Use of a sucnr1 gene expression inhibitor in the preparation of a product for preventing or treating retained placenta in a dairy cow

Abstract

The application discloses application of a SUCNR1 gene expression inhibitor in preparation of a product for preventing or treating a dairy cow dystocia. The application provides application of a substance taking a SUCNR1 protein as an inhibition target and / or a substance taking a SUCNR1 gene as an inhibition target in preparation of a product. The application of the product is as follows: promoting trophoblast cell apoptosis; inhibiting macrophage M2 type polarization; promoting trophoblast cell apoptosis by inhibiting macrophage M2 type polarization. The inventors of the application find that silencing the SUCNR1 gene can inhibit a PI3K signal pathway, thereby inhibiting macrophage M2 type polarization, thereby promoting trophoblast cell apoptosis, thereby helping to solve the problem of dystocia.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of SUCNR1 gene expression inhibitors in the preparation of products for the prevention or treatment of retained placenta in dairy cows. Background Technology

[0002] Retained placenta (RP) is a common reproductive disorder in dairy cows. Epidemiological surveys show that in 2023, the average incidence of RP in large-scale dairy farms in my country was 6.2%, while in areas with poor farming practices and conditions, the incidence could be as high as 20.5%. The placenta, retained in the uterus, decomposes and putrefies, leading to secondary infections and causing puerperal metritis or endometritis. This not only significantly reduces milk production but also greatly affects the reproductive performance of dairy cows. Simultaneously, RP significantly increases the incidence of other secondary diseases, such as abomasal displacement, milk fever, ketosis, and mastitis, causing many dairy cows to be culled prematurely, resulting in substantial economic losses for the dairy industry. The occurrence of RP is a complex biological process involving the adhesion of the maternal placenta and fetal placenta within the uterus after calving. Its causes mainly include hormonal metabolic disorders, uterine atony, incomplete decomposition of the extracellular matrix of placental tissue, immune cell dysfunction, and inhibition of trophoblast cell apoptosis. Despite extensive research into the causes of this disease in recent years, the key mechanisms underlying its pathogenesis remain unclear. Summary of the Invention

[0003] The purpose of this invention is to provide the application of SUCNR1 gene expression inhibitors in the preparation of products for the prevention or treatment of retained placenta in dairy cows.

[0004] This invention provides the application of substances that target the SUCNR1 protein and / or the SUCNR1 gene in the preparation of products; the purpose of the products is to promote apoptosis of trophoblast cells.

[0005] The present invention also provides the application of substances that target the SUCNR1 protein and / or substances that target the SUCNR1 gene in the preparation of products; the products are used to inhibit macrophage M2 polarization.

[0006] The present invention also provides the application of substances that target the SUCNR1 protein and / or substances that target the SUCNR1 gene in the preparation of products; the purpose of the products is to promote trophoblast cell apoptosis by inhibiting macrophage M2 polarization.

[0007] The present invention also provides the application of substances targeting SUCNR1 protein and / or SUCNR1 gene as inhibitory targets in the preparation of products; the purpose of the products is to inhibit the "inhibitory effect of macrophage M2 polarization on trophoblast cell apoptosis" by inhibiting macrophage M2 polarization.

[0008] The present invention also provides the application of substances targeting the SUCNR1 protein and / or the SUCNR1 gene as inhibitory targets in the preparation of products; the products are used to inhibit macrophage M2 polarization by inhibiting the PI3K signaling pathway.

[0009] The present invention also provides the use of substances that target the SUCNR1 protein and / or substances that target the SUCNR1 gene in the preparation of products; the products are used for the prevention and / or treatment of retained placenta in dairy cows.

[0010] Specifically, the substance that targets the SUCNR1 gene is a siRNA that targets the SUCNR1 gene.

[0011] The siRNA is si-Sucnr1-2, si-Sucnr1-1, or si-Sucnr1-3.

[0012] The si-Sucnr1-2 is an siRNA composed of the nucleic acid molecules shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0013] The si-Sucnr1-1 is an siRNA composed of the nucleic acid molecules shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0014] The si-Sucnr1-3 is an siRNA composed of the nucleic acid molecules shown in SEQ ID NO: 5 and SEQ ID NO: 6.

[0015] Specifically, the trophoblast cells are bovine trophoblast cells.

[0016] Specifically, the macrophages are bovine macrophages.

[0017] Specifically, the trophoblast cells are trophoblast cells derived from the placenta.

[0018] Specifically, the cow in question is a Holstein dairy cow.

[0019] Specifically, the placenta is a Holstein cow placenta.

[0020] The inhibition of macrophage M2 polarization is manifested by downregulating the abundance of M2 polarization marker proteins in macrophages. The inhibition of macrophage M2 polarization is manifested by downregulating the expression levels of M2 polarization marker genes in macrophages. The M2 polarization marker proteins are CD206 protein and / or CD163 protein and / or TGF-β protein and / or ARG-1 protein. The M2 polarization marker genes are CD206 gene and / or ARG-1 gene and / or IL-10 gene and / or TGF-β gene. The CD206 protein is bovine CD206 protein. The CD163 protein is bovine CD163 protein. The TGF-β protein is bovine TGF-β protein. The ARG-1 protein is bovine ARG-1 protein. The CD206 gene is bovine CD206 gene. The ARG-1 gene is bovine ARG-1 gene. The IL-10 gene is bovine IL-10 gene. The TGF-β gene is the bovine TGF-β gene.

[0021] The promotion of bovine trophoblast cell apoptosis is manifested by upregulating the abundance of pro-apoptotic proteins and / or downregulating the abundance of anti-apoptotic proteins in bovine trophoblast cells. The promotion of bovine trophoblast cell apoptosis is manifested by upregulating the expression of pro-apoptotic genes and / or downregulating the expression of anti-apoptotic genes in bovine trophoblast cells. The pro-apoptotic protein is BAX protein and / or CASP3 protein. The anti-apoptotic protein is BCL2 protein. The pro-apoptotic gene is CASP3 gene and / or CASP8 gene and / or BAX gene. The anti-apoptotic gene is BCL2 gene. The BAX protein is bovine BAX protein. The CASP3 protein is bovine CASP3 protein. The BCL2 protein is bovine BCL2 protein. The CASP3 gene is bovine CASP3 gene. The CASP8 gene is bovine CASP8 gene. The BAX gene is bovine BAX gene. The BCL2 gene is bovine BCL2 gene.

[0022] The SUCNR1 protein is bovine SUCNR1 protein. The SUCNR1 gene is bovine SUCNR1 gene.

[0023] Bovine SUCNR1 protein, also known as Bos taurus succinate receptor 1.

[0024] The bovine SUCNR1 protein is encoded by the bovine SUCNR1 gene.

[0025] Gene ID of bovine SUCNR1 gene: 539622.

[0026] Specifically, the bovine SUCNR1 gene is shown in NCBI Reference Sequence: XM_003585693.6 (26-DEC-2024).

[0027] Specifically, the bovine SUCNR1 protein is shown in NCBI Reference Sequence: XP_003585741.2 (26-DEC-2024).

[0028] Bovine CD206 protein, also known as Bos taurus mannose receptor C-type 1.

[0029] The bovine CD206 protein is encoded by the bovine CD206 gene.

[0030] Gene ID of bovine CD206 gene: 787578.

[0031] Specifically, the bovine CD206 gene is shown in NCBI Reference Sequence: XM_003586772.6 (26-DEC-2024).

[0032] Specifically, bovine CD206 protein is shown in XP_003586820.2 (26-DEC-2024).

[0033] Bovine CD163 protein, also known as Bos taurus CD163 molecule.

[0034] The bovine CD163 protein is encoded by the bovine CD163 gene.

[0035] Gene ID of bovine CD163 gene: 533844.

[0036] Specifically, the bovine CD163 gene is shown in NCBI Reference Sequence: NM_001163413.1 (02-APR-2025).

[0037] Specifically, bovine CD163 protein is shown in NCBI Reference Sequence: NP_001156885.1 (02-APR-2025).

[0038] Bovine TGF-β protein, also known as Bos taurus transforming growth factor beta 1.

[0039] Bovine TGF-β protein is encoded by the bovine TGF-β gene.

[0040] Gene ID of bovine TGF-β gene: 282089.

[0041] Specifically, the bovine TGF-β gene is shown in NCBI Reference Sequence: NM_001166068.1 (20-JUN-2025).

[0042] Specifically, bovine TGF-β protein is shown in NCBI Reference Sequence: NP_001159540.1 (20-JUN-2025).

[0043] Bovine ARG-1 protein, also known as Bos taurus arginase 1.

[0044] The bovine ARG-1 protein is encoded by the bovine ARG-1 gene.

[0045] Gene ID of bovine ARG-1 gene: 513608.

[0046] Specifically, the bovine ARG-1 gene is shown in NCBI Reference Sequence: NM_001046154.1 (22-JUN-2025).

[0047] Specifically, bovine ARG-1 protein is shown in NCBI Reference Sequence: NP_001039619.1 (22-JUN-2025).

[0048] Bovine IL-10 protein, also known as Bos taurus interleukin 10.

[0049] Bovine IL-10 protein is encoded by the bovine IL-10 gene.

[0050] Gene ID of bovine IL-10 gene: 281246.

[0051] Specifically, the bovine IL-10 gene is shown in NCBI Reference Sequence: NM_174088.1 (27-APR-2025).

[0052] Specifically, bovine IL-10 protein is shown in NCBI Reference Sequence: NP_776513.1 (27-APR-2025).

[0053] Bovine BAX protein, also known as Bos taurus BCL2 associated X, apoptosis regulator.

[0054] Bovine BAX protein is encoded by the bovine BAX gene.

[0055] Gene ID of bovine BAX gene: 280730.

[0056] Specifically, the bovine BAX gene is shown in NCBI Reference Sequence: NM_173894.1 (02-APR-2025).

[0057] Specifically, bovine BAX protein is shown in NCBI Reference Sequence: NP_776319.1 (02-APR-2025).

[0058] Bovine CASP3 protein is also known as Bos taurus caspase 3.

[0059] The bovine CASP3 protein is encoded by the bovine CASP3 gene.

[0060] Gene ID of bovine CASP3 gene: 408016.

[0061] Specifically, the bovine CASP3 gene is shown in NCBI Reference Sequence: NM_001077840.1 (28-APR-2025).

[0062] Specifically, bovine CASP3 protein is shown in NCBI Reference Sequence: NP_001071308.1 (28-APR-2025).

[0063] Bovine BCL2 protein is also known as Bos taurus BCL2 apoptosis regulator.

[0064] Bovine BCL2 protein is encoded by the bovine BCL2 gene.

[0065] Gene ID of bovine BCL2 gene: 281020.

[0066] Specifically, the bovine BCL2 gene is shown in NCBI Reference Sequence: NM_001166486.1 (27-APR-2025).

[0067] Specifically, bovine BCL2 protein is shown in NCBI Reference Sequence: NP_001159958.1 (27-APR-2025).

[0068] Bovine CASP8 protein, also known as Bos taurus caspase 8.

[0069] The bovine CASP8 protein is encoded by the bovine CASP8 gene.

[0070] Gene ID of bovine CASP8 gene: 507481.

[0071] Specifically, the bovine CASP8 gene is shown in NCBI Reference Sequence: XM_005202615.5 (26-DEC-2024).

[0072] Specifically, bovine CASP8 protein is shown in NCBI Reference Sequence: XP_005202672.1 (26-DEC-2024).

[0073] Succinate is an important metabolic intermediate in the tricarboxylic acid cycle. Succinate receptor 1 (SUCNR1) is the only known specific receptor for succinate, located on the cell membrane and specifically binding to succinate. Macrophage M2 polarization inhibits trophoblast cell apoptosis, leading to retained placenta. Using biochemical, molecular biological, cell biological, and bioinformatics research methods, the inventors of this invention found reduced apoptosis in placental tissue from cows with retained placenta, and significantly increased succinate content and SUCNR1 abundance (including protein and mRNA abundance). Furthermore, the inventors discovered that silencing the SUCNR1 gene can inhibit the PI3K signaling pathway, thereby suppressing macrophage M2 polarization and promoting trophoblast cell apoptosis, thus helping to resolve the problem of retained placenta. Attached Figure Description

[0074] Figure 1 The result diagram is from Example 1.

[0075] Figure 2 This is a result diagram of Example 2.

[0076] Figure 3 This is a result diagram of Example 3.

[0077] Figure 4 This is a result diagram of Example 4.

[0078] Figure 5 This is a result diagram of Example 5.

[0079] Figure 6 This is a result diagram of Example 6.

[0080] Figure 7 The result diagram is shown in Example 7. Detailed Implementation

[0081] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0082] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. LPS and IFN-γ induce macrophage M1 polarization. IL-4 and IL-10 induce macrophage M2 polarization. Opti-MEM serum-depleted culture medium: Thermo Fisher Scientific, catalog number 31985070. RNAFit transfection reagent: Hanbio Biotechnology. 1% penicillin-streptomycin: Gibco Scientific, catalog number 15140122. LY294002 (PI3K specific inhibitor): CAS number 154447-36-6, MedChemExpress, catalog number HY-10108. Unless otherwise specified, all quantitative experiments in the following examples were performed in triplicate, and the results were averaged. Unless otherwise specified, cell culture conditions were: 37℃, 5% CO2. RPMI-1640 complete medium: RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. DMEM-F12 complete medium: DMEM-F12 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. IL-4 solution was obtained by dissolving IL-4 in sterile PBS buffer containing 0.1% BSA. IL-10 solution was obtained by dissolving IL-10 in sterile PBS buffer containing 0.1% BSA. LY294002 solution was obtained by dissolving LY294002 in DMSO.

[0083] Bovine trophoblast cells (BTCs) are described in the following literature: Exosomes derived from placental trophoblast cells regulate endometrial epithelial receptivity in dairy cows during pregnancy; Journal of Reproduction and Development, Vol. 68, No 1, 2022.

[0084] Bovine macrophages (BoMac) are described in the following literature: Novel Secreted Protein of Mycoplasma bovis MbovP280 Induces Macrophage Apoptosis ThroughCRYAB; Frontiers in Immunology; February 2021, Volume 12, Article 619362.

[0085] Example 1: Reduced apoptosis level in placental tissue cells of cows with retained placenta. Source of placental tissue samples from dairy cows: Placental tissue samples were collected from cows of similar parity (2-4 parities) and body condition within 30 minutes of calving. Cows were divided into a healthy group (n=6) and a group with retained placenta (n=6) based on whether the placenta had been expelled within 12 hours postpartum. The healthy group was denoted as Control, and the group with retained placenta was denoted as RP.

[0086] 1. Organize TUNEL examination Apoptosis in placental tissue was detected using the TUNEL BrightGreen Apoptosis Detection Kit (A112, Vazyme, Nanjing, China) and the EdUTP TUNEL Apoptosis Assay Kit (C1182, Beyotime, Shanghai, China). For fluorescent TUNEL staining, FITC-12-dUTP was used to label the ends of fragmented nuclear DNA, and apoptotic cells were identified by the green fluorescence signal in the nucleus. The fluorescence signal was observed using a confocal fluorescence microscope (A1HD25, NIKON, Tokyo, Japan). For DAB-based TUNEL staining, cells with fragmented nuclear chromatin and DAB-positive brown staining of the nucleus were identified as apoptotic cells, and the images were observed using an optical microscope (ECLIPSE Ci, NIKON, Tokyo, Japan).

[0087] The results of fluorescent TUNEL staining are shown in Figure 1 A (i) and Figure 1 B(i). The results showed that, compared with the healthy group, the number of green fluorescent positive cell nuclei in the placental region of the retained placenta group was significantly reduced, and the intensity of the green fluorescence signal was significantly weakened, suggesting a decreased level of apoptosis in this region. The results of DAB-modified TUNEL staining are shown in [Figure number missing]. Figure 1 A(ii) and Figure 1 B(ii). The results further confirmed that the proportion of brownish-red, nuclear-positive apoptotic cells in the placental tissue of dairy cows in the retained placenta group was significantly lower than that in the healthy group.

[0088] 2. Protein immunoblotting assay (Western blot) To further clarify the molecular changes related to apoptosis in placental tissue, apoptosis-related proteins were detected. Placental tissue was collected, total protein was extracted, and Western blot was performed. The primary antibody used for Western blot was either BAX polyclonal antibody (50599-2-Ig, Proteintech, Wuhan, China), or Anti-Bcl-2 Rabbit pAb (WL01556, WANLEIBIO, Shenyang, China), or Anti-Caspase-3 / Cleaved Caspase-3 Rabbit pAb (WL02117, WANLEIBIO, Shenyang, China), or β-actin antibody (AF7018, Affinity Biosciences, Jiangsu, China). The secondary antibody used for Western blot was HRP-labeled goat anti-rabbit IgG (H+L) secondary antibody (RGAR001, Proteintech, Wuhan, China). The chromogenic reagent used for Western blot was an enhanced chemiluminescence reagent (MA0186, MeilunBio, Dalian, China). Data was acquired using an imaging system (5200Multi, Tanon, Shanghai, China). ImageJ software (version 1.53, National Institutes of Health, Bethesda, USA) was used for density analysis of the band grayscale values. β-actin on the same membrane was used as an internal reference for normalization, and the relative abundance of the target protein was calculated.

[0089] See results Figure 1 C and Figure 1 D. The results showed that, compared with the healthy group, the abundance of pro-apoptotic proteins (BAX and cleaved caspase-3) in the placental tissue of cows with retained placenta was significantly reduced, while the abundance of anti-apoptotic protein (BCL2) in the placental tissue of cows with retained placenta was significantly increased.

[0090] 3. Quantitative real-time PCR (qRT-PCR) Placental tissue was collected, total RNA was extracted, and cDNA was obtained by reverse transcription. Using cDNA as a template and β-actin as an internal reference gene, target genes (CASP3, CASP8, CASP9, BAX, or BCL2) were detected by qRT-PCR. The relative expression levels of the target genes were obtained using the 2^-ΔΔCt method. Primers for detecting the target genes and internal reference genes are shown in Table 1.

[0091] Table 1

[0092] The qRT-PCR test results are shown below. Figure 1 E. Compared with the healthy group, the relative expression levels of apoptosis-related genes (CASP3, CASP8, CASP9, and BAX genes) in the placental tissue of dairy cows with retained placenta were significantly downregulated, while the relative expression level of anti-apoptotic gene (BCL2 gene) in the placental tissue of dairy cows with retained placenta was significantly increased.

[0093] 4. Tissue immunofluorescence Immunofluorescence detection was performed using CK7 as a marker for trophoblast cells. Placental tissue was harvested, and paraffin sections with a thickness of 4-5 μm were prepared for immunofluorescence staining. The primary antibodies used for immunofluorescence staining were Cytokeratin 7 Monoclonal antibody (66483-1-Ig, ProteinTech) and BAX Polyclonal antibody (50599-2-Ig, ProteinTech, Wuhan, China). A fluorescent secondary antibody targeting the primary antibody was used for immunofluorescence. Cell nuclei were counterstained with DAPI. Observation and image acquisition were performed using confocal fluorescence microscopy. Images of all sections were acquired under the same parameters, and five non-overlapping fields were randomly selected from each sample. Quantitative analysis of the target protein fluorescence signal was performed using ImageJ software.

[0094] See results Figure 1 F and Figure 1 G (the ordinate index is the relative BAX fluorescence index, defined as the ratio of the BAX fluorescence integral density in the CK7-positive region to the area of ​​that CK7-positive region within the same field of view, i.e., the relative fluorescence intensity of BAX per unit area of ​​the trophoblast region). The results showed that the relative BAX fluorescence index in the placental tissue of dairy cows in the retained placenta group was significantly lower than that in the healthy group.

[0095] The results of Example 1 showed that, compared with the healthy group, the apoptosis level of placental tissue cells in the cows with retained placenta was significantly inhibited.

[0096] Example 2: Succinic acid accumulation in placental tissue of retained placenta in dairy cows Source of placental tissue samples from dairy cows: Placental tissue samples were collected from cows of similar parity (2-4 parities) and body condition within 30 minutes of calving. Cows were divided into a healthy group (n=6) and a group with retained placenta (n=6) based on whether the placenta had been expelled within 12 hours postpartum. The healthy group was denoted as Control, and the group with retained placenta was denoted as RP.

[0097] 1. LC-MS / MS liquid chromatography-tandem mass spectrometry 50 mg of placental tissue was weighed and added to pre-cooled methanol / acetonitrile / water extraction buffer (2:2:1, v / v / v) and a stable isotope internal standard, followed by thorough homogenization at low temperature. The sample was vortexed, incubated on ice, and centrifuged at 4°C, and the supernatant was collected. The supernatant was concentrated under vacuum, reconstituted with the initial mobile phase, filtered through a 0.22 μm filter, and then analyzed. Polar metabolites were detected using a Vanquish UHPLC system (Thermo Fisher, USA) coupled with an Orbitrap Exploris 120 mass spectrometer, with chromatographic separation using a BEH Amide column. Data were acquired in both positive and negative ion electrospray ionization modes. A standard curve was established using succinic acid as a standard, and quantification was performed using the internal standard method. The succinic acid concentration in the sample was calculated based on the peak area ratio and the standard curve, and finally converted to the succinic acid content per unit mass of tissue. Metabolite annotation was performed based on the BiotreeDB (v3.0) database. After data preprocessing and metabolite annotation, multivariate statistical methods were used to analyze the dataset. Variable importance inprojection (VIP) values ​​were calculated using orthogonal partial least squares discriminant analysis (OPLS-DA) to evaluate the contribution of each metabolite to the separation between groups. P Metabolites with a VIP value <0.05 and a VIP value >1 were defined as statistically significant differential metabolites.

[0098] The OPLS-DA score plot showed a clear separation between the retained placenta group and the healthy group at the metabolic profile level. Figure 2 A). Volcano plot analysis revealed differential metabolites, in which... P After using <0.05 and VIP>1 as screening criteria, a total of 474 differentially expressed metabolites were identified. Among them, the abundance of 329 metabolites was increased in the retained placenta group, while the abundance of the remaining metabolites was decreased. Figure 2 B). Further screening using fold change thresholds >1.5 or <0.67 yielded 292 differentially expressed metabolic features. Metabolic pathway enrichment analysis indicated that these differentially expressed metabolites mainly involved lipid metabolism, amino acid metabolism, carbohydrate metabolism, and cofactor and vitamin metabolism pathways. Figure 2 C). Notably, succinic acid was identified as a differentially enriched metabolite in a significant pathway ( Figure 2 D), and it is also among the top 25 metabolites in VIP score rankings ( Figure 2 E), suggesting that it may be involved in RP-related metabolic abnormalities.

[0099] 2. Enzyme-linked immunosorbent assay (ELISA) Take 50 mg of placental tissue sample, add pre-cooled sample diluent, and prepare a 10% tissue homogenate at a ratio of 1:9 (m:v). Homogenize thoroughly on ice, centrifuge at 4°C, and collect the supernatant as the test sample. Prepare a succinic acid standard concentration gradient according to the kit instructions, and set up blank wells, standard wells, and sample wells. After adding the standard or sample to each well, add the enzyme-labeled reagent, incubate at 37°C, wash the plate, and then add chromogenic solution A and chromogenic solution B sequentially for color development in the dark. After terminating the reaction, measure the absorbance value at 450 nm. Calculate the succinic acid concentration in the sample based on the standard curve, and convert it to the succinic acid content per unit mass of tissue by combining the homogenization factor and tissue mass.

[0100] See results Figure 2 F. Compared with the healthy group, the succinic acid level in the placental tissue of dairy cows with retained placenta was significantly increased.

[0101] Example 3: Increased SUCNR1 levels in placental tissue from retained placenta in dairy cows Source of placental tissue samples from dairy cows: Placental tissue samples were collected from cows of similar parity (2-4 parities) and body condition within 30 minutes of calving. Cows were divided into a healthy group (n=6) and a group with retained placenta (n=6) based on whether the placenta had been expelled within 12 hours postpartum. The healthy group was denoted as Control, and the group with retained placenta was denoted as RP.

[0102] 1. Protein immunoblotting assay (Western blot) Placental tissue was collected, and total protein was extracted for Western blot analysis. The primary antibody used for Western blot was either SUCNR1 / GPR91 Antibody (NBP1-00861, NOVUS Biologicals, Centennial, CO, USA) or β-actin antibody (AF7018, Affinity Biosciences, Jiangsu, China). The secondary antibody used was HRP-labeled goat anti-rabbit IgG (H+L) (RGAR001, Proteintech, Wuhan, China). The chromogenic reagent used for Western blot was an enhanced chemiluminescence reagent (MA0186, MeilunBio, Dalian, China). Imaging was performed using a 5200Multi (Tanon, Shanghai, China). Image density analysis of the band gray values ​​was performed using ImageJ software (version 1.53, National Institutes of Health, Bethesda, USA). β-actin on the same membrane was used as an internal control for normalization, and the relative abundance of the target protein was calculated.

[0103] See results Figure 3 A and Figure 3 B. Compared with the healthy group, the SUCNR1 protein level in the placental tissue of dairy cows with retained placenta was significantly increased.

[0104] 2. Quantitative real-time PCR (qRT-PCR) Placental tissue was collected, total RNA was extracted, and cDNA was obtained by reverse transcription. Using cDNA as a template and β-actin as an internal reference gene, qRT-PCR was used for detection. SUCNR1 Genes were obtained using the 2^-ΔΔCt method. SUCNR1 Relative gene expression levels. Detection. SUCNR1 Primers for the gene and internal reference gene are shown in Table 2.

[0105] Table 2

[0106] See results Figure 3 C. Compared with the healthy group, the abundance of SUCNR1 mRNA in the placental tissue of dairy cows with retained placenta was significantly increased.

[0107] 3. Tissue immunofluorescence Placental tissue was harvested and prepared into paraffin sections with a thickness of 4-5 μm, followed by immunofluorescence staining. The primary antibody used for immunofluorescence staining was either SUCNR1 antibody (NBP1-00861, NOVUS Biologicals, Centennial, CO, USA) or CD206 antibody (18704-1-AP, Proteintech, Wuhan, China). A fluorescent secondary antibody targeting the primary antibody was used for immunofluorescence. Cell nuclei were counterstained with DAPI. Observation and image acquisition were performed using a confocal fluorescence microscope. Images of all sections were acquired under the same parameters, and five non-overlapping fields were randomly selected from each sample. Quantitative analysis of the target protein fluorescence signal was performed using ImageJ software. CD206 positivity indicates M2 type macrophages.

[0108] See results Figure 3 D and Figure 3 E. Results showed that SUCNR1 expression was enhanced in M2 macrophages in the placental tissue of the retained placenta group, and CD206 expression was also increased. + SUCNR1 + The proportion of double-positive cells was significantly higher in the control group than in the healthy group.

[0109] The results of this embodiment show that SUCNR1 is upregulated in placental tissue of retained placenta in dairy cows and is mainly associated with increased infiltration of M2 macrophages, suggesting that SUCNR1 may be involved in the regulation of local macrophage polarization in the placenta during retained placenta.

[0110] Example 4: Preparation and Screening of siRNA I. Preparation of siRNA The siRNAs are si-Sucnr1-1, si-Sucnr1-2, si-Sucnr1-3, or siRNA-NC.

[0111] si-Sucnr1-1 consists of a positive strand and a negative strand. Both the positive and negative strands are single-stranded nucleic acids. Positive strand (SEQ ID NO: 1): 5'-CUGCUGUUGUUUUUGGCUATT-3'. Negative strand (SEQ ID NO: 2): 5'-UAGCCAAAAACAACAGCAGTT-3'.

[0112] si-Sucnr1-2 consists of a positive strand and a negative strand. Both the positive and negative strands are single-stranded nucleic acids. Positive strand (SEQ ID NO: 3): 5'-GAUAUCUGCUCAUGAACUATT-3'. Negative strand (SEQ ID NO: 4): 5'-UAGUUCAUGAGCAGAUAUCTT-3'.

[0113] si-Sucnr1-3 consists of a positive strand and a negative strand. Both the positive and negative strands are single-stranded nucleic acids. Positive strand (SEQ ID NO: 5): 5'-CAAUCAAAGCUUCAUUUAUTT-3'. Negative strand (SEQ ID NO: 6): 5'-AUAAAUGAAGCUUUGAUUGTT-3'.

[0114] siRNA-NC consists of a positive strand and a reverse strand. Both the positive and reverse strands are single-stranded nucleic acids. Positive strand: 5'-UUCUCCGAACGUGUCACGUTT-3'. Reverse strand: 5'-ACGUGACACGUUCGGAGAATT-3'.

[0115] II. Comparison of the effects of different siRNAs The tested siRNAs were si-Sucnr1-1, si-Sucnr1-2, si-Sucnr1-3, or siRNA-NC prepared in step one.

[0116] 1. Collect bovine macrophages and resuspend them in RPMI-1640 complete medium to obtain a cell suspension. In a 6-well plate, seed each well with 2 mL of the cell suspension (1 × 10⁻⁶ cells / well). 5 (1 cell / well), cultured until cell confluence reaches 30%–50%.

[0117] 2. Mix 5 μL of the test siRNA aqueous solution, 10 μL of RNAFit transfection reagent, and 85 μL of Opti-MEM serum-depleted medium, and incubate at room temperature for 10 min to obtain 100 μL of transfection complex. The concentration of the test siRNA in the aqueous solution is 20 μM.

[0118] 3. After completing step 1, aspirate the culture supernatant, add 1.9 mL of RPMI-1640 medium containing 10% fetal bovine serum and 100 μL of the transfection complex prepared in step 2 to each well, mix gently, and then incubate for 48 hours.

[0119] 4. After completing step 3, collect cells, extract total protein, and perform Western blot.

[0120] The primary antibodies used in Western blot were either SUCNR1 antibody or β-actin antibody. SUCNR1 antibody (SUCNR1 / GPR91 Antibody, rabbit-derived): NOVUS Biologicals, catalog number NBP1-00861. β-actin antibody (Rabbit polyclonal antibody to beta Actin): Affinity Biosciences, catalog number AF7018.

[0121] The secondary antibody used in Western blot was an HRP-labeled goat anti-rabbit antibody. The full name of the HRP-labeled goat anti-rabbit antibody is Multi-rAb® HRP-Goat Anti-Rabbit Recombinant Secondary Antibody (H+L), manufactured by Proteintech, with product catalog number RGAR001.

[0122] The colorimetric reagent used in Western blot was ECL luminescent liquid (MeilunBio, catalog number MA0186).

[0123] Data was acquired using an imaging system (5200Multi, Tanon, Shanghai, China). ImageJ software (version 1.53, National Institutes of Health, Bethesda, USA) was used to analyze the density of the band gray values. β-actin on the same membrane was used as an internal reference for normalization, and the relative abundance of the target protein was calculated.

[0124] The experiment was repeated three times, and the average value of the results was taken.

[0125] See the example image. Figure 4 A, the statistical results are shown in [reference]. Figure 4 B.

[0126] 5. After completing step 3, collect cells, extract total RNA, and reverse transcribe to obtain cDNA. Use the cDNA as a template and detect it using qRT-PCR. SUCNR1 Genes and β-actin gene. Normalization was performed using the β-actin gene as an internal reference, and the 2^-ΔΔCt method was used to calculate the result. SUCNR1 Relative gene expression levels. Detection. SUCNR1 Primers for the gene and internal reference gene are shown in Table 3. Three replicate experiments were performed, and the average value was taken. Results are shown below. Figure 4 C.

[0127] Table 3

[0128] The results showed that si-Sucnr1-2 was silenced. SUCNR1 Genes are the most efficient.

[0129] The si-Sucnr1 used in subsequent embodiments is si-Sucnr1-2.

[0130] Example 5: Inhibition of M2 polarization-related markers in bovine macrophages by silencing the SUCNR1 gene. I. Grouping Processing 1. Collect bovine macrophages and resuspend them in RPMI-1640 complete medium to obtain a cell suspension. In a 6-well plate, seed each well with 2 mL of the cell suspension (1 × 10⁻⁶ cells / well). 5 (1 cell / well), cultured until cell confluence reaches 30%–50%.

[0131] 2. Preparation of transfection complex Mix 5 μL of the test siRNA solution, 10 μL of RNAFit transfection reagent, and 85 μL of Opti-MEM serum-depleted medium, and incubate at room temperature for 10 min to obtain 100 μL of transfection complex. The concentration of the test siRNA in the aqueous solution was 20 μM. When the test siRNA was siRNA-NC, the resulting transfection complex was the siRNA-NC transfection complex. When the test siRNA was si-Sucnr1, the resulting transfection complex was the si-Sucnr1 transfection complex.

[0132] 3. After completing step 1, group the data and set up 3 duplicate holes in each group.

[0133] Group 1 (si-NC): Discard the culture supernatant, add 1.9 mL of RPMI-1640 medium containing 10% fetal bovine serum and 100 μL of siRNA-NC transfection complex to each well, and incubate for 48 hours; then, add 2 μL of 10 μg / mL IL-4 solution and 2 μL of 10 μg / mL IL-10 solution to each well, and incubate for 24 hours; then, add 20 μL of water to each well, and incubate for 24 hours.

[0134] Group 2 (si-NC+succ): Discard the culture supernatant, add 1.9 mL of RPMI-1640 medium containing 10% fetal bovine serum and 100 μL of siRNA-NC transfection complex to each well, and incubate for 48 hours; then, add 2 μL of 10 μg / mL IL-4 solution and 2 μL of 10 μg / mL IL-10 solution to each well, and incubate for 24 hours; then, add 20 μL of 100 mM succinic acid aqueous solution to each well, and incubate for 24 hours.

[0135] Group 3 (si-Sucnr1): Discard the culture supernatant, add 1.9 mL of RPMI-1640 medium containing 10% fetal bovine serum and 100 μL of si-Sucnr1 transfection complex to each well, and incubate for 48 hours; then, add 2 μL of 10 μg / mL IL-4 solution and 2 μL of 10 μg / mL IL-10 solution to each well, and incubate for 24 hours; then, add 20 μL of water to each well, and incubate for 24 hours.

[0136] Group 4 (si-Sucnr1+succ): Discard the culture supernatant, add 1.9 mL of RPMI-1640 medium containing 10% fetal bovine serum and 100 μL of si-Sucnr1 transfection complex to each well, and incubate for 48 hours; then, add 2 μL of 10 μg / mL IL-4 solution and 2 μL of 10 μg / mL IL-10 solution to each well, and incubate for 24 hours; then, add 20 μL of 100 mM succinic acid aqueous solution to each well, and incubate for 24 hours.

[0137] 2. After completing step one, collect cells, extract total protein, and perform Western blot.

[0138] The primary antibodies used in Western blot were: CD206 antibody, CD163 antibody, TGF-β antibody, ARG-1 antibody, or β-actin antibody. CD206 antibody (CD206 polyclonal antibody, rabbit-derived): Proteintech, catalog number 18704-1-AP. CD163 antibody (CD163 Rabbit pAb): Bioss, catalog number bs-2527R. TGF-β antibody (Rabbit polyclonal antibody to TGF beta 1): Affinity Biosciences, catalog number AF1027. ARG-1 antibody (Arginase-1 polyclonal antibody, rabbit-derived): Proteintech, catalog number 16001-1-AP. β-actin antibody (Rabbit polyclonal antibody to beta Actin): Affinity Biosciences, catalog number AF7018.

[0139] The secondary antibody used in Western blot was an HRP-labeled goat anti-rabbit antibody. The full name of the HRP-labeled goat anti-rabbit antibody is Multi-rAb® HRP-Goat Anti-Rabbit Recombinant Secondary Antibody (H+L), manufactured by Proteintech, with product catalog number RGAR001.

[0140] The colorimetric reagent used in Western blot was ECL luminescent liquid (MeilunBio, catalog number MA0186).

[0141] Data was acquired using an imaging system (5200Multi, Tanon, Shanghai, China). ImageJ software (version 1.53, National Institutes of Health, Bethesda, USA) was used to analyze the density of the band gray values. β-actin on the same membrane was used as an internal reference for normalization, and the relative abundance of the target protein was calculated.

[0142] The experiment was repeated three times, and the average value of the results was taken.

[0143] See the example image. Figure 5 A, the statistical results are shown in [reference]. Figure 5 B. The results showed that, compared with the si-NC group, the abundance of multiple M2 macrophage-related marker proteins (CD206, CD163, TGF-β, ARG-1) in bovine macrophages was significantly upregulated in the si-NC+succ treatment group; compared with the si-NC+succ treatment group, the abundance of multiple M2 macrophage-related marker proteins (CD206, CD163, TGF-β, ARG-1) in bovine macrophages was significantly downregulated in the si-Sucnr1+succ treatment group.

[0144] III. After completing step one, cells were collected, total RNA was extracted, and qRT-PCR was performed. The target genes were CD206, IL-10, Arg1, and TGF-β. Normalization was performed using β-actin as an internal reference gene, and the relative expression levels of each target gene were calculated using the 2^-ΔΔCt method. Primers for detecting each target gene and internal reference gene are shown in Table 4. Three replicate experiments were performed, and the average results were taken. Results are shown in [Table 4]. Figure 5 C. Further results showed that SUCNR1 deletion significantly reduced the mRNA expression levels of M2 polarization-related marker genes such as CD206, ARG-1, IL-10, and TGF-β.

[0145] Table 4

[0146] The results of this embodiment indicate that SUCNR1 is involved in the maintenance and regulation of the M2 polarization-related phenotype of bovine macrophages, suggesting that it plays a promoting role in the M2 polarization of bovine macrophages; silencing the SUCNR1 gene leads to a decrease in the abundance of M2 polarization markers in bovine macrophages (decreased protein abundance and decreased mRNA abundance).

[0147] Example 6: SUCNR1 knockdown inhibits succinate-induced PI3K signaling pathway activation I. Grouping Processing Same as step one in Example 5.

[0148] 2. After completing step one, collect cells, extract total protein, and perform Western blot.

[0149] The primary antibodies used in Western blot were: AKT antibody, p-AKT antibody, PI3K antibody, p-PI3K antibody, mTOR antibody, p-mTOR antibody, or β-actin antibody. AKT antibody (AKT Monoclonal antibody; mouse-derived): Proteintech, catalog number 60203-2-Ig. p-AKT antibody (Phospho-AKT (Ser473) Monoclonal antibody; mouse-derived): Proteintech, catalog number 66444-1-Ig. PI3K antibody (Rabbit polyclonal antibody to PI3K p85 alpha): Affinity Biosciences, catalog number AF6241. p-PI3K antibody (Rabbit polyclonal antibody to Phospho-PI3K p85alpha (Tyr607)): ​​Affinity Biosciences, catalog number AF3241. mTOR antibody (mTOR Monoclonal antibody; mouse-derived): Proteintech, catalog number 66888-1-Ig. p-mTOR antibody (Phospho-mTOR (Ser2448) Monoclonal antibody; mouse-derived): Proteintech, catalog number 67778-1-Ig. β-actin antibody (Rabbit polyclonal antibody to beta Actin): Affinity Biosciences, catalog number AF7018.

[0150] The secondary antibodies used in Western blotting were rabbit-derived and mouse-derived. The rabbit-derived secondary antibody was Multi-rAb® HRP-Goat Anti-Rabbit Recombinant Secondary Antibody (H+L), from Proteintech, catalog number RGAR001. The mouse-derived secondary antibody was also Multi-rAb® HRP-Goat Anti-Mouse Recombinant Secondary Antibody (H+L), from Proteintech, catalog number RGAM001.

[0151] The colorimetric reagent used in Western blot was ECL luminescent liquid (MeilunBio, catalog number MA0186).

[0152] Data was acquired using an imaging system (5200Multi, Tanon, Shanghai, China). ImageJ software (version 1.53, National Institutes of Health, Bethesda, USA) was used to analyze the density of the band gray values. β-actin on the same membrane was used as an internal reference for normalization, and the relative abundance of the target protein was calculated.

[0153] Set up three repeated trials and take the average of the results.

[0154] See example photos Figure 6 A. Statistical results are shown in [reference needed]. Figure 6 The results showed that exogenous succinic acid treatment significantly enhanced the phosphorylation levels of AKT, PI3K, and mTOR in bovine macrophages, while knockdown of the SUCNR1 gene significantly inhibited this promoting effect.

[0155] III. Grouping Processing 1. Collect bovine macrophages and resuspend them in RPMI-1640 complete medium to obtain a cell suspension. In a 6-well plate, seed each well with 2 mL of the cell suspension (1 × 10⁻⁶ cells / well). 5 (1 cell / well), cultured until cell confluence reaches 30%–50%.

[0156] 2. Preparation of transfection complex Same as step 2 of Example 5.

[0157] 3. After completing step 1, group the data and set up 3 duplicate holes in each group.

[0158] Group 1: Discard the culture supernatant, add 1.9 mL of RPMI-1640 medium containing 10% fetal bovine serum and 100 μL of siRNA-NC transfection complex to each well, and incubate for 48 hours; then, add 2 μL of 10 μg / mL IL-4 solution and 2 μL of 10 μg / mL IL-10 solution to each well, and incubate for 24 hours; then, add 2 μL of water to each well, and incubate for 2 hours; finally, add 20 μL of 100 mM succinic acid aqueous solution to each well, and incubate for 24 hours.

[0159] Group 2: Discard the culture supernatant, add 1.9 mL of RPMI-1640 medium containing 10% fetal bovine serum and 100 μL of siRNA-NC transfection complex to each well, and incubate for 48 hours; then, add 2 μL of 10 μg / mL IL-4 solution and 2 μL of 10 μg / mL IL-10 solution to each well, and incubate for 24 hours; then, add 2 μL of 10 mM LY294002 solution to each well, and incubate for 2 hours; finally, add 20 μL of 100 mM succinic acid aqueous solution to each well, and incubate for 24 hours.

[0160] Group 3: Discard the culture supernatant, add 1.9 mL of RPMI-1640 medium containing 10% fetal bovine serum and 100 μL of si-Sucnr1 transfection complex to each well, and incubate for 48 hours; then, add 2 μL of 10 μg / mL IL-4 solution and 2 μL of 10 μg / mL IL-10 solution to each well, and incubate for 24 hours; then, add 2 μL of water to each well, and incubate for 2 hours; finally, add 20 μL of 100 mM succinic acid aqueous solution to each well, and incubate for 24 hours.

[0161] Group 4: Discard the culture supernatant, add 1.9 mL of RPMI-1640 medium containing 10% fetal bovine serum and 100 μL of si-Sucnr1 transfection complex to each well, and incubate for 48 hours; then, add 2 μL of 10 μg / mL IL-4 solution and 2 μL of 10 μg / mL IL-10 solution to each well, and incubate for 24 hours; then, add 2 μL of 10 mM LY294002 solution to each well, and incubate for 2 hours; finally, add 20 μL of 100 mM succinic acid aqueous solution to each well, and incubate for 24 hours.

[0162] IV. After completing step three, cells were collected, total RNA was extracted, and qRT-PCR was performed. The target genes were CD206, IL-10, Arg1, TGF-β, and CD163. Normalization was performed using β-actin as an internal reference gene, and the relative expression levels of each target gene were calculated using the 2^-ΔΔCt method. Primers for detecting each target gene and internal reference gene are shown in Table 5. Three replicate experiments were performed, and the average value was taken.

[0163] Table 5

[0164] See results Figure 6 C to Figure 6G. The results showed that LY294002 treatment significantly decreased the expression of M2 polarization-related marker genes in bovine macrophages induced by succinate, including CD206, CD163, Arg-1, IL-10, and TGF-β. The results of this example indicate that the PI3K signaling pathway plays a key mediating role in succinate-induced M2 polarization of bovine macrophages, suggesting that the succinate-SUCNR1 axis promotes M2 phenotype polarization in bovine macrophages by activating the downstream PI3K / AKT / mTOR pathway. The results also suggest that SUCNR1 partially regulates M2 polarization through the PI3K pathway.

[0165] Example 7: Succinate-SUCNR1 axis-induced conditioned medium for bovine macrophages inhibits apoptosis in bovine trophoblast cells. See flowchart Figure 7 A.

[0166] I. Preparation of Conditioned Culture Media 1. Collect bovine feeder cells and resuspend them in DMEM-F12 complete medium to obtain a cell suspension. In a 6-well plate, seed each well with 2 mL of the cell suspension (2 × 10⁶ cells / well). 5 (1 cell / well) and cultured until the cell confluence reaches 70%–80%; then, add 20 μL of water to each well and culture for 24 hours; then, centrifuge at 4°C and 1000×g for 15 minutes, collect the supernatant, filter it through a 0.22 μm pore size filter membrane, and collect the filtrate, which is the conditioned medium 1.

[0167] 2. Collect bovine feeder cells and resuspend them in DMEM-F12 complete medium to obtain a cell suspension. In a 6-well plate, seed each well with 2 mL of the cell suspension (2 × 10⁶ cells / well). 5 (1 cell / well) and cultured until the cell confluence reaches 70%–80%; then, add 20 μL of 100 mM succinic acid aqueous solution to each well and culture for 24 hours; then, centrifuge at 4°C and 1000 × g for 15 minutes, collect the supernatant, filter it through a 0.22 μm pore size filter membrane, and collect the filtrate, which is the conditioned medium 2.

[0168] 3. Collect bovine macrophages and resuspend them in RPMI-1640 complete medium to obtain a cell suspension. In a 6-well plate, seed each well with 2 mL of the cell suspension (1 × 10⁻⁶ cells / well). 5 (1 cell / well), cultured until cell confluence reaches 30%–50%.

[0169] 4. Preparation of transfection complex Same as step 2 of Example 5.

[0170] 5. After completing step 3, discard the culture supernatant, add 1.9 mL of RPMI-1640 medium containing 10% fetal bovine serum and 100 μL of siRNA-NC transfection complex to each well, and incubate for 48 hours; then, add 2 μL of 100 μg / mL LPS aqueous solution and 2 μL of 100 μg / mL IFN-γ aqueous solution to each well, and incubate for 24 hours, discarding the culture supernatant; then, add 2 mL of RPMI-1640 complete medium to each well, and incubate for 24 hours; then, add 20 μL of water to each well, and incubate for 24 hours; then, centrifuge at 1000×g for 15 minutes at 4℃, collect the supernatant, filter it through a 0.22 μm pore size filter membrane, and collect the filtrate, which is the conditioned medium 3.

[0171] 6. After completing step 3, discard the culture supernatant, add 1.9 mL of RPMI-1640 medium containing 10% fetal bovine serum and 100 μL of siRNA-NC transfection complex to each well, and incubate for 48 hours; then, add 2 μL of 10 μg / mL IL-4 solution and 2 μL of 10 μg / mL IL-10 solution to each well, and incubate for 24 hours, discarding the culture supernatant; then, add 2 mL of RPMI-1640 complete medium to each well, and incubate for 24 hours; then, add 20 μL of water to each well, and incubate for 24 hours; then, centrifuge at 1000×g for 15 minutes at 4℃, collect the supernatant, filter it through a 0.22 μm pore size filter membrane, and collect the filtrate, which is the conditioned medium 4.

[0172] 7. After completing step 3, discard the culture supernatant, add 1.9 mL of RPMI-1640 medium containing 10% fetal bovine serum and 100 μL of siRNA-NC transfection complex to each well, and incubate for 48 hours; then, add 2 μL of 10 μg / mL IL-4 solution and 2 μL of 10 μg / mL IL-10 solution to each well, and incubate for 24 hours, discarding the culture supernatant; then, add 2 mL of RPMI-1640 complete medium to each well, and incubate for 24 hours; then, add 20 μL of 100 mM succinic acid aqueous solution to each well, and incubate for 24 hours; then, centrifuge at 1000×g for 15 minutes at 4℃, collect the supernatant, filter it through a 0.22 μm pore size filter membrane, and collect the filtrate, which is the conditioned medium 5.

[0173] 8. After completing step 3, discard the culture supernatant, add 1.9 mL of RPMI-1640 medium containing 10% fetal bovine serum and 100 μL of si-Sucnr1 transfection complex to each well, and incubate for 48 hours; then, add 2 μL of 10 μg / mL IL-4 solution and 2 μL of 10 μg / mL IL-10 solution to each well, and incubate for 24 hours, discarding the culture supernatant; then, add 2 mL of RPMI-1640 complete medium to each well, and incubate for 24 hours; then, add 20 μL of water to each well, and incubate for 24 hours; then, centrifuge at 4℃ and 1000×g for 15 minutes, collect the supernatant, filter it through a 0.22 μm pore size filter membrane, and collect the filtrate, which is the conditioned medium 6.

[0174] 9. After completing step 3, discard the culture supernatant, add 1.9 mL of RPMI-1640 medium containing 10% fetal bovine serum and 100 μL of si-Sucnr1 transfection complex to each well, and incubate for 48 hours; then, add 2 μL of 10 μg / mL IL-4 solution and 2 μL of 10 μg / mL IL-10 solution to each well, and incubate for 24 hours, discarding the culture supernatant; then, add 2 mL of RPMI-1640 complete medium to each well, and incubate for 24 hours; then, add 20 μL of 100 mM succinic acid aqueous solution to each well, and incubate for 24 hours; then, centrifuge at 4℃ and 1000×g for 15 minutes, collect the supernatant, filter it through a 0.22 μm pore size filter membrane, and collect the filtrate, which is the conditioned medium 7.

[0175] II. Culture of bovine trophoblast cells The test culture media were conditioned media 1 to conditioned media 7 prepared in step one.

[0176] Bovine feeder cells were collected and resuspended in DMEM-F12 complete medium to obtain a cell suspension. 2 mL of the cell suspension (2 × 10⁶ cells / well) was seeded into each well of a 6-well plate. 5 (1 cell / well), culture until the cell confluence reaches 70%–80%; then, discard the culture supernatant, add 2 mL of the test medium to each well, and culture for 24 hours.

[0177] Each group had 3 replicates. When using conditioned medium 1, this group was designated Ctrl-BTCs. When using conditioned medium 2, this group was designated Succ-BTCs. When using conditioned medium 3, this group was designated si-Ctrl-M1-CM. When using conditioned medium 4, this group was designated si-Ctrl-M2-CM. When using conditioned medium 5, this group was designated si-Ctrl-Succ-M2-CM. When using conditioned medium 6, this group was designated si-Sucnr1-M2-CM. When using conditioned medium 7, this group was designated si-Sucnr1-Succ-M2-CM.

[0178] III. Apoptosis levels in bovine trophoblast cells were detected using Annexin V-FITC / PI double staining combined with flow cytometry. After completing step two, cells were collected and analyzed using the Annexin V-FITC apoptosis detection kit (C1062, Beyotime, Shanghai, China) according to the manufacturer's instructions. Flow cytometry (LSRFortessa, Becton Dickinson, USA) was used to detect apoptosis. The results were analyzed using FlowJo software (version 10.9, BD, USA), and the proportions of early and late apoptotic cells were statistically analyzed.

[0179] See exemplary flow graph Figure 7 B, the statistical results are shown in [reference]. Figure 7 C. Compared with the Ctrl-BTCs group, the apoptosis level in the si-Ctrl-M1-CM group was significantly increased, while the apoptosis level in the si-Ctrl-M2-CM group was significantly decreased. Compared with the si-Ctrl-M2-CM group, the apoptosis level in the si-Ctrl-Succ-M2-CM group was further decreased. Compared with the si-Ctrl-M2-CM group, the apoptosis level in the si-Sucnr1-M2-CM group was significantly increased. Compared with the si-Ctrl-Succ-M2-CM group, the apoptosis level in the si-Sucnr1-Succ-M2-CM group was significantly increased.

[0180] IV. Protein Immunoblot Assay (Western blot) After completing step two, cells were collected, total protein was extracted, and Western blot was performed.

[0181] The primary antibodies used in Western blot were: BAX antibody, BCL2 antibody, Cleaved Caspase-3 antibody, or β-actin antibody. BAX antibody (BAX Polyclonal antibody): Proteintech, catalog number 50599-2-Ig. BCL2 antibody (Anti-Bcl-2 Rabbit pAb): Wanleibio, catalog number WL01556. Cleaved Caspase-3 antibody (Anti-Caspase-3 / Cleaved Caspase-3 Rabbit pAb): Wanleibio, catalog number WL02117. β-actin antibody (Rabbit polyclonal antibody to betaActin): Affinity Biosciences, catalog number AF7018.

[0182] The secondary antibody used in the Western blot was Multi-rAb® HRP-Goat Anti-Rabbit Recombinant Secondary Antibody (H+L), Proteintech, catalog number RGAR001.

[0183] The colorimetric reagent used in Western blot was an enhanced chemiluminescence reagent (MA0186, MeilunBio, Dalian, China).

[0184] Data was acquired using an imaging system (5200Multi, Tanon, Shanghai, China). ImageJ software (version 1.53, National Institutes of Health, Bethesda, USA) was used to analyze the density of the band gray values. β-actin on the same membrane was used as an internal reference for normalization, and the relative abundance of the target protein was calculated.

[0185] See results Figure 7 D and Figure 7E. Compared with the Ctrl-BTCs group, the si-Ctrl-M1-CM group showed significantly increased abundance of BAX and cleaved caspase-3 proteins, and significantly decreased abundance of BCL2 protein. Compared with the Ctrl-BTCs group, the si-Ctrl-M2-CM group showed significantly decreased abundance of BAX and cleaved caspase-3 proteins, and significantly increased abundance of BCL2 protein. Compared with the si-Ctrl-M2-CM group, the si-Ctrl-Succ-M2-CM group showed further decreased abundance of BAX and cleaved caspase-3 proteins, and further increased abundance of BCL2 protein. Compared with the si-Ctrl-M2-CM group, the si-Sucnr1-M2-CM group showed significantly increased abundance of BAX and cleaved caspase-3 proteins, and significantly decreased abundance of BCL2 protein. Compared with the si-Ctrl-Succ-M2-CM group, the abundance of BAX and cleaved caspase-3 proteins was significantly increased, while the abundance of BCL2 protein was significantly decreased in the si-Sucnr1-Succ-M2-CM group.

[0186] V. Quantitative Real-Time PCR (qRT-PCR) After completing step three, cells were collected, total RNA was extracted, and qRT-PCR was performed. The target genes were CASP3, CASP8, BAX, and BCL2. Normalization was performed using β-actin as an internal reference gene, and the relative expression levels of each target gene were calculated using the 2^-ΔΔCt method. Primers for detecting each target gene and the internal reference gene are shown in Table 6. Three replicate experiments were performed, and the results were averaged.

[0187] Table 6

[0188] See results Figure 7 F to Figure 7 Compared with the Ctrl-BTCs group, the relative expression levels of CASP3, CASP8, and BAX genes were significantly upregulated in the si-Ctrl-M1-CM group, while the relative expression level of BCL2 gene was significantly downregulated. Compared with the Ctrl-BTCs group, the relative expression levels of CASP3, CASP8, and BAX genes were significantly downregulated in the si-Ctrl-M2-CM group, while the relative expression level of BCL2 gene was significantly upregulated. Compared with the si-Ctrl-M2-CM group, the relative expression levels of CASP3, CASP8, and BAX genes were significantly upregulated in the si-Sucnr1-M2-CM group, while the relative expression level of BCL2 gene was significantly downregulated.

[0189] In the above results, there were no significant differences between conditioned medium 1 and conditioned medium 2 treatments. The results indicate that succinic acid has no direct effect on bovine trophoblast cell apoptosis; no significant changes were observed in the apoptosis levels or the transcriptional levels of apoptosis-related genes such as CASP3, CASP8, BAX, and BCL2 between the two treatments. These results suggest that succinic acid does not directly act on bovine trophoblast cell apoptosis, but rather indirectly inhibits it by enhancing the M2 polarization-related anti-apoptotic program of bovine macrophages. The results also indicate that knocking down SUCNR1 weakens the M2-induced anti-apoptotic effect of bovine macrophages on bovine trophoblast cells.

[0190] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The application of substances that target the SUCNR1 protein and / or substances that target the SUCNR1 gene in the preparation of a product; the product is used to promote apoptosis of trophoblast cells.

2. The application of substances that target SUCNR1 protein and / or substances that target the SUCNR1 gene in the preparation of the product; the purpose of the product is to inhibit macrophage M2 polarization.

3. The application of substances that target SUCNR1 protein and / or substances that target the SUCNR1 gene in the preparation of the product; the purpose of the product is to promote trophoblast cell apoptosis by inhibiting macrophage M2 polarization.

4. The application of substances that target SUCNR1 protein and / or substances that target the SUCNR1 gene in the preparation of the product; the product is used to inhibit "the inhibitory effect of macrophage M2 polarization on trophoblast cell apoptosis" by inhibiting macrophage M2 polarization.

5. The application of substances that target the SUCNR1 protein and / or substances that target the SUCNR1 gene in the preparation of the product; the purpose of the product is to inhibit macrophage M2 polarization by inhibiting the PI3K signaling pathway.

6. The use of substances that target the SUCNR1 protein and / or substances that target the SUCNR1 gene in the preparation of products; the purpose of the products is to prevent and / or treat retained placenta in dairy cows.

7. The application as described in any one of claims 1 to 6, characterized in that: The substance that targets the SUCNR1 gene is siRNA that targets the SUCNR1 gene.

8. The application as described in claim 7, characterized in that: The siRNA is si-Sucnr1-2, si-Sucnr1-1, or si-Sucnr1-3; si-Sucnr1-2 is composed of the nucleic acid molecules shown in SEQ ID NO: 3 and SEQ ID NO: 4; si-Sucnr1-1 is composed of the nucleic acid molecules shown in SEQ ID NO: 1 and SEQ ID NO: 2; si-Sucnr1-3 is composed of the nucleic acid molecules shown in SEQ ID NO: 5 and SEQ ID NO:

6.

9. The application as described in claim 1, 3, or 4, characterized in that: The trophoblast cells are bovine trophoblast cells.

10. The application as described in claim 2, 3, 4, or 5, characterized in that: The macrophages mentioned are bovine macrophages.

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