CN111407883B - New use of IFN-lambda 3 in toxoplasma gondii infection - Google Patents

Abstract

The invention belongs to the technical field of biomedical treatment, and relates to a novel application of IFN-lambda 3 in toxoplasma gondii infection. The invention discloses an application of IFN-lambda 3 in preparing a medicament for treating or preventing toxoplasma gondii infection. The invention also discloses a composition for preventing or treating toxoplasma gondii infection, which is characterized in that the active ingredient of the composition is IFN-lambda 3. The invention also provides a use of a composition for the preparation of a medicament for the prophylaxis or treatment of toxoplasma gondii infection, wherein the composition comprises IFN-lambda 3 and one or more pharmaceutically acceptable carriers. The invention provides a new way and a new method for treating toxoplasma gondii infection, and solves the problem that bad pregnancy caused by toxoplasma gondii infection cannot be treated in the prior art.

Description

Novel uses of IFN-λ3 in Toxoplasma gondii infection

Technical field

The invention belongs to the field of biomedical technology and relates to the new use of IFN-λ3 in Toxoplasma gondii infection. Specifically, it relates to the application of IFN-λ3 in the preparation of medicines for treating or preventing Toxoplasma gondii infection, a method for preventing or treating Toxoplasma gondii infection. A composition for Toxoplasma gondii infection, and the use of a composition in preparing a medicament for preventing or treating Toxoplasma gondii infection.

Background technique

Toxoplasma gondii is an important opportunistic pathogenic protozoa. Individuals with normal immunity who are infected with Toxoplasma gondii often have a latent infection state. For patients with impaired immune function or immunodeficiency, such as patients with AIDS, organ transplantation, and malignant tumors, Toxoplasma infection is an important cause of death. Infection in pregnant women can affect the development of the fetus, leading to miscarriage, fetal abnormalities, stillbirth, premature delivery, birth defects and other congenital toxoplasmosis. In recent years, with the rapid increase in urban population, the growing number of pets (especially cats), and people's preference for eating wild animals and other bad eating habits, more and more people are exposed to the risk of Toxoplasma gondii infection. According to statistics, about 1 billion people around the world are infected with Toxoplasma gondii, of which the infection rate among pregnant women is 10% to 27.5%. Every year, about 90,000 newborns are threatened by Toxoplasma gondii infection. As a country with a large population, how to effectively prevent and treat congenital toxoplasmosis is a huge challenge for China.

Interferon (Interferon, IFN) is mainly divided into three types: type I interferon (IFN-α and IFN-β), type II interferon (IFN-γ) and type III interferon (IFN-λ). Type I IFN (IFN-α and IFN-β) is mainly antiviral and induces the expression of multiple interferon-stimulated genes (ISGs) through related signaling pathways, thereby exerting antiviral effects. Type II interferon (IFN-γ) is an important cytokine that inhibits the proliferation of Toxoplasma gondii. After Toxoplasma gondii tachyzoites invade the body, they stimulate macrophages to produce IL-12, which in turn activates NK cells and T cells to secrete IFN-γ. IFN-γ then induces the expression of IFN-γ-inducible genes, which then directly kills the tachyzoites parasitizing the host cells. However, during pregnancy, the production and excessive secretion of IFN-γ are the main factors leading to adverse pregnancy. IFN-γ induces miscarriage by recruiting CD49b ⁺ NK and regulating the expression of Ly-49 receptor on NK cells. In the rat abortion model, IFN-γ up-regulates tumornecrosis factor-α (TNF-α) and down-regulates the expression of Matrix metalloproteinases-2 and-9 (MMP-2 and MMP-9), further aggravating abortion. In human pregnancy, the role of IFN-γ in adverse pregnancy has also been confirmed. Fetal-derived T cells promote uterine contraction by secreting IFN-γ and TNF-α, thus leading to premature birth. Therefore, IFN-γ cannot protect the mother against Toxoplasma infection during pregnancy and maintain normal pregnancy.

IFN-λ (type III interferon) has a relatively independent and specific ability to resist pathogenic infections. The IFN-λ receptor is composed of interleukin-28 receptor alpha (IL-28Rα) and interleukin-10 receptor beta (IL-10Rβ). IL-10Rβ is widely present in cells and tissues. , IL-28Rα is mainly expressed on the surface of epithelial cells, neutrophils and hepatocytes. The limitation of receptor expression means that IFN-λ plays a relatively independent and specific anti-pathogen effect in certain tissues. The human IFN-λ family consists of IFN-λ1, IFN-λ2, IFN-λ3, and IFN-λ4, while mice have only two functional IFN-λs, IFN-λ2 and IFN-λ3. In the study of rotavirus, researchers found that IFN-λ2 can significantly inhibit rotavirus replication in the intestines of young mice, and IFN-λ receptor knockout promotes rotavirus proliferation in mice. Researchers such as Muir first proposed that IFN-λ1a can inhibit the proliferation of hepatitis C virus (HCV). IFN-λ1a can quickly inhibit HCV replication (within 12 hours) and achieve a similar effect to IFN-α within 24 hours. However, the proportion of patients experiencing complications such as thrombocytopenia and neutropenia has significantly decreased. Therefore, IFN-λ1a is expected to replace IFN-α in the clinical treatment of HCV. Rebecca L. et al. used Aspergillus fumigatus (Af) as a model to study antifungal immune responses and found that depletion of CCR2 ⁺ monocytes reduced the ability of neutrophils to inhibit the growth of invasive fungi. IFN-λ2/3 directly acts on neutrophils to activate their antifungal response, and mice with neutrophil-specific deletion of IFN-λ receptors succumb to invasive aspergillosis. Neutrophil CCR2-depleted mice can be effectively treated by adoptive transfer of CCR2 ⁺ monocytes or IFN-λ2/3. Therefore, IFN-λ2/3 is a key regulator of neutrophils and exerts antifungal effects. Cryptosporidium parvum (C.parvum), as an important opportunistic pathogenic protozoa, causes clinical manifestations mainly including diarrhea. Exogenous IFN-λ3 can reduce the insect load in intestinal epithelial cells (Intestinal Epithelial Cells, IECs), restore transepithelial electrical resistance (TEER), and resist C. parvum invasion.

Although a large amount of experimental data supports the ability of IFN-λ2/3 to inhibit the proliferation of viruses, fungi and Cryptosporidium parvum, whether IFN-λ3 can inhibit the proliferation of Toxoplasma needs further study.

Contents of the invention

The object of the present invention is to provide the application of IFN-λ3 in the preparation of medicines for the treatment or prevention of Toxoplasma gondii infection, a composition for the prevention or treatment of Toxoplasma gondii infection, and the use of IFN-λ3 in the preparation of medicines for the prevention or treatment of Toxoplasma gondii infection. Application of drugs for Toxoplasma gondii infection to solve problems in the existing technology.

In view of the above-mentioned purpose of the invention, embodiments of the present invention provide the application of IFN-λ3 in the preparation of drugs for treating or preventing Toxoplasma gondii infection.

Furthermore, the IFN-λ3 intervention up-regulated the expression of CK functional molecules in the placenta and inhibited the proliferation of Toxoplasma gondii.

An embodiment of the present invention also provides a composition for preventing or treating Toxoplasma gondii infection, characterized in that the active ingredient of the composition is IFN-λ3.

Further, the composition includes IFN-λ3 and one or more pharmaceutically acceptable carriers.

Wherein, the carrier includes pharmaceutically acceptable diluents, excipients, fillers, adhesives, absorption promoters, surfactants and synergists.

Embodiments of the present invention further provide the use of a composition in preparing a medicament for preventing or treating Toxoplasma gondii infection, characterized in that the composition includes IFN-λ3 and one or more pharmaceutically acceptable accepted carrier.

Further, the carrier includes pharmaceutically acceptable diluents, excipients, fillers, adhesives, absorption promoters, surfactants and synergists.

The beneficial effects of the above technical solutions of the present invention are as follows:

(1) Embodiments of the present invention prepare multiple groups of pregnant mouse models, and experiments on multiple groups of pregnant mouse models prove that IFN-λ3 intervention can significantly reduce the abortion rate caused by Toxoplasma gondii infection and alleviate the damage to the mouse placenta caused by Toxoplasma gondii infection. Damage and up-regulate the expression of functional molecules such as CK in the placenta; at the same time, the present invention detects the expression of Toxoplasma SAG1 in each cell culture model group by PCR after co-culturing Toxoplasma gondii and JEG-3 cells, verifying that IFN-λ3 intervention can inhibit gondii, thus the embodiments of the present invention verify that IFN-λ3 intervention can treat adverse pregnancy caused by Toxoplasma gondii and inhibit the proliferation of Toxoplasma gondii, which provides a theoretical basis for the treatment of Toxoplasma gondii infection.

(2) The present invention provides the application of IFN-λ3 in inhibiting Toxoplasma gondii infection, a drug for preventing or treating Toxoplasma gondii infection, providing new ways and methods for the treatment of Toxoplasma gondii infection, and solving current problems. The problem of adverse pregnancy caused by Toxoplasma gondii infection cannot be treated with existing technology.

Description of the drawings

Figure 1 is a diagram of the miscarriage status of the pregnant rats in the uninfected Toxoplasma gondii group, the pregnant rats infected with Toxoplasma gondii group, and the Toxoplasma gondii infected group after the pregnant rats were pretreated with IFN-λ3 for 24 hours in Example 1 of the present invention. ;

Figure 2 shows the placenta of mice in Example 2 of the present invention in the group of pregnant mice not infected with Toxoplasma gondii, the group of pregnant mice infected with Toxoplasma gondii, and the group of pregnant mice infected with Toxoplasma gondii after being pretreated with IFN-λ3 for 24 hours. Damage diagram;

Figure 3 shows the placental CK of the pregnant rats in the uninfected Toxoplasma gondii group, the pregnant rats infected with Toxoplasma gondii group, and the pregnant rats in the Toxoplasma gondii infected group after pretreatment with IFN-λ3 for 24 hours in Example 3 of the present invention. Expression situation diagram;

Figure 4 shows the co-culture of Toxoplasma gondii and JEG-3 cells in Example 4 of the present invention. The cells are not infected with Toxoplasma gondii, the cells are infected with Toxoplasma gondii, and the cells are co-stimulated with Toxoplasma gondii and IFN-λ3. Group, cells were pretreated with IFN-λ for 324h, and then infected with Toxoplasma gondii group, cells were treated with Toxoplasma gondii and IFN-γ, cells were pretreated with IFN-γ for 24h, and then infected with Toxoplasma gondii group SAG1 expression diagram.

Detailed ways

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to specific embodiments.

Experimental Materials

1.1. JEG-3 cell line was purchased from Thermo Fisher Scientific.

1.2. Trizol reagent was purchased from Invitrogen Company of the United States.

1.3. Reverse transcription reagents were purchased from Bio-Rad Company of the United States.

1.4. SYBR Green Master Mix and TaqMan Universal Master Mix were purchased from ThermoFisher Scientific Company of the United States.

1.5. The primers were synthesized by Sigma in the United States.

1.6. Fetal bovine serum was purchased from Hyclone Company.

1.7. Trypsin and MEM were purchased from Thermo Fisher Scientific Company in the United States.

Preparing pregnant mice for experiments

Divide at least 24 male rats and 48 female rats into 24 groups. Each group includes 2 female rats and 1 male rat. The 2 female rats and 1 male rat will be caged together at 5 pm the night before. Detect the white vaginal suppository at 7 o'clock every day. If vaginal plug is detected, the pregnancy period of the mouse is determined to be E0.5 (embryonic day 0.5).

24 groups of pregnant mice were divided into different groups and model groups were prepared. The specific groups are as follows:

(1) Pregnant mice are not infected with Toxoplasma gondii group (normal pregnancy group);

(2) Pregnant rats infected with Toxoplasma gondii group;

(3) Pregnant mice were pretreated with IFN-λ3 for 24 hours and then infected with Toxoplasma gondii group;

Examples 1 to 3 of the present invention Prepare the above three groups of pregnant rats, each group has at least four pregnant rats (the pregnancy period of each pregnant rat is the same), and conduct the experiments of the following Examples 1 to 3.

Example 1. IFN-λ3 intervention can significantly reduce the abortion rate caused by Toxoplasma gondii infection

Pregnant mice were injected with IFN-λ3 (2 μg/ml) on the 9.5th day of pregnancy (E9.5). On the E10.5 day, the pregnant mice were infected with Toxoplasma gondii. Subsequently, pregnant mice were injected with IFN-λ3 every day until 18.5 days of gestation. Monitor the changes in the weight of pregnant rats, observe the abortion rate of pregnant rats, the weight and size of fetal rats. When infected with Toxoplasma gondii in the second trimester, the weight of pregnant mice showed a downward trend compared with their own body weight. The mice were killed on day E18.5, and the size of the fetal mice was measured by measuring the length of the top and rump (mm) × the diameter of the occipital and frontal diameter (mm ² ), and the abortion rate was observed at the same time. Collect various tissue samples from mother rats and fetal rats for subsequent experiments.

The results are shown in Figure 1. Compared with the group of pregnant rats not infected with Toxoplasma gondii (normal pregnancy group), Toxoplasma infection in the group of pregnant rats infected with Toxoplasma gondii resulted in an abortion rate of about 58.9%. After IFN-λ3 intervention, the abortion rate of pregnant rats in the group infected with Toxoplasma gondii was significantly reduced after 24 hours of IFN-λ3 pretreatment.

These results suggest that IFN-λ3 can improve adverse pregnancy outcomes caused by Toxoplasma gondii infection.

Example 2. IFN-λ3 can reduce the damage to mouse placenta caused by Toxoplasma gondii infection.

After pregnant mice were infected with Toxoplasma gondii on day E10.5, mouse placentas were collected on day E18.5, fixed in formalin and stained with HE to observe the structural changes of the placenta.

Among them, the method of HE staining is as follows: rinse the slides with tap water for 3 minutes. Rinse with distilled water for 1 minute. Place the slides in pre-cooled 3% Triton X-100 solution at 4°C for permeabilization for 5 minutes. Hematoxylin staining for 10 minutes. Rinse with tap water for 30 seconds. Differentiation solution (2% hydrochloric acid ethanol) for 20 s. Rinse with tap water for 5-10 minutes. Eosin staining for 30s-2min. Dehydrate in gradients of 50%, 70%, 80%, 95% and absolute ethanol for 1-3 minutes. Xylene I and II become transparent for 1-3 minutes. The slides were sealed with neutral resin and images were taken under a microscope.

It can be seen from the HE staining results that the normal structure of the normal mouse placenta is divided into the decidua (DE), junctional zone (JZ) and labyrinth zone (LZ). Compared with the group of pregnant mice not infected with Toxoplasma gondii (normal pregnancy group), the Toxoplasma gondii infection in the group of pregnant mice infected with Toxoplasma gondii destroyed the normal structure of the placenta, which was manifested in the thinning of the decidua layer and the connecting zone, accompanied by the placental The number of cells in the labyrinth area was significantly reduced, as shown in Figure 2, suggesting reduced placental nutrient supply and gas exchange impairment. After IFN-λ3 intervention, pregnant mice were pretreated with IFN-λ3 for 24 hours. In the group infected with Toxoplasma gondii, the thickness of the decidua layer and junction zone was close to that of the normal mouse placenta. At the same time, the number of cells in the labyrinth zone was obvious. Increase.

These results indicate that Toxoplasma gondii infection can destroy the normal structure of the mouse placenta, and IFN-λ3 can significantly reduce placental damage.

Example 3. IFN-λ3 intervention can upregulate the expression of functional molecules such as CK in the placenta.

Cytokeratin (CK) is a marker protein of mouse placental trophoblast cells.

Cell immunofluorescence detection method: Collect mouse placenta tissue and make 6-8 micron sections. When staining, sections were allowed to dry at room temperature for 15 minutes. Then soak in PBS for 10 minutes to remove OCT; permeabilize with 0.5% Triton , place in a 4°C refrigerator and incubate overnight. After washing three times with PBS, drop the diluted fluorescent secondary antibody on the glass slide and incubate at room temperature in the dark for 90 minutes. After washing three times with PBS, Hoechst dye was dropped on the slide and incubated at room temperature in the dark for 15 min. After washing three times with PBS, the slides were mounted with 50% glycerol. Images were taken using confocal laser.

In the normal pregnancy group (pregnant mice not infected with Toxoplasma gondii), a large amount of CK expression was observed in the placenta of mice. However, in the group of pregnant mice infected with Toxoplasma gondii, the number of CK in the mouse placenta decreased significantly, indicating that Toxoplasma can Reduce the number of trophoblast cells in mouse placenta. As shown in Figure 3, pregnant rats were pretreated with IFN-λ3 for 24 hours and then infected with Toxoplasma gondii. That is, after IFN-λ3 intervention, CK expression increased significantly, suggesting that IFN-λ3 can reduce placental damage and improve placental function. .

Example 4. IFN-λ3 intervention inhibits the proliferation of Toxoplasma gondii

In this example, in vitro experiments using cell culture were used to verify that IFN-λ3 intervention inhibits the proliferation of Toxoplasma gondii ; among them, the cell model group used in this example can be divided into the following six groups: Cells are not infected with Toxoplasma gondii Group, cells infected with T. gondii group, cells co-stimulated with T. gondii and IFN-λ3, cells pretreated with IFN-λ3 for 24 hours, infected with T. gondii group, cells with T. gondii and IFN- In the γ co-treated group, the cells were pretreated with IFN-γ for 24 hours and then infected with Toxoplasma gondii.

The method for obtaining the JEG-3 cell strain infected by Toxoplasma gondii is as follows: culture the JEG-3 cell strain in a cell culture flask, and use MEM complete medium containing 10% fetal bovine serum (containing 100 μg/mL streptomycin). and 100 U/mL penicillin), and cultured in a cell culture incubator at 37°C with 5% _CO2 . Change the culture medium of cells every other day, and perform cell passage or conduct subsequent experiments when the cells reach about 80% confluence. 2×10 ⁵ /well JEG-3 cells were plated in a 6-well plate. After culturing in a cell culture incubator for 24 hours, Toxoplasma gondii infected the JEG-3 cell line at a multiplicity of infection (MOI) of 2.

After JEG-3 cells were pretreated with IFN-λ3 and co-cultured with Toxoplasma gondii, the expression of SAG1 was detected by real-time PCR. The method for detecting the number of Toxoplasma gondii is as follows: collect tissues or cells, add an appropriate weight of tissues or cells to Trizol for lysis, extract RNA, use a reverse transcription kit, and reverse transcribe into cDNA with Oligo(dT)18. Store frozen at -80℃. After infecting JEG3 cells with 10-fold dilution of Toxoplasma gondii, the extracted RNA was reverse transcribed into cDNA and used as a template to amplify on a fluorescence quantitative PCR instrument to develop a standard curve. Reaction conditions: 95℃, 3min pre-denaturation; 40 cycles: 95℃, 15s; 60℃, 1min. Corresponding to the standard curve, the calculated relative amount of Toxoplasma gondii SAG1.

After JEG-3 cells were pretreated with IFN-λ3 and co-cultured with Toxoplasma gondii, the expression of SAG1 was detected by real-time PCR. The results are as follows: IFN-γ co-acted with Toxoplasma gondii on JEG-3 cells in vitro. After co-culture for 48 hours, the cell culture supernatant was collected, and the expression of Toxoplasma SAG1 was detected by Real-time PCR. The results showed that the group treated with cells, Toxoplasma gondii and IFN-γ was used as a positive control group, and IFN-γ had the ability to inhibit the proliferation of Toxoplasma gondii. After the cells were pretreated with IFN-γ for 24 hours, the proliferation of Toxoplasma gondii in the Toxoplasma gondii infection group (another positive control group) was inhibited. Similarly, IFN-λ3 can significantly reduce the expression of SAG1 in the group of cells stimulated by Toxoplasma gondii and IFN-λ3. After pretreatment with IFN-λ3, as shown in Figure 4, after cells were pretreated with IFN-λ for 324h, the expression of Toxoplasma SAG1 in the Toxoplasma gondii infection group further decreased.

These results indicate that IFN-λ3 can inhibit the proliferation of Toxoplasma gondii in vitro.

The above is the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications can also be made. should be regarded as the protection scope of the present invention.

Claims (3)

1. Use of ifn- λ3 for the preparation of a medicament for the treatment or prevention of toxoplasma gondii infection, characterized in that: the IFN-lambda 3 intervenes in up-regulating expression of CK functional molecules in placenta and inhibiting proliferation of toxoplasma gondii.
2. 2. Use of a composition comprising IFN- λ3 and one or more pharmaceutically acceptable carriers for the manufacture of a medicament for the prevention or treatment of toxoplasma gondii infection.
3. 3. Use of a composition according to claim 2 for the manufacture of a medicament for the prevention or treatment of toxoplasma gondii infection, wherein the carrier comprises pharmaceutically acceptable diluents, excipients, fillers, binders, absorption enhancing agents, surfactants and synergists.