Application of SphK1 as target for treating placental dysfunction of gestational diabetes mellitus

Abstract

The invention discloses application of SphK1 as a target spot for treating placental dysfunction of gestational diabetes mellitus. Researches find that in a high-glucose environment, the expression of SphK1 protein in human chorionic trophoblast cells is remarkably reduced, and the cell proliferation and migration ability is seriously damaged along with the reduction of the expression of SphK1 protein in the human chorionic trophoblast cells. By constructing an SphK1 overexpression cell model, it is proved that up-regulation of SphK1 expression can effectively reverse cell function damage induced by high glucose, and the proliferation and migration ability of the cell can be recovered. Further, through gene knock-down and exogenous S1P supplementary experiments, the key effect of the SphK1 and the metabolites thereof in protecting the trophoblast function is verified. The invention provides a new theoretical explanation for the pathological mechanism of GDM, and reveals the potential value of developing GDM therapeutic drugs or diagnostic tools by taking SphK1 as a target.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and in particular relates to the application of SphK1 as a target for treating placental dysfunction in gestational diabetes mellitus. Background Technology

[0002] Gestational diabetes mellitus (GDM) refers to impaired glucose tolerance that first occurs or is discovered during pregnancy, and it is one of the most common metabolic disorders of pregnancy. According to global standardized estimates, the current global average prevalence of GDM is approximately 14.2%, and its incidence is continuously rising due to changes in modern lifestyles and the increasing obese population. GDM poses a serious threat to maternal and fetal health, significantly increasing the risk of short-term complications such as gestational hypertension, preeclampsia, macrosomia, and dystocia, as well as leading to an increased incidence of adverse pregnancy outcomes such as spontaneous abortion, premature birth, stillbirth, and congenital malformations. Furthermore, GDM may significantly increase the long-term risk of the mother developing type 2 diabetes and cardiovascular disease. At the placental level, the pathophysiological changes in GDM are mainly manifested as placental trophoblast dysfunction induced by a high-glucose environment, specifically including impaired cell proliferation, invasion, and differentiation, as well as increased apoptosis. As a key organ for maternal-fetal material exchange during pregnancy, placental dysfunction plays a central role in the development and progression of GDM. Existing studies have shown that there are significant metabolic disorders and abnormal signaling pathways in the placental tissue of GDM patients, such as enhanced inflammatory response, increased oxidative stress levels, and dysregulation of multiple cell signal transduction pathways.

[0003] Currently, the clinical management of GDM mainly relies on dietary control, exercise intervention, and insulin therapy. However, these strategies have not completely corrected GDM-related placental dysfunction, and some patients still face the risk of adverse pregnancy outcomes. Therefore, in-depth exploration of the molecular mechanisms of placental trophoblast dysfunction in the context of GDM and identification of key regulatory targets are of significant clinical importance for developing new therapeutic intervention strategies.

[0004] The human chorionic trophoblast cell line (HTR-8 / SVneo) is a classic in vitro model for studying the pathological mechanisms of gastrointestinal dysplasia (GDM). This cell line retains many biological characteristics of normal trophoblast cells, including proliferation, migration, and invasion capabilities, and can effectively mimic the behavior of trophoblast cells in vivo. In GDM-related research, HTR-8 / SVneo cells are widely used to investigate the effects of high glucose environments on trophoblast cell function and its potential mechanisms. Studies have shown that high glucose stimulation (such as 30 mM D-glucose) can significantly induce a series of functional abnormalities in HTR-8 / SVneo cells, including inhibited proliferation, arrested cell cycle progression, increased release of inflammatory factors, and impaired glucose uptake. These abnormal phenotypes are highly consistent with the trophoblast dysfunction observed in placental tissue of GDM patients, making HTR-8 / SVneo cells an ideal in vitro model for studying the pathogenesis of GDM.

[0005] Sphingosine kinase 1 (SphK1) is a key rate-limiting enzyme in the sphingolipid metabolism pathway. Its gene is located on human chromosome 17, q25.1, and encodes a 384-amino acid protein. SphK1 is primarily distributed in the cytoplasm. Upon activation, it translocates to the cell membrane and catalyzes the phosphorylation of sphingosine to sphingosine-1-phosphate (S1P). S1P is an important bioactive lipid mediator, acting as an intracellular second messenger to regulate cell proliferation, survival, and apoptosis. It can also bind to five G protein-coupled receptors (S1PR1–5) on the cell membrane via autocrine or paracrine pathways, thereby activating multiple downstream signal transduction pathways.

[0006] In recent years, research has gradually revealed the important role of SphK1 and its signaling pathway in the regulation of glucose homeostasis. For example, in diabetic nephropathy, high glucose can activate the SphK1 / S1P pathway, inducing upregulation of fibronectin (FN) expression in glomerular mesangial cells, thereby promoting the progression of renal fibrosis. Simultaneously, SphK1 also participates in the regulation of insulin signaling and glucose metabolism. In the KK / Ay diabetic mouse model, overexpression of SphK1 can enhance the phosphorylation levels of key proteins such as Akt in the insulin signaling pathway, improving glycemic homeostasis. These findings suggest that SphK1 plays a crucial role in metabolic regulation and high glucose-induced cell damage.

[0007] In summary, SphK1, as a key regulator of sphingolipid metabolism, may play an important role in the development and progression of placental dysfunction in gastrointestinal dysplasia (GDM). Under high glucose conditions, alterations in SphK1 expression and activity may lead to abnormal S1P levels, thereby participating in the pathological process of GDM by regulating trophoblast cell proliferation, apoptosis, inflammatory responses, and energy metabolism. However, the specific molecular mechanisms by which SphK1 regulates trophoblast cell function in GDM, especially its direct impact on the biological phenotype of HTR-8 / SVneo cells, are not fully understood and require further clarification. This invention aims to systematically explore the regulatory role of SphK1 on the high glucose-induced biological phenotype of HTR-8 / SVneo cells, providing experimental evidence for developing novel therapeutic strategies for GDM. Summary of the Invention

[0008] To achieve the above objectives, the present invention provides the use of an SphK1 agonist in the preparation of a medicament for the prevention and / or treatment of placental dysfunction associated with gestational diabetes mellitus.

[0009] The present invention also provides the use of the SphK1 gene or its encoded protein in the preparation of a medicament for improving the biological function of human chorionic trophoblast cells under conditions of gestational diabetes mellitus, wherein the biological function includes cell proliferation capacity and / or cell migration capacity.

[0010] The present invention also provides a method for improving the function of human chorionic trophoblast cells under high glucose conditions, the method comprising the step of upregulating the expression and / or activity of SphK1 in the cells.

[0011] Furthermore, the step of upregulating SphK1 expression and / or activity is achieved by introducing a SphK1 overexpression vector into the cells.

[0012] Furthermore, the SphK1 overexpression vector is a recombinant lentiviral vector.

[0013] The present invention also provides a pharmaceutical composition comprising an effective dose of an SphK1 agonist and a pharmaceutically acceptable carrier for the prevention and / or treatment of gestational diabetes.

[0014] The present invention also provides an in vitro method for screening candidate drugs for the prevention and / or treatment of gestational diabetes mellitus, the method comprising the following steps: a) Culture human chorionic trophoblast cells under high glucose conditions; b) Treat the cells with the candidate drug; c) Detect the expression level and / or activity of SphK1 in the cells; If the candidate drug can significantly increase the expression level and / or activity of SphK1 in the cells, it indicates that the candidate drug is a potential drug for the prevention and / or treatment of GDM.

[0015] Furthermore, in step c), the cell's proliferative capacity and / or migration capacity are further detected.

[0016] The present invention also provides a diagnostic reagent or kit for assessing the risk of gestational diabetes or placental function, wherein the reagent or kit is used to detect the protein expression level or gene expression level of SphK1 in human placental tissue or isolated trophoblast cells.

[0017] The present invention also provides the use of a combination of an SphK1 agonist and insulin in the preparation of a medicament for treating gestational diabetes.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects: This invention demonstrates through a series of experiments that SphK1 plays a crucial role in high glucose-induced dysfunction of human chorionic trophoblast cells. Studies show that a high glucose environment leads to a significant downregulation of SphK1 expression, resulting in severely impaired cell proliferation and migration; while overexpression of SphK1 can effectively reverse this damage and improve cell proliferation and migration.

[0019] This discovery not only reveals a new mechanism of placental dysfunction in GDM, but more importantly, it establishes SphK1 as a potential target for the treatment of GDM, providing an important theoretical basis and experimental foundation for the development of novel treatment strategies for GDM. Attached Figure Description

[0020] Figure 1 The results show the effects of high glucose on the proliferation and migration of HTR-8 / SVneo cells. In this figure, A is a bar chart of cell proliferation as determined by the CCK-8 assay; B and D are typical microscopic images of cells at 0 and 24 hours after the cell scratch assay; C and E are typical microscopic images and bar charts of cells migrating as determined by the Transwell assay.

[0021] Figure 2 The figures show the results of SphK1 protein expression after high glucose induction. A is a typical band pattern of SphK1 protein expression detected by Western blotting; B is a bar chart of SphK1 protein expression levels; and C is a typical fluorescence micrograph of SphK1 expression detected by immunofluorescence (green fluorescence) and DAPI nuclear staining (blue).

[0022] Figure 3The results are shown in the figure for validating the infection efficiency and overexpression effect of SphK1 overexpression lentivirus. A is a typical fluorescence micrograph of mCherry red fluorescent protein expression after lentiviral infection; B is a typical band diagram of SphK1 protein expression in the overexpression group and control group detected by Western blotting; C is a bar chart of SphK1 overexpression levels.

[0023] Figure 4 This figure shows the results of SphK1 overexpression in rescuing high glucose-induced cell functional damage. A is a bar chart of cell proliferation recovery as detected by the CCK-8 assay; B and D are typical microscopic images of the cell scratch assay and their healing rate statistics; C and E are typical microscopic images of the Transwell assay and their migration cell count statistics.

[0024] Figure 5 The results of SphK1 gene knockdown efficiency verification are shown in Figure A. A typical fluorescence micrograph of mCherry red fluorescent protein expression after siRNA transfection, used to indicate transfection efficiency; B is a typical band diagram of SphK1 protein expression in the negative control group (si-NC) and the SphK1 knockdown group (si-SphK1) cells detected by Western blotting; C is a bar chart of SphK1 protein expression level.

[0025] Figure 6 The results show the effect of SphK1 knockdown on the function of HTR-8 / SVneo cells under high glucose conditions. In the figure, A is a bar chart of cell proliferation capacity as detected by the CCK-8 assay; B and C are typical microscopic images of the cell scratch assay at 0 and 24 hours and their healing rate statistics; D and E are typical microscopic images of the Transwell cell migration assay and their migrating cell count statistics. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] All raw materials used in this invention are not particularly restricted in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0028] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0029] The "SphK1 gene" mentioned in this article, short for Sphingosine kinase-1 (Gene ID: 8877), is a key rate-limiting enzyme in catalyzing the phosphorylation of sphingosine (Sph) to produce sphingosine 1-phosphate (S1P). The SphK1 gene maintains the metabolic balance among ceramides (Cer), Sph, and S1P within cells, regulating cellular biological behavior. The "recombinant lentiviral vector" refers to a lentivirus carrying the SphK1 coding sequence used as a gene delivery vector, capable of efficiently infecting both dividing and non-dividing cells and integrating the target gene SphK1 into the host cell genome, thereby achieving stable and long-term overexpression of the SphK1 gene.

[0030] Example 1: Effects of a high-glucose environment on the proliferation and migration of HTR-8 / SVneo cells 1. Cell culture and establishment of a high glucose model The RPMI-1640 complete culture medium formula used for the HTR-8 / SVneo cell line is: RPMI-1640 basal medium + 10% fetal bovine serum + 0.5% penicillin-streptomycin (100×). Culture conditions: 37℃, 5% CO2 constant temperature and humidity incubator.

[0031] HTR-8 / SVneo cells in logarithmic growth phase and in good condition were digested with trypsin, centrifuged, and resuspended into a single-cell suspension. These suspensions were then seeded at appropriate densities in well plates and pre-cultured in RPMI-1640 complete medium for 24 hours until cells were fully adherent and 80%-90% confluence. The old medium was discarded, and the cells were washed twice with PBS before being replaced with RPMI-1640 basal medium containing 30 mM D-glucose to establish a hyperglycemic injury model in the HTR-8 / SVneo cell line. A normal glucose concentration (5.5 mM D-glucose) group was established as a control, and a 30 mM mannitol group was established as a hyperosmolar control to eliminate the influence of osmotic pressure changes on the cells.

[0032] 2. CCK-8 assay for cell proliferation capacity Approximately 2×10 4 Cells were seeded into 96-well plates, with 5 replicates per group. After pre-culturing in 100 μL of RPMI-1640 complete medium for 24 hours, high glucose stimulation was administered for 24 and 48 hours, respectively. The medium was discarded, and after rinsing with PBS, 100 μL of CCK-8 working solution (10 μL CCK-8 solution + 90 μL RPMI-1640 basal medium) was added to each well, and the plates were incubated in the dark for 1 hour. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader.

[0033] The results are as follows Figure 1As shown in Figure A, compared with the normal glucose group, cell proliferation decreased after 24 and 48 hours of high glucose stimulation, while the mannitol hyperosmolar control group showed no significant difference from the normal group, indicating that high glucose specifically inhibits cell proliferation.

[0034] 3. Cell scratch assay to detect migration ability Draw a reference line on the bottom of the 6-well plate with a marker, and inoculate approximately 8 × 10⁸ cm⁻¹ per well. 4 Cells were pre-cultured in 2 mL of RPMI-1640 complete medium for 24 hours, followed by high glucose stimulation for 48 hours. Using a 200 μL sterile pipette tip perpendicular to the bottom of the plate, a scratch was made perpendicular to the reference line with uniform force. The old medium was discarded, and the cells were rinsed twice with PBS. 2 mL of RPMI-1640 basal medium was added to each well, and scratch images of the same location at 0 hours and 24 hours were collected under an inverted microscope.

[0035] The results are as follows Figure 1 B and Figure 1 As shown in Figure D, high glucose induces a decrease in cell migration ability in the HTR-8 / SVneo cell line.

[0036] 4. Transwell experiments to verify transferability Add 700 μL of RPMI-1640 complete culture medium to a 24-well plate as a chemotherapeutic agent, and carefully place it into a Transwell chamber, avoiding the formation of air bubbles; select cells stimulated with high glucose for 48 hours as the experimental group, resuspend the cells in RPMI-1640 basal culture, and add 300 μL of cell suspension (containing 2 × 10⁶ cells / mL) to the upper chamber. 4 (100 cells), continue culturing for 24 hours; after migration, wipe off the unmigrated cells from the upper chamber membrane surface, fix with 4% paraformaldehyde at room temperature for 30 min, stain with 0.1% crystal violet solution at room temperature for 30 min, wash with PBS and place in a ventilated area to air dry; select random fields of view for image acquisition under an inverted microscope, and count the migrating cells using ImageJ.

[0037] The results are as follows Figure 1 C and Figure 1 As shown in E, high glucose induces a decrease in cell migration ability in the HTR-8 / SVneo cell line.

[0038] Example 2: Effect of high glucose on SphK1 protein expression in HTR-8 / SVneo cells 1. Western blot detection of SphK1 protein expression level (1) Cell protein extraction: Pre-treat cells, discard old culture medium, rinse twice with pre-cooled PBS, add appropriate amount of protein lysis buffer (RIPA complete lysis buffer + 1% protease inhibitor + 2% phosphatase inhibitor), then collect cells with a cell scraper and transfer them to EP tubes, place them on a 4℃ 360° rotating mixer to react fully for 45 minutes, and finally centrifuge (4℃, 12000 rpm, 15 minutes), and transfer the protein supernatant to a new EP tube for later use. (2) Protein concentration determination: Add 8 protein standards to the 96-well plate in sequence, prepare the target protein sample according to the BCA kit instructions, preheat the microplate reader for 30 minutes, measure the absorbance (OD value) of each well at a wavelength of 562 nm, plot the standard concentration curve and calculate the protein concentration.

[0039] (3) Electrophoresis: Add loading buffer to the target protein sample in proportion, heat in a boiling water bath for 10 minutes to denature it fully; prepare a 10% SDS-PAGE gel according to the target molecule, put it into the electrophoresis tank, pour in the electrophoresis buffer, add 5 μL of the sample to be tested to each sample well in sequence, connect the electrodes correctly, adjust the voltage to 80 V until the bromophenol blue indicator enters the separating gel, and then adjust the voltage to 120 V constant voltage until the sample is completely separated.

[0040] (4) Transfer membrane: Activate the PVDF membrane with anhydrous methanol and make a "sandwich" structure according to the order of anode-sponge-filter paper-PAGE gel-PVDF membrane-filter paper-sponge-cathode. Avoid generating air bubbles between each layer. Place it in the transfer tank, pour in pre-cooled transfer buffer, and run at a constant current of 300mA for 60 minutes after correctly connecting the electrodes. Block the PVDF membrane with TBST containing 5% skim milk for 2 hours.

[0041] (5) Antibody incubation: Prepare primary antibodies SphK1 (Immunoway / YN0035) and GAPDH (Proteintech / 60004–10-Ig) at a ratio of 1:1000 with antibody dilution buffer. Add the primary antibody working solution to the corresponding antibody incubation box and incubate overnight on a shaker at 4°C. Recover the primary antibody and wash the membrane with TBST for 10 minutes. Repeat 3 times. Prepare the secondary antibody working solution of the corresponding species with TBST and add it to the corresponding antibody incubation box. Incubate at room temperature for 2 hours and wash the membrane with TBST for 10 minutes. Repeat 3 times.

[0042] (6) Development: Turn on the eBlOT electronic tablet imager, place the PVDF film upside down in the sample chamber of the imager, add an appropriate amount of developing solution, cover the lid, and select a suitable exposure time for development; use Image-J software to analyze the gray value of the strip.

[0043] The results are as follows Figure 2 A and Figure 2As shown in Figure B, the protein expression level of SphK1 was significantly reduced in the high glucose-induced HTR-8 / SVneo cell line.

[0044] 2. Detection of SphK1 expression level by cellular immunofluorescence Place poly-L-lysine-coated cell spreaders into 24-well plates, seeding approximately 2 × 10⁶ cells per well. 4 Cells were pre-cultured in 500 μL RPMI-1640 complete medium for 24 hours, followed by high glucose stimulation for 48 hours. The old medium was discarded, and the cells were washed twice with PBS. After fixing with pre-chilled anhydrous methanol (-20°C), residual methanol was washed away with PBS. Blocking buffer (1% FBS + 0.3% Triton + 98.7%) was then added. Block with PBS at room temperature for 2 hours; prepare primary antibody SphK1 (Immunoway / YN0035) at a 1:200 ratio using antibody dilution buffer, add the primary antibody working solution to the corresponding sample well, incubate overnight at 4°C on a shaker, recover the primary antibody, wash with pre-cooled PBS for 10 minutes, repeat 3 times; add the corresponding species' fluorescent secondary antibody working solution to the corresponding sample well, incubate at room temperature in the dark for 2 hours, wash with pre-cooled PBS in the dark for 10 minutes, repeat 3 times; add 10 μL of anti-fluorescence quenching mounting medium (containing DAPI) to a glass slide, remove the slide with sterile ophthalmic forceps, invert it onto the glass slide to avoid air bubbles; finally, select random fields of view for image acquisition under an inverted microscope.

[0045] The results are as follows Figure 2 As shown in C, the fluorescence signal of SphK1 is weakened in the high glucose-induced HTR-8 / SVneo cell line.

[0046] Example 3: Construction and identification of SphK1 overexpression lentivirus The construction and packaging of the SphK1 overexpression recombinant lentiviral vector were completed by Weinan Biotechnology Co., Ltd. Cell count was approximately 2 × 10⁻⁶. 4 Cells were seeded into 6-well plates and pre-cultured in 2 mL of RPMI-1640 complete medium for 24 hours; the old medium was discarded, and the cells were washed twice with PBS. Cells were then seeded according to the multiple of infection (MOI=100) and viral titer (1×10⁻⁶). 8 (PFU / mL) 100 μL of the virus stock solution was added to 2 mL of RPMI-1640 complete medium, then added to the sample wells. The mixture was gently mixed to ensure adequate virus contact with the cells. After 12 hours of infection, the medium was replaced with fresh RPMI-1640 complete medium. After 72 hours of infection, the red fluorescent protein (mCherry) signal was observed under an inverted fluorescence microscope. Results are as follows: Figure 3 As shown in (A), the infection efficiency is approximately 90%.

[0047] 72 hours after viral infection, the medium was replaced with RPMI-1640 complete medium containing 2 μg / mL puromycin. HTR-8 / SVneo cell lines that stably overexpress SphK1 were screened, cell proteins were extracted, and SphK1 expression was detected by Western blot.

[0048] The results are as follows Figure 3 As shown in (B, C), compared with the control group, the protein expression level of SphK1 in the experimental group infected with SphK1 overexpressing lentivirus was significantly increased. Therefore, in subsequent examples, it was used to interfere with the expression of the SphK1 gene.

[0049] Example 4: The rescue effect of SphK1 overexpression on high glucose-induced functional impairment of HTR-8 / SVneo cells Following the experimental protocol of Example 3, the HTR-8 / SVneo cell line was infected with a virus, specifically an overexpression fragment of SphK1 was applied to enhance SphK1 expression, named OE-SphK1. The experimental groups included the NC group (treated with 30mM D-glucose) and the OE-SphK1 group (treated with 30mM D-glucose). Cell function assays were performed using the same procedures as in Example 1.

[0050] The results of the CCK-8 experiment showed that ( Figure 4 A) Under high glucose conditions, cell proliferation was significantly inhibited in the NC group, while cell proliferation in the OE-SphK1 group was increased compared to the NC group, approaching the level of the normal glucose control group. Scratch assay ( Figure 4 B, Figure 4 D) and Transwell experiment ( Figure 4 C, Figure 4 E) The results consistently showed that under high glucose conditions, the cell migration ability of the OE-SphK1 group was significantly better than that of the NC group. Scratch healing rate was improved, and the number of transwell cells increased. This indicates that SphK1 overexpression effectively reversed the decline in HTR-8 / SVneo cell line proliferation and migration induced by high glucose.

[0051] Example 5: Effects of SphK1 gene knockdown on HTR-8 / SVneo cell function under high glucose conditions To verify the key role of SphK1 in high glucose-induced trophoblast dysfunction from a reverse perspective, we used small interfering RNA (siRNA) technology to specifically knock down the expression of endogenous SphK1 in HTR-8 / SVneo cells and systematically evaluated its effects on cell function.

[0052] 1. Design and transfection of SphK1-specific siRNA To minimize off-target effects and ensure knockdown efficiency, two different siRNA sequences (named si-SphK1-1 and si-SphK1-2) were designed and synthesized targeting the coding sequence of the human SphK1 gene (Gene ID: 8877), along with a sequence-independent negative control siRNA (si-NC). All siRNAs were labeled with mCherry red fluorescent markers to facilitate real-time monitoring of transfection efficiency.

[0053] HTR-8 / SVneo cells in logarithmic growth phase were fed at approximately 5 × 10⁻⁶ cells per cell line. 4 Cells were seeded at a density of [number] cells / well in 24-well plates and cultured in antibiotic-free complete medium for 24 hours until cell confluence reached 60%-70%. Following the liposome transfection reagent instructions, 50 nM siRNA and the transfection reagent complex were added to the cells separately. Six hours after transfection, the medium was replaced with fresh complete medium. After another 72 hours of culturing, the cells were observed under an inverted fluorescence microscope. Figure 5 As shown in Figure A, a significant mCherry red fluorescence signal can be observed, and the transfection efficiency is approximately 90%, indicating that the siRNA was successfully introduced into the cells.

[0054] 2. Verification of SphK1 knockdown efficiency Seventy-two hours after transfection, cells from each group were collected, total protein was extracted, and the SphK1 protein expression level was detected by Western blotting to verify the knockdown efficiency. The experimental procedure was the same as in Example 2. Results are as follows: Figure 5 B protein band diagram and Figure 5 The bar chart for C shows that, compared with the si-NC control group, the SphK1 protein expression level in cells transfected with si-SphK1-1 was significantly knocked down, with a decrease of approximately 70% (p<0.001); while si-SphK1-2 also showed a significant knockdown effect, but the efficiency was slightly lower than that of si-SphK1-1. Therefore, subsequent functional experiments used si-SphK1-1 (referred to as the si-SphK1 group) with higher knockdown efficiency.

[0055] 3. Effects of SphK1 knockdown on cell function under high glucose environment Based on verifying the knockdown efficiency, the effects of SphK1 knockdown on cell function in a high-glucose environment were further investigated. Cells transfected with si-NC or si-SphK1 were treated with medium containing 30 mM D-glucose for 48 hours after the medium was replaced with fresh medium to simulate the high-glucose pathological environment of GDM, followed by a series of functional tests.

[0056] 3.1 Cell proliferation capacity detection Cell proliferation was detected using the CCK-8 assay. Figure 6 Results showed that, under high glucose conditions, the cell proliferation activity of the si-SphK1 group was significantly reduced compared with that of the si-NC group, indicating that knockdown of SphK1 exacerbated the inhibition of cell proliferation by high glucose.

[0057] 3.2 Cell migration ability detection Cell migration ability was assessed using scratch assays and Transwell assays. Scratch assay ( Figure 6 (B, 6C) showed that after 24 hours of culture, the scratch healing rate of the si-SphK1 group was significantly lower than that of the si-NC group. Transwell assay ( Figure 6 The results (D, 6E) were consistent with this, showing that the number of cells that migrated across the membrane in the si-SphK1 group was significantly reduced compared to the si-NC group.

[0058] Functional experiments showed that knocking down SphK1 expression under high glucose stress significantly exacerbated the inhibition of trophoblast proliferation and reduced migration ability. This reverse genetic evidence corroborates the results of the overexpression experiment (Example 4), confirming from both positive and negative perspectives that SphK1 is a key factor in maintaining the normal biological function of trophoblasts under high glucose conditions, and that its downregulation directly participates in and exacerbates GDM-related placental cell dysfunction.

[0059] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of an SphK1 agonist in the preparation of a medicament for the prevention and / or treatment of placental dysfunction associated with gestational diabetes mellitus.

2. The use of a SphK1 gene or its encoded protein in the preparation of a medicament for improving the biological function of human chorionic trophoblast cells under conditions of gestational diabetes mellitus, wherein the biological function includes cell proliferation capacity and / or cell migration capacity.

3. A method for improving the function of human chorionic trophoblast cells under high-glucose conditions, characterized in that, The method includes the step of upregulating the expression and / or activity of SphK1 in the cells.

4. The method according to claim 3, characterized in that, The step of upregulating SphK1 expression and / or activity is achieved by introducing a SphK1 overexpression vector into the cells.

5. The method according to claim 4, characterized in that, The SphK1 overexpression vector is a recombinant lentiviral vector.

6. A pharmaceutical composition, characterized in that, Contains an effective dose of SphK1 agonist and a pharmaceutically acceptable carrier for the prevention and / or treatment of gestational diabetes.

7. An in vitro method for screening candidate drugs for the prevention and / or treatment of gestational diabetes mellitus, characterized in that, The method includes the following steps: a) Culture human chorionic trophoblast cells under high glucose conditions; b) Treat the cells with the candidate drug; c) Detect the expression level and / or activity of SphK1 in the cells; If the candidate drug can significantly increase the expression level and / or activity of SphK1 in the cells, it indicates that the candidate drug is a potential drug for the prevention and / or treatment of GDM.

8. The method according to claim 7, characterized in that, In step c), the cell’s proliferative capacity and / or migration capacity are further detected.

9. A diagnostic reagent or kit for assessing the risk of gestational diabetes or placental function, characterized in that, The reagents or kits are used to detect the protein expression level or gene expression level of SphK1 in human placental tissue or isolated trophoblast cells.

10. The use of a combination of an SphK1 agonist and insulin in the preparation of a drug for treating gestational diabetes mellitus.

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