Application of vitamin D3 in improvement of trophoblast proliferation inhibition

Abstract

The invention discloses application of vitamin D3 in preparation of a product for improving trophoblast proliferation inhibition. The research proves that the vitamin D3 can obviously improve the proliferation inhibition of palmitic acid on human trophoblast cells; the vitamin D3 can reduce palmitic acid induced cell active oxygen level up-regulation; vitamin D3 can up-regulate expression of Nrf-2, HO-1 mRNA and protein in cells induced by palmitic acid. Vitamin D3 can improve palmitic acid induced human trophoblast cell proliferation inhibition by activating an Nrf2 / HO-1 pathway. The invention provides a new product for improving the proliferation inhibition of palmitic acid on human trophoblast cells, and has a good clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of vitamin D3 in improving the inhibition of trophoblast proliferation. Background Art

[0002] Trophoblasts refer to cells with trophoblastic functions, which come from the trophoblast outside the embryo. Trophoblast cells grow rapidly and form many hair-like protrusions on the surface of the embryonic sac, called "villi". Trophoblasts are mainly found in many parts of the human body, such as embryos, placentas, and certain glands. Trophoblasts play an important role in organisms. They are mainly responsible for providing nutrients and oxygen to early embryos to ensure the normal development of embryos. Especially in the placenta, trophoblasts provide the fetus with necessary nutrients and oxygen through communication with maternal blood. In addition, these cells are also involved in maintaining hormonal balance during pregnancy to ensure the smooth progress of pregnancy. Trophoblasts have a high degree of proliferation and differentiation ability. They can meet the specific needs of the embryo or placenta by dividing and differentiating. During the formation of the placenta, trophoblasts undergo a series of morphological and functional changes to adapt to different stages of placental development. These changes ensure the effective function of the placenta, thereby supporting the normal growth of the fetus.

[0003] Overweight and obesity during pregnancy can increase the risk of pregnancy complications. Palmitic acid (PA) is one of the main components of saturated fatty acids in food. Studies have shown that the concentration of palmitic acid in the blood of obese people is significantly increased. Studies have shown that excessive palmitic acid concentration in pregnant women can affect oocyte maturation and fertilized egg implantation, increasing the risk of pregnancy complications in pregnant women, and animal experiments have the same evidence. In addition, in vitro studies have shown that high concentrations of palmitic acid can cause human trophoblasts to produce excessive reactive oxygen species (ROS), causing oxidative stress, damaging trophoblasts, and even affecting fetal development. Reactive oxygen induces increased expression of nuclear factor erythroid 2-related factor 2 (Nrf-2) and its downstream gene heme oxygenase 1 (HO-1). When oxidative stress occurs in the body, Nrf2 expression can be increased to reduce the damage caused by oxidative stress.

[0004] Vitamin D is a fat-soluble vitamin. Active vitamin D3 has antioxidant effects and can regulate cell proliferation. Under oxidative stress, vitamin D3 can increase the expression of Nrf2 protein to alleviate oxidative stress. Nrf2 can further increase HO-1 expression to alleviate oxidative stress. However, there are few reports on vitamin D3 regulating palmitic acid-induced inhibition of human trophoblast proliferation. Summary of the invention

[0005] The purpose of the present invention is to address the above-mentioned problems and provide an application of vitamin D3 in the preparation of a product for improving the inhibition of trophoblast proliferation.

[0006] The trophoblast proliferation inhibition is palmitic acid-induced trophoblast proliferation inhibition.

[0007] Preferably, the above-mentioned application technology scheme is the use of vitamin D3 in the preparation of a product for improving palmitic acid-induced trophoblast proliferation inhibition during pregnancy.

[0008] Preferably, the above-mentioned application technology scheme is the use of vitamin D3 in the preparation of a product for improving palmitic acid-induced trophoblast proliferation inhibition in obese people.

[0009] In the above application technology scheme, vitamin D3 improves the inhibition of trophoblast proliferation by activating the Nrf2 / HO-1 pathway.

[0010] In the above application technology scheme, vitamin D3 improves the oxidative stress and cell proliferation inhibition of trophoblast cells.

[0011] In the above application technology scheme, vitamin D3 increases the expression levels of Nrf2 and HO-1.

[0012] The product described is a drug.

[0013] The medicament further includes a pharmaceutically acceptable carrier.

[0014] The beneficial effects of the present invention are:

[0015] The present invention study confirmed that vitamin D3 can significantly improve the inhibition of palmitic acid on the proliferation of human trophoblast cells (the improvement is about 11.5%; t = 8.79, P = 0.001); vitamin D3 can reduce the increase in the level of cellular reactive oxygen induced by palmitic acid (the reduction is 19.8%; t = 7.84, P < 0.001); vitamin D3 can upregulate the expression of Nrf-2 and HO-1 mRNA and protein in palmitic acid-induced cells. Vitamin D3 may improve the inhibition of human trophoblast cell proliferation induced by palmitic acid by activating the Nrf2 / HO-1 pathway. The present invention provides a new product for improving the inhibition of palmitic acid on the proliferation of human trophoblast cells, and has good prospects for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1The results of detecting the proliferation ability of HTR8 / SVneo cells by CCK-8 and EdU method are shown: (A) Compared with the control group, the change of HTR-8 / SVneo cell proliferation ability showed statistical difference starting from 200μM PA (P<0.05), and was negatively correlated with the concentration of PA; (B) Different concentrations of vitamin D3 had no effect on the proliferation ability of HTR-8 / SVneo cells; (C) Compared with the 400μM PA group, 1μM and 10μM vitamin D3 could improve the inhibitory effect of PA on the proliferation of HTR-8 / SVneo cells ( # P<0.05); (D) EdU staining results showed that compared with the control group, the proliferation ability of HTR-8 / SVneo cells in the 400μM PA group was significantly inhibited (* P<0.05). Compared with the 400μM PA group, 1μM and 10μM vitamin D3 treatments could improve the inhibitory effect of PA on the proliferation of HTR-8 / SVneo cells ( # Ρ<0.05); (E) Figure 1 The fluorescence images of D were statistically analyzed; the data are expressed as mean ± standard deviation (n = 3).

[0017] Figure 2 The results of detecting ROS activity in HTR8 / SVneo cells using a reactive oxygen species assay kit are shown: (A) Compared with the control group, the ROS levels in the 400 μM and 600 μM PA groups were significantly increased, with statistical differences (P<0.05, n=6-8); (B) Different concentrations of vitamin D3 had no effect on ROS generation in HTR-8 / SVneo cells (n=6-7); (C) Compared with the 400 μM PA group, 1 μM vitamin D3 reduced the increase in ROS generation in HTR-8 / SVneo cells caused by PA ( # P<0.05, n=6-8); (D) ROS staining of HTR-8 / SVneo cells showed that 1 μM vitamin D3 could reduce the increase in ROS production in HTR-8 / SVneo cells caused by PA compared with the 400 μM PA group ( # P<0.05, n=3). (E) Figure 2 The fluorescence images of D were subjected to statistical analysis; the data were expressed as mean ± standard deviation.

[0018] Figure 3Figure 2 shows the results of fluorescence quantitative PCR detection of Nrf2, HO-1, NOX4 and NOX5 gene expression: mRNA expression levels of Nrf2 (A), HO1 (B), NOX4 (C) and NOX5 (D) after HTR8 / SVneo cells were treated with 400 μM PA or 400 μM PA+1 μM Vitamin D3 for 24 h; (E) Nrf2 and HO-1 protein band expression after HTR8 / SVneo cells were treated with 400 μM PA or 400 μM PA+1 μM Vitamin D3 for 24 h; (F) Grayscale analysis statistical chart of Nrf2 and HO-1 protein expression, using β-actin as an internal reference; Data are expressed as mean ± SD (*P < 0.05, compared with the control group; # P<0.05, compared with 400 μM PA group, n=3).

[0019] Figure 4 The results showed that silencing the Nrf2 gene could eliminate the improvement effect of vitamin D3 on the inhibition of HTR8 / SVneo cell proliferation induced by palmitic acid: HTR8 / SVneo cells were transfected with si-Nrf2 or control siRNA for 24 hours, and then treated with 1 μM vitamin D3 and 400 μM palmitic acid for 24 hours: (A) Western blot was used to detect the bands and relative protein expression levels of Nrf2 and HO-1 (si-NC group *p<0.05, si-NC+1μMVD3+400μM palmitic acid group #p<0.05, n=3); (B) CCK-8 method was used to determine cell viability ( a,b,c P<0.05, n=3); data are expressed as mean±standard deviation. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0021] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0022] Research method of the embodiment of the present invention: Human trophoblast cells HTR8 / SVneo were cultured in vitro, and the CCK-8 method and EdU staining were used to detect the inhibitory effect of vitamin D3 on HTR8 / SVneo cell proliferation. A reactive oxygen species (ROS) kit was used to detect the effect of vitamin D3 on palmitic acid-induced reactive oxygen species in HTR8 / SVneo cells. Polymerase chain reaction (qPCR) was used to detect the mRNA expression of nuclear factor erythroid2-related factor 2 (Nrf-2), heme oxygenase-1 (HO-1), nitrogen oxide 4 (NOX4) and nitrogen oxide 5 (NOX5) genes. Western Blot was used to detect the expression of Nrf-2 and HO-1 proteins. Lipofectamine was used. TM siRNA-Nrf2 was transfected with 2000 transfection reagent to construct HTR8 / SVneo cells with Nrf2 knockdown, and the transfection efficiency was detected by Western Blot. The negative control group (siRNA-NC group) and transfection group (siRNA-Nrf2 group) cells were treated with 1μM vitamin D3 and 400μM palmitic acid for 24h, and the CCK-8 method was used to further verify whether knocking down Nrf2 inhibited vitamin D3 to improve palmitic acid-induced HTR8 / SVneo cell proliferation inhibition.

[0023] The results of the present invention show that palmitic acid can inhibit the proliferation of HTR8 / SVneo cells (IC 50=364.1μM). Vitamin D3 (1μM) can significantly improve the proliferation inhibition of HTR8 / SVneo cells induced by palmitic acid (400μM) (the improvement is about 11.5%; t=8.79, P=0.001); Vitamin D3 can reduce the increase in cellular reactive oxygen species induced by palmitic acid (the decrease is 19.8%; t=7.84, P<0.001); Vitamin D3 can upregulate the expression of Nrf-2 and H in cells induced by palmitic acid. The expression levels of Nrf2 and HO-1 proteins in cells were significantly decreased after siRNA-Nrf2 transfection (the decrease was about 54.9%; t=12.08, Ρ<0.001; 69.7%; t=12.15, Ρ<0.001). The results of CCK-8 showed that transfection of siRNA-Nrf2 significantly weakened the improvement effect of vitamin D3 on the inhibition of palmitic acid-induced cell proliferation (the reduction was about 13.1%, t=4.33, P=0.012).

[0024] The specific research experiments are as follows:

[0025] Example 1

[0026] 1 Materials and methods

[0027] 1.1 Main Reagents

[0028] Human trophoblast HTR8 / SVneo cell line was purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.; fetal bovine serum and RPMI-1640 medium were purchased from Gibco, USA; trypsin and penicillin-streptomycin double antibody were purchased from Biosharp; vitamin D3 powder was purchased from Chongqing Mengbio; palmitic acid solution was purchased from Xi'an Kunchuang Technology Development Co., Ltd.; CCK-8 kit was purchased from Japan dojindo; EdU cell proliferation detection kit was purchased from Guangzhou Ruibo Biotechnology Co., Ltd.; reactive oxygen species detection kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; TRIzol, RNA reverse transcription kit, TBGreen premix Ex Taq II were purchased from TAKARA; PCR primers and siRNA-Nrf2 were purchased from Shanghai Sangon Co., Ltd., China; Nrf2, HO-1 and β-Actin primary antibodies were purchased from CST.

[0029] 1.2 Cell culture

[0030] Human trophoblast cells HTR8 / SVneo were selected, and RPMI-1640 (containing 10% fetal bovine serum and 1% penicillin-streptomycin) was selected as the culture medium, and placed in a cell culture incubator containing 5% CO2 and 37°C. In subsequent experiments, cells in the logarithmic growth phase were used, and HTR8 / Svneo cells were treated with gradient concentrations of palmitic acid (100μM, 200μM, 400μM, 600μM), gradient concentrations of vitamin D3 (0.1μM, 1μM, 10μM), and gradient concentrations of vitamin D3 (0.1μM, 1μM, 10μM) and 400μM palmitic acid for 24h.

[0031] 1.3 Cell proliferation assay

[0032] Take cells in the logarithmic growth phase, digest them with trypsin, count them and dilute the cell concentration to 1×10 5 / ml were inoculated in 96-well plates. After the cells were cultured for 12 hours and adhered to the wall, gradient concentrations of palmitic acid (0μM, 100μM, 200μM, 400μM, 600μM) and gradient concentrations of vitamin D3 (0.1μM, 1μM, 10μM) were added respectively; after the experimental concentration of palmitic acid was determined to be 400μM according to IC50, it was co-cultured with gradient concentrations of vitamin D3 (0μM, 0.1μM, 1μM, 10μM) for 24 hours. Subsequently, 10μl of CCK-8 reagent was added to each well and placed in a cell culture incubator for continuous culture for about 2 hours. The absorbance value of the 96-well plate was measured at a wavelength of 450nm by a fluorescent microplate reader.

[0033] Cell viability was tested using the EdU cell proliferation assay kit. Cells in the logarithmic growth phase were taken and 1×10 4 Cells were seeded in a 96-well plate. After the cells were cultured for 12 hours and adhered to the wall, a mixture of 400 μM PA and gradient concentrations of vitamin D3 (0 μM, 0.1 μM, 1 μM, 10 μM) was added and co-cultured for 24 hours. EdU working solution was prepared, and 100 μl of 50 μM EdU culture medium was added to each well and incubated for 2 hours. 4% paraformaldehyde was fixed at room temperature for 30 minutes, and the paraformaldehyde was discarded; 0.5% TritonX-100 was used for permeabilization at room temperature for 15 minutes. Then 100 μl of 1X After incubating the staining reaction solution in the dark for 30 minutes, 100 μl of 1X Hoechst 33342 reaction solution was added to each well. After incubating in the dark for 30 minutes, the staining images were obtained using a fluorescence microscope.

[0034] 1.4 Detection of Cellular Reactive Oxygen Species by Reactive Oxygen Species Detection Kit

[0035] Take cells in logarithmic growth phase and inoculate them in 6-well plates. Use gradient concentrations of palmitic acid (100μM, 200μM, 400μM, 600μM), gradient concentrations of vitamin D3 (0.1μM, 1μM, 10μM), and gradient concentrations of vitamin D3 (0.1μM, 1μM, 10μM) and 400μM PA for co-culture. Digest and collect cells in 15ml EP tubes. Dilute DCFH-DA 1:1000 and resuspend the cells, incubate in an incubator for 20 minutes. Wash the cells thoroughly three times and measure the reactive oxygen activity using a fluorescent microplate reader.

[0036] Cells were seeded in a 48-well plate and co-cultured with 400 μM palmitic acid and vitamin D3 (0 μM, 1 μM) for 24 hours after adherence. DCFH-DA diluted 1:1000 was added to each well and incubated for 20 minutes. The cells were washed three times and the reactive oxygen species staining images were obtained under a fluorescence microscope.

[0037] 1.5 RNA extraction and quantitative PCR

[0038] Total RNA was extracted from HTR8 / Svneo cells using Trizol, and RNA concentration was determined using a NanoDrop 2000 spectrophotometer. cDNA was synthesized from 1-2 μg RNA using PrimeScript RT Master Mix. Relative mRNA levels were amplified using the TBGreenpremix Ex Taq II kit and a real-time fluorescence quantitative PCR instrument, with GAPDH as an internal reference and the 2-ΔΔCT method used to calculate relative expression. The primer sequences used are shown in Table 1.

[0039] Table 1. Primers used in real-time qPCR

[0040]

[0041]

[0042] 1.6 Western Blot

[0043] Proteins were extracted from HTR8 / SVneo cells using IP cell lysate containing protease inhibitors. BCA protein detection kit was used to detect protein concentration. 50 μl of protein solution was added to each well of SDS-PAGE gel and electrophoresed at 100V constant voltage for 1 hour. The protein was then transferred to PVDF membrane under 260mA constant current conditions. 5% skim milk powder was prepared for blocking at room temperature for 1 hour and washed 3 times with TBST. Then, it was incubated with antibodies against Nrf2 (1:1000), HO-1 (1:1000) and β-Actin (1:1000) on a shaker at 4°C overnight. After washing 3 times in TBST the next day, goat anti-rabbit fluorescent secondary antibody was used for incubation at room temperature for 1 hour. Then, a dual-color infrared laser imaging system was used for development. The protein band intensity was quantified using ImageJ software to calculate protein expression.

[0044] 1.7 siRNA-Nrf2 transfection

[0045] HTR8 / SVneo cells were cultured in 6-well culture plates until the cell density reached 40%-50% of the plate area. 4 μl of Lipofectamine TM 2000 transfection reagent was diluted with 50μl serum-free medium and mixed and allowed to stand at room temperature for 5 minutes. 5μl siRNA-Nrf2 was diluted with 200μl serum-free medium and mixed and allowed to stand for 5 minutes in each well. Then the two dilutions were mixed and allowed to stand at room temperature for 20 minutes and then added to the 6-well plate and the cells were cultured for 24 hours. The transfection efficiency was detected by Western Blot. The target sequences used in this study are shown in Table 2.

[0046] Table 2. Sequences of Nrf2-siRNA

[0047]

[0048] 1.8 CCK-8 assay to determine cell viability after siRNA-Nrf2 transfection

[0049] After digestion, the cells transfected with siRNA-Nrf2 and siRNA-NC were counted with a cell counting plate and diluted to a cell concentration of 1×105 / ml. Then, 100 μl of the diluted cell suspension (1×104 cells) was inoculated into a 96-well plate and cultured in a 5% CO2, 37°C cell culture incubator for 12 hours. After the cells were cultured for 12 hours and adhered to the wall, vitamin D3 solution and palmitic acid were added respectively. The cells were divided into si-NC group, si-NC+400μM palmitic acid group, si-NC+1μM vitamin D3 group, si-NC+400μM palmitic acid+1μM vitamin D3 group, si-Nrf2 group, and si-Nrf2+400μM palmitic acid+1μM vitamin D3 group and cultured in a cell culture incubator for 24 hours. After 24 hours, remove the culture medium in the 96-well plate and add 100 μl of complete culture medium to each well, then add 10 μl of CCK-8 reagent to each well and culture in a 5% CO2, 37°C cell culture incubator for 2 hours. Use a red-hot syringe needle to remove the bubbles in the 96 wells, and use a fluorescent microplate reader to measure the absorbance value of the 96-well plate at a wavelength of 450 nm. (The whole process of adding CCK-8 reagent and after adding it should be protected from light).

[0050] 1.9 Statistical analysis

[0051] All data are expressed as Mean ± SD. Graph Pad Software 8.0 was used for analysis, and differences between groups were analyzed using one-way ANOVA, followed by Sidak multiple comparisons for intra-group comparisons (>2). P < 0.05 was considered statistically significant.

[0052] 2 Results

[0053] 2.1 Vitamin D3 alleviates the proliferation inhibition of HTR8 / SVneo cells induced by palmitic acid

[0054] HTR8 / SVneo cells were treated with gradient concentrations of palmitic acid (0 μM, 100 μM, 200 μM, 400 μM, 600 μM) to determine the IC50 value. Figure 1 As shown in A, after 24h of intervention of HTR8 / SVneo cells with palmitic acid at different concentrations, compared with the control group (palmitic acid concentration of 0μM), the changes in the proliferation ability of HTR-8 / SVneo cells in the 200μM palmitic acid group (t=15.81, Ρ<0.001), 400μM palmitic acid group (t=28.97, Ρ<0.001) and 600μM palmitic acid group (t=35.70, Ρ<0.001) were statistically different and negatively correlated. The calculated IC50 value was 364.1μM, so 400μM palmitic acid with a concentration close to it was selected in subsequent experiments.

[0055] To determine whether vitamin D3 improves the proliferation inhibition of HTR-8 / SVneo cells induced by palmitic acid, firstly, Figure 1 As shown in Figure B, HTR-8 / SVneo cells were cultured with gradient concentrations of vitamin D3 (0μM, 0.1μM, 1μM, 10μM) for 24 hours. Compared with the control group of 0μM vitamin D3, the 0.1μM, 1μM and 10μM vitamin D3 groups had no significant effect on the proliferation ability of HTR-8 / SVneo cells. Subsequently, 0μM, 0.1μM, 1μM and 10μM vitamin D3 were added to 400μM palmitic acid to culture cells for 24h. Compared with the 400μM palmitic acid group, 1μM (t=8.79, P=0.001) and 10μM (t=7.97, P=0.001) vitamin D3 can significantly improve the inhibition of 400μM palmitic acid on the proliferation of HTR-8 / SVneo cells, and the amplitude is about 11.5% ( Figure 1 C) Do the following to the cells: Figure 1 EdU staining test was performed after treatment with C. The results showed that compared with the control group, the cell proliferation ability of the 400μM palmitic acid group was significantly inhibited (t=24.34, P<0.001); compared with the 400μM palmitic acid group, the cell proliferation ability was partially restored after treatment with 1μM (t=7.54, P=0.002) and 10μM (t=8.77, P=0.001) vitamin D3 ( Figure 1 D, blue fluorescence represents cell nuclei, and red fluorescence represents the number of proliferating cells). Figure 1 E is the image of Figure 1 The above experiments showed that vitamin D3 can alleviate the proliferation inhibition of HTR-8 / SVneo cells induced by 400 μM palmitic acid.

[0056] 2.2 Vitamin D3 reduces palmitic acid-induced oxidative stress in HTR8 / SVneo cells

[0057] To clarify the regulatory effect of palmitic acid on oxidative stress in HTR8 / SVneo cells, Figure 2As shown in A, after using palmitic acid of different concentration gradients to intervene HTR8 / SVneo cells for 24 hours, the reactive oxygen levels of cells were detected using a reactive oxygen detection kit. Compared with the control group (400μM palmitic acid), the reactive oxygen levels of cells in the 400μM (t=19.30, Ρ<0.001) and 600μM (t=20.16, Ρ<0.001) palmitic acid groups were significantly increased, with statistical differences, and the increase was 106.5% and 109.9%, respectively. At the same time, after treating cells with different gradient concentrations of vitamin D3 (0μM, 0.1μM, 1μM, 10μM) for 24 hours, the results showed that vitamin D3 had no significant effect on the generation of reactive oxygen species in cells ( Figure 2 B). The cells treated with 400 μM palmitic acid were further treated with 1 μM vitamin D3. The results showed that 1 μM vitamin D3 could reduce the high reactive oxygen levels in HTR-8 / SVneo cells induced by 400 μM palmitic acid, with a statistical difference (t=7.84, Ρ<0.001), and the reduction was 19.8% ( Figure 2 C) In addition, the cells were subjected to Figure 2 After the same treatment as in C, the cells were stained with reactive oxygen species and detected by fluorescence. Compared with the 400 μM palmitic acid group, the reactive oxygen species level in the 1 μM vitamin D3 + 400 μM palmitic acid group was significantly reduced, with a statistical difference (t = 3.33, P = 0.03), which also showed that vitamin D3 (1 μM) had an improving effect on the oxidative stress environment of HTR-8 / SVneo cells induced by 400 μM palmitic acid ( Figure 2 D, Green fluorescence indicates the level of reactive oxygen species). Figure 2 E is the image obtained using ImageJ software Figure 2 Statistical graph obtained after analyzing the fluorescence of D. In summary, vitamin D3 can alleviate palmitic acid-induced oxidative stress in HTR-8 / SVneo cells.

[0058] 2.3 Vitamin D3 improves palmitic acid-induced oxidative stress in HTR8 / SVneo cells by regulating the Nrf2 / HO-1 signaling pathway

[0059] After treating cells with 1 μM vitamin D3 and 400 μM palmitic acid, we found that compared with the control group, the mRNA levels of Nrf2 (t=3.03, P=0.039) and HO-1 (t=4.18, P=0.014) in the 400 μM palmitic acid group were significantly increased. After treatment with 1 μM vitamin D3, the mRNA levels of Nrf2 (t=13.27, P<0.001) and HO-1 (t=11.84, P<0.001) increased further, and the differences were statistically significant ( Figure 3A, B). In addition, compared with the control group, there was no statistically significant change in the mRNA levels of NOX4 and NOX5 in the 400μM palmitic acid group and the 1μM vitamin D3 + 400μM palmitic acid group ( Figure 3 C, D). Furthermore, WB results showed that compared with the control group, the protein expression levels of Nrf2 (t = 4.20, P = 0.014) and HO-1 (t = 5.58, P = 0.005) in the 400 μM palmitic acid group were significantly increased, and the protein expression levels of Nrf2 (t = 3.17, P = 0.034) and HO-1 (t = 2.95, P = 0.042) were further increased after treatment with 1 μM vitamin D3, and the differences were statistically significant ( Figure 3 E, 3F). The above studies indicate that vitamin D3 may regulate palmitic acid-induced cellular oxidative stress by activating the Nrf-2 / HO-1 pathway.

[0060] 2.4 Knockdown of Nrf2 gene expression weakened the protective effect of vitamin D3 on HTR8 / SVneo cells treated with palmitic acid

[0061] To further confirm that vitamin D3 improves palmitic acid-induced inhibition of HTR8 / SVneo cell proliferation by activating the Nrf2 / HO-1 pathway, HTR8 / SVneo cells were transfected with siRNA-Nrf2 and siRNA-NC for 24 h, and then 1 μM vitamin D3 + 400 μM palmitic acid were used to intervene in HTR8 / SVneo cells for 24 h. Figure 4 As shown in A, the expression levels of Nrf2 and HO-1 proteins were significantly reduced after transfection with si-Nrf2. Compared with the 1μM vitamin D3+400μM palmitic acid+si-NC group, the expression levels of Nrf2 and HO-1 proteins in the 1μM vitamin D3+400μM palmitic acid+si-Nrf2 group were significantly reduced (t=12.08, P<0.001; t=12.08, P<0.001). CCK-8 results ( Figure 4 B) shows that the protective effect of vitamin D3 on the proliferation ability of HTR8 / SVneo cells treated with palmitic acid was significantly reduced after transfection of si-Nrf2 (t=4.33, P=0.012).

[0062] 3 Analysis

[0063] Normal placental trophoblast cell function is crucial for maintaining normal maternal and fetal blood circulation and immune regulation. Its dysfunction increases the risk of gestational diabetes and preeclampsia in pregnant women, and even leads to fetal growth restriction. Obesity in pregnant women can cause oxidative stress in the placenta, leading to placental dysfunction. Palmitic acid is a common saturated fatty acid, and palmitic acid additives are often used to establish obesity models in cell experiments. In this study, we selected human trophoblast cells HTR8 / SVneo as an in vitro experimental cell model, and treated HTR8 / SVneo cells with palmitic acid to establish an in vitro obesity model.

[0064] Vitamin D is a natural antioxidant known for its cell-protective effects. Vitamin D3 is a fat-soluble vitamin that is mainly produced in the skin and further converted into active vitamin D3 in the liver and kidneys. Studies have shown that vitamin D3 improves fetal growth and development in pregnant women with preeclampsia by regulating placental amino acid transport. Studies have also shown that vitamin D3 can enhance the remodeling ability of incomplete spiral arteries in placental explants. Vitamin D3 can also enhance the immune response of the placenta. However, the effect of vitamin D3 on placental dysfunction caused by maternal obesity is still unclear. In the present study, PA was used to simulate the maternal obesity model in vitro. At the same time, in HTR8 / SVneo cells treated with 400μM PA, the addition of 1μM vitamin D3 can improve the 400μM PA-induced HTR8 / SVneo cell proliferation inhibition and ROS generation increase. qPCR and WB experiments showed that after PA acted on HTR8 / SVneo cells, the mRNA and protein expression levels of Nrf2 and HO-1 were significantly increased, and the difference was statistically significant compared with the control group. After vitamin D3 acted on HTR8 / SVneo cells, the mRNA and protein expression levels of Nrf2 and HO-1 were further increased, but the difference in the mRNA expression levels of NOX4 and NOX5 was not statistically significant. Therefore, the present study found that vitamin D3 improves PA-induced oxidative stress and cell proliferation inhibition in human trophoblast HTR8 / SVneo cells by activating the Nrf2 / HO-1 pathway.

[0065] Nrf2 is a key transcription factor that coordinates the expression of genes encoding antioxidant and detoxification enzymes. It is mainly regulated by Kelch-like epichlorohydrin-associated protein 1 (Keap1) and plays an important role in multiple aspects of cell and organism resistance to oxidative stress. Nrf2 regulates oxidative stress response through multiple mechanisms, and it is directly involved in inducing the expression of detoxification enzymes. After being synthesized in the cytoplasm, Nrf2 is transferred to the nucleus and forms a heterodimer with musculoaponeurotic fibrosarcoma protein (MAF) K, G and F. The dimer recognizes and binds to the antioxidant response element (ARE) enhancer sequence. Nrf2 / ARE protects cells from oxidative stress damage by activating heme oxygenase 1, NAD(P)H quinone oxidoreductase 1 and glutathione peroxidase. The present study showed that compared with the control group, 400 μM PA increased the mRNA and protein expression levels of Nrf2 and HO-1 in cells, while after 1 μM vitamin D3 combined with 400 μM PA intervention, Nrf2,

[0066] The mRNA and protein expressions of HO-1 were further increased. Therefore, the activation of Nrf2 / HO-1 pathway may be the result of vitamin

[0067] One of the potential molecular mechanisms by which D3 improves PA-induced inhibition of human trophoblast cell proliferation.

Claims

1. Application of vitamin D3 in the preparation of products for improving the inhibition of trophoblast proliferation.

2. The use according to claim 1, characterized in that: The trophoblast proliferation inhibition is palmitic acid-induced trophoblast proliferation inhibition.

3. The use according to claim 2, characterized in that: Application of vitamin D3 in the preparation of products for improving palmitic acid-induced inhibition of trophoblast proliferation during pregnancy.

4. The use according to claim 2, characterized in that: Application of vitamin D3 in the preparation of products for improving palmitic acid-induced trophoblast proliferation inhibition in obese people.

5. The use according to claim 1, characterized in that: Vitamin D3 improves trophoblast proliferation inhibition via activation of the Nrf2 / HO-1 pathway.

6. The use according to claim 5, characterized in that: Vitamin D3 improves oxidative stress and cell proliferation inhibition in trophoblast cells.

7. The use according to claim 5, characterized in that: Vitamin D3 increases the expression levels of Nrf2 and HO-1.

8. The use according to claim 1, characterized in that: The product described is a drug.

9. The use according to claim 8, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

Images