, the pharmaceutically acceptable carrier is one or more of a solvent, a dispersant, a suspending aid, a surfactant, a thickener, a preservative, a solid binder or a lubricant.
[0011] Preferably, the dosage form of the drug is a solution, injection or oral preparation.
[0012] The present invention also provides a medicine for preventing or treating tuberculosis, which comprises L-citrulline.
[0013] Preferably, the drug further comprises a pharmaceutically acceptable carrier.
[0014] Preferably, the pharmaceutically acceptable carrier is one or more of a solvent, a dispersant, a suspending aid, a surfactant, a thickener, a preservative, a solid binder or a lubricant.
[0015] Compared with the prior art, the present invention has the following advantages and technical effects:
[0016] The present invention validates the significant technical effect of L-citrulline in anti-tuberculosis treatment using a zebrafish-Mycobacterium marinum infection model. After five days of continuous treatment with 250-500 mM L-citrulline, the bacterial load of Mycobacterium marinum in infected zebrafish was significantly reduced compared to the control group, and colony-forming unit (CFU) detection showed that bacterial proliferation was significantly inhibited. Furthermore, the average survival time of zebrafish in the L-citrulline-treated group was significantly prolonged compared to the control group, with the survival curve showing statistically significant differences. Further mechanistic studies have shown that Cit treatment reduces inflammatory cytokine levels, exerting an anti-inflammatory effect.
[0017] This technical result demonstrates for the first time that L-citrulline can reduce the pathogen load in the host and prolong the survival of the infection model, providing a novel metabolite intervention strategy for anti-tuberculosis treatment. Compared with traditional antibiotic treatment, L-citrulline, as an endogenous amino acid metabolite, has potential advantages in host immune regulation.
[0018] The present invention can anticipate that L-citrulline can play a positive role in the prevention and treatment of tuberculosis, can effectively enhance the host immune defense, reduce the survival and spread of tuberculosis bacteria in the body, is expected to shorten the course of tuberculosis treatment, improve the treatment effect, and provide a new reference and basis for clinical treatment.
[0019] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 The results of the effect of L-citrulline on the bacterial load of zebrafish in Example 1 are as follows;
[0021] Figure 2 The results of the effect of L-citrulline on the survival time of zebrafish in Example 2 are as follows;
[0022] Figure 3 The statistical results of the mRNA expression levels of TNF-α, IL-6, IL-1β, and IL-10 in zebrafish in the control group and the L-citrulline treatment group of Example 1 are shown, wherein: Figure 3 a in the table is the statistical result of TNF-α mRNA expression level. Figure 3 b in the figure is the statistical result of IL-6 mRNA expression level. Figure 3 c in the figure is the statistical result of IL-1β mRNA expression level. Figure 3 The d in the figure is the statistical result of IL-10 mRNA expression level. DETAILED DESCRIPTION
[0023] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.
[0024] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.
[0025] Source of test materials:
[0026] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in this field and can be purchased through commercial channels.
[0027] Example 1
[0028] Effects of L-citrulline on bacterial load in zebrafish
[0029] AB type wild zebrafish fertilized eggs were selected and incubated at 28.5°C in embryo culture medium containing 0.5 mg / L methylene blue. Three days after fertilization, 250 CFU of Mycobacterium marinum bacterial solution were injected to establish an infection model. After injection, the zebrafish were divided into a control group and an L-citrulline treatment group. In the L-citrulline treatment group, zebrafish were placed in culture solutions containing 100 μM, 250 μM, and 500 μmL L-citrulline, respectively. The solution was changed every day and the culture was continued for 5 days. The control group was placed in a culture solution without L-citrulline, and the culture solution was changed every day and the culture was continued for 5 days. The bacterial load in zebrafish was detected using the CFU method, and the results are as follows. Figure 1 .
[0030] Depend on Figure 1 It can be seen that compared with the control group, the bacterial load in the L-citrulline treatment groups (250 μm and 500 μm) was significantly reduced (P<0.001, P<0.01).
[0031] Example 2
[0032] Effects of L-citrulline on the survival time of zebrafish
[0033] AB type wild zebrafish fertilized eggs were selected and incubated at 28.5°C in embryo culture medium containing 0.5 mg / L methylene blue. Three days after fertilization, 250 CFU of Mycobacterium marinum bacterial solution were injected to establish an infection model. After injection, the zebrafish were divided into a control group and an L-citrulline treatment group. In the L-citrulline treatment group, zebrafish were placed in culture solutions containing 100 μM, 250 μM, and 500 μM L-citrulline, respectively, and the solution was replaced every day. The control group was placed in a culture solution without L-citrulline, and the culture solution was replaced every day. The survival time of the zebrafish was recorded and a survival curve was drawn. The results are shown in Figure 2. Figure 2 .
[0034] Depend on Figure 2 It can be seen that the average survival time of zebrafish in the L-citrulline treatment group was significantly prolonged, which was statistically significant compared with the control group (P<0.05).
[0035] The effect was verified through the following tests:
[0036] On the fifth day, 500 μM samples from the L-citrulline-treated and control groups described in Example 1 were collected and assayed for mRNA expression levels of TNF-α, IL-6, IL-1β, and IL-10. Twenty fish were included in each group, with three replicates for each L-citrulline-treated and control groups. The collected zebrafish larvae samples were placed in a 1.5 mL enzyme-free centrifuge tube, gently centrifuged, and the water removed by aspiration. 1 mL of Trizol was then added.
[0037] RNA extraction
[0038] (1) Precool the centrifuge to 4°C, centrifuge the phase lock gel tube for 1 minute, transfer the solution to a phase lock gel tube, add 400 μL of chloroform, shake and mix until milky white, and let it stand for 2 minutes. Centrifuge at 13000 rpm at 4°C for 15 minutes. The solution will separate into three layers.
[0039] (2) Transfer the supernatant (about 500 μL) to a new EP tube, add 450 μL of isopropanol, mix thoroughly, let stand at room temperature for 30 min (or -80°C for 10 min), and centrifuge at 13,000 rpm at 4°C for 40 min;
[0040] (3) Cleaning the RNA precipitate: Add 1 mL of anhydrous ethanol to the precipitate and centrifuge at 13,000 rpm at 4°C for 5 min. The precipitate is visible to the naked eye. During centrifugation, pay attention to the orientation of the EP tube, with the open side facing inward and the precipitate on the outside. Aspirate the liquid from the inside to avoid touching the precipitate.
[0041] (4) Secondary washing: Add 300 μL of 70% ethanol (prepared with DEPC water) to the precipitate and centrifuge at 13,000 rpm at 4°C for 5 min. Repeat this step once to completely remove residual salt.
[0042] (5) Carefully aspirate the liquid portion using a pipette, place the EP tube in a clean bench with the lid open and air dry for 5-10 minutes. Be careful to avoid over-drying, which may cause RNA to become difficult to dissolve. Add 20-30 μL of DEPC water according to the amount of precipitation and pipette repeatedly to ensure that the RNA is completely dissolved.
[0043] (6) RNA purity assessment: Pipette 1 μL of sample and use DEPC water as a blank control. Record the absorbance values of A260, A280, and A230. Calculate the A260 / A280 ratio (ideal range 1.8-2.0) and the A260 / A230 ratio (ideal range >2.0).
[0044] Reverse transcription
[0045] (1) Sample dilution: Calculate the required amount of DEPC water based on the initial RNA concentration and dilute the RNA sample to 800-1000 ng / μL;
[0046] (2) Removal of genomic DNA: Prepare the following mixture (Premix) in an RNase-free centrifuge tube: 4 μL of 4×gDNAwiper and 16 μL of RNase-free HO. Gently pipette to mix and incubate at 42°C for 2 min.
[0047] (3) Prepare the reverse transcription reaction system: 5×HiScriptⅡqRT Super Mix 4μL, Premix 16μL, and mix thoroughly by gently pipetting. Set the program to 37℃ for 15min, 85℃ for 5s, and store at 4℃.
[0048] qPCR amplification
[0049] Prepare a 10 μL reaction system according to the table below. Set up three technical replicates for each target gene and reference gene (ef1α) for each sample. Before the experiment, equilibrate all reagents (SYBR Green premix, primers, and cDNA template) at room temperature for 15 minutes, vortex to mix, and then centrifuge briefly. After sample addition, tightly cap the reaction tube and centrifuge briefly to ensure that the liquid accumulates at the bottom of the tube. Use nuclease-free consumables throughout the entire process.
[0050] A two-step amplification protocol was used: initial denaturation at 95°C for 30 seconds, followed by denaturation at 95°C for 5 seconds and annealing / extension at 60°C for 30 seconds, for 40 cycles; and melting curve analysis was performed from 65°C to 95°C (at a heating rate of 0.5°C / s). The reaction system is shown in Table 1.
[0051] Table 1 Reaction system
[0052]
[0053] The synthesized cDNA was used as a template for RT-PCR reaction, and the housekeeping gene ef1α was used as an internal reference. The detection results were expressed as Ct values. The Ct values of the target and internal reference genes were averaged over three replicate wells. The relative expression of the target gene was expressed as 2 -△△C t calculation, that is, △Ct=Ct target gene-Ct internal reference gene, △△Ct=△Ct 实验组 -△Ct 对照组 GraphPadPrism 9 was used for data analysis and graphing. Figure 3 .
[0054] Depend on Figure 3 It can be seen that Cit treatment significantly reduced the expression levels of TNF-α and IL-1β (P<0.05, P<0.01).
[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.
Claims
1. Use of L-citrulline in the preparation of a medicament for preventing or treating tuberculosis.
2. The application according to claim 1, characterized in that The medicament comprises an effective amount of L-citrulline.
3. The use according to any one of claims 1-2, characterized in that: The drug further includes a pharmaceutically acceptable carrier.
4. The application according to claim 3, characterized in that The pharmaceutically acceptable carrier is one or more of a solvent, a dispersant, a suspending aid, a surfactant, a thickener, a preservative, a solid binder or a lubricant.
5. The use according to any one of claims 1 to 4, characterized in that: The dosage form of the medicine is solution, injection or oral preparation.
6. A drug for preventing or treating tuberculosis, characterized in that: Such drugs include L-citrulline.
7. The drug according to claim 6, characterized in that The drug further includes a pharmaceutically acceptable carrier.
8. The drug according to claim 7, characterized in that The pharmaceutically acceptable carrier is one or more of a solvent, a dispersant, a suspending aid, a surfactant, a thickener, a preservative, a solid binder or a lubricant.