Application of tectoridin B in preparation of medicine for preventing and / or treating toxoplasma gondii infection

Abstract

The invention belongs to the technical field of medicine preparation, and particularly relates to application of tectoridin B in preparation of a medicine for preventing and / or treating toxoplasma gondii infection. According to the application disclosed by the invention, the tectoridin B is used for inhibiting the toxoplasma gondii GFP-RH, and an experiment shows that the tectoridin B with the concentration of 0.025-1.6 mu M has a remarkable inhibition effect on the proliferation of the toxoplasma gondii GFP-RH in an SH-SY5Y cell. And the median inhibitory concentration (EC50) of the tectoridin B to the toxoplasma gondii GFP-RH strain is 0.25 mu M. And a potential drug target is provided for treatment or prevention of toxoplasmosis.

Description

Technical Field

[0001] The invention belongs to the technical field of drug preparation, and in particular relates to the application of tectoriformis glycoside B in the preparation of drugs for preventing and / or treating Toxoplasma gondii infection. Background Art

[0002] Toxoplasmosis, also known as toxoplasmosis, is a zoonosis caused by infection with Toxoplasma gondii, which mainly affects the eyes, brain, heart, liver, lymphatic organs, etc. Toxoplasma infection during pregnancy can cause fatal central nervous system lesions in the fetus (such as hydrocephalus, intracranial calcification, microcephaly, etc.), and can also form tissue cysts in neurons, causing latent infection and leading to abnormal neurodevelopment.

[0003] Current therapeutic compounds are poorly tolerated, develop resistance, have limited efficacy, and require long-term treatment. In this context, finding new therapeutic targets is crucial for drug discovery. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a safe and highly effective drug for treating toxoplasmosis infection, and specifically to use of irisin B in the preparation of a drug for preventing and / or treating toxoplasmosis infection.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the use of tectoriformis glycoside B in inhibiting the proliferation of Toxoplasma gondii. The concentration of tectoriformis glycoside B in inhibiting the proliferation of Toxoplasma gondii is 0.025-1.6 μM.

[0007] The present invention also provides the use of tectoriusin B in preparing a medicine for preventing and / or treating Toxoplasma gondii infection.

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

[0009] Preferably, tectoriformis B inhibits the expression of PDCD5 protein, triggers mitochondrial autophagy, and interferes with the proliferation of Toxoplasma gondii.

[0010] The present invention also provides the use of tectoriformis B in preparing a medicine for inhibiting the expression of PDCD5 protein.

[0011] The present invention also provides the use of tectoriusin B in preparing a medicine for activating mitochondrial autophagy.

[0012] The present invention also provides a medicine for preventing Toxoplasma gondii infection, comprising tectoriformis B and pharmaceutically acceptable excipients.

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

[0014] The present invention also provides a medicine for treating Toxoplasma gondii infection, comprising tectoriformis B and pharmaceutically acceptable excipients.

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

[0016] The present invention provides the use of tectoriformis B in the preparation of a drug for preventing and / or treating Toxoplasma infection. The present invention uses tectoriformis B to inhibit Toxoplasma GFP-RH. Experiments show that 0.025 to 1.6 μM tectoriformis B has a significant inhibitory effect on the proliferation of Toxoplasma GFP-RH in SH-SY5Y cells. The half inhibitory concentration (EC 50) of tectoriformis B on Toxoplasma GFP-RH strain is 2.377 μM. 50 ) is 0.25μM. Through various methods, the molecular mechanism of irisin B in inhibiting Toxoplasma infection was studied. It was found that irisin B inhibited the expression of PDCD5 protein and triggered mitochondrial autophagy, thereby interfering with the proliferation of Toxoplasma. In vivo infection experiments were also conducted on mice, and it was found that irisin B can indeed inhibit the proliferation of Toxoplasma and reduce the mortality of Toxoplasma-infected mice. It provides potential drug targets for the treatment or prevention of Toxoplasma. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Effects of different concentrations of tectoriusin B on SH-SY5Y cell viability;

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

[0019] Figure 3 Immunofluorescence staining was used to detect the effect of tectorigenin B on Toxoplasma gondii infection and proliferation (the upper figure shows the observation results under immunofluorescence microscope, the lower left figure shows the proportion of cells with different division times, and the lower right figure shows the percentage of infected cells);

[0020] Figure 4 To explore the molecular mechanism of tectorigenin B in inhibiting Toxoplasma gondii infection by Western blotting;

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

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

[0023] Figure 7 To detect the molecular mechanism of tectorigenin B in inhibiting Toxoplasma gondii infection by FACS method;

[0024] Figure 8 To detect the molecular mechanism of tectorigenin B in inhibiting the proliferation of Toxoplasma gondii by FACS method;

[0025] Fig. 9 Immunofluorescence staining was used to verify the molecular mechanism of irisin B inhibiting Toxoplasma infection by stimulating mitochondrial autophagy (the upper picture shows the observation results under an immunofluorescence microscope, the lower left picture shows the proportion of cells with different division times, and the lower right picture shows the percentage of infected cells);

[0026] Fig.10 The effect of tectorius glycoside B on the number of Toxoplasma gondii bradyzoites in mice;

[0027] Fig.11 The effect of tectorius glycoside B on the size of Toxoplasma bradyzoites in mice;

[0028] Fig.12 This is the effect of irisin B on the survival of mice infected with Toxoplasma gondii. DETAILED DESCRIPTION

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

[0030] Sources of raw materials and reagents used in the examples of the present invention:

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

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

[0033] 1× phosphate buffered saline (1× PBS buffer) was purchased from Shanghai Solebow Biotechnology Co., Ltd. with the catalog number P1020.

[0034] Trypsin cell digestion solution (containing EDTA, 0.25%: 0.02%) was purchased from Jiangsu KeyGen Biotech Co., Ltd. with the catalog number VC2005.

[0035] Cytotoxicity Assay Kit (CellTiter AQueous One Solution Cell Proliferation Assay) was purchased from Promega Corporation, USA, with the catalog number G3581.

[0036] BCA protein concentration determination kit (BCA Protein Assay Kit) was purchased from Guangzhou Kangrun Biotechnology Co., Ltd. with the catalog number E162-01.

[0037] Anti-TP3 antibody (Anti-Toxoplasma gondiiAntibody (TP3) SC-52255), anti-Parkin antibody (anti-ParkinAntibody (PRK8): SC-32282), and anti-α-Tubulin antibody (anti-alphaTubulinAntibody (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] Anti-p62 / SQSTM1 antibody (Anti-p62 / SQSTM1 Antibody P0067) was purchased from Sigma, Germany.

[0040] Iristectorin B was purchased from MEC (MedChemExpress) in the United States with the product number HY-N6819.

[0041] Control siRNA (SC-37007) and PDCD5 siRNA (h) (SC-97752) were 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, catalog number: 2582539.

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

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

[0046] Example 1

[0047] Screening of tectoriusin B concentration

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

[0049] 0.5×10 4 SH-SY5Y cells were seeded at 100 μg / mL in a 96-well plate and cultured at 37°C and 5% CO2. The next day, when the cell confluence reached about 80%, the cells were treated with different concentrations of Iristectorin B (0, 0.25, 0.5, 1, 2, 4, 5, 10, 20 μM), and three replicate wells were prepared for each sample. After 24 hours, 20 μL of CellTiter was added to each well. AQueous OneSolution reagent was incubated in the dark at 37°C and 5% CO2 for 1 hour, 10% SDS was added to terminate the reaction, and the absorbance was measured at a wavelength of 490 nm. The effect of irisin B on cell viability was calculated based on the absorbance value. The results are shown in Figure 1 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 blank control group;

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

[0054] Figure 1 The results showed that Iristectorin B below 1 μM had no toxicity to SH-SY5Y cells, and its half inhibitory concentration (IC50 ) was 9.94 μM, and the range of 0.25 to 1 μM was selected to see whether irisin B would affect the proliferation of Toxoplasma gondii when it had no effect on the host cells themselves.

[0055] Example 2

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

[0057] 0.8×10 6 SH-SY5Y cells with a density of 100 cells / mL were inoculated in a 60 mm culture dish and cultured at 37°C and 5% CO2. The next day, when the cells grew to about 80%, SH-SY5Y cells 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 h. The number of cells inoculated was increased to 5 times (MOI5, 4×10 6 The cells were infected with the GFP-RH strain of Toxoplasma gondii (ng / mL) for 24 h. The genomic DNA was extracted and the DNA concentration was unified to 50 ng / mL. The extracted DNA was used for real-time fluorescence quantitative PCR to detect the repeating unit of the Toxoplasma B1 gene. The primers were synthesized by Sangon Biotechnology. The primer sequence information is shown in Table 1. After the sample determination was completed, the number of parasites was quantified according to the standard curve.

[0058] Standard curve establishment method: 1×10 7 Toxoplasma gondii GFP-RH strain was diluted with DEPC water to a final concentration of 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 1×10 1 / mL, extract genomic DNA, and unify the DNA concentration to 50ng / mL. Use real-time fluorescence quantitative PCR to detect the repeating unit of Toxoplasma gondii B1 gene, and draw a standard curve based on the results.

[0059] The experimental results are as follows Figure 2 As shown in the results, Iristectorin B in the concentration range of 0.025 to 1.6 μM significantly inhibited the proliferation of Toxoplasma gondii GFP-RH strain in SH-SY5Y cells. The inhibitory effect was concentration-dependent. The half inhibitory concentration (EC 50 ) is 0.25μM.

[0060] Table 1 Primer sequences

[0061]

[0062] Example 3

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

[0064] The experiment was divided into: experimental group and control group. The experimental group was Iristectorin B pretreatment group; the control group was added with corresponding volume of EMEM / F-12K culture medium.

[0065] 0.5×10 4 SH-SY5Y cells at 100 / mL were inoculated in a 12-well plate covered with a cell slide and cultured at 37°C and 5% CO2. The next day, when the cells grew to about 70%, SH-SY5Y cells were pretreated with 0.05 μM Iristectorin B for 4 h. The number of cells inoculated was increased to 5 times (MOI5, 2.5×10 4 / mL) of Toxoplasma gondii RFP-RH strain. After 2 hours of invasion, wash the non-invaded free tachyzoites with 1×PBS, and change to fresh EMEM / F-12K medium for further culture for 22 hours. Then fix with 4% paraformaldehyde and wash three times with PBST containing 0.3% Triton X-100, 5 min / time. Block with 1% BSA at room temperature for 30 minutes. Anti-α-Tubulin antibody (anti-alphaTubulinAntibody(B-7):SC-5286) was diluted 1:250 and incubated at 4°C overnight. The primary antibody was recovered the next day and washed three times with PBST, 5 min / time. The secondary antibody (goat anti-mouse IgG, Alexa Fluor TM 488, A11029), incubated at room temperature in the dark for 2 h. Washed three times with PBST, 10 min / time. After staining and sealing, observed and photographed under an immunofluorescence microscope, and counted the Toxoplasma infection rate and Toxoplasma proliferation per 100 cells.

[0066] The experimental results are as follows Figure 3 As shown, in the control group (CTL), more than 50% of the tachyzoites will divide three times in 24 hours, and about 10% of the tachyzoites will divide four times to produce 16 tachyzoites, while in the experimental group (Iristectorin B), less than 20% of the parasites will divide three times, and about 40% will divide once; more than 60% of the cells in the control group were infected with Toxoplasma gondii, while after pretreatment with Iristectorin B, parasites could be observed in only about 50% of the cells, indicating that Iristectorin B can significantly inhibit the proliferation and infection of Toxoplasma gondii.

[0067] Example 4

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

[0069] 0.8×10 6 SH-SY5Y cells with a density of 10 cells / mL were inoculated into a 60 mm culture dish and cultured at 37°C and 5% CO2. The next day, when the cells grew to about 80%, SH-SY5Y cells were pretreated with 0.05 μM Iristectorin B for 4 h. The number of cells inoculated was increased to 5 times the number (MOI5, 4.0×10 6 Cells were infected with the Toxoplasma gondii GFP-RH strain (1000 cells / mL). After 2 hours of invasion, the non-invaded free tachyzoites were washed with 1×PBS and cultured in fresh EMEM / F-12K medium for 22 hours. Proteins were extracted and the expression levels of PDCD5, mitochondrial autophagy-related proteins (ACO2, PINK1, Parkin, P62, LC3), and Toxoplasma surface protein TP3 were detected by western blotting; α-Tubulin was used as an internal reference.

[0070] The experimental results are as follows Figure 4 As shown in the figure, the expression level of PDCD5 protein increased in Toxoplasma infection, and mitophagy was inhibited (PINK1 and Parkin protein levels decreased, the expression of selective autophagy receptor protein p62 was upregulated, and the classic autophagy marker LC3 was reduced). Iristectorin B reversed the above effects, reduced the expression level of PDCD5 protein, and reduced the expression of ACO2, a key enzyme in the mitochondrial tricarboxylic acid cycle, indicating that mitochondrial function was impaired and mitophagy was activated. The expression of Toxoplasma surface protein TP3 was inhibited. This shows that Iristectorin B inhibits Toxoplasma proliferation and may induce mitophagy by inhibiting PDCD5 expression.

[0071] Example 5

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

[0073] 0.8×10 6 SH-SY5Y cells of 100 μg / mL were inoculated into a 6-well cell plate and cultured at 37°C and 5% CO2. The next day, when the cells grew to about 70%, prepare 6 1.5 mL EP tubes A, B, C, D, E, and F. Add 1200 μL of 1.5 mL EP tube to tube A. Medium+72μL RNAiMAX Reagent; add 300 μL to tube B Medium + 3μL siCTL (30pmol); add 300μL to tube C Medium + 3μL siPDCD5 (30pmol); add 300μL to tube D Medium + 3 μL pcDNA3.1 (30 pmol); add 300 μL to tube E Medium + 3μL pcDNA3.1-PDCD5 (30pmol). 300μL of each of AB, AC, AD, and AE tubes were mixed at a ratio of 1:1. 1200μL of F tube was added Medium, incubate at room temperature for 5 min. Add 600 μL of the mixture to 6-well and incubate at 37°C for 24 h. Then increase the number of cells to 5 times the number of cells (MOI5, 4.0×10 6 The cells were infected with the Toxoplasma gondii GFP-RH strain (1000 cells / mL). After 2 hours of invasion, the uninvaded free tachyzoites were washed with 1×PBS and replaced with fresh EMEM / F-12K medium for 22 hours. mRNA was extracted with Trizol, and after synthesizing cDNA, the expression levels of PDCD5 and SAG1 genes were detected by RT-PCR, and HPRT-1 was used as an internal reference.

[0074] The experimental results are as follows Figure 5 As shown in the figure, when the PDCD5 gene is silenced, the expression level of the Toxoplasma surface gene SAG1 is downregulated; when the PDCD5 gene is overexpressed, the expression level of the Toxoplasma surface gene SAG1 is increased, indicating that PDCD5 contributes to the intracellular proliferation of Toxoplasma.

[0075] Example 6

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

[0077] 0.8×10 6 SH-SY5Y cells of 100 μg / mL were inoculated into a 6-well cell plate and cultured at 37°C and 5% CO2. The next day, when the cells grew to about 70%, prepare 6 1.5 mL EP tubes A, B, C, D, E, and F. Add 1200 μL of 1.5 mL EP tube to tube A. Medium+72μL RNAiMAX Reagent; Add to tube B: 300 μL Medium + 3μL siCTL (30pmol); add 300μL to tube C Medium + 3μL siPDCD5 (30pmol); add 300μL to tube D Medium + 3 μL pcDNA3.1 (30 pmol); add 300 μL to tube E Medium + 3μL pcDNA3.1-PDCD5 (30pmol). 300μL of each of AB, AC, AD, and AE tubes were mixed at a ratio of 1:1. 1200μL of F tube was added Medium, incubate at room temperature for 5 min. Add 600 μL of the mixture to 6-well and incubate at 37°C for 24 h. Then increase the number of cells to 5 times the number of cells (MOI5, 4.0×10 6 Cells were infected with the GFP-RH strain of Toxoplasma gondii (1000 cells / mL). After 2 hours of invasion, the free tachyzoites that did not invade were washed with 1×PBS and fresh EMEM / F-12K medium was used to continue culturing for 22 hours. Proteins were extracted and the expression levels of PDCD5 and Toxoplasma surface protein TP3 were detected by western blotting; α-Tubulin was used as an internal reference.

[0078] The experimental results are as follows Figure 6 As shown in the figure, when the PDCD5 gene is silenced, the expression level of Toxoplasma surface protein TP3 is downregulated; when the PDCD5 gene is overexpressed, the expression level of Toxoplasma surface gene TP3 is increased, indicating that PDCD5 contributes to the intracellular proliferation of Toxoplasma.

[0079] Example 7

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

[0081] 0.5×10 4 SH-SY5Y cells were inoculated into a 12-well plate at 37°C and 5% CO2. The next day, when the cells grew to about 70%, 6 1.5 mL EP tubes A, B, C, D, E, and F were prepared. Tube A: 600 μL Medium+36μL RNAiMAX Reagent; Tube B: 150 μL Medium+1.5μL siCTL (30pmol); C tube 150μL Medium+1.5μL siPDCD5 (30pmol); D tube: 150μL Medium+1.5μL pcDNA3.1 (30pmol); E tube: 150μL Medium + 1.5 μL pcDNA3.1-PDCD5 (30 pmol). 150 μL of each of the two tubes AB, AC, AD, and AE were mixed at a ratio of 1:1, and 600 μL of the medium was added to the F tube. Medium, incubate at room temperature for 5 min. Add 300 μL of the mixture to 6-well and incubate at 37°C for 24 h. Increase the number of cells to 5 times the number of cells (MOI5, 2.5×10 4 Cells were infected with the Toxoplasma gondii GFP-RH strain (1000 cells / mL). After 1 h (for Toxoplasma invasion) or 2 h of invasion, the uninfected free tachyzoites were washed with 1×PBS, and fresh EMEM / F-12K medium was used to continue culturing for 22 h (for Toxoplasma proliferation). 0.25% trypsin-EDTA was added to digest the cells for 3 min, and the digestion was terminated with EMEM / F-12K medium. The cell pellet was collected by centrifugation at 1,000 rpm for 3 min, and the cells were washed twice with FACS buffer (PBS containing 1% BSA). The data were analyzed by FACScan (BD Bio-science), and the median fluorescence intensity value (MFI) was recorded.

[0082] Without any treatment, the median fluorescence intensity (MFI) of GFP in the CTL group was 26.4% after infection for 1 h. After PDCD5 gene silencing, the MFI of GFP decreased to 19.8%, while after PDCD5 gene overexpression, the MFI of GFP increased to 34.7% ( Figure 7 ). At 24h of infection, GFP with an MFI higher than 67% could be detected in the MOI5 group. In the PDCD5 gene silencing group, the MFI of GFP decreased to 40.5%, while the MFI of GFP increased to 82.1% when the PDCD5 gene was overexpressed ( Figure 8 ). This indicates that PDCD5 is indeed essential for the proliferation and infection of Toxoplasma gondii, and irisin B blocks the intracellular infection and growth of Toxoplasma gondii by inhibiting the expression of PDCD5 gene.

[0083] Example 8

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

[0085] 0.5×10 4 SH-SY5Y cells at 100 / mL were inoculated in a 12-well plate covered with a cell slide and cultured at 37°C and 5% CO2. After 24 hours, when the cells grew to about 70%, the cells were pretreated with autophagy inhibitors 3-MA (5 μM), Wortmannin (1 μM) and Baf-A1 (10 nM) for 4 hours, and then the number of inoculated cells was increased to 5 times (MOI5, 2.5×10 4 / mL) of Toxoplasma gondii GFP-RH strain to infect cells. After 2 hours of invasion, wash the non-invaded free tachyzoites with 1×PBS, and replace with fresh EMEM / F-12K medium for further culture for 22 hours. Then fix with 4% paraformaldehyde and wash three times with PBST containing 0.3% Triton X-100, 5 min / time. Block with 1% BSA at room temperature for 30 minutes. Anti-α-Tubulin antibody (anti-alpha TubulinAntibody(B-7):SC-5286) was diluted 1:250 and incubated at 4°C overnight. The primary antibody was recovered the next day and washed three times with PBST, 5 min / time. The secondary antibody (goat anti-mouse IgG, Alexa Fluor TM 568, A11031), incubated at room temperature in the dark for 2 h. Washed three times with PBST, 10 min / time. After staining and sealing, the slides were observed and photographed under an immunofluorescence microscope, and the infection rate and proliferation of Toxoplasma gondii per 100 cells were counted. No autophagy inhibitor was added to the control (Con) group.

[0086] The experimental results are as follows Fig. 9 As shown, in the control (Con) group, about 30% of the tachyzoites will divide three times in 24 hours to produce 8 tachyzoites in one vesicle. In the autophagy inhibitor treatment group, more than half of the tachyzoites can divide three times in 24 hours, and even about 20% of the worms will divide four times to produce 16 tachyzoites. In addition, about 60% of the cells in the control group were infected with Toxoplasma gondii, and after pretreatment with autophagy inhibitors, parasites can be observed in 75-80% of the cells. This shows that inhibiting mitochondrial autophagy will promote Toxoplasma intracellular infection and proliferation. It shows that irisin B interferes with Toxoplasma intracellular proliferation by inducing mitochondrial autophagy.

[0087] Example 9

[0088] Iridoside B inhibits Toxoplasma gondii infection and proliferation in mice

[0089] The experiment was divided into a control group (CTL) and an experimental group (Iristectorin B), each group consisted of three mice. After infection with Toxoplasma gondii, the experimental group was continuously administered with Iristectorin B for one week, while the control group was continuously administered with an equal volume of PBS for one week.

[0090] Six C57BL / 6J mice of similar weight, similar age (6-8 weeks), and same sex (male) were randomly divided into two groups. The route of human infection with Toxoplasma was simulated by gavage of 80 ME-49 Toxoplasma bradyzoites to each mouse. One week later, Iristectorin B was administered once a day to the experimental group mice at a dose of 30 mg / kg by intraperitoneal injection for one week. The control group mice were given an equal volume of PBS buffer in the same way at the same time. After the experiment, the mice were killed by cervical dislocation and the brain tissue was collected. 1 / 4 of the brain tissue was ground in 300 μL PBS buffer. Take 10 μL to observe the state of ME-49 bradyzoites under a microscope and count them. Compare the differences between the control group and the experimental group.

[0091] The number of bradyzoites in the experimental group of mice treated with Iristectorin B for one week was Fig.10 ),size( Fig.11 ) were significantly lower than those in the control group. It was confirmed that irisin B can indeed inhibit the proliferation of Toxoplasma gondii.

[0092] Example 10

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

[0094] The experiment was divided into a control group (CTL) and an experimental group (Iristectorin B), each group consisted of 3 mice. After infection with Toxoplasma gondii, the experimental group was continuously administered with Iristectorin B for one week, while the control group was continuously administered with an equal volume of PBS for one week.

[0095] Twelve C57BL / 6J mice of similar weight, similar age (6-8 weeks), and same sex (male) were randomly divided into two groups. Each mouse was gavaged with 80 ME-49 Toxoplasma bradyzoites to simulate the path of human infection with Toxoplasma. One week later, Iristectorin B was administered once a day to the experimental group mice at a dose of 30 mg / kg by intraperitoneal injection for one week. The control group mice were given an equal volume of PBS buffer in the same way at the same time. The survival of the mice was recorded within 15 days.

[0096] The experimental results are as follows Fig.12 All mice in the control group died within 7 to 10 days, while two mice in the experimental group that were given Iristectorin B for one week still survived on the 15th day, which was significantly different from the control group. This shows that Iristectorin B can indeed inhibit the proliferation of Toxoplasma gondii.

[0097] The present invention provides the use of tectoriformis B in the preparation of a drug for preventing and / or treating toxoplasmosis infection. The present invention uses tectoriformis B to inhibit the tachyzoites RH of Toxoplasma gondii. Experiments have shown that 0.025 to 1.6 μM tectoriformis B has a significant inhibitory effect on the proliferation of the RH strain of Toxoplasma gondii in SH-SY5Y cells. The half inhibitory concentration (EC 50) of tectoriformis B on the GFP-RH strain of Toxoplasma gondii is 1.34, which is 1.38. 50 ) is 0.25μM. When studying the molecular mechanism of irisin B in inhibiting Toxoplasma infection through various methods, it was found that irisin B inhibits the expression of PDCD5 protein and triggers mitochondrial autophagy, thereby interfering with the proliferation of Toxoplasma. The inhibition of Toxoplasma infection in mice by irisin B was also studied, and it was found that irisin B can indeed inhibit the proliferation of Toxoplasma and reduce the mortality rate of mice. It provides potential drug targets for the treatment or prevention of toxoplasmosis.

[0098] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The use of tiridin B in inhibiting the proliferation of Toxoplasma gondii, characterized in that: The concentration of irisin B in inhibiting the proliferation of Toxoplasma gondii is 0.025-1.6 μM.

2. Use of irisin B in the preparation of drugs for preventing and / or treating Toxoplasma gondii infection.

3. The use according to claim 2, characterized in that: The concentration of tectorius glycoside B in the drug is 0.025-1.6 μM.

4. The use according to claim 2 or 3, characterized in that: Iridoside B inhibits the expression of PDCD5 protein, triggers mitochondrial autophagy, and interferes with the proliferation of Toxoplasma gondii.

5. Application of irisin B in the preparation of drugs for inhibiting PDCD5 protein expression.

6. Application of irisin B in the preparation of drugs for activating mitochondrial autophagy.

7. A drug for preventing Toxoplasma gondii infection, characterized in that: The invention comprises tectoriformis glycoside B and pharmaceutically acceptable excipients.

8. The drug according to claim 7, characterized in that The concentration of tectoriusin B in the drug is 0.025-1.6 μM.

9. A drug for treating Toxoplasma gondii infection, characterized in that: The invention comprises tectoriformis glycoside B and pharmaceutically acceptable excipients.

10. The drug according to claim 9, characterized in that The concentration of tectoriusin B in the drug is 0.025-1.6 μM.

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