Use of a protopanaxatriol in the preparation of a type 2 streptococcus suis capsular inhibitor

Abstract

The present application belongs to the technical field of medicine and pharmacy, and in particular relates to an application of proto-prosolan in preparation of a Streptococcus suis type 2 capsule inhibitor. The present application discloses that proto-prosolan can significantly weaken the pathogenicity and immune escape ability of Streptococcus suis type 2 by inhibiting capsule synthesis, and then effectively plays an anti-infection role. Compared with vaccines, antibodies and phage enzymes and other highly limited anti-capsule strategies specific to serotypes, proto-prosolan, as an anti-toxicity inhibitor of Streptococcus suis type 2, has potential advantages such as broad spectrum, and has important scientific significance and application value for the research and development of new anti-drugs in the future. The proto-prosolan can be applied to the preparation of a Streptococcus suis type 2 capsule inhibitor and a drug for preventing and treating Streptococcus suis type 2 infection.

Description

Technical Field

[0001] This invention relates to the field of medical and pharmaceutical technology, specifically to the application of protopanaxadiol in the preparation of a type 2 streptococcal capsular inhibitor. Background Technology

[0002] Streptococcus suis serotype 2 is an important zoonotic pathogen that primarily infects pigs, but can also be transmitted to humans through contact. Human infection is commonly caused by occupational exposure in slaughtering and processing industries, and can lead to meningitis, septicemia, and toxic shock syndrome. It has a rapid onset and high mortality rate, making it a significant global public health problem.

[0003] Streptococcus suis type 2 (S. suis) is the major serotype of Streptococcus suis infection, and its pathogenicity is mainly influenced by a series of virulence factors. Among these, the capsule is one of the main virulence factors of Streptococcus suis, a structure mainly composed of polysaccharides covering the outer layer of the bacterial cell wall, with sialic acid as a major component. The capsule not only assists the pathogen in resisting various environmental stresses in the external environment but also helps it evade recognition by the host's immune system, making it a key factor influencing the pathogen's pathogenicity. Therefore, the capsule could serve as a potential target for the treatment of Streptococcus suis type 2 infection.

[0004] Protopanaxadiol, also known as 20(S)-protopanaxadiol (CAS No.: 34080-08-5), is one of the main active metabolites of ginsenosides in vivo, belonging to the dammarane-type tetracyclic triterpenoids. Its structural characteristic is the S configuration at the C-20 position, which is key to its biological activity. As an aglycone of proto-ginsenosides (such as Rb1 and Rg1), it is more easily absorbed and directly exerts its pharmacological effects. Studies have shown that protopanaxadiol possesses broad biological activities, including significant antitumor, anti-inflammatory, antioxidant, and neuroprotective effects. It can induce tumor cell apoptosis, inhibit proliferation and metastasis through multiple signaling pathways, and also has the potential to protect the nervous system and improve cognitive function, making it an important lead compound for new drug development. However, there are currently no reports of protopanaxadiol as a capsular inhibitor of Streptococcus suis type 2. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] To address the aforementioned technical problems, according to one aspect of the present invention, the present invention provides the following technical solution:

[0007] Application of protopanaxadiol in the preparation of a capsular inhibitor of Streptococcus suis type 2.

[0008] As a preferred embodiment of the application of protopanaxadiol in the preparation of a type 2 streptococcal capsular inhibitor according to the present invention, the protopanaxadiol inhibits capsular synthesis by inhibiting the production of type 2 streptococcal capsular polysaccharide.

[0009] As a preferred embodiment of the application of protopanaxadiol in the preparation of a type 2 streptococcal capsular inhibitor according to the present invention, wherein the type 2 streptococcal capsular inhibitor targets type 2 streptococcus suis O5ZYH33.

[0010] As a preferred embodiment of the application of protopanaxadiol in the preparation of a type 2 Streptococcus suis capsule inhibitor according to the present invention, the protopanaxadiol prevents and treats type 2 Streptococcus suis infection by inhibiting the synthesis of type 2 Streptococcus suis capsule.

[0011] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention discloses that protopanaxadiol can significantly weaken the pathogenicity and immune evasion ability of Streptococcus suis type 2 by inhibiting capsule synthesis, thereby exerting an effective anti-infection effect. Compared with anti-capsule strategies such as vaccines, antibodies, and phage enzymes, which are highly limited by serotype specificity, protopanaxadiol, as an antiviral inhibitor of Streptococcus suis type 2, has potential advantages such as broad spectrum. It has important scientific significance and application value for the development of new anti-antibiotic alternatives in the future, and can be applied to the preparation of Streptococcus suis type 2 capsule inhibitors, drugs for the prevention and treatment of Streptococcus suis type 2 infection, etc. Attached Figure Description

[0012] To more clearly illustrate the technical solutions of the embodiments of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and detailed embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0013] Figure 1 For the sialic acid content detection results provided in Example 1 of the present invention, **: P<0.01;

[0014] Figure 2 For the determination of crude sugar content of capsular polysaccharide provided in Example 2 of the present invention, ns: no statistical difference; **: P<0.01;

[0015] Figure 3 The image shows a transmission electron microscope image of the capsule morphology provided in Example 3 of the present invention, where A is the solvent control group, B is the original ginsenoside treatment group, and the red arrow points to the capsule.

[0016] Figure 4 The growth curve measurement results of Streptococcus suis type 2 05ZYH33 provided in Example 4 of the present invention;

[0017] Figure 5 The cytotoxicity analysis of protopanaxadiol provided in Example 5 of the present invention is shown in Figures A and B, which correspond to the cytotoxicity analysis results of HEp-2 and RAW264.7 cells, respectively. ns: no statistical difference.

[0018] Figure 6 The results of the epithelial cell adhesion test provided in Example 6 of the present invention; ns: no statistical difference; *: P<0.05;

[0019] Figure 7 The results of the macrophage phagocytosis assay provided in Example 7 of this invention; ns: no statistical difference; *: P<0.05; **: P<0.01;

[0020] Figure 8 The results of the analysis of the protective effect of protopanaxadiol against larvae of the giant wax moth caused by Streptococcus suis type 2, provided in Example 8 of the present invention.

[0021] All statistical analyses were performed using GraphPad Prism software (version 10.1.2). 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. Time-series tests (log-rank test) were used for survival analysis. Detailed Implementation

[0022] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0023] Secondly, the present invention is described in detail with reference to the schematic diagrams. When detailing the embodiments of the present invention, for ease of explanation, the cross-sectional views illustrating the device structure may be partially enlarged, not according to the usual scale. Furthermore, the schematic diagrams are merely examples and should not limit the scope of protection of the present invention. In addition, actual fabrication should include three-dimensional spatial dimensions of length, width, and depth.

[0024] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0025] This invention discloses a novel use of protopanaxadiol, namely its application in the preparation of a type 2 Streptococcus suis capsule inhibitor. The protopanaxadiol reduces the pathogenicity of Streptococcus suis by inhibiting capsule synthesis, thereby enhancing the host's immune recognition of the bacteria and preventing type 2 Streptococcus suis infection.

[0026] The embodiments of this invention reveal, through experiments such as sialic acid content detection, phenol-sulfuric acid method for quantitative capsular polysaccharide content, negative staining electron microscopy observation, growth curve, cytotoxicity assessment, epithelial cell adhesion test, macrophage phagocytosis test, and survival analysis of large wax moth larvae, that protopanaxadiol can significantly weaken the pathogenicity and immune evasion ability of Streptococcus suis type 2 by inhibiting capsular synthesis.

[0027] The type 2 Streptococcus suis used in this embodiment of the invention is specifically type 2 Streptococcus suis O5ZYH33, which is preserved in the Pharmacology and Toxicology Laboratory of the Institute of Zoonoses, Jilin University.

[0028] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0029] Example 1: Determination and analysis of sialic acid content:

[0030] Overnight cultured Streptococcus suis type 2 05ZYH33 was inoculated into 3 mL of THY medium at a dilution of 1:100. A control group (0, with an equal volume of DMSO solvent added) and drug-treated groups (previously treated with ginsenosides at final concentrations of 16, 32, and 64 μg / mL, purchased from Chengdu Deruike Biotechnology Co., Ltd.) were established and cultured until OD... 600 = 1.0. Centrifuge 2 mL of bacterial culture at 12000 × g for 5 min at 4 °C, wash three times with sterile PBS, collect the bacterial cells and freeze at -80 °C for 10 h. Add 100 μL of 0.4 M sulfuric acid aqueous solution to the sample to resuspend the bacterial precipitate, freeze again at -80 °C for 1 h, centrifuge at 12000 × g for 10 min, take 50 μL of supernatant as the sample, and use a sialic acid kit to detect the content;

[0031] The results are as follows Figure 1 As shown, protopanaxadiol can dose-dependently inhibit the content of sialic acid, the main component of the capsular polysaccharide of Streptococcus suis type 2, and a statistically significant difference is achieved at 16 μg / mL.

[0032] Example 2: Determination of crude sugar content of capsular polysaccharides:

[0033] Overnight cultured Streptococcus suis type 2 05ZYH33 was inoculated into 20 mL of THY medium containing (16-64 μg / mL) protopanaxadiol. A control group with an equal amount of DMSO was prepared. 05ZYH33 was cultured at 37℃ 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 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 OD using an ELISA reader. 490 The content of capsular polysaccharides was calculated using a self-made glucose standard curve.

[0034] The results are as follows Figure 2 As shown, protopanaxadiol can inhibit capsular polysaccharide synthesis in a dose-dependent manner from Streptococcus suis type 2 05ZYH33: it showed significant inhibition at 32 μg / mL.

[0035] Example 3: Transmission electron microscopy observation of capsule structure:

[0036] Overnight cultured Streptococcus suis type 2 05ZYH33 was multiplied at a ratio of 1:100 to the logarithmic growth phase (OD). 600 =0.6), with an equal volume of DMSO as the drug solvent control. The drug-treated group was cultured with a final concentration of 32 μg / mL protopanaxadiol. The bacterial cells were collected by centrifugation and washed twice with PBS. After discarding the supernatant, 2 mL of electron microscopy fixative was added to the bacterial cell pellet and incubated overnight at 4°C. The samples were embedded in LR white resin and further prepared into ultrathin sections. The samples loaded with copper mesh were stained with 2% uranium acetate and lead citrate. The bacterial surface capsule structure was observed using a transmission electron microscope.

[0037] The results are as follows Figure 3 As shown in the figure, compared with the solvent control group, the surface capsule of Streptococcus suis type 2 in the original ginsenoside triol treatment group was significantly thinner.

[0038] Example 4: Determination of growth curve of Streptococcus suis type 2:

[0039] Single colonies of Streptococcus suis type 2 (05ZYH33) were picked and cultured in THY liquid medium overnight at 37°C and 200 rpm in a constant temperature shaker. The culture was then expanded to OD at a ratio of 1:100. 600=0.3, the bacterial culture was aliquoted into four 50 mL Erlenmeyer flasks, 20 mL in each, and different concentrations of protopanaxadiol (0, 32, 64, and 128 μg / mL) were added sequentially (group 0 was treated with an equal volume of DMSO as a drug solvent control). The flasks were incubated at 37℃ and 200 rpm, and the OD of each sample was measured every 1 hour. 600 The value continues until growth reaches a plateau.

[0040] The results are as follows Figure 4 As shown, there was no significant difference in bacterial growth between the groups treated with different concentrations of protopanaxadiol and the untreated group, indicating that protopanaxadiol does not affect the growth of Streptococcus suis type 2 O5ZYH33 within the effective concentration range.

[0041] Example 5: Analysis of the toxic effects of protopanaxadiol on HEp-2 and RAW264.7 cells:

[0042] The potential toxicity of protopanaxadiol to HEp-2 and RAW264.7 cells (purchased from the American Culture Collection and preserved in our laboratory) was determined using a CCK-8 assay kit. Cells in culture flasks were digested with trypsin, the cell suspension was diluted, and the cells were counted at 5 × 10⁻⁶. 4 Cells were seeded at a density of 100 cells per well in 96-well plates and cultured overnight in a cell culture incubator (37°C, 5% CO2) to allow cell adhesion. The next day, the culture medium was replaced and protopanaxadiol was added to a final concentration of 0, 8, 16, 32, and 64 μg / mL (with an equal volume of DMSO as a drug solvent control). Wells containing cells and wells without cells were set up as controls. Three replicates were performed per well. The plates were incubated at 37°C for 10 h. After incubation, 100 μL of culture medium was discarded from each well of the 96-well plate, and 10 μL of CCK-8 solution was added. The plates were incubated at 37°C for 1 h, and the OD was measured using a microplate reader. 450 Absorbance, record the data and calculate;

[0043] The results are as follows Figure 5 As shown, compared with the solvent control group, protopanaxadiol did not have significant cytotoxicity on HEp-2 and RAW264.7 cells.

[0044] Example 6: Adhesion test of Streptococcus suis type 2 to HEp-2 epithelial cells:

[0045] HEp-2 cells were seeded in 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. Then, they were pre-cultured to the logarithmic growth phase (OD). 600Streptococcus suis type 2 (0.6 μg / mL) 05ZYH33 was used to infect cells at an MOI of 50. A control group (0, with an equal volume of DMSO) and drug-treated groups (16, 32, and 64 μg / mL) were set up, with three parallel wells in each group. After incubation at 37°C in a 5% CO2 incubator for 2 h, cells were washed three times with sterile PBS to remove unadhered bacteria, then sterile water was added to lyse the cells for 5 min. The samples were then diluted with PBS and plated for counting.

[0046] The results are as follows Figure 6 As shown, compared with the solvent control group, the treatment with protopanaxadiol increased the adhesion rate of Streptococcus suis type 2 05ZYH33 in a dose-dependent manner, with the adhesion rate at 64 μg / mL increasing by about 1.6 times.

[0047] Example 7: Phagocytosis assay of RAW264.7 macrophages against Streptococcus suis type 2:

[0048] RAW 264.7 cells were seeded in 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. Then, *Streptococcus suis* type 2 (05ZYH33) pre-cultured to the logarithmic growth phase (OD=0.6) was used to infect the cells at an MOI of 20. A control group (0, with an equal volume of DMSO) and drug-treated groups (16, 32, and 64 μg / mL) were set up, with three parallel wells in each group. After incubation for 1 h in a 37°C incubator containing 5% CO2, the cells were washed three times with sterile PBS to remove unadhered bacteria, and then replaced with DMEM medium containing 100 μg / mL to kill extracellular bacteria. After 1 h of incubation, the cells were washed three times with sterile PBS, and sterile water was added for 5 min to lyse the cells. The samples were then diluted with PBS and plated for counting.

[0049] The results are as follows Figure 7 As shown, protopanaxadiol dose-dependently increased the phagocytic rate of macrophages by approximately 5 times compared to the control group.

[0050] Example 8: Analysis of the protective effect of protopanaxadiol against Streptococcus suis larvae infected with Streptococcus suis type 2:

[0051] This study used *Streptococcus suis* larvae (purchased from Tianjin Huiyude Biotechnology Co., Ltd.) as experimental animals. Overnight cultured *Streptococcus suis* type 2 05ZYH33 was expanded into 20 mL of THY medium and cultured at 37°C until OD500. 600 =0.6~0.8, centrifuge to collect bacterial cells, wash with sterile PBS and resuspend the bacterial culture in PBS. Inoculate the right side of the last pair of abdominal feet with 10 μL (3 × 10⁻⁶) of the solution. 8CFUs were used to inoculate bacteria. After infection, animals were randomly divided into two groups (solvent control, 25 mg / kg protopanaxadiol) and blank control group, which were injected with an equal volume of PBS. Ten animals were in each group. The right side of the second to last pair of abdominal feet was given 10 μL of blank solvent (the animal experiment used biological solvents as solvents: 45% physiological saline, 5% Tween 80, 40% polyethylene glycol 400, 10% DMSO) or protopanaxadiol solution (prepared using biological solvents). The animals were incubated at 37°C, and the results were recorded every 8 hours.

[0052] The results are as follows Figure 8 As shown, compared with the solvent control group, treatment with 25 mg / kg protopanaxadiol can increase the survival rate of giant wax moth larvae by 20%, indicating that protopanaxadiol has a good protective effect on the giant wax moth larvae model infected with Streptococcus suis type 2.

[0053] In summary, the embodiments of the present invention have demonstrated through experiments such as sialic acid content detection, capsular polysaccharide crude sugar content determination, bacterial capsule negative staining electron microscopy observation, growth curve determination, cytotoxicity analysis, cell adhesion, cell phagocytosis, and survival rate analysis of *Streptococcus suis* larvae that protopanaxadiol can significantly weaken the pathogenicity of *Streptococcus suis* type 2 by inhibiting capsule synthesis, thereby exerting an effective anti-infective effect. This compound has no antibacterial activity against *Streptococcus suis* type 2 within the tested concentration range and has no obvious drug toxicity to mammalian cells.

[0054] Based on this, protopanaxadiol can be used to prepare a capsular inhibitor of Streptococcus suis type 2 or a drug for treating Streptococcus suis type 2 infectious diseases.

[0055] Although the present invention has been described above with reference to embodiments, various modifications can be made and components can be replaced with equivalents without departing from the scope of the invention. In particular, as long as there is no structural conflict, the features in the disclosed embodiments can be combined with each other in any manner. The lack of an exhaustive description of these combinations in this specification is merely for the sake of brevity and resource conservation. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. The application of protopanaxadiol in the preparation of a capsular inhibitor of Streptococcus suis type 2.

2. The application of protopanaxadiol according to claim 1 in the preparation of a type 2 streptococcal capsular inhibitor, characterized in that, The protopanaxadiol inhibits capsule synthesis by suppressing the production of capsular polysaccharides in Streptococcus suis type 2.

3. The application of protopanaxadiol according to claim 1 in the preparation of a type 2 streptococcal capsular inhibitor, characterized in that, The type 2 streptococcus capsular inhibitor targets type 2 streptococcus suis O5ZYH33.

4. The application of protopanaxadiol according to claim 1 in the preparation of a type 2 streptococcal capsular inhibitor, characterized in that, The protopanaxadiol prevents and treats Streptococcus suis infection by inhibiting the synthesis of Streptococcus suis capsules.

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