阅读说明：本技术 芫花素在制备抗单增李斯特杆菌感染药物中的应用 (Application of genkwanin in preparation of drug for resisting listeria monocytogenes infection ) 是由 冯海华 徐蕾 王建锋 卢葛锦 邓旭明 于 2019-12-06 设计创作，主要内容包括：本发明提供了芫花素在制备抗单增李斯特杆菌感染药物中的应用,通过兔红细胞溶血试验、对J774细胞的保护作用实验及小鼠感染实验治疗学实验,证明芫花素在极低浓度下可以有效抑制单核细胞增生性李斯特杆菌溶血素的成孔活性,同时能够抑制单增李斯特菌对J774细胞介导的细胞毒性作用并对单增李斯特菌引起的小鼠感染有较好的治疗作用。本发明用芫花素治疗单核增生性李斯特菌感染,较传统的抗生素治疗相比不易诱导耐药性的产生且对细菌的选择压力较低,试验中未见显著的毒性作用,且具有较高的治愈率。因此芫花素可以用于新药开发,并对药物靶标确认有着重要意义。(The invention provides an application of genkwanin in preparation of a drug for resisting listeria monocytogenes infection, and through rabbit erythrocyte hemolysis test, J774 cell protection effect experiment and mouse infection experiment therapentic experiment, the genkwanin is proved to be capable of effectively inhibiting pore-forming activity of listeria monocytogenes hemolysis under extremely low concentration, simultaneously inhibiting J774 cell mediated cytotoxicity effect of listeria monocytogenes and having better treatment effect on mouse infection caused by listeria monocytogenes. The invention uses genkwanin to treat the listeria monocytogenes infection, is not easy to induce the generation of drug resistance and has lower selective pressure on bacteria compared with the traditional antibiotic treatment, has no obvious toxic effect in the test and has higher cure rate. Therefore, the genkwanin can be used for developing new drugs and has important significance for drug target confirmation.)

1. Application of genkwanin in preparing medicine for resisting Listeria monocytogenes infection is provided.

2. The use of claim 1, wherein said listeria monocytogenes infection is a bacterial infection by a listeria bacterium, and wherein said bacterial infection is an infection by a listeria monocytogenes bacterium.

3. The use as claimed in claim 1, wherein the genkwanin medicine is pharmaceutically acceptable vehicle, such as injection, capsule, tablet or powder for injection.

Technical Field

The invention relates to application of genkwanin in preparation of a drug for resisting Listeria monocytogenes infection, belonging to the field of medicines.

Background

Genkwanin (Genkwanin) is a flavonoid compound extracted from plant Genkwanin, and has been reported to have multiple biological activities such as anti-inflammation and antioxidation, but no related report for treating listeriosis monocytogenes exists at present.

Listeria monocytogenes is an important facultative anaerobic, zoonosis intracellular parasitic pathogenic bacterium and is a pathogenic bacterium of Listeria diseases. The environment is widely distributed, is resistant to high temperature, high salt and high pH value, can be proliferated in water, oil, fertilizer, animal feed, dairy products and refrigerators, and can infect people and livestock through feces-oral transmission, drinking water and other ways. As an opportunistic pathogen, Listeria monocytogenes mainly infects the old, pregnant women and people with low immunity, and is clinically manifested as gastroenteritis, meningitis, abortion, septicemia, even death and the like. The pathogenicity of the listeria monocytogenes is closely related to the secretion and activity of virulence factors thereof, wherein the listeria monocytogenes secreted hemolysin LLO can help listeria monocytogenes escape from phagocytes when internalizing invading cells, so that the bacteria are prevented from being cleared by target cells, and the pathogenicity of listeria monocytogenes with hemolysin knocked out is significantly lower than that of wild type listeria monocytogenes. Therefore, listeriolysin can be studied as a potential target for the development of anti-viral drugs. Compared with the traditional antibiotics, the antitoxic drug not only can play a good role in treatment, but also has the characteristics of low toxicity, low selection pressure and the like, is not easy to induce pathogenic bacteria to generate drug resistance, is more green and safe, and is a potential antibiotic substitute. The invention discovers for the first time that genkwanin can inhibit the pathogenicity of Listeria monocytogenes by inhibiting the pore-forming activity of Listeriolysin in a targeted manner.

Disclosure of Invention

The invention discovers the pore-forming activity of genkwanin for inhibiting listeria monocytogenes hemolysin

The invention discovers the effect of genkwanin in inhibiting J774 cell-mediated cytotoxicity of listeria monocytogenes

The invention discovers that the genkwanin has better treatment effect on mouse infection caused by Listeria monocytogenes

The application of the preferred genkwanin in the technical scheme of the invention in preparing the drug for resisting the listeria monocytogenes infection

The invention can prepare the genkwanin and pharmaceutically acceptable auxiliary materials into various dosage forms

The invention preferably prepares the genkwanin into the dosage forms of injection, capsule, tablet, powder injection and the like

Compared with the treatment with antibiotics, the treatment with the genkwa essence has the advantages of no drug resistance and high cure rate

Detailed Description

Through rabbit erythrocyte hemolysis test, J774 cell protection effect experiment and mouse infection experiment therapeutic experiment, the genkwanin is proved to be capable of efficiently inhibiting the pore-forming activity of Listeria monocytogenes hemolysin and the inhibiting effect on Listeria pathogenicity.

1. Hemolysis test

Purified listeriolysin was added to 96-well plates for two-fold serial dilutions, 10 per well⁷Rabbit red blood cells, incubated at 37 ℃ for 30 minutes and centrifuged. The highest dilution factor at which the erythrocytes were lysed was taken as the haemolytic titer (Hemolysis titre). The hemolytic titers of listeria monocytogenes hemolysin without and with different concentrations of genkwanin are shown in table 1.

TABLE 1 inhibitory Effect of Genkwanin on pore-forming Activity of Listeria monocytogenes hemolysin

2. Protective effect of genkwanin on J774 cells

J774 cells were plated in 96-well plates (30000 per well) and cultured in DMEM complete media at 37 ℃ with 5% CO₂After culturing for 12 hours under the conditions of (1), adding listeria monocytogenes (bacteria: cells: 80:1) into each hole, adding different gradients of drugs genkwanin, continuously culturing for 5 hours, centrifuging (1000rpm, 10min), taking supernatant, and detecting the activity of lactate dehydrogenase in the supernatant to detect the survival rate of the cells. The results (table 2) show that genkwanin significantly inhibits the cytotoxic effect of listeria monocytogenes on J774 cells, and is concentration-dependent.

TABLE 2 Genkwanin inhibits J774 cell-mediated cytotoxic effects of Listeria monocytogenes

3. Experimental and therapeutic study on mouse Listeria monocytogenes infection

3.1 establishment of mouse Listeria monocytogenes infection model

Female BALB/c mice weighing 18-20g were injected intraperitoneally with 200. mu.L (10)⁷CFU/ml) Listeria monocytogenes suspension, and establishing a mouse Listeria infection model.

3.2 protection rate test

Mice were randomly divided into two groups of 40 mice each after inoculation with listeria monocytogenes. The genkwanin treatment group mice were administered 100mg/kg (50 μ L) of genkwanin (dissolved in DMSO solvent) by subcutaneous injection once every 12 hours. Infection control groups were given 50 μ L of DMSO solvent subcutaneously at the same time point. After dosing according to the dosing schedule, mice were recorded for 48h, 72h and 96h of listeria infection, respectively. The results show (table 3) that the mortality rate of mouse listeria monocytogenes infection is remarkably reduced after the treatment by the genkwanin administration.

TABLE 3 Genkwanin reduces mortality from Listeria monocytogenes infection in mice