Use of inhibiting MST1 / 2 in the treatment of diseases infected with polymyxin-resistant bacteria

Abstract

The present invention relates to the field of medical technology, and in particular to the application of inhibiting MST1 / 2 or knocking out MST1 / 2 in macrophages in the treatment of diseases infected with polymyxin-resistant bacteria. The present invention finds that intraperitoneal injection of the MST1 / 2 inhibitor XMU-MP-1 can significantly improve the survival rate of mice infected with Escherichia coli carrying the transferable polymyxin gene (mcr)-3; and inhibiting MST1 / 2 or knocking out MST1 / 2 in macrophages can significantly reduce the tissue bacterial load of mice and improve the inflammatory response level of mice; using the MST1 / 2 inhibitor or knocking out MST1 / 2 in macrophages to treat diseases related to mcr-3 Escherichia coli infection can effectively improve the inflammatory response level of mice and promote the clearance of mcr-3 Escherichia coli in the body; the present invention provides a new option and idea for the current treatment of diseases related to transferable polymyxin-resistant bacterial infection.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of inhibiting MST1 / 2 in the treatment of diseases infected with polymyxin-resistant bacteria. Background Art

[0002] With the widespread use of antibiotics in animal husbandry and animal treatment, bacterial resistance has gradually emerged and intensified, posing a significant challenge to the clinical treatment of infectious diseases and the development of new drugs. In recent years, the number of antibiotics available to treat drug-resistant bacterial infections has been decreasing. Among them, polymyxins are the "last line of defense" against drug-resistant Gram-negative infections.

[0003] Colistin is a narrow-spectrum antimicrobial agent originating from polymyxobacteria. Over the past decade, the emergence of multidrug-resistant Gram-negative bacteria has led to a significant increase in colistin use. Transferable colistin resistance genes (mcr) have also emerged and rapidly spread among Gram-negative bacteria, leading to the emergence of colistin resistance in already multidrug-resistant bacteria and compromising antibiotic therapy. Ten plasmid-transmitted mcr gene variants have been reported, with mcr-1 and mcr-3 being the most prevalent. Our group has previously demonstrated that, compared with mice infected with bacteria without mcr-3, mice infected with bacteria carrying mcr-3 exhibit a deficient inflammatory response and reduced bacterial clearance from tissues during the initial infection phase. Ultimately, mice infected with bacteria carrying mcr-3 have higher mortality rates than mice infected with bacteria without mcr-3. Therefore, finding ways to reduce antibiotic use is crucial to prevent the further development of antibiotic resistance.

[0004] Macrophages are a crucial component of the body's innate immunity, crucial for clearing pathogens and initiating immune responses. Therefore, enhancing the immune clearance of pathogens by the body's own macrophages could help reduce antibiotic use in the treatment of bacterial infections. MST1 / 2, a key component of the Hippo pathway, was first discovered in Drosophila. MST1 / 2 phosphorylates downstream LATS1 / 2, which in turn phosphorylates YAP / TAZ. By preventing nuclear translocation, MST1 / 2 inhibits the function of YAP / TAZ as transcriptional regulators, regulating physiological processes such as cell proliferation, differentiation, and organ size. Furthermore, MST1 / 2 plays a crucial role in innate immunity. Studies have shown that MST1 / 2 not only regulates the production of inflammatory cytokines and type I interferons, impacting macrophage phagocytosis and clearance of pathogens, but also modulates the differentiation of regulatory and effector T cells, further contributing to maintaining homeostasis. However, there are currently no reports of the use of MST1 / 2 inhibitors or MST1 / 2 knockout in macrophages for the treatment of infections with polymyxin-resistant bacteria.

[0005] In summary, it is urgent to solve the problem of polymyxin resistance. Finding adjuvants or methods that can assist antibiotic treatment and enhance the immune response to pathogenic microorganisms is crucial to combating bacterial infections and reducing the use of antibiotics. Summary of the Invention

[0006] In view of this, the present invention provides an application of inhibiting MST1 / 2 or knocking out MST1 / 2 in macrophages in the treatment of diseases infected with polymyxin-resistant bacteria.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides the use of inhibition, knockdown or knockout of MST1 / 2 in any of the following:

[0009] (I) Preparation of drugs for use against polymyxin-resistant bacteria; and / or

[0010] (II) Preparation of drugs for treating diseases caused by polymyxin-resistant bacterial infections; and / or

[0011] (III), preparing antibiotic adjuvants; and / or

[0012] (IV) preparing a formulation that reduces the emergence of polymyxin resistance genes.

[0013] In some embodiments of the present invention, the inhibition is performed using an inhibitor, including but not limited to XMU-MP-1.

[0014] In some specific embodiments of the present invention, the polymyxin-resistant bacteria include Escherichia coli containing the mcr-3 gene.

[0015] In some specific embodiments of the present invention, the resistance to polymyxin-resistant bacteria includes reducing the bacterial load in animal tissues, and the reducing the bacterial load in animal tissues includes promoting the clearance of mcr-3 Escherichia coli in the animal body.

[0016] In some embodiments of the invention, the treatment comprises promoting the clearance of mcr-3 E. coli from the body.

[0017] In some specific embodiments of the present invention, the treatment comprises increasing the level of inflammatory response in the animal, and the increasing the level of inflammatory response in the animal comprises increasing the level of IL-1β and / or TNF-α in the animal's liver.

[0018] In some embodiments of the invention, the treating comprises increasing the survival rate of the animal.

[0019] In some specific embodiments of the present invention, the animal comprises a polymyxin-resistant bacterial infection model, wherein the polymyxin-resistant bacterial infection model is a mouse intraperitoneal injection of 5×10 8 or 8×10 8 CFU of polymyxin-resistant bacteria, including mcr-3 Escherichia coli.

[0020] The present invention found that in mice infected with E. coli carrying mcr-3, intraperitoneal injection of the MST1 / 2 inhibitor XMU-MP-1 can significantly improve the survival rate of mice; and inhibiting MST1 / 2 or knocking out MST1 / 2 in macrophages can significantly reduce the tissue bacterial load of mice and improve the inflammatory response level of mice; using MST1 / 2 inhibitors or knocking out MST1 / 2 in macrophages to treat diseases related to mcr-3 E. coli infection can effectively improve the inflammatory response level of mice and promote the clearance of mcr-3 E. coli in the body.

[0021] The present invention has the beneficial effects of applying MST1 / 2 inhibitors or macrophage MST1 / 2 knockout strategies to drug development or treatment research, enhancing the body's immune response in mcr-3-related infections, promoting clearance of mcr-3 E. coli, and assisting with antibiotic therapy, further reducing antibiotic use while achieving effective therapeutic effects. This invention provides a novel option and approach for the current treatment of mcr-3-related infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0023] Figure 1 It was shown that intraperitoneal injection of MST1 / 2 inhibitors reduced the mortality of mice infected with mcr-3 Escherichia coli;

[0024] Figure 2 The results show that intraperitoneal injection of MST1 / 2 inhibitors or knockout of MST1 / 2 in macrophages reduced the tissue bacterial load of mice infected with mcr-3 Escherichia coli. The data are mean ± standard deviation, and the statistical method was t-test.

[0025] Figure 3 Intraperitoneal injection of MST1 / 2 inhibitors or knockout of MST1 / 2 in macrophages increased the level of IL-1β in the livers of mice infected with mcr-3 Escherichia coli. The ● group represents the WT group, the ■ group represents the XMU-MP-1 group, and the ▲ group represents the MST1 / 2 cKO group. Data are mean ± SD, and the statistical method was t-test.

[0026] Figure 4 The results showed that intraperitoneal injection of MST1 / 2 inhibitors or knockout of MST1 / 2 in macrophages increased the level of TNF-α in the liver of mice infected with mcr-3 Escherichia coli. Among them, the ● group is the WT group, the ■ group is the XMU-MP-1 group, and the ▲ group is the MST1 / 2 cKO group. The data are all mean ± standard deviation, and the statistical method is t test. DETAILED DESCRIPTION

[0027] The present invention discloses the use of inhibiting MST1 / 2 or knocking out MST1 / 2 in macrophages in the treatment of diseases infected with polymyxin-resistant bacteria. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve this. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0028] Gene-edited mice have been widely used in scientific research. The LysM Cre ;Mst1 / 2 flox / flox The mice were gene-edited mice with myeloid cell-specific knockout of MST1 / 2.

[0029] The present invention provides an application of inhibiting MST1 / 2 or knocking out MST1 / 2 in macrophages in the treatment of diseases infected with polymyxin-resistant bacteria. The MST1 / 2 inhibitor XMU-MP-1 used is purchased from MedChemExpress with the product number HY-100526. The knockout mice used are LysM Cre Mice and Mst1 / 2 flox / flox Mouse breeding, LysM Cre Mice and Mst1 / 2 flox / flox All mice were purchased from Jackson Labs. The Escherichia coli DH5α-pHSG299-mcr3 strain (mcr-3 E. coli, previously published in DOI 10.1002 / advs.202101336) was constructed and maintained in our laboratory. Unless otherwise noted, the reagents, methods, and equipment used in this article are conventional in the art.

[0030] The present invention will be further described below in conjunction with the embodiments:

[0031] Example 1

[0032] 1. Experimental Animals

[0033] Twenty-four 6-8 week old female C57BL / 6J mice were randomly divided into two groups, with 12 mice in each group:

[0034] mcr-3 Escherichia coli infection group (E. coli group): mice were intraperitoneally injected with 8×10 8 At the same time as CFU of mcr-3 Escherichia coli, the same volume of DMSO was intraperitoneally injected as a solvent control for XMU-MP-1;

[0035] mcr-3 E. coli infection XMU-MP-1 treatment group (E. coli + XMU-MP-1 group): mice were intraperitoneally injected with 8×10 8 At the same time, 2 mg / kg XMU-MP-1 was injected intraperitoneally with 100 CFU of mcr-3 Escherichia coli.

[0036] 2. mcr-3 Escherichia coli infection model

[0037] The E. coli group was intraperitoneally injected with 8×10 8 CFU of mcr-3 Escherichia coli, and the same volume of DMSO was injected intraperitoneally as a solvent control of XMU-MP-1, and DMSO was given again after 24 h;

[0038] The E. coli + XMU-MP-1 group was intraperitoneally injected with 8×10 8CFU of mcr-3 E. coli and 2 mg / kg XMU-MP-1, and XMU-MP-1 was given again 24 h later;

[0039] The mortality of mice was recorded for 48 consecutive hours, and the survival curve was drawn.

[0040] 3. Experimental Results

[0041] MST1 / 2 inhibitors 8×10 8 The effect of mcr-3 CFU Escherichia coli infection on the mortality of mice (the results are shown in Table 1, Figure 1 XMU-MP-1 significantly reduced the mortality of mice infected with mcr-3 E. coli. The solid black line represents the survival rate of mice infected with mcr-3 E. coli, while the dashed black line represents the survival rate of mice infected with mcr-3 E. coli and treated with XMU-MP-1.

[0042] Table 1 Number of mice deaths at different time points

[0043]

[0044]

[0045] The results showed that MST1 / 2 inhibitors could effectively improve the survival rate of mice infected with mcr-3 Escherichia coli.

[0046] Example 2

[0047] 1. Experimental Animals

[0048] Take 6-8 week old female C57BL / 6J mice (including 8 Mst1 / 2 flox / flox mice and 4 LysM Cre ;Mst1 / 2 flox / flox mouse), Mst1 / 2 flox / flox Mice were randomly divided into 2 groups, 4 in each group;

[0049] LysM Cre ;Mst1 / 2 flox / flox Mice, 4 per group;

[0050] mcr-3 Escherichia coli infection control group (WT group): Mst1 / 2 flox / flox Mice were intraperitoneally injected with 5×10 8 CFU of mcr-3 Escherichia coli, and the same volume of DMSO was injected intraperitoneally as a solvent control for XMU-MP-1;

[0051] mcr-3 Escherichia coli infection XMU-MP-1 treatment group (WT+XMU-MP-1 group): Mst1 / 2 flox / floxMice were intraperitoneally injected with 5×10 8 CFU of mcr-3 E. coli were simultaneously injected intraperitoneally with 2 mg / kg XMU-MP-1;

[0052] mcr-3 Escherichia coli infection MST1 / 2 knockout group (MST1 / 2 cKO group): LysM Cre ;Mst1 / 2 flox / flox Mice were intraperitoneally injected with 5×10 8 CFU of mcr-3 Escherichia coli, and the same volume of DMSO was injected intraperitoneally as a solvent control of XMU-MP-1.

[0053] 2. mcr-3 Escherichia coli infection model

[0054] WT group was treated with Mst1 / 2 flox / flox Mice were intraperitoneally injected with 5×10 8 CFU of mcr-3 Escherichia coli, and the same volume of DMSO was injected intraperitoneally as a solvent control of XMU-MP-1, and DMSO was given again after 24 h;

[0055] Mst1 / 2 was used in the WT+XMU-MP-1 group flox / flox Mice were injected intraperitoneally with 5×10 8 CFU of mcr-3 E. coli and 2 mg / kg XMU-MP-1, and XMU-MP-1 was given again 24 h later;

[0056] LysM was used in the MST1 / 2 cKO group. Cre ;Mst1 / 2 flox / flox Mice were intraperitoneally injected with 5×10 8 CFU of mcr-3 Escherichia coli, and the same volume of DMSO was injected intraperitoneally as a solvent control of XMU-MP-1, and DMSO was given again after 24 h;

[0057] 48 hours after infection, the heart, liver, spleen, lungs, and kidneys were aseptically removed for homogenization, and the bacterial load in the tissues was detected by bacterial titration. The liver was removed for RNA extraction and the levels of inflammatory factors were detected.

[0058] 3. Experimental Results

[0059] 1. Effect of inhibiting MST1 / 2 or knocking out macrophage MST1 / 2 on the bacterial load in mouse tissues infected with mcr-3 E. coli. After homogenization of the tissues, the supernatant was diluted serially and the bacterial load in the mouse tissues was detected by plate counting (the results are shown in Table 2). Figure 2Compared with the WT group, bacterial loads in the heart, liver, and spleen tissues of the XMU-MP-1 group were significantly decreased (P < 0.05). The bacterial loads in the liver and spleen tissues of the MST1 / 2 cKO group were also significantly decreased (P < 0.05). Compared with the WT group, bacterial loads in the lung and kidney tissues of both the XMU-MP-1 and MST1 / 2 cKO groups showed a downward trend, but no statistical differences were found (P > 0.05). In the figure, * indicates a statistically significant difference between the XMU-MP-1 group and the WT group (P < 0.05); # indicates a statistically significant difference between the MST1 / 2 cKO group and the WT group (P < 0.05).

[0060] Table 2 Tissue bacterial load (CFU / g)

[0061]

[0062]

[0063] The results showed that inhibiting MST1 / 2 or knocking out macrophage MST1 / 2 could reduce the tissue bacterial load of mice infected with mcr-3 Escherichia coli.

[0064] 2. Effects of MST1 / 2 inhibition or macrophage MST1 / 2 knockout on the immune response of mice infected with mcr-3 E. coli. RNA was extracted from mouse liver tissue, and the levels of inflammatory factors were detected by qRT-PCR (results are shown in Tables 3 and 4). The levels of IL-1β are shown in Tables 3 and 4. Figure 3 Compared with the WT group (● group in the figure), the IL-1β mRNA content in the XMU-MP-1 group (■ group in the figure) was significantly increased (P < 0.05), and the MST1 / 2 cKO group (▲ group in the figure) was significantly increased but without statistical difference (P > 0.05). Figure 4 Compared with the WT group (● group in the figure), the TNF-α mRNA level in the XMU-MP-1 group (■ group in the figure) was significantly increased (P < 0.05). In the MST1 / 2 cKO group (▲ group in the figure), the mRNA level was significantly increased, but the difference was not statistically significant (P > 0.05). * indicates a statistically significant difference between the XMU-MP-1 group and the WT group (P < 0.05).

[0065] Table 3 Relative content of IL-1β mRNA (×10 -2 )

[0066]

[0067] Table 4 Relative content of TNF-α mRNA (×10 -2 )

[0068]

[0069] The results showed that inhibition of MST1 / 2 or knockout of macrophage MST1 / 2 could increase the levels of IL-1β and TNF-α in the liver of mice infected with mcr-3 Escherichia coli.

[0070] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of MST1 / 2 inhibition in any of the following: (I) Preparation of drugs for use against polymyxin-resistant bacteria; and / or (II) Preparation of drugs for treating diseases caused by polymyxin-resistant bacterial infections; The inhibition adopts an inhibitor, and the inhibitor is XMU-MP-1; The polymyxin-resistant bacteria contain mcr-3 genes of Escherichia coli.

2. The use according to claim 1, characterized in that The anti-polymyxin-resistant bacteria include reducing the bacterial load in animal tissues.

3. The use according to claim 2, characterized in that The method of reducing the bacterial load in animal tissues includes promoting mcr-3 Elimination of E. coli.

4. The use according to claim 1, wherein The treatment involves promoting mcr-3 Elimination of E. coli.

5. The use according to claim 1, characterized in that The treatment involves increasing the level of an inflammatory response in the animal.

6. The use according to claim 5, characterized in that The increasing of the inflammatory response level of the animal includes increasing the IL-1β content and / or TNF-α content in the animal's liver.

7. The use according to claim 1, characterized in that The treatment includes increasing the survival rate of the animal.

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