CN113786399A

CN113786399A - Application of glabridin in preparation of salmonella III type secretion system inhibitor

Info

Publication number: CN113786399A
Application number: CN202111190671.5A
Authority: CN
Inventors: 吕强华; 邓旭明; 宋泽宇; 王建锋; 邓彦宏; 王琳; 冯海华; 邱家章; 周永林
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2021-10-13
Filing date: 2021-10-13
Publication date: 2021-12-14

Abstract

The invention relates to the application of glabridin in the preparation of Salmonella type III secretion system inhibitor. The protective effect of licorice on the host cell invasion and damage mediated by Salmonella was evaluated by the Salmonella infection HeLa cell model, and the glabridin did not affect the growth of bacteria within the effective concentration range, indicating that the selective pressure of licorice on bacteria was small, thus Reduced the risk of drug resistance; in vivo experiments established a Salmonella infection mouse model by oral gavage, and found that licorice significantly reduced the mortality of Salmonella-infected mice and the colonization number of target organs, proving that licorice is effective against Salmonella infection Has a protective effect. Due to the abuse of traditional antibiotics and the continuous enhancement of bacterial resistance, glabridin has the characteristics of good therapeutic effect and no drug resistance and drug residues. Therefore, glabridin is of great significance for the preparation of anti-Salmonella infection drugs.

Description

Application of glabridin in preparation of salmonella III type secretion system inhibitor

Technical Field

The invention relates to application of glabridin in preparation of a salmonella III type secretion system inhibitor, belonging to the technical field of medical pharmacy.

Background

Salmonella (Salmonella) belongs to gram-negative facultative anaerobes, is divided into two species of Salmonella bangolensis (s.bongori) and Salmonella enterica (s.enterica), and also divided into 6 subspecies of intestinal subspecies (enterica), saramae (salamae), arizona (arizonae), dispariosa (diaarizona), hatton (houtenae) and indian (indica), mainly comprises over 2600 serotypes, and is one of the important pathogenic bacteria causing food-borne diseases. Salmonella is the second most common zoonosis pathogen in the european union, and most serotypes are derived from animal food. Salmonella infection can cause gastroenteritis type, cold-like type, septicemia type, cold type and cholera type food poisoning, and the main symptoms are acute gastroenteritis, accompanied with symptoms of fever, chills, nausea, headache, general debilitation, diarrhea, emesis, etc. Almost half of the cases of food-borne infections occurring annually in france are caused by salmonella; in the united states, salmonella causes about 120 million diseases, 23000 hospitalizations, and 450 deaths per year. In 2013 to 2017, the number of cases of salmonella enteritidis caused by eating polluted egg products and poultry meat in European countries is increased, huge economic loss is caused to the livestock breeding industry, and public health and food safety are seriously threatened.

Although antibiotics are the most commonly used method for treating and controlling bacterial infections, most antibiotics are critical elements directly affecting bacterial survival and thereby killing bacteria, and induce bacterial resistance due to their extremely selective pressure. The III type secretion system (T3SS) is mainly encoded by two virulence islands of SPI-1 and SPI-2, is responsible for secretion and transportation of effector proteins, and plays a key role in the aspects of inflammatory reaction, apoptosis, systemic infection and the like mediated by various stages of bacterial infection. Among them, SPI-1 plays a decisive role in the invasion of epithelial cells by Salmonella. Therefore, T3SS is used as a drug target, the anti-infection purpose is achieved through a non-sterilization mode, the natural selective pressure of bacteria can be reduced, and the generation of multiple drug resistance genes is avoided.

Glabradine (Glabradin) is an isoflavanoid compound widely found in the plant Glycyrrhiza glabra. Modern pharmacological research shows that the compound has a plurality of pharmacological effects of resisting oxidation, resisting tumor, resisting inflammation, reducing blood sugar and blood fat, resisting atherosclerosis, protecting the nervous system and the cardiovascular system and the like. At present, no report of the effect of glabridin in inhibiting the function of a salmonella III type secretion system and preparing a medicine for resisting salmonella infection is seen at home and abroad.

Disclosure of Invention

The glabridin CAS number of the invention is 59870-68-7, the molecular formula is C₂₀H₂₀O₁₄And the molecular weight is 324.37.

The chemical structural formula of glabridin is as follows:

the research takes a salmonella non-life essential component III type secretion system as an object, constructs a salmonella III type secretion system report system, and establishes a III type secretion system inhibitor screening platform. Through a large number of screening and identification, the glabridin is found to obviously inhibit the function of a salmonella III type secretion system under the condition of not influencing the growth of bacteria, inhibit the invasion of salmonella into HeLa cells and cell damage mediated by the III type secretion system, and have good protection effect on mice infected by salmonella. In conclusion, the research provides a basis for clinically preparing the salmonella III type secretion system inhibitor, researching and developing the anti-salmonella III type secretion system medicament and lays a good foundation for researching and developing innovative medicaments for salmonella infection of livestock and poultry in China.

Drawings

FIG. 1: glabridin inhibits translocation of effector protein SipA and further inhibits function of T3SS (blue indicates normal transport function of effector protein SipA; green indicates that effector protein SipA cannot normally transport)

FIG. 2: glabridin did not affect the growth of Salmonella typhimurium (time on the horizontal axis and measured value on the vertical axis)

FIG. 3: glabridin inhibited invasion of Salmonella into HeLa cells (horizontal axis represents Glabridin concentration and vertical axis represents cell invasion rate)

FIG. 4: effect of Glabridin on protection of Salmonella-mediated HeLa cell injury (concentration of Glabridin on horizontal axis and LDH Release amount on vertical axis)

FIG. 5 Glabridin decreases the number of colonizations of target organs of mice infected with Salmonella (horizontal axis indicates different treatment groups and vertical axis indicates the number of colonizations)

Detailed Description

The present invention is further illustrated by the following examples, which do not limit the invention in any way, and any modifications or changes that can be easily made by a person skilled in the art without departing from the technical solution of the invention will fall within the scope of the claims of the invention.

Example 1

Application of glabridin in preparing salmonella III type secretion system inhibitor in any pharmaceutically acceptable carrier.

Example 2

Glabridin is used as salmonella III type secretion system inhibitor for preparing medicine for treating infectious diseases.

Example 3

Glabridin is used as Salmonella III type secretion system inhibitor for treating infectious diseases caused by bacteria, especially human and livestock infections caused by Salmonella, including typhoid, paratyphoid, gastroenteritis, pullorum disease, etc.

1. Effect of Glabridin on Salmonella type III secretion System function

Firstly, a SipA-beta-lactamase-TEM report plasmid is constructed by a molecular cloning method and is transferred into salmonella. After incubating and culturing the natural compound and salmonella containing a reporter plasmid for 3h, infecting cells for 2h by MOI (equal to 50), adding a CCF4-AM substrate and incubating for 45min in dark. T3SS inhibitors were screened by different fluorescence ratios under immunofluorescence microscopy.

And (4) conclusion: the SipA-beta-lactamase-TEM infected Hela cells show blue fluorescence, which indicates that T3SS effector protein SipA can be normally transported to HeLa cells, and when key gene invA of a T3SS system is knocked out, the normal function is lost, and the HeLa cells show green fluorescence. As a result, the glabridin-treated group was found to be mostly green fluorescent in the visual field, indicating that the glabridin-treated group significantly inhibited the transport of the Salmonella T3SS effector protein SipA (see figure 1).

2. Effect of Glabridin on Salmonella growth

Selecting single colony of Salmonella, culturing in LB liquid medium (0.3M NaCl) overnight (37 deg.C, 200rpm), and expanding to 600nm absorbance (OD) at the next day according to a ratio of 1:100_600nm) About 0.3, the bacterial liquid is measured and evenly distributed into five conical bottles with the volume of 50ml (about 20ml in each bottle), and glabridin with different concentrations (0, 4, 8, 16 and 32 mug/ml) is respectively added. The culture was continued at 37 ℃ and 200rpm, and the OD of each sample was measured every 0.5h_600nmUntil the growth reaches the plateau stage.

And (4) conclusion: compared with the group without drug, the growth state of the bacteria of the glabridin treatment group (4-32 mug/ml) is not abnormal, which indicates that the glabridin does not influence the normal growth of the salmonella within the effective concentration range (see figure 2).

3 Effect of Glabridin on Salmonella invasion into host cells

The effect of syringaldehyde on internalization of salmonella into host cells was determined by the gentamicin protection assay. 24 well plates 4X 10 inoculate per well⁵HeLa cells were cultured overnight in a blank control group, a.DELTA.invA-SL 1344 group and a glabridin-treated group at different concentrations. Overnight cultured SL1344 and Δ invA-SL1344 were incubated at 1:20 for amplification at 37 ℃ and 200rpm for 4h. Determination of OD_600nmThen diluting the bacterial liquid by using a DMEM medium, infecting cells by MOI (100), centrifuging at 1000rpm for 10min, then culturing for 1h in an incubator at 37 ℃, washing for three times by using PBS (phosphate buffered saline), and then continuously incubating for 1h by using a DMEM medium containing 100 mu g/ml gentamicin to kill extracellular bacteria which do not enter host cells. Washing with PBS for three times, permeabilizing with 0.2% saponin 500 μ l/well for 10min, diluting, plating, incubating at 37 deg.C for 12 hr, and counting colonies.

And (4) conclusion: glabridin can obviously inhibit the invasion of salmonella to HeLa cells within the concentration range of 4-32 mug/ml (shown in figure 3).

Protective effect of 4-glabridin on salmonella-mediated HeLa cell injury

HeLa cells were suspended in DMEM supplemented with 10% fetal bovine serum in 96-well plates at 2X 10⁴Density culture overnight per well. Salmonella SL1344 and Δ invA-SL1344 were cultured overnight in LB broth containing 0.3M NaCl, after pretreatment with glabridin at various concentrations for 4h, the cells were infected with MOI 100, three replicates per group, and after infection for 6h, cell culture supernatants were centrifuged at 1000rpm for 10min in new 96-well plates, and the LDH release rates of each group were calculated by measuring the absorbance at 492nm according to the instructions for the Lactate Dehydrogenase (LDH) kit.

And (4) conclusion: glabridin inhibited salmonella-mediated HeLa cell damage in a dose-dependent manner compared to the control group (see figure 4).

4. Glabridin reduces colony colonization of salmonella infected mouse target organ

The streptomycin pretreated mice are randomly divided into a healthy control group, an infected group (SL1344 infected) and an infected treatment group (glabridin treatment after SL1344 infection), wherein the healthy control group is not subjected to any treatment and is separately bred in an isolated way; after infection treatment, the glabridin (100mg/kg body weight/time, 2 times/day, 4 days) is orally taken for treatment, and after infection treatment, the normal saline with the same volume is orally taken. The liver, spleen and cecal tissues of each group of mice are taken 96h after infection, weighed and ground to prepare tissue homogenate, diluted in multiple times, smeared on an LB solid agar plate containing streptomycin resistance, cultured at 37 ℃ for 12h, and then colonies are counted.

And (4) conclusion: the colony colonization numbers of the liver, spleen and caecum of mice in the glabridin treatment group are obviously lower than those of the infected group, and have no obvious difference with those of the healthy group (see figure 5).

In conclusion, the research uses salmonella T3SS as a target to screen inhibitors, and evaluates the in vivo and in vitro effects of glabridin to be screened. In vitro test, a HeLa cell model is infected by salmonella, the protection effect of glabridin on salmonella-mediated cell invasion and injury is evaluated, meanwhile, glabridin does not influence the normal growth of salmonella in an effective concentration range, and the inhibition effect of glabridin on salmonella T3SS is realized through a non-bacteriostatic or bactericidal mechanism; a salmonella-infected mouse model is established in vivo in an oral gavage mode, and the glabridin is found to be capable of obviously reducing the colony planting number of target organs (liver, spleen and caecum) of a salmonella-infected mouse. The research provides theoretical basis and thinking for preparation of drugs such as inhibitors and the like taking a salmonella III type secretion system as a target.

Claims

1. Application of glabridin in preparing salmonella III type secretion system inhibitor is provided.

2. The use of claim 1, wherein glabridin is used to inhibit a biological function of a salmonella type III secretion system.

3. The use of claim 1, wherein glabridin inhibits salmonella type III secretory system function and reduces salmonella pathogenicity.