Application of TRAF6 inhibitor in treating congenital toxoplasmosis
By inhibiting TRAF6 using the TRAF6 inhibitor C25-140, the treatment problem of congenital toxoplasmosis was solved and the development of fetal mice was significantly improved.
Patent Information
- Application Number
- CN202510866895.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-26
- Publication Date
- 2025-10-10
AI Technical Summary
The existing technology lacks drugs that can effectively treat congenital toxoplasmosis.
TRAF6 inhibitors, particularly C25-140, are used to improve congenital toxoplasmosis by inhibiting TRAF6.
TRAF6 inhibitors significantly improved congenital toxoplasmosis, reduced fetal mortality and developmental disorders, and increased fetal growth, weight, and size.
Smart Images
Figure CN120754099A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technology, and in particular to the use of a TRAF6 inhibitor in treating congenital toxoplasmosis. Background Art
[0002] Toxoplasma gondii is a zoonotic apicomplexan protozoan that poses a significant threat to human health. It is estimated that approximately one-third of the global population is infected with this pathogen. Primary infection during pregnancy can cause severe damage to the fetus through the maternal-fetal interface, leading to congenital toxoplasmosis such as miscarriage, premature birth, and stillbirth. The maternal-fetal interface is composed of a variety of immune cells, including NK cells, macrophages, T cells, and dendritic cells, which work synergistically to maintain immune homeostasis, maternal-fetal tolerance, and anti-pathogen defense. Imbalances in immune tolerance at the maternal-fetal interface have been shown to be a significant cause of congenital toxoplasmosis.
[0003] Toxoplasma gondii is a widespread zoonosis. Primary infection during pregnancy can cross the maternal-fetal interface, resulting in serious consequences: First, it can directly lead to miscarriage, stillbirth, or premature birth; second, it can cause congenital toxoplasmosis, resulting in severe multisystem damage to the fetus and newborn. These include central nervous system lesions such as intracranial calcification, hydrocephalus or microcephaly, epilepsy, and psychomotor retardation; ocular lesions such as retinochoroiditis (which can cause visual impairment or even blindness) and strabismus; and sensorineural hearing loss, hepatosplenomegaly, jaundice, pneumonia, and myocarditis. Neonatal infection can present with severe systemic symptoms and even death, and survivors may suffer from long-term neurocognitive and behavioral problems. Given the lack of effective treatments for congenital toxoplasmosis, the disease poses a significant challenge to maternal and child health and public health. Summary of the Invention
[0004] The purpose of the present invention is to solve the problem that there is a lack of effective drugs for treating congenital toxoplasmosis in the prior art.
[0005] In order to achieve the above object, the present invention adopts the following technical solutions:
[0006] The invention relates to the use of TRAF6 as a target in the preparation of a drug for treating congenital toxoplasmosis, wherein the drug inhibits TRAF6 to achieve the purpose of improving congenital toxoplasmosis.
[0007] Preferably, the TRAF6 is a member of the E3 ubiquitin ligase family with a molecular weight of 60 kDa.
[0008] The present application also provides the use of TRAF6 inhibitors in the preparation of drugs for treating congenital toxoplasmosis.
[0009] The present application also provides a drug for treating congenital toxoplasmosis, wherein the drug comprises a TRAF6 inhibitor.
[0010] Preferably, the TRAF6 inhibitor is C25-140.
[0011] Preferably, the medicine further comprises medically acceptable excipients.
[0012] This application also provides the use of TRAF6 as a detection target in the preparation of products for detecting congenital toxoplasmosis.
[0013] Preferably, the product is a detection reagent or a detection kit, and the detection reagent or the detection kit is used to detect the expression of TRAF6.
[0014] The present application also provides a detection kit for detecting congenital toxoplasmosis, wherein the detection kit comprises a reagent for detecting TRAF6 expression.
[0015] Preferably, the kit further comprises other medically acceptable reagents.
[0016] Compared with the prior art, this application has the following beneficial effects:
[0017] The present invention demonstrates that T. gondii infection upregulates TRAF6 expression in mouse placental tissue by constructing a mouse model of congenital toxoplasmosis. An in vitro model using T. gondii-derived protein to stimulate macrophages confirmed that T. gondii-derived protein can promote TRAF6 expression. A TRAF6 inhibitor can partially reverse the stimulatory effect of T. gondii-derived protein on TRAF6. Injecting T. gondii-infected pregnant mice with a TRAF6 inhibitor confirmed that inhibiting TRAF6 expression significantly improves congenital toxoplasmosis. This provides a new approach and drug for the treatment of congenital toxoplasmosis. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is the corresponding figure in Example 1 of the present application, which is used to demonstrate the construction of a congenital toxoplasmosis mouse model, wherein: Figure 1 A: Representative images of mouse placenta and fetuses: After pregnant mice in the PBS and infection groups were euthanized at G17.5, the mouse placenta and fetuses were observed. Figure 1 BC: Fetal size and weight were assessed. Fetal size was determined by multiplying crown-rump length by occipitofrontal diameter. OF: occipitofrontal diameter; CRL: crown-rump length. Figure 1 D: HE staining shows changes in mouse placental structure. Placental tissues from the control and infected groups were collected at G17.5 and HE staining was performed to observe the structure of the mouse placenta.
[0019] Figure 2This is the corresponding figure in Example 2 of the present application, used to show the expression of TLR4 and TRAF6 in mouse placenta tissue detected by Western blot. Compared with the control group, *: P < 0.05.
[0020] Figure 3 This is the corresponding figure in Example 3 of this application, wherein: Figure 3 A: Western blot analysis of the effect of Toxoplasma gondii antigen (5 μg / mL) on the expression of TLR4 and TRAF6 in macrophages. Compared with the control group, *: P < 0.05. Figure 3 BC: Cell immunofluorescence assay to detect the effect of Toxoplasma gondii antigen (5 μg / mL) on the expression of TLR4 and TRAF6. *: P < 0.05.
[0021] Figure 4 This is the corresponding figure in Example 4 of the present invention, used to demonstrate the effect of TRAF6 inhibitor (C25-140) on TRAF6 expression induced by Toxoplasma gondii antigen detected by Western blot. *: P < 0.05.
[0022] Figure 5 This is the corresponding figure in Example 5 of the present invention, used to demonstrate the effect of TRAF6 inhibitors on congenital toxoplasmosis, wherein: Figure 5 A: Fetal weight was measured. Pregnant mice were intraperitoneally injected with PBS or the TRAF6 inhibitor C25-140 (10 mg / kg) at G7.5. 300 Toxoplasma gondii tachyzoites were injected intraperitoneally at G8.5. Fetal weight was measured at G17.5. Figure 5 B: Fetal size measurement: Pregnant mice were intraperitoneally injected with PBS or the TRAF6 inhibitor C25-140 (10 mg / kg) at G7.5, and 300 Toxoplasma gondii tachyzoites were injected intraperitoneally at G8.5. Fetal size was measured at G17.5. DETAILED DESCRIPTION
[0023] The present invention is further described in detail below with reference to specific embodiments.
[0024] The present application provides the use of TRAF6 as a target in the preparation of a product for treating congenital toxoplasmosis, thereby achieving the purpose of improving congenital toxoplasmosis by inhibiting TRAF6.
[0025] The TRAF6 mentioned above is transcription activating factor 3 (TRAF6), a member of the E3 ubiquitin ligase family with a molecular weight of 60 kDa. It is rich in a basicleucine zipper (bZIP) structure and encodes 181 amino acids. TRAF6 and its family members can regulate gene transcription by binding to the common DNA sequence "TGAC GTCA" in vivo. TRAF6 can regulate cell proliferation and mediate cellular stress responses. In this application, a congenital toxoplasmosis model was constructed to explore the role of TRAF6 in congenital toxoplasmosis. By constructing an in vitro cell model, the pathological mechanism of TRAF6 in congenital toxoplasmosis was explored. This study provides a new research perspective for congenital toxoplasmosis, may provide theoretical support and experimental basis for clinical practice, and promote the development and application of new therapies.
[0026] Based on the above application, the present application also provides the use of a TRAF6 inhibitor in the preparation of a drug for treating congenital toxoplasmosis, wherein the TRAF6 inhibitor is C25-140.
[0027] Based on the above application, the present application also provides a drug for treating congenital toxoplasmosis, which includes a TRAF6 inhibitor.
[0028] The present application also provides the use of TRAF6 as a detection target in the preparation of a product for detecting congenital toxoplasmosis, wherein the product is a detection reagent or a detection kit, and the detection reagent or the detection kit is used to detect the expression of TRAF6.
[0029] Based on the above application, the present application also provides a detection kit for detecting congenital toxoplasmosis. The detection kit contains a reagent for detecting TRAF6 expression. In other embodiments, the kit also includes other medically acceptable reagents.
[0030] The above contents are described below with reference to specific embodiments:
[0031] 1. Experimental materials and sources:
[0032]
[0033] 2. Example 1: Construction of a mouse model of congenital toxoplasmosis:
[0034] Pregnant mice were intraperitoneally injected with 300 Toxoplasma gondii tachyzoites on G8.5 day and euthanized on G17.5 day to observe the effect of Toxoplasma infection on fetal development. Fetal development was assessed by fetal size (CRL×OF) and fetal weight.
[0035] The specific construction method is as follows:
[0036] 6-8 week old C57BL / 6 mice were mated overnight at a 1:2 male to female ratio, and the next morning before 8:00 am, if white plugs were found on the vulva of the female mice, the female mice were marked as the 0.5th day of gestation (G0.5). On G8.5, the pregnant mice were injected intraperitoneally with a solution of 300 T. gondii (RH strain) tachyzoites in PBS to construct a mouse model of congenital toxoplasmosis, while the normal group was injected intraperitoneally with the same volume of PBS solution. On G17.5, the pregnant mice were euthanized using CO2 asphyxiation. The pregnancy outcome was observed, and the size and weight of the fetal mice were measured, and all the mouse placentas were collected for subsequent experiments. The development of the fetal mice was evaluated according to the weight and size of the fetal mice, and the size of the fetal mice was measured by the product of the crown-rump length (CRL) and the occipitofrontal diameter (OF).
[0037] The results are shown in Figure 1 , please refer to Figure 1 A-C, T. gondii infection caused fetal mouse death and obvious intrauterine growth retardation. Figure 1 D, T. gondii infection can destroy the structure of the mouse placenta, with hemorrhage, necrosis and vacuolization in the decidua area (De). The above figures verify that the mouse model of congenital toxoplasmosis in this embodiment is successfully constructed.
[0038] III. Example 2: Verification of T. gondii infection promoting placental TRAF6 expression
[0039] First, the mouse model of congenital toxoplasmosis was constructed according to Example 1, and the mouse placentas were collected on G17.5 to detect TRAF6 expression.
[0040] The Western blot detection results show the expression of TRAF6 in mouse placental tissue, please refer to Figure 2 , and infection can up-regulate the expression of TRAF6 in mouse placental tissue.
[0041] IV. Example 3: Verification of T. gondii antigen inducing macrophage expression of TRAF6
[0042] First, the macrophage cell line (Raw264.7 cells) was stimulated with T. gondii antigens (TgAg, 5 μg / mL) for 24 h, and then the cells were collected to detect TRAF6 expression;
[0043] The Western blot detection results show that after antigen stimulation, the expression of TRAF6 in macrophages increases compared with the control group, indicating that the antigen can significantly promote the expression of TRAF6 Figure 3A). In addition, similar results were also observed by immunofluorescence staining, green fluorescence (TLR4) and magenta (TRAF6) were significantly up-regulated after T. gondii antigen treatment Figure 3 B). These data indicated that antigen could promote macrophage expression of TRAF6 in vitro, which was consistent with the results of animal models.
[0044] V. Example 4: Verification of TRAF6 inhibitor inhibiting TRAF6 up-regulation induced by parasite-derived antigen
[0045] In this embodiment, Raw264.7 cells were seeded in 6-well plates and cultured to 70-80% confluence. After pre-stimulation of cells with C25-140 (20 μM) for 2 h, the cells were stimulated with antigen (5 μg / mL) for 24 h, and then collected for detection of TRAF6 expression.
[0046] Experimental groups (n = 3): Control group (CON): no treatment; C25-140 group: C25-140 (20 μM) stimulation group; TgAg group: 5 μg / mL antigen stimulation group; C25-140 + TgAg group: C25-140 and antigen co-stimulation group.
[0047] The results of Western blot detection showed that TRAF6 expression was up-regulated after antigen stimulation. However, when TRAF6 inhibitor (C25-140) was added, TRAF6 expression was inhibited. Compared with the simple antigen stimulation group, the TRAF6 expression in the C25-140 and TgAg co-stimulation group was significantly down-regulated, indicating that the TRAF6 inhibitor could inhibit the up-regulation of TRAF6 induced by antigen.
[0048] VI. Example 5: Verification of TRAF6 inhibitor improving congenital toxoplasmosis
[0049] In this embodiment, pregnant mice were injected intraperitoneally with PBS or TRAF6 inhibitor C25-140 (10 mg / kg) at G7.5, and infected with T. gondii at G8.5, and the development of fetal mice was evaluated at G17.5 to study the effect of TRAF6 inhibitor on congenital toxoplasmosis. TRAF6 inhibitor could up-regulate the body weight of fetal mice Figure 5 A) and the size of fetal mice Figure 5 B). Therefore, the TRAF6 inhibitor (C25-140) could significantly improve congenital toxoplasmosis.
[0050] The following are specific experiments applied in the above embodiments:
[0051] Experiment 1: Protein extraction
[0052] (1) Fresh mouse placental tissue or cultured cells were taken and rinsed with pre-cooled PBS for 3 times to remove residual blood or culture medium.
[0053] (2) Grind the tissue or collect the cell pellet and centrifuge at 1000×g for 5 min at 4°C.
[0054] (3) Add pre-cooled RIPA lysis buffer (containing 1% protease inhibitors), and lyse tissue samples at a ratio of 1:10, and cell samples at a ratio of 1×10 6 cells / 100 μL lysate.
[0055] (4) Lyse on ice for 15 min, centrifuge at 12,000 × g for 15 min at 4°C, and collect the supernatant, which is the total protein solution.
[0056] Experiment 2: Western blotting
[0057] (1) Clean the glass plate with tap water and let it dry naturally. Then align the two glass plates and insert them vertically into the glue rack to fix them. Check them repeatedly to avoid glue leakage.
[0058] (3) Pour the prepared separation gel into the glass plate and leave it at room temperature for about 40 minutes. After the separation gel solidifies, slowly add the concentrated gel and insert the comb.
[0059] (4) The extracted protein sample was thoroughly mixed with 2× Loading Buffer at a ratio of 1:1 and then boiled in a metal bath at 100°C for 10 min.
[0060] (5) Slowly add the sample into the sample well using a sample needle, and use the pre-stained protein marker as a reference for protein size.
[0061] (6) Inject freshly prepared electrophoresis buffer into the electrophoresis tank, making sure that the liquid surface completely covers the gel. After electrophoresis at 60V constant voltage for 30 minutes, adjust the voltage to 120V constant voltage. Wait until the bromophenol blue indicator reaches the bottom of the gel and turn off the electrophoresis.
[0062] (7) Prepare 1× transfer buffer and pre-cool it at 4°C.
[0063] (8) Cut the PVDF membrane to the appropriate size, mark it, and activate it in methanol for 2 minutes. Assemble the PVDF, gel, and filter paper into a transfer "sandwich" structure in order, place it in the electrophoresis tank, add pre-cooled 1× transfer buffer, and transfer the membrane at a constant current of 350mA for 90 minutes.
[0064] (9) Remove the PVDF membrane and rinse it in TBST solution 1-2 times.
[0065] (10) Block the PVDF membrane with 5% skim milk at room temperature for 1-2 h.
[0066] (11) Dilute the primary antibody according to the recommended ratio in the instructions, and incubate the PVDF membrane with the primary antibody in a shaking incubator at 4°C overnight.
[0067] (12) The next day, the PVDF membrane was washed four times with 1× TBST for 10 min each time. The PVDF membrane was incubated with the secondary antibody on a shaker at room temperature for 1 h and then washed four times with 1× TBST for 10 min each time.
[0068] (13) ECL development and grayscale analysis were performed using ImageJ software.
[0069] Experiment 3: HE staining
[0070] (1) Fixation: Mouse placental tissue was fixed with 10% neutral formalin to preserve its original structure.
[0071] (2) Embedding and sectioning: After paraffin embedding, the mouse placenta paraffin block was cut into 4 μm thick sections and attached to a glass slide.
[0072] (3) Dewaxing and hydration: Bake the paraffin sections to be stained in a 60°C oven for 30 min. Place the baked sections in xylene I and II for 5 min each. Soak the dewaxed sections in 100%, 90%, 80%, and 70% ethanol for 5 min each, followed by rinsing with distilled water for 5 min to perform a gradient hydration.
[0073] (4) Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 5 minutes, and the cell nuclei will appear blue.
[0074] (5) Differentiation and blueing: Use 1% hydrochloric acid ethanol to differentiate and remove excess staining, and then return to blueing in running water.
[0075] (6) Eosin staining: Immerse the sections in eosin solution for 1 minute, and the cytoplasm and interstitium will appear pink.
[0076] (7) Dehydration and mounting: The sections were rapidly dehydrated with gradient ethanol (70%→100%), transparentized in xylene for 2 min, and mounted with neutral gum.
[0077] Experiment 4: Immunofluorescence staining
[0078] (1) Fixation: The culture medium was removed and the treated Raw264.7 cells in the 24-well plate were fixed in 4% paraformaldehyde for 30 min, and then washed three times with PBS at room temperature.
[0079] (2) Permeabilization: Add 200 μL of 0.1% Triton-X to each well to permeabilize the cells for 5 min at room temperature.
[0080] (3) Blocking: Add 250 μL of 5% BSA to each well and block at room temperature for 1 hour.
[0081] (4) Primary antibody incubation: Dilute the primary antibody according to the instructions of the primary antibody, add an appropriate amount of antibody onto the slide, and incubate at 4°C overnight.
[0082] (5) Elute the primary antibody: wash with PBS on a shaker for 5 min each time.
[0083] (6) Secondary antibody incubation: dilute the secondary antibody according to the instructions, add the fluorescent secondary antibody to the slide, and incubate at 37°C for 90 min in the dark.
[0084] (7) DAPI staining: dilute the Hoechest nuclear dye with PBS, add Hoechest to the slide, and incubate at room temperature for 30 min in the dark.
[0085] (8) Elute the secondary antibody: wash with PBS 3 times for 5 min each time in the dark.
[0086] (9) Mounting: remove the slide from the 24-well plate and place it on a glass slide with glycerol, apply nail polish around the slide, and incubate at 4°C overnight.
[0087] (10) Take pictures: take pictures using a fluorescence microscope.
[0088] In summary, the present application verifies that TRAF6 is highly expressed in the mouse placental tissue in animal models such as congenital toxoplasmosis. At the same time, T. gondii antigens promote the expression of TRAF6 protein in macrophages. TRAF6 inhibitors stimulate macrophages, which can partially reverse the promotion of TRAF6 by T. gondii antigens. The use of TRAF6 inhibitors can significantly improve congenital toxoplasmosis. Through the verification experiment of this embodiment, it can be seen that T. gondii and its antigens promote the expression of TRAF6. It is verified that the TRAF6 inhibitor significantly improves the effect of congenital toxoplasmosis. Thus, a new direction and drug for treating congenital toxoplasmosis is provided.
Claims
1. Use of TRAF6 as a target in the preparation of a drug for treating congenital toxoplasmosis, characterized by: The drug achieves the purpose of improving congenital toxoplasmosis by inhibiting TRAF6.
2. The use of TRAF6 as a target in the preparation of a drug for treating congenital toxoplasmosis according to claim 1, characterized in that: The TRAF6 is a member of the E3 ubiquitin ligase family with a molecular weight of 60 kDa.
3. Application of TRAF6 inhibitors in the preparation and treatment of congenital toxoplasmosis.
4. A drug for treating congenital toxoplasmosis, characterized in that: The drugs include TRAF6 inhibitors.
5. Use of the TRAF6 inhibitor according to claim 4 in the preparation of a drug for treating congenital toxoplasmosis, characterized in that: The TRAF6 inhibitor is C25-140.
6. The drug for treating congenital toxoplasmosis according to claim 5, characterized in that: The medicine further comprises medically acceptable excipients.
7. Application of TRAF6 as a detection target in the preparation of products for detecting congenital toxoplasmosis.
8. Use of TRAF6 as a detection target in the preparation of a product for detecting congenital toxoplasmosis according to claim 7, characterized in that: The product is a detection reagent or a detection kit, and the detection reagent or the detection kit is used to detect the expression of TRAF6.
9. A detection kit, characterized in that: The detection kit is used for detecting congenital toxoplasmosis, and contains a reagent for detecting TRAF6 expression.
10. A detection kit according to claim 9, characterized in that: The kit also includes other medically acceptable reagents.