Use of tectorigenin B in the preparation of a drug for preventing and / or treating toxoplasma infection

Abstract

The application belongs to the technical field of medicine preparation, and particularly relates to application of Irisolidon B in preparation of medicine for preventing and / or treating toxoplasma infection. The application utilizes Irisolidon B to inhibit toxoplasma GFP-RH, and it is found through experiments that 0.025-1.6 muM of Irisolidon B has a significant inhibitory effect on the proliferation of toxoplasma GFP-RH in SH-SY5Y cells. The half inhibitory concentration (EC 50 ) of Irisolidon B on toxoplasma GFP-RH is 0.25 muM. The application provides a potential drug target for the treatment or prevention of toxoplasmosis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, and particularly relates to the application of irisin B in the preparation of drugs for the prevention and / or treatment of toxoplasmosis infection. Background Technology

[0002] Toxoplasmosis, also known as toxoplasmosis, is a zoonotic disease caused by infection with Toxoplasma gondii, primarily affecting organs such as the eyes, brain, heart, liver, and lymph nodes. During pregnancy, toxoplasmosis infection can cause fatal central nervous system disorders in the fetus (such as hydrocephalus, intracranial calcification, and microcephaly), and can also lead to the formation of tissue cysts in neurons, resulting in latent infection and abnormal neurodevelopment.

[0003] Current therapeutic compounds are poorly tolerated, leading to increased resistance and limited efficacy, necessitating long-term treatment. In this context, identifying new therapeutic targets is crucial for drug discovery. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a safe and highly effective medicine for treating toxoplasmosis infection, specifically relating to the use of irisin B in the preparation of medicines for the prevention and / or treatment of toxoplasmosis infection.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides the application of irisinoside B in inhibiting the proliferation of Toxoplasma gondii, wherein the concentration of irisinoside B in inhibiting the proliferation of Toxoplasma gondii is 0.025–1.6 μM.

[0007] The present invention also provides the use of irisin B in the preparation of medicaments for the prevention and / or treatment of toxoplasmosis infection.

[0008] Preferably, the concentration of irisinoside B in the drug is 0.025–1.6 μM.

[0009] Preferably, irisin B interferes with the proliferation of Toxoplasma gondii by inhibiting PDCD5 protein expression, triggering mitophagy.

[0010] The present invention also provides the application of irisin B in the preparation of drugs that inhibit PDCD5 protein expression.

[0011] This invention also provides the application of irisin B in the preparation of drugs that activate mitophagy.

[0012] The present invention also provides a drug for preventing toxoplasmosis infection, comprising iridoside B and pharmaceutically acceptable excipients.

[0013] Preferably, the concentration of irisinoside B in the drug is 0.025–1.6 μM.

[0014] The present invention also provides a medicament for treating toxoplasmosis infection, comprising iridoside B and pharmaceutically acceptable excipients.

[0015] Preferably, the concentration of irisinoside B in the drug is 0.025–1.6 μM.

[0016] This invention provides the application of irisin B in the preparation of drugs for the prevention and / or treatment of Toxoplasma gondii infection. This invention utilizes irisin B to inhibit Toxoplasma gondii GFP-RH. Experiments showed that 0.025–1.6 μM irisin B significantly inhibited the proliferation of Toxoplasma gondii GFP-RH in SH-SY5Y cells. The half-maximal inhibitory concentration (EC50) of irisin B against Toxoplasma gondii GFP-RH strains is also described. 50 The concentration was 0.25 μM. Multiple methods were used to investigate the molecular mechanism by which irisinoside B inhibits Toxoplasma gondii infection. The study found that irisinoside B interferes with Toxoplasma gondii proliferation by inhibiting PDCD5 protein expression and inducing mitophagy. In vivo infection experiments in mice also showed that irisinoside B can indeed inhibit Toxoplasma gondii proliferation and reduce the mortality rate of mice infected with Toxoplasma gondii. This provides a potential drug target for the treatment or prevention of Toxoplasma gondii. Attached Figure Description

[0017] Figure 1 Effects of different concentrations of irisin B on the viability of SH-SY5Y cells;

[0018] Figure 2 The inhibitory effect of different concentrations of irisin B on Toxoplasma gondii;

[0019] Figure 3 The effect of iridoside B on Toxoplasma gondii infection and proliferation was detected by immunofluorescence staining (the top figure shows the observation results under an immunofluorescence microscope, the bottom left figure shows the proportion of cells at different division numbers, and the bottom right figure shows the percentage of infected cells).

[0020] Figure 4 To explore the molecular mechanism by which irisin B inhibits Toxoplasma gondii infection using Western blotting;

[0021] Figure 5 To verify by RT-PCR that irisin B interferes with intracellular infection and proliferation of Toxoplasma gondii by inhibiting PDCD5 protein expression;

[0022] Figure 6 To verify by Western blotting that irisin B interferes with intracellular infection and proliferation of Toxoplasma gondii by inhibiting PDCD5 protein expression;

[0023] Figure 7 To investigate the molecular mechanism by which irisin B inhibits Toxoplasma gondii infection using the FACS method;

[0024] Figure 8 To detect the molecular mechanism by which irisin B inhibits Toxoplasma gondii proliferation using the FACS method;

[0025] Figure 9 To verify the molecular mechanism by which iridoside B inhibits Toxoplasma gondii infection by stimulating mitophagy using immunofluorescence staining (the top image shows the observation results under an immunofluorescence microscope, the bottom left image shows the proportion of cells at different division numbers, and the bottom right image shows the percentage of infected cells);

[0026] Figure 10 The effect of iridoside B on the number of Toxoplasma gondii bradyzoites in mice.

[0027] Figure 11 The effect of iridoside B on the size of Toxoplasma gondii bradyzoites in mice;

[0028] Figure 12 The effect of irisin B on the survival of mice infected with Toxoplasma gondii. Detailed Implementation

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] The sources of raw materials and reagents used in the embodiments of this invention are as follows:

[0031] Human neuroblastoma cells SH-SY5Y (SH-SY5Y cells) were purchased from Beina Biotechnology.

[0032] SH-SY5Y dedicated culture medium (87% EMEM / F-12K + 10% FBS + 1% L-glutamine + 1% sodium pyruvate + 1% NEAA) was purchased from Beina Biotechnology.

[0033] 1× phosphate buffer (1× PBS buffer) was purchased from Shanghai Solarbio Biotechnology Co., Ltd., catalog number P1020.

[0034] The pancreatic enzyme cell digestion solution (containing EDTA, 0.25% and 0.02%) was purchased from Jiangsu Kaiji Biotechnology Co., Ltd., product number VC2005.

[0035] Cell Toxicity Assay Kit (CellTiter) The AQueous One Solution CellProliferation Assay was purchased from Promega, USA, product number G3581.

[0036] The BCA Protein Assay Kit was purchased from Guangzhou Kangrun Biotechnology Co., Ltd., catalog number E162-01.

[0037] Anti-TP3 antibody (Anti-Toxoplasma gondii Antibody (TP3) SC-52255), anti-Parkin antibody (anti-Parkin Antibody (PRK8): SC-32282), and anti-alpha-Tubulin antibody (anti-alphaTubulin Antibody (B-7): SC-5286) were purchased from Santa Cruz Biotechnology, USA.

[0038] Anti-PDCD5 antibody (anti-PDCD5 Polyclonal antibody 12456-1-AP), anti-PINK1 antibody (Anti-PINK1 Polyclonal antibody 23274-1-AP), anti-LC3 antibody (Anti-LC3 Polyclonal antibody 14600-1-AP), and anti-ACO2 antibody (Anti-Aconitase 2 Polyclonal antibody 11134-1-AP) were all purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0039] The anti-p62 / SQSTM1 antibody (Anti-p62 / SQSTM1 Antibody P0067) was purchased from Sigma-Aldrich, Germany.

[0040] Iristectorin B was purchased from MedChemExpress in the United States, catalog number HY-N6819.

[0041] Control siRNA (SC-37007) and PDCD5 siRNA (h) (SC-97752) were both purchased from Santa Cruz Biotechnology, USA.

[0042] The pcDNA3.1 and pcDNA3.1-PDCD5 plasmids were provided by Wuhan Hewu Biotechnology Co., Ltd.

[0043] RNAiMAX Reagent was purchased from Invitrogen, USA, product number: 2582539.

[0044] I(1×) Reduced Serum Medium was purchased from Gibco, USA, product number: 2276963.

[0045] The tachyzoites of Toxoplasma gondii GFP-RH strain, RFP-RH strain, and ME-49 bradyzoites were all provided by Professor Guang-Ho Cha of the Department of Infectious Biology at Chungnam National University, South Korea.

[0046] Example 1

[0047] Iris glycoside B concentration screening

[0048] The experiment was divided into three groups: experimental group, control group, and blank control group. The experimental group was pretreated with different concentrations of iris glycoside B; the control group was treated with the corresponding volume of EMEM / F-12K medium; and the blank control group was not inoculated with cells and contained only EMEM / F-12K medium.

[0049] 0.5×10 4 SH-SY5Y cells at a density of 10 cells / mL were seeded in 96-well plates and cultured at 37°C and 5% CO2. On the second day, when cell confluence reached approximately 80%, cells were treated with different concentrations of Iristectorin B (0, 0.25, 0.5, 1, 2, 4, 5, 10, 20 μM), with three replicates per sample. After 24 hours, 20 μL of CellTiter was added to each well. The AQueous OneSolution reagent was incubated at 37°C and 5% CO2 in the dark for 1 hour. The reaction was terminated by adding 10% SDS, and the absorbance was measured at 490 nm. The effect of irisin B on cell viability was calculated based on the absorbance values, and the results are as follows: Figure 1 As shown.

[0050] Cell viability % = (A 实验 -A 空白 ) / (A 对照 -A 空白 )×100

[0051] A 实验 : Absorbance of the experimental group;

[0052] A 空白 :Absorbance of the blank control group;

[0053] A 对照 : Absorbance of the control group;

[0054] Figure 1 The results showed that Iristectorin B at concentrations below 1 μM had no toxicity to SH-SY5Y cells, and its half-maximal inhibitory concentration (IC50) for SH-SY5Y cell growth was [not specified].50 The concentration was 9.94 μM. The range of 0.25–1 μM was selected to investigate whether irisin B would affect the proliferation of Toxoplasma gondii without affecting the host cells themselves.

[0055] Example 2

[0056] Experiment on the inhibition of Toxoplasma gondii proliferation by irisin B

[0057] 0.8×10 6 SH-SY5Y cells were seeded at 1 / mL in 60mm culture dishes and cultured at 37℃ and 5% CO2. The next day, when the cells reached approximately 80% confluence, they were pretreated with different concentrations of Iristectorin B (0, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 μM) for 4 hours. The cell number was then increased fivefold (MOI 5, 4 × 10⁶ cells / mL). 6 Cells were infected with the Toxoplasma gondii GFP-RH strain (number of cells / mL) for 24 h. Genomic DNA was extracted and the DNA concentration was standardized to 50 ng / mL. The extracted DNA was used to detect the repeat units of the Toxoplasma gondii B1 gene using real-time quantitative PCR. Primers were synthesized by Sangon Biotech, and the primer sequence information is shown in Table 1. After the sample was measured, the number of parasites was quantified according to the standard curve.

[0058] Standard curve establishment method: 1×10 7 Toxoplasma gondii GFP-RH strain, cells / mL, was serially diluted with DEPC water to a final concentration of 1×10⁻⁶. 7 1×10 6 1×10 5 1×10 4 1×10 3 1×10 2 1×10 1 Genomic DNA was extracted at a concentration of 50 ng / mL. Real-time quantitative PCR was used to detect repeat units of the Toxoplasma gondii B1 gene, and a standard curve was plotted based on the results.

[0059] Experimental results are as follows Figure 2 As shown, Iristectorin B, in the concentration range of 0.025–1.6 μM, significantly inhibited the proliferation of Toxoplasma gondii GFP-RH strain in SH-SY5Y cells in a concentration-dependent manner. The half-maximal inhibitory concentration (EC50) of Iristectorin B against Toxoplasma gondii GFP-RH strain was also indicated. 50 The value is 0.25 μM.

[0060] Table 1 Primer sequences

[0061]

[0062] Example 3

[0063] Irisoside B inhibits Toxoplasma gondii infection and proliferation (immunofluorescence staining method)

[0064] The experiment was divided into an experimental group and a control group. The experimental group was pretreated with Iristectorin B, while the control group was treated with the corresponding volume of EMEM / F-12K medium.

[0065] 0.5×10 4 SH-SY5Y cells were seeded at a density of 10 cells / mL in 12-well plates plated with cell spreaders and cultured at 37°C and 5% CO2. On the second day, when the cells reached approximately 70% confluence, they were pretreated with 0.05 μM ristectorin B for 4 hours. The seeded cell number was increased to a 5-fold increase (MOI 5, 2.5 × 10⁻⁶). 4 Cells were infected with Toxoplasma gondii RFP-RH strain (number of cells / mL). Two hours after invasion, uninvaded free tachyzoites were washed with 1×PBS, and the cells were cultured in fresh EMEM / F-12K medium for another 22 hours. The cells were then fixed with 4% paraformaldehyde, washed three times with PBST containing 0.3% Triton X-100, 5 min each time, and blocked with 1% BSA at room temperature for 30 min. Anti-α-Tubulin antibody (anti-alphaTubulinAntibody(B-7):SC-5286) was diluted 1:250 and incubated overnight at 4°C. The primary antibody was recovered the next day, and the cells were washed three times with PBST, 5 min each time. The secondary antibody (goat anti-mouse IgG, Alexa Fluor) was diluted 1:500. TM 488, A11029), incubated at room temperature in the dark for 2 hours. Washed three times with PBST, 10 min each time. After staining and mounting, observed and photographed under an immunofluorescence microscope, and the infection rate and proliferation of Toxoplasma gondii per 100 cells were counted.

[0066] Experimental results are as follows Figure 3 As shown, in the control group (CTL), more than 50% of tachyzoites divided 3 times within 24 hours, and about 10% of tachyzoites divided 4 times to produce 16 tachyzoites. In contrast, in the experimental group (Iristectorin B), less than 20% of the parasites divided 3 times, and about 40% divided once. In the control group, more than 60% of the cells were infected with Toxoplasma gondii, while after Iristectorin B pretreatment, only about 50% of the cells showed the presence of parasites. This indicates that Iristectorin B can significantly inhibit the proliferation and infection of Toxoplasma gondii.

[0067] Example 4

[0068] Molecular mechanism of irisin B inhibiting Toxoplasma gondii infection and proliferation (Western blotting method)

[0069] 0.8×10 6 SH-SY5Y cells were seeded at a density of 10 cells / mL into 60 mm culture dishes and cultured at 37°C and 5% CO2. On the second day, when the cells reached approximately 80% confluence, they were pretreated with 0.05 μM Iristectorin B for 4 h. The seeded cell number was then increased fivefold (MOI 5, 4.0 × 10⁻⁶). 6 Cells were infected with the Toxoplasma gondii GFP-RH strain (number of cells / mL). Two hours after invasion, uninvaded free tachyzoites were washed away with 1×PBS, and the cells were cultured in fresh EMEM / F-12K medium for another 22 hours. Proteins were extracted, and the expression levels of PDCD5, mitophagy-related proteins (ACO2, PINK1, Parkin, P62, LC3), and the Toxoplasma gondii surface protein TP3 were detected by Western blotting; α-Tubulin was used as an internal control.

[0070] Experimental results are as follows Figure 4 As shown, Toxoplasma gondii infection increases PDCD5 protein expression and inhibits mitophagy (PINK1 and Parkin protein levels decrease, selective autophagy receptor protein p62 expression is upregulated, and the classic autophagy marker LC3 is reduced). Iristectorin B reverses these effects, decreasing PDCD5 protein expression and reducing the expression of ACO2, a key enzyme in the mitochondrial tricarboxylic acid cycle, indicating impaired mitochondrial function and activation of mitophagy. The expression of the Toxoplasma gondii surface protein TP3 is also inhibited. This suggests that Iristectorin B's inhibition of Toxoplasma gondii proliferation may induce mitophagy by suppressing PDCD5 expression.

[0071] Example 5

[0072] Molecular mechanism of irisin B inhibiting Toxoplasma gondii infection and proliferation (RT-PCR method)

[0073] 0.8×10 6 SH-SY5Y cells at a density of 10 cells / mL were seeded into 6-well cell culture plates and cultured at 37°C and 5% CO2. On the second day, when the cells reached approximately 70% confluence, six 1.5mL EP tubes (A, B, C, D, E, and F) were prepared. 1200μL of [unspecified substance] was added to tube A. Medium + 72μL RNAiMAX Reagent; Add 300 μL to tube B. Medium + 3 μL siCTL (30 pmol); Add 300 μL to tube C Medium + 3 μL SiPDCD5 (30 pmol); Add 300 μL to tube D Medium + 3 μL pcDNA3.1 (30 pmol); add 300 μL to tube E. Medium + 3 μL pcDNA3.1-PDCD5 (30 pmol). Mix 300 μL each of tubes AB, AC, AD, and AE in a 1:1 ratio until homogeneous. Add 1200 μL to tube F. Medium, incubate at room temperature for 5 min. Add 600 μL of the mixture to a 6-well container and incubate at 37°C for 24 h. Then seed cells at a 5-fold increase in number (MOI 5, 4.0 × 10⁻⁶). 6 Cells were infected with the Toxoplasma gondii GFP-RH strain (number of cells / mL). After 2 hours of invasion, uninvaded free tachyzoites were washed away with 1×PBS, and the cells were cultured in fresh EMEM / F-12K medium for another 22 hours. mRNA was extracted using Trizol, cNDA was synthesized, and the expression levels of PDCD5 and SAG1 genes were detected by RT-PCR. HPRT-1 was used as an internal control.

[0074] Experimental results are as follows Figure 5 As shown, silencing the PDCD5 gene downregulates the expression level of the Toxoplasma gondii surface gene SAG1; overexpression of the PDCD5 gene increases the expression level of the Toxoplasma gondii surface gene SAG1. This indicates that PDCD5 contributes to the intracellular proliferation of Toxoplasma gondii.

[0075] Example 6

[0076] Molecular mechanism of irisin B inhibiting Toxoplasma gondii infection and proliferation (Western blotting method)

[0077] 0.8×10 6 SH-SY5Y cells at a density of 10 cells / mL were seeded into 6-well cell culture plates and cultured at 37°C and 5% CO2. On the second day, when the cells reached approximately 70% confluence, six 1.5mL EP tubes (A, B, C, D, E, and F) were prepared. 1200μL of [unspecified substance] was added to tube A. Medium + 72μL RNAiMAX Reagent; Add 300 μL to tube B. Medium + 3 μL siCTL (30 pmol); Add 300 μL to tube C. Medium + 3 μL SiPDCD5 (30 pmol); Add 300 μL to tube D. Medium + 3 μL pcDNA3.1 (30 pmol); Add 300 μL to tube E. Medium + 3 μL pcDNA3.1-PDCD5 (30 pmol). Mix 300 μL each of tubes AB, AC, AD, and AE in a 1:1 ratio until homogeneous. Add 1200 μL to tube F. Medium, incubate at room temperature for 5 min. Add 600 μL of the mixture to a 6-well container and incubate at 37°C for 24 h. Then seed cells at a 5-fold increase in number (MOI 5, 4.0 × 10⁻⁶). 6 Cells were infected with the Toxoplasma gondii GFP-RH strain (number of cells / mL). After 2 hours of invasion, uninvaded free tachyzoites were washed away with 1×PBS, and the cells were cultured in fresh EMEM / F-12K medium for another 22 hours. Proteins were extracted, and the expression levels of PDCD5 and Toxoplasma gondii surface protein TP3 were detected by Western blotting; α-Tubulin was used as an internal control.

[0078] Experimental results are as follows Figure 6 As shown, silencing the PDCD5 gene downregulates the expression level of the Toxoplasma gondii surface protein TP3; overexpression of the PDCD5 gene increases the expression level of the Toxoplasma gondii surface gene TP3. This indicates that PDCD5 contributes to the intracellular proliferation of Toxoplasma gondii.

[0079] Example 7

[0080] Molecular mechanism of irisin B inhibiting Toxoplasma gondii infection and proliferation (FACS method)

[0081] 0.5×10 4 SH-SY5Y cells at a density of 10 cells / mL were seeded in 12-well plates and cultured at 37°C and 5% CO2. On the second day, when the cells reached approximately 70% confluence, six 1.5mL EP tubes (A, B, C, D, E, and F) were prepared. Tube A contained 600μL of cells. Medium + 36μL RNAiMAX Reagent; Tube B: 150μL Medium + 1.5 μL SiCTL (30 pmol); C tube 150 μL Medium + 1.5 μL siPDCD5 (30 pmol); D tube: 150 μL Medium+1.5μL pcDNA3.1 (30pmol); E tube: 150μL Medium + 1.5 μL pcDNA3.1-PDCD5 (30 pmol). Mix 150 μL each of tubes AB, AC, AD, and AE in a 1:1 ratio until homogeneous. Add 600 μL to tube F. Medium, incubate at room temperature for 5 min. Add 300 μL of the mixture to a 6-well container and incubate at 37°C for 24 h. Seed cells at a 5-fold increase in number (MOI 5, 2.5 × 10⁻⁶). 4 Cells were infected with the Toxoplasma gondii GFP-RH strain (number of cells / mL). After 1 h (for Toxoplasma gondii invasion) or 2 h, uninfected free tachyzoites were washed off with 1×PBS, and the cells were cultured in fresh EMEM / F-12K medium for 22 h (for Toxoplasma gondii proliferation). Cells were digested with 0.25% trypsin-EDTA for 3 min, digestion was stopped with EMEM / F-12K medium, and the cell pellet was collected by centrifugation at 1,000 rpm for 3 min. The cells were then washed twice with FACS buffer (PBS containing 1% BSA), and the data were analyzed using FACScan (BDBio-science), recording the median fluorescence intensity (MFI).

[0082] Without any treatment, 1 hour after infection, the CTL group showed a median fluorescence intensity (MFI) of GFP of 26.4%. PDCD5 gene silencing reduced the MFI of GFP to 19.8%; while PDCD5 gene overexpression increased the MFI of GFP to 34.7%. Figure 7 At 24 hours after infection, GFP showed a mean fractional free (MFI) higher than 67% in the MOI5 group. In the PDCD5 gene silencing group, the MFI of GFP decreased to 40.5%, while in the PDCD5 gene overexpression group, the MFI of GFP increased to 82.1%. Figure 8 This indicates that PDCD5 is indeed crucial for Toxoplasma gondii proliferation and infection, and that irisin B inhibits intracellular infection and growth of Toxoplasma gondii by suppressing PDCD5 gene expression.

[0083] Example 8

[0084] Molecular mechanism of irisin B inhibiting Toxoplasma gondii infection and proliferation (immunofluorescence staining method)

[0085] 0.5×10 4 SH-SY5Y cells were seeded at a density of 1 / mL in 12-well plates coated with cell spreaders and cultured at 37°C and 5% CO2. After 24 hours, when the cells reached approximately 70% confluence, they were pretreated for 4 hours with the autophagy inhibitors 3-MA (5 μM), Wortmannin (1 μM), and Baf-A1 (10 nM), respectively. The seeded cell number was then increased fivefold (MOI 5, 2.5 × 10⁻⁶). 4Cells were infected with the Toxoplasma gondii GFP-RH strain (number of cells / mL). Two hours after invasion, uninvaded free tachyzoites were washed with 1×PBS, and the cells were cultured in fresh EMEM / F-12K medium for another 22 hours. The cells were then fixed with 4% paraformaldehyde, washed three times with PBST containing 0.3% Triton X-100, 5 min each time, and blocked with 1% BSA at room temperature for 30 min. Anti-alpha Tubulin antibody (anti-alpha Tubulin Antibody (B-7): SC-5286) was diluted 1:250 and incubated overnight at 4°C. The primary antibody was recovered the next day, and the cells were washed three times with PBST, 5 min each time. The secondary antibody (goat anti-mouse IgG, Alexa Fluor) was diluted 1:500. TM 568, A11031), incubated at room temperature in the dark for 2 hours. Washed three times with PBST, 10 min each time. After staining and mounting, observed and photographed under an immunofluorescence microscope, and the Toxoplasma gondii infection rate and Toxoplasma gondii proliferation were counted per 100 cells. The control (Con) group did not receive any autophagy inhibitors.

[0086] Experimental results are as follows Figure 9 As shown, in the control (Con) group, approximately 30% of tachyzoites divided three times within 24 hours, producing eight tachyzoites within a single vesicle. In the autophagy inhibitor treatment group, more than half of the tachyzoites were able to divide three times within 24 hours, with approximately 20% of the parasites even dividing four times to produce 16 tachyzoites. Furthermore, approximately 60% of the cells in the control group were infected with *Toxoplasma gondii*, while after autophagy inhibitor pretreatment, parasites were observed in 75-80% of the cells. This indicates that inhibiting mitophagy promotes intracellular infection and proliferation of *Toxoplasma gondii*. It also suggests that iridoside B interferes with *Toxoplasma gondii* intracellular proliferation by inducing mitophagy.

[0087] Example 9

[0088] Iris glycoside B inhibits Toxoplasma gondii infection and proliferation in mice.

[0089] The experiment consisted of two groups: a control group (CTL) and an experimental group (Iristectorin B), with three mice in each group. The experimental group was treated with Iristectorin B for one week after infection with Toxoplasma gondii, while the control group was treated with an equal volume of PBS for one week.

[0090] Six C57BL / 6J mice of similar weight, age (6–8 weeks), and sex (male) were randomly divided into two groups. Each mouse was administered 80 ME-49 Toxoplasma gondii bradyzoites via gavage to mimic the human infection pathway. One week later, the experimental group mice were given Iristectorin B at a dose of 30 mg / kg via intraperitoneal injection once daily for another week. The control group mice were given an equal volume of PBS buffer at the same time and in the same manner. At the end of the experiment, the mice were euthanized by cervical dislocation, and brain tissue was collected. One-quarter of the brain tissue was ground in 300 μL of PBS buffer. 10 μL of the solution was examined under a microscope to observe and count the ME-49 bradyzoites. Differences between the control and experimental groups were compared.

[0091] The number of bradyzoites in mice in the experimental group that received Iristectorin B for one week was... Figure 10 ),size( Figure 11 The levels of irisin B were significantly lower than those in the control group. This confirms that irisin B can indeed inhibit the proliferation of Toxoplasma gondii.

[0092] Example 10

[0093] Effect of irisin B on the survival rate of mice infected with Toxoplasma gondii

[0094] The experiment consisted of two groups: a control group (CTL) and an experimental group (Iristectorin B), with three mice in each group. The experimental group was administered Iristectorin B for one week after infection with Toxoplasma gondii, while the control group was administered an equal volume of PBS for one week.

[0095] Twelve C57BL / 6J mice of similar weight, age (6–8 weeks), and sex (male) were randomly divided into two groups. Each mouse was administered 80 ME-49 Toxoplasma gondii bradyzoites via gavage to mimic the human Toxoplasma gondii infection pathway. One week later, the experimental group mice were given Iristectorin B at a dose of 30 mg / kg via intraperitoneal injection once daily for another week. The control group mice were given an equal volume of PBS buffer at the same time and in the same manner. The survival rate of the mice was recorded over 15 days.

[0096] Experimental results are as follows Figure 12 In the control group, all mice died within 7-10 days. In the experimental group, mice treated with Iristectorin B for one week still had two surviving mice on day 15, a significant difference compared to the control group. This confirms that Iristectorin B can indeed inhibit the proliferation of Toxoplasma gondii.

[0097] This invention provides the application of irisin B in the preparation of drugs for the prevention and / or treatment of toxoplasmosis infection. This invention utilizes irisin B to inhibit the tachyzoite RH of *Toxoplasma gondii*. Experiments showed that 0.025–1.6 μM irisin B significantly inhibited the proliferation of *Toxoplasma gondii* RH strains in SH-SY5Y cells. The half-maximal inhibitory concentration (EC50) of irisin B against *Toxoplasma gondii* GFP-RH strains is also described. 50 The concentration was 0.25 μM. Multiple methods were used to investigate the molecular mechanism by which irisinoside B inhibits Toxoplasma gondii infection. It was found that irisinoside B interferes with Toxoplasma gondii proliferation by inhibiting PDCD5 protein expression and inducing mitophagy. Further studies investigated the inhibition of Toxoplasma gondii infection in mice, finding that irisinoside B indeed inhibited Toxoplasma gondii proliferation and reduced mouse mortality. This provides a potential drug target for the treatment or prevention of toxoplasmosis.

[0098] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of irisinoside B in the preparation of drugs for the prevention and / or treatment of toxoplasmosis infection.

2. The application according to claim 1, characterized in that, The concentration of irisinoside B in the drug ranges from 0.025 to 1.6 μM.

3. The application according to claim 1 or 2, characterized in that, Iris glycoside B interferes with Toxoplasma gondii proliferation by inhibiting PDCD5 protein expression, triggering mitophagy.

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