Application of macleaya cordata extract as salmonella type III secretion system inhibitor

Abstract

The invention provides a novel application of a macleaya cordata extract as a salmonella type III secretion system (T3SS) inhibitor. Through construction of a chick infection model and in-vitro experiments, the extract is proved to be capable of remarkably reducing the bacterial load of infected chick liver and spleen tissues, relieving tissue pathological damage and reducing inflammatory cell infiltration. Mechanism research shows that the macleaya cordata extract can down-regulate the expression of T3SS key virulence protein SipA in a dose-dependent manner, interfere the function of a secretion system, and further block the secretion and expression of virulence factors. The invention discloses the potential application of the macleaya cordata extract in the aspect of resisting salmonella toxicity for the first time, and provides a development strategy and a technical basis of a T3SS inhibitor from a natural product.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, and particularly relates to application of an extract of bocconia to preparation of a Salmonella type III secretion system inhibitor. BACKGROUND

[0002] Salmonella is a kind of gram-negative facultative intracellular pathogen, belongs to the Enterobacteriaceae family, is widely distributed in nature and can infect human and animal hosts, and causes systemic diseases such as typhoid fever, gastroenteritis and septicemia. Epidemiological data shows that Salmonella is the primary pathogen of bacterial foodborne diseases worldwide, and its incidence rate and disease burden are significantly higher than those of other common foodborne pathogens (such as Escherichia coli and Campylobacter jejuni). At present, more than 2700 serotypes of Salmonella have been identified, and its genetic diversity brings great challenges to prevention and control. Clinically, antibiotics (such as fluoroquinolones and beta-lactams) are still the main means for treating Salmonella infection. However, due to antibiotic abuse and selective pressure, the prevalence rate of multi-drug resistant (MDR) strains is increasing year by year, and it is urgent to develop new anti-infection strategies based on non-killing mechanisms. Anti-virulence strategy is one of the hotspots of current anti-infection strategy research. This strategy targets bacterial virulence factors and achieves the purpose of anti-infection without affecting bacterial growth, and is not easy to produce selective pressure on bacteria, which is an important direction of new anti-infection strategy research. Anti-virulence therapy is an intervention strategy targeting pathogen virulence factors rather than their survival essential pathways, which can reduce the damage to the host microbiota and the selective pressure of drug resistance by specifically inhibiting the pathogenicity (such as adhesion, invasion or immune escape) of bacteria rather than directly killing bacteria. The type III secretion system (T3SS) of Salmonella, which is encoded by pathogenic islands SPI-1 and SPI-2, is a key molecular device for the bacteria to break through the host barrier and survive intracellularly. Due to its high conservation and decisive role in virulence, it has become an ideal target for the development of anti-virulence drugs. Salmonella T3SS can secrete various effector proteins such as SipA, SipB, SipC, SseG and SseJ, which can help the bacteria invade host cells and proliferate intracellularly, complete the infection and pathogenic process. Therefore, the development of anti-virulence strategies targeting Salmonella T3SS can provide a candidate solution for the treatment of drug-resistant Salmonella infection.

[0003] Natural compounds are valuable resources for drug development. Macleaya cordata is a perennial herb of the Papaveraceae family, and its medicinal value has been recorded in traditional medicine. Modern pharmacological studies have shown that the main active ingredients of M. cordata are benzophenanthridine alkaloids (such as sanguinarine and chelerythrine), which have broad-spectrum biological activities, including anti-inflammatory, analgesic, and antibacterial effects. In vitro experiments have confirmed that its extract shows inhibitory effects on pathogenic microorganisms such as Staphylococcus aureus (gram-positive bacteria), Escherichia coli (gram-negative bacteria), and Leptospira. In addition, sanguinarine can reduce the release of TNF-α, IL-6, and other pro-inflammatory factors by inhibiting the NF-κB and MAPK signaling pathways. In a colitis model, it can reduce the intestinal mucosal damage score (p<0.01), and show selective cytotoxicity to various cancer cells (such as HeLa, MCF-7). Chelerythrine can inhibit the growth of Trichomonas vaginalis by inducing the mitochondrial apoptosis pathway. Although M. cordata alkaloids have effects on various pathogenic microorganisms, their effects on Salmonella (especially T3SS-dependent virulence) have not been clearly reported. Therefore, the present invention focuses on M. cordata extract rich in sanguinarine and chelerythrine to explore the role and mechanism of M. cordata in combating Salmonella infection, with the aim of providing a potential alternative drug with broad application prospects for the development of anti-virulence strategies for Salmonella infection in clinical practice. SUMMARY

[0004] The M. cordata extract described in the present invention is purchased from Hunan Hanqing Biotechnology Co., Ltd. Its preparation process is as follows: the M. cordata fruit pods are crushed, and 0.5% sulfuric acid solution is used for stirring extraction at 70°C for 4 times, with 1.5 hours of immersion each time, a liquid-to-solid ratio of 20 mL / g for the first immersion, and a liquid-to-solid ratio of 10 mL / g for the other three immersions; the initial extract is heated and refluxed in 90% ethanol twice, with a liquid-to-solid ratio of 5 mL / g for each immersion, and an immersion time of 1 hour; the extract from the first immersion is decolorized using 0.1% activated carbon, concentrated, adjusted to a pH of 2-3, and crystallized at low temperature for 1.5 hours; after filtration and drying, the M. cordata extract is obtained.

[0005] The present study constructed a Salmonella chicken embryo infection model, and the results showed that the M. cordata extract could effectively treat Salmonella pullorum (SP C79-13) infection and reduce histopathological damage. Through trichloroacetic acid (TCA) precipitation and immunoblotting experiments, it was further confirmed that the M. cordata extract could target Salmonella virulence factors and exhibit an inhibitory effect on their virulence.

[0006] The positive effects of the present invention are: The application provides the use of the extract of the plant of the genus Boerhaavia in the development of T3SS inhibitors, and discloses that the extract of the plant of the genus Boerhaavia can inhibit the secretion and expression of T3SS effector protein SipA, and thus inhibit the function of the T3SS of Salmonella. BRIEF DESCRIPTION OF DRAWINGS

[0007] Table 1 Clinical treatment effect of the extract of the plant of the genus Boerhaavia on the chicks against Salmonella infection Figure 1 Histopathological analysis of target organs of the chicks infected with Salmonella by the extract of the plant of the genus Boerhaavia Figure 2 Colonization of target organs of the chicks infected with Salmonella by the extract of the plant of the genus Boerhaavia Figure 3 Effect of the extract of the plant of the genus Boerhaavia on the growth of Salmonella Figure 4 Effect of the extract of the plant of the genus Boerhaavia on the secretion of Salmonella effector proteins Figure 5 Effect of the extract of the plant of the genus Boerhaavia on the expression of Salmonella effector proteins DETAILED DESCRIPTION

[0008] The present application is further described by the following examples, which do not limit the present application in any manner, and any modification or change made by those skilled in the art without departing from the technical solutions of the present application will fall within the scope of the claims of the present application.

[0009] 1. Protective effect of Boerhaavia on chicks 1.1 Construction of a Salmonella chick infection model One-day-old healthy chicks were adaptively fed in a specified facility for 3 days, and the environmental temperature and humidity, feeding density, ventilation, and free feeding and drinking conditions were ensured to be appropriate during the feeding period. After the adaptive feeding, the healthy chicks were randomly divided into 5 cages, with 10 chicks in each cage. The specific grouping is as follows: (1) Healthy control group: under the specified conditions, the chicks were conventionally fed for 9 days without any treatment; (2) SP C79-13 infection model group: under the specified conditions, the chicks were conventionally fed for 4 days, and then attacked by bacteria for 4 days, and then conventionally fed for 5 days; (3) Drug treatment group: under the specified conditions, the chicks were conventionally fed for 4 days, and then attacked by bacteria, and then given the extract of the plant of the genus Boerhaavia (1 mg / mL) by gavage at a dose of 2 mg / kg for 4 days, and then conventionally fed for one day; The next day, all the chicks were sacrificed by cervical dislocation according to the aseptic operation procedure.

[0010] 1.2 Clinical symptom observation test The diet and water of each group of chicks were observed and recorded daily, and the mental state, clinical manifestations, and fecal state of each group of chicks were observed and recorded at the following time points: before administration, before challenge, after challenge, and before necropsy. The results were expressed as the number of chicks with typical symptoms per total number of chicks in each group (Table 1).

[0011] Table 1 Clinical treatment effect of Boerhaave extract on chicks infected with Salmonella

[0012] Conclusion: Compared with the healthy control group, the SPC79-13 infected model group of chicks had severe dehydration, rapid weight loss, small body size, unsteady standing, decreased water intake, and refusal to eat during the test period. The signs of chicks treated with Boerhaave extract were significantly improved, and the diet and water intake were also improved to varying degrees, indicating that Boerhaave extract had an effective therapeutic effect on chicken salmonella infection, and the drug itself had no negative impact on the normal growth of chicks.

[0013] 1.3 Target organ bacterial load and histopathological analysis The chicks were euthanized by cervical dislocation according to aseptic operation procedures, and the liver and spleen of the euthanized chicks were aseptically collected and embedded in paraffin to prepare sections for hematoxylin-eosin (HE) staining (Fig. Figure 1 ). Another part of the tissue was weighed, ground, and prepared into a tissue homogenate, which was diluted with PBS and spread on LB agar plates. After 12 hours of incubation at 37°C, the number of colonies was counted (Fig. Figure 2 ).

[0014] Conclusion: Boerhaave extract can significantly reduce the bacterial load of Salmonella in chicks and alleviate pathological damage.

[0015] 2. Growth curve test The overnight activated SPC79-13 bacterial solution was expanded to a new LB medium at a ratio of 1:20, and incubated at 37°C, 180 rpm in a horizontal shaker until the OD 600nm value reached 0.3, and then aliquoted (30 mL / bottle). The culture was added with gradient diluted Boerhaave extract, and the OD 600nm values of different cultures were measured every hour using a UV spectrophotometer. After 7 hours of continuous measurement, the OD 600nm values at different time points were used to draw a growth curve.

[0016] Conclusion: Boerhaave extract at 0-64 μg / mL had no significant effect on the growth of Salmonella SPC79-13 (Fig. Figure 3 ).

[0017] 3. Effects of Macleaya cordata extracts on secretion and expression of Salmonella effectors Salmonella was inoculated into LB liquid medium containing 0.3 M NaCl and incubated overnight at 37 ℃, 200 rpm. The next day, the culture was diluted 1:20 to OD 600nm about 0.3, and different concentrations of Macleaya cordata extracts were added (0, 8, 16, and 32 μg / mL). The culture was incubated at 37 ℃, 200 rpm for 5 hours. The supernatant was treated with trichloroacetic acid for 8 hours, and SDS-loading buffer was added and boiled. The secretion of effectors was analyzed by SDS-PAGE and Western blotting (Fig. 3). Figure 4 The bacterial cells were collected by centrifugation, and an appropriate amount of bacterial cells was added to SDS-loading buffer and boiled for 10 minutes. Figure 5

[0018] Conclusion: After pretreatment with Macleaya cordata extracts, the secretion and expression of SipA, an invasion-related effector protein of Salmonella SPC79-13, decreased. This result indicates that Macleaya cordata extracts affect the function of the T3SS of Salmonella by inhibiting the secretion and expression of effectors of the T3SS.​

Claims

1. Use of an extract of Oxyria sinensis in the preparation of an inhibitor of the type III secretion system of Salmonella.

2. Use according to claim 1, wherein The extract of Oxyria sinensis inhibits the function of the type III secretion system of Salmonella by inhibiting the expression of Salmonella effector proteins.

3. The use according to claim 2, characterized in that, The Salmonella effector proteins include at least SipA.

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