Use of levosimendan in the preparation of inhibitors of salmonella typhimurium cdsH

Abstract

The application belongs to the technical field of biological medicine, and provides application of levosimendan in preparation of a Salmonella typhimurium CDSH inhibitor. The application verifies that levosimendan can inhibit CDSH enzyme activity, reduce the tolerance of S. Typhimurium, and enhance the curative effect of gentamicin through CDSH enzyme activity inhibition experiments, lead acetate test paper strip detection experiments, H2S fluorescent probe detection experiments, gentamicin stress experiments, and Galleria mellonella infection model experiments, so that levosimendan can be used for preparing the CDSH inhibitor, and then used for preparing a drug for treating infectious diseases caused by S. Typhimurium.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of levosimendan in the preparation of CDSH inhibitors of Salmonella typhimurium. Background Technology

[0002] Salmonella infection is a major public health burden, and its antibiotic resistance has made the treatment of salmonellosis difficult. For example, the formation of resistance to first-line treatment drugs such as ceftriaxone and azithromycin often leads to clinical treatment failure, thereby increasing medical costs and mortality. More importantly, the development of bacterial tolerance has also promoted the development of drug resistance.

[0003] Endogenously produced hydrogen sulfide (H2S) in bacteria can help them resist antibiotic stress through multiple mechanisms, leading to clinical treatment failure. This H2S-mediated defense mechanism is one of the main causes of bacterial tolerance. Furthermore, the H2S-mediated tolerance mechanism also provides a target for developing novel antibacterial strategies. In Salmonella Typhimurium, the degradation of L-cysteine ​​by cysteine ​​desulfhydrase (CDSH) is one of the pathways for H2S production. Therefore, developing CDSH inhibitors to reduce bacterial tolerance and enhance antibiotic efficacy is a novel strategy for combating Salmonella infections.

[0004] Levosimendan, molecular formula C 14 H 12 N6O, with a molecular weight of 280.28, is a novel calcium sensitizer-like positive inotropic drug. As a calcium sensitizer, it primarily works by increasing the responsiveness of cardiac contractile proteins to calcium. 2+ Levosimendan enhances myocardial contractility by activating adenosine triphosphate (ATP)-sensitive potassium channels to dilate coronary and peripheral blood vessels without increasing myocardial oxygen consumption. This drug is suitable for short-term treatment of acute decompensated heart failure that does not respond well to conventional treatments. However, there are no reports in the existing technology regarding levosimendan for the treatment of bacterial infections. Summary of the Invention

[0005] The purpose of this invention is to provide the application of levosimendan in the preparation of CDSH inhibitors for Salmonella typhimurium, aiming to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] On the one hand, the present invention provides the application of levosimendan in the preparation of a CDSH inhibitor of Salmonella Typhimurium, wherein the Salmonella Typhimurium is S. Typhimurium SL1344.

[0008] On the other hand, the present invention provides the use of levosimendan in the preparation of drugs for the prevention or treatment of Salmonella typhimurium infection, wherein the Salmonella typhimurium is S. typhimurium SL1344.

[0009] On the other hand, the present invention provides the use of levosimendan and gentamicin in combination in the preparation of a drug for the prevention or treatment of Salmonella typhimurium infection, wherein the Salmonella typhimurium is S. typhimurium SL1344, the dosage of levosimendan is 20 mg / kg, and the dosage of gentamicin is 4 mg / kg.

[0010] Compared with the prior art, the specific beneficial effects of the present invention are as follows:

[0011] This invention verifies through CDSH enzyme activity inhibition experiments, lead acetate test strip detection experiments, H2S fluorescent probe detection experiments, gentamicin stress experiments, and large wax moth infection model experiments that levosimendan can reduce S. typhimurium tolerance and enhance the efficacy of gentamicin by inhibiting CDSH enzyme activity. Therefore, levosimendan can be used to prepare CDSH inhibitors, and further used to prepare drugs for treating infectious diseases caused by Salmonella typhimurium. Attached Figure Description

[0012] Figure 1 This is a diagram showing the inhibition of CDSH enzyme activity by levosimendan provided in Example 1 of the present invention;

[0013] Figure 2 Visual image of the inhibition of Salmonella Typhimurium H2S by levosimendan provided in Embodiment 2 of the present invention;

[0014] Figure 3 This is a graph showing the effect of levosimendan on H2S content in bacteria, provided in Example 3 of the present invention.

[0015] Figure 4 This is a quantitative graph of the number of surviving bacteria under gentamicin stress provided by levosimendan in Example 4 of the present invention;

[0016] Figure 5 The survival rate of the *Hemiberlesia lataniae* infection model before and after treatment with levosimendan alone or in combination with gentamicin antibiotics, as provided in Example 5 of this invention;

[0017] In the attached figures, statistical analysis was performed using GraphPad Prism 9.0. All experiments were independently repeated three times. Data are expressed as mean ± standard deviation (SD). The significance of differences between groups was assessed using one-way ANOVA, and the significance level was indicated by an asterisk in the results. ). Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0019] The levosimendan of this invention has the molecular formula C 14 H 12 N6O, with a molecular weight of 280.28, has the following molecular structure:

[0020] ;

[0021] The Salmonella Typhimurium in this embodiment of the invention is specifically S. Typhimurium SL1344, which was donated by Professor Qiu Jiazhang of Jilin University.

[0022] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0023] Example 1: Assay on the inhibition of CDSH enzyme activity by levosimendan:

[0024] Preparation and purification of CDSH protein: The cdsH gene fragment was amplified from the bacterial genome of *S. typhimurium* SL1344 and constructed into the pCold-SUMO plasmid (purchased from Changsha Aibiwei Biotechnology Co., Ltd.) by enzyme digestion and ligation to obtain the recombinant plasmid pCold-SUMO-cdsH. This plasmid was transformed into competent expression cells, plated, cultured, and single colonies containing the recombinant plasmid pCold-SUMO-cdsH were picked and inoculated into LB liquid medium containing 100 μg / mL ampicillin. The cells were cultured overnight at 37°C and 180 rpm with shaking. The following day, after expansion, the cells were cultured to mid-logarithmic growth. IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.1 mM, and expression was induced for 24 h at 20°C and 180 rpm. After induction, the bacterial pellet was collected and resuspended in pre-chilled lysis buffer (20 mM Tris-HCl, pH 8.5, containing 300 mM... The lysis buffer, which had been disrupted by ultrasonication, was centrifuged at 4°C and 12,000 rpm for 30 min in NaCl and 5 mM β-mercaptoethanol. The supernatant was collected and then purified using a Ni-NTA affinity chromatography column.

[0025] The CDSH enzyme activity inhibition assay was performed in 96-well plates. The CDSH enzyme activity was reflected by the characteristic that the enzyme catalyzes the production of PbS (lead sulfide) from L-cysteine ​​via H2S. The total reaction volume was 200 μL, containing 50 μM pyridoxal phosphate (PLP) and 0.25 mg / mL purified CDSH protein. Different concentrations of levosimendan (0, 50, 100, 200, 400 μM) were pre-incubated with CDSH enzyme in reaction buffer for 15 minutes. Then, 10 mM L-cysteine ​​and 0.4 mM lead acetate were added to the reaction buffer to initiate the reaction. After reacting at 37°C for 30 minutes, the absorbance of the reaction system was measured at 390 nm. The enzyme activity of the untreated group served as a 100% control, and the relative enzyme activity of each concentration group was calculated. The results are as follows: Figure 1 As shown, CDSH enzyme activity was significantly inhibited with increasing levosimendan concentration, confirming that levosimendan can be used as an effective small molecule inhibitor of this enzyme.

[0026] Example 2: Detection of levosimendan's inhibition of intrabacterial H2S production using lead acetate test strips:

[0027] First, overnight cultured *Salmonella typhimurium* was expanded at a ratio of 1:100 into fresh M9 medium containing 0.5 mM L-cysteine. Then, 200 μM or 400 μM levosimendan was added. Lead acetate test strips were placed on the inner wall of glass test tubes to detect bacterial H2S production. The samples were then incubated at 37°C in a shaker for 3 hours. The lead acetate test strips were then removed to observe the amount of bacterial H2S produced. The results are as follows: Figure 2 As shown, 200 μM or 400 μM of levosimendan can inhibit the production of H2S.

[0028] Example 3: Determination of the effect of levosimendan on H2S content in bacteria:

[0029] The amount of H2S produced in Salmonella was determined using the WSP-5 fluorescent probe. First, overnight cultured Salmonella Typhimurium was inoculated into fresh M9 medium containing 0.5 mM L-cysteine ​​at a 1:100 expansion ratio. The inoculated bacterial culture was then placed in a shaker at 37 ℃. Experimental groups were given 50 μM, 100 μM, 200 μM, and 400 μM levosimendan, while the control group received no levosimendan. After 3 h of culture, bacterial cells in the logarithmic growth phase were collected, and a final concentration of 10 μM WSP-5 fluorescent probe was added. The cells were incubated at 37 ℃ in the dark for 40 min. After incubation, the cells were washed twice with PBS and resuspended in PBS. Quantitative fluorescence measurement was performed using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 524 nm. The fluorescence intensity directly reflects the relative level of intracellular H2S.

[0030] The results are as follows Figure 3 As shown, the intracellular WSP-5 fluorescence signal intensity decreased with increasing levosimendan concentration, demonstrating that levosimendan can effectively inhibit CDSH-mediated hydrogen sulfide generation.

[0031] Example 4: Quantitative determination of the number of surviving Salmonella Typhimurium under gentamicin stress after treatment with a combination of levosimendan and gentamicin:

[0032] Cultures of Salmonella Typhimurium strain were inoculated at a ratio of 1:50 into M9 medium containing 0.5 mM L-cysteine, and different concentrations of levosimendan (50 μM, 100 μM, 200 μM, and 400 μM) were added. The culture was incubated in a constant temperature shaking incubator for 3 h, and then 60 μg / mL of gentamicin was added for stress incubation. The culture was continued in a constant temperature shaking incubator for another 3 h. After incubation, the bacterial cells were collected by centrifugation, washed twice with PBS to remove residual drug, and the bacterial culture was serially diluted and spread on plates. The plates were then incubated overnight at 37 °C, and the number of surviving colonies (CFU / mL) was accurately counted.

[0033] The results are as follows Figure 4 As shown, in the gentamicin stress experiment, the number of surviving colonies in the combined treatment groups of levosimendan (200, 400 μM) and gentamicin was significantly lower than that in the antibiotic-only treatment groups after 3 h, indicating that levosimendan can enhance the killing ability of gentamicin against Salmonella typhimurium.

[0034] Example 5: Experimental analysis of the effect of levosimendan on gentamicin treatment of Salmonella typhimurium-infected large wax moth model:

[0035] A Salmonella typhimurium infection model was established in the large wax moth (purchased from Tianjin Huiyude Biotechnology Co., Ltd.) to evaluate the protective effect of the combined use of levosimendan and gentamicin. First, bacterial cells in the logarithmic growth phase were collected by centrifugation, and then... 5 To establish an infection model, bacterial suspension was injected into the second abdominal leg of each larva using a microsyringe at a dose of CFU / larva. The larvae were randomly divided into three groups: gentamicin monotherapy group (4 mg / kg gentamicin), solvent control group (PBS), levosimendan monotherapy group (20 mg / kg levosimendan), and levosimendan combined with gentamicin group (20 mg / kg levosimendan + 4 mg / kg gentamicin) to compare the survival rate under different treatments. All treatment groups were incubated at 37 ℃, and the survival of larvae was accurately recorded every 6 h. Kaplan-Meier survival curves were plotted.

[0036] The results are as follows Figure 5As shown, the survival rate of the solvent control group was 0%, the survival rate of the levosimendan monotherapy group was about 10%, and the combined treatment group of levosimendan and gentamicin showed better efficacy, with a significant increase in larval survival rate (about 40%), demonstrating that levosimendan combined with gentamicin has significant synergistic therapeutic efficacy in the in vivo model.

[0037] In summary, the embodiments of the present invention demonstrate that levosimendan can reduce the tolerance of Salmonella typhimurium by inhibiting CDSH enzyme activity, providing a new approach and potential lead compound for the prevention and treatment of Salmonella infection, and verifying that levosimendan can have a synergistic therapeutic effect when used in combination with gentamicin.

[0038] Therefore, levosimendan can be used as a CDSH inhibitor to prepare drugs for the prevention or treatment of infectious diseases caused by Salmonella typhimurium. Levosimendan can also be used in combination with gentamicin to prepare drugs for the prevention or treatment of infectious diseases caused by Salmonella typhimurium.

[0039] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of levosimendan in the preparation of CDSH inhibitors for Salmonella typhimurium, characterized in that, The Salmonella Typhimurium in question is S. Typhimurium SL1344.

2. The application of levosimendan in the preparation of drugs for the prevention or treatment of Salmonella typhimurium infection, characterized in that, The Salmonella Typhimurium in question is S. Typhimurium SL1344.

3. The application of levosimendan according to claim 2 in the preparation of drugs for the prevention or treatment of Salmonella typhimurium infection, characterized in that, The levosimendan reduces Salmonella typhimurium tolerance by inhibiting CDSH enzyme activity, thereby preventing or treating Salmonella typhimurium infection.

4. The application of levosimendan and gentamicin in the preparation of drugs for the prevention or treatment of Salmonella typhimurium infection, characterized in that, The Salmonella Typhimurium is S. Typhimurium SL1344, the dosage of levosimendan is 20 mg / kg, and the dosage of gentamicin is 4 mg / kg.

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