Application of PPAR gamma agonist in treatment of bad pregnancy caused by toxoplasma gondii infection
The problem of adverse pregnancy caused by Toxoplasma infection in Toxoplasma gondii was solved by increasing PPARγ expression by using PPARγ agonists such as rosiglitazone, which significantly improved pregnancy outcomes.
Patent Information
- Application Number
- CN202411622993.6
- 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
The effect of PPARγ on adverse pregnancy outcomes caused by Toxoplasma gondii infection is not known in the prior art.
PPARγ is used as a target and PPARγ agonists such as rosiglitazone are used to improve adverse pregnancy caused by Toxoplasma gondii infection.
By increasing PPARγ expression, PPARγ agonists can significantly improve adverse pregnancy outcomes caused by Toxoplasma infection.
Smart Images
Figure CN119592676A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biomedicine, and in particular to application of a PPARγ agonist in treating adverse pregnancy caused by Toxoplasma gondii infection. Background Art
[0002] Toxoplasma gondii (T. gondii) is a parasitic protozoan that is widely spread around the world, infecting about one-third of the world's population. With the increase in the number of cat owners, the incidence of T. gondii infection has increased significantly. Although individuals with normal immunity are often in a latent state of infection and do not show clinical symptoms, the first infection of pregnant women with T. gondii during pregnancy often has adverse effects on the fetus, resulting in serious physiological defects in the fetus, such as hearing and vision loss and mental retardation. The adverse pregnancy outcomes caused by T. gondii infection are mainly caused by the imbalance of immune tolerance at the maternal-fetal interface composed of placental trophoblast cells and maternal decidua. The maternal-fetal interface contains a large number of decidual immune cells (DICs), including natural killer (NK) cells, macrophages, T cells and dendritic cells, which play an indispensable role in pathogen clearance and immune tolerance regulation. Existing studies have found that Toxoplasma gondii infection inhibits the function and number of regulatory T cells (Tregs), destroys maternal-fetal immune tolerance, and thus leads to adverse pregnancy outcomes. However, in addition to Tregs, other DICs are also associated with immune imbalance at the maternal-fetal interface after Toxoplasma gondii infection.
[0003] Decidual macrophages are the main cell population at the maternal-fetal interface, accounting for 20%-30% of DICs, and are mainly involved in pathogen phagocytosis and antigen presentation. Experimental evidence shows that Toxoplasma gondii infection induces a large number of macrophages to accumulate in peripheral infected tissues. At the same time, Toxoplasma gondii infection activates macrophages to produce a variety of proinflammatory mediators, including tumor necrosis factor-alpha (TNF-α) and inducible nitric oxide synthase (iNOS), thereby biasing decidual macrophages toward the M1 proinflammatory state. Changes in the number and function of macrophages are closely related to maternal-fetal immune tolerance. In mice lacking immunosuppressive molecules such as B7 homolog 4 (B7-H4), leukocyte immunoglobulin-like receptor B4 (LILRB4), and T-cell immunoglobulin domain and mucin domain containing protein-3 (Tim-3), decidual macrophages showed a more obvious polarization toward M1 macrophages, leading to worse pregnancy outcomes. However, these immunosuppressive molecules are not specifically expressed on the surface of decidual macrophages. Therefore, the functional molecules and signaling mechanisms involved in the response of decidual macrophages to T. gondii infection remain unclear. Summary of the invention
[0004] The purpose of the present invention is to solve the technical problem that the prior art is unclear about the effect of PPARγ on adverse pregnancy outcomes caused by Toxoplasma gondii infection.
[0005] In order to achieve the above object, the present invention adopts the following technical solutions:
[0006] Application of PPARγ as a target in the preparation of drugs for treating adverse pregnancy caused by Toxoplasma gondii.
[0007] Preferably, the drug uses the PPARγ as an activation target to improve adverse pregnancy caused by Toxoplasma gondii.
[0008] Preferably, the drug comprises a PPARγ agonist.
[0009] Preferably, the PPARγ agonist comprises rosiglitazone.
[0010] The present application also provides a drug for treating adverse pregnancy caused by Toxoplasma gondii, wherein the drug includes a PPARγ agonist, and the PPARγ agonist treats adverse pregnancy caused by Toxoplasma gondii by increasing PPARγ expression.
[0011] Preferably, the PPARγ agonist is rosiglitazone.
[0012] The present application also provides the use of PPARγ as a target in the preparation of a product for detecting adverse pregnancy caused by Toxoplasma gondii.
[0013] Preferably, the product is a detection reagent or a detection kit, and the detection kit comprises a reagent for detecting the expression of PPARγ.
[0014] Preferably, the detection reagent or detection kit also includes other medically acceptable reagents.
[0015] Compared with the prior art, this application has the following beneficial effects:
[0016] The embodiments of the present invention verified that T.gondii infection down-regulated PPARγ expression by studying the effect of T.gondii infection on PPARγ expression in placental tissue in pregnant mice. Using T.gondii antigen to stimulate macrophage cell lines, peritoneal macrophages and bone marrow-derived macrophages in vitro models, it was verified that T.gondii antigen can inhibit and promote macrophage PPARγ expression. PPARγ agonists can partially reverse the inhibitory effect of T.gondii antigen on PPARγ. Using PPARγ agonists to inject T.gondii-infected pregnant mice, it was verified that promoting PPARγ 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
[0017] Figure 1 In the examples of the present invention, Toxoplasma infection down-regulates PPARγ expression in placental tissue.
[0018] Figure 1 A: Fetal mouse image; *: P < 0.05, each data represents a single fetal mouse (n = 5-7 mice);
[0019] Figure 1 B: Flow cytometry was used to detect the decidual macrophages (CD11b + ) percentage;
[0020] Figure 1 C: Immunohistochemistry was used to detect the expression of PPARγ in the placenta tissue of mice in the PBS group and the Toxoplasma gondii infection group. Scale bar: 100μm or 50μm;
[0021] Figure 1 D: Western blot was used to detect the expression of PPARγ in mouse placenta tissue (n=8 mice).
[0022] Data analysis was performed using two-tailed unpaired Student's t-test. *: P < 0.05
[0023] Figure 2 . Toxoplasma gondii antigen inhibits the expression of PPARγ in macrophages.
[0024] Figure 2 A: PPARγ expression was determined by Western blot. The values in the figure represent 3 independent repeated experiments. *: P<0.05.
[0025] Figure 2 B: Immunofluorescence detection of PPARγ expression. Scale bar: 15 μm.
[0026] Figure 3 . Toxoplasma gondii antigen inhibits the expression of PPARγ in peritoneal macrophages. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparisons test. *: P < 0.05.
[0027] Figure 4 . Toxoplasma gondii antigen inhibits PPARγ expression in BMDMs.
[0028] Figure 4 A: Experimental operation flow chart.
[0029] Figure 4 B: Western blot was used to detect the expression of PPARγ in BMDMs. The data in the figure represent 3 independent repeated experiments. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparisons test. *: P < 0.05.
[0030] Figure 4 C: Confocal microscopy was used to detect the expression of PPARγ in mouse BMDMs. Scale bar: 10 μm or 15 μm.
[0031] Figure 5 .Western blot was used to detect the effect of rosiglitazone on PPARγ expression.
[0032] Data were analyzed using two-way ANOVA with Sidak's multiple comparisons test. *: P < 0.05, data represent the results of 3 independent repeated experiments, TgAg: T.gondiiantigens.
[0033] Figure 6 Effect of rosiglitazone on adverse pregnancy outcomes caused by Toxoplasma gondii infection.
[0034] The fetal weight and size comparison chart were measured. Data analysis was performed using two-way ANOVA with Sidak's multiple comparisons test. *: P < 0.05. DETAILED DESCRIPTION
[0035] The present invention is further described in detail below in conjunction with specific embodiments.
[0036] The following is an explanation of the above content in combination with specific verification experiments:
[0037] Example 1: Verification of the effect of Toxoplasma gondii infection on PPARγ expression
[0038] First, a mouse model of abortion caused by Toxoplasma infection was established. 300 Toxoplasma tachyzoites were intraperitoneally injected into pregnant mice at G8.5 days. The mice were euthanized at G17.5 days to observe the effect of Toxoplasma infection on fetal development. Fetal development was evaluated by fetal size (CRL×OF) and fetal weight. Figure 1 A, Toxoplasma infection causes fetal mortality and significant intrauterine growth retardation in mice.
[0039] Flow cytometry was used to detect the decidual macrophages (CD11b + ) percentage, the results are as follows Figure 1 As shown in B, Toxoplasma infection can increase the proportion of decidual macrophages in the mouse placenta;
[0040] To detect PPARγ expression in mouse placenta tissue using histochemistry, please refer to Figure 1 C, Toxoplasma infection can lead to decreased PPARγ expression in placental tissue.
[0041] Western blot was used to detect the expression of PPARγ in mouse placenta tissue. Figure 1 D, Toxoplasma infection leads to downregulation of PPARγ expression in placental tissue.
[0042] In summary, this example verifies that Toxoplasma gondii can inhibit the expression of PPARγ in the placenta.
[0043] Example 2: Verification of Toxoplasma gondii antigen inhibiting macrophage PPARγ expression
[0044] First, Raw264.7 cells were treated with IL-4 (20 ng / mL) for 1 h, and then stimulated with TgAg (T. gondii antigens) for 24 h, and then the cells were collected to detect PPARγ expression;
[0045] Western blot results showed that after TgAg treatment, the P values were less than 0.05 (P = 0.0268) compared with the control group, indicating that TgAg treatment significantly reduced the expression level of PPARγ ( Figure 2 A). In addition, similar results were observed by immunofluorescence staining. The green fluorescence (PPARγ) was significantly reduced after Toxoplasma gondii antigen treatment ( Figure 2 B). These data indicate that TgAg can inhibit PPARγ expression in vitro, consistent with the results in animal models.
[0046] Example 3: Verification of T. gondii antigen inhibiting PPARγ expression in peritoneal macrophages
[0047] In this embodiment, peritoneal macrophages were isolated from wild-type mice to study the effect of TgAg on downstream signaling pathways.
[0048] First, peritoneal macrophages isolated from wild-type mice were pretreated with IL-4 (20 ng / mL) for 1 h, and then the cells were stimulated with 5 μg / mL TgAg for 24 h, and the cells were collected for detection. The detection results in this example are consistent with those in Example 2. Compared with the group without TgAg treatment, the expression of PPARγ in peritoneal macrophages of wild-type mice in the TgAg group was significantly decreased, with a P value of <0.05, and the difference was statistically significant ( Figure 3 ), indicating that TgAg can inhibit the expression of PPARγ in mouse peritoneal macrophages.
[0049] That is, Toxoplasma gondii antigen affects the expression level of PPARγ.
[0050] Example 4: Verification of T. gondii antigen inhibiting PPARγ expression in bone marrow-derived macrophages
[0051] In this example, bone marrow-derived macrophages (BMDMs) were extracted from wild-type cells to study the effect of TgAg on PPARγ expression.
[0052] For details, please refer to Figure 4A, Mature BMDMs were successfully induced on the eighth day. Subsequently, BMDMs were pretreated with IL-4 (20 ng / mL) for 1 h, and then stimulated with TgAg (5 μg / mL) for 24 h, and the cells were collected for detection. Figure 4 As shown in Figure B, consistent with the results of the peritoneal macrophage study in Example 3, TgAg can significantly inhibit the expression level of PPARγ in wild-type mouse BMDMs, with P values less than 0.05 (P value is 0.0013), and the difference is statistically significant ( Figure 4 B). Cell immunofluorescence also confirmed the above conclusion. After TgAg treatment, the number of fluorescence decreased significantly ( Figure 4 C).
[0053] The experimental results of BMDMs further proved that TgAg inhibited PPARγ expression.
[0054] Example 5: PPARγ agonist rosiglitazone partially reverses the inhibitory effect of Toxoplasma gondii antigen on PPARγ
[0055] In this example, rosiglitazone was used as an agonist of PPARγ to detect downstream protein expression. Through preliminary experiments, 1 μM was used as the optimal stimulation concentration. Specifically, Raw264.7 cells were pretreated with IL-4 (20 ng / mL) for 1 h, and then stimulated with TgAg (5 μg / mL) and / or rosiglitazone (1 μM) for 24 h, and then the cells were collected for detection.
[0056] Western blot detection showed that after 24h of rosiglitazone (RSG) stimulation, the expression level of PPARγ increased, indicating that rosiglitazone promoted the expression of PPARγ. After 24h of TgAg induction, the expression level of PPARγ increased in TgAg+IL-4+RSG compared with TgAg+IL-4, P<0.05, which was statistically significant ( Figure 5 ). Therefore, the PPARγ agonist rosiglitazone partially reversed the inhibitory effect of Toxoplasma gondii antigen on PPARγ.
[0057] Example 6: PPARγ agonist rosiglitazone significantly improves adverse pregnancy outcomes caused by Toxoplasma gondii infection
[0058] On the 8.5th day of pregnancy, the pregnant mice were injected with Toxoplasma gondii tachyzoites intraperitoneally, and on the 9.5th day of pregnancy, the PPARγ agonist rosiglitazone was injected through the tail vein. The pregnancy outcome was observed on the 18.5th day of pregnancy, and the weight and size of the fetuses were measured. Rosiglitazone can significantly increase the size of the fetuses ( Figure 6 A) and fetal weight ( Figure 6 B) Therefore, the PPARγ agonist rosiglitazone significantly improved adverse pregnancy outcomes caused by Toxoplasma gondii infection.
[0059] Some of the laboratory reagents and consumables and experimental steps in the above examples are as follows:
[0060] 1. Laboratory reagents and consumables
[0061] (1) Anti-GAPDH antibody (Proteintech, USA)
[0062] (2) Anti-PPARγ (CST, USA)
[0063] (3) Green fluorescent secondary antibody Alexa 488, Red fluorescent secondary antibody Alexa 568 (Invitrogen, USA)
[0064] (4) HRP-labeled goat anti-mouse antibody, HRP-labeled goat anti-rabbit antibody (Proteintech, USA)
[0065] (5) Anti-Hoechst 33342 antibody (Thermo Fisher Scientific, USA)
[0066] (6) Rosiglitazone (MCE, USA)
[0067] (7) DMEM culture medium (Gibco, USA)
[0068] (8) Fetal bovine serum (Shanghai Yikesai Biological Products Co., Ltd.)
[0069] (9) Tris, glycine, SDS (Shanghai Sangon Biotechnology Co., Ltd.)
[0070] (10) HEPES, DTT (BBI Life Sciences)
[0071] (11) PMSF, ammonium persulfate (Shanghai Beyotime Company)
[0072] (12) Tween-20, DMSO (Shanghai Sangon Biotechnology Co., Ltd.)
[0073] (13) Sodium chloride, isopropyl alcohol (Xilong Scientific Co., Ltd.)
[0074] (14) Methanol, anhydrous ethanol, xylene, chloroform (Shanghai Lingfeng Chemical Reagent Co., Ltd.)
[0075] (15) Acrylamide / Bis (Sigma Aldrich, USA)
[0076] (16) 35mm culture dish, 60mm culture dish, 100mm culture dish, slide (Wuxi Nice Biotechnology Co., Ltd.)
[0077] (17) 6-well culture plate, 24-well culture plate, 15 mL centrifuge tube, 50 mL centrifuge tube (Wuxi Nice Biotechnology Co., Ltd.)
[0078] (18) 500mL, 1000mL measuring cylinder (Beijing BOMEX Company)
[0079] (19) 15mL ultrafiltration centrifuge tube (Millipore, USA)
[0080] (20) Cryogenic tube (China Biosharp Company)
[0081] (21) PVDF membrane (Merk, Germany)
[0082] (22) Small transfer filter paper (Bio-Rad, USA)
[0083] (23) Plastic sheet (Bio-Rad, USA)
[0084] (24) Plastic film (Wuxi Xinhuguang Plastic Industry Co., Ltd.)
[0085] (25) Adhesive slides and cover glass (Jiangsu Shitai Experimental Equipment Co., Ltd.)
[0086] (26) PAP water blocking pen (Abcam, USA)
[0087] (27) Wet box (Zhongshan Jinqiao Co., Ltd.)
[0088] (28) Pipette (Eppendorf, USA)
[0089] (29) ECL developer (Dalian Meilun Biotechnology Co., Ltd.)
[0090] (30) BluePlusProteinMarker (Beijing Quanshijin Biotechnology Co., Ltd.)
[0091] (31) Precision Plus Protein TM Dual Color Standards (Bio-Rad, USA)
[0092] (32) CD11b / 32 blocking antibody, anti-mouseTrem2 flow cytometry antibody (R&D Company, USA)
[0093] (33) APC-CD11b flow cytometry antibody, APC-IgG2b isotype control (eBioscience, USA)
[0094] (34) Millex-GP filter (Merck Millipore, Germany)
[0095] (35) Multiple immunofluorescence staining kit (Aibixin (Shanghai) Biotechnology Co., Ltd.)
[0096] (36) Recombinant mouse IL-4 (PeproTech, USA)
[0097] 2. Construction of animal models
[0098] 6-8 weeks old 25-30g C57BL / 6J mice were mated in cages at a ratio of 2:1 between female and male mice. The next morning, vaginal plugging was found as gestation 0.5 (G0.5). Pregnant mice on G8.5 (6-9 mice per group) were intraperitoneally injected with Toxoplasma gondii tachyzoites (300 / 200μL PBS) or PBS. All pregnant mice were euthanized at G17.5, and the placentas and fetuses were collected for subsequent experiments.
[0099] 3. Western blot detection
[0100] (1) Cleaning the glass plate: Use an appropriate amount of detergent to clean the glass plate, then rinse it with double distilled water and let it dry naturally.
[0101] (2) Leak detection: Assemble thin and thick glass plates and check for leakage.
[0102] (3) Glue preparation: Select the required separation gel concentration according to the molecular weight of the target protein, add the required reagents into a clean beaker, mix well and then pour the gel. Wait for 40 minutes for solidification before preparing the concentrated gel.
[0103] (4) Sample loading: Pour the prepared electrophoresis buffer into the electrophoresis tank, and then load the protein samples in order, with a voltage of 60V. When bromophenol blue runs to the separation gel, increase the voltage to 120V until bromophenol blue runs to the bottom of the gel.
[0104] (5) Transfer: Cut the PVDF membrane according to the size of the gel, soak the PVDF membrane in methanol for 2 min, and then put it into double distilled water for depolarization. Prepare transfer clips, sponges and filter paper, and arrange them in the order of sponge, filter paper, PVDF membrane, filter paper, and sponge. Put the transfer clips into the transfer tank with the black transfer surface corresponding to the black surface of the transfer tank. Set the transfer conditions to 350V for 60min.
[0105] (6) Blocking: Prepare 5% skim milk in advance, cut the PVDF membrane according to the size of the gel, put it in the skim milk, and block it for 2 hours.
[0106] (7) Primary antibody incubation: dilute the antibody according to the ratio with 1× TBST and incubate at 4°C overnight.
[0107] (8) Cleaning the primary antibody: Wash with 1× TBST for 10 min each time, 4 times.
[0108] (9) Secondary antibody incubation: Prepare secondary antibody according to the species of the primary antibody and incubate at room temperature for 1 h.
[0109] (10) Washing the secondary antibody: Use 1× TBST for washing, 10 min each time, 4 times.
[0110] (11) Development: Prepare a developer in a 1:1 ratio, mix well, and then drop it onto the membrane for development.
[0111] 4. Extraction of primary mouse macrophages
[0112] Mouse peritoneal macrophages:
[0113] (1) 6-8 week old C57BL / 6 mice were selected, euthanized, disinfected by immersion in 75% alcohol, and then fixed on a sterile dissection table.
[0114] (2) Cut open the mouse abdomen, hold the syringe in the right hand and pierce the mouse subcutaneously into the abdominal cavity, inject 5 mL of PBS containing 2% FBS, and massage the mouse abdomen for 3-5 minutes.
[0115] (3) Use a sterile Pasteur pipette to collect the mouse peritoneal lavage fluid, draw it back into a 15 mL centrifuge tube, centrifuge it at 300 g, 4°C for 5 min, discard the supernatant, add DMEM medium to resuspend the precipitate, wash it several times, then add DMEM medium containing 10% FBS to resuspend it, inoculate it into a 60 mm culture dish, and culture it in an incubator containing 5% CO2 for 2-4 h. Then wash away the non-adherent cells, and the adherent cells are mouse peritoneal macrophages, and add complete medium to continue culturing.
[0116] 5. Mouse bone marrow-derived macrophages
[0117] (1) Adult C57BL / 6 mice aged 6-8 weeks were euthanized and immersed in 75% alcohol, then fixed on an operating table.
[0118] (2) Use surgical scissors to separate and remove the mouse's tibia and femur, fully peel off the excess muscle tissue, then wash them in PBS for 2-3 times, and finally transfer them to complete culture medium containing 10% FBS.
[0119] (3) Cut open the femur and tibia at both ends, inject 1 mL of complete culture medium, and continuously flush bone marrow cells from one end of the bone into a 50 mL centrifuge tube.
[0120] (4) Add 5 times the volume of red blood cell lysis buffer to the above solution, fully lyse for 15 minutes, and centrifuge at 100g for 15 minutes to obtain a cell pellet.
[0121] (5) After discarding the supernatant, add complete medium again to resuspend, then filter with a 200-mesh filter and centrifuge again at 100 g for 15 min. Repeat twice.
[0122] (6) After the final wash, add complete medium containing 40 ng / mL M-CSF to resuspend the cells and inoculate them in a 10 cm culture dish. Change the medium by half on the third and sixth days of culture, and finally culture until the eighth day to obtain mature bone marrow-derived macrophages.
[0123] 6. Cell Immunofluorescence
[0124] (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.
[0125] (2) Wash three times with PBS at room temperature.
[0126] (3) Permeabilization: Use 0.1% Triton-X for permeabilization at room temperature for 5 minutes, and add 200 μL to each well.
[0127] (4) Blocking: Block with 5% BSA prepared in PBS at room temperature for 1 hour, adding 250 μL to each well.
[0128] (5) 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.
[0129] (6) Wash the primary antibody by gently shaking with PBS on a shaker for 5 min each time.
[0130] (7) Secondary antibody incubation: Dilute the secondary antibody according to the instructions, add fluorescent secondary antibody onto the slide, and incubate at 37°C in the dark for 90 min.
[0131] (8) DAPI staining: Dilute the nuclear dye Hoechest with PBS, add an appropriate amount of the diluted dye to the slide, and incubate at room temperature in the dark for 30 minutes.
[0132] (9) Washing the secondary antibody: Wash with PBS three times for 5 min each time in a dark environment.
[0133] (10) Sealing: Remove the slide from the 24-well plate and place it on a glass slide with glycerol. Apply nail polish around the slide and keep it moist at 4°C overnight.
[0134] 7. Multiplex Immunofluorescence (TSA)
[0135] (1) The grouped cells were fixed with 4% paraformaldehyde at room temperature for 15 min.
[0136] (2) Wash twice with PBS, 1 min each time, and then permeabilize the cells with 0.1% Triton-X at room temperature for 5 min.
[0137] (3) Wash the cells with PBS for 1 min. Repeat this step once and then add 200 μL of 5% BSA to each well for blocking for 20 min.
[0138] (4) After removing the blocking solution, incubate the primary antibody diluted according to the instructions at 200 μL per well and incubate at room temperature for 1 h.
[0139] (5) Wash the cells twice with 1× TBST, 3 min each time.
[0140] (6) After washing, add the anti-rabbit secondary antibody in the kit to the 24-well plate, completely immersing the cells, and incubate at room temperature for 10 min.
[0141] (7) Wash the cells twice with 1× TBST, each time for 3 min.
[0142] (8) Prepare the signal amplification solution (1:100 dilution) in advance and incubate at room temperature with shaking for 10 min.
[0143] (9) Wash the cells three times with 1× TBST, each time for 3 min.
[0144] (10) Preheat the antibody elution buffer at 37°C in advance, add a few drops of elution buffer to cover the cells, and after elution for 3 min, remove the elution buffer and add 300 μL of elution buffer to each well and elute at 37°C for 1 h.
[0145] (11) Wash the cells once with double distilled water and then wash the cells with 1× TBST for 2 min.
[0146] (12) Block again with 5% BSA for 20 min at room temperature.
[0147] (13) After one round of staining is completed, repeat steps 4-11.
[0148] (14) After all staining is completed, perform nuclear staining by adding 1× DAPI in the kit into a 24-well plate, immersing the sample, and incubating at room temperature for 5 min.
[0149] (15) After staining, the cells were washed three times with 1× TBST for 2 min each time.
[0150] (16) Remove the slide from the 24-well plate and place it face down on a glass slide with anti-quenching agent. Seal the slide with clear nail polish.
[0151] (17) Observe the staining under a confocal microscope.
[0152] 8. Immunohistochemistry
[0153] (1) Prepare paraffin sections.
[0154] (2) Place the slide in a 72°C oven and bake for 1 hour.
[0155] (3) Dewax the sample by placing it in xylene, changing the mixture every ten minutes, for 2-3 times. Place the sample in gradient alcohol solutions of 100%, 100%, 95%, and 75%, with each gradient solution lasting 2 minutes.
[0156] (4) Wash with PBS containing 1‰ Tween for 2 min each time, for 5 times.
[0157] (5) High-pressure repair: preheat the antigen repair solution, place the slide in the boiling antigen repair solution, heat it in a microwave oven to a boiling or sub-boiling state for 10 minutes, and cool it for 30 minutes.
[0158] (6) Wash with the above PBS 3-5 times, 2 min each time.
[0159] (7) Use a PAP pen to draw a circle around the tissue, add 3% hydrogen peroxide, and incubate at room temperature for 10 minutes.
[0160] (8) Wash with PBS 3-5 times, 2 min each time.
[0161] (9) Add 5% BSA to cover the tissue for blocking for 1 hour.
[0162] (10) Add the diluted primary antibody according to the ratio specified in the antibody manual onto the tissue slide and incubate at 37°C for 1 h.
[0163] (11) Wash with PBS 3-5 times, 2 min each time.
[0164] (12) Add secondary antibody and incubate at room temperature for 30 min.
[0165] (13) Wash with PBS 3-5 times, 2 min each time.
[0166] (14) Add DAB color developing solution and rinse with tap water after 5 minutes to stop color development and rinse for 3-5 minutes.
[0167] (15) Use hydrochloric acid alcohol to differentiate for 30 seconds.
[0168] (16) Rinse with tap water for 3-5 minutes to return to blue, then rinse with running water for 3-5 minutes.
[0169] (17) Incubate the slides in 90% ethanol for 30 s, 100% ethanol for 30 s, and xylene for 30 s. Repeat each step twice.
[0170] (18) Finally, use neutral resin for sealing.
[0171] 9. Flow Cytometry
[0172] (1) Collect mouse placental tissue.
[0173] (2) Add 3 mL of medium and grind the mouse placenta tissue thoroughly using a grinding rod to prepare a cell suspension.
[0174] (3) Filter using a 300-mesh filter.
[0175] (4) Collect the filtered cell suspension and centrifuge at 4°C, 200g for 5 min.
[0176] (5) After centrifugation, remove the supernatant and add 5 times the volume of red blood cell lysis buffer. Mix thoroughly and let stand for 10 minutes.
[0177] (6) After standing, centrifuge at 4°C, 200 g for 10 min and discard the supernatant.
[0178] (7) Resuspend the tube by adding 5 mL of basal medium and centrifuge at 4°C, 200 g for 5 min.
[0179] (8) After centrifugation, discard the supernatant, add 1 mL of basal culture medium to resuspend, and take out 1×10 6 The cells were placed in a 15 mL centrifuge tube and centrifuged again at 4°C for 10 min.
[0180] (9) After centrifugation, add 100 μL of Staining buffer.
[0181] (10) CD16 / 32 blocking antibody was added and incubated for 15 min.
[0182] (11) Add flow cytometry antibodies, empty tubes, and isotype controls, and incubate at 4°C in the dark for 30 min.
[0183] (12) Add 1 mL of Staining Buffer and wash twice, then centrifuge at 300 g for 5 min at 4 °C.
[0184] (13) Finally, add 200 μL of Staining buffer to resuspend the cells and detect them using a flow cytometer.
[0185] The following are some of the experimental materials and their sources used in the above examples of this application:
[0186] In combination with the above specific embodiments, the present application provides the use of PPARγ as a target in the preparation of a drug for treating adverse pregnancy caused by Toxoplasma gondii, wherein the drug uses PPARγ as an activation target to improve adverse pregnancy caused by Toxoplasma gondii.
[0187] In one embodiment, the drug comprises a PPARγ agonist, and the PPARγ agonist includes rosiglitazone.
[0188] Based on the above content, the present application also provides a drug for treating adverse pregnancy caused by Toxoplasma gondii, wherein the drug comprises a PPARγ agonist, and preferably, the PPARγ agonist is rosiglitazone.
[0189] In addition, the present application also provides the use of PPARγ as a detection 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.
[0190] In one embodiment, the product further comprises other medically acceptable agents.
[0191] Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that promotes placental growth, controls trophoblast differentiation, oxidative pathways and barrier generation, and maintains normal pregnancy. Activation of PPARγ can inhibit the production of inflammatory cytokines, reduce inflammation and maintain immune balance, thereby promoting fetal growth. In this application, by constructing Toxoplasma gondii-infected and wild-type mouse pregnancy models, the effects of Toxoplasma gondii infection on pregnancy outcomes and PPARγ were explored. By constructing an in vitro cell model, the pathological mechanism of PPARγ and its downstream pathway molecules during Toxoplasma gondii infection was explored. Through this study, a new research perspective is provided for Toxoplasma gondii infection-induced adverse pregnancy outcomes, which may provide theoretical support and experimental basis for clinical practice, and promote the development and application of new therapies.
[0192] In summary, the present application verifies through specific embodiments that in animal models such as pregnant mice infected with T.gondii, T.gondii infection can inhibit the expression of PPARγ in mouse placental tissue. At the same time, T.gondii antigens inhibit the expression of PPARγ protein in macrophage cell lines, peritoneal macrophages and bone marrow-derived macrophages. PPARγ agonists stimulate macrophages and can partially reverse the inhibitory effect of T.gondii antigens on PPARγ. The use of PPARγ agonists can significantly improve adverse pregnancy caused by Toxoplasma infection. Through the verification experiment of this embodiment, it can be seen that T.gondii and its antigens inhibit PPARγ expression. It is verified that PPARγ agonists 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.
Claims
1. Application of PPARγ as a target in the preparation of drugs for the treatment of adverse pregnancy caused by Toxoplasma gondii.
2. The use of PPARγ as a target in the preparation of a drug for treating adverse pregnancy caused by Toxoplasma gondii according to claim 1, characterized in that: The drug uses the PPARγ as an activation target to improve adverse pregnancy caused by Toxoplasma gondii.
3. The use of PPARγ as a target in the preparation of a drug for treating adverse pregnancy caused by Toxoplasma gondii according to claim 1, characterized in that: The medicine comprises a PPARγ agonist.
4. The use of PPARγ as a target in the preparation of a drug for treating adverse pregnancy caused by Toxoplasma gondii according to claim 3, characterized in that: The PPARγ agonists include rosiglitazone.
5. A drug for treating adverse pregnancy caused by Toxoplasma gondii, characterized in that: The medicine comprises a PPARγ agonist, and the PPARγ agonist is used to treat adverse pregnancy caused by Toxoplasma gondii by increasing the expression of PPARγ.
6. The drug for treating adverse pregnancy caused by Toxoplasma gondii according to claim 5, characterized in that: The PPARγ agonist is rosiglitazone.
7. Application of PPARγ as a target in the preparation of products for detecting adverse pregnancy caused by Toxoplasma gondii.
8. The use of PPARγ 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 is a detection reagent or a detection kit, and the detection reagent or the detection kit comprises a reagent for detecting the expression of PPARγ.
9. The use of PPARγ 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 kit also includes other medically acceptable reagents.