Application of RACK1 protein K106 site beta-hydroxybutyrylation modification level as a biomarker for evaluating the risk of neural tube defects associated with gestational diabetes mellitus

Abstract

The application discloses application of a RACK1 protein K106 site beta-hydroxybutyrylation modification level as a biomarker for evaluating a risk of a neural tube malformation related to maternal gestational diabetes, and relates to the fields of biological medicine and epigenetics. The application discloses for the first time a molecular mechanism of failure of neural tube closure of an embryo under a maternal gestational diabetes state: high sugar reduces RACK1 K106 site beta-hydroxybutyrylation modification by down-regulating CBP / p300, weakens RACK1-Vangl2 interaction, inhibits a PCP signal path, and causes abnormal neural tube closure. Based on the mechanism, the application establishes for the first time that the RACK1 K106 site beta-hydroxybutyrylation modification level is negatively correlated with a risk of occurrence of a neural tube malformation related to maternal gestational diabetes. The application provides a novel epigenetic biomarker and a theoretical basis for early risk evaluation, prenatal screening and auxiliary diagnosis of a neural tube malformation related to gestational diabetes.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and epigenetics, and in particular to the application of the β-hydroxybutyrylation modification level of the RACK1 protein K106 site as a biomarker for assessing the risk of neural tube defects associated with gestational diabetes mellitus. Background Technology

[0002] Neural tube defects (NTDs) are a group of serious congenital structural birth defects caused by abnormal closure of the neural tube in early embryonic development. In humans, the main clinical types include anencephaly, spina bifida, and encephalocele. Severe NTDs can lead to fetal death and place a significant burden on affected families and healthcare systems. Globally, the prevalence of NTDs is estimated at approximately 1.9 per 1,000 live births, with significant geographical variations closely related to socioeconomic status and public health policies.

[0003] Although folic acid supplementation during the perinatal period has been proven to effectively reduce the risk of NTDs by up to 70%, a significant number of cases cannot be prevented by folic acid supplementation, suggesting the existence of other important etiological mechanisms. Gestational diabetes mellitus (GDM) is one such clearly established, non-genetic, high-risk environmental factor independent of folic acid metabolism. Studies have shown that fetuses born to mothers with diabetes have a 2-5 times higher risk of birth defects than the general population. Meanwhile, diabetes is also considered one of the important environmental factors leading to NTDs, but its underlying pathogenic mechanisms are not yet fully understood.

[0004] Hyperglycemia during pregnancy disrupts the embryonic metabolic environment, leading to oxidative stress and lipid metabolism disorders. These disturbances have been shown to induce excessive apoptosis of neuroepithelial cells, thereby disrupting the dynamic balance required for normal neural tube closure. However, explaining the pathogenesis of GDM-related NTDs solely from the perspective of oxidative stress and apoptosis is significantly insufficient. Antioxidant interventions have not achieved ideal results in clinical translation, suggesting the existence of other key mechanisms independent of oxidative stress. Furthermore, abnormal alterations in various intermediate metabolites under GDM conditions may exert broader regulatory effects through epigenetic pathways, an area lacking systematic research. In recent years, epigenetic regulation, particularly post-translational modifications of proteins directly driven by specific metabolites, has become a cutting-edge perspective for elucidating the mechanisms of developmental disorders caused by metabolic diseases. However, a deep understanding of how GDM interferes with epigenetic regulation during embryonic neural tube development through metabolic disorders remains lacking.

[0005] β-hydroxybutyrate (bhb) is an important ketone body whose levels are significantly altered under metabolic states such as starvation, ketogenic diets, or diabetes. In recent years, a novel lysine post-translational modification mediated by bhb, β-hydroxybutyrylation (Kbhb), has been identified. This modification is enriched on histones and plays a crucial role in regulating gene transcription and metabolic reprogramming. Subsequent studies have shown that Kbhb may exert neuroprotective effects in neurological diseases by inducing oxidative stress and inflammatory responses. However, existing research on Kbhb modification mainly focuses on histones. Little is known about non-histone proteins, particularly those involved in signaling pathways, the biological functions of this modification during development, and their upstream catalytic enzyme systems. In particular, whether Kbhb modification participates in the regulation of neural tube development in embryos with GDM, and its specific molecular mechanisms, remain unexplored.

[0006] From a developmental biology perspective, neural tube closure is a highly coordinated spatiotemporal process involving multiple key cellular events such as neuroepithelial cell proliferation, polarization, and directional migration. Among these, the planar cell polarity (PCP) pathway is considered the master regulator of neural tube closure in vertebrates, primarily due to its crucial role in coordinating cell polarization and cytoskeleton remodeling during elongation. Genetic evidence indicates that disruption of the core PCP pathway genes Celsr1 and Vangl2 leads to severe neural tube defects in mice. Proper targeting and localization of the core membrane protein Vangl2 on the cell membrane is a prerequisite for its normal PCP signal transduction. Previous studies have clearly shown that the protein RACK1 (Receptor for Activated C Kinase 1) is an essential interacting protein for the proper membrane localization of Vangl2 and activation of the PCP pathway. RACK1 binds to Vangl2 through its WD40 repeat domain, assembling the PCP signaling complex and coordinating cell polarization establishment and directional movement. Conversely, loss of RACK1 function or disruption of RACK1-Vangl2 interaction directly leads to Vangl2 membrane localization failure, cell polarity disorder, and ultimately NTDs.

[0007] However, it remains unclear whether metabolic disorders in the context of GDM affect RACK1 function through post-translational modifications, thereby disrupting this key protein-protein interaction in the PCP signaling pathway and leading to neural tube defects.

[0008] In summary, the existing technologies have at least the following shortcomings: (1) the molecular mechanism of GDM-induced NTDs has not been fully elucidated, and there is a lack of systematic research from the perspective of metabolism and modification; (2) the specific functions and molecular mechanisms of non-histone Kbhb modification during embryonic development are still lacking; (3) there are no reports on whether RACK1 protein has functional Kbhb modification and the relationship between this modification and the risk of GDM-related NTDs; (4) there is a lack of biomarkers based on protein post-translational modifications for assessing the risk of GDM-related NTDs in clinical practice. Therefore, developing risk assessment biomarkers targeting β-hydroxybutyrylation modification at the K106 site of RACK1 protein has important clinical significance and application value. Summary of the Invention

[0009] The purpose of this invention is to provide the application of the β-hydroxybutyrylation modification level of the RACK1 protein K106 site as a biomarker for assessing the risk of gestational diabetes mellitus-associated neural tube defects (GDM), in order to solve the technical problem in the prior art of lacking effective epigenetic biomarkers for GDM-associated neural tube defects, which makes it impossible to conduct early risk assessment and prenatal screening for this disease.

[0010] Specifically, the molecular mechanisms by which gestational diabetes mellitus (GDM) leads to neural tube defects (NTDs) have not been fully elucidated by existing technologies. There is a lack of biomarkers based on epigenetic mechanisms. Biomarkers such as alpha-fetoprotein (AFP) used for NTD risk assessment lack specificity for GDM and cannot reflect the epigenetic mechanisms of the disease. The changes in non-histone β-hydroxybutyrylation modification during embryonic development and its relationship with disease risk have not been reported. The association between the level of β-hydroxybutyrylation modification at the K106 site of RACK1 protein and the risk of GDM-related neural tube defects has not been revealed. There is a lack of risk assessment kits that use β-hydroxybutyrylation modification at the K106 site of RACK1 protein as a detection target.

[0011] To achieve the above objectives, the present invention provides the following technical solution: the application of the β-hydroxybutyrylation modification level of the RACK1 protein K106 site as a biomarker for assessing the risk of neural tube defects associated with gestational diabetes mellitus.

[0012] Furthermore, the evaluation criteria are as follows: the level of β-hydroxybutyrylation modification at the K106 site of the RACK1 protein is negatively correlated with the risk of neural tube defects associated with gestational diabetes mellitus, that is, a decrease in the level of this modification indicates an increased risk of neural tube defects.

[0013] Furthermore, the gestational diabetes mellitus-associated neural tube defects are caused by abnormal glucose metabolism in the embryonic brain tissue, leading to changes in β-hydroxybutyrate levels, which in turn reduces the β-hydroxybutyrylation modification level of the RACK1 protein K106 site via CBP / p300.

[0014] Furthermore, the CBP / p300 is an upstream enzyme that catalyzes the β-hydroxybutyrylation modification of the RACK1 protein at the K106 site.

[0015] Furthermore, the detection methods for the β-hydroxybutyrylation modification level at the K106 site of the RACK1 protein include immunoblotting, immunoprecipitation, mass spectrometry, and antibody detection methods that specifically recognize K106bhb modification.

[0016] Furthermore, the test samples for the application are derived from embryonic tissue, amniotic fluid, or maternal blood.

[0017] Furthermore, the reagent for detecting the level of β-hydroxybutyrylation modification at the K106 site of the RACK1 protein is used in the preparation of a kit for assessing the risk of neural tube defects associated with gestational diabetes mellitus.

[0018] Furthermore, the kit contains an antibody that specifically recognizes the β-hydroxybutyrylation modification at the K106 site of the RACK1 protein.

[0019] Furthermore, the kit also includes reagents for detecting an internal reference protein, including β-actin.

[0020] Furthermore, the assessment is used for prenatal screening or auxiliary diagnosis of gestational diabetes mellitus-related neural tube defects.

[0021] Compared with the prior art, the present invention has the following advantages and effects:

[0022] (1) The application of the β-hydroxybutyrylation modification level of the RACK1 protein at K106 site provided by this invention as a biomarker for assessing the risk of neural tube defects associated with gestational diabetes mellitus (GDM) is the first to provide an epigenetic biomarker based on a complete molecular mechanism, and the first to reveal the complete molecular mechanism from metabolic disorder to neural tube closure failure in GDM: abnormal glucose metabolism - altered β-hydroxybutyrate levels - mediated by upstream catalytic enzyme CBP / p300 - decreased β-hydroxybutyrylation modification level of the RACK1 protein at K106 site - impaired interaction between RACK1 and Vangl2 protein - failure of Vangl2 membrane localization - instability of planar cell polarity signaling pathway - failure of embryonic neural tube closure. Based on this mechanism, this invention establishes the β-hydroxybutyrylation modification level of the RACK1 protein at K106 site as a risk assessment biomarker for the first time. The selection of this biomarker has a clear mechanistic basis, rather than empirical screening.

[0023] (2) Existing research on β-hydroxybutyrylation modification mainly focuses on histones. This invention is the first to identify a functional Kbhb modification site (K106) on non-histone RACK1 and associate it with disease risk assessment, thus expanding the research field and application scope of Kbhb modification.

[0024] (3) This invention discovers that CBP / p300 is a key upstream catalytic enzyme mediating β-hydroxybutyrylation modification of the RACK1 protein at the K106 site, and that the modification activity of CBP / p300 at the RACK1 K106 site is reduced in gestational diabetes mellitus. This discovery provides a theoretical basis for understanding the molecular basis of this biomarker change. There is a negative correlation between the level of β-hydroxybutyrylation modification at the RACK1 protein K106 site and the risk of neural tube defects associated with gestational diabetes mellitus, i.e., a decrease in modification level directly indicates an increased risk. This association is based on complete causal chain verification and has high reliability and reproducibility.

[0025] (4) Changes in β-hydroxybutyrylation modification at the K106 site of the RACK1 protein occur early in embryonic development, earlier than the formation of structural malformations. Therefore, this biomarker has a unique advantage in early risk assessment and can provide a valuable time window for clinical intervention. Changes in β-hydroxybutyrylation modification at the K106 site of the RACK1 protein are directly driven by metabolic disorders in gestational diabetes mellitus and have good disease specificity, which helps to distinguish gestational diabetes mellitus-related NTDs from NTDs caused by other reasons.

[0026] (5) The level of β-hydroxybutyrylation modification at the K106 site of RACK1 protein can be detected by a variety of mature techniques such as immunoblotting, immunoprecipitation, mass spectrometry or specific antibody detection, which has good operability and scalability.

[0027] (6) This invention provides a novel molecular target for prenatal screening of gestational diabetes mellitus-related neural tube defects. It can be used in combination with existing screening indicators such as alpha-fetoprotein to improve the sensitivity and specificity of screening.

[0028] (7) Based on the biomarkers disclosed in this invention, diagnostic kits, test strips or related testing equipment for assessing the risk of neural tube defects associated with gestational diabetes can be developed, which have good prospects for industrial transformation. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0030] Figure 1 A schematic flowchart of the experimental process provided in the embodiments of the present invention;

[0031] Figure 2 The image provided in this embodiment of the invention shows a stereomicroscopic observation of the neural tube closure morphology of mouse embryos; where A is a congenital embryo; B is an NTD embryo exhibiting spina bifida; C is an NTD embryo exhibiting incomplete closure; and D is an NTD embryo exhibiting anencephaly.

[0032] Figure 3 The following are the results of Western Blot detection of RACK1 K106bhb modification level in mouse embryonic brain tissue provided in this embodiment of the invention; wherein A is the Western Blot detection of β-hydroxybutyrylation modification level of RACK1 K106 site in mouse embryonic brain tissue; and B is the Western Blot detection of total RACK1 protein expression level in mouse embryonic brain tissue.

[0033] Figure 4 The images provided in this embodiment of the invention show the detection results of RACK1-Vangl2 interaction and Vangl2 membrane localization; where A is a Western blotting image of RACK1-Vangl2 interaction; and B is a Vangl2 membrane localization detection result image.

[0034] Figure 5 Figure 1 shows the qPCR detection and Western Blot verification results provided in this embodiment of the invention; where A is a bar chart of the relative expression levels of CBP mRNA and p300 mRNA in the embryonic brain tissue of the GDM model group and the normal control group; B is a Western Blot detection figure of CBP in mouse embryonic brain tissue of the GDM model group and the normal control group; and C is a Western Blot detection figure of p300 in embryonic brain tissue of the GDM model group and the normal control group.

[0035] Figure 6Figures showing the validation results of the in vitro cell model provided in this embodiment of the invention are as follows: A is a Western Blot diagram of the total RACK1 protein expression level in HT-22 cells; B is a Western Blot diagram of the β-hydroxybutyrylation modification level at the RACK1 K106 site; C is a Western Blot diagram of the interaction between Vangl2 and RACK1 detected by immunoprecipitation; D is a Western Blot diagram of the effect of the RACK1 K106R mutant on the RACK1-Vangl2 interaction; E is a validation diagram of the effect of the RACK1 K106R mutant on Vangl2 binding to RACK1; F is a Western Blot diagram of the effect of high glucose treatment on RHOA protein expression; G is a Western Blot diagram of the effect of high glucose treatment on Ror1 protein expression; H is a Western Blot diagram of the effect of high glucose treatment on AP-1 protein expression; I is a Western Blot diagram of the effect of high glucose treatment on CBP protein expression; J is a Western Blot diagram of the effect of high glucose treatment on p300 protein expression.

[0036] Figure 7 A complete molecular mechanism diagram of embryonic neural tube closure failure in gestational diabetes mellitus provided in an embodiment of the present invention. Detailed Implementation

[0037] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0038] Unless otherwise specified, the experimental methods used in the following examples are all conventional experimental methods; the materials and reagents used are all available through commercial channels unless otherwise specified.

[0039] As attached Figure 1 As shown:

[0040] Example 1:

[0041] This embodiment provides the construction of a mouse model of embryonic NTDs induced by maternal gestational diabetes mellitus.

[0042] 1. Establishment of experimental animals and models

[0043] SPF-grade female / male ICR mice, 6-8 weeks old, weighing 28±2g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After one week of acclimatization, the mice were mated in a 2:1 female-to-male ratio. Mice with vaginal plugs detected the following morning were defined as gestational day 0.5 (E0.5).

[0044] The pregnant mice were randomly divided into two groups of 30 each:

[0045] Normal control group: fed with normal feed, and injected intraperitoneally with an equal volume of citrate buffer (pH 4.5) at E5.5.

[0046] GDM model group: fed a high-fat diet, and injected streptozotocin (STZ, 200 mg / kg) into the peritoneum at E5.5 to induce a maternal gestational diabetes model.

[0047] Blood glucose levels in pregnant mice were measured at E8.5, E9.5, and E10.5. Mice with blood glucose levels ≥16.7 mmol / L for three consecutive days were considered to have successfully developed GDM model.

[0048] 2. Embryonic tissue collection and morphological observation of embryonic neural tube closure

[0049] Pregnant mice were sacrificed at E10.5 (the critical period for neural tube closure), and embryos were removed by cesarean section. The neural tube closure status of the embryos was observed and recorded under a stereomicroscope. Embryos were classified according to the degree of neural tube closure as follows:

[0050] Normal (CON) embryo: The neural tube is completely closed from the point of closure to both ends of the head and tail;

[0051] Neural tube defects (NTDs) in embryos: The neural tube fails to close, is only partially closed or not closed at all, and has open defects, including spina bifida, encephalocele, etc.

[0052] Embryonic brain tissue (including forebrain, midbrain and hindbrain regions) was collected. Some tissues were immediately fixed with 4% paraformaldehyde for histological examination, and some tissues were flash-frozen in liquid nitrogen and stored at -80°C for later use.

[0053] 3. Experimental Results

[0054] Stereoscopic observation revealed that the neural tube of embryos in the normal control group was intact and morphologically normal; embryos in the GDM model group showed significant neural tube closure failure, manifested as spina bifida, brain defects, and encephalocele. Figure 2 As shown.

[0055] HE staining revealed the outline of embryonic tissue, and the results showed that the neural tube of the NTD embryo was not closed.

[0056] The incidence of NTDs in each group of embryos was calculated. The statistical results showed that: a total of 376 embryos were collected in the normal control group, of which 4 cases of NTDs were found, with an incidence of 1.06% (4 / 376); a total of 387 embryos were collected in the GDM model group, of which 68 cases of NTDs were found, with an incidence of 17.57% (68 / 387), which was significantly higher than that in the normal control group (P<0.001).

[0057] Example 2:

[0058] This embodiment provides the detection of RACK1 protein and the β-hydroxybutyrylation modification level of RACK1 protein at the K106 site in the embryonic brain tissue of a mouse model of maternal gestational diabetes mellitus.

[0059] 1. Protein extraction and quantification

[0060] Embryonic brain tissue stored at -80℃ in Example 1 was randomly selected from 6 embryos in each group, with approximately 50 mg of each sample added. 500 μL of IRIPA lysis buffer (containing 1× protease inhibitor and 1× phosphatase inhibitor) was added, and the tissue was homogenized on ice and centrifuged at 4℃ (12000 rpm, 15 min). The supernatant was collected. Protein concentration was determined using the BCA method.

[0061] 2. Preparation of specific antibodies against RACK1 K106bhb

[0062] Four SPF-grade New Zealand white rabbits were immunized multiple times, and small amounts of serum were collected for ELISA testing to preliminarily assess the titer and specificity of the antiserum. Rabbits immunized with RACK1 K106bhb peptide A were designated R1 and R2, and rabbits immunized with RACK1 K106bhb peptide B were designated R3 and R4. Endogenous RACK1 K106bhb protein was detected in the immunized rabbit serum using Western blotting (WB), with Anti-RACK1Rabbit mAb added as an unmodified control. WB results showed that bands with a theoretical molecular weight of approximately 35 kDa of the target protein were detectable in both R1 and R3. Based on the combined results of ELISA and WB, serum from rabbits R1 and R3 was selected for purification.

[0063] Sufficient rabbit serum was collected and purified using Protein A and immunogenic peptide columns via affinity chromatography. The purified antibodies R1 and R3 were labeled Ab1 and Ab3, respectively. The purified antibodies were then analyzed by ELISA, Dot Blot, and Western blotting. ELISA results showed that A1 and Ab3 had a recognition ability for at least one modified peptide at a dilution of 1:50K, and were more than 10 times stronger than their recognition ability for unmodified negative peptides. Dot blot results showed that Ab1 and Ab3 exhibited strong binding to modified peptides but virtually no binding to unmodified negative peptides.

[0064] The positive antibodies Ab1 and Ab3 from the Dot / ELISA results were analyzed by Western blotting using Mouse spleen, Mouse brain, C2C12, and NIH-3T3 lysis buffers. Ab1 showed a band of approximately 35 kDa in both C2C12 and NIH-3T3 lysis buffers; this band represents the target protein RACK1 K106bhb. The specific peptide sequences are shown in Table 1.

[0065] Table 1. Peptide Information

[0066] RACK1 K106bhb modified peptide A CTRRFVGHT-(β-HydroxyButyryl)KDVLS β-hydroxybutyrylation 1703.88 RACK1 K106bhb modified peptide B CRRFVGHT-(β-HydroxyButyryl)KDVL β-hydroxybutyrylation 1515.80 RACK1 unmodified peptide CTRRFVGHTKDVLS / 1617.84

[0067] 3. Western Blot analysis of RACK1 K106bhb modification level

[0068] An equal amount of protein (30 μg) was subjected to SDS-PAGE electrophoresis (12% separating gel), transferred to a PVDF membrane (0.45 μm pore size), and blocked with 5% skim milk powder at room temperature for 1 h. The following primary antibodies (all diluted with antibody dilution buffer) were then added and incubated overnight at 4°C:

[0069] Anti-RACK1 K106bhb specific antibody (1:1000).

[0070] Anti-RACK1 total protein antibody (1:10000).

[0071] Anti-β-Actin antibody (1:5000, internal control).

[0072] The following day, after washing the membrane (3 washes with TBST, 10 min each), HRP-labeled secondary antibody (goat anti-rabbit IgG, 1:5000) was added, and the membrane was incubated at room temperature for 1 h. ECL chemiluminescence imaging was then performed, and images were taken using a gel imaging system. Gray-scale analysis was performed using ImageJ software. The expression level of RACK1K106bhb was detected using β-Actin as an internal reference, with the normal control group normalized to 1.00 as a reference. All experiments were independently repeated three times. Data are expressed as mean ± SD, and t-tests were used for comparisons between groups.

[0073] 3. Experimental Results

[0074] As attached Figure 3 As shown:

[0075] Western blot analysis showed that the RACK1 / β-Actin ratio in the embryonic brain tissue of the GDM model group was 1.07±0.02, which was not significantly different from that of the normal control group (1.00±0.08) (P>0.05).

[0076] In the normal control group, the level of β-hydroxybutyrylation modification of RACK1 protein at the K106 site was high in the embryonic brain tissue; in the GDM model group, the level of RACK1 K106bhb modification was significantly reduced in the embryonic brain tissue.

[0077] Quantitative analysis showed that the RACK1 K106bhb / β-Actin ratio in the GDM model group was 0.44±0.11, which was only 0.44±0.14 times that of the normal control group (1±0.18) (P<0.05), and the difference was statistically significant.

[0078] The above results indicate that during gestational diabetes mellitus, the expression level of RACK1 protein in embryonic brain tissue did not change significantly, but the β-hydroxybutyrylation modification level at the K106 site of RACK1 protein was significantly reduced.

[0079] Example 3:

[0080] This embodiment provides the detection of RACK1-Vangl2 interaction and Vangl2 membrane localization.

[0081] 1. Detection of RACK1-Vangl2 interaction by co-immunoprecipitation (Co-IP)

[0082] Embryonic brain tissue protein samples (6 animals per group) from Example 1 were collected, with 500 μg of total protein per sample. Anti-RACK1 antibody (4 μg) and Protein A / G agarose beads (20 μL) were added, and the samples were incubated overnight at 4°C by rotation. The next day, the samples were washed with RIPA lysis buffer, and 30 µL of LDS-PAGE loading buffer was added. After boiling denaturation, Western blotting was performed, and the Vangl2 content in the immunoprecipitation complex was detected using anti-Vangl2 antibody (1:1000).

[0083] 2. Immunofluorescence detection of Vangl2 membrane localization

[0084] Brain tissue from E10.5 embryos was sectioned in paraffin (5 μm thick) and stained with immunofluorescence: the primary antibody was anti-Vangl2 antibody (1:200), and the secondary antibody was Alexa Fluor 594-labeled goat anti-rabbit IgG. Cell nuclei were counterstained with DAPI, and the localization of Vangl2 on the cell membrane was observed under a confocal microscope.

[0085] 3. Experimental Results

[0086] As attached Figure 4 As shown:

[0087] Immunoprecipitation results showed that obvious co-precipitated bands of RACK1 and Vangl2 were visible in the normal control group, indicating that there was a significant protein interaction between RACK1 and Vangl2 in the embryonic brain tissue of the normal control group. The amount of RACK1-Vangl2 co-precipitation was significantly reduced in the GDM model group, and grayscale analysis showed that its interaction intensity was about 64.5% of that in the control group, indicating that the interaction between RACK1 and Vangl2 in the embryonic brain tissue of the GDM model group was significantly weakened.

[0088] Immunofluorescence results showed that Vangl2 protein in embryonic neuroepithelial cells of the normal control group was continuously linearly distributed on the cell membrane with clear localization; Vangl2 protein localization in embryonic neuroepithelial cells of the GDM model group was significantly reduced, and its localization on the cell membrane was abnormal, with some cells showing abnormal accumulation of Vangl2 in the cytoplasm.

[0089] The above results indicate that the reduced level of RACK1 K106bhb modification under GDM conditions leads to impaired RACK1-Vangl2 interaction, which in turn disrupts the normal localization of Vangl2 in the cell membrane.

[0090] Example 4:

[0091] This embodiment provides verification of the expression level of the upstream acyltransferase CBP / p300 in the GDM state.

[0092] 1. Experimental Methods

[0093] Embryonic brain tissue samples (6 animals per group) from Example 1 were taken, and total RNA and total protein were extracted. Real-time quantitative PCR (qPCR) was used for preliminary screening, and Western Blot was used to verify the expression changes of CBP / p300 at the protein level. β-Actin was used as an internal control, and the normal control group sample was calibrated to 1.0.

[0094] qPCR detection (screening):

[0095] Total RNA was extracted from the brain tissue of each group of embryos and reverse transcribed into cDNA.

[0096] qPCR amplification was performed using the SYBR Green method;

[0097] The primer sequences are as follows:

[0098] CBP: positive 5'-GGCTTCTCCGCGAATGACAA-3';

[0099] Reverse 5'-GTTTGGACGCAGCATCTGGA-3';

[0100] p300: positive 5'-TTCAGCCAAGCGGCCTAAA-3';

[0101] Reverse 5'-CGCCACCATTGGTTAGTCCC-3';

[0102] Internal reference (β-Actin): positive 5'-GTGCTATGTTGCTCTAGACTTCG-3';

[0103] Reverse 5'-ATGCCACAGGATTCCATACC-3';

[0104] Reaction conditions: 95℃ pre-denaturation for 60s; 95℃ for 15s, 60℃ for 15s, 40 cycles;

[0105] Use 2 −ΔΔCt The relative expression levels were calculated using a method to screen out molecules with significant changes in expression.

[0106] Western Blot validation:

[0107] Total protein was extracted from the embryonic brain tissue of each group, and after quantification, it was separated by SDS-PAGE electrophoresis, as in Example 2.

[0108] After transfer, the following antibodies were incubated:

[0109] Anti-CBP antibody (1:1000);

[0110] Anti-p300 antibody (1:1000);

[0111] Anti-β-Actin antibody (1:5000, internal control);

[0112] ECL was developed after incubation with the secondary antibody, and the gray values ​​of the bands were quantitatively analyzed using ImageJ software.

[0113] 2. Experimental Results

[0114] As attached Figure 5 As shown:

[0115] qPCR screening results:

[0116] The relative expression levels of CBP mRNA in the embryonic brain tissue of the GDM model group were 0.80±0.05 (n=6) and p300 mRNA were 0.72±0.05; in the normal control group, the relative expression levels of CBP mRNA and p300 mRNA were 1.00±0.05 and 1.00±0.03, respectively. Compared with the normal control group, the expression levels of CBP and p300 mRNA in the embryonic brain tissue of the GDM model group were significantly decreased (P<0.05).

[0117] Western Blot validation results:

[0118] Compared with the normal control group (1.12±0.11), the protein expression level of CBP in the embryonic brain tissue of the GDM model group (0.54±0.07) was significantly reduced (P<0.05); compared with the normal control group (2.04±0.09), the protein expression level of p300 in the embryonic brain tissue of the GDM model group (1.16±0.10) was also significantly reduced (P<0.01), which is consistent with the trend of qPCR results.

[0119] The above results indicate that CBP / p300 is an upstream enzyme that promotes β-hydroxybutyrylation modification of the RACK1 protein at the K106 site. Under GDM conditions, the expression of CBP / p300 at both the mRNA and protein levels was significantly downregulated, resulting in a decrease in its activity for β-hydroxybutyrylation modification of the RACK1 K106 site.

[0120] Example 5:

[0121] This embodiment provides in vitro cell model validation.

[0122] 1. Materials and Methods

[0123] The mouse hippocampal neuron cell line HT-22 (purchased from Guangzhou Genio Biotechnology Co., Ltd.) was used for all in vitro experiments in this embodiment. Cells were cultured in high-glucose DMEM medium (containing 4.5 g / L glucose, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin / streptomycin (Pronoseu), and incubated at 37°C in a 5% CO2 incubator. Cells were passaged every 2-3 days, and cells in the logarithmic growth phase were used for further processing.

[0124] Based on the experimental objectives, the cells were grouped and treated as follows:

[0125] Control group: cultured in conventional high-glucose DMEM medium.

[0126] High Glucose (HG) treatment group: The medium was replaced with DMEM containing 125 mM glucose and treated for 24-48 hours.

[0127] Mutant group (RACK1 K106R): pCDNA3.1-RACK1-K106R mutant plasmid (constructed using QuikChange II site-directed mutagenesis kit and confirmed by DNA sequencing) was transfected into HT-22 cells via liposome transfection, resulting in a stable G418 (400 µg / mL) HT-22 cell line.

[0128] 2. Protein interaction detection (immunoprecipitation, Co-IP)

[0129] HT-22 cells from each group were collected and lysed on ice for 30 minutes using RIPA lysis buffer containing protease and phosphatase inhibitors. The supernatant was collected by centrifugation at 12,000 g for 15 minutes at 4°C. An equal volume of total protein (500 μg) was added, along with 4 μg of anti-Vangl2 antibody and 20 μL of Protein A / G agarose beads. The mixture was incubated overnight at 4°C by rotation. The precipitate complex was washed the next day, denatured by boiling in SDS loading buffer, and then subjected to SDS-PAGE. Western blot analysis was performed using anti-RACK1 and anti-Vangl2 antibodies, respectively.

[0130] 3. Protein expression detection (Western blot)

[0131] Total protein (30 μg) was extracted from each group of cells and subjected to Western blotting according to the method described in Example 2. Primary antibodies included: anti-RACK1, anti-Vangl2, anti-Ror1, anti-RHOA, anti-AP1 (c-Jun / c-Fos), anti-CBP, anti-p300, and anti-β-Actin (internal control). Gray-scale analysis was performed as in Example 2.

[0132] 4. Experimental Results

[0133] As attached Figure 6 As shown:

[0134] 1) High glucose treatment reduces RACK1 K106bhb levels and weakens RACK1-Vangl2 interaction:

[0135] Western blot results showed that, compared with the control group, there was no significant change in the total RACK1 protein expression in HT-22 cells treated with high glucose. However, the RACK1 K106bhb modification level was significantly decreased compared with the control group (1.076±0.102) (0.745±0.066) (P<0.01). Co-IP experiments indicated that the amount of the RACK1-Vangl2 immunoprecipitation complex was significantly reduced after high glucose treatment, suggesting that high glucose inhibits their interaction.

[0136] 2) The RACK1 K106 mutation disrupts its binding to Vangl2:

[0137] A RACK1 K106R (simulated deacylation) mutant was constructed. Co-IP results showed that the amount of co-precipitation of RACK1 and Vangl2 was reduced by about 70% compared with the wild-type RACK1 transfection group. The K106R mutation significantly weakened the interaction between RACK1 and Vangl2 (compared with wild-type RACK1), further confirming that the K106 site plays a key role in maintaining the formation of the RACK1-Vangl2 complex.

[0138] 3) High glucose levels induce NTD-related phenotypes by inhibiting the PCP signaling pathway:

[0139] Western blot analysis of key downstream molecules in the PCP signaling pathway revealed that the expression levels of Ror1, RHOA, and AP1 (c-Jun / c-Fos) proteins in the high-glucose treatment group were significantly lower than those in the control group (P<0.01 or P<0.05), indicating that high glucose inhibits PCP signaling pathway activity. This is consistent with the cellular model characteristics of neural tube defects (NTDs). Specific results are shown in Table 2.

[0140] Table 2 Comparison of relative expression levels of key proteins in the PCP signaling pathway between the control group and the high glucose group

[0141] Ror1 / β-Actin (relative multiple) 0.96±0.05 0.31±0.05** RHOA / β-Actin (relative multiple) 1.00±0.07 0.33±0.06** AP1 / β-Actin (relative fold) 0.96±0.05 0.63±0.10**

[0142] Note: ** indicates statistical significance (P < 0.01, extremely significant difference).

[0143] 4) CBP / P300-mediated RACK1 K106bhb modification:

[0144] Western blot analysis was performed on HT-22 cells treated with high glucose. The results showed that, compared with the control group (1.55±0.17), the expression level of CBP protein in the high glucose-treated group (0.74±0.11) was significantly decreased (P<0.01); the expression level of p300 protein in the high glucose-treated group (0.11±0.04) was also significantly decreased compared with the control group (1.02±0.03). CBP / p300 protein expression levels were positively correlated with RACK1 K106bhb modification levels. Combined with the aforementioned result of significantly reduced K106bhb modification levels in the RACK1 K106R mutant, this suggests that CBP / P300 may be involved in mediating RACK1 K106bhb modification.

[0145] Example 6:

[0146] This embodiment provides a summary of mechanism verification.

[0147] Based on the results of the above embodiments, this invention reveals for the first time the complete molecular mechanism of neural tube failure in embryos during gestational diabetes mellitus, as shown in the appendix. Figure 7 As shown:

[0148] Gestational diabetes mellitus → abnormal glucose metabolism → altered β-hydroxybutyrate levels → mediated by upstream catalytic enzyme CBP / p300 → decreased β-hydroxybutyrylation modification at the K106 site of RACK1 protein → impaired interaction between RACK1 and Vangl2 protein → failure of Vangl2 membrane localization → instability of planar cell polarity signaling pathways → failure of neural tube closure in the embryo, leading to neural tube defects.

[0149] Based on the above mechanism, this invention establishes for the first time a negative correlation between the level of β-hydroxybutyrylation modification at the K106 site of the RACK1 protein and the risk of neural tube defects associated with gestational diabetes mellitus (GDM), i.e., a decreased modification level indicates an increased risk of neural tube defects. This biomarker can be used for early risk assessment, prenatal screening, and auxiliary diagnosis of GDM-related NTDs.

[0150] As attached Figure 1 The diagram shown is a schematic representation of the complete molecular mechanism described in this invention.

[0151] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. Application of the β-hydroxybutyrylation modification level of RACK1 protein at K106 site as a biomarker for assessing the risk of maternal gestational diabetes mellitus-related neural tube defects.

2. The application according to claim 1, characterized in that, The evaluation criteria were as follows: the level of β-hydroxybutyrylation modification at the K106 site of the RACK1 protein was negatively correlated with the risk of neural tube defects associated with gestational diabetes mellitus, and a decrease in the modification level indicated an increased risk of neural tube defects.

3. The application according to claim 1, characterized in that, The gestational diabetes mellitus-associated neural tube defects are caused by abnormal glucose metabolism in the embryonic brain tissue, leading to changes in β-hydroxybutyrate levels, which in turn reduces the level of β-hydroxybutyrylation modification at the K106 site of the RACK1 protein through CBP / p300.

4. The application according to claim 3, characterized in that, The CBP / p300 is an upstream enzyme that catalyzes the β-hydroxybutyrylation modification of the RACK1 protein at the K106 site.

5. The application according to claim 1, characterized in that, The methods for detecting the β-hydroxybutyrylation modification level at the K106 site of the RACK1 protein include immunoblotting, immunoprecipitation, mass spectrometry, and antibody detection methods that specifically recognize RACK1K106bhb modification.

6. The application according to claim 1, characterized in that, The application of reagents for detecting the level of β-hydroxybutyrylation modification at the K106 site of the RACK1 protein in the preparation of kits for assessing the risk of neural tube defects associated with gestational diabetes mellitus.

7. The application according to claim 6, characterized in that, The kit contains an antibody that specifically recognizes the β-hydroxybutyrylation modification at the K106 site of the RACK1 protein.

8. The application according to claim 7, characterized in that, The kit also includes reagents for detecting an internal reference protein, including β-actin.

9. The application according to claim 1, characterized in that, The assessment is used for prenatal screening or auxiliary diagnosis of gestational diabetes mellitus-associated neural tube defects.

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