Application of epimedin A in preparation of streptococcus suis type 2 capsule inhibitor

Abstract

The invention discloses application of epimedin A in preparation of a streptococcus suis type 2 capsule inhibitor, and belongs to the technical field of biological medicine. The compound can effectively inhibit the synthesis function of the streptococcus suis type 2 capsule, further enhances the removal effect of a host on the streptococcus suis type 2, reduces the pathogenicity of bacteria, and provides a new candidate compound and idea for preparing a capsule inhibitor of the streptococcus suis type 2 and researching and developing a novel medicine for treating streptococcus suis type 2 infection.

Description

Technical Field

[0001] This invention relates to the medical use of chopogonin A, and in particular discloses the application of chopogonin A in the preparation of Streptococcus suis type 2 capsule inhibitor, belonging to the field of biomedical technology. Background Technology

[0002] Streptococcus suis ( Streptococcus suis Streptococcus suis is a Gram-positive bacterium that seriously threatens animal health, commonly causing bacteremia, endocarditis, and arthritis in pigs. Simultaneously, as a zoonotic pathogen, humans can be infected through contact with infected pigs or consumption of contaminated pork products, potentially leading to serious diseases such as meningitis, septicemia, and pneumonia. Based on the immunogenicity of its capsular polysaccharide, Streptococcus suis can be classified into 29 serotypes, among which serotype 2 is the most widespread and virulent. Most human infections caused by Streptococcus suis are caused by serotype 2, and its spread poses a significant threat to property and public health. Currently, drug resistance in Streptococcus suis serotype 2 is increasingly becoming a challenge.

[0003] The capsule is a recognized key virulence factor in *Streptococcus suis*. This polysaccharide structure encapsulating the bacterial cell surface can suppress the host's innate immune response (including phagocytosis and cytokine release) by masking bacterial surface antigens, thereby helping bacteria evade recognition and clearance by the host's immune system. This is the first step in establishing infection. Multiple studies have revealed the crucial role of the capsule in the pathogenicity of *Streptococcus suis*. For example, mutant strains lacking the capsule completely lose their pathogenicity and can be rapidly cleared from the host. Notably, capsule synthesis is not essential for bacterial growth; therefore, compared to traditional antibiotics, capsule inhibitors have no bactericidal effect and do not exert selective pressure on bacterial growth, thus making them less likely to induce drug resistance. Therefore, directly targeting capsule biosynthesis to weaken bacterial pathogenicity has become a highly attractive therapeutic strategy for controlling *Streptococcus suis* infection.

[0004] Chaohuoding A is Epimedium (Epimedium plant of the Berberidaceae family). Epimedium brevicornu The main active ingredient of Maxim's dried leaves is also a flavonoid compound with a relatively high content in medicinal materials. Ascodone A has shown good pharmacological activities in anti-osteoporosis, anti-inflammation, anti-oxidation, anti-Alzheimer's disease, and neuroprotection. However, there are no reports on the treatment of bacterial infections with ascodone A. Summary of the Invention

[0005] The application of the disclosed chamomile A in the preparation of Streptococcus suis type 2 capsule inhibitor aims to weaken the pathogenicity of Streptococcus suis by directly targeting capsule biosynthesis and solving the drug resistance problem of Streptococcus suis type 2.

[0006] The molecular formula of the morning glory tincture A described in this invention is C39 H 50 O 20 With a molecular weight of 838.80, the molecular structure of Astragalus membranaceus A is as follows: .

[0007] The application of the present invention, namely, chomosine A, in the preparation of a Streptococcus suis type 2 capsule inhibitor, inhibits capsule biosynthesis by reducing the transcriptional level of genes in the Streptococcus suis type 2 capsule biosynthesis gene cluster; the genes include csp2E , cps2F , cps2G , cps2H , csp2k , cps2J as well as cps2N .

[0008] Another object of the present invention is to provide the application of ascorbic acid A in the preparation of drugs against Streptococcus suis type 2 infection. Ascorbic acid A enhances the host's clearance of bacteria by inhibiting the synthesis of Streptococcus suis type 2 capsules, thereby reducing the pathogenicity of the bacteria.

[0009] The oxytocin A described in this invention can be used as a capsular inhibitor of Streptococcus suis type 2 for application in any pharmaceutically acceptable form of drug carrier. The chopoxetine A described in this invention is used as a component in the preparation of a medicament for treating Streptococcus suis type 2 infectious diseases. The medicament may also include other active ingredients and pharmaceutically acceptable adjuvants.

[0010] This invention observed that astragalus extract A had no significant inhibitory effect on the growth of Streptococcus suis type 2 through growth curve determination. The inhibitory effect of astragalus extract A on the capsule of Streptococcus suis type 2 was verified using a phenol-sulfuric acid method for capsular polysaccharide quantification and transmission electron microscopy. qRT-PCR real-time fluorescence quantitative assay demonstrated that astragalus extract A inhibited the transcriptional level of the gene clusters involved in the capsule synthesis of Streptococcus suis type 2, thereby reducing capsule synthesis. The mouse peritoneal macrophage phagocytosis assay and whole blood killing assay showed that astragalus extract A increased the sensitivity of the bacteria to the host's innate immunity by inhibiting the capsule of Streptococcus suis type 2. The survival rate of mice verified the in vivo therapeutic effect of astragalus extract A on Streptococcus suis type 2 infection.

[0011] The positive effects of this invention are as follows: it discloses that astragaloside A can effectively inhibit the synthetic function of streptococcus suis type 2 capsule, thereby enhancing the host's clearance of the bacteria and reducing the pathogenicity of the bacteria, providing new candidate compounds and ideas for the preparation of streptococcus suis type 2 capsule inhibitors and the development of novel drugs for the treatment of streptococcus suis type 2 infection. Attached Figure Description

[0012] Figure 1 The growth curve provided in Embodiment 1 of the present invention; Figure 2 The results of capsular polysaccharide content determination provided in Example 2 of the present invention; Figure 3 The image shown is a transmission electron microscope image of the capsule morphology provided in Example 3 of the present invention, wherein a is the solvent control group and b is the group treated with Astragalus membranaceus A. Figure 4 The transcriptional level of the capsule synthesis gene cluster of Streptococcus suis type 2 05ZYH33 provided in Example 4 of this invention; Figure 5 The results of the cell phagocytosis experiment provided in Example 5 of the present invention; Figure 6 The whole blood killing test results provided in Embodiment 6 of the present invention; Figure 7 The mouse survival rate results provided in Example 7 of this invention; All statistical analyses were performed using GraphPad Prism software (version 8.0.2). Unless otherwise stated, all quantitative data were derived from at least three independent experiments and are expressed as mean ± standard error (mean ± SEM). Unpaired two-tailed t-tests were used for comparisons between two groups; one-way or two-way ANOVA was used for comparisons among multiple groups, supplemented by appropriate post-hoc tests; log-rank tests were used for survival analysis. P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01). Detailed Implementation

[0013] 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.

[0014] The molecular formula of Astragalus membranaceus A is C 39 H 50 O 20 With a molecular weight of 838.80, the molecular structure of Astragalus membranaceus A is as follows: .

[0015] The Streptococcus suis type 2 strain used in this embodiment of the invention is specifically Streptococcus suis type 2 05ZYH33, which is preserved in the Pharmacology and Toxicology Laboratory of the Institute of Zoonoses, Jilin University.

[0016] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0017] Example 1: Determination of growth curve: To determine the effect of ascorbic acid A on the growth of Streptococcus suis type 2, the absorbance of Streptococcus suis type 2 05ZYH33 culture medium at different time points was measured using a UV spectrophotometer to quantify bacterial density. Overnight Streptococcus suis type 2 05ZYH33 culture was added to fresh culture medium at a ratio of 1:100, with different concentrations (4-64 μg / mL) of ascorbic acid A added. DMSO was used as a control. The cultures were incubated in a shaker at 37°C, and the absorbance (OD) of the bacterial culture was measured at regular intervals. 600 ), collect data and plot bacterial growth curves.

[0018] The results are as follows Figure 1 As shown, compared with the DMSO treatment group, there was no significant difference in bacterial growth status in the treatment groups with added (4, 8, 16, 32, 64 μg / mL) oxadixyl A, indicating that oxadixyl A does not affect the normal growth of Streptococcus suis type 2 05ZYH33 within the effective concentration range (8-64 μg / mL) for inhibiting the capsule.

[0019] Example 2, Determination of capsular polysaccharide content: Overnight cultured Streptococcus suis type 2 05ZYH33 was inoculated into 20 mL of THY medium containing (8-64 μg / mL) oxytocin A. A sample with added DMSO served as a positive control. Streptococcus suis was cultured at 37°C until OD500. 600 =0.6-0.8, centrifuge and discard the supernatant. The collected bacteria are suspended in glycine buffer containing lysozyme (0.1 mol / L, pH = 9.2) and cultured at 37℃ for 8 h. Then, take 1 mL of the lysis supernatant and treat it with proteinase K at 60℃ for 2 h. Add CaCl2 to a final concentration of 0.1 M and stir for 1 h. Then, add anhydrous ethanol to a final concentration of 25% and incubate at 4℃ for 2 h. Centrifuge to precipitate the nucleic acid in the supernatant. Then, take the supernatant after centrifugation and add anhydrous ethanol to a final concentration of 80% and store at 4℃ for 18 h to precipitate the capsular polysaccharide. The content of capsular polysaccharide is determined by the phenol-sulfuric acid method: resuspend the polysaccharide in 800 μL of distilled water, take 200 μL of the above solution, add 200 μL of 8% phenol and 1 mL of concentrated sulfuric acid, react at 45℃ for 30 min, and measure the absorbance OD at a wavelength of 490 nm. 490 The content of capsular polysaccharides was calculated using a self-made glucose standard curve.

[0020] The results are as follows Figure 2 As shown, it can be seen that chomosine A can inhibit the synthesis of capsular polysaccharides of Streptococcus suis type 2 05ZYH33 in a dose-dependent manner: an inhibitory trend is observed at 8 μg / mL, and significant inhibition is shown at 32 μg / mL.

[0021] Example 3: Observation of the inhibitory effect of chamomile A on the capsule of Streptococcus suis type 2: An overnight culture of Streptococcus suis type 2 05ZYH33 was added to THY medium at a ratio of 1:100. Streptococcus suis type 2 with 64 μg / mL of cymoxanil A was cultured in THY medium until the logarithmic growth phase (OD2). 600 =0.6-0.8), and samples with DMSO solvent without ascorbic acid A were added as controls. The bacterial pellet was collected by centrifugation at 3000 g for 10 min. The pellet was washed three times with PBS (pH 7.4) and fixed overnight with 2.5% glutaraldehyde. The samples were treated with 1% osmium tetroxide for 2 h, and then continuously diluted with ethanol for dehydration. The dehydrated cells were embedded in epoxy resin and their capsule morphology was observed using an HT-7700 transmission electron microscope.

[0022] The results are as follows Figure 3 As shown, it can be seen that compared with the solvent control group without added drugs ( Figure 3 (a) After treatment with 64 μg / mL of phorbol A, the capsule of Streptococcus suis type 2 05ZYH33 was significantly thinned, and its structural integrity was disrupted. Figure 3 (b)

[0023] Example 4: RNA extraction and qRT-PCR quantitative detection: Overnight culture of Streptococcus suis type 2 05ZYH33 was added to fresh THY medium at a ratio of 1:100, with different concentrations (16 μg / mL, 32 μg / mL) of ascorbic acid A added. A sample containing DMSO solvent without ascorbic acid A was used as a control. The culture was incubated until the logarithmic growth phase (OD2). 600 =0.6-0.8), centrifuge to collect bacterial pellet, resuspend in PBS, treat in a 95℃ metal bath for 40 min, then incubate with 20 mg / mL lysozyme at 37℃ for 2 h. Total RNA was extracted from the treated bacterial culture sample using the TRIzol method, and cDNA was synthesized by reverse transcription. The glucosyltransferase gene was analyzed using a real-time quantitative PCR kit. csp2E rhamnosyltransferase gene cps2F galactosyltransferase gene cps2G , cps2H , csp2k N-acetylglucosamine glycosyltransferase gene cps2J sialyl glycosyltransferase gene cps2N Quantitative analysis was performed using specific primers targeting 16S rRNA as internal controls. Amplification data were analyzed using a comparative critical threshold (2). −ΔΔCΤ The calculation results are obtained using the method.

[0024] The results are as follows Figure 4As shown, it can be seen that chamomile A, within the effective concentration range of 16 and 32 μg / mL for inhibiting the capsule of Streptococcus suis type 2, inhibits the capsule synthesis gene cluster of Streptococcus suis type 2 05ZYH33. csp2E , cps2F , cps2G , cps2H , csp2k , cps2J as well as cps2N It has a significant inhibitory effect on transcriptional levels.

[0025] Example 5, Cell phagocytosis assay: Primary peritoneal macrophages from C57 mice were prepared using a 3% mercaptoacetate broth induction method. In short, C57 mice were intraperitoneally injected with 3% mercaptoacetate broth, recruiting a large number of macrophage precursors (monocytes) into the peritoneal cavity, where they differentiated into macrophages. These macrophages were then collected and purified via peritoneal lavage with PBS. The collected primary peritoneal macrophages from C57 mice were seeded into 24-well plates at a density of 5 × 10⁶ cells per well. 5 Cells were cultured overnight in a 37°C incubator containing 5% CO2. Pre-cultured Streptococcus suis type 2 05ZYH33 was then used to infect the cells at an MOI of 20, followed by incubation with 16-64 μg / mL of gentamicin A. A control group without gentamicin A was added to DMSO solvent. The cells were then centrifuged at 1000 g for 10 min for simultaneous infection. After 1 h of incubation, the cells were washed three times with PBS and incubated for 1 h in fresh medium containing 100 μg / mL gentamicin to kill extracellular bacteria. The cells were then washed, lysed, serially diluted, and plated on THY agar for colony-forming units (CFU). The phagocytic rate was defined as the ratio of phagocytosed bacteria to the total number of bacteria measured before the addition of gentamicin.

[0026] The results are as follows Figure 5 As shown, 60 min after infection, 2.05% of wild-type 05ZYH33 were phagocytosed by mouse peritoneal macrophages. Ascorbic acid A increased the phagocytic rate of mouse peritoneal macrophages in a dose-dependent manner. Treatment with 64 μg / mL ascorbic acid A increased the phagocytic rate to 4.24%, which was twice that of the untreated group.

[0027] Example 6: Whole Blood Killing Test Streptococcus suis type 2 05ZYH33 was cultured in fresh THY medium supplemented with DMSO or (8-32 μg / mL) ascorbic acid A for 4 h, then centrifuged at 5000 r for 10 min to collect the bacterial cells. The cells were washed twice in sterile 0.01M PBS, and the bacterial concentration was diluted to 1×10⁻⁶. 8CFU / mL, then 40 μL of bacterial suspension was added to 500 μL of whole pig blood, and 8-32 μg / mL of chomodine A was added for treatment. DMSO solvent was added as a drug-free control. The mixture was incubated at 37°C for 3 h. Samples were taken and diluted every 1 h, spread on THY agar plates, and incubated overnight at 37°C. The number of surviving bacteria was counted and the survival rate was the ratio of the initial colony count.

[0028] The results are as follows Figure 6 As shown, treatment with chopogonin A can effectively reduce the resistance of Streptococcus suis type 2 05ZYH33 to blood-killing. After 3 h of whole blood killing, 32 μg / mL chopogonin A reduced the survival rate of untreated 05ZYH33 from 36.8% to 17.2%.

[0029] Example 7, Mouse survival rate test: Streptococcus suis type 2 05ZYH33 was cultured in fresh THY medium supplemented with DMSO or 64 μg / mL ascorbic acid A for 4 h. The bacterial pellet was collected by centrifugation, washed three times with PBS, and resuspended at 2×10⁻⁶. 9 CFU / mL bacterial suspension was administered to female ICR mice (6-8 weeks old) after 3 days of acclimatization with free access to food and water. The mice were then fasted for 12 hours prior to infection, followed by an intraperitoneal injection of 2×10⁻⁶ CFU / mL bacterial suspension. 9 CFU of Streptococcus suis was administered to mice in two groups: a blank control group was injected with an equal volume of PBS, a drug-treated group was administered the drug intraperitoneally three times daily at a dose of 50 mg / kg, and the untreated infection group and the blank control group were injected with the same volume of physiological saline. Mice mortality was recorded within 72 h after inoculation.

[0030] The results are as follows Figure 7 As shown, the survival rate of mice in the untreated group was 15% within 72 h, while treatment with chopogonin A (50 mg / kg) increased the survival rate to 46%.

[0031] In summary, the embodiments of this invention verify that ascorbic acid A can effectively inhibit the biosynthesis of the capsule by reducing the transcriptional level of related genes in the capsule biosynthesis gene cluster of Streptococcus suis type 2, manifested as a significant reduction in capsule polysaccharide content and a significant thinning of capsule thickness. The inhibition of capsule synthesis weakens the bacteria's ability to evade host immune clearance, making it easier for them to be cleared by the innate immune system. Further in vivo experiments have demonstrated that ascorbic acid A treatment can significantly improve the survival rate of infected mice.

[0032] Based on this, Ascodone A can be used as a capsular inhibitor of Streptococcus suis type 2 for application in any pharmaceutically acceptable form of drug carrier, providing a new reference for the research of drugs against Streptococcus suis type 2 infection. It can also be used as a component in the preparation of drugs for the treatment of Streptococcus suis type 2 infectious diseases, and the drugs may also include other active ingredients and pharmaceutically acceptable adjuvants.

[0033] 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 chomodine A in the preparation of Streptococcus suis type 2 capsular inhibitor, characterized in that: The aforementioned cytosine A inhibits capsule biosynthesis by reducing the transcriptional level of genes in the streptococcus suis type 2 capsule biosynthesis gene cluster; The genes include csp2E , cps2F , cps2G , cps2H , csp2k , cps2J as well as cps2N .

2. The application of the astragaloside A according to claim 1 in the preparation of a streptococcal type 2 capsular inhibitor, characterized in that: The Streptococcus suis type 2 capsular inhibitor targets Streptococcus suis type 2 05ZYH33.

3. The application of chopogonin A in the preparation of drugs against Streptococcus suis type 2 infection, characterized in that, Ascorbic acid A enhances the host's clearance of bacteria and reduces bacterial pathogenicity by inhibiting the synthesis of Streptococcus suis type 2 capsules.

4. The application of chomodine A in the preparation of Streptococcus suis type 2 capsular inhibitor, characterized in that: Any pharmaceutically acceptable form of drug carrier.

5. A component of chamomile A used in the preparation of drugs for treating Streptococcus suis type 2 infectious diseases, characterized in that: The drug also includes other active ingredients and pharmaceutically acceptable adjuvants.

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