Application of mulberry leaf water extract in preventing and treating adverse pregnancy caused by PM2.5

Abstract

The present invention belongs to the field of medical technology and specifically relates to the use of a mulberry leaf aqueous extract in improving adverse pregnancy outcomes caused by PM2.5. The mulberry leaf aqueous extract can effectively improve adverse pregnancy outcomes caused by PM2.5; the mulberry leaf aqueous extract prevents and treats adverse pregnancy outcomes caused by PM2.5 by reducing the biological damage to trophoblast cells caused by PM2.5.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to an application of a mulberry leaf water extract in improving adverse pregnancy outcomes caused by PM2.5. Background Art

[0002] Particulate matter (PM) is one of the most widespread and harmful air pollutants in the air. It is composed of a mixture of airborne particulate matter and various chemicals (such as organic chemicals, transition metal oxides, acids, sulfates, etc.). PM2.5 is defined as atmospheric particulate matter with an aerodynamic diameter of less than 2.5μm. PM2.5 has been proven to enter the alveoli through inhalation, deposit on the alveolar walls, and eventually enter the systemic blood circulation, negatively affecting major organs including the lungs, cardiovascular system, immune system, nervous system, and reproductive system. In addition, as more and more studies have found that this respirable particle has multiple adverse effects on humans and animals, the International Agency for Research on Cancer has listed it as a human carcinogen.

[0003] Due to the toxic effects of PM2.5, there is increasing evidence that exposure to PM2.5 during pregnancy can lead to various adverse pregnancy outcomes, such as placental dysfunction, ovarian dysfunction, impaired fetal development, reduced birth weight, premature birth, reduced gestational age, congenital disabilities, and stillbirth. Exposure to PM2.5 during pregnancy can also have adverse effects on the health of offspring, such as cardiovascular disease, respiratory damage, and neurodevelopmental disorders. Previous studies have found that urban PM2.5 affects placental function, such as placental thrombosis, chorioamnionitis, trophoblast formation and invasion deficiency, and adversely affects fetal growth by interfering with maternal-fetal interaction. PM2.5 exposure during pregnancy can also cause abnormal changes in the DNA methylation spectrum of the placental genome, mainly concentrated in genes related to reproductive development, immune regulation, and material metabolism. In addition, et al. demonstrated that PM2.5 can cause placental trophoblast cell toxicity, impaired hormone regulation, inflammation, and oxidative stress.

[0004] Currently, research on the treatment of PM2.5-induced cell or tissue damage is still in its infancy. Studies have shown that rosiglitazone inhibits the cytotoxicity of PM2.5 on A549 cells, and that melatonin can prevent PM2.5-induced lung damage by reducing ferroptosis in lung epithelial cells. Several traditional Chinese medicines, such as astragaloside IV, wolfberry polysaccharides, and Dengshi Qingmai Decoction, can protect the lungs and cardiovascular system from PM2.5 damage. However, pregnant women are particularly vulnerable to medication side effects, which may affect fetal development and even cause physical deformities. Therefore, exploring treatments for adverse pregnancy outcomes caused by PM2.5 is crucial.

[0005] Mulberry leaves have the effects of dispelling wind-heat, clearing the lungs and moistening dryness, clearing the liver and improving eyesight. Rich in nutrients such as protein, minerals, and vitamins, as well as numerous bioactive ingredients such as polysaccharides, polyphenols, flavonoids, and alkaloids, mulberry leaves possess significant medicinal value. Mulberry leaves are a food that can be used for both medical and dietary purposes. Moderate consumption by pregnant women not only provides nutritional support but also enhances disease resistance and promotes fetal growth and development. However, whether mulberry leaves can alleviate adverse pregnancy outcomes caused by air pollutants, particularly PM2.5, has yet to be studied. Summary of the Invention

[0006] In response to the above technical problems, the present invention provides an application of a mulberry leaf water extract in preventing and treating adverse pregnancy caused by PM2.5.

[0007] Furthermore, mulberry leaf water extract can effectively improve adverse pregnancy outcomes caused by PM2.5.

[0008] Furthermore, mulberry leaf water extract can effectively improve placental structural damage caused by PM2.5, and the improvement of placental structural damage caused by PM2.5 includes reduced placental vacuolar structure, tighter cell arrangement and improved vascular development.

[0009] Furthermore, mulberry leaf water extract prevents and treats adverse pregnancy outcomes caused by PM2.5 by reducing the damage to the biological functions of trophoblasts caused by PM2.5.

[0010] Furthermore, mulberry leaf water extract can enhance the weakened trophoblast proliferation activity caused by PM2.5.

[0011] Furthermore, mulberry leaf water extract can reduce the apoptosis of trophoblasts caused by PM2.5.

[0012] Furthermore, mulberry leaf water extract can improve the weakened migration ability of trophoblasts caused by PM2.5.

[0013] Furthermore, mulberry leaf water extract can improve the weakened trophoblast invasion ability caused by PM2.5.

[0014] Furthermore, mulberry leaf water extract can improve the weakened tube-forming ability of trophoblasts caused by PM2.5.

[0015] The beneficial effects of the present invention are: providing an application of a mulberry leaf water extract in preventing and treating adverse pregnancy outcomes caused by PM2.5; the mulberry leaf water extract can effectively improve adverse pregnancy outcomes caused by PM2.5; and the mulberry leaf water extract prevents and treats adverse pregnancy outcomes caused by PM2.5 by reducing the biological function damage of trophoblasts caused by PM2.5. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1This is a comparison chart of the number of fetuses, fetal weight, fetal mortality rate and placenta weight after pregnant mice were treated with different experiments;

[0017] Figure 2 HE staining under a microscope to observe the placenta structures with different treatments;

[0018] Figure 3 CCK8 was used to detect the proliferation activity of cells treated with different methods;

[0019] Figure 4 Flow cytometry was used to detect cell apoptosis under different treatments;

[0020] Figure 5 The migration ability of cells treated with different methods was tested by scratch assay;

[0021] Figure 6 The invasion ability of cells with different treatments was tested for invasion assay;

[0022] Figure 7 The tube formation experiment was used to detect the ability of cells treated with different methods to form tubes. DETAILED DESCRIPTION

[0023] 1. Collection and preparation of PM2.5 particles.

[0024] This study used a Dandong Better BTPM-AS1 ambient air particulate sampler and collected PM2.5 particles in the Jinan area. After sampling, a 6 cm x 6 cm area of ​​filter membrane was cut, added to 100 mL of ultrapure water, and ultrasonically vibrated for 120 minutes. The filter was then filtered through eight layers of gauze. The filtrate was freeze-dried overnight at -20°C in a freeze dryer to obtain PM2.5 particles, which were then stored at -80°C in the dark.

[0025] 2. Preparation of mulberry leaf aqueous extract.

[0026] Take 1 g of mulberry leaf powder (passed through No. 3 sieve), weigh accurately, add 20 mL of water, weigh, reflux extract for 1 hour, cool to room temperature, make up the weight loss to 20 mL, and filter to obtain a mulberry leaf water extract with a final concentration of 50 mg / mL.

[0027] 3. Construct a PM2.5 exposure pregnant mouse model.

[0028] Thirty SPF-grade C57 pregnant mice of similar weight were purchased from the Animal Research Center of Shandong University and randomly divided into three groups (10 mice / group): PBS control group, PM2.5 treatment group, and PM2.5 + mulberry leaf aqueous extract group. The average tidal volume of C57 mice was approximately 0.15 mL, the respiratory rate of each mouse was approximately 180 breaths / min, and the total daily air intake was 0.15 × 180 × 60 × 24 = 40500 mL = 0.041 m 3According to reports, the PM2.5 exposure of pregnant women in Jinan in 2020 was about 64μg / m 3 Therefore, under the uncertainty factor of 100, the daily inhalation of PM2.5 during the entire pregnancy period is 0.041m 3 ×64 μg / m 3 =2.624μg, and the total PM2.5 intake during the entire pregnancy is approximately 2.624μg×20×100=5248μg. 5248μg PM2.5 was weighed and dissolved in 60μL PBS buffer to prepare a PM2.5 suspension. Pregnant mice were intratracheally instilled with 20μL of PM2.5 suspension on gestational days 1.5d, 7.5d, and 12.5d, respectively, and the PBS control group was intratracheally instilled with an equal amount of PBS (n=10 per group). Referring to the literature, the PM2.5+mulberry leaf water extract group was gavaged daily with 10μL of 50mg / mL mulberry leaf water extract starting on gestational day 1. All mice were euthanized on gestational day 17.5d.

[0029] The results are as follows Figure 1 As shown in Figures A and B, pregnant mice exposed to PM2.5 pulmonary gavage experienced severe adverse pregnancy outcomes, including a decrease in fetal number and weight, increased fetal mortality, and reduced placental weight. Oral gavage with mulberry leaf extract effectively reversed the PM2.5-induced adverse pregnancy outcomes, with an increase in fetal number and weight, a decrease in fetal mortality, and an increase in placental weight.

[0030] Through animal experiments, we can clearly see that mulberry leaf water extract can effectively improve the adverse pregnancy outcomes of pregnant mice caused by PM2.5.

[0031] 4. HE staining of placental tissue.

[0032] The placenta is a vital organ for the exchange of substances between the fetus and the mother. During pregnancy, it performs crucial functions such as substance exchange, immune defense, and metabolic regulation. Numerous studies have shown that structural damage to the placenta can lead to adverse pregnancy outcomes such as fetal growth restriction, birth defects, and premature birth. To investigate changes in placental structure in this example, HE staining was used to observe the placental structure.

[0033] Sections were dehydrated in a gradient of xylene and ethanol, then stained with hematoxylin for 3-8 minutes. After rinsing with running water, sections were differentiated with 1% hydrochloric acid and alcohol for 1 minute, followed by blueing with 0.6% ammonia solution. Sections were stained with eosin solution for 2 minutes, then dehydrated in a gradient of xylene and ethanol, air-dried, and mounted with neutral gum. Sections were observed under a microscope, and representative images were taken.

[0034] like Figure 2As shown, PM2.5 caused significant damage to the structure of the mouse placenta, with numerous vacuoles, loosely arranged cells, poor placental vascular development, and a significant reduction in the effective vascular area. This suggests that PM2.5 reduces the area of ​​the placenta responsible for maternal-fetal exchange, a key factor in PM2.5-related adverse pregnancy outcomes. Treatment with mulberry leaf aqueous extract in pregnant mice showed significant improvement in placental damage, with fewer vacuoles, more tightly packed cells, and significantly improved vascular development compared to the PM2.5 group.

[0035] This shows that mulberry leaf water extract can effectively improve placental structural damage caused by PM2.5.

[0036] 5. Cell culture.

[0037] Placental trophoblast is the most important functional cell in placental tissue, anchored on the surface of uterine decidua in the early stage of placenta formation, on the one hand, by migration and invasion ability, placenta is implanted on the endometrium, on the other hand, after invading into the maternal spiral artery, by forming tubular lumen, complete the recasting of uterine spiral artery, provide maternal blood for growing fetus. The normal proliferation, apoptosis, migration, invasion and tube-forming biological function of trophoblast are the necessary conditions for normal growth of placenta and fetus. If abnormal, self-tapping spiral recasting abnormality and placental blood flow deficiency will be caused, resulting in the generation of multiple adverse pregnancy outcomes. In order to explore the effect of mulberry leaf water extract, the present invention selects commonly used placental trophoblast cell line JEG-3, and utilizes CCK8 experimental detection cell proliferation activity, utilizes flow cytometer to detect cell apoptosis situation, utilizes scratch test to detect cell migration ability, utilizes Transwell experimental detection cell invasion ability, utilizes tube-forming test to detect cell angiogenesis ability.

[0038] Human trophoblast cell line JEG-3 was cultured in 1640 medium (10% fetal bovine serum and 1% penicillin and streptomycin). The cells were divided into the following groups: control group, mulberry leaf aqueous extract group, PM2.5 treatment group, and PM2.5 + mulberry leaf aqueous extract group. The PM2.5 treatment group was treated with 100 μg / mL PM2.5; the PM2.5 + mulberry leaf aqueous extract group was pre-treated with 1 mg / mL mulberry leaf aqueous extract for 1 hour before the addition of 100 μg / mL PM2.5; the control group was treated with the same amount of PBS solution. Various cell biological functions were measured after 24 hours of treatment.

[0039] 6. CCK8 was used to detect cell proliferation activity.

[0040] JEG-3 cells were seeded in 96-well plates, with 8,000 cells per well. After overnight culture, PM2.5 and mulberry leaf aqueous extract were added. 24 hours later, 10% CCK8 solution was added to each well, and the absorbance at 450 nm was measured using a microplate reader.

[0041] like Figure 3 As shown in the results, PM2.5 caused a significant decrease in the proliferation activity of JEG-3 cells. Mulberry leaf water extract itself had no effect on cell proliferation activity, but it could effectively improve the cell proliferation activity that was reduced by PM2.5. This experiment proves that mulberry leaf water extract can effectively improve the cell proliferation activity that was reduced by PM2.5.

[0042] 7. Detect cell apoptosis by flow cytometry.

[0043] The Annexin V-FITC / PI apoptosis kit (Shanghai Biotech Co., Ltd.) was used to prepare cell suspensions, with approximately 6 × 10 4 5 μL of Annexin V-FITC and PI were added to 100 μL of cells, respectively, and 400 μL of buffer was added after staining in the dark at room temperature for 15 min. Cell apoptosis was detected by flow cytometry.

[0044] like Figure 4 As shown in the results, PM2.5 caused a significant increase in the apoptosis level of JEG-3 cells. Mulberry leaf water extract itself did not affect cell apoptosis, but after adding mulberry leaf water extract, PM2.5-induced cell apoptosis was reduced. This experiment proves that mulberry leaf water extract can reduce PM2.5-induced trophoblast apoptosis.

[0045] 8. Scratch test to detect cell migration ability.

[0046] The cells to be tested were seeded into six-well plates (1×10 5 After adherent growth, the bottom of the well plate was gently scratched with a pipette tip, and a clear gap was visible under a microscope. After treatment according to different experimental requirements, the wells were incubated at 37°C for 24 hours, observed and photographed under a microscope, and the cell migration area was calculated using ImageJ.

[0047] like Figure 5 As shown, scratch tests clearly show that PM2.5 causes a decrease in the migration area of ​​JEG-3 cells, while mulberry leaf extract has no effect on cell migration. Adding mulberry leaf extract significantly reverses the reduction in cell migration area caused by PM2.5. This experiment demonstrates that mulberry leaf extract can enhance the weakened migration ability of trophoblast cells caused by PM2.5.

[0048] 9. Invasion assay was used to detect cell invasion ability.

[0049] A Transwell insert (8 μm pores, Corning, USA) was placed in a 24-well plate. 60 μL (1 mg / mL) of Matrigel matrix (Corning, USA) was added to the upper chamber. The Matrigel was allowed to solidify at 37°C for 1 hour. 1×10 5Cells were suspended in 100 μL of serum-free culture medium and added to the upper chamber. Then, 600 μL of complete culture medium supplemented with 10% fetal bovine serum was added to the lower chamber. After incubation at 37°C for 12 hours, the cells and matrix were gently scraped from the chamber using a cotton swab. Each chamber was then fixed with 4% paraformaldehyde, washed, and stained with 1% crystal violet for 30 minutes. Cells invading the bottom of the chamber were observed microscopically, and representative images were taken.

[0050] like Figure 6 As shown, the Transwell assay clearly shows that PM2.5 reduces the invasive ability of JEG-3 cells. Mulberry leaf water extract itself does not affect cell invasiveness, but it significantly improves the weakened invasiveness of JEG-3 cells induced by PM2.5. This experiment demonstrates that mulberry leaf water extract can improve the weakened invasiveness of trophoblast cells induced by PM2.5.

[0051] 10. Tube formation experiment detects the ability of cells to form tube lumens.

[0052] Add 50 μL of Matrigel to a 96-well plate and incubate at 37°C for 1 hour. After the Matrigel solidifies, seed the plates with cells from the different treatment groups, at a rate of 30,000 cells per well. After incubation at 37°C for 4 hours, photograph the plates under a microscope to observe the formation of tubes and capture representative images.

[0053] like Figure 7 As shown in the results, PM2.5 significantly reduced the tube-forming ability of JEG-3 cells. Mulberry leaf water extract itself did not affect the tube-forming ability of cells, but it could improve the reduced tube-forming ability caused by PM2.5. This experiment proves that mulberry leaf water extract can improve the weakened tube-forming ability of cells caused by PM2.5.

[0054] 11. Statistical analysis was performed using GraphPad Prism 8.0 software. All experiments were repeated at least three times. Comparisons between two groups were performed using the independent sample t-test. Comparisons between multiple groups were performed using one-way analysis of variance. Pairwise comparisons were performed using the least significant difference t-test. P < 0.05 was considered statistically significant.

[0055] In summary, PM2.5 reduced the proliferation, migration, invasion, and tube formation abilities of JEG-3 cells and increased apoptosis. Mulberry leaf extract itself had no effect on the biological functions of JEG-3 cells, but it was able to reverse the PM2.5-induced damage to cell biological functions.

Claims

1. The application of mulberry leaf water extract as the sole active ingredient in the preparation of drugs for preventing and treating adverse pregnancy caused by PM2.

5.

2. Use of the mulberry leaf water extract as claimed in claim 1 as the sole active ingredient in the preparation of a drug for preventing and treating adverse pregnancy caused by PM2.5, characterized in that: Mulberry leaf water extract improves adverse pregnancy outcomes caused by PM2.

5.

3. Use of the mulberry leaf water extract as claimed in claim 1 as the sole active ingredient in the preparation of a drug for preventing and treating adverse pregnancy caused by PM2.5, characterized in that: Mulberry leaf water extract improves placental structural damage caused by PM2.5, and the improvement of placental structural damage caused by PM2.5 includes reduced placental vacuolar structure, tighter cell arrangement and improved vascular development.

4. Use of the mulberry leaf water extract as claimed in claim 1 as the sole active ingredient in the preparation of a drug for preventing and treating adverse pregnancy caused by PM2.5, characterized in that: Mulberry leaf water extract prevents and treats adverse pregnancy outcomes caused by PM2.5 by reducing the damage to the biological functions of trophoblasts caused by PM2.

5.

5. Use of the mulberry leaf water extract as claimed in claim 4 as the sole active ingredient in the preparation of a drug for preventing and treating adverse pregnancy caused by PM2.5, characterized in that: Mulberry leaf water extract increases the weakening of trophoblast proliferation activity caused by PM2.

5.

6. Use of the mulberry leaf water extract as claimed in claim 4 as the sole active ingredient in the preparation of a drug for preventing and treating adverse pregnancy caused by PM2.5, characterized in that: Mulberry leaf water extract reduces PM2.5-induced trophoblast apoptosis.

7. Use of the mulberry leaf water extract as claimed in claim 4 as the sole active ingredient in the preparation of a drug for preventing and treating adverse pregnancy caused by PM2.5, characterized in that: Mulberry leaf water extract enhances the weakened migration ability of trophoblast cells induced by PM2.

5.

8. Use of the mulberry leaf water extract as claimed in claim 4 as the sole active ingredient in the preparation of a drug for preventing and treating adverse pregnancy caused by PM2.5, characterized in that: Mulberry leaf water extract can improve the weakened trophoblast invasion ability caused by PM2.

5.

9. Use of the mulberry leaf aqueous extract as claimed in claim 4 as the sole active ingredient in the preparation of a drug for preventing and treating adverse pregnancy caused by PM2.5, characterized in that: Mulberry leaf water extract increases the weakening of trophoblast tube-forming ability caused by PM2.5.

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