CN115068460B - Application of L-alanine in preparation of anti-infective drug - Google Patents

Application of L-alanine in preparation of anti-infective drug Download PDF

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CN115068460B
CN115068460B CN202210571294.8A CN202210571294A CN115068460B CN 115068460 B CN115068460 B CN 115068460B CN 202210571294 A CN202210571294 A CN 202210571294A CN 115068460 B CN115068460 B CN 115068460B
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李惠
彭博
彭宣宪
蒋明
陈新海
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Sun Yat Sen University
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Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of L-alanine in preparation of anti-infective drugs. Proved by researches, the L-alanine can improve the resistance of organisms to bacterial infection, thereby preventing and treating diseases caused by bacterial infection. The mechanism is that the L-alanine can activate macrophages in the body, promote the phagocytosis of the L-alanine to bacteria, improve the immunity of the body, and rapidly remove the bacteria in the body, thereby improving the survival rate of the infected organisms. On the other hand, L-alanine has been widely used in foods and medicines, and has high safety.

Description

Application of L-alanine in preparation of anti-infective drug
Technical Field
The invention belongs to the technical field of biological medicine. More particularly, the application of L-alanine in preparing anti-infective drugs.
Background
L-Alanine (L-Alanine) is a basic unit constituting a protein, and is one of 20 amino acids constituting a human protein. The L-alanine can be used as food additive, and can be added into various foods and beverages such as bread, water ice, fruit tea, milk product, carbonated beverage, water ice, etc., to improve nutritive value of food; the taste of the artificial synthetic sweetener can be improved, the sweetness is enhanced, and the dosage is reduced; can be used as flavoring agent to increase flavoring effect of flavoring agent. L-alanine can also be used as humectant, skin conditioner and antistatic agent in cosmetics, and can strengthen skin horny layer and improve skin moisture retention. In addition, L-alanine is an important raw material for synthesizing VB6, is one of the components of nutrient essence-supplementing amino acid nutrient transfusion, is the main component of amino acid injection-800 mainly used for treating liver and encephalopathy and promoting hepatic coma patients to wake up quickly, is also a good medicament for promoting urination, and has wide application in medical treatment. For example, chinese patent application CN104586869a provides for the use of the small molecule metabolite alanine in improving antibiotic susceptibility, but experimental model results show that alanine alone does not have a significant inhibitory effect on bacterial infection.
Wherein bacterial infection is a disease caused by invasion of pathogenic bacteria or conditional pathogenic bacteria into a host for growth and reproduction, production of toxins and other metabolites, and bacterial infection control mainly depends on antibiotics and the anti-infection immunity of the host. Antibiotics can act on specific links of DNA, RNA and protein synthesis systems of bacteria, interfere with the metabolic action of bacteria, thereby impeding the vital activities of bacteria or inhibiting the growth of bacteria, such as β -lactam antibiotics (penicillins, cephalosporins, etc.), fluoroquinolone antibiotics (ciprofloxacin, ofloxacin, etc.), macrolide antibiotics (roxithromycin, clarithromycin), aminoglycoside antibiotics (amikacin, etc.), etc. However, antibiotics commonly used in the prior art are abused, bacteria can gradually generate drug resistance under the selective pressure of the antibiotics, so that the bacteria are insensitive to the antibiotics, and the treatment of bacterial infection is more difficult. Therefore, it is of great importance to provide a drug that enhances the host's resistance to bacterial infection.
Disclosure of Invention
The invention aims to overcome the defect and the defect that the existing bacterial infection antibiotics have drug resistance in treatment, and provides a novel application of L-alanine for improving host resistance to bacterial infection in preparation of anti-infective drugs.
The above object of the present invention is achieved by the following technical scheme:
according to the invention, the research shows that the L-alanine can improve the resistance of organisms to bacterial infection, so that diseases caused by bacterial infection can be prevented and treated. Experiments prove that after the organism is infected by bacteria after being treated by L-alanine or is treated by L-alanine after being infected, the organism can activate macrophages in the body, promote the phagocytosis of the organism to the bacteria, improve the immunity of the organism, and quickly remove the bacteria in the organism, thereby improving the survival rate of the infected organism.
Thus, the invention claims the use of L-alanine for the preparation of an anti-infective agent. Preferably, the L-alanine also includes pharmaceutically acceptable salts and hydrates thereof.
Further, the infection is caused by bacterial infection, including gram-negative bacteria and gram-positive bacteria.
Preferably, the gram-negative bacteria are vibrio parahaemolyticus, escherichia coli, klebsiella pneumoniae or pseudomonas aeruginosa.
Preferably, the gram positive bacterium is staphylococcus aureus or streptococcus b.
Still further, the bacteria also include clinically resistant bacteria of the above bacteria.
Further, the L-alanine increases the phagocytic capacity of macrophages against bacteria.
The medicine can be used for treating bacterial infection of animals, livestock and poultry and the like, such as mammals, rodents and the like, besides being used for treating bacterial infection of mice and human beings. Examples of other animals include horses, dogs, cats, and the like.
Still further, the anti-infection includes preventing and treating diseases caused by bacterial infection.
Further, the anti-infective drug also contains pharmaceutically acceptable auxiliary materials.
Further, the anti-infective medicament is in the form of oral administration, injection or inhalation.
The invention has the following beneficial effects:
proved by researches, the L-alanine can improve the resistance of organisms to bacterial infection, thereby preventing and treating diseases caused by bacterial infection. The mechanism is that the L-alanine can activate macrophages in the body, promote the phagocytosis of the L-alanine to bacteria, improve the immunity of the body, and rapidly remove the bacteria in the body, thereby improving the survival rate of the infected organisms. On the other hand, L-alanine has been widely used in foods and medicines, and has high safety.
Drawings
FIG. 1 is a statistical graph of the survival rate (A) of mice infected with Vibrio parahaemolyticus after alanine pretreatment and the residual amount (B) of Vibrio parahaemolyticus in the body organ in example 1.
FIG. 2 is a statistical plot of survival data of mice infected with various clinically resistant bacteria after alanine pretreatment in example 2.
FIG. 3 is a statistical chart showing the residual amounts of organ bacteria in vivo after alanine pretreatment in example 2, after infection with various clinically resistant bacteria.
FIG. 4 shows the phagocytosis of bacteria by alanine-pretreated macrophages in example 3. FIGS. 4A and 4D are statistical graphs of phagocytosis numbers of macrophages against various bacteria after alanine pretreatment; FIG. 4B is a graph showing the statistics of phagocytosis of E.coli by macrophages after alanine pretreatment at 4 ℃; FIG. 4C is a statistical plot of phagocyte phagocytic bacterial count after antibiotic treatment of phagocytic bacteria (panel A).
FIG. 5 is a statistical plot of macrophage phagocytic bacteria data for alanine pre-treatment at various concentrations in example 3.
FIG. 6 shows phagocytosis of bacteria by isolated macrophages in alanine pre-treated mice in example 4. FIG. 6A is a statistical plot of the number of isolated macrophage phagocytic bacteria in alanine pre-treated mice; FIG. 6B is a statistical plot of the number of phagocytic bacteria by isolated macrophages in alanine pre-treated C57BL/10ScN mice.
FIG. 7 is a statistical plot of survival of mice treated with alanine or not after infection with various bacteria in example 5.
FIG. 8 is a statistical chart showing the bacterial numbers of the internal organs of the mice treated with alanine after infection with various bacteria in example 5.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
TABLE 1 abbreviations for strains used in the invention
Reagents and materials used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 alanine pretreatment can increase mice resistance to Vibrio parahaemolyticus infection
1.1 Ala pretreatment can improve survival rate of mice infected with Vibrio parahaemolyticus
(1) Experimental method
Mice (5-6 weeks old, weight 21-25 g) were acclimatized for one week and then randomly divided into 6 groups, 3 of which were experimental groups and 3 of which were corresponding control groups, each group of 10 mice. The mice were injected by tail vein at a dose of 0.375g/kg alanine, 2 times a day, 12 hours apart, for 3 consecutive days; the control group was injected with an equal volume of physiological saline in the same manner. 1 hour after the last injection, each mouse was challenged with Vibrio Parahaemolyticus (VP) by intraperitoneal injection, and the amount of the challenged agent was respectively as follows for each 2 groups (experimental group and corresponding control group): 2X 10 8 Bacteria/mouse, 3×10 8 Bacteria/mice and 4X 10 8 Bacteria/mice. Mice were continuously observed for 48 hours, and the death of the mice was counted and the mortality was calculated. The calculation formula is as follows: the number of dead mice per group/total number of mice per group x 100%.
(2) Experimental results
Survival of 3 doses of Vibrio parahaemolyticus infected mice and survival of mice pre-treated with alanine and re-challenged are shown in FIG. 1A. As can be seen from the figure: 2X 10 8 The survival rate of the bacteria/dose infected mice is 60%, and the mice infected with the dose are all survived after being pretreated with alanine, which shows that the survival rate of the mice treated with alanine is improved by 40%; 3X 10 8 The survival rate of the bacteria/dose infected mice is 50%, and the mice infected with alanine are treated firstly and then all survive, which shows that the survival rate of the mice treated with alanine is improved by 50%; 4X 10 8 Bacteria/agentThe survival rate of the mice infected by the amount is 30%, and the survival rate of the mice infected by the dosage is improved to 90% after the mice are treated by alanine, which shows that the survival rate of the mice treated by the alanine is improved by 60%.
The above results demonstrate that mice previously treated with alanine have improved resistance to infection by Vibrio parahaemolyticus.
1.2 increased clearance of organs of mice from Vibrio parahaemolyticus after alanine pretreatment
(1) Experimental method
Mice (5-6 weeks old, weight 21-25 g) were acclimatized for one week and then randomly divided into 2 groups of 5 mice each. The experimental group was injected with mice via tail vein at a dose of 0.375g/kg of alanine 2 times a day, 12 hours apart, continuously for 3 days, and the control group was injected with an equal volume of physiological saline in the same manner. 1 hour after the last injection, each mouse was intraperitoneally injected with 4X 10 Vibrio parahaemolyticus 8 Mice were euthanized after 6 hours of dislocation and dissected under sterile conditions to obtain the liver, spleen and kidneys of the mice. The 50 mg of the obtained viscera are fully homogenized in a homogenizer by using 1ml of physiological saline, after the homogenate is diluted with different concentrations, 10 microliter points are respectively taken out on LB solid medium, and the culture is carried out in a 28 ℃ incubator until monoclonal appears, bacteria are counted, and then the number is taken as the logarithm with the bottom of 10, and statistical data are obtained.
(2) Experimental results
As a result of the experiment, referring to FIG. 1B, it can be seen that the number of residual bacteria in the liver, spleen and kidney of the mice which were treated with alanine and then infected with Vibrio parahaemolyticus was 38.95%,9.7% and 34.02%, respectively, of the corresponding organs of the mice directly infected with Vibrio parahaemolyticus, i.e., the number of residual bacteria was reduced by 2.57 times, 10.3 times and 2.94 times, respectively. This result shows that the mice re-infected with Vibrio parahaemolyticus after treatment with alanine had significantly reduced bacterial numbers, whether liver, spleen or kidney, compared to mice re-infected with Vibrio parahaemolyticus after treatment with no alanine.
The above results demonstrate that the scavenging ability of the organs in the body of mice treated with alanine in advance was increased against Vibrio parahaemolyticus.
EXAMPLE 2 alanine pretreatment can increase mice' resistance to various bacterial infections
2.1 Ala pretreatment can improve survival rate of mice infected with various bacteria
(1) Experimental method
Mice (5-6 weeks old, 21-25 g weight) were acclimatized for one week, and randomly divided into 2 groups of 20 animals each for each bacterium. The experimental group was injected with mice via tail vein at a dose of 0.375g/kg of alanine 2 times a day, 12 hours apart, continuously for 3 days, and the control group was injected with an equal volume of physiological saline in the same manner. 1 hour after the last injection, each 2 groups of mice were challenged with different bacteria by intraperitoneal injection according to the following doses, each with the following amounts:
escherichia coli, EC-Y6 and EC-Y9 (2X 10) 8 Bacteria/mice), EC-17 (1X 10) 7 Bacteria/mice); pseudomonas aeruginosa, PA-001 (6X 10) 6 Bacteria/mouse), PA-22 and PA-24 (1X 10) 7 Bacteria/mice); staphylococcus aureus, S.aureus (5X 10) 9 Bacteria/mouse), MRSA-13 and MRSA-15 (8X 10) 9 Bacteria/mice); klebsiella pneumoniae, KP-0118 and KP-00367 (1X 10) 8 Bacteria/mice).
Mice were continuously observed for 48 hours after challenge, and the death of the mice was counted and the mortality calculated. The calculation formula is as follows: the number of dead mice per group/total number of mice per group x 100%.
(2) Experimental results
As a result of the experiment, referring to fig. 2, it can be seen that the mortality rate of mice infected with different bacteria after alanine treatment was reduced:
for clinical E.coli, the survival rate of EC-Y6 was increased by 90% (from 10% untreated to 100% alanine treated), the survival rate of EC-Y9 was increased by 40% (from total untreated deaths to 40% alanine treated), and the survival rate of EC-Y17 was increased by 75% (from 20% untreated to 95% alanine treated).
For clinical P.aeruginosa, the survival rate of PA-001 was increased by 45% (from 40% untreated to 85% alanine treated), the survival rate of PA-22 was increased by 80% (from total untreated deaths to 80% alanine treated), and the survival rate of PA-24 was increased by 30% (from 10% untreated to 40% alanine treated).
For staphylococcus aureus, the survival rate of S.aureau was increased by 50% (from 40% untreated to 90% alanine treated), the survival rate of methicillin-resistant staphylococcus aureus MRSA-13 was increased by 5% (from 95% untreated to 100% alanine treated), and the survival rate of methicillin-resistant staphylococcus aureus MRSA-15 was increased by 15% (from 85% untreated to 100% alanine treated).
For Klebsiella pneumoniae, the survival rate of KP-0118 was increased by 40% (from 10% of untreated to 50% of alanine treatment), and the survival rate of KP-0367 was increased by 50% (from 20% of untreated to 70% of alanine treatment).
The above results demonstrate that mice previously treated with alanine are resistant to multiple pathogenic bacterial infections.
2.2 increase in the clearance of organs of mice to various bacteria after alanine pretreatment
(1) Experimental method
Mice (5-6 weeks old, weight 21-25 g) were acclimatized for one week, and divided into 2 groups of 5 for each bacteria. The mice were injected by tail vein at a dose of 0.375g/kg alanine, 2 times a day, 12 hours apart, for 3 consecutive days. The control group was injected with an equal volume of physiological saline in the same manner. 1 hour after the last injection, each 2 groups of mice were intraperitoneally injected with different kinds of bacteria at the same dose of 2.1.
Taking out liver, spleen and kidney of mice infected with different bacteria respectively according to the method of example 1.2, homogenizing, gradient diluting, and plating on LB solid plate, and culturing at 37deg.C; after the bacteria grow out, the bacteria are counted, and the number of bacteria remained in the organs of the mice of the experimental group and the control group is counted, and then the number is taken as the base 10 logarithm.
(2) Experimental results
As a result of the experiment, referring to fig. 3, it can be seen from the graph that the bacterial count of each tissue and organ in the mice after alanine addition was significantly smaller than that of the control group. The specific cases are as follows:
mice which were first treated with alanine and then infected with different bacteria had a 4-110 fold decrease in the number of residual bacteria in the liver compared to mice treated without alanine, each bacteria in the following specific cases: EC-Y6 (110 times), EC-Y9 (30.5 times), EC-17 (4.9 times), PA-001 (65.5 times), PA-22 (75.08 times), PA-24 (23.2 times), S.aureus (4 times), MRSA-13 (3.6 times), MRSA-15 (8.89 times), KP-0118 (6.5 times), KP-00367 (6.2 times);
compared with mice which are not treated by alanine, the number of residual bacteria in the spleen is reduced by 2.6-76 times, and the specific conditions of the reduction times of each bacteria are as follows: EC-Y6 (75.3 times), EC-Y9 (27.9 times), EC-17 (5.2 times), PA-001 (28.2 times), PA-22 (53.63 times), PA-24 (17.8 times), S.aureus (2.6 times), MRSA-13 (3.1 times), MRSA-15 (6.22 times), KP-0118 (12.7 times), KP-00367 (7.8 times);
compared with mice which are not treated by alanine, the number of residual bacteria in the kidneys is reduced by 3.6-58 times, and the specific conditions of the reduction times of each bacteria are as follows: EC-Y6 (93.5 times), EC-Y9 (57.3 times), EC-17 (3.6 times), PA-001 (38.2 times), PA-22 (47.08 times), PA-24 (18 times), S.aureus (10.8 times), MRSA-13 (3.8 times), MRSA-15 (9.6 times), KP-0118 (5.8 times), KP-00367 (8.4 times).
The above results indicate that mice re-infected with bacteria after treatment with alanine showed a significant decrease in the number of each bacteria compared to mice re-infected with bacteria after treatment without alanine, whether liver, spleen or kidney. Demonstrating that the ability of the organs to clear different bacteria in the body of mice treated with alanine in advance is significantly increased, and that alanine has the universality of promoting the mice to clear infected bacteria.
Example 3 alanine can promote phagocytic ability of macrophages against bacteria and has alanine concentration dependence
L-alanine can promote the removal of infectious bacteria from mice and improve the anti-infective ability of mice, probably because L-alanine stimulates the phagocytic ability of macrophages of the organism, thereby improving the removal of pathogenic bacteria by the host. Now, the change of the phagocytic capacity of the RAW264.7 cells to bacteria after exogenous alanine is added is studied by taking the mouse macrophage-RAW 264.7 as a study object.
Macrophages can be evaluated for their ability to phagocytose bacteria using 2 methods: the 1 st is that after the phagocytic bacteria of the macrophage are completed, the phagocytic bacteria can be released by breaking the macrophage, then the bacteria are diluted, and the number of the bacteria is counted by adopting a plate counting method. The influence of alanine on the phagocytic capacity of macrophages can be known by comparing the change of the bacterial number phagocytosed by the macrophages before and after alanine treatment; the 2 nd is the mean fluorescence intensity of the fluorescence-labeled bacteria entering the macrophages, i.e. MFI (Mean fluorescence intensity, MFI), measured by flow cytometry with fluorescence-labeled bacteria. The greater the MFI, the greater the number of bacteria phagocytosed by the macrophages, i.e. the greater the phagocytic capacity. The effect of alanine on phagocytic capacity of macrophages can be known by comparing the MFI changes of the macrophages before and after alanine treatment.
3.1 alanine promotes phagocytic ability of phagocytic cells to bacteria
(1) Experimental method
RAW264.7 cell culture: after resuscitating the murine macrophage cell line-RAW 264.7, it was cultured in complete medium of DMEM (low sugar) containing 10% (v/v) of enhanced calf serum, 100mg/L penicillin and 100mg/L streptomycin in at 5% CO 2 And a cell incubator with 100% humidity and 37 ℃ for regular passage, wherein the passage ratio is 1:5-1:10, and the passage time is 2-3 days. The experiment was performed with cells in the logarithmic growth phase, and the biological experiment was repeated three times.
RAW264.7 phagocytosis assay: after digestion with cell RAW264.7, cell counts were performed and re-plated to maintain consistent cell numbers for each test group, with 5X 10 cells per well of the six well plate 6 A cell; standing the incubator for 2-3 hours, changing the complete culture medium into a DMEM culture medium of 0.5% calf serum for overnight starvation treatment, changing the complete culture medium into a DMEM culture medium without serum, and simultaneously adding 40mM alanine for treatment for 1.5 hours; then according to bacteria: different bacteria are respectively added into the cells according to the ratio of 100:1, the cells are gently shaken uniformly, and a horizontal rotating head centrifuge is used for 2000rpm,Centrifuging at 4deg.C for 5min to allow bacteria to settle to the bottom and combine with macrophage, which is beneficial to phagocytosis; after 1.5 hours incubation in incubator, stopping phagocytosis with pre-chilled PBS, washing off bacteria not adsorbed or phagocytized by cells with PBS, and repeating the washing for 4 times; cells were treated with 1% triton100 for 20 min to break up, bacteria were diluted in plates and counted.
(2) Experimental results
Referring to FIG. 4A, it can be seen that the number of Vibrio parahaemolyticus phagocytes increases 1.77-fold after the treatment with alanine (the number of bacteria phagocytosed by phagocytes after the treatment with alanine divided by the number of bacteria phagocytosed by phagocytes not treated with alanine), the number of Escherichia coli phagocytes increases 1.79-fold, the number of Staphylococcus aureus phagocytes increases 1.86-fold, the number of Escherichia coli EC-Y17 phagocytes increases 2-fold, the number of Pseudomonas aeruginosa PA-001 phagocytes increases 1.94-fold, and the number of Streptococcus hemolyticus phagocytes increases 1.66-fold.
The above results demonstrate that phagocytic bacteria are significantly more abundant than those phagocytosed by non-alanine treated phagocytes after alanine treatment.
3.2 exclusion experiments
To exclude the results from bacteria adhering to the surface of phagocytes or from bacteria not washed clean adhering to the surface of phagocytes, the following two exclusion experiments were performed:
first, macrophages are treated with alanine at different concentrations at 4℃at which temperature they adhere to but do not endocytose the bacteria, and then E.coli containing green fluorescent protein (Green fluorescent protein, GFP for short) is added. As a result, it was found that the MFI of macrophages was not increased compared to the control after treatment with alanine at different concentrations (FIG. 4B). This result precludes the possibility of macrophages increasing MFI by increasing adhesion to bacteria, indicating the specificity of this phagocytosis.
Secondly, to confirm that bacteria adhered to the surfaces of phagocytes in the experimental process are cleaned, after the phagocytes are completely phagocytized, antibiotics (100 micrograms/milliliter of ampicillin and gentamicin respectively) are added to the phagocytes after the phagocytes are cleaned, the bacteria adhered to the surfaces of the macrophages are bred for 1 hour (the aim of cleaning the bacteria adhered to the surfaces of the macrophages), then the cells are broken, the bacteria are diluted, and the number of the bacteria phagocytized by the macrophages is counted, so that the result is shown as 4C. The results demonstrate that although phagocytic results after antibiotic treatment (fig. 4C) are different from phagocytic results without antibiotic treatment (fig. 4A), the results are substantially consistent, indicating that the use of PBS to wash the surface of phagocytes against adherent bacteria is viable.
On the basis, the conditions of clinical multi-drug resistant escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa and MRSA marked by fluorescein isothiocyanate (Fluorescein isothiocyanate, FITC) after the RAW264.7 cells are treated by alanine are studied, and the MFI of the fluorescence marked bacteria entering macrophages is detected by a flow cytometer, so that the ability of the macrophages to phagocytize the bacteria is judged. By comparing MFI values of phagocytic bacteria of macrophages before and after alanine treatment, the effect of alanine on phagocytic capacity of phagocytes can be assessed. As a result, it was found (FIG. 4D) that when macrophages were treated with alanine, the ability to phagocytise Escherichia coli Y6 was increased 1.2-fold, and the ability to phagocytise Escherichia coli Y9 was increased 1.26-fold; the ability to phagocytose Klebsiella pneumoniae 0118 is increased by 1.3 times, and the ability to phagocytose Klebsiella pneumoniae 0367 is increased by 1.18 times; the capability of phagocytizing the pseudomonas aeruginosa 22 is increased by 1.24 times, and the capability of phagocytizing the pseudomonas aeruginosa 24 is increased by 1.22 times; the ability to phagocytose both MRSA13 and 15 was increased by a factor of 1.18.
3.3 alanine enhancing macrophage phagocytic bacteria with alanine concentration gradient
(1) Experimental method
Phagocytes were prepared by the procedure of example 3.1, treated with 10mM and 40mM alanine, respectively, and then incubated with fluorescein isothiocyanate (Fluorescein isothiocyanate, FITC) -labeled different bacteria, respectively, in an incubator for 1.5 hours, respectively. Phagocytes treated with the corresponding alanine concentrations served as controls. MFI of macrophages to bacteria after treatment with alanine at different concentrations was measured using a flow cytometer.
(2) Experimental results
The results are seen in fig. 5, which shows:
for VP bacteria, the MFI increased by a factor of 1.7 after treatment of macrophages with 10mM alanine, and by a factor of 2.1 after treatment of macrophages with 40mM alanine;
for E.coli, the MFI increased 1.33-fold after treatment of macrophages with 10mM alanine, and 1.45-fold after treatment of macrophages with 40mM alanine;
for staphylococcus aureus, MFI increased 1.84-fold after treatment of macrophages with 10mM alanine, 2.13-fold after treatment of macrophages with 40mM alanine;
for clinical E.coli Y17, the MFI increased 1.6-fold after treatment of macrophages with 10mM alanine, and 1.9-fold after treatment of macrophages with 40mM alanine;
for pseudomonas aeruginosa, the MFI increased 1.60-fold after treatment of macrophages with 10mM alanine, and 1.86-fold after treatment of macrophages with 40mM alanine;
for hemolytic streptococcus, the MFI increased 1.58-fold after treatment of macrophages with 10mM alanine, and 1.74-fold after treatment of macrophages with 40mM alanine;
for klebsiella pneumoniae 0118, the MFI increased 1.08-fold after treatment of macrophages with 10mM alanine, and 1.27-fold after treatment of macrophages with 40mM alanine.
The results show that exogenous alanine can enhance macrophage-mediated phagocytosis in vitro and has alanine concentration dependence. Phagocytic bacteria, including gram negative or gram positive bacteria, demonstrate the prevalence of alanine to enhance macrophage phagocytosis.
Example 4 alanine can increase the ability of mice to phagocytose bacteria by macrophages in the abdominal cavity and is time dependent
4.1 phagocytosis of macrophages by L-alanine in vivo
(1) Experimental method
To further confirm whether L-alanine can promote macrophages in vivoPhagocytosis, i.v. injection of L-alanine (0.375 g/kg) into mice was performed twice daily, at 12 hours intervals, for three consecutive days, while mice injected with the same volume of physiological saline were used as a control group. Then separating the mouse abdominal macrophages, performing cell count, and paving 5×10 cells in each of six holes 6 A cell; phagocytic function of phagocytes against bacteria was then examined as in example 3.1.
(2) Experimental results
As a result, referring to fig. 6A, it can be seen from the graph that the number of macrophages isolated from mice treated with alanine, the number of Vibrio parahaemolyticus phagocytized (the number of bacteria phagocytosed by macrophages isolated from mice injected with alanine divided by the number of bacteria phagocytosed by macrophages isolated from mice injected with physiological saline) increased by 1.51 times, the number of escherichia coli phagocytized by 1.19 times, the number of staphylococcus aureus phagocytosed by 1.16 times, the number of escherichia coli EC-Y17 phagocytized by 1.3 times, the number of pseudomonas aeruginosa PA001 phagocytized by 1.2 times, the number of streptococcus haemolyticus phagocytized by 1.29 times, and the number of klebsiella pneumoniae phagocytized by 1.29 times.
The above results demonstrate that mice treated with alanine have significantly more macrophages to phagocytose bacteria than those without alanine.
4.2C57BL/10ScN mouse experiments
(1) Experimental method
Experiments were further performed using C57BL/10ScN mice, which are mice deficient in the tlr4 gene, which have a defect in response to LPS stimulation, because the tlr4 gene is deleted, resulting in the inability of the gene mRNA and protein to synthesize upon LPS stimulation. Experimental procedure was the same as 4.1, and the number of macrophages isolated from alanine treated mice was determined for phagocytosis of clinical E.coli, klebsiella pneumoniae and Pseudomonas aeruginosa.
(2) Experimental results
As a result, referring to FIG. 6B, it can be seen that when C57BL/10ScN mice were used, then the fluorescence of phagocytic bacteria, i.e., the number of phagocytic bacteria, of macrophages was similar in both alanine-injected mice and normal saline-injected mice, indicating that alanine did not increase the phagocytic capacity of macrophages in mice when the tlr4 gene was deleted.
EXAMPLE 5 alanine can increase resistance of mice infected with multiple resistant bacteria to infectious bacteria
5.1 alanine can increase survival of mice infected with multiple resistant bacteria
(1) Experimental method
Mice (5-6 weeks old, weight 21-25 g) were acclimatized for one week and then randomly divided into groups, each group being infected with one bacteria (i.p. injection). The doses of bacterial infection are as follows: vibrio parahaemolyticus (4×10) 8 Bacteria/mice); escherichia coli EC-Y6 and EC-Y9 (2X 10) 8 Bacteria/mice), pseudomonas aeruginosa PA-22 and PA-24 (1X 10) 7 Bacteria/mouse), klebsiella pneumoniae KP-0118 and KP-0367 (1×10) 8 Bacteria/mice); staphylococcus aureus MRSA-13 and-15 (8X 10) 9 Bacteria/mice). Mice were then subdivided into 2 groups of 10 mice each. A total of 5 injections of 0.75g/kg L-alanine (experimental group) or equivalent sterile saline (control group) were administered at 1, 4, 7, 10 and 20 hours after bacterial infection, respectively. The mice were then observed for 2 days, survival recorded, and survival calculated.
(2) Experimental results
As a result, referring to fig. 7, it can be seen from the graph that the survival rate of the mice injected with alanine after bacterial infection is significantly improved as compared with that of the mice injected with physiological saline. For the vibrio parahaemolyticus virus-challenged mice, the survival rate is improved by 30 percent (the 0 percent of physiological saline group is increased to 30 percent of alanine treatment group); for Escherichia coli EC-Y6-challenged mice, the survival rate is improved by 20% (from 0% of physiological saline group to 20% of injected alanine group); for Escherichia coli EC-Y9-challenged mice, the survival rate is improved by 10% (from 0% of physiological saline group to 10% of injected alanine group); for the pseudomonas aeruginosa 22 virus-challenged mice, the survival rate is improved by 10 percent (the increase from 0 percent of the physiological saline group to 10 percent of the alanine group); for the pseudomonas aeruginosa 24 challenge mice, the survival rate is improved by 10 percent (the survival rate is increased from 20 percent of the physiological saline group to 20 percent of the alanine group); for Klebsiella pneumoniae 0118-challenged mice, the survival rate is improved by 10 percent (10 percent of physiological saline group is increased to 20 percent of alanine group injection); for klebsiella pneumoniae 0367-challenged mice, the survival rate is improved by 20% (the increase from 20% of physiological saline group to 40% of injected alanine group); for MRSA13 challenged mice, the survival rate is improved by 5% (from 95% of physiological saline group to 100% of alanine group); the survival rate of MRSA15 challenged mice was improved by 15% (from 85% in saline group to 100% in alanine group).
The above results demonstrate that alanine increased the resistance of mice to multi-resistant bacteria, thereby increasing survival when the mice were infected with the bacteria.
5.2 alanine can increase the bacterial clearance in mice infected with multiple resistant bacteria
(1) Experimental method
The 5.1 mice were dislocation sacrificed and dissected in a sterile environment to obtain the liver, spleen and kidneys of the mice. The 50 mg of the obtained viscera were thoroughly homogenized with 1mL of physiological saline in a homogenizer, the homogenates were subjected to double dilution, 10. Mu.L of each diluted homogenate was spotted on LB solid medium, and the culture was carried out in a 37℃incubator until the occurrence of monoclonal, and the number of bacteria was counted. The SPSS statistical software was used to calculate whether there was a difference in bacterial count between the saline-injected control group and the alanine-injected experimental group.
(2) Experimental results
As a result, referring to fig. 8, it can be seen from the graph that the number of bacteria in each tissue and organ of mice treated with alanine after bacterial infection was significantly smaller than that of mice treated with no alanine in the control group. The specific cases are as follows:
alanine injection mice after bacterial infection showed a fold decrease in the number of residual bacteria in the liver compared to normal saline injection mice: VP (8.75 times), EC-Y6 (5.87 times), EC-Y9 (5.01 times), PA-22 (6.19 times), PA-24 (6.71 times) KP-0118 (3.89 times), KP-00367 (34.79 times), MRSA-13 (31.19 times), MRSA-15 (11.21 times); the number of residual bacteria in the spleen was reduced by multiple times compared with mice not treated with alanine: VP (8.68 times), EC-Y6 (4.51 times), EC-Y9 (32.67 times), PA-22 (3.27 times), PA-24 (14.79 times) KP-0118 (8.23 times), KP-00367 (1.99 times), MRSA-13 (12.51 times), MRSA-15 (3.68 times); the number of residual bacteria in the kidneys was reduced by multiple times compared with mice not treated with alanine: VP (3.2 times), EC-Y6 (15.48 times), EC-Y9 (3.68 times), PA-22 (6.38 times), PA-24 (9.51 times) KP-0118 (8.5 times), KP-00367 (2 times), MRSA-13 (3 times), MRSA-15 (4.88 times).
The above results demonstrate that mice infected with multidrug resistant bacteria are treated with alanine and the organs have improved clearance of the infected bacteria.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (5)

  1. The use of l-alanine as sole active ingredient in the manufacture of a medicament for the prevention of an infection, characterized in that the infection is caused by a bacterial infection, said bacteria being vibrio parahaemolyticus, escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, staphylococcus aureus or streptococcus b.
  2. 2. The use according to claim 1, wherein the L-alanine further comprises a pharmaceutically acceptable salt thereof.
  3. 3. The use according to claim 1 or 2, wherein the L-alanine increases the phagocytic capacity of macrophages against bacteria.
  4. 4. The use according to claim 1 or 2, wherein the infection-preventing medicament further comprises a pharmaceutically acceptable adjuvant.
  5. 5. The use according to claim 1 or 2, wherein the infection-preventing agent is in the form of an oral, injectable or inhalant preparation.
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CN104586869A (en) * 2014-12-31 2015-05-06 中山大学 Small molecule metabolite for improving effect of antibiotic to eliminate pathogenic bacteria
CN111803481A (en) * 2020-09-01 2020-10-23 上海市肺科医院 Application of L-alanine in preparing medicine for preventing and treating tuberculosis

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CN104586869A (en) * 2014-12-31 2015-05-06 中山大学 Small molecule metabolite for improving effect of antibiotic to eliminate pathogenic bacteria
CN111803481A (en) * 2020-09-01 2020-10-23 上海市肺科医院 Application of L-alanine in preparing medicine for preventing and treating tuberculosis

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