Application of P-STAT6 agonist in treatment of poor pregnancy caused by toxoplasma gondii infection
By targeting P-STAT6 and increasing its expression with P-STAT6 agonist, the lack of effective treatment of congenital toxoplasmosis in the prior art has been solved, and the adverse pregnancy outcomes caused by toxoplasmosis infection have been significantly improved.
Patent Information
- Application Number
- CN202411623127.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-14
- Publication Date
- 2025-03-11
AI Technical Summary
There is a lack of effective treatment methods for preventing and treating congenital toxoplasmosis in the prior art, especially in the problem of adverse pregnancy caused by pregnant women after infection with Toxoplasma gondii.
By targeting P-STAT6, the expression of P-STAT6 is increased using P-STAT6 agonist to treat adverse pregnancy caused by Toxoplasma gondii infection.
The adverse pregnancy outcome caused by Toxoplasma infection was significantly improved, and the fetal development status was improved by increasing the expression of P-STAT6 in placental tissue.
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Figure CN119592677A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technology, and in particular to application of P-STAT6 in treating adverse pregnancy caused by Toxoplasma gondii infection. Background Art
[0002] Toxoplasma gondii is an important opportunistic pathogenic protozoan. Individuals with normal immunity are mostly latently infected, while pregnant women infected with Toxoplasma can affect fetal development, leading to congenital toxoplasmosis such as miscarriage, malformation, stillbirth, premature birth and birth defects. In recent years, with the rapid increase in urban population, the growing number of pet owners (especially cats), and people's bad eating habits such as eating wild animals, more than 30% of the world's population is exposed to the risk of Toxoplasma infection, among which the infection rate of pregnant women is 10% to 27.5%, and about 90,000 newborns are threatened by Toxoplasma infection every year. In China, according to serological surveys, the infection rate of the population is 5% to 20%, and in some areas it is as high as more than 30%. Moreover, with the full implementation of the "three-child policy", the proportion of clinically elderly mothers and precious children will continue to increase. Therefore, as a populous country, how to prevent and control congenital toxoplasmosis and protect the reproductive health of the people is a huge challenge we face. Summary of the invention
[0003] The purpose of the present invention is to solve the technical problem that there is a lack of treatment methods for preventing and treating congenital toxoplasmosis in the prior art.
[0004] In order to achieve the above object, the present invention adopts the following technical solutions:
[0005] Application of P-STAT6 as a target in the preparation of drugs for the treatment of adverse pregnancy.
[0006] Preferably, the adverse pregnancy is adverse pregnancy caused by Toxoplasma gondii.
[0007] Preferably, the Toxoplasma gondii is Toxoplasma gondii.
[0008] Preferably, the drug targets P-STAT6 to improve adverse pregnancy caused by Toxoplasma gondii infection.
[0009] Based on the application of P-STAT6 as a target, the present application provides a drug for treating adverse pregnancy caused by Toxoplasma gondii, wherein the drug includes a P-STAT6 agonist, and the drug treats adverse pregnancy caused by Toxoplasma gondii by increasing the expression of P-STAT6.
[0010] Preferably, the medicine also includes other medically acceptable agents.
[0011] The present application also provides the use of P-STAT6 as a target in the preparation of a product for detecting adverse pregnancy caused by Toxoplasma gondii, wherein the product is a detection reagent or a detection kit.
[0012] Preferably, the product comprises a reagent for detecting the expression of P-STAT6.
[0013] Preferably, the product also includes other medically acceptable agents.
[0014] Compared with the prior art, this application has the following beneficial effects:
[0015] The embodiments of the present invention verified that T.gondii infection down-regulates P-STAT6 expression through a study on the effect of T.gondii infection on the expression of P-STAT6 in placental tissue in pregnant mice. The in vitro model of macrophage cell lines, peritoneal macrophages and bone marrow-derived macrophages stimulated by T.gondii antigens verified that T.gondii antigens can inhibit and promote the expression of P-STAT6 in macrophages. P-STAT6 agonists can partially reverse the inhibitory effect of T.gondii antigens on P-STAT6. Using P-STAT6 agonists on T.gondii-infected pregnant mice, it was verified that promoting P-STAT6 expression can significantly improve adverse pregnancy caused by Toxoplasma infection. Thus, a new direction and drug are provided for the treatment of adverse pregnancy caused by Toxoplasma gondii. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 In the examples of the present invention, Toxoplasma infection down-regulates the expression of P-STAT6 in placental tissue.
[0017] Figure 1 A: Fetal mouse picture; on G8.5 day, pregnant mice were intraperitoneally injected with Toxoplasma tachyzoites (300), and euthanized on G17.5 day to observe the effect of Toxoplasma infection on fetal size and weight (n=5-7 mice).
[0018] Figure 1 B: Flow cytometry was used to detect the decidual macrophages (CD11b + ) percentage;
[0019] Figure 1 C: Western blot was used to detect the expression of STAT6 and P-STAT6 in mouse placenta tissue (n = 8 mice);
[0020] Figure 1 D: Immunohistochemistry was used to detect the expression of P-STAT6 in mouse placenta tissue. Scale bar: 100 μm or 50 μm;
[0021] Data were analyzed using two-tailed unpaired Student's t-test. *: P < 0.05.
[0022] Figure 2 . Toxoplasma gondii antigen inhibits the expression of P-STAT6 in macrophages.
[0023] Figure 2 A: Western blot was used to detect the expression of STAT6 and P-STAT6 in macrophages. Raw264.7 cells were pretreated with IL-4 (20 ng / mL) for 1 h, and then stimulated with Toxoplasma gondii antigen for 24 h. Then the cells were collected to detect the expression of STAT6 and P-STAT6.
[0024] Figure 2 B: Immunofluorescence detection of P-STAT6 expression in macrophages. Raw264.7 cells were pretreated with IL-4 (20 ng / mL) for 1 h, and then stimulated with Toxoplasma gondii antigen for 24 h. The cells were then collected to detect the expression of P-STAT6. Scale bar: 15 μm.
[0025] TgAg: T. gondii antigens. Data were analyzed using one-way ANOVA with Tukey's multiple comparisons test. *: P < 0.05.
[0026] Figure 3 . Toxoplasma gondii antigen inhibits P-STAT6 expression in peritoneal macrophages.
[0027] Western blot was used to detect the expression of STAT6 and P-STAT6 in peritoneal macrophages. Peritoneal macrophages isolated from wild-type mice were cultured with IL-4 (20 ng / mL) for 1 h, and then stimulated with Toxoplasma gondii antigen (5 μg / mL) for 24 h, and then the cells were collected to detect the expression of P-STAT6.
[0028] One-way ANOVA with Tukey's multiple comparisons test was used for statistical analysis. *: P < 0.05.
[0029] Figure 4 . Toxoplasma gondii antigen inhibits the expression of P-STAT6 in bone marrow-derived macrophages.
[0030] Figure 4 A: Experimental operation flow chart. Mouse BMDMs were extracted according to the scheme described in the schematic diagram for subsequent experiments;
[0031] Figure 4 B: Western blot detection of STAT6 and P-STAT6 expression in bone marrow-derived macrophages. Mouse bone marrow-derived macrophages were isolated and stimulated with IL-4 (20 ng / mL) in vitro for 1 h, and then stimulated with Toxoplasma gondii antigen (5 μg / mL) for 24 h, and then the cells were collected to detect STAT6 and P-STAT6 expression.
[0032] Figure 4 It also includes: using confocal microscopy to detect the expression of P-STAT6 in mouse bone marrow-derived macrophages. Mouse bone marrow-derived macrophages were isolated and stimulated with IL-4 (20 ng / mL) in vitro for 1 h, and then stimulated with Toxoplasma gondii antigen (5 μg / mL) for 24 h. Scale bar: 10 μm or 15 μm.
[0033] Figure 4 The data in B were analyzed using one-way ANOVA with Tukey's multiple comparisons test. *: P < 0.05.
[0034] Figure 5 .P-STAT6 agonists significantly improve adverse pregnancy outcomes caused by Toxoplasma gondii infection.
[0035] On 8.5 days of pregnancy, Toxoplasma tachyzoites were injected into the abdominal cavity of pregnant mice, on 9.5 days of pregnancy, P-STAT6 agonist was injected through the tail vein, and the pregnancy outcome was observed on 17.5 days of pregnancy.
[0036] Figure 5 A: Calculate the abortion rate of pregnant mice;
[0037] Figure 5 B: Measure the size of the fetus;
[0038] Figure 5 C: Measure the weight of fetal mice;
[0039] Data were analyzed using two-tailed unpaired Student's t-test. *: P < 0.05. DETAILED DESCRIPTION
[0040] In this application, a pregnancy model of wild-type mice infected with Toxoplasma gondii was constructed to explore the effects of Toxoplasma gondii infection on pregnancy outcomes and P-STAT6. By constructing an in vitro cell model, the pathological mechanism of P-STAT6 in the adverse pregnancy caused by Toxoplasma gondii infection was explored. Through this study, a new research perspective is provided for the adverse pregnancy outcomes induced by Toxoplasma gondii infection, which may provide theoretical support and experimental basis for clinical practice and promote the development and application of new therapies.
[0041] Specifically, the present application provides the use of P-STAT6 as a target in the preparation of a drug for treating adverse pregnancy, wherein the adverse pregnancy is adverse pregnancy caused by Toxoplasma gondii, and specifically, the Toxoplasma gondii is Toxoplasma gondii.
[0042] In one embodiment, the drug targets P-STAT6 to improve adverse pregnancy caused by Toxoplasma gondii infection.
[0043] Based on the application of P-STAT6 as a target, the present application also provides a drug for treating adverse pregnancy caused by Toxoplasma gondii, wherein the drug includes a P-STAT6 agonist, and the drug increases the expression of P-STAT6 to treat adverse pregnancy caused by Toxoplasma gondii. In one embodiment, the P-STAT6 agonist is recombinant mouse IL-4 (PeproTech, USA).
[0044] In one embodiment, the medicament further comprises other medically acceptable agents.
[0045] The present application also provides the use of P-STAT6 as a target in the preparation of a product for detecting adverse pregnancy caused by Toxoplasma gondii, wherein the product is a detection reagent or a detection kit, and the product includes a reagent for detecting the expression of P-STAT6. In one embodiment, the product also includes other medically acceptable reagents.
[0046] The following is an explanation of the above content in combination with specific verification experiments:
[0047] Example 1: Effects of Toxoplasma gondii infection on the expression of STAT6 and P-STAT6
[0048] Infection with Toxoplasma gondii during pregnancy can lead to adverse pregnancy outcomes such as miscarriage, stillbirth or intrauterine growth retardation of the fetus. The present invention constructed a mouse model of miscarriage caused by Toxoplasma gondii infection and found that Toxoplasma gondii infection can cause fetal death and significant intrauterine growth retardation ( Figure 1 A).
[0049] The present invention uses flow cytometry to analyze the decidual macrophages (CD11b + ) in the placental tissue, and found that Toxoplasma infection can increase the proportion of decidual macrophages in the mouse placenta ( Figure 1 B).
[0050] Western blot was used to detect the expression of STAT6 and P-STAT6 in mouse placental tissue, showing that Toxoplasma infection downregulated the expression of P-STAT6 in placental tissue ( Figure 1 C).
[0051] The expression of P-STAT6 in mouse placental tissue was detected by histochemistry, showing that Toxoplasma gondii infection down-regulated the expression of P-STAT6 in placental tissue ( Figure 1 D) Therefore, Toxoplasma gondii can inhibit P-STAT6 expression in the placenta.
[0052] Example 2: Toxoplasma gondii antigen inhibits macrophage P-STAT6 expression
[0053] In this example, the effect of Toxoplasma gondii antigen on the expression of P-STAT6 in macrophages was further investigated in vitro. First, IL-4 (20 ng / mL) was used to treat the macrophage cell line (Raw264.7 cells) for 1 h. The results of Western blot showed that the expression of P-STAT6 in macrophages was downregulated after Toxoplasma gondii antigen treatment. Compared with the control group, the difference was statistically significant, indicating that Toxoplasma gondii antigen treatment significantly reduced the expression level of STAT6 ( Figure 2 A). Immunofluorescence staining also confirmed that Toxoplasma gondii antigen can downregulate the expression of P-STAT6 in macrophages ( Figure 2 B). Western blot and immunofluorescence staining results confirmed that Toxoplasma gondii antigen could inhibit the expression of P-STAT6 in vitro.
[0054] Example 3: Toxoplasma gondii antigen inhibits P-STAT6 expression in peritoneal macrophages
[0055] The effect of Toxoplasma gondii antigen on the expression of STAT6 and P-STAT6 in peritoneal macrophages was investigated by isolating mouse peritoneal macrophages and stimulating them with Toxoplasma gondii antigen in vitro. Peritoneal macrophages were first pretreated with IL-4 (20 ng / mL) for 1 hour, and then Toxoplasma gondii antigen (5 μg / mL) was used to stimulate peritoneal macrophages for 24 hours. Compared with the group without Toxoplasma gondii antigen treatment, the expression of P-STAT6 in peritoneal macrophages of mice in the Toxoplasma antigen group was significantly decreased, with a P value less than 0.05, and the difference was statistically significant ( Figure 3 ), indicating that Toxoplasma gondii antigen can reduce the expression of P-STAT6 in mouse peritoneal macrophages. Therefore, Toxoplasma gondii antigen can inhibit the expression of P-STAT6.
[0056] Example 4: Toxoplasma gondii antigen inhibits P-STAT6 expression in bone marrow-derived macrophages
[0057] Bone marrow-derived macrophages were isolated from mice to investigate the effect of Toxoplasma gondii antigen on the expression of STAT6 and P-STAT6 in bone marrow-derived macrophages. Figure 4The scheme in A successfully induced mature bone marrow-derived macrophages on the eighth day. After pre-treating bone marrow-derived macrophages with IL-4 (20 ng / mL) for 1 hour, Toxoplasma gondii antigen (5 μg / mL) continued to stimulate and induce for 24 hours. In this example, it was found that Toxoplasma gondii antigen down-regulated the expression of P-STAT6 in bone marrow-derived macrophages. Compared with the untreated control group, the P value was less than 0.05, and the difference was statistically significant ( Figure 4 B). Cell immunofluorescence also confirmed these results. After Toxoplasma gondii antigen treatment, the amount of P-STAT6 fluorescence was significantly reduced ( Figure 4 ). Using an in vitro cell model of bone marrow-derived macrophages, the inhibitory effect of Toxoplasma gondii antigen on P-STAT6 expression was further confirmed.
[0058] Example 5: P-STAT6 agonist significantly improves adverse pregnancy outcomes caused by Toxoplasma infection
[0059] On the 8.5th day of pregnancy, 300 Toxoplasma tachyzoites were injected into the abdominal cavity of pregnant mice, and the P-STAT6 agonist was injected through the tail vein on the 9.5th day of pregnancy. The pregnancy outcome was observed on the 18.5th day of pregnancy. The effect of Toxoplasma infection on fetal development was evaluated by measuring the weight and size of the fetuses. P-STAT6 agonists can significantly improve the abortion rate ( Figure 5 A), while significantly increasing the size of the fetus ( Figure 5 B) and fetal weight ( Figure 5 C). Therefore, P-STAT6 agonists significantly improve adverse pregnancy outcomes caused by Toxoplasma gondii infection.
[0060] Some of the experimental steps in the above examples are as follows:
[0061] 1. Experimental Materials and Sources
[0062] (1) Anti-GAPDH antibody (Proteintech, USA)
[0063] (2) Anti-P-STAT6 (CST, USA)
[0064] (3) Anti-STAT6 (CST, USA)
[0065] (4) Red fluorescent secondary antibody Alexa 568 and green fluorescent secondary antibody Alexa 488 (Invitrogen, USA)
[0066] (5) HRP-goat anti-rabbit antibody and HRP-goat anti-mouse antibody (Proteintech, USA)
[0067] (6) Anti-Hoechst 33342 antibody (Thermo Fisher Scientific, USA)
[0068] (7) DMEM culture medium (Gibco, USA)
[0069] (8) Fetal bovine serum (Shanghai Yikesai Biological Products Co., Ltd.)
[0070] (9) Sodium chloride, isopropyl alcohol (Xilong Scientific Co., Ltd.)
[0071] (10) Acrylamide / Bis (Sigma Aldrich, USA)
[0072] (11) Tris, formaldehyde, glycine and SDS (Shanghai Sangon Biotechnology Co., Ltd.)
[0073] (12) HEPES and DTT (BBI Life Sciences)
[0074] (13) Ammonium persulfate (Shanghai Beyotime Company)
[0075] (14) DMSO and Tween-20 (Shanghai Sangon Biotechnology Co., Ltd.)
[0076] (15) Methanol, anhydrous ethanol, xylene and chloroform (Shanghai Lingfeng Chemical Reagent Co., Ltd.)
[0077] (16) 15 mL centrifuge tubes, 50 mL centrifuge tubes, 6-well culture plates and 24-well culture plates (Wuxi Nice Biotechnology Co., Ltd.)
[0078] (17) Slides, 35 mm culture dishes, 60 mm culture dishes and 100 mm culture dishes (Wuxi Nice Biotechnology Co., Ltd.)
[0079] (18) 500mL, 1000mL measuring cylinder (Beijing BOMEX Company)
[0080] (19) Cryogenic tube (China Biosharp Company)
[0081] (20) PVDF membrane (Merk, Germany)
[0082] (21) Small transfer filter paper (Bio-Rad, USA)
[0083] (22) Plastic sheet (Bio-Rad, USA)
[0084] (23) Plastic film (Wuxi Xinhuguang Plastic Industry Co., Ltd.)
[0085] (24) Adhesive slides and cover slips (Jiangsu Shitai Experimental Equipment Co., Ltd.)
[0086] (25) PAP water blocking pen (Abcam, USA)
[0087] (26) Wet box (Zhongshan Jinqiao Co., Ltd.)
[0088] (27) Pipettes of various specifications (Eppendorf, USA)
[0089] (28) Multiple immunofluorescence staining kit (Aibixin (Shanghai) Biotechnology Co., Ltd.)
[0090] (29) ECL developer (Dalian Meilun Biotechnology Co., Ltd.)
[0091] (30) Blue Plus Protein Marker (Beijing Quanshijin Biotechnology Co., Ltd.)
[0092] (31) Precision Plus ProteinTM Dual Color Standards (Bio-Rad, USA)
[0093] (32) CD11b / 32 blocking antibody, APC-IgG2b isotype control and APC-CD11b flow cytometry antibody (eBioscience, USA)
[0094] (33) Millex-GP filter (Merck Millipore, Germany)
[0095] (34) Recombinant mouse IL-4 (PeproTech, USA)
[0096] 2. Construction of animal models
[0097] C57BL / 6J mice (6-8 weeks; 25-30 g) were co-housed in a ratio of 2:1 between female and male mice. The female mice were checked for vaginal plugs the next morning. If vaginal plugs were found, they were considered to be gestation 0.5 (G0.5). On G8.5, pregnant mice were divided into two groups, with 6-9 mice in each group. The experimental group was intraperitoneally injected with Toxoplasma gondii tachyzoites (300 / 200 μL PBS), while the control group was intraperitoneally injected with 200 μL PBS. P-STAT6 agonists were injected through the tail vein on gestational day 9.5, and the pregnancy outcomes were observed on G17.5. All pregnant mice were euthanized on G17.5, and the placentas and fetuses were collected for subsequent experiments.
[0098] 3. Western blot
[0099] (1) The cultured RAW264.7 cells were placed in a protein lysis buffer containing phosphatase inhibitors and protease inhibitors and placed on ice for 15 min;
[0100] (2) Ultrasonic lysis for 10 seconds, centrifugation at 6216 × g for 1 minute, and collection of the supernatant;
[0101] (3) Mix the supernatant with 2× loading buffer at a ratio of 1:1;
[0102] (4) Denature the protein in a metal bath for 10 min and centrifuge for 5 min;
[0103] (5) Clean the glass plate and pour the prepared separation gel into the glass plate, flatten it with isopropanol, and let it stand for 45 minutes;
[0104] (6) Carefully pour out the isopropanol and rinse with sterile water;
[0105] (7) Pour the prepared concentrated gel into the glass plate, insert a comb that matches the glass plate, and let it stand for 45 minutes;
[0106] (8) Observe whether the concentrated gel has solidified. If so, carefully remove the comb and place the glass plate in an electrophoresis tank filled with a newly configured electrophoresis buffer. Add markers that match the protein molecular weight and different groups of heat-denatured proteins to the corresponding wells. Apply 60V voltage. When bromophenol blue runs to the separation gel, increase the voltage to 120V until bromophenol blue runs to the bottom of the gel.
[0107] (9) Prepare new transfer buffer and pre-cool it in a 4°C refrigerator. Cut the PVDF membrane according to the size of the gel, soak the PVDF membrane in methanol for 2 min, and then depolarize it in double distilled water.
[0108] (10) Prepare the transfer tongs, PVDF membrane, sponge and filter paper and place them in the following order: sponge, filter paper, PVDF membrane, filter paper and sponge. Place the transfer tongs in the transfer tank with the black side facing the black side of the transfer tank. Set the transfer conditions to 350V for 60min.
[0109] (11) After transfer, the PVDF membrane was washed once with TBST and then blocked with 5% skim milk at room temperature for 2 h.
[0110] (12) Incubate the PVDF membrane in the primary antibody at 4°C overnight;
[0111] (13) Wash the PVDF membrane 5 times with 1× TBST, 10 min each time;
[0112] (14) Place the PVDF membrane in the secondary antibody and incubate at room temperature for 1 h;
[0113] (15) Wash the PVDF membrane 5 times with 1× TBST, 10 min each time;
[0114] (16) Add ECL developer and develop the image under a developer.
[0115] 4. Extraction of Mouse Bone Marrow-derived Macrophages
[0116] (1) Adult C57BL / 6 mice (6-8 weeks) were euthanized, immersed in 75% alcohol, and fixed on the operating table.
[0117] (2) Use surgical scissors to separate the mouse tibia and femur, fully peel off the excess muscle tissue, wash them in PBS for 2-3 times, and transfer them to complete culture medium (containing 10% FBS).
[0118] (3) Use surgical scissors to cut open both ends of the femur and tibia, use a 1 mL syringe to absorb complete culture medium, and continuously flush bone marrow cells at one end of the bone into a 50 mL centrifuge tube.
[0119] (4) Add 5 volumes of red blood cell lysis buffer to the 50 mL centrifuge tube containing bone marrow cells, centrifuge at room temperature for 15 min, and centrifuge at 100 g for 15 min. Discard the supernatant and collect the cell pellet.
[0120] (5) Add complete medium to the cell pellet to resuspend the cells, filter with a 200-mesh filter, and centrifuge at 100 g for 15 min. Repeat this process twice.
[0121] (6) After the last wash, complete medium (containing 40 ng / mL M-CSF and 10% FBS) was added to the cell pellet to resuspend the cells and inoculate them in a 10 cm culture dish. Half of the medium was replaced on the 3rd and 6th days of culture, and mature bone marrow-derived macrophages were obtained on the 8th day of culture.
[0122] 5. Extraction of Mouse Peritoneal Macrophages
[0123] (1) Adult C57BL / 6 mice (6-8 weeks) were euthanized, immersed in 75% alcohol, and fixed on the operating table.
[0124] (2) Cut open the mouse abdomen with surgical scissors, inject 5 mL of PBS solution containing 2% FBS into the mouse peritoneal cavity, and massage the mouse abdomen for 3-5 minutes.
[0125] (3) Use a sterile Pasteur pipette to collect mouse peritoneal lavage fluid into a 15 mL centrifuge tube, centrifuge at 4°C, 300 g for 5 min, discard the supernatant, add DMEM medium to resuspend the precipitate, wash several times, add DMEM complete medium containing 10% FBS to resuspend, inoculate the cell fluid into a 60 mm culture dish, place in a 5% CO2 incubator and culture for 2-4 hours, then discard the non-adherent cells. The adherent cells are the mouse peritoneal macrophages, and complete medium is added to continue the culture.
[0126] 6. Cell Immunofluorescence
[0127] (1) Add pre-cooled 4% paraformaldehyde to a 24-well plate for 15 min and wash with TBST;
[0128] (2) Antigen heat repair was performed using citric acid repair solution (pH = 6.8) or Tris-EDTA repair solution (pH = 9.0), microwaved at medium heat for 20 min, cooled for 20 min, and washed with PBS;
[0129] (3) Add 5% BSA and shake at room temperature for 30 min for blocking;
[0130] (4) Add diluted primary antibody and incubate overnight at 4°C, then wash with TBST;
[0131] (5) Add secondary antibody and incubate at room temperature for 10 min, then wash with TBST;
[0132] (6) Add TSA single-color fluorescent dye and incubate for 10 min, then wash with TBST;
[0133] (7) Wash with citric acid repair solution (pH = 6.8) or Tris-EDTA repair solution (pH = 9.0), microwave for 20 min, cool for 20 min, and wash with PBS;
[0134] (8) Add 5% BSA and shake at room temperature for 30 min;
[0135] (9) Add the diluted second primary antibody and incubate overnight at 4°C, then wash with TBST;
[0136] (10) Add secondary antibody and incubate at room temperature for 10 min, then wash with TBST;
[0137] (11) Add the second TSA single-color fluorescent dye and incubate for 10 min, then wash with TBST;
[0138] (12) Wash with citric acid repair solution (pH = 6.8) or Tris-EDTA repair solution (pH = 9.0), microwave for 20 min, cool for 20 min, and wash with PBS;
[0139] (13) Add 5% BSA and shake at room temperature for 30 min;
[0140] (14) Add the diluted third primary antibody and incubate at 4°C overnight, then wash with TBST;
[0141] (15) Add secondary antibody and incubate at room temperature for 10 min, then wash with TBST;
[0142] (16) Add the third TSA single-color fluorescent dye and incubate for 10 min, then wash with TBST;
[0143] (17) Wash with citric acid repair solution (pH = 6.8) or Tris-EDTA repair solution (pH = 9.0), microwave for 20 min, cool for 20 min, and wash with PBS;
[0144] (18) Add diluted DAPI solution for nuclear staining;
[0145] (19) Use tweezers to remove the slides from the 24-well plate, seal them with antifade mounting medium, and photograph them using a laser confocal microscope.
[0146] 7. Flow Cytometry
[0147] (1) On gestational day 17.5, placental tissues of mice were collected;
[0148] (2) Add 3 mL of basal culture medium to the collected placental tissue, and grind the mouse placental tissue thoroughly with a grinding rod to prepare a cell suspension. Filter the cell suspension using a 300-mesh filter to remove tissue blocks and connective tissue;
[0149] (3) Collect the filtered cell suspension and centrifuge at 200g at 4°C for 5 min;
[0150] (4) Discard the supernatant, add 5 times the volume of red blood cell lysis buffer, mix thoroughly, and let stand at room temperature for 10 minutes. Centrifuge at 4°C, 200g for 10 minutes and discard the supernatant;
[0151] (5) Resuspend with 5 mL of basal medium and centrifuge at 200 g for 5 min at 4 °C;
[0152] (6) Discard the supernatant, add 1 mL of basal medium to resuspend, count the cells, and take out 1×10 6 The cells were placed in a 15 mL centrifuge tube and centrifuged at 4 °C for 10 min;
[0153] (7) Resuspend the cells with 100 μL of staining buffer, add CD16 / 32 blocking antibody, and incubate at 4°C for 15 min;
[0154] (8) Add CD11b flow cytometry antibody to the cell fluid, set up an empty staining tube and an isotype control, and incubate at 4°C in the dark for 30 min;
[0155] (9) Wash twice with 1 mL of staining buffer and centrifuge at 300 g for 5 min at 4 °C;
[0156] (10) Add 200 μL of staining buffer to resuspend the cells and detect them using a flow cytometer.
[0157] 8. Immunohistochemistry
[0158] (1) Place the tissue paraffin sections in a 72°C oven for 1 h;
[0159] (2) Dewax with 3 cylinders of fresh xylene for 15 min each, and then wash with PBS;
[0160] (3) Use 100%, 100%, 95%, and 75% gradient alcohol to make it transparent;
[0161] (4) Antigen heat repair was performed using citric acid repair solution (pH = 6.8) or Tris-EDTA repair solution (pH = 9.0), microwaved at medium heat for 20 min, cooled for 20 min, and washed with PBS;
[0162] (5) Use an immunohistochemistry pen to draw a circle outside the tissue, add enough endogenous peroxidase blocker inside the circle for 10 min, and wash with PBS;
[0163] (6) Add 5% BSA to block the sections for 1 h;
[0164] (7) After adding the primary antibody, the sections were incubated at 4°C overnight and washed with PBS;
[0165] (8) After adding the secondary antibody, incubate at room temperature for 30 min and wash with PBS;
[0166] (9) Add 3-3′-diaminobenzidine (DAB) for 6–8 min to develop color, and tap water turns blue for 3–5 min;
[0167] (10) After counterstaining with hematoxylin for 20 min, hydrochloric acid alcohol differentiation was used;
[0168] (11) Using the recovered repair fluid to return to blue for 3 to 5 minutes;
[0169] (12) After dehydration with gradient alcohol, xylene was used for transparency;
[0170] (13) Use neutral resin to seal the slides and photograph them using a Leica microscope.
[0171] In summary, the embodiments of the present invention verify that Toxoplasma gondii infection can inhibit the expression of P-STAT6 in mouse placental tissue by constructing animal models such as pregnant mice infected with Toxoplasma gondii. At the same time, Toxoplasma gondii antigens inhibit the expression of P-STAT6 protein in macrophage cell lines, peritoneal macrophages and bone marrow-derived macrophages. The use of P-STAT6 agonists can significantly improve adverse pregnancy caused by Toxoplasma gondii infection. Through the verification experiment of this embodiment, it can be seen that Toxoplasma gondii and its antigens inhibit P-STAT6 expression. It is verified that P-STAT6 agonists significantly improve adverse pregnancy caused by Toxoplasma gondii infection. Thus, a new direction and drug are provided for the treatment of adverse pregnancy caused by Toxoplasma gondii.
Claims
1. Application of P-STAT6 as a target in the preparation of drugs for the treatment of adverse pregnancy.
2. The use of P-STAT6 as a target in the preparation of a drug for treating adverse pregnancy according to claim 1, characterized in that: The adverse pregnancy is the adverse pregnancy caused by Toxoplasma gondii.
3. The use of P-STAT6 as a target in the preparation of a drug for treating adverse pregnancy according to claim 2, characterized in that: The Toxoplasma gondii is Toxoplasma gondii.
4. The use of P-STAT6 as a target in the preparation of a drug for treating adverse pregnancy according to claim 1, characterized in that: The drug targets P-STAT6 to improve adverse pregnancy caused by Toxoplasma gondii infection.
5. A drug for treating adverse pregnancy caused by Toxoplasma gondii, characterized in that: The medicine includes a P-STAT6 agonist, and the medicine is used to treat adverse pregnancy caused by Toxoplasma gondii by increasing the expression of P-STAT6.
6. The drug for treating adverse pregnancy caused by Toxoplasma gondii according to claim 5, characterized in that: The medicine also includes other medically acceptable agents.
7. Use of P-STAT6 as a target in the preparation of a product for detecting adverse pregnancy caused by Toxoplasma gondii, characterized in that: The product is a detection reagent or a detection kit.
8. The use of P-STAT6 as a target in the preparation of a product for detecting adverse pregnancy caused by Toxoplasma gondii according to claim 7, characterized in that: The product includes a reagent for detecting the expression of P-STAT6.
9. The use of P-STAT6 as a target in the preparation of a product for detecting adverse pregnancy caused by Toxoplasma gondii according to claim 8, characterized in that: The product may also include other medically acceptable agents.