CN111803481A - Application of L-alanine in the preparation of drugs for preventing and treating tuberculosis - Google Patents

Abstract

The invention discloses application of L-alanine in preparing a medicament for preventing and treating tuberculosis. According to the invention, through modes of C57BL/6 mice and SCID mice infection models, macrophage in vitro infection models, exogenous metabolite addition and the like, L-alanine is proved to protect a host against tubercle bacillus infection through natural immune cells, L-alanine promotes macrophage antibacterial peptide generation and limits survival of tubercle bacillus in cells, and L-alanine is expected to assist the conventional antitubercular drugs, so that the treatment course can be shortened, the curative effect can be improved, and a reference is provided for clinical treatment.

Description

Application of L-alanine in the preparation of drugs for preventing and treating tuberculosis

technical field

The invention relates to the field of tuberculosis prevention and treatment, in particular to the application of L-alanine in the preparation of medicines for preventing and treating tuberculosis.

Background technique

Tuberculosis, caused by Mycobacterium Tuberculosis (MTB) infection, is one of the top ten causes of death worldwide and the most fatal infectious disease. In my country, the situation is also severe, and it is the country with the second highest burden of tuberculosis in the world. The recommended treatment regimen for tuberculosis is a combination of multiple anti-tuberculosis drugs for 6-9 months, which leads to poor medication compliance and further promotes the development of multidrug-resistant tuberculosis (MDR-TB). At present, MDR-TB has posed a threat to global public health. The cure rate of MDR-TB patients treated with conventional regimens is lower than 50%, and even some patients will aggravate. Host-directed therapy (HDT) is different from the previous anti-tuberculosis drugs that directly kill tuberculosis, but regulates the host's immune function and fights tuberculosis infection. Several clinical studies have shown that HDT drug adjuvant therapy can effectively shorten the medication time of patients, especially for MDR-TB patients. Since HDT drugs all target the classical anti-tuberculosis immune pathway of the host, HDT-resistant tuberculosis rarely occurs. Although HDT drug adjuvant therapy has many advantages, especially bringing more choices for drug-resistant TB patients, HDT is still an emerging treatment option in the field of TB, and it has been confirmed that the mechanism of action is clear and it has host protective effects in TB. HDT drugs are also very limited. Therefore, it is urgent to find HDT drugs that can directly target the host's anti-tuberculosis immune pathway, and it has high clinical significance.

The host immune response to TB begins with phagocytosis of TB by alveolar macrophages and dendritic cells followed by direct clearance through autophagosome maturation, antimicrobial peptide production, and reactive oxygen species/reactive nitrogen species (ROS/RNS) production intracellular tuberculosis. Antibacterial peptides (AMPs) are generally positively charged peptides composed of 12-50 amino acids. AMPs are amphiphilic, with both water-transporting and hydrophilic regions, which help AMPs bind to the bacterial outer wall. There are two major families of antimicrobial peptides in mammals: Cathelicidins and Defensins. Antimicrobial peptides can directly limit the growth of Mycobacterium tuberculosis through various mechanisms. For example, antimicrobial peptide LL-37 can interfere with the function of the bacterial membrane of Mycobacterium tuberculosis and disrupt the structure of the bacterial wall to kill Mycobacterium tuberculosis. It has been found that active vitamin D31,25-dihydroxyvitamin D3 can bind to vitamin D receptors and initiate the expression of antimicrobial peptides. Clinical trials have shown that a histidine deacetylase inhibitor-sodium phenylbutyrate (PBA) plus vitamin D is expected to cure tuberculosis patients, especially drug-resistant tuberculosis patients.

In recent years, the impact of metabolic small molecule substances on diseases, especially on tumors and intestinal flora, has been a research hotspot of scientists around the world. More and more data show that the occurrence and development of most diseases are accompanied by local or global metabolism. abnormal. Recently, several consecutive international high-impact articles have reported small molecule metabolites that have protective therapeutic effects on the host in diseases such as pancreatic cancer, pathogenic enterobacteria, and intestinal cancer. Metabolites directly affect disease outcome. However, in tuberculosis caused by Mycobacterium tuberculosis infection, small molecule metabolites have been used for research in the direction of disease diagnosis, and their effects on the outcome of tuberculosis disease are still poorly understood.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the application of L-alanine in the preparation of drugs for preventing and treating tuberculosis, aiming at the problems of the prior art.

The invention provides the application of L-alanine in the preparation of drugs for preventing and treating tuberculosis.

Further, the medicine also includes pharmaceutically acceptable excipients.

Further, the drug protects the host against tuberculosis infection through innate immune cells.

Further, the drug promotes the production of antimicrobial peptides in macrophages and limits the intracellular survival of Mycobacterium tuberculosis.

Further, the drug can improve the ability of macrophages to clear multidrug-resistant tuberculosis.

Further, the drug can assist anti-tuberculosis drugs.

The present invention utilizes C57BL/6 mouse and SCID mouse infection model to analyze the effect of L-alanine on tuberculosis.

The invention utilizes the macrophage in vitro infection model to analyze the effect of L-alanine on the macrophage anti-tuberculosis immune function.

The present invention adopts the above technical scheme, compared with the prior art, has the following technical effects:

The present invention proves that L-alanine protects the host against tuberculosis infection, L-alanine through innate immune cells through infection models of C57BL/6 mice and SCID mice, in vitro infection models of macrophages, and exogenous addition of metabolites. L-alanine promotes the production of antimicrobial peptides in macrophages and limits the intracellular survival of tuberculosis. L-alanine can improve the clearance of multidrug-resistant tuberculosis by macrophages. L-alanine is expected to assist current anti-tuberculosis drugs and can Shorten the course of treatment, improve the curative effect, and provide reference for clinical treatment.

Description of drawings

Fig. 1 is the lung tissue section diagram (1-A) and the lung CFU statistical result diagram (1-B) of the C57BL/6 mouse infection model experiment in Example 1 of the present invention;

Fig. 2 is the survival curve diagram of SCID mouse infection model experiment in the embodiment of the present invention 1;

Fig. 3 is the Q-PCR result figure in the embodiment of the present invention 2;

FIG. 4 is a graph showing the results of the intracellular survival experiment of the macrophage in vitro infection model in Example 2 of the present invention.

Detailed ways

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

It should be noted that the embodiments of the present invention and the features of the embodiments may be combined with each other under the condition of no conflict.

The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but it is not intended to limit the present invention.

Example 1

C57BL/6 mouse and SCID mouse infection model to analyze the effect of L-alanine on tuberculosis.

(1) C57BL/6 mouse infection model experiment: Wild-type 6-8 week old C57BL/6 female mice, 8 in each group, were infected with Mycobacterium tuberculosis H37Rv through the respiratory tract, about 200 CFU/mouse. The control group was given drinking water, the L-alanine group was supplemented with L-alanine (30 mg/mL), and the D-alanine group was added with D-alanine (30 mg/mL) as a drug control. ), the mice were killed by cervical dislocation 30 days after infection, and the lung tissue was isolated to detect the following indicators: 1) Lung CFU: Collect lung tissue under aseptic conditions, dilute 10-fold and 100-fold after homogenization, and inoculate 0.1 ml of each Mie's 7H10 medium was cultured at 37°C for 4 weeks, the growth of MTB was observed, the number of colonies (CFU) was counted, and the amount of bacteria in lung tissue was calculated. 2) Histopathological detection of lung: Collect lung tissue, fix it with 4% paraformaldehyde, dehydrate, soak in wax, embed, slice at 4 μm, stain with H&E, and observe whether the tissue has edema, inflammatory cell infiltration, hemorrhage, etc. pathological damage. The results are shown in Figure 1.

(2) SCID mouse infection model experiment: Wild-type 6-8 week old SCID female mice, 16 in each group, were aerosol-infected with Mycobacterium tuberculosis H37Rv, about 200 CFU/mouse. The control group was given drinking water, and the L-alanine group was added L-alanine (30 mg/mL) to the drinking water. The survival of the mice was continuously observed, and the survival curve was drawn. The results are shown in Figure 2.

The results showed that the pathological changes in the lungs of mice infected with M. With a smaller load of bacteria (Fig. 1-B), it can be seen that L-alanine can significantly improve the host's ability to resist Mycobacterium tuberculosis. Supplementation with L-alanine can significantly improve the survival rate and survival time of SCID mice (Figure 2). Since SCID mice are adaptive immune response-deficient mice, they only retain complete innate immune function. It can be seen that L- Alanine enhances the host's ability to resist tuberculosis infection mainly by affecting the stage of innate immune response in mice.

Example 2

In vitro infection model of macrophages to analyze the effect of L-alanine on the immune function of macrophages.

(1) Influence of antimicrobial peptide production: Take mouse peritoneal macrophages or mouse immortalized bone marrow-derived macrophages (iBMDM), add L at a final concentration of 0, 0.1 and 1 mM - H37Rv infection was administered 12 hours after alanine treatment, MOI=5. After 0, 12, and 24 hours of infection, the supernatant was removed, the cells were lysed with 1 ml TRIZOL, RNA was extracted, and the changes in the expression levels of related molecules such as antimicrobial peptides were detected by RT-PCR to determine the effect of L-alanine on the production of antimicrobial peptides in macrophages. Impact. The experimental results are shown in Figure 3.

(2) Intracellular survival experiment: Take wild-type mouse peritoneal primary macrophages, culture them in complete 1640 medium (containing 10% FBS + 1% penicillin-streptomycin) at 37 degrees for 4 hours, and replace the cells after they adhere to the wall. No 1640 medium, L-alanine was added to make the final concentration of 0 and 1 mM, respectively, 12 hours later, the H37Rv strain and the clinically isolated Mycobacterium tuberculosis drug-resistant strain (MDR) kept in the laboratory were infected respectively, MOI=2 . After 2 hours of infection, the supernatant was removed, the cells were repeatedly washed with PBS three times to remove extracellular bacteria, and the number of bacteria entering the cells was counted by CFU. Or after washing, add 1640 medium containing gentamicin and 0 and 1 mM L-alanine to continue culturing for 24 hours, and then detect the survival of intracellular bacteria by CFU.

The results showed that in mouse peritoneal macrophages and iBMDM cells, L-alanine could significantly promote the expression of the antimicrobial peptides Camp and Defb4 genes by RT-PCR (Figure 3A-D).

L-alanine stimulation was given to mouse peritoneal macrophages, and then the cells were infected with the standard tuberculosis strain H37Rv (Fig. 4A) and the multidrug-resistant strain MDR (Fig. 4B), and it was found that L-alanine can also promote macrophages Intracellular clearance capacity of phages against Mycobacterium tuberculosis.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the embodiments and protection scope of the present invention. Those skilled in the art should be aware of the equivalents made by using the description and illustrations of the present invention. The solutions obtained by substitutions and obvious changes shall all be included in the protection scope of the present invention.

Claims (6)

1. Application of L-alanine in the preparation of drugs for preventing and treating tuberculosis. 2. The application of L-alanine according to claim 1 in the preparation of a medicament for preventing and treating tuberculosis, wherein the medicament also comprises a pharmaceutically acceptable adjuvant. 3. The application of L-alanine according to claim 1 in the preparation of a drug for preventing and treating tuberculosis, wherein the drug protects the host against tuberculosis infection by innate immune cells. 4. The application of L-alanine according to claim 1 in the preparation of a drug for preventing and treating tuberculosis, wherein the drug promotes the production of antibacterial peptides in macrophages and limits the intracellular survival of tuberculosis bacteria. 5. The application of L-alanine according to claim 1 in the preparation of a drug for preventing and treating tuberculosis, wherein the drug can improve the scavenging effect of macrophages on multidrug-resistant tuberculosis. 6. The application of L-alanine according to claim 1 in the preparation of a drug for preventing and treating tuberculosis, wherein the drug can assist anti-tuberculosis drugs.

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