Application of pterostilbene in preparation of medicine for treating mouse ovarian injury caused by aflatoxin

Abstract

The invention relates to the technical field of biological medicines, and discloses application of pterostilbene in preparation of a medicine for treating mouse ovarian injury caused by aflatoxin. The invention not only discloses that pterostilbene is used as a novel target spot for treating the ovarian injury caused by the AFB1, but also clarifies the action effect of the pterostilbene in treating the ovarian injury caused by the AFB1, and has important theoretical significance and application value.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to use of pterostilbene in preparing a medicine for treating ovarian damage in mice caused by aflatoxin. Background Art

[0002] Mycotoxins are important environmental pathogenic factors for intestinal and ovarian diseases in lactating mammals. Mycotoxins can not only directly damage the ovaries, but also affect ovarian gene expression and seriously affect ovarian health. AFB1, as a type of mycotoxin, is seriously harmful to mammals. AFB1 is considered to be one of the most dangerous mycotoxins to human and animal health because it is highly carcinogenic and mutagenic. AFB1 is the most toxic mycotoxin, with reproductive toxicity, hepatotoxicity, kidney toxicity, immunotoxicity, etc., which seriously endangers the reproductive function of livestock and poultry.

[0003] In recent years, pan-apoptosis has attracted much attention as a unique programmed inflammatory cell death mode. Cell death can be executed through different subprograms. Pan-apoptosis (PANoptos is) is a unique programmed inflammatory cell death mode that combines the main features of pyroptosis, apoptosis, and necrosis, and can be mediated by a multi-protein pan-apoptotic complex assembled by integrating components of other cell death modes. Increasing evidence shows that pan-apoptosis occurs in response to a variety of stimuli such as viral or bacterial infection, cytokine storm, and cancer. Almost all cases of pan-apoptosis involve the assembly of a complex multi-protein complex (PANoptosome), which is essential for initiating cell death and sensing PAMPs, DAMPs, or other risk factors.

[0004] PTE is a natural polyphenol compound derived from plants such as rosewood, blueberry, grape and palm tree, and has a wide range of pharmacological effects, such as anti-cancer, anti-inflammatory, anti-diabetic, antioxidant and cardiovascular protection. PTE is a methylated derivative of resveratrol, and its bioavailability and half-life are several times that of resveratrol. In addition, PTE can also reduce oxidative stress and inflammation caused by particulate matter in keratinocytes. PTE can alleviate hydrogen peroxide-induced oxidative stress in preimplantation mouse embryos through the Nrf2 signaling pathway. PTE may regulate pan-apoptosis-related proteins through its own excellent properties such as antioxidant and anti-inflammatory effects, thereby alleviating AFB1-induced ovarian toxicity. Summary of the invention

[0005] The purpose of the present invention is to solve the problems existing in the prior art and to propose the use of pterostilbene in preparing a medicine for treating ovarian damage in mice caused by aflatoxin.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] Use of pterostilbene in preparing a medicine for treating ovarian damage in mice caused by aflatoxin.

[0008] Preferably, the drug is a preparation prepared by using pterostilbene as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0009] The term "pharmaceutically acceptable" refers to a carrier, vehicle, diluent, excipient, and / or the salt or ester formed therefrom that is generally chemically or physically compatible with other ingredients constituting a drug dosage form and physiologically compatible with the receptor.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The present invention not only reveals that pterostilbene is a novel target for treating ovarian damage caused by AFB1, but also clarifies the effect of pterostilbene in treating ovarian damage caused by AFB1, which has important theoretical significance and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The following is the expression of mRNA of the pan-apoptotic markers in the ovaries of each group of mice in Example 1.

[0013] Figure 2 This is the HE staining of the ovaries of each group of mice in Example 2.

[0014] Figure 3 The figure shows the changes in sex hormones of mice in different groups in Example 3. DETAILED DESCRIPTION

[0015] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0016] In order to verify the effect of pterostilbene (PTE) in the preparation of drugs for treating ovarian damage in mice caused by aflatoxin B1 (AFB1), the following experiments were conducted:

[0017] Example 1: Verification of the effect of PTE on the level of ovarian pan-apoptosis induced by AFB1

[0018] 1. Construction of mouse ovarian injury model

[0019] Forty 6-8 week old mice were selected and randomly divided into 4 groups after one week of adaptive feeding, each with 10 mice, including blank control group (Control), pterostilbene control group (PTE), model group (AFB1), and rescue group (AFB1+PTE). Mice in the AFB1 modeling group and PTE rescue group were intraperitoneally injected with 0.75 mg / kg AFB1 solution for 14 consecutive days to induce ovarian damage in mice. Mice in the AZT rescue group were also intraperitoneally administered with 50 mg / kg PTE for 14 consecutive days. On the 15th day of the experiment, the mice were killed by cervical dislocation and the mouse ovaries were taken for subsequent experiments.

[0020] 2. Real-time fluorescence quantitative PCR detection of the expression of genes related to apoptosis, inflammation, and oxidative stress in the ovary

[0021] The total RNA of mouse colon tissue was extracted using the TR I zo l method, and then the extracted total RNA was reverse transcribed into cDNA for future use. Real-time fluorescence quantitative PCR (qPCR) primers were designed for the gene sequences of pan-apoptotic markers such as GSDME, AIM2, MLKL, RIPK1, caspase-3, and CFLAR (Table 1), and the expression of these genes in mouse ovaries was detected by qPCR. Figure 1 As shown, the mRNA expression levels of pan-apoptosis markers in the AFB1 group were significantly upregulated, indicating that the pan-apoptosis level expression in the ovarian tissue of mice with AFB1-induced ovarian damage was significantly upregulated, while the relevant indicators of the AFB1+PTE group were significantly lower than those of the AFB1 group, and ovarian damage was alleviated.

[0022] Table 1 qPCR primer sequences

[0023]

[0024]

[0025] Example 2: Verification of the effect of PTE on AFB1-induced pathological manifestations

[0026] 1. Construction of mouse ovarian injury model, the construction method is the same as that in Example 1;

[0027] 2. Evaluation of Ovarian Pathological Manifestations in Mice

[0028] 1. Effect of PTE on ovarian histopathology in mice with ovarian injury

[0029] The paraffin sections of mouse colon tissue were prepared and stained with hematoxylin and eosin (HE) according to the following steps.

[0030] 1) The method of dehydration and paraffin embedding of mouse colon tissue is as described in Example 1;

[0031] 2) Paraffin sections of mouse colon tissue were cut into 4 μm sections and baked in a 60°C oven for 1 hour;

[0032] 3) Dewax the sections in xylene for 10 minutes and dehydrate them in 100%-95%-90%-80%-70%-50% graded alcohol for 3 minutes each;

[0033] 4) Place the slices in double distilled water and let stand for 5 minutes, repeat once;

[0034] 5) Immerse the sections in hematoxylin stain and let stand for 5 minutes;

[0035] 6) Rinse the sections with tap water to remove unattached dye;

[0036] 7) Immerse the slices in hydrochloric acid ethanol solution for 2 seconds;

[0037] 8) Immerse the slices in ammonia water for 3 seconds;

[0038] 9) Soak the sections in eosin stain for 3 minutes;

[0039] 10) Rinse the slices with tap water to remove unattached dyes.

[0040] 11) Soak the sections in 75%-80%-90%-96%-100% ethanol for 5 minutes and in xylene for 10 minutes;

[0041] 12) Add a drop of neutral resin to the transparent slice and cover it with a coverslip to seal.

[0042] like Figure 2 The HE staining results show that the ovarian follicles of the control group and PTE group mice developed normally. Compared with the mice in other groups, the ovarian follicles of the AFB1 group mice were dysplastic, follicular atresia, large follicles and corpora lutea coexisted, and the number of corpora lutea increased.

[0043] Example 3: Verification of the effect of PTE on the expression of sex hormones in ovarian tissue of mice with ovarian damage

[0044] 1. Construction of mouse ovarian injury model, the construction method is the same as that in Example 1;

[0045] 2. Evaluation of Ovarian Sex Hormone Expression in Mice:

[0046] 1. Detection of inflammatory factors in mouse serum by enzyme-linked immunosorbent assay

[0047] Mouse E2 ELISA Kit (Ruixin biotech, RXJ203008M) and mouse AMH ELISA Kit (Ruixinbiotech, RXJ203291M) were used to separate mouse serum and detect the content of sex hormone E2 and AMH therein according to the following steps:

[0048] 1) Collect blood samples from mice in each group, wait for blood samples to agglutinate for 30 minutes, centrifuge at 1000 g for 10 minutes, and draw serum samples from the upper layer;

[0049] 2) Add 1X washing solution (300 μl / well) to soak the ELISA plate, let it stand for 30 seconds, then discard the liquid in the well and pat the ELISA plate dry;

[0050] 3) Add 100 μl / well of each gradient standard and serum sample and incubate at room temperature for 2 hours;

[0051] 4) Discard the liquid in the wells, add 1X washing solution (300 μl / well) to wash the plate three times, and pat the ELISA plate dry;

[0052] 5) Add the detection antibody to the ELISA plate at 100 μl / well and incubate at room temperature for 2 hours;

[0053] 6) Discard the liquid in the wells, add 1X washing solution (300 μl / well) to wash the plate three times, and pat the ELISA plate dry;

[0054] 7) Add the enzyme conjugate to the ELISA plate at 100 μl / well and incubate at room temperature for 20 minutes;

[0055] 8) Discard the liquid in the wells, add 1X washing solution (300 μl / well) to wash the plate three times, and pat the ELISA plate dry;

[0056] 9) 10 minutes before use, return the substrate solution to room temperature, add the substrate solution to the ELISA plate, 100 μl / well, and incubate at room temperature in the dark for 20 minutes;

[0057] 10) Add 50 μl / well stop solution to the ELISA plate and gently shake the plate until the color is evenly developed;

[0058] 11) Read the absorbance at 450 nm within 10 minutes.

[0059] like Figure 3 As shown in the figure, the levels of E2 and AMH in the serum of the AFB1 group were significantly lower than those in the other groups. After treatment with PTE, the levels of E2 and AMH in the serum of the AFB1+PTE group were effectively relieved compared with the AFB1 group.

[0060] In summary, the present invention illustrates that AFB1 induces ovarian damage in mice by activating pan-apoptosis, and pterostilbene regulates pan-apoptosis-related proteins, especially the expression of RIPK1-PANoptosome, by acting on antioxidant and anti-inflammatory effects, thereby effectively alleviating AFB1-induced ovarian toxicity, indicating that pterostilbene has important research and application prospects in the fields of food safety and reproductive health. This invention not only reveals a new target for treating ovarian damage caused by AFB1, but also illustrates the effect of pterostilbene in treating ovarian damage caused by AFB1, which has important theoretical significance and application value.

[0061] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. Use of pterostilbene in the preparation of a drug for treating ovarian damage in mice caused by aflatoxin.

2. The use of pterostilbene according to claim 1 in the preparation of a drug for treating ovarian damage in mice caused by aflatoxin, characterized in that: The medicine is a preparation prepared by taking pterostilbene as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

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