Application of TRIM21 as biomarker in screening or preparing preparation for reducing miscarriage risk related to ciprofloxacin exposure

Abstract

The invention relates to the technical field of biological medicine, in particular to application of TRIM21 serving as a biomarker in screening or preparing a preparation for reducing ciprofloxacin exposure related abortion risk. The problems that in the prior art, key action links and molecular mechanisms of pregnancy loss caused by exposure of environment or non-therapeutic dose ciprofloxacin are not clear, and objective detection markers capable of prompting risk states in the early stage of pregnancy and relatively standard and repeatable detection methods are lacked are solved. The invention discloses an application of TRIM21 as a biomarker in screening or preparing a preparation for reducing miscarriage risk related to ciprofloxacin exposure, the pregnancy risk caused by ciprofloxacin exposure can be recognized in an early stage by detecting the expression level of TRIM21, and a candidate preparation with a protective effect is further screened. The invention provides a new thought and target for developing a novel medicine for preventing abortion.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of TRIM21 as a biomarker in screening or preparing agents that reduce the risk of ciprofloxacin exposure-related miscarriage. Background Technology

[0002] Miscarriage (especially early pregnancy loss) is one of the most common adverse pregnancy outcomes in obstetrics and gynecology, clinically manifested as embryonic arrest, spontaneous expulsion, or the need for surgical evacuation. Its causes involve multiple factors, including embryonic chromosomal abnormalities, uterine anatomical abnormalities, endocrine and metabolic abnormalities, infections, immune abnormalities, and coagulation abnormalities. However, a significant proportion of cases lack clear etiological evidence, making it difficult to implement timely and targeted interventions and risk management.

[0003] Besides traditional causes, the impact of environmental exposure factors on pregnancy outcomes is receiving increasing attention. Antibiotics, as widely used and emitted active compounds, can enter the environmental media through medical treatment, animal husbandry, and wastewater discharge, leading to low-dose exposure in the general population under non-therapeutic conditions. Unlike inert pollutants, antibiotics have definite biological activity, and their environmental residues may affect tissue homeostasis. Ciprofloxacin is a commonly used fluoroquinolone antibacterial drug. Previous literature has reported its detection in some water and environmental samples and its correlation with biological processes such as cellular stress response, mitochondrial function alteration, and cell proliferation and apoptosis regulation. However, whether non-therapeutic doses of ciprofloxacin exposure in the environment increases the risk of pregnancy loss remains unclear and lacks a clear and coherent chain of evidence. Specifically: First, current research has not yet clarified the key mechanisms and molecular pathways by which environmental or non-therapeutic doses of ciprofloxacin exposure affect pregnancy loss. Given this lack of clarity, related risk interventions are mostly limited to general supportive care or empirical strategies, making it difficult to develop targeted intervention approaches and screening criteria that match the risks associated with ciprofloxacin exposure. This restricts the development and translational application of strategies to reduce exposure-related pregnancy loss. Second, because the aforementioned mechanisms and pathways are not yet fully understood, current technologies struggle to identify biomarkers from pregnancy-related tissues or peripheral samples that stably reflect the risk status associated with ciprofloxacin exposure. Furthermore, there is a lack of standardized, reproducible detection methods and quality control points for establishing such biomarkers. Therefore, clinical and research-level assessments remain largely focused on post-exposure diagnosis and management, lacking objective detection methods that can detect potential risks from environmental or non-therapeutic dose exposure early in pregnancy, hindering proactive risk identification. Summary of the Invention

[0004] To address the aforementioned problems, this invention aims to provide the application of TRIM21 as a biomarker in screening or preparing agents that reduce the risk of ciprofloxacin exposure-related miscarriage. By establishing a detection and evaluation system with TRIM21 expression level as the core indicator, the regulatory effect of candidate agents on TRIM21 expression is correlated with changes in ciprofloxacin exposure-related miscarriage risk, thereby enabling early detection of exposure-related risks and providing objective evidence for the screening, preparation, and quality control of agents that reduce the risk of ciprofloxacin exposure-related miscarriage.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: On the one hand, TRIM21 is used as a biomarker in screening agents that reduce the risk of miscarriage associated with ciprofloxacin exposure.

[0006] Secondly, the application of TRIM21 as a biomarker in the preparation of agents that reduce the risk of miscarriage associated with ciprofloxacin exposure.

[0007] Thirdly, the expression level of TRIM21 protein is positively correlated with the risk of miscarriage caused by ciprofloxacin exposure.

[0008] Fourthly, a kit for screening or preparing a method to reduce the risk of ciprofloxacin exposure-related miscarriage, said kit comprising a detection reagent for detecting TRIM21 expression levels.

[0009] Fifthly, the application of reagents used to knock down Trim21 expression in the preparation of formulations for inhibiting placental aging and reducing the risk of ciprofloxacin exposure-related miscarriage.

[0010] Sixthly, the reagent used to knock down Trim21 expression was a combination of fenenostat and quininosteg, with fenenostat at a dose of 50 mg / kg / 2d and quininosteg at a dose of 10 mg / kg / 2d.

[0011] Compared with existing technologies, the application of TRIM21 as a biomarker in screening or preparing agents to reduce the risk of ciprofloxacin exposure-related miscarriage has brought the following significant effects: 1. This invention clarifies the mechanism and key steps of ciprofloxacin (CIP) environmental / non-therapeutic dose exposure leading to pregnancy loss: through epidemiological studies, mouse models, and cell experiments, it reveals that CIP can induce placental and trophoblast cell senescence and thus trigger miscarriage; and it clarifies that at the molecular level, CIP upregulates TRIM21, promotes TRIM21-mediated MFF ubiquitination and degradation, and leads to MFF downregulation and mitochondrial dysfunction, providing a clear mechanistic basis and evaluation pathway for subsequent targeted intervention and formulation screening; 2. This invention introduces TRIM21 as a detectable biomarker for the risk associated with ciprofloxacin exposure, so that the screening of candidate formulations no longer depends on the ex post facto judgment of simply observing the miscarriage outcome, but can use changes in TRIM21 at the cellular and tissue levels as an early readout, forming a clearer basis for targeted screening, improving R&D efficiency and translatability. 3. This invention proposes using TRIM21 expression level as one of the detectable indicators of ciprofloxacin exposure-related placental damage / aging status, and it can be used to evaluate the effects of candidate intervention programs. By detecting changes in TRIM21 expression before and after treatment with candidate programs, and combining this with comparative analysis of embryonic resorption and placental aging and mitochondrial function-related indicators, the effects of candidate programs can be quantitatively evaluated and compared. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 In the table, a is a schematic diagram of the case-control study of HC and UM; b is the CIP level (μg / mL) in urine samples of HC and UM (n=50); c is the univariate and multivariate logistic regression analysis of the association between urine CIP level and miscarriage; d is a schematic diagram of the CIP-exposed pregnant mouse model; pregnant mice were administered CIP by gavage for 13 consecutive days at doses of 0, 0.45, 12.5, 160, or 320 mg / kg / d (n=6 per group); e is the weight gain of CIP-exposed pregnant mice over gestation days; f is the weight gain of CIP-exposed pregnant mice on day 14 relative to their initial weight on day 1 (n=6 per group); gh is a representative image of embryo resorption in CIP-exposed pregnant mice (indicated by red arrows) (g) and its mean miscarriage rate (h) (n=6 per group). Figure 2 In the table, a) is the directed acyclic plot analysis of the influence of potential confounding factors (pink) on the association between the primary cause (CIP exposure, green) and the outcome (unexplained miscarriage, blue) analyzed by DAGitty software; b) is the association between urinary CIP levels and miscarriage assessed by stratified analysis (all interactions P > 0.05); c) is the initial body weight of CIP-exposed pregnant mice on day 1 (n=6 per group). Figure 3In the table, a represents the CCK8 assay results of HTR-8 / SVneo cells treated with 0, 10, 20, or 40 μg / mL CIP at 0, 12, 24, 36, or 48 hours (n=3 independent experiments); b and c represent SA-β-galactosidase staining and quantification of positive cells in HTR-8 / SVneo cells treated with 0, 10, 20, or 40 μg / mL CIP (n=3 independent experiments); de represents the Western spectral density of p16, p21, p38, and β-gal proteins in HTR-8 / SVneo cells treated with 0, 10, 20, or 40 μg / mL CIP. blot analysis and relative quantification (n=3 independent experiments); fg: SA-β-galactosidase staining and positive intensity quantification of chorionic villus tissue from healthy controls and patients with unexplained miscarriage (n=12); hi: Western blot analysis and relative quantification of p16, p21, p38, and β-gal protein levels in chorionic villus tissue from healthy controls and patients with unexplained miscarriage (n=12); j: Forest plot multivariate logistic regression analysis (including 95% confidence interval, n=12) of the association between p16, p21, p38, and β-gal protein levels in chorionic villus tissue and miscarriage; kl: SA-β-galactosidase staining and positive intensity quantification of placental tissue from CIP-exposed mice (n=6 per group); mn: Western blot analysis and relative quantification of p16, p21, p38, and β-gal protein levels in placental tissue from CIP-exposed mice (n=6); o: Schematic diagram of the CIP-exposed mouse model with D+Q intervention: pregnant mice were given saline or 160 mg / kg / d. CIP was administered by gavage, and mice were given 5 mg / kg D + 50 mg / kg Q every 3 days (solvent: 10% PEG-400); pq represents embryo resorption (indicated by red arrows) and mean abortion rate (n=6 per group) in the 160 mg / kg / d CIP exposure combined with D+Q intervention group; r represents the quantitative intensity of SA-β-galactosidase staining in placental tissue of mice in the 160 mg / kg / d CIP exposure combined with D+Q intervention group (n=6 per group); st represents the Western blot analysis and relative quantification of p16, p21, p38 and β-gal protein levels in placental tissue of mice in the 160 mg / kg / d CIP exposure combined with D+Q intervention group (n=6 per group). Figure 4In the table, a) is the Venn intersection plot of differentially expressed proteins with a fold change >1.3-fold and p<0.05 among the three groups: chorionic villus tissue from unexplained abortion and healthy controls, placental tissue from mice exposed to CIP at 160 mg / kg / d and 0 mg / kg / d, and HTR-8 / SVneo cells exposed to CIP at 40 μg / mL and 0 μg / mL; b) is the RT-qPCR analysis of MFF mRNA levels in HTR-8 / SVneo cells treated with 0, 10, 20, or 40 μg / mL CIP (n=3 independent experiments); c) is the Western blot analysis and relative quantification of MFF protein levels in HTR-8 / SVneo cells treated with 0, 10, 20, or 40 μg / mL CIP (n=3 independent experiments); d) is the CCK8 cell viability analysis of MFF-overexpressing HTR-8 / SVneo cells exposed to 40 μg / mL CIP at 0, 12, 24, 36, or 48 hours (n=3 independent experiments); ef) is the 40 SA-β-galactosidase staining and quantification of positive cells in MFF-overexpressing HTR-8 / SVneo cells exposed to 40 μg / mL CIP (n=3 independent experiments); gh represents Western blot analysis and relative quantification of p16, p21, p38, and β-gal protein levels in MFF-overexpressing HTR-8 / SVneo cells exposed to 40 μg / mL CIP (n=3 independent experiments); i represents the relative mitochondrial DNA copy number in MFF-overexpressing HTR-8 / SVneo cells exposed to 40 μg / mL CIP (n=3 independent experiments); j represents the NAD+ / NADH ratio in MFF-overexpressing HTR-8 / SVneo cells exposed to 40 μg / mL CIP (n=3 independent experiments); k represents the representative fluorescence signal image and relative quantification of mitochondrial membrane potential in MFF-overexpressing HTR-8 / SVneo cells exposed to 40 μg / mL CIP (n=3 independent experiments); l represents the relative mitochondrial DNA copy number in MFF-overexpressing HTR-8 / SVneo cells exposed to 40 μg / mL CIP (n=3 independent experiments); l represents the relative mitochondrial DNA copy number in MFF-overexpressing HTR-8 / SVneo cells exposed to 40 μg / mL CIP (n=3 independent experiments); Representative images and relative quantification of reactive oxygen species (ROS) in MFF-overexpressing HTR-8 / SVneo cells under CIP exposure (n=3 independent experiments); m represents the representative images and relative quantification of mitochondrial ROS in MFF-overexpressing HTR-8 / SVneo cells under 40 μg / mL CIP exposure (n=3 independent experiments); no represents the Western blot analysis and relative quantification of MFF protein levels in villous tissues of healthy controls and patients with unexplained miscarriage (n=12); p represents the relative mitochondrial DNA copy number in villous tissues of healthy controls and patients with unexplained miscarriage (n=12); q represents the NAD+ / NADH ratio in villous tissues of healthy controls and patients with unexplained miscarriage (n=12); r represents the mitochondrial membrane potential level in villous tissues of healthy controls and patients with unexplained miscarriage (n=12). Figure 5 In the table, a) RT-qPCR analysis of MFF mRNA levels in MFF-overexpressing or knockdown HTR-8 / SVneo cells (n=3 independent experiments); b) Western blot analysis and relative quantification of MFF protein levels in MFF-overexpressing or knockdown HTR-8 / SVneo cells (n=3 independent experiments); cd) CCK8 assay analysis of MFF viability in MFF-overexpressing (c) or knockdown (d) HTR-8 / SVneo cells at 0, 12, 24, 36, or 48 hours (n=3 independent experiments); e) MFF in villous tissue from healthy controls and patients with unexplained miscarriage. RT-qPCR analysis of mRNA levels (n=12 per group); fi: Pearson correlation analysis of urinary CIP levels with relative mitochondrial DNA copy number, NAD+ / NADH ratio, mitochondrial membrane potential, and MFF protein levels in chorionic villus tissue of healthy controls and patients with unexplained miscarriage (n=12 per group); jl: Pearson correlation analysis of MFF protein levels with relative mitochondrial DNA copy number, NAD+ / NADH ratio, and mitochondrial membrane potential in chorionic villus tissue of healthy controls and patients with unexplained miscarriage (n=12 per group); mp: Pearson correlation analysis of MFF protein levels with p16, p21, p38, and β-gal protein levels in chorionic villus tissue of healthy controls and patients with unexplained miscarriage (n=12 per group). Figure 6In the table, ab represents the Western blot analysis and relative quantification of murine Mff protein levels in placental tissue of CIP-exposed mice (n=6); c represents the RT-qPCR analysis of Mff mRNA levels in placental tissue of CIP-exposed mice (n=6); d is a schematic diagram of the CIP-exposed mouse model with Mff overexpression: pregnant mice were treated with saline or 160 mg / kg / d CIP, and simultaneously given 10 mg / kg pcDNA3.1-Mff every 3 days to overexpress Mff (with empty vector pcDNA3.1 as a control); ef represents the embryo resorption status (indicated by red arrow, e) and mean abortion rate (f) of mice in the 160 mg / kg / d CIP exposure combined with Mff overexpression group (n=6 per group); gh represents the SA-β-galactosidase staining and relative quantification of positive intensity in placental tissue of mice in the 160 mg / kg / d CIP exposure combined with Mff overexpression group (n=6 per group); ij represents the 160 mg / kg / d CIP exposure combined with Mff overexpression group. Western blot analysis and relative quantification of murine Mff, p16, p21, p38 and β-gal protein levels in placental tissue of mice exposed to CIP combined with Mff overexpression (n=6 per group); k is the relative mitochondrial DNA copy number in placental tissue of mice exposed to CIP combined with Mff overexpression (n=6); l is the NAD+ / NADH ratio in placental tissue of mice exposed to CIP combined with Mff overexpression (n=6); m is the mitochondrial membrane potential level in placental tissue of mice exposed to CIP combined with Mff overexpression (n=6). Figure 7In the above, ab represents the residual MFF protein level and its relative quantification in HTR-8 / SVneo cells exposed to 40 μg / mL CIP after treatment with 10 μM CHX for 10 hours (n=3 independent experiments); c represents the MFF protein level and its relative quantification in HTR-8 / SVneo cells exposed to 40 μg / mL CIP after treatment with 10 μM MG132 or 50 μM CQ (n=3 independent experiments); de represents the MFF-ubiquitinated protein level and its relative quantification in HTR-8 / SVneo cells exposed to 40 μg / mL CIP precipitated by MFF antibody in 40 μg / mL CIP precipitated by 40 μg / mL CIP precipitated by 40 μg / mL CIP precipitated by 40 μg / mL CIP precipitated by 40 μg / mL CIP precipitated by 40 μg / mL CIP precipitated by 40 μg / mL CIP precipitated by 40 μg / mL CIP precipitated by 40 μg / mL CIP precipitated by 40 μg / mL CIP precipitated by 40 μg / mL CIP precipitated by 40 μg / mL CX ... The levels and relative quantifications of TRIM21 protein in CIP-exposed HTR-8 / SVneo cells (n=3 independent experiments); ij represents the levels of MFF protein precipitated by TRIM21 antibody in 40 μg / mL CIP-exposed HTR-8 / SVneo cells, and the levels and relative quantifications of MFF and TRIM21 protein in cell lysates (n=3 independent experiments); ko represents the levels of TRIM21 and MFF-ubiquitinated protein precipitated by MFF antibody in TRIM21-knockdown 40 μg / mL CIP-exposed HTR-8 / SVneo cells, and the levels and relative quantifications of MFF and TRIM21 protein in cell lysates (n=3 independent experiments). Figure 8 In the figures, ab represents the protein levels of TRIM21 in MFF(a) and MFF in TRIM21(b) during the IP assay; c represents the MFF mRNA level in MFF-overexpressing or knocked-down HTR-8 / SVneo cells; d represents the MFF protein level and its relative quantification in MFF-overexpressing or knocked-down HTR-8 / SVneo cells; ef represents the ability of viTRIM21-overexpressing (e) or knocked-down (f) HTR-8 / SVneo cells to survive at 0, 12, 24, 36, or 48 h; gh represents the SA-β-galactose staining in TRIM21-overexpressing or knocked-down HTR-8 / SVneo cells, and the quantification of SA-β-galactose-positive cells; ij represents the protein levels of P16, P21, and P38, and the β-gal in TRIM21-overexpressing or knocked-down HTR-8 / SVneo cells and their relative quantification. Figure 9In the table, a represents the CCK8 assay results of TRIM21 knockdown HTR-8 / SVneo cells exposed to 40 μg / mL CIP at 0, 12, 24, 36, or 48 hours (n=3 independent experiments); bc represents SA-β-galactosidase staining and quantification of positive cells in TRIM21 knockdown HTR-8 / SVneo cells exposed to 40 μg / mL CIP (n=3 independent experiments); de represents the levels and relative quantification of p16, p21, p38, and β-gal proteins in TRIM21 knockdown HTR-8 / SVneo cells exposed to 40 μg / mL CIP (n=3 independent experiments); f represents the relative mitochondrial DNA copy number in TRIM21 knockdown HTR-8 / SVneo cells exposed to 40 μg / mL CIP (n=3 independent experiments); g represents the relative mitochondrial DNA copy number in TRIM21 knockdown HTR-8 / SVneo cells exposed to 40 μg / mL CIP (n=3 independent experiments); The relative NAD+ / NADH ratio of TRIM21 knocked-down HTR-8 / SVneo cells under CIP exposure (n=3 independent experiments); h is the relative quantitative level of mitochondrial membrane potential in TRIM21 knocked-down HTR-8 / SVneo cells under 40 μg / mL CIP exposure (n=3 independent experiments); i is the relative quantitative level of reactive oxygen species (ROS) in TRIM21 knocked-down HTR-8 / SVneo cells under 40 μg / mL CIP exposure (n=3 independent experiments); j is the relative quantitative level of mitochondrial ROS in TRIM21 knocked-down HTR-8 / SVneo cells under 40 μg / mL CIP exposure (n=3 independent experiments). Figure 10 In the table, ab represents the TRIM21 protein level and its relative quantification in the villus tissue of healthy controls and patients with unexplained miscarriage (n=12); cg represents the MFF-ubiquitinated protein and TRIM21 protein levels precipitated by MFF antibody in the villus tissue of healthy controls and patients with unexplained miscarriage, as well as the MFF and TRIM21 protein levels and their relative quantification in the tissue lysate (n=12 per group); h represents the forest plot multivariate logistic regression analysis (including 95% confidence interval, n=12) of the association between TRIM21 protein level in villus tissue and miscarriage. Figure 11In the table, a represents the level of murine Trim21 mRNA in placental tissue of CIP-exposed mice (n=6); bc represents the level and relative quantification of murine Trim21 protein in placental tissue of CIP-exposed mice (n=6); d represents the levels of Trim21 protein and Mff-ubiquitinated protein precipitated by Mff antibody in placental tissue of 160 mg / kg / d CIP-exposed mice, and the levels and relative quantification of Mff and Trim21 protein in tissue lysate (n=6 per group); e is a schematic diagram of the Trim21 knockdown CIP-exposed mouse model: pregnant mice were treated with saline or 160 mg / kg / d CIP, and simultaneously given 10 mg / kg AS-Trim21 every 3 days (AS-NC as the control); fg represents the embryo resorption status (indicated by red arrows) and mean miscarriage rate of mice in the 160 mg / kg / d CIP exposure combined with Trim21 knockdown group (n=6 per group); h represents the 160 mg / kg / d... The levels of Mff-ubiquitinated protein and Trim21 protein precipitated by Mff antibody in placental tissue of mice exposed to CIP combined with Trim21 knockdown, and the levels and relative quantification of Mff and Trim21 protein in tissue lysate (n=6 per group); ij represents SA-β-galactosidase staining and positive intensity quantification in placental tissue of mice exposed to CIP combined with Trim21 knockdown at 160 mg / kg / d (n=6 per group); kl represents the levels and relative quantification of p16, p21, p38, and β-gal proteins in placental tissue of mice exposed to CIP combined with Trim21 knockdown at 160 mg / kg / d (n=6 per group); m represents the relative mitochondrial DNA copy number in placental tissue of mice exposed to CIP combined with Trim21 knockdown at 160 mg / kg / d (n=6); n represents the NAD+ / NADH ratio in placental tissue of mice exposed to CIP combined with Trim21 knockdown at 160 mg / kg / d (n=6); o represents the NAD+ / NADH ratio in placental tissue of mice exposed to CIP combined with Trim21 knockdown at 160 mg / kg / d (n=6); CIP exposure combined with Trim21 knockdown of placental tissue in mice (n=6) showed mitochondrial membrane potential levels. Detailed Implementation

[0014] This application proposes the use of TRIM21 as a biomarker in screening or preparing agents to reduce the risk of ciprofloxacin exposure-related miscarriage. It primarily addresses the shortcomings of existing technologies, such as the unclear key mechanisms and molecular pathways of pregnancy loss caused by environmental or non-therapeutic doses of ciprofloxacin exposure, the lack of objective biomarkers that can indicate risk status in early pregnancy, and the absence of standardized and reproducible detection methods. As shown in the figure, based on evidence from epidemiological studies, mouse models, and cell experiments, this application establishes a detection and assessment approach centered on TRIM21 expression levels: under ciprofloxacin exposure conditions, changes in TRIM21 expression in trophoblast cells, placental tissue, or villous tissue are detected and compared with placental aging-related phenotypes and pregnancy loss-related outcomes to evaluate the efficacy and application value of candidate interventions. Furthermore, candidates that can reduce or inhibit abnormally elevated TRIM21 expression levels can be used as the basis for screening or preparing agents to reduce the risk of ciprofloxacin exposure-related miscarriage. This allows for earlier risk identification and intervention evaluation, providing objective evidence for candidate agent screening, efficacy verification, and quality control during the preparation process.

[0015] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims. Example 1

[0016] method chemicals Ciprofloxacin hydrochloride (CIP, Macklin, C861180-25g) was dissolved in ddH2O to prepare a 5 mg / mL CIP stock solution. Cell counting kit-8 was purchased from Abcam, Cambridge, UK (ab228554). β-galactosidase (SA-β-gal) staining kit was purchased from Beyotime Biotechnology, Shanghai, China (C0602). Cell ROS detection kit was purchased from Abcam, Cambridge, UK (ab113851). Fluorescent probe MitoSOX™ Red was purchased from AbMole (M19992). Enhanced mitochondrial membrane potential detection kit (JC-1) was purchased from Beyotime Biotechnology, Shanghai, China (C2003S). Tissue mitochondrial isolation kit was purchased from Beyotime Biotechnology, Shanghai, China (C3606). FastPure blood / clock / tissue / bacterial DNA extraction mini-scale kit was purchased from Vazyme Biotechnology Co., Ltd. (DC112-01 / 02). The coenzyme I (NAD+ / NADH) assay kit was purchased from Addison (ADS-W-FM001). The protein synthesis inhibitor cyclohexylimide was purchased from Abcam, Cambridge, UK (ab120093).

[0017] chorionic villus tissue and urine samples Chorionic villus sampling and urine samples were collected from 50 women with unexplained abortion (UM group) and 50 women who voluntarily terminated their pregnancies due to unwanted pregnancy (HC group) at Shenzhen Maternity & Child Health Hospital affiliated with Southern Medical University, aged 25 to 30 years. All women were 6–10 weeks pregnant. Exclusion criteria included uterine abnormalities, chromosomal karyotype abnormalities, autoimmune abnormalities, antiphospholipid antibody syndrome, endocrine or metabolic disorders, polycystic ovary syndrome, preeclampsia or eclampsia, etc. Pregnancy was determined by a professional physician based on amenorrhea, morning sickness, β-hCG levels, early pregnancy factors, etc. Miscarriage was defined as loss of embryo or fetus weighing <500 g or before 20 weeks of gestation, including spontaneous abortion or voluntary abortion. The HC group had a history of successful pregnancy. The HC and UM women did not receive any treatment. The characteristics of these HC and UM women are listed in Table S1, including age, gestational age, BMI, education level, household income, smoking and alcohol consumption in the three months prior to the abortion procedure. All information was obtained from medical records. A piece measuring approximately 2 × 0.5 × 0.5 cm was manually dissected from the fetal side of the placenta. 3 Chorionic villus tissue of varying sizes was collected, and the maternal decidua was removed. After washing with sterile saline, the chorionic villus tissue samples were immediately frozen in liquid nitrogen and stored at -80 °C for RNA or protein extraction. Single-point midstream urine samples were collected from these women on the same day of the abortion procedure using polypropylene containers (n = 50 per group). The experimental protocol was approved by the Ethics Committee of Shenzhen Maternity & Child Health Hospital, Southern Medical University. All participants signed written informed consent forms before enrollment.

[0018] Table S1 shows potential confounding factors for the HC and UM groups.

[0019] a HC: Healthy control group. b UM: unexplained miscarriage group. c Student's t-test or Chi-square test. d mean ± standard deviation (n = 50 in each UM or HC group). e number of women f BMI: Body Mass Index. HPLC-MS / MS determination of CIP levels in urine samples CIP levels in urine samples were determined using HPLC-MS / MS. In short, human urine samples were mixed with an internal standard (ciprofloxacin-d8) and extracted using an Oasis Prime HLB extraction column (1 cc / 30 mg, Waters, Milford, Massachusetts, USA). The extracts were then separated using an Agilent 1200 series rapid resolution LC system (Agilent Technologies, Santa Clara, CA, USA) containing a reversed-phase ZORBAX SB-Aq C18 column (2.1 x 100 mm, 1.8 μm, Agilent Technologies) and a mobile phase of water and acetonitrile containing 0.1% formic acid. Subsequently, the samples were analyzed using an Agilent 6410 B triple quadrupole mass spectrometer (Agilent Technologies) equipped with an electrospray ionization source. Mass spectrometry conditions were as follows: ESI positive ion mode, nitrogen drying gas temperature 300°C, flow rate 10 L / min, nebulizer pressure 30 psi, capillary voltage 4000 V. Mass spectrometry data were acquired and processed using MassHunter workstation software B.01.03 and B.01.04 (Agilent Technologies). The limit of detection (LOD) for CIP in urine samples was 0.047 ng / mL. CIP levels in reagent blanks were all below the LOD. CIP was detected in 40% of patients in the HC group (n = 50) and 50% of patients in the UM group (n = 50). Calibration criteria were linear below 100 ng / mL (n = 3 per concentration), with a coefficient of determination of 0.9950 and an accuracy of 95%. Spike recoveries ranged from 85.3% to 97.6%, with relative standard deviations below 10.0%.

[0020] Mice and Experimental Design Pregnant C57BL / 6 mice (Charles River Company, Beijing, China) were randomly assigned to different groups (n = 6 per group). The day of vaginal plug appearance was considered day 1 of pregnancy (D1), and this was further confirmed by weighing the mice daily. Pregnant mice were treated with CIP daily. To explore the effect of real-environmental exposure dose (REED) of CIP on miscarriage, the actual daily intake dose of CIP in humans was calculated. One study showed that the CIP intake of residents of the Canary Islands, Spain, was approximately 37.5 μg / kg / day. This dose can be considered as the representative real-environmental exposure dose (REED) in humans under unconscious environmental exposure. Based on the body surface area coefficient (mice / human = 12), the REED of CIP in mice corresponds to 0.45 mg / kg / day (37.5 μg / kg / day x 12) of CIP. Clinically, patients with advanced chronic kidney disease may take 500-1000 mg / day of CIP, corresponding to 85-170 mg / kg / day (500 mg / day / 70 kg x 12) of CIP in mice. In another model study using C57BL / 6 mice with aortic aneurysm and dissection, exposure to 100 mg / kg / day of CIP for four weeks increased susceptibility to aortic dissection and rupture. Considering the dosage used in the literature “Effect of ciprofloxacin on susceptibility to aortic dissection and rupture in mice,” the short lifespan of mice, and the feasibility of mouse model experiments, the CIP dosages used in this mouse model were selected as 0, 0.45, 12.5, 160, or 320 mg / kg / day, corresponding to 0 (control), 1, 28, 356, or 712 times the REED of CIP, respectively.

[0021] Model 1: A mouse model of CIP exposure during pregnancy was established: ① Control group, given an equal volume of saline; ② 1-fold REED CIP group, given 0.45 mg / kg / day of CIP; ③ 28-fold REED CIP group, given 12.5 mg / kg / day of CIP; ④ 356-fold REED CIP group, given 160 mg / kg / day of CIP; ⑤ 712-fold REED CIP group, given 320 mg / kg / day of CIP. From day 1 to day 13, mice were administered CIP or an equal volume of saline via gavage daily.

[0022] Model 2 established a miscarriage intervention model by administering CIP-exposed pregnant mice D+Q (dasatinib and quercetin, cell senescence inhibitors, Aladdin): ① Control group, given an equal volume of saline; ② 160 mg / kg / day CIP in saline; ③ 160 mg / kg / day CIP and an equal volume of 10% PEG-400 as a solvent; ④ 160 mg / kg / day CIP and 5 mg / kg / 3 days of 10% PEG-400 as D and 50 mg / kg / 3 days of 10% PEG-400 as Q. From D1 to D13, mice were administered CIP or an equal volume of saline daily by gavage. Simultaneously, from D1 to D13, every three days, mice were administered 5 mg / kg D and 50 mg / kg Q of 10% PEG-400 or an equal volume of 10% PEG-400 by gavage.

[0023] Model 3 constructed another miscarriage intervention model by administering CIP to pregnant mice exposed to pcDNA3.1-Mff (overexpressing murine Mff): ① 160 mg / kg / d CIP and 10 mg / kg / 3d empty vector pcDNA3.1, and ② 160 mg / kg / d CIP and 10 mg / kg / 3d pcDNA3.1-Mff. Pregnant mice were administered CIP or an equal volume of saline by gavage daily from day 1 to day 13. Simultaneously, from day 1 to day 13, pcDNA3.1 (empty vector as control) or pcDNA3.1-Mff (plasmid overexpressing murine Mff) was administered intraperitoneally every three days.

[0024] Model 4, in another miscarriage intervention model, CIP-exposed pregnant mice were given AS-Trim 21 (knockdown of mouse Trim 21): ① 160 mg / kg / d CIP and AS-NC, and ② 160 mg / kg / d CIP and 10 mg / kg / 3d AS-Trim 21. Pregnant mice were administered CIP or an equal volume of saline by gavage daily from day 1 to day 13. Simultaneously, from day 1 to day 13, AS-NC (siRNA control) or AS-Trim21 (siRNA knockdown of mouse Trim21) was administered intraperitoneally every three days.

[0025] Model 5 established a miscarriage treatment model by administering F+Q (Fimepinostat and Quisinostat, Trim 21 inhibitors, FDA-approved drugs) to CIP-exposed pregnant mice: ① 160 mg / kg / day of CIP, and ② 160 mg / kg / day of CIP plus 50 mg / kg / 2 days of F and 10 mg / kg / 2 days of Q. From day 1 to day 13, mice were administered CIP or an equal volume of saline via gavage daily. Simultaneously, from day 1 to day 13, mice were also administered 50 mg / kg / 2 days of Fimepinostat (APExBIO, A4097) in saline via gavage every other day, and alternated with Fimepinostat by intraperitoneal injection of 10 mg / kg / 2 days of Quisinostat (APExBIO, A4090).

[0026] For models 1-5, mice were sacrificed on day 14 by injection of sodium pentobarbital (100 mg / kg), and uteruses were collected. Embryo resorption was defined as smaller in size or darker in color compared to healthy pink viable embryos. The abortion rate per mouse and the mean abortion rate per group were calculated as: (number of resorbed embryos) / (total number of normal embryos + number of resorbed embryos). RNA and protein were extracted from one placenta randomly selected from each mouse for RT-qPCR and Western blot analysis, respectively. This animal project was authorized by the Ethics Committee of the Eighth Affiliated Hospital of Sun Yat-sen University.

[0027] Cell culture Human HTR-8 / SVneo trophoblast cells (immortized with SVneo virus, purchased from Hunan Fenghui Biotechnology Co., Ltd., CL0164; Research Resource Identifier: CVCL_7162) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) (Gibco, Invitrogen), 100 U / mL penicillin, and 100 ng / mL streptomycin in a humidified incubator at 37°C and 5% CO2. The cell line was confirmed to be free of contamination by mycoplasma detection and STR identification. Under real-world exposure conditions, the average CIP level detected in pregnant women's urine samples was 73.5 μg / mg creatinine. The mean minimum inhibitory concentrations (MICs) of CIP against Salmonella Typhi and Salmonella Paratyphi A were 1.20 and 1.97 μg / mL, respectively. The normal CIP dose for antibacterial treatment is 50–400 μg / mL. Human primary vascular smooth muscle cells exposed to 110 μg / mL CIP experience accelerated angiotensin II-induced senescence. HT1197 and HT1376 cells exposed to 50–100 μg / mL CIP exhibited significant cytotoxic effects. Based on the dosage and preliminary experiments used in the literature “Ciprofloxacin and epirubicin synergistically induce apoptosis in human urothelial cancer cell lines,” this study constructed a CIP-exposed human trophoblast cell model by treating HTR-8 / SVneo cells at 37°C for 24 hours with 0, 10, 20, or 40 μg / mL CIP.

[0028] High-throughput mRNA sequencing and data processing Two random pairs of UM vs HC female chorionic villus tissue (0.1 g), 160 vs 0 mg / kg / d CIP-exposed mouse placental tissue (0.1 g), and 40 vs 0 μg / mL CIP-treated human trophoblast HTR-8 / SVneo cells (5 × 10^6 cells) were used for mRNA sequencing. High-throughput mRNA sequencing was performed on the HiSeq 2000 sequencing platform (BGI-Shenzhen) following BGI's commercial standard workflow (https: / / www.bgi.com / ). In short, total RNA was extracted using Trizol reagent (Thermo Fisher Scientific). The process included rRNA removal, double-stranded cDNA synthesis, end repair, degradation of one strand, and enrichment of the other strand by quantitative reverse transcription PCR (RT-qPCR). Library quality was confirmed by sequencing. Differentially expressed mRNAs (DEGs) were selected based on a fold change > 1.3 and p < 0.05, and were generated from read counts using Dr.Tom (biosys.bgi.com), an online bioinformatics platform provided by BGI Genomics. Human and mouse DEGs were searched in the Ensembl database (Homo sapiens or house mouse, NCBI_GCF_000001635.27_GRCm39) to determine their genomic locations. These DEGs were used for gene ontology (GO) and KEGG analysis, generating GO and KEGGs respectively.

[0029] Proteome sequencing Two random pairs of UM vs HC female chorionic villus tissue (0.1 g), 160 vs 0 mg / kg / d CIP-exposed mouse placental tissue (0.1 g), and 40 vs 0 μg / mL CIP-treated human trophoblast HTR-8 / SVneo cells (5 × 10^6 cells) were used for proteomic sequencing (Novogene, China). Proteomic sequencing was performed on an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific). Briefly, proteins were extracted and their concentrations were determined using the Bradford protein assay (Bio-Rad, USA). After digestion with Trypsin Gold (Promega), shotgun proteomics analysis was performed using an EASY-nLC 1200U HPLC system (Thermo Fisher Scientific) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific) in data-dependent acquisition mode. Differentially expressed proteins (DEPs) were selected based on a fold change >1.3 and p <0.05, and were generated from read counts using Novogene's online bioinformatics platform (https: / / magic.novogene.com / customer / main# / loginNew). DEPs were searched in the UniProt mouse (Mus_musculus_uniprot_2022_1_27.fasta, 86492 sequences) and Homo sapiens (homo_sapiens_uniprot_2022_1_27.fasta, 203711 sequences) databases to determine their genomic locations. These DEPs were used for gene ontology (GO) and KEGG analysis, generating GO and KEGG results respectively.

[0030] IP-MS analysis of MFF-binding proteins MFF-binding proteins were analyzed by Novogene Genetics (Beijing, China) using an EASY-nLC 1200 UHPLC system (Thermo Fisher Scientific) coupled with a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific). In short, MFF-binding proteins were separated and digested overnight at 37°C with trypsin (10 ng / μL). Samples were desalted, lyophilized, and resuspended in loading buffer A (0.1% formic acid, wt / vol). Subsequently, samples were eluted on a reversed-phase column with loading buffer B (0.1% formic acid in 80% acetonitrile, wt / vol) and injected into the mass spectrometer at a constant flow rate of 600 nL / min. The eluted peptides were analyzed by mass spectrometry, scanning m / z 300–1500 in positive ion mode at a spray voltage of 2.3 kV. The primary mass spectrometry resolution was set to 60,000 (at 200 m / z). The 40 most abundant precursor ions were selected from the full MS scan for analysis at a resolution of 15,000 (at 200 m / z). Raw mass spectrometry files were identified using MaxQuant (www.maxquant.org). Proteins were identified by searching the UniProt database (www.uniprot.org).

[0031] Cell transfection Empty vector pcDNA3.1 (catalog number V790-20) was purchased from Thermo Fisher Scientific. The cDNA used to construct the MFF (pcDNA3.1-MFF) and TRIM21 (pcDNA3.1-TRIM21) overexpression plasmids was synthesized and constructed by Addgene. The corresponding RNA sequences were obtained from the National Center for Biotechnology Information (NCBI) database (Gene Bank, Homo sapiens, GRCh38.p14; sequences shown in Table S3). Empty vector pcDNA3.1 was used as a negative control. Si-MFF, si-TRIM21, and si-NC (negative controls) were custom-made by Thermo Fisher (sequences shown in Table S2). HTR-8 / SVneo cells (1 × 10^6 cells / well) were seeded in 6-well plates and cultured to 80% confluence. Following the manufacturer's protocol, 1 μg of plasmid or 50 nM siRNA was transfected into feeder cells for 24 hours using turbofect transfection reagent (R0531, Thermo Scientific). Transfection efficiency was verified by RT-qPCR and WB.

[0032] Table S2 shows the DNA sequences used to construct the pcDNA3.1 overexpression plasmid.

[0033] Table S3 shows the RNA sequences of siRNAs used for cell transfection.

[0034] Cell vitality Cell viability was assessed using the Cell Counting Kit-8 (CCK8, ab228554, Abcam, Cambridge, UK). Cells (5 × 10^3 cells per well, triple replicates per group) were seeded in 96-well plates and treated with 0, 10, 20, or 40 μg / mL CIP for 0, 12, 24, 36, or 48 hours, respectively. In another experiment, HTR-8 / SVneo cells (5 × 10^3 cells per well, triple replicates per group) were seeded in 96-well plates and treated with either 1 μg plasmid or 50 nM siRNA, followed by co-treatment with 0 or 40 μg / mL CIP for 0, 12, 24, 36, or 48 hours. Subsequently, 10 μL of incubation reagent and 90 μL of DMEM / F12 medium were added according to the manufacturer's protocol. The 96-well plates were completely covered with aluminum foil to protect them from light. The plates were then incubated at 37°C for 1 hour. Absorbance was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA), with cell culture medium as background. Cell proliferation was represented by changes in absorbance at 450 nm. All experiments were repeated three times.

[0035] β-galactosidase activity assay β-galactosidase activity was determined using a aging-associated β-galactosidase (SA-β-gal) staining kit (Beyotime Biotechnology, Shanghai, China). Female chorionic villus tissue, various mouse placental tissues, and HTR-8 / SVneo cells under different treatments were used for β-galactosidase activity assays. Frozen sections of female chorionic villus tissue or mouse placental tissue and cells were fixed with 1.0 mL of fixative for 15 minutes at room temperature. The staining mixture was added to the frozen sections or cells and further incubated overnight at 37°C. The next day, cells were washed with PBS and observed under a microscope. SA-β-gal positive cells (blue-green) were counted in 15 random fields, and the results are expressed as the percentage of total tissue positivity or the percentage of total cells.

[0036] ROS (Reactive Oxygen Species) Measurement Intracellular ROS levels were measured using a Cell ROS Detection Kit (ROS, ab113851, Abcam, Cambridge, UK). ROS levels in HTR-8 / SVneo cells treated with 0 or 40 μg / mL CIP were determined under conditions of co-overexpression of Mff or co-knockdown of Trim 21. Cells (5 × 10^3 cells per well) were incubated at 37°C in the dark with 10 μM DCFH-DA (final concentration) for 30 min. Cells were thoroughly washed three times with PBS to remove excess DCFH-DA. Subsequently, the resulting cells were observed under a fluorescence microscope (Leica, Germany). DCFDA fluorescence of all samples was excited at 480 nm and detected at 525 nm, and quantified using Image J1.43 U (NIH Image J System, Bethesda, MD). All experiments were repeated three times. ROS levels are expressed as mean fluorescence intensity and normalized relative to control levels.

[0037] Mitochondrial ROS Measurement Mitosolic ROS levels in cells were detected using the fluorescent probe MitoSOX™ Red (AbMole). In short, cells were seeded in confocal dishes, treated with 1 μg plasmid or 50 nM siRNA and co-treated with 0 or 40 μg / mL CIP, and then incubated with 1–2 mL of 5 µM MitoSOX Red at 37°C in the dark for 10 min. Subsequently, the cells were gently washed three times with pre-warmed appropriate buffer. Finally, the cells were counterstained with DAPI staining solution; mitochondrial ROS levels were then observed under a confocal laser scanning microscope. Mitosolic ROS levels are expressed as mean fluorescence intensity and normalized relative to control levels.

[0038] Mitochondrial membrane potential (MMP) measurement MMP levels were determined using the JC-1 fluorescent probe. HTR-8 / SVneo cells were treated with 1 μg plasmid or 50 nM siRNA and co-treated with 0 or 40 μg / mL CIP. After washing twice with PBS, cells were incubated at 37°C with 1 mL of JC-1 staining working solution for 20 min. After washing twice with JC-1 buffer, changes in cellular MMPs were observed under a fluorescence microscope (Leica, Germany). JC-1 is a positively charged dye that accumulates in the inner mitochondrial membrane. Red fluorescence (excitation 525 nm, emission 590 nm) indicates healthy cells; green fluorescence (excitation 490 nm, emission 530 nm) indicates mitochondrial depolarization due to a decrease in membrane potential. MMP levels were expressed as the ratio of red to green fluorescence intensity, normalized relative to the control group.

[0039] MMP levels were also determined in female chorionic villus tissue and mouse placental tissue. First, tissue mitochondria were isolated using a tissue mitochondrial isolation kit (Beyotime Biotechnology, Shanghai, China). In short, female chorionic villus tissue and mouse placental tissue were homogenized with mitochondrial isolation medium A. After centrifugation at 4°C, 1000 g for 10 minutes, the supernatant was discarded; the precipitate was then collected by further centrifugation at 4°C, 11,000 g for 10 minutes. MMP levels were determined using an enhanced mitochondrial membrane potential detection kit (JC-1) (Beyotime Biotechnology, Shanghai, China). The levels were expressed as the ratio of red to green fluorescence intensity, standardized against the control group.

[0040] Mitochondrial DNA copy number Total mitochondrial DNA was extracted using the FastPure Blood / Clock / Tissue / Bacterial DNA Extraction Mini Kit (Vazyme Biotech Co., Ltd.) according to the manufacturer's instructions. mtDNA was quantified by quantitative real-time PCR using iTaq Universal SYBR Green (Bio-Rad) on a Bio-Rad CFX Connect with primers specific to mitochondrial proteins encoding 12S and nucleoactin. Primer sequences are shown in Table S4.

[0041] Table S4. Primer sequences used for various PCR detections.

[0042] NAD+ and NADH measurements Total intracellular NAD+ and NADH levels were measured using a Coenzyme I (NAD+ / NADH) assay kit (AIDISHENG, catalog number ADS-W-FM001) according to its instructions. In short, the assays were performed on HTR-8 / SVneo cells treated with 0 or 40 μg / mL CIP and co-overexpressing Mff or co-knockdown Trim 21, HC and UM female chorionic villus tissue, CIP-exposed mouse placental tissue, and CIP-exposed mouse placental tissue treated with Mboat1 overexpression plasmid, AS-Trim 21, or F+Q. NAD+ extraction was performed as follows: Cells (5 x 10^7) or tissues (0.1 g) were incubated on ice with 1 mL of extraction buffer A at 60 °C for 30 min, followed by centrifugation at 4 °C, 12,000 rpm for 10 min. The supernatant was then mixed with extraction buffer B to neutral pH. NAD+ was collected by centrifugation at 4 °C, 12,000 rpm for 5 min. NADH extraction was performed as follows: Cells (5 x 10^7) or tissue (0.1 g) were incubated on ice with 1 mL of extraction buffer B at 60 °C for 30 min, followed by centrifugation at 4 °C and 12,000 rpm for 10 min. The supernatant was mixed with extraction buffer A to neutral pH. NADH was then collected by centrifugation at 4 °C and 12,000 rpm for 5 min. Subsequently, NAD+ or NADH was mixed with reagents one through four in the kit, and absorbance was measured at 450 nm using a microplate reader, with a second measurement after 30 min. NAD+ and NADH concentrations were calculated based on a standard curve. NAD+ and NADH concentrations are expressed as nmol / 10^4 cells or nmol / g tissue, and normalized relative to control levels.

[0043] Quantitative reverse transcription PCR (RT-qPCR) Total RNA was extracted from various HTR-8 / SVneo cells, female chorionic villus tissue, and mouse placental tissue using Trizol (Invitrogen, Carlsbad, USA). RNA quality and quantity were assessed using a NanoDrop 2000 UV spectrophotometer (Thermo Fischer Scientific, Waltham, USA); RNA integrity index (RIN) was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). RNA concentrations ranged from 500 to 1500 ng / μL, with RIN ≥ 9. RNA purity was high, with A260 / 280 values ​​between 1.8 and 2.2. The isolated RNA (800 ng) was converted to cDNA using a first-strand cDNA synthesis kit (Invitrogen). The cDNA was then amplified using 20 μL of SYBR Green Supermix (Takara, Kyoto, Japan). The RT-qPCR program was as follows: 95 °C pre-denaturation for 30 seconds, cycling at 95 °C for 10 seconds for a total of 40 cycles, followed by melting at 95 °C for 15 seconds, 60 °C for 60 seconds, and then 95 °C for 15 seconds. Specific primer sequences are shown in Table S4. The mRNA level of GAPDH remained unchanged during the experiments and was used as a standardized internal control for all mRNA detections. All experiments were repeated three times. The mRNA expression level was 2... -ΔΔCt Where Ct is the cycle threshold, ΔCt = target gene Ct - average GAPDH (Ct), ΔΔCt = sample group ΔCt - average control group ΔCt.

[0044] Western blot analysis Total protein was extracted from human trophoblast HTR-8 / SVneo cells, female chorionic villus tissue, and mouse placental tissue using RIPA lysis buffer (Thermo Fisher Scientific) and quantified using the Pierce BCA Protein Assay Kit (Pierce). Proteins (10–30 μg per well, equal amounts within groups but different amounts between groups for better comparison) were separated on 10% or 12% SDS-PAGE gels and transferred to pre-equilibrated polyvinylidene fluoride membranes (PVDF, Amersham Biosciences, Buckinghamshire, UK). After blocking with TBST (10 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20) containing 5% bovine serum albumin (BSA, Sigma-Aldrich) for 1 hour at room temperature, the membranes were incubated overnight with primary antibody at 4 °C. Primary antibodies included anti-MFF (17090-1-AP, Proteintech, 1:1000 dilution), anti-TRIM21 (PS03101S, Abmart, 1:1000 dilution), anti-GLB1 (R244445, Zen BioScience, 1:1000 dilution), anti-P38 (200782, Zen BioScience, 1:1000 dilution), anti-P21 (R25235, Zen BioScience, 1:1000 dilution), anti-P16 (R23897, Zen BioScience, 1:1000 dilution), and anti-GAPDH (5174, CST, 1:1000 dilution). The PVDF membrane was washed three times with TBST and then incubated with secondary antibodies at room temperature for 1 hour. Secondary antibodies included goat anti-rabbit IgG (ab205718, Abcam, 1:10000 dilution) and goat anti-mouse IgG (ab6789, Abcam, 1:10000 dilution). The PVDF membrane was then washed three times, and protein detection was performed using enhanced chemiluminescence (Amersham Corporation, Arlington Heights, IL, USA). The intensity of each band was quantified using Image J. All experiments were repeated three times. The band density values ​​in both experimental and control groups were normalized to their corresponding GAPDH band (sample control, ratio / GAPDH%).

[0045] Protein stability testing MFF protein stability was measured in cells treated with the protein synthesis inhibitor cycloheximide (CHX, Abcam, ab120093) to reflect protein degradation. For CIP-exposed cells co-treated with CHX, cells were first treated with 40 μg / mL CIP for 48 h, then incubated with 10 μM CHX for 0, 2, 4, 6, or 8 h. Cells were then collected, and proteins in the cell lysate supernatant were analyzed by Western blotting. Protein band intensity was quantified using ImageJ. These experiments were performed independently three times.

[0046] The impact of CIP on protein degradation pathways Protein degradation assays were performed as previously described. Cells were treated with 10 μM MG132 (a proteasome inhibitor) or 50 μM CQ (a lysosomal inhibitor) for 6 hours to inhibit protein degradation. For CIP-exposed feeder cells co-treated with MG132 or CQ and CHX, cells were first treated with 40 μg / mL CIP for 48 hours and then transferred to fresh culture medium. They were then incubated with 10 μM CHX for 4 hours, followed by incubation with 10 μM MG132 or 50 μM CQ for another 6 hours. Total protein was then extracted, and the remaining protein was analyzed by Western blotting. These experiments were performed independently three times.

[0047] Co-immunoprecipitation (Co-IP) analysis HTR-8 / SVneo cells (1 × 10^7) were lysed on ice using IP lysis buffer (Thermo Fisher Scientific). Villous or placental tissue (approximately 0.2 g) was mixed with IP lysis buffer and homogenized in a homogenizer. Proteins in the cell lysates were quantified using the Pierce BCA Protein Assay Kit. The lysates were incubated overnight at 4°C with MFF antibody (17090-1-AP, Proteintech, 1:200 dilution) or TRIM21 antibody (PS03101S, Abmart, 1:200 dilution) as a negative control, followed by incubation with Protein A / G magnetic beads (HY-K0202, MedChemExpress) for an additional 6 hours to form bead-immunoprecipitation complexes. Subsequently, the magnetic beads were washed five times with IP washing buffer, and the immunoprecipitated proteins were harvested by boiling them in 1 × SDS loading buffer at 98°C for 10 minutes for subsequent Western blot analysis.

[0048] ubiquitination analysis Cell and tissue lysates (1 × 10^7 cells in 0.1 mL IP lysis buffer or 0.2 g tissue in 1 mL IP lysis buffer) were gently incubated overnight at 4°C with anti-MFF (17090-1-AP, Proteintech, 1:200 dilution) using a gentle rotational incubation method, with an equal volume of IgG antibody (ab172730, Abcam, 1:200 dilution) as a negative control. The mixture was then gently incubated at 4°C with 40 μL Protein A / G Magnetic Beads (B23201, MCE, USA) for 4 hours. Afterward, the magnetic beads were separated and thoroughly washed with IP lysis buffer, then mixed with SDS loading buffer and boiled at 95°C for 10 minutes. Subsequently, the immunoprecipitated proteins were separated by 10% SDS-PAGE, and ubiquitinated MFFs were detected by Western blotting using anti-ubiquitin antibody (1:5000 dilution, ab179434, Abcam).

[0049] 3. Results Part 1: CIP exposure induces miscarriage.

[0050] Epidemiological analysis shows that CIP exposure is associated with unexplained miscarriage in women.

[0051] To explore the potential association between CIP exposure and unexplained miscarriage (UM), urine samples were newly collected from UM patients and their matched healthy controls (HC) who voluntarily chose to terminate their pregnancies due to unwanted pregnancy (n = 50). Figure 1 a) Known causes were excluded, such as autoimmune abnormalities, uterine abnormalities or cervical insufficiency, symptoms of endocrine or metabolic disorders, luteal phase defects, and antiphospholipid antibody syndrome. To account for the influence of potential confounding factors, several variables, such as baseline characteristics, clinical information, and lifestyle, were collected from medical records based on previous research and their potential association with miscarriage (Table S1). These variables did not show statistically significant differences between the two groups (Table S1).

[0052] The level of CIP in urine samples was detected by HPLC-MS / MS. The average level of CIP in urine samples of the UM group was 0.277 ng / mL, and that of the HC group was 0.093 ng / mL. Figure 1 b). The CIP level in urine samples from the UM group was significantly higher than that in the HC group ( Figure 1 b). In the unadjusted model, univariate logistic regression analysis showed that higher urinary CIP levels (OR = 27.235, 95% CI, 2.942–252.158) were positively correlated with miscarriage. Figure 1c). Based on directed acyclic graph analysis, variables such as age, BMI, education level, family income, smoking, and alcohol consumption were considered potential confounding factors for miscarriage. Figure 2 a). To account for its impact, a multivariate logistic regression model analysis was performed after adjusting for all these confounding factors. The results showed that CIP levels were still associated with miscarriage (adjusted OR = 134.409 in urine samples, 95% CI 4.78 - 3773.07). Figure 1 C). To further examine whether the estimated association differed across subgroups, stratified analysis showed that the stratification factor did not significantly alter the association between CIP levels and miscarriage (all interaction p-values ​​> 0.05). Figure 2 (b) confirms the robustness of these results. In summary, all statistical analyses confirm a positive correlation between CIP exposure and unexplained miscarriage.

[0053] Mouse model experiments confirmed that CIP exposure induces abortion in mice.

[0054] Subsequently, the causal relationship between CIP exposure and potential induction of miscarriage was investigated. To this end, a CIP-exposed pregnant mouse model was established. Pregnant mice were administered CIP by gavage daily for 13 consecutive days (from D1 to D13) at doses of 0, 0.45, 12.5, 160, or 320 mg / kg / day. Figure 1 d). As validation, exposure to ≥12.5 mg / kg / d CIP reduced the weight gain in CIP-exposed mice ( Figure 1 ef, Figure 2 c), increase the number of embryos resorbed ( Figure 1 g), and increased mouse abortion rate ( Figure 1 (h), indicating that exposure to high levels of CIP during pregnancy can induce abortion in mice.

[0055] Part Two: CIP exposure induces miscarriage by promoting aging.

[0056] mRNA and protein sequencing suggest that aging may be involved in CIP exposure-induced miscarriage.

[0057] To investigate which cell phenotypes might be involved in CIP exposure-induced miscarriage, high-throughput transcriptome sequencing was performed to screen for the most significantly altered cell phenotypes. First, two pairs of HC and UM female villus tissues were randomly selected for transcriptome sequencing. GO and KEGG analyses of differentially expressed mRNAs (DEGs) showed significant enrichment of senescence-related GO entries (cellular senescence, senescence, cellular senescence, inflammatory response) and the KEGG pathway (cellular senescence). Second, mouse placental tissues exposed to 160 mg / kg / d CIP (with unexposed tissues as controls) were also used for transcriptome sequencing. GO and KEGG analyses of these DEGs also showed significant enrichment of senescence-related GO entries (cellular senescence) and the KEGG pathway (cellular senescence). Third, trophoblast cells in placental villus tissue are crucial for healthy female pregnancies; HTR-8 / SVneo cells have been widely used as a trophoblast cell model in various miscarriage studies. HTR-8 / SVneo cells treated with 40 vs 0 μg / mL CIP were used for transcriptome sequencing. GO and KEGG analyses of these DEGs revealed that CIP exposure altered GO entries (cellular senescence and cell senescence) and KEGG pathways (cellular senescence) associated with cellular senescence. Therefore, these transcriptome sequencing analyses suggest that senescence may be involved in CIP exposure-induced miscarriage.

[0058] In addition to transcriptome sequencing, these villous tissues, mouse placental tissues, and HTR-8 / SVneo cells were also used for proteome sequencing. GO and KEGG analyses of differentially expressed proteins (DEPs) in UM vs HC villous tissues showed significant enrichment of senescence-related GO entries (cellular senescence) and the KEGG pathway (MAPK signaling pathway). GO and KEGG analyses of DEPs in placental tissues from mice exposed to 160 vs 0 mg / kg / d CIP also showed significant enrichment of senescence-related GO entries (senescence and inflammation) and the KEGG pathway (Wnt signaling pathway). Simultaneously, GO and KEGG analyses of DEPs in HTR-8 / SVneo cells exposed to 40 vs 0 μg / mL CIP also showed alterations in senescence-related GO entries (senescence) and the KEGG pathway (oxidative phosphorylation). Therefore, these proteome sequencing analyses also suggest that senescence may be involved in CIP exposure-induced miscarriage. In conclusion, both transcriptome and proteome sequencing analyses indicate that CIP exposure may alter senescence and induce miscarriage.

[0059] Experiments using trophoblast cells, female chorionic villus tissue, and mouse models have confirmed that CIP exposure induces miscarriage by causing aging.

[0060] To confirm this experimentally, firstly, in CIP-exposed HTR-8 / SVneo cells, CIP exposure inhibited cell viability ( Figure 3a) and increased the level of SA-β-galactosidase-positive cells ( Figure 3 bc). Simultaneously, CIP exposure also upregulated the protein levels of cellular senescence-related proteins (P16, P21, P38, and β-gal) in CIP-exposed HTR-8 / SVneo cells. Figure 3 These data support the view that CIP exposure leads to trophoblast cell senescence.

[0061] Secondly, in the female villous tissue, the level of SA-β-galactosidase-positive cells in the villous tissue of the UM group ( Figure 3 The protein levels of fg) and P16, P21, P38 and β-gal were higher than those in the HC group. Figure 3 (hi), indicating a higher level of senescence in the umbilical region (UM) villous tissue. Logistic regression analysis showed that higher levels of P16, P21, P38, and β-gal proteins were associated with miscarriage. Figure 3 (j) indicates that higher levels of senescence in chorionic villi are associated with unexplained miscarriage. Furthermore, Pearson correlation analysis showed a positive correlation between urinary CIP levels and the levels of SA-β-galactosidase-positive cells, P16, P21, P38, or β-gal protein in UM chorionic villi. Data points were clearly separated between the UM and HC groups. In conclusion, these results suggest a positive correlation between CIP exposure, chorionic villi senescence, and unexplained miscarriage.

[0062] Third, in mouse placental tissue, CIP exposure reduced the level of SA-β-galactosidase-positive cells in mouse placental tissue ( Figure 3 kl) and the protein levels of murine P16, P21, P38 and β-gal ( Figure 3 The levels of mn were all higher. The amino acid sequences of P16, P21, P38, and β-gal were conserved in humans, mice, monkeys, pigs, and cattle (Table S5). Therefore, these results confirm that CIP exposure leads to placental aging in mice and induces abortion. To further verify the role of aging in CIP-induced abortion in mice, a mouse abortion intervention model was constructed in which CIP-exposed pregnant mice were orally administered 5 mg / kg / 3d D and 50 mg / kg / 3d Q (D+Q). Figure 3 D+Q is a typical aging inhibitor and has been widely used in various cell and animal experiments. D+Q treatment significantly reduced embryo resorption and miscarriage rates in CIP-exposed mice. Figure 3 pq). This treatment also effectively restored (i.e. reduced) the level of SA-β-galactosidase-positive cells in mouse placental tissue. Figure 3 r) and the protein levels of murine P16, P21, P38 and β-gal ( Figure 3In summary, these results confirm that inhibiting placental aging in mice can effectively reduce miscarriage in this CIP-exposed mouse model. In other words, CIP exposure induces miscarriage in mice by causing placental aging.

[0063] In summary, experiments based on CIP exposure in trophoblast cells, female HC and UM villous tissue, and two CIP-exposed mouse models confirmed that CIP exposure induces miscarriage by causing senescence.

[0064] Part 3: CIP exposure induces aging by downregulating MFF.

[0065] CIP downregulation of MFF leads to senescence of human trophoblast cells.

[0066] Subsequently, the study explored which proteins might play a key role in trophoblast cell senescence induced by CIP exposure. In the intersection of three proteomic datasets, the MFF protein with the lowest expression level was identified ( Figure 4 a). MFF is known to be an important protein in mitochondrial division, participating in mitochondrial division and function. However, whether it is involved in trophoblast cell senescence induced by CIP exposure is completely unknown. To explore its novel function, we first examined its mRNA and protein levels in CIP-exposed trophoblast cells. CIP exposure decreased MFF protein levels but did not alter its mRNA levels (a). Figure 4 bc), which is consistent with the finding that there are significant differences in the proteome dataset but not in the transcriptome dataset. Subsequently, overexpression of MFF ( Figure 5 ab) Promotes cell vitality ( Figure 5 c); and knocking down MFF ( Figure 5 ab) Inhibits cell viability ( Figure 5 d). Simultaneously, MFF overexpression downregulated MFF, while knockdown of MFF upregulated the levels of SA-β-galactosidase-positive cells and the protein levels of P16, P21, P38, and β-gal in trophoblast cells. Therefore, these results indicate that CIP downregulated MFF expression levels, and that MFF itself can effectively inhibit trophoblast cell senescence. Furthermore, co-overexpression of MFF in CIP-exposed trophoblast cells restored (i.e., increased) cell viability. Figure 4 d) and (i.e., reduce) the level of SA-β-galactosidase-positive cells ( Figure 4 ef) and the protein levels of P16, P21, P38 and β-gal ( Figure 4 The results indicate that co-overexpression of MFF can alleviate trophoblast cell senescence induced by CIP exposure. In summary, these results support the view that MFF can inhibit trophoblast cell senescence; CIP exposure downregulates MFF expression levels, thereby inducing trophoblast cell senescence.

[0067] Mitochondrial free radicals (MFFs) are known to be key regulatory molecules for maintaining mitochondrial function. Furthermore, mitochondrial dysfunction has been shown to be associated with aging. However, whether CIPs and MFFs may co-regulate mitochondrial function in trophoblast cells remains unknown. CIP exposure reduces the copy number of mitochondrial DNA (mtDNA). Figure 4 i) NAD+ / NADH ratio ( Figure 4 j) and mitochondrial membrane potential (MMP) level Figure 4 k), and increased total cellular ROS (k), and increased total cellular ROS (k) Figure 4 l) and mitochondrial ROS levels ( Figure 4 This indicates that CIP exposure leads to mitochondrial dysfunction. However, co-overexpression of MFF reversed all these changes and rescued mitochondrial function in CIP-exposed trophoblast cells. Figure 4 Therefore, these results support the idea that MFF can effectively maintain mitochondrial function; CIP exposure downregulates MFF expression levels, thereby causing mitochondrial dysfunction, which may further induce trophoblast cell senescence.

[0068] CIP exposure, lower MFF protein levels, mitochondrial dysfunction, and villous tissue aging are associated with unexplained miscarriage in women.

[0069] Secondly, in the female villous tissue, the MFF protein level in the UM group was significantly lower than that in the HC group. Figure 4 No. However, its mRNA level remained almost unchanged in both groups. Figure 5 e). Meanwhile, the mtDNA copy number in the chorionic villus tissue of the UM group ( Figure 4 p), NAD+ / NADH ratio ( Figure 4 q) and MMP level ( Figure 4 The levels of MFF protein, mtDNA copy number, NAD+ / NADH ratio, and MMP were all lower than those in the HC group. Therefore, mitochondrial function maintained by MFF was disrupted in the UM group villous tissue. Logistic regression analysis showed that lower MFF protein levels, mtDNA copy number, NAD+ / NADH ratio, and MMP levels were associated with miscarriage. Pearson correlation analysis further showed that urinary CIP levels were negatively correlated with MFF protein levels, mtDNA copy number, NAD+ / NADH ratio, and MMP levels in UM villous tissue, and exhibited a linear relationship. Figure 5 Meanwhile, the MFF protein level in UM villous tissue was positively correlated with mtDNA copy number, NAD+ / NADH ratio, and MMP level, and showed a linear relationship. Figure 5 Furthermore, the MFF protein level in UM villous tissue was negatively correlated with and linearly related to the protein levels of P16, P21, P38, and β-gal. Figure 5(mp). All data points in the UM and HC groups were relatively separated. In summary, these results confirm a close association between CIP exposure, lower MFF protein levels, mitochondrial dysfunction, aging, and unexplained miscarriage.

[0070] CIP exposure downregulates Mff protein levels, leading to placental mitochondrial dysfunction and aging, thereby inducing miscarriage in a CIP-exposed mouse model.

[0071] Third, in mouse models, CIP-exposed mouse placental tissue showed lower levels of murine Mff protein. Figure 6 ab). However, its mRNA level remained almost unchanged ( Figure 6 c). The amino acid sequence of MFF is conserved in humans, mice, monkeys, pigs, and cattle (Table S5). To further investigate the role of MFF in CIP-induced aging and miscarriage, another mouse intervention model was constructed in which CIP-exposed mice were intraperitoneally injected with an MFF overexpression plasmid, with an empty vector as a control. Figure 6 d). Overexpression of Mff reduced embryo resorption and alleviated miscarriage in CIP-exposed mice. Figure 6 Overexpression of Mff also restored (i.e. reduced) the level of SA-β-galactosidase-positive cells in placental tissue of CIP-exposed mice. Figure 6 gh) and the protein levels of Mff, P16, P21, P38 and β-gal ( Figure 6 Furthermore, overexpression of Mff also restored (i.e. increased) mtDNA copy number, NAD+ / NADH ratio, and MMP levels in placental tissue of CIP-exposed mice. Figure 6 In summary, these results indicate that overexpression of Mff can restore mitochondrial function, alleviate placental aging, and mitigate miscarriage in CIP-exposed mice. In other words, CIP exposure downregulates Mff protein levels, thereby causing mitochondrial dysfunction and placental aging, which in turn induces miscarriage in mice.

[0072] Part IV: CIP promotes TRIM21-mediated MFF ubiquitination degradation.

[0073] CIP promotes TRIM21-mediated MFF ubiquitination degradation, leading to senescence in human trophoblast cells.

[0074] Since CIP exposure downregulated MFF protein levels rather than mRNA levels, the underlying mechanism was subsequently explored. Protein stability assays showed that CIP exposure promoted the degradation of MFF protein. Figure 7(ab). Typically, proteins are degraded via the ubiquitination-proteasome pathway or the autophagy-lysosome pathway, which can be specifically blocked by the proteasome inhibitor MG132 or the lysosome inhibitor CQ, respectively. In CIP-exposed trophoblast cells, treatment with MG132 (but not CQ) significantly increased the accumulation of MFF protein. Figure 7 c). Simultaneously, CIP exposure also increased the level of ubiquitinated MFF (MFF-Ub, an intermediate product of MFF ubiquitination degradation). Figure 7 The results showed that CIP exposure promoted the ubiquitination and degradation of MFF.

[0075] Since the E3 ubiquitin ligase of MFF remains unknown, attempts were subsequently made to identify it. MFF IP-MS experiments showed that an E3 ubiquitin ligase, TRIM21, was the protein that interacted most strongly with MFF. Figure 7 f). In the intersection of the three proteomic sequencing datasets, TRIM21 protein levels were also highly expressed in all three datasets. Figure 4 a). Therefore, TRIM21 was chosen, and its ligase function was explored in subsequent studies. IP analysis confirmed that TRIM21 and MFF protein interact ( Figure 8 ab). IP experiments showed that overexpression of TRIM21 ( Figure 8(cd) upregulated MFF-Ub protein levels and decreased MFF protein levels in cell lysates; while knockdown of TRIM21 produced the opposite result. To further identify the potential ligase active site of TRIM21, Cys-16 was identified as a possible active site for TRIM21 based on Uniprot analysis. Therefore, Cys-16 was mutated to Ala to construct the mutant TRIM21 (TRIM21-mt). For ease of detection, a His tag was added to MFF to construct His-MFF, and a Flag tag was added to wild-type or mutant TRIM21 to construct Flag-TRIM21-wt or Flag-TRIM21-mt. IP experiments using a limited but equal amount of His antibody (anti-His-MFF) showed that, compared to Flag-TRIM21-wt, overexpression of Flag-TRIM21-mt decreased MFF-Ub protein levels and increased MFF protein levels in cell lysates. As a control, overexpression of Flag-TRIM21-mt and Flag-TRIM21-wt yielded similar protein levels in cell lysates. However, His-MFF was downgraded to similar Flag-TRIM21-mt and Flag-TRIM21-wt protein levels. These results indicate that mutations at the TRIM21 active site reduce MFF ubiquitination but do not affect its binding to MFF. Therefore, these results confirm that TRIM21 is an E3 ubiquitin ligase of MFF and promotes its ubiquitination and degradation.

[0076] Next, the combined roles of CIP and TRIM21 in MFF ubiquitination and degradation were investigated. First, it was explored whether CIP might regulate TRIM21 expression levels. It was found that CIP exposure upregulated both the mRNA and protein levels of TRIM21. Figure 7 gh), and promoted the formation of the TRIM21 and MFF complex ( Figure 7 Furthermore, co-transfection experiments showed that co-knockdown of TRIM21 in CIP-exposed trophoblast cells restored (i.e. reduced) MFF-Ub protein levels and (i.e. increased) MFF protein levels. Figure 7 In summary, these results indicate that CIP exposure upregulates TRIM21 protein levels, promotes TRIM21-mediated MFF ubiquitination and degradation, and ultimately downregulates MFF protein levels in CIP-exposed trophoblast cells.

[0077] Subsequently, the study further explored whether TRIM21 is involved in CIP-induced mitochondrial dysfunction and senescence in trophoblast cells. Overexpression of TRIM21 inhibited mitochondrial function, while knockdown of TRIM21 promoted trophoblast cell viability. Figure 8ef). Overexpression of TRIM21 upregulates, while knockdown of TRIM21 downregulates the level of SA-β-galactosidase-positive cells in trophoblast cells, as well as the protein levels of β-gal, P38, P21, and P16. Figure 8 gj). Co-transfection experiments also showed that knocking down TRIM21 in CIP-exposed trophoblast cells could restore (i.e. increase) cell viability. Figure 9 a) and (i.e., reducing) the level of SA-β-galactosidase-positive cells ( Figure 9 bc) and the protein levels of P16, P21, P38 and β-gal ( Figure 9 Furthermore, co-knockdown of TRIM21 also restored (i.e. increased) the mtDNA copy number in CIP-exposed trophoblast cells. Figure 9 f) NAD+ / NADH levels ( Figure 9 g) and MMP ( Figure 9 h, Figure 10 a), and (i.e., reduced) total cellular ROS ( Figure 9 i) and mitochondrial ROS levels ( Figure 9 These results indicate that CIP exposure upregulates TRIM21 protein levels, leading to mitochondrial dysfunction and senescence in CIP-exposed trophoblast cells.

[0078] Validation of TRIM21 / MFF in female villous tissue.

[0079] Then, the TRIM21-mediated MFF ubiquitination degradation in female chorionic villus tissue was investigated. The TRIM21 protein level in the UM group was higher than that in the HC group. Figure 10 ab). IP experiments using limited but equal amounts of MFF antibody showed that the UM group had higher levels of TRIM21 protein and MFF-Ub in the villous tissue, and lower levels of MFF protein in the cell lysate. Figure 10 cg). Logistic regression analysis showed that higher TRIM21 protein levels were associated with miscarriage ( Figure 10 h). Furthermore, Pearson correlation analysis showed that urinary CIP levels were positively correlated with TRIM21 or MFF-Ub protein levels in umbilical villous tissue; TRIM21 and MFF-Ub protein levels were positively correlated in the UM group; TRIM21 protein levels in the UM group were negatively correlated with mtDNA copy number, NAD+ / NADH ratio, and MMP levels, and positively correlated with P16, P21, P38, and β-gal protein levels. Data points were clearly separated between the UM and HC groups. In conclusion, these results confirm a close association between CIP exposure, TRIM21 and MFF-Ub protein levels in villous tissue, mitochondrial dysfunction, aging, and unexplained miscarriage.

[0080] Validate the murine Trim21 / Mff in a CIP-exposed mouse model.

[0081] Subsequently, the role of Trim21 in this CIP-exposed mouse model was also verified. The amino acid sequence of TRIM21 is conserved in humans, mice, monkeys, pigs, and cattle. Higher levels of mouse-derived Trim21 protein were observed in placental tissue from CIP-exposed mice. Figure 11 ab). IP experiments using limited but equal amounts of Mff antibody showed that CIP-exposed mouse placental tissue had higher levels of Mff-downward Trim21 protein and Mff-Ub, and lower levels of Mff protein in the lysate. Figure 11 To verify the role of Trim21 in CIP-induced abortion, another abortion intervention model was constructed, in which pregnant mice exposed to CIP received intraperitoneal injection of AS-Trim21 (an antisense oligonucleotide of Trim21). AS-Trim21 can complementary pair with Trim21 mRNA, thereby reducing the level of Trim21 protein in mouse trophoblast cells. Figure 11 e). Knocking down Trim21 reduced embryo resorption and alleviated miscarriage in this CIP-exposed mouse model. Figure 11 fg). IP experiments using limited but equal amounts of Mff antibody showed that knocking down Trim21 restored (i.e. reduced) Mff-downgraded Trim21 protein levels, Trim21 protein levels in tissue lysates, and Mff-Ub levels, and (i.e. increased) Mff protein levels in tissue lysates. Figure 11 h). Knocking down Trim21 also restored (i.e. reduced) the level of SA-β-galactosidase-positive cells in placental tissue of CIP-exposed mice. Figure 11 The protein levels of P16, P21, P38, and β-gal (ij) and P21, P38, and β-gal (ij) Figure 11 Furthermore, knocking down Trim21 also restored (i.e. increased) mtDNA copy number, NAD+ / NADH ratio, and MMP levels. Figure 11 In summary, these results indicate that CIP exposure upregulates Trim21 protein levels, promotes Trim21-mediated Mff ubiquitination and degradation, leading to placental mitochondrial dysfunction and senescence, thereby inducing abortion in this CIP-exposed mouse model.

[0082] Part Five: Treatment for Miscarriage To date, there are no clinically available MFF agonists. Fimepinostat and quinsinostat (Fimepinostat + Quisinostat) have been reported as clinical inhibitors of TRIM21 and have been widely used in various cell and animal experiments. To validate their function, treatment with F+Q was found to downregulate Trim21 protein levels in mouse trophoblast cells. A mouse abortion treatment model was then constructed in which CIP-exposed pregnant mice were orally administered F and intraperitoneally injected with Q. In this model, F+Q treatment downregulated Trim21 protein levels and upregulated MFF protein levels in mouse placental tissue, reduced embryo resorption, and decreased the abortion rate in mice. Furthermore, IP experiments using limited but equal amounts of MFF antibody showed that this treatment also restored (i.e., reduced) MFF-downregulated Trim21 protein levels, Trim21 protein levels in tissue lysates, and Mff-Ub levels, but (i.e., increased) MFF protein levels in tissue lysates. This treatment also restored (i.e. reduced) the level of SA-β-galactosidase-positive cells and the protein levels of P16, P21, P38, and β-gal in placental tissue of CIP-exposed mice. Furthermore, this treatment restored (i.e. increased) mtDNA copy number, NAD+ / NADH ratio, and MMP levels. In summary, these results indicate that therapeutic downregulation of Trim21 can effectively restore mitochondrial function, inhibit placental aging, and reduce abortion in mice, providing an important biological target for the treatment of unexplained abortion.

[0083] A novel approach to CIP exposure-induced trophoblast cell senescence This study found that CIP exposure leads to trophoblast cell senescence by promoting TRIM21-mediated MFF ubiquitination. CIP exposure downregulates MFF protein levels. MFF protein is degraded via the ubiquitination-proteasome pathway. TRIM21, a novel E3 ligase for MFF, was further identified. CIP exposure upregulates TRIM21 expression, thereby promoting TRIM21-mediated MFF ubiquitination and degradation, and downregulating MFF protein levels. Subsequently, the downregulated MFF induces various mitochondrial dysfunctions, such as decreased mtDNA copy number and NAD+ / NADH ratio, and increased total ROS and mitochondrial ROS levels, which further contribute to cellular senescence, including increased SA-β-galactosidase-positive cell levels and protein levels of β-gal, P38, P21, and P16. Trophoblast cell senescence subsequently induces abortion. The cellular mechanisms are consistent with those observed in umbilical cord tissue of female UM women and placental tissue of CIP-exposed mice. Reducing placental aging, supplementing with mouse Mff, and knocking down mouse Trim21 can all effectively alleviate placental aging in mice and reduce the miscarriage rate in CIP-exposed mice. Therefore, this study provides a new pathway and biological mechanism by which CIP exposure leads to aging and subsequently induces miscarriage.

Claims

1. Application of TRIM21 as a biomarker in screening agents to reduce the risk of ciprofloxacin exposure-related miscarriage.

2. Application of TRIM21 as a biomarker in the preparation of formulations that reduce the risk of ciprofloxacin exposure-related miscarriage.

3. In the application according to any one of claims 1-2, the expression level of TRIM21 protein is positively correlated with the risk of miscarriage associated with ciprofloxacin exposure.

4. A kit for screening or preparing a method to reduce the risk of ciprofloxacin exposure-related miscarriage, characterized in that, The kit contains a detection reagent for detecting the expression level of TRIM21 as described in claim 1.

5. Application of reagents for knocking down Trim21 expression in the preparation of formulations for inhibiting placental aging and reducing the risk of ciprofloxacin exposure-related miscarriage.

6. In the application according to claim 5, the reagent used to knock down Trim21 expression is a combination of phenornostat and quinsinostat, wherein, The dosage of feminostal is 50 mg / kg / 2 days, and the dosage of quinsinostat is 10 mg / kg / 2 days.

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