Use of mtor in the preparation of a drug for treating congenital toxoplasmosis
By inhibiting the MTOR signaling pathway and using MTOR inhibitors such as rapamycin to treat congenital toxoplasmosis, the toxicity and drug resistance problems of existing drugs have been solved, significantly improving intrauterine growth restriction in fetal mice and providing a safe and effective treatment option.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANTONG UNIV
- Filing Date
- 2026-03-25
- Publication Date
- 2026-06-16
AI Technical Summary
Existing drugs for treating congenital toxoplasmosis carry risks of bone marrow suppression, teratogenicity, and drug resistance. Furthermore, traditional drugs are ineffective against intra-tissue cysts, are prone to relapse, and are difficult to effectively control the inflammatory response caused by toxoplasmosis infection.
Using mTOR as a target, drugs for treating congenital toxoplasmosis are prepared by inhibiting mTOR expression or by using mTOR inhibitors such as rapamycin, thereby inhibiting the mTOR signaling pathway to improve the disease.
It significantly improved intrauterine growth restriction in fetal mice, reduced the risk of adverse pregnancy outcomes, avoided the toxicity and drug resistance of traditional drugs, and provided a safe and effective treatment option.
Smart Images
Figure CN122208762A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of biomedical technology, and in particular to the application of mTOR in the preparation of drugs for the treatment of congenital toxoplasmosis. Background Technology
[0002] Toxoplasma gondii is an obligate intracellular parasitic protozoan that can infect almost all warm-blooded animals. Approximately one-third of the global population carries a latent infection. Transmission primarily occurs through consumption of undercooked meat containing cysts or pseudocysts, contact with environments contaminated with cat feces, or mother-to-child transmission. Congenital toxoplasmosis is a major risk associated with primary infection during pregnancy, potentially leading to fetal death, premature birth, and severe neurological damage in newborns (such as hydrocephalus and choroiditis). Current clinical treatment mainly involves combination drug regimens; for example, pyrimethamine combined with sulfadiazine can inhibit parasite DNA synthesis and proliferation. Spiramycin is the first-line treatment for pregnant women to reduce the risk of fetal infection.
[0003] Existing drugs have significant limitations: First, pyrimethamine and sulfadiazine are prone to causing toxic reactions such as bone marrow suppression and liver and kidney damage, and pyrimethamine is teratogenic and contraindicated in early pregnancy; second, these drugs mainly target the trophozoite stage and are ineffective against intracellular cysts, leading to frequent relapses after discontinuation and requiring long-term maintenance therapy; furthermore, the emergence of drug-resistant strains and the inability of drugs to alleviate excessive inflammatory responses (such as pneumonia) caused by Toxoplasma gondii infection further limit efficacy. Therefore, the development of novel, highly effective, and low-toxicity treatment strategies is urgently needed. Summary of the Invention
[0004] The purpose of this application is to address the problems of bone marrow suppression, teratogenicity, and drug resistance risks associated with existing drugs for Toxoplasma gondii.
[0005] To achieve the above objectives, this application provides the following technical solution.
[0006] This application also provides the application of mTOR as a target in the preparation of drugs for the treatment of congenital toxoplasmosis.
[0007] Preferably, the drug improves congenital toxoplasmosis by inhibiting the expression of mTOR.
[0008] This application also provides the use of MTOR inhibitors in the preparation of drugs for the treatment of congenital toxoplasmosis.
[0009] Preferably, the mTOR inhibitor is rapamycin.
[0010] This application also provides a drug for treating congenital toxoplasmosis, wherein the drug achieves the therapeutic effect by inhibiting the expression of mTOR.
[0011] Preferably, the drug includes an mTOR inhibitor.
[0012] Preferably, the drug also includes other medically acceptable excipients.
[0013] This application also provides the application of MTOR as a detection target in the preparation of products for diagnosing congenital toxoplasmosis.
[0014] Preferably, the product is a detection reagent or detection kit, and the product includes reagents for detecting mTOR expression or phosphorylation levels.
[0015] Compared with the prior art, this application has at least the following beneficial effects:
[0016] This application is the first to demonstrate that MTOR can serve as a key therapeutic target for congenital toxoplasmosis, clarifying that toxoplasmosis infection significantly upregulates MTOR expression in placental tissue and macrophages, providing a new direction for targeted therapy of this disease.
[0017] In specific verification experiments, this application uses an MTOR inhibitor (rapamycin) for intervention, which avoids the clinical shortcomings of traditional drugs such as bone marrow suppression, liver and kidney damage, teratogenicity, easy relapse and drug resistance, and is especially suitable for safe treatment of toxoplasmosis infection during pregnancy.
[0018] Through specific verification experiments, this application demonstrates that MTOR inhibitors can significantly increase the crown-rump length, occipital-frontal diameter, and body weight of fetal mice in a congenital toxoplasmosis model, effectively reverse intrauterine growth restriction caused by Toxoplasma gondii infection, and reduce the risk of adverse pregnancy outcomes.
[0019] This application uses both in vivo placental tissue testing and in vitro cell experiments to verify that Toxoplasma gondii and its secreted and excreted antigens can activate the mTOR pathway, and that inhibiting mTOR can directly improve the disease. The mechanism is clear and the data are reliable. Attached Figure Description
[0020] Figure 1 The accompanying drawing in Embodiment 1 of this application illustrates the construction of a mouse model of congenital toxoplasmosis. Figure 1 A: Representative images of embryos and fetal mice. Figure 1 B: Size of a fetal mouse. Figure 1 C: Fetal mouse weight. PBS group: Pregnant mice injected with an equal volume of PBS solution; T. gondii group: Pregnant mice infected with *Toxoplasma gondii*. Data are expressed as mean ± SD and were statistically analyzed using a two-tailed unpaired Student's t-test.
[0021] Figure 2The accompanying figure in Embodiment 2 of this application illustrates the effect of Toxoplasma gondii infection on the mTOR signaling pathway. Figure 2 A. At GD17.5, placental tissues from mice in the PBS and T. gondii groups were collected, and the expression of MTOR protein in the placental tissues was detected by Western Blot (n=4 pregnant mice / group). PBS group: pregnant mice injected with an equal volume of PBS solution; T. gondii group: pregnant mice infected with Toxoplasma gondii. Figure 2 B. After stimulating Raw264.7 cells with Toxoplasma gondii secreted excretory antigens (TgAg, 5 μg / mL) for 48 hours, Western Blot analysis was performed on the expression of MTOR protein. Figure 2 After stimulating Raw264.7 cells with C and TgAg (5 μg / mL) for 48 hours, the expression of mTOR (red) in Raw264.7 cells was analyzed by immunofluorescence. Cell nuclei were counterstained with DAPI (blue), and quantitative analysis was performed using ImageJ software. Data are expressed as mean ± SD, and statistical analysis was performed using the two-tailed unpaired Student's t-test.
[0022] Figure 3 The accompanying drawing in Embodiment 3 of the present invention is used to illustrate the effect of the MTOR inhibitor rapamycin on congenital toxoplasmosis. Figure 3 A: Representative images of embryos and fetal mice. Figure 3 B: Size of a fetal mouse. Figure 3 C: Fetal rat weight. Infection group: Pregnant rats infected with *Toxoplasma gondii*. MTOR inhibitor intervention group: Pregnant rats infected with *Toxoplasma gondii* after intraperitoneal injection of MTOR inhibitor. Data are expressed as mean ± SD and were statistically analyzed using a two-tailed unpaired Student's t-test. Detailed Implementation
[0023] This application discloses the use of mTOR as a target in the preparation of drugs for treating congenital toxoplasmosis. The drugs improve congenital toxoplasmosis by inhibiting mTOR expression.
[0024] The mTOR signaling pathway plays a crucial role in the physiological and pathological processes of the placenta; its dysregulation is closely associated with various adverse pregnancy outcomes. Studies have shown that the placental mTOR signaling pathway is activated in human fetal growth restriction and its animal models. Multiple studies have confirmed that *Toxoplasma gondii* infection enhances the host mTOR signaling pathway and its dependent mRNA translation, a mechanism believed to benefit host cell survival and *Toxoplasma gondii* replication. Therefore, inhibiting mTOR activity may enhance the host's immune response against *Toxoplasma gondii* and improve congenital toxoplasmosis.
[0025] MTOR is the mammalian target of rapamycin, belonging to the phosphatidylinositol 3-kinase-associated kinase (PIKK) family.
[0026] Furthermore, this application also provides the use of mTOR inhibitors in the preparation of drugs for treating congenital toxoplasmosis. In one embodiment, the mTOR inhibitor is rapamycin, which has a molecular weight of 914.2 Da and a chemical formula of C. 51 H 79 NO 13 The chemical structural formula of the rapamycin is as follows: Figure 1 As shown.
[0027] Based on the above applications, this application provides a drug for treating congenital toxoplasmosis, wherein the drug achieves the therapeutic effect by inhibiting mTOR expression.
[0028] In one embodiment, the drug includes an MTOR inhibitor, preferably rapamycin.
[0029] The drug also includes other medically acceptable excipients.
[0030] Furthermore, this application also provides the application of mTOR as a detection target in the preparation of products for diagnosing congenital toxoplasmosis. In one embodiment, the product is a detection reagent or detection kit, which includes reagents for detecting the expression or phosphorylation level of mTOR.
[0031] The above content will be explained in conjunction with specific verification experiments below.
[0032] I. Experimental Materials and Sources
[0033]
[0034] II. Verification Experiment
[0035] 1. Example 1: Construction of a mouse model of congenital toxoplasmosis
[0036] Adult C57BL / 6 mice aged 6–8 weeks and weighing 20–22 g were selected to construct an animal model.
[0037] A. Animal Model Construction and Grouping
[0038] Mice were housed together at 17:00 at a female-to-male ratio of 2:1. The vaginal plugs of the female mice were examined at 7:00 the following morning. Female mice with white, waxy plugs were marked as gestation day 0.5 (GD 0.5) and included in the experiment.
[0039] Experimental grouping: All pregnant mice were intraperitoneally injected at GD 8.5, and the groups were as follows:
[0040] Control group: 200 μL PBS solution.
[0041] Infection group: 200 μL PBS solution containing Toxoplasma gondii tachyzoites (1500 tachyzoites / mL).
[0042] B. Sample collection and fetal mouse development assessment
[0043] (1) Sample collection: All pregnant mice were euthanized by CO2 asphyxiation at GD 17.5, and all fetuses and placentas were removed for subsequent experimental analysis.
[0044] (2) Morphological measurement:
[0045] 1) Fetal mouse size: Measure the crown-rump length (CRL) and occipital-frontal diameter (OF), and express it as the product of the two (CRL × OF).
[0046] 2) Fetal rat weight.
[0047] The results are as follows Figure 1 As shown, Toxoplasma gondii infection caused significant intrauterine growth retardation in fetal mice. Specifically, the size of fetal mice in the infected group (…) Figure 1 B) and fetal mouse weight ( Figure 1 C) All were significantly downregulated. The above figures verify the successful construction of the congenital toxoplasmosis mouse model in this embodiment.
[0048] Example 2: Verification of Toxoplasma gondii infection activating the mTOR signaling pathway
[0049] Using the congenital toxoplasmosis mouse model constructed in Example 1, placental samples were collected at GD 17.5 to detect mTOR expression. Western blot results showed that mTOR levels were significantly upregulated in placental tissue of infected mice. Figure 2 A).
[0050] Raw264.7 cells were stimulated in vitro with TgAg (5 μg / mL) for 48 hours, and mTOR expression was analyzed by Western blot. The results showed that mTOR expression was significantly increased after TgAg stimulation. Figure 2 B). Immunofluorescence staining further confirmed that mTOR expression was upregulated after TgAg stimulation ( Figure 2 C).
[0051] Example 3: Validation of mTOR inhibitors in improving congenital toxoplasmosis
[0052] Pregnant rats were intraperitoneally injected with the mTOR inhibitor rapamycin (2 mg / kg) at GD 7.5 and infected with Toxoplasma gondii at GD 8.5. Fetal development was assessed at GD 17.5. Results showed that the fetal size and weight of the rapamycin intervention group were significantly higher than those of the infection group. Figure 3 (A-3C). Therefore, the mTOR inhibitor rapamycin can significantly improve congenital toxoplasmosis.
[0053] III. Some Experimental Steps
[0054] 1. The method for preparing the excretory and secreted antigens of Toxoplasma gondii is as follows:
[0055] 1) Place 1×10 7 / mL of Toxoplasma gondii tachyzoites (RH strain) were inoculated into 24-well plates, and serum-free RPMI-1640 medium was added to a final volume of 2 mL. The 24-well plates were then placed in a CO2 incubator (37°C) and incubated for 3 hours.
[0056] 2) Collect the culture supernatant, centrifuge at 4°C (300 × g) for 10 minutes, transfer the supernatant to a 50 mL ultrafiltration centrifuge tube (5 kDa), add sterile PBS, and centrifuge at 4°C (1100 × g) for 40 minutes.
[0057] 3) Discard the filtrate, add sterile PBS to the ultrafiltration tube to resuspend the precipitate, and repeat the above centrifugation steps until the solution is clear.
[0058] 4) The collected solution was sterilized by filtration through a 0.22 μm filter membrane to obtain soluble Toxoplasma gondii antigen. The antigen was treated with an endotoxin removal kit to remove endotoxins, and the protein concentration was determined using the BCA method. Finally, the antigen was aliquoted and stored at -80℃.
[0059] 2. The steps for Western blot are as follows:
[0060] 1) Protein extraction
[0061] Before the experiment, pre-chill the centrifuge and start the ice maker. Wash placental tissue or cells twice with pre-chilled PBS. Prepare protein lysis working solution at a ratio of RIPA lysis buffer, PMSF, and phosphatase inhibitor of 100:1:1. For tissue samples, add 150–250 μL of lysis buffer per 20 mg and mechanically homogenize using a glass homogenizer; for cell samples, discard the culture medium and add 100 μL of lysis buffer to the cell pellet for lysis. After complete lysis, centrifuge at 12400 ×g for 5 minutes at 4°C, collect the supernatant, add 2× protein loading buffer, and heat in a 100°C metal bath for 10 minutes. The obtained samples can be used directly for subsequent experiments or stored at -80°C.
[0062] 2) Western Blot Detection
[0063] (1) Preparation of adhesive: Prepare a suitable concentration of separating adhesive (8%-10%), quickly pour it into the gap between the glass plates, let it stand for about 40 minutes until it is completely solidified, pour 5% concentrated adhesive into the upper layer, and let it stand for 30 minutes.
[0064] (2) Sample loading and electrophoresis: After fully resuspending the prepared protein samples and protein markers, add them to the sample loading wells in sequence. Pour electrophoresis buffer into the electrophoresis tank, set the initial voltage to 60 V, and after the bromophenol blue indicator enters the separating gel, increase the voltage to 120 V and continue electrophoresis until the bromophenol blue reaches the bottom of the gel.
[0065] (3) Transfer: Cut a PVDF membrane to an appropriate size, activate it with methanol for 2 minutes, and then rinse the PVDF membrane with ddH2O water. Carefully remove air bubbles to prepare a transfer "sandwich" structure, place it in the transfer tank, add freshly prepared transfer buffer, and transfer the membrane at a constant current of 350 mA for 60 minutes.
[0066] (4) Sealing: After the transfer is completed, place the PVDF membrane in 5% skim milk prepared with 1×TBST and seal at room temperature for 2 hours.
[0067] (5) Antibody incubation and development: Dilute the primary antibody at an appropriate ratio and incubate the PVDF membrane overnight at 4°C. Wash the PVDF membrane three times with 1×TBST, 15 minutes each time. Add the secondary antibody of the corresponding species, incubate at room temperature for 1 hour, and wash the PVDF membrane three times with 1×TBST, 15 minutes each time. Mix equal volumes of ECL chemiluminescence developing solution A and solution B and apply the mixture to the membrane for development and image acquisition.
[0068] 3. Immunofluorescence staining
[0069] 1) Fixation: Discard the culture medium, add 4% paraformaldehyde, and fix Raw264.7 cells at room temperature for 30 minutes. After fixation, wash the cells three times with PBS for 10 minutes each time.
[0070] 2) Permeation and Blocking: Add 200 μL of 0.1% Triton X-100 to each well and permeate at room temperature for 5 minutes. Discard the permeate and add 250 μL of 5% BSA blocking solution to each well, then block at room temperature for 1 hour.
[0071] 3) Primary antibody incubation: Dilute the primary antibody according to the recommended ratio in the instructions, carefully drop the diluted primary antibody onto the cell slide, and incubate overnight in a humidified chamber at 4°C.
[0072] 4) Secondary antibody and nuclear staining: After incubation with primary antibody, wash the slide three times with PBS on a shaker for 10 minutes each time. Add diluted fluorescent secondary antibody, taking care to avoid light, and incubate at 37°C in the dark for 90 minutes. Then, add diluted Hoechst nuclear dye and stain at room temperature in the dark for 30 minutes.
[0073] 5) Mounting and Imaging: After staining, wash the slides three times with PBS for 10 minutes each time under dark conditions. Remove the slides and carefully mount them, cell side down, onto a glass slide with an anti-fluorescence quenching mounting medium. Seal the edges with colorless nail polish. After the mounting medium has solidified, observe and acquire images under a fluorescence microscope.
[0074] In summary, this application, through specific embodiments, verifies that mTOR is highly expressed in mouse placental tissue in animal models such as congenital toxoplasmosis. Simultaneously, TgAg promotes mTOR protein expression in macrophages. The verification experiments in this embodiment demonstrate that *Toxoplasma gondii* and its antigens promote mTOR expression. The use of mTOR inhibitors can significantly improve congenital toxoplasmosis, verifying the significant ameliorative effect of mTOR inhibitors on congenital toxoplasmosis. Therefore, this provides a new direction and drug for the treatment of congenital toxoplasmosis.
Claims
1. Application of mTOR as a target in the preparation of drugs for the treatment of congenital toxoplasmosis.
2. The application of mTOR as a target in the preparation of drugs for treating congenital toxoplasmosis according to claim 1, characterized in that: The drug aims to improve congenital toxoplasmosis by inhibiting the expression of mTOR.
3. Application of mTOR inhibitors in the preparation of drugs for the treatment of congenital toxoplasmosis.
4. The use of the mTOR inhibitor according to claim 3 in the preparation of a drug for treating congenital toxoplasmosis, characterized in that: The mTOR inhibitor is rapamycin.
5. A drug for treating congenital toxoplasmosis, characterized in that: The drug treats congenital toxoplasmosis by inhibiting the expression of mTOR.
6. A drug for treating congenital toxoplasmosis according to claim 5, characterized in that: The drug includes an MTOR inhibitor.
7. A drug for treating congenital toxoplasmosis according to claim 6, characterized in that: The drug also includes other medically acceptable excipients.
8. Application of mTOR as a detection target in the preparation of products for diagnosing congenital toxoplasmosis.
9. The application of MTOR as a detection target according to claim 8 in the preparation of products for diagnosing congenital toxoplasmosis, characterized in that: The product is a detection reagent or detection kit, which includes reagents for detecting mTOR expression or phosphorylation levels.