Application of 4-GBA in preparation of medicine for improving infection endurance capacity of sepsis patient

Abstract

The invention relates to application of 4-GBA in preparation of a medicine for improving infection endurance capacity of a sepsis patient, 4-GBA is used for treating mice infected with sepsis, compared with control mice, the survival rate of the mice treated by the 4-GBA is far larger than that of the control mice, and experiments prove that the 4-GBA has a protection effect on sepsis infection. The mechanism of 4-GBA for improving infection endurance capacity of sepsis patients is researched, and after 4-GBA treatment, the content of liver and kidney functional markers in peripheral blood serum of mice infected with sepsis is remarkably reduced, the content of DAMPs (injury related molecular patterns) related molecules in peripheral blood serum of mice infected with sepsis is remarkably reduced, and the application of 4-GBA in the treatment of the mice infected with sepsis in the treatment of the infection endurance capacity of the patients infected with sepsis in the treatment of the infection endurance capacity of the patients infected with sepsis in the treatment of the patients infected with sepsis in the treatment of the patients infected with sepsis. And the product does not have in-vivo and in-vitro sterilization functions and body immune regulation functions. The result shows that 4-GBA can improve the infection endurance capacity of the sepsis patient, and provides a new direction and a new drug for treatment of the sepsis patient.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a preparation method and application of a drug 4-GBA for improving the infection tolerance of sepsis patients. Background Art

[0002] Sepsis is characterized by high morbidity and mortality. The treatment options recommended by the "2021 International Guidelines for the Management of Sepsis and Septic Shock" include: early fluid resuscitation, early anti-infection, hemodynamic management, mechanical assisted ventilation, etc. The above treatment options reduce the mortality rate of patients with sepsis to 20%, but cannot further reduce the mortality rate of patients with sepsis. In patients with sepsis, drug-resistant bacterial infection is an important cause of death in patients. At present, the proportion of bacterial resistance caused by the abuse of antibiotics has reached 20% to 60%, and only 12 new antibiotics have been launched in the past 20 years. The speed of research and development of new antibiotics is much lower than the speed of bacterial resistance. The field of sepsis research is facing a severe situation in which the mortality rate is increasing instead of decreasing.

[0003] The human body's anti-infection response to bacterial infection includes two mechanisms: resistance to infection and tolerance to infection. The resistance mechanism is driven by the immune response and aims to kill and eliminate pathogens. Immunomodulators belong to the resistance mechanism. At present, the main immunomodulators are: corticosteroids, anti-TNF-α, GM-CSF / G-CSF, INF-γ, immunoglobulins, etc. The above-mentioned immunomodulators have not been able to obtain definite effects in large-scale clinical randomized controlled trials, so they are not recommended by the guidelines. The tolerance mechanism aims to slow down the pathological damage caused by pathogens or / and immune responses to the host. So far, the tolerance molecules with "positive" regulatory effects reported include: ferritin heavy chain in hepatocytes, autophagy-related proteins in the lungs, insulin-like growth factor in white adipose tissue, aryl hydrocarbon receptor in splenic dendritic cells, amphiregulin produced by natural lymphocytes 2 or regulatory T cells in the lungs, macrophage nuclear factor erythroid 2-related factor 2, and NADPH oxidase subunit Cybb. The above molecules can alleviate target organ damage, increase the survival rate of animals in different infection models and improve prognosis through different regulatory mechanisms without affecting the pathogen load. However, there is a lack of effective regulators for the above molecules.

[0004] Guanidinobutyric acid (4-GBA) is widely distributed in various tissues and organs of the human body. There are no reports on the regulation of infection tolerance of sepsis patients by 4-GBA. Summary of the invention

[0005] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the present invention provides the use of 4-GBA in the preparation of a drug for improving the infection tolerance of sepsis patients.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] Application of 4-GBA in the preparation of drugs for improving infection tolerance of sepsis patients.

[0008] Furthermore, the drug enhances the patient's infection tolerance by reducing DAMPs-related molecules.

[0009] Furthermore, the DAMPs-related molecules include interleukin-1α, interleukin-1β, tumor necrosis factor-β, and interleukin-6.

[0010] Furthermore, the drug is administered by intragastric or intraperitoneal injection.

[0011] Furthermore, the effective dosage of the drug is 50 mg / Kg to 200 mg / Kg.

[0012] Regarding the drug, preferably, the drug also includes a pharmaceutically acceptable carrier, such carriers include (but are not limited to): diluents, buffers, suspensions, emulsions, granules, encapsulations, excipients, fillers, adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents or adsorption carriers.

[0013] The drug of the present invention can be prepared into various dosage forms as required, including but not limited to tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories.

[0014] The administration route of the drug of the present invention is not limited as long as it can exert the desired therapeutic or preventive effect, including but not limited to oral, intravenous injection, intramuscular injection, subcutaneous injection, sublingual dissolution, rectal irrigation, nasal spray, oral spray, local skin or systemic transdermal administration.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] 1. The present invention discloses the use of 4-GBA in the preparation of a drug for improving the infection tolerance of sepsis patients. By using 4-GBA to treat mice infected with sepsis, the survival rate of mice treated with 4-GBA is much higher than that of control mice. The experiment confirms that 4-GBA has a protective effect on sepsis infection.

[0017] 2. The present invention studies the mechanism of 4-GBA improving the infection tolerance of sepsis patients. After treatment with 4-GBA, the content of liver function biomarker alanine aminotransferase and kidney function biomarker creatinine in the peripheral blood serum of mice infected with sepsis was significantly reduced; and after treatment with 4-GBA, the content of damage-associated molecular pattern (DAMPs) related molecules interleukin-1α (IL-1α), interleukin-1β (IL-1β), tumor necrosis factor-β (TNF-β), and interleukin-6 (IL-6) in the peripheral blood serum of mice infected with sepsis was significantly reduced. 4-GBA does not have in vivo and in vitro bactericidal function and regulates the body's immune function. It shows that 4-GBA can improve the infection tolerance of sepsis patients.

[0018] 3. The present invention discloses the use of 4-GBA in the preparation of a drug for improving the infection tolerance of sepsis patients, and clarifies that 4-GBA can reduce liver and kidney function and improve the survival rate of septic mice, which provides a new direction and new drug for the treatment of sepsis patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific embodiments of the present invention, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below.

[0020] Figure 1 This is the result of Example 1, which shows the effect of 4-GBA on the survival of mice infected with sepsis.

[0021] Figure 2 This is the result of bacterial plating of tissues and organs of septic mice infected with E. coli by 4-GBA in Example 2.

[0022] Figure 3 The results of the in vitro antibacterial experiment of 4-GBA in Example 3 are shown.

[0023] Figure 4 This is the experimental result of Example 4 on the subtypes of peripheral blood immune cells of 4-GBA-infected E. coli-infected septic mice.

[0024] Figure 5 This is the result of Example 4 4-GBA detection of liver and kidney function biomarkers in E. coli infected septic mice.

[0025] Figure 6 This is the result of Example 5 on the effects of 4-GBA on the damage-associated molecular patterns (DAMPs) in the peripheral blood serum of septic mice infected with E. coli. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but it should not be understood as limiting the scope of protection of the present invention. Any equivalent changes in form made based on the concept of the present invention should be regarded as the scope of the present invention.

[0027] The present invention provides a general and / or specific description of the materials and test methods used in the experiment. The test methods or test methods involved are all conventional methods unless otherwise specified; the reagents or instruments used are all conventional products available on the market, prepared or used in a conventional manner, unless the manufacturer is specified.

[0028] Sources

[0029] The drug guanidine butyric acid (4-GBA) used in the experiment was purchased from Sigma.

[0030] C57BL / 6 mice were purchased from the Experimental Animal Center of Army Medical University.

[0031] 4-GBA: 4-guanidinobutyric acid, molecular formula: NH=C(NH2)NHCH2CH2CH2COOH. Molecular weight: 145.16. Molecular structure formula is as follows.

[0032]

[0033] Example 1 Mouse survival experiment

[0034] Prepare 4-GBA drug solution, weigh an appropriate amount of 4-GBA powder and dissolve it in 1 ml of normal saline, and prepare 4-GBA drug solutions with drug concentrations of 0 mg / ml, 10 mg / ml, 20 mg / ml, and 40 mg / ml respectively. The final oral drug volume for mice is 100 ul.

[0035] C57BL / 6J male mice aged 6 to 8 weeks and weighing 18 to 22 g were selected; the mice were divided into a control group (0 mg / Kg), a 4-GBA low-dose group (50 mg / Kg), a 4-GBA medium-dose group (100 mg / Kg), and a 4-GBA high-dose group (200 mg / Kg), with 8 mice in each group; the control group (0 mg / Kg), the 4-GBA low-dose group (50 mg / Kg), the 4-GBA medium-dose group (100 mg / Kg), and the 4-GBA high-dose group (200 mg / Kg) were orally administered with 100 ul of 4-GBA drug solution with drug concentrations of 0 mg / ml, 10 mg / ml, 20 mg / ml, and 40 mg / ml by intraperitoneal injection of E. coli (1×10 7 CFU / mouse), and the survival of mice was observed and recorded every hour.

[0036] The survival of mice in the 4-GBA control group, low-dose group, medium-dose group, and high-dose group can be found in Figure 1 , it can be seen from the figure that oral administration of 4-GBA in the high-dose group (200 mg / Kg) 2 hours in advance can improve the survival rate of mice compared with the control group (0 mg / Kg). P < 0.01, there is a statistical difference between the control group (0 mg / Kg) and the 4-GBA high-dose group (200 mg / Kg), indicating that 4-GBA can improve the survival rate of septic mice.

[0037] Example 2: Tissue and organ bacterial plating experiment

[0038] C57BL / 6J male mice aged 6 to 8 weeks and weighing 18 to 22 g were selected. The mice were divided into a control group, an E. coli group, and an E. coli + 4-GBA (200 mg / Kg) group, with 4 mice in each group. The control group and the E. coli group were orally gavaged with 100 ul of a 0 mg / ml 4-GBA drug solution 2 hours in advance; the E. coli + 4-GBA (200 mg / Kg) group was orally gavaged with 100 ul of a 40 mg / ml 4-GBA drug solution 2 hours in advance, and intraperitoneally injected with E. coli (1×10 7 CFU / mouse); 12 hours later, the heart, liver, spleen, lung, kidney, peripheral blood, and peritoneal lavage fluid of the mice were taken under sterile conditions for bacterial plating and incubated in a 37°C incubator for 12 hours. The bacterial colonies were observed and counted.

[0039] Mouse tissue bacterial plating results are shown in Figure 2 , it can be seen from the figure that 4-GBA cannot reduce the bacterial load in the heart, liver, spleen, lung, kidney, peripheral blood (BLF), and peritoneal lavage fluid (PLF). P>0.05, there is no statistical difference between the 4-GBA+E.coli group and the E.coli group, indicating that 4-GBA does not have a bactericidal effect in vivo.

[0040] Example 3 In vitro antibacterial experiment

[0041] Thaw the E. coli strain frozen in -80℃ refrigerator at room temperature. Use a bacterial inoculation loop to spread E. coli on the bacterial culture dish; incubate in a 37℃ incubator for 12 hours to generate colonies, pick out a single colony with a cotton swab, and prepare a bacterial suspension with 3mL of normal saline; use a McFarland turbidimeter to detect the bacterial concentration, and prepare it to 0.5 McFarland unit (bacterial count is about 1.5×10 8 cfu / mL). Dilute 1000 times with LB broth, add 50ul to 96 wells, and finally obtain a bacterial suspension concentration of about 5.0×10 5cfu / mL. Weigh an appropriate amount of guanidine butyric acid (4-GBA), prepare 4-GBA with physiological saline to an initial concentration of 40mM, dilute the drug with LB broth by two-fold dilution, and the drug concentrations after dilution are 20mM, 10mM, 5mM, 2.5mM, 1.25mM, 0.625mM, 0.3125mM, and 0mM, respectively. Take a 96-well plate, set up drug control, blank control, and positive control; 50ul drug + 50ul diluted bacterial suspension in each well, 4 replicates per group; after oscillation and mixing, incubate at 37°C, 5% CO2 for 16 hours. Detect the OD value at 600nm with an enzyme marker.

[0042] The results of the in vitro antibacterial test of 4-GBA are shown in Figure 3 ,It can be seen from the figure that 4-GBA cannot inhibit the growth of E. coli, indicating that 4-GBA does not have a bactericidal effect in vitro.

[0043] Example 4 Detection of peripheral blood immune cell subtypes and peripheral blood serum liver and kidney function biomarkers

[0044] C57BL / 6J male mice aged 6 to 8 weeks and weighing 18 to 22 g were selected. There were 4 mice in each group, including the control group, the E. coli group, and the E. coli + 4-GBA (200 mg / Kg) group. The control group and the E. coli group were orally gavaged with 100 ul of 0 mg / ml 4-GBA drug solution 2 hours in advance; the E. coli + 4-GBA (200 mg / Kg) group was orally gavaged with 100 ul of 40 mg / ml 4-GBA drug solution 2 hours in advance, and E. coli (1×10 7 CFU / mouse). After 12 hours, peripheral venous blood was collected from mice by orbital blood sampling. Blood was collected using anticoagulant tubes, split red, stained with flow cytometry antibodies, and cell subtypes were detected on a flow cytometer. At the same time, ELISA was used to detect the expression levels of alanine aminotransferase (ALT), a liver function biomarker, and creatinine (Cre), a kidney function biomarker, in serum.

[0045] The results of the experiment on the effect of 4-GBA on the subtypes of peripheral blood immune cells in mice with sepsis infected with E. coli are shown in Figure 4 As can be seen from the figure, oral administration of 4-GBA (200 mg / Kg) + E. coli group 2 hours in advance can reduce the percentage of peripheral blood NK cells, CD4+T cells, CD8+T cells, DC cells, Macrophage, and Neutrophils immune cell subtypes compared with the E. coli group. P>0.05, there is no statistical difference between the 4-GBA+E. coli group and the E. coli group. This shows that 4-GBA does not have the function of regulating the body's immunity.

[0046] The results of 4-GBA detection of liver and kidney function biomarkers in E. coli infected septic mice are shown in Figure 5 , it can be seen from the figure that oral administration of 4-GBA (200mg / Kg) + E. coli 2 hours in advance can reduce the liver function biomarker alanine aminotransferase (ALT) and kidney function biomarker creatinine (Cre) compared with the E. coli group. P < 0.01, there is a statistical difference between the 4-GBA + E. coli group and the E. coli group. This shows that 4-GBA can protect the host organs.

[0047] Example 5 Detection of Damage Related Molecular Patterns (DAMPs) Related Molecules in Peripheral Blood Serum

[0048] C57BL / 6J male mice aged 6 to 8 weeks and weighing 18 to 22 g were selected; there were 4 mice in each group, including the control group, the E. coli group, and the E. coli + 4-GBA (200 mg / Kg) group. The control group and the E. coli group were orally gavaged with 100 ul of 0 mg / ml 4-GBA drug solution 2 hours in advance. The E. coli + 4-GBA (200 mg / Kg) group was orally gavaged with 100 ul of 40 mg / ml 4-GBA drug solution 2 hours in advance. E. coli (1×10 7 CFU / mouse). After 12 hours, peripheral venous blood of mice was collected using a coagulation tube. Serum was separated by centrifugation at 4°C and 3000prm for 15 minutes. The expression levels of DAMPs-related molecules interleukin-1α (IL-1α), interleukin-1β (IL-1β), tumor necrosis factor-β (TNF-β), and interleukin-6 (IL-6) in peripheral blood serum were detected using a multifactor detection kit.

[0049] The results were analyzed using GraphPad Prism 8.02 software. The t test was used between two groups. The ANAVA analysis of variance was used between multiple groups. Survival analysis was performed using Mantel-Cox. The test level was α = 0.05, *P < 0.05, there was a statistical difference. **P < 0.01, statistically significant difference. ns, P > 0.05, no statistical difference.

[0050] The results of 4-GBA on the damage-associated molecular patterns in the peripheral blood serum of E. coli-infected septic mice are as follows Figure 6, it can be seen from the figure that oral administration of 4-GBA (200 mg / Kg) + E. coli group 2 hours in advance can reduce the DAMPs related molecules interleukin-1α (IL-1α), interleukin-1β (IL-1β), tumor necrosis factor-β (TNF-β), interleukin-6 (IL-6) in peripheral blood serum compared with E. coli group. P < 0.05, there is a statistical difference between the 4-GBA + E. coli group and the E. coli group. This shows that 4-GBA can improve the survival rate of septic mice by inhibiting the molecules related to damage-associated molecular patterns.

[0051] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

Application of 1.4-GBA in the preparation of drugs for improving infection tolerance of sepsis patients.

2. The use according to claim 1, characterized in that: The drug enhances the patient's infection tolerance by reducing DAMPs-related molecules.

3. The use according to claim 2, characterized in that: The DAMPs-related molecules include interleukin-1α, interleukin-1β, tumor necrosis factor-β, and interleukin-6.

4. The use according to claim 2 or 3, characterized in that: The drug is administered by intragastric or intraperitoneal injection.

5. The use according to claim 4, characterized in that: The effective dosage of the drug is 50 mg / Kg to 200 mg / Kg.

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