Tanshinone IIA induces oxidative stress in trophoblast cells and enhances copper dependent death

Tanshinone IIA induces oxidative stress in trophoblast cells and enhances copper dependent death | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Tanshinone IIA induces oxidative stress in trophoblast cells and enhances copper dependent death Juemeng Du, Xuanyin Wang, Yuerui Zeng, Jingjing Feng, Lisha Yang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5772434/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract Background Oxidative stress occurs in trophoblast cells during the development of tubal pregnancy (TP), compared to normal pregnancy. It has been demonstrated that Previous studies have shown that Tanshinone IIA (TSA) can increase reactive oxygen species (ROS) levels and exacerbate oxidative stress in tumor cells, while its effects on trophoblast cells or affects cuproptosis pathways remain unclear. Method We collected chorionic tissue from patients with normal intrauterine pregnancies (NP) or TP to detect the expression of related proteins. HTR-8/SVneo cells were cultured in vitro and treated with Elesclomol, CuCl2 and/or TSA, tetrathiomolybdate (TTM). The expressions of proteins such as DLAT, DLST, FDX1, Lipo-DLAT, Lipo-DLST, Bax, and Bcl-2 were measured. Mitochondrial membrane potential, cell apoptosis, and cell function were also assessed. Result The concentration of TSA added to HTR8-SVneo cells was 30 µM. The protein expression of DLAT, DLST, Lipo-DLAT, Lipo-DLST monomers, FDX1 and Bcl-2/ Bax was downregulated by the addition of Elesclomol and CuCl2 intervention in the cells. Meanwhile, the levels of reactive oxygen species (ROS) increased, mitochondrial membrane potential decreased, cell apoptosis increased, and cell invasion and migration were attenuated. The addition of TSA enhanced these effects, while the addition of TTM mitigated them. Conclusion TSA can promote oxidative stress in HTR-8/SVneo cells, leading to cell apoptosis. This process can be reversed by copper chelator. Biological sciences/Drug discovery Health sciences/Diseases Health sciences/Medical research ectopic pregnancy Tanshinone IIA oxidative stress copper dependent Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Highlights Tanshinone IIA (TSA) impacts cuproptosis pathways in trophoblast cells. TSA increases the production of reactive oxygen species (ROS) and induces mitochondrial apoptosis，which contributes to cell damage and dysfunction. The oxidative stress and apoptosis in trophoblast cells may be involved in the development of tubal pregnancy. This process could potentially be targeted by copper-related inhibitors to prevent or mitigate the condition. Introduction Ectopic pregnancy is a common condition in early pregnancy, most frequently occurring in the fallopian tube, known as TP. Untimely diagnosis and treatment can seriously jeopardize the health of pregnant women and lead to death. Currently, the main treatment involves surgical removal of the fallopian tube (salpingectomy) or removal of the ectopic pregnancy tissue while preserving the fallopian tube (salpingostomy). However, surgical treatment also increases the risk of pelvic inflammatory disease, recurrence of ectopic pregnancy, and may reduce the chances of natural conception[1]. The main alternative is to inhibit trophoblast proliferation with MTX[2]. However, MTX is reproductively toxic[3] and may also impair liver function and cause gastrointestinal reaction. Early ectopic pregnancy occurs when trophoblast cells grow in an unsuitable environment, where insufficient blood supply and local hypoxia result in elevated ROS production[4]. Excess ROS damages cellular structures, proteins, and DNA, triggering inflammation and cell death, which subsequently disrupts normal embryo implantation and development. This oxidative stress exacerbates local tissue damage and further worsens the condition of ectopic pregnancy[5]. Salvia miltiorrhiza , a traditional Chinese herbal medicine, has been used in Asian countries for centuries. TSA, a fat-soluble active ingredient extracted from Salvia miltiorrhiza , is a terpenoid with diverse pharmacological properties, including antioxidant, antibacterial[6], anti-inflammatory[7], antitumor, and antithrombotic activities[8]. The water-soluble derivatives of Salvia miltiorrhiza have been approved by the National Medical Products Administration (NMPA, China) for the treatment of cardiovascular diseases. The main pharmacological effects are mediated through the regulation of inflammatory pathways, oxidative stress responses, and apoptotic signaling. In normal cells, TSA primarily exerts protective effects[9], whereas in tumor cells and other pathological cells, it mainly promotes ROS production, induces ferroptosis[10] to enhance cellular oxidative stress, or mediates apoptosis and inhibits cellular proliferation through the PI3K/AKT/mTOR pathway, JNK signaling, and other related pathways. Tsvetkov et al. identified a novel form of programmed cell death termed “cuproptosis”[11]. Intracellular Cu 2+ is reduced to the more toxic Cu + by FDX1 through an indirect mechanism. LIAS links the lipoyl group to DLAT and DLST, and Cu + binds to the lipoylated DLAT and DLST through disulfide bonds, inducing oligomerization. DLAT and DLST are core components of the Pyruvate Dehydrogenase Complex (PDC) and α-Ketoglutarate Dehydrogenase Complex (KGDC), respectively, which are essential for the TCA cycle. Therefore, alterations in DLAT and DLST ultimately lead to cell death, and their depletion also results in the accumulation of ROS[12]. Cuproptosis relies on mitochondrial respiration, and inhibitors of electron transport chain complexes I and II can attenuate it. Experimental results demonstrate that cuproptosis cannot be inhibited by known cell death inhibitors but can be reversed by copper chelators. Materials and Methods Ethical Statement The study was approved by the Research Medical Ethics Committee of the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine (Guangzhou, China) (JY2024-149). Written informed consent was obtained from all patients and healthy volunteers prior to their participation. The study was conducted in accordance with the 1964 Declaration of Helsinki and its later amendments. Inclusion and Exclusion Criteria Patients in all groups were free from primary diseases, including cardiac, hepatic, and renal insufficiency, as well as hematologic, neoplastic, and immune system diseases. Additionally, patients with a history of drug therapy within the last 3 months, or those who were smokers and/or alcoholics, were excluded from the study. Patients diagnosed with TP based on β-hCG levels and transvaginal ultrasound findings, showing a gestational sac within the fallopian tube, were included in the TP group. Pathologic examination of intrauterine tissues confirmed the absence of chorionic tissue[13]. Patients with ectopic pregnancies located outside the fallopian tube or those who were hemodynamically unstable on admission were excluded from the study. Patients with NP, confirmed by transvaginal ultrasound (TVUS), were included in the control group. Pathologic examination of uterine tissues confirmed the presence of chorionic tissue in the sections. Study Cohort The study population included patients with tubal pregnancy (TP; n = 10) and women with normal intrauterine pregnancy (NP; n = 10). Clinical data of the participants are summarized in Table 1. Human chorionic villi were collected from the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine. Tissue samples were divided into two portions: one was stored at -80°C for protein expression analysis, and the other was fixed in 4% paraformaldehyde (PFA) and embedded in paraffin. Table 1. Clinical data of the participants. Parameter Normal Pregnancy (n=10) tubal pregnancy (n=10) p-value Age (years) 27.1 ± 3.4 (22–34) 28.7 ± 3.4 (24–35) 0.28 BMI (kg/m²) 23.8 ± 3.3 (20–29) 23.3 ± 3.3 (18–28) 0.72 Gestational age (days) 47.1 ± 4.6 (42–53) 48.8 ± 4.1 (44–56) 0.37 Previous ectopic 0 [0–0] 0 [0–0.25] 0.5 Serum hCG (IU/L)* 10,076 [5,450–17,543] 1,286 [918–1,678] <0.001 Data presented as mean ± SD (range) for normally distributed variables (age, BMI, gestational age) or median [IQR] for non-normally distributed variables (hCG). Categorical data shown as n (%). BMI, body mass index. *p < 0.05 considered statistically significant. Ce ll Lines, Reagents, and Antibodies HTR-8/SVneo cells [14] were purchased from the American Type Culture Collection (CRL-3271). This cell line contains two cell populations: trophoblast and stromal/mesenchymal cells [15]. The cells were cultured in RPMI-1640 medium (Gibco, 11875093) supplemented with 10% fetal bovine serum (FBS; Gibco, A5670701) and antibiotics (penicillin, 100 IU/mL; streptomycin, 100 μg/mL; NCM Biotech, C100C5) at 37°C in a 5% CO2 atmosphere. Tanshinone IIA (568-72-9) and Elesclomol (STA-4783) were purchased from MedChemExpress. TTM (323446) was obtained from Sigma-Aldrich. Antibodies against DLAT (13426-1-AP), FDX1 (82957-2-RR), Bax (50599-2-Ig), Bcl2 (12789-1-AP), and β-actin (20536-1-AP) were purchased from Proteintech. Antibodies against DLST (ab177934), lipoic acid (ab58724), and LIAS (ab246917) were purchased from Abcam. The cells were divided into the following groups: NC (negative control), TSA (Tanshinone IIA treatment), MOD (model group treated with 5 μM CuCl 2 and 50 nM Elesclomol), MOD+TSA (MOD group with Tanshinone IIA treatment), and MOD+TSA+TTM (MOD+TSA group with 20 μM TTM treatment) [16–18].The concentrations of Elesclomol (50 nM) and CuCl₂ (5 μM) were selected based on previous studies demonstrating cuproptosis induction in various cell types at these ranges [11,19-20]. CCK8 cell viability assay Cell viability was determined using the Cell Counting Kit-8 (CCK-8; Solarbio, CA12120) according to the manufacturer’s instructions. Cells were seeded in 96-well plates at a density of 1×10^4 cells per well and treated according to the experimental groups, with three replicate wells per group. After treatment, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C for 1.5 hours. Absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, USA). Immunofluorescence Staining (IF) Cell culture chamber slides were placed in 12-well plates, and cells were seeded at a density of 4×10^4 cells per well. After grouping, cells were fixed on the slides with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 20 minutes, and blocked with BSA. Subsequently, the cells were incubated overnight with a primary antibody (anti-FDX1 antibody, 1:500 dilution), followed by incubation with a FITC-conjugated secondary antibody (Proteintech, SA00003-2, 1:200 dilution) for 2 hours at room temperature. After washing, antifading mounting medium containing DAPI (Solarbio, S2110) was added dropwise to the slides. The slides were coverslipped, and images were captured using a fluorescence microscope (Olympus, Japan). Immunohistochemistry (IHC) Tissues were fixed in 4% paraformaldehyde for 24h, embedded in paraffin, and the wax blocks were made into paraffin sections, followed by dewaxing hydration operation, restore antigenic epitopes using Tris-EDTA antigen retrieval solution (Beyotime P0084) at 37℃, blocking non-specific binding sites with 20% goat serum (Solarbio SL038), and then primary antibody(anti-FDX1 antibody 1:200, anti-Bax antibody 1:200, anti-Bcl 2 antibody 1:200) was used at 4°C overnight incubation, reaction using HRP-labeled secondary antibody, color development with DAB color solution (proteintech PK10006), and scanning imaging using Digital Pathology Slice Scanning System (3DHistech Pannoramic 250). Western blot Cells or tissues were lysed using RIPA Lysis Buffer (Beyotime, P0013B) on ice for 30 minutes, followed by centrifugation to collect the supernatant. The supernatant was mixed with loading buffer (Beyotime, P0015L) and heated at 100°C for 5 minutes. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (MCE, IPVH00010). The membranes were blocked with 5% skimmed milk for 2 hours at room temperature to prevent non-specific binding. Subsequently, the membranes were incubated overnight at 4°C with primary antibodies, including anti-FDX1 (1:1000), anti-Bax (1:2000), anti-Bcl-2 (1:2000), anti-β-actin (1:10000), anti-Lipoic Acid (1:10000), and anti-LIAS (1:2500). After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG or anti-mouse IgG for 1 hour at room temperature. Chemiluminescent reactions were performed using an ECL substrate (Tanon, 180-5001), and images were captured using a multifunctional imager (Bio-Rad Laboratories, Hercules, USA). Protein band intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, USA). ROS and Mitochondrial Membrane Potential (MMP) Detection Cells were seeded in 12-well plates at a density of 4×10^4 cells per well and treated according to the experimental groups. A solution of dichloro-dihydro-fluorescein diacetate (DCFH-DA; Beyotime, S0035S) or JC-1 fluorescent probe (Solarbio, M8650) was prepared according to the manufacturer’s instructions. The cells were incubated with the probes at 37°C for the indicated time in the dark, followed by three washes with PBS. Images were captured using a fluorescence microscope (Olympus, Japan). Flow Cytometry for Apoptosis or ROS Detection Cells were seeded into 6-well plates at a density of 5×10^5 cells per well and treated according to the experimental groups. Working solutions of Annexin V-FITC and PI (MULTI SCIENCES, AP101) or DCFH-DA were prepared according to the manufacturer’s instructions. After treatment, the cells were harvested and resuspended in the working solutions. For apoptosis detection, the cells were incubated with Annexin V-FITC and PI at 37°C in the dark for 5 minutes. For ROS detection, the cells were incubated with DCFH-DA at 37°C in the dark for 20 minutes. Following incubation, the cells were analyzed using a FACSCanto II flow cytometer (BD Biosciences, USA). Cell Invasion Assay Matrigel matrix gel (Corning, 356234) was added to the upper chambers of Transwell inserts (Corning, 3422) and allowed to solidify. HTR-8/SVneo cells were suspended in medium containing the drug and 1% fetal bovine serum (FBS), and 1×10^5 cells were seeded into the upper chambers. The lower chambers were filled with medium containing 10% FBS. After incubation according to the experimental groups, cells on the lower surface of the membrane were fixed with 4% paraformaldehyde (PFA) and stained with crystal violet. Images were captured using a microscope (Olympus, Japan) and analyzed with ImageJ software. Wound-Healing Assay Cells were seeded into 6-well plates at a density of 4×10^4 cells per well and cultured until they reached 80% confluence. A sterile pipette tip was used to create a wound in the cell monolayer. The cells were rinsed twice with RPMI-1640 medium, and images were captured at 0 hours using a microscope (Olympus, Japan). After wounding, the cells were incubated for 24 hours according to the experimental groups. Following the intervention, the cells were cultured for an additional 24 hours, and images were captured again for analysis using ImageJ software. Statistical Analysis .All statistical analyses were performed using SPSS 27.0 (IBM, USA) and GraphPad Prism 8.0 (GraphPad Software, USA). Normality was assessed by Shapiro-Wilk testing. Continuous variables with normal distribution were expressed as mean ± standard deviation and analyzed using independent t-tests (two-group comparisons) or one-way ANOVA with Tukey’s post-hoc test (multi-group comparisons), while non-normally distributed data were presented as median [interquartile range] and evaluated with Mann-Whitney U tests or Kruskal-Wallis tests with Bonferroni’s correction, respectively. P-value < 0.05 was considered statistically significant. Results Cuproptosis in Chorionic Villi of Patients with Tubal Pregnancy We collected samples from 20 patients aged between 22 and 35 years, including 10 cases of NP and 10 cases of TP ( Table 1 ). The tissues were obtained from chorionic villi in the uterine cavity (NP group) or from TP. Protein expression and immunohistochemical analyses were performed ( Figure 1 ). The results showed that the levels of FDX1, DLST, and DLAT were significantly higher in normal chorionic villi compared to those from TP, with only DLAT showing statistical significance. Immunohistochemical staining revealed that trophoblast cells expressed higher levels of LIAS, FDX1, Bax, and Bcl2 compared to stromal cells within the chorionic villi. Compared to the NP group, TP tissues exhibited increased Bax expression and decreased expression of LIAS, FDX1, and Bcl2. The reduced expression of FDX1 and Bcl2, combined with elevated Bax levels in TP tissues, suggests enhanced apoptosis in trophoblast cells, potentially associated with cuproptosis. Effect of Tanshinone IIA on HTR8-SVneo Cells To determine the optimal concentration of tanshinone IIA (TSA) for intervention, cells were treated with various concentrations of TSA for 12 or 24 hours, and cell viability was assessed using the CCK-8 assay. The results showed that concentrations of 30-40 μM reduced cell viability by 50% ( Figure 2a ). Based on these findings, 30-40 μM TSA was selected for subsequent experiments. After 24 hours of treatment, the levels of lipoic acid synthetase (LIAS) and ferredoxin 1 (FDX1) were measured ( Figure 2b, 2c ). Compared to the untreated group, TSA treatment decreased LIAS levels and increased FDX1 expression; however, these changes were not statistically significant. Effect of Tanshinone IIA on Cuproptosis and Cell Function in HTR8-SVneo Cells Trophoblast cells in early pregnancy exhibit migratory and invasive capabilities, which contribute to the invasion of the fallopian tube and its eventual rupture in tubal pregnancies. To further investigate the effects of cuproptosis and tanshinone IIA (TSA) on cell function, cells were treated according to the same grouping method. The results showed that cuproptosis induced by Elesclomol and CuCl 2 significantly inhibited the invasion and migration abilities of the cells. However, the combination of TSA with cuproptosis further suppressed these cellular functions, and tetrathiomolybdate (TTM) partially reversed this effect ( Figure 3 ). Effect of Tanshinone IIA on Oxidative Stress in HTR8-SVneo Cells To further explore the effects of cuproptosis and tanshinone IIA (TSA) on oxidative stress, we used fluorescent probes to detect intracellular reactive oxygen species (ROS), apoptosis, and mitochondrial membrane potential ( Figure 4 ). The results showed that cuproptosis induced by Elesclomol and CuCl 2 significantly increased intracellular ROS levels and promoted apoptosis. However, the combination of TSA with cuproptosis further enhanced these effects, and tetrathiomolybdate (TTM) partially reversed this phenomenon . Effect of Tanshinone IIA on Cuproptosis-related Protein Expression in HTR8-SVneo Cells Western blotting and immunofluorescence were performed to detect the expression levels of acylated dihydrolipoamide acetyltransferase (lip-DLAT), acylated dihydrolipoamide S-acetyltransferase (lip-DLST), FDX1, Bax, and Bcl2 proteins in the cells. The results showed that cuproptosis induced by Elesclomol and CuCl 2 downregulated the expression of lip-DLAT, lip-DLST, FDX1, and Bcl 2 , while Bax expression was upregulated. Combined treatment with tanshinone IIA (TSA) further enhanced these changes, and tetrathiomolybdate (TTM) partially reversed the effects ( Figure 5 ). Discussion Oxidative stress occurs during normal pregnancy, where increased energy demands and elevated oxygen consumption enhance mitochondrial activity, subsequently increasing ROS production. As crucial signaling molecules, ROS participate in cellular proliferation, migration, and angiogenesis[21]. A balanced ROS level, supported by an efficient antioxidant defense system, maintains redox homeostasis, ensuring normal fetal development and maternal health[22]. However, excessive ROS can cause cellular damage, lipid peroxidation, DNA oxidation, and protein modification, leading to cellular and tissue dysfunction[18]. Studies have shown that excessive ROS plays a significant role in the pathophysiology of pregnancy complications, including miscarriage, preeclampsia[22], intrauterine growth restriction, preterm premature rupture of membranes, and TP[23]. ROS can alter the oxidation state of intracellular copper ions, increasing their reactivity. Oxidative stress increases mitochondrial membrane permeability, depletes antioxidants such as glutathione (GSH) and metallothionein (MT), and impairs the cell’s ability to detoxify copper ions[24]. This reduces copper-binding capacity, elevates free copper levels, and ultimately triggers cuproptosis. Additionally, oxidative stress activates transcription factors such as NF-κB or directly modifies copper transport proteins such as CTR1 and ATP7A/B[25], leading to increased copper uptake or reduced copper efflux, which promotes copper accumulation and cuproptosis. Moreover, cuproptosis disrupts the mitochondrial tricarboxylic acid (TCA) cycle, further exacerbating oxidative stress[26]. FDX1, an iron-sulfur cluster (ISC) protein, plays a crucial role in mitochondrial metabolism and the cuproptosis pathway. It facilitates electron transfer from NADPH to downstream enzymes via iron-sulfur clusters (2Fe-2S) [27]. Additionally, FDX1 promotes the deposition of copper ions into mitochondria and interacts with lipoylases to induce cytosolic lipoylation, disrupting their normal function and leading to cell death [28]. According to a study by Tsvetkov et al. published in Science, knockdown of FDX1 reduces lipoylation of tricarboxylic acid cycle components DLAT and DLST, partially reversing cuproptosis. However, FDX1 expression tends to decrease during cuproptosis [11]. Our findings indicate that, in the absence of drug treatment, trophoblast cells in chorionic villi tissues from patients with tubal pregnancy exhibit higher levels of apoptosis and cuproptosis-associated protein expression compared to those from patients with normal intrauterine pregnancy. This suggests that trophoblast cells in tubal pregnancy are more prone to apoptosis and cuproptosis. The antitumor activity of tanshinone IIA (TSA) has been demonstrated in various cancer cell lines, with its role in mediating apoptosis through increased reactive oxygen species (ROS) levels being particularly significant. Previous studies have shown that TSA reduces glutathione (GSH) and cysteine levels while increasing ROS levels in BGC-823 and NCI-H87 cells [29], and alters mitochondrial membrane potential in chronic myeloid leukemia (CML) cells, thereby inducing intrinsic apoptosis [30]. It was also also suggested that Sodium Tanshinone IIA Sulfonate inhibits the release of High Mobility Group Box 1 protein (HMGB1) from hypoxic trophoblasts, thereby reducing inflammatory responses and oxidative stress, protecting endothelial cell function[31]. In the present study, we investigated the effects of TSA on tubal pregnancy (TP) trophoblast cells using HTR-8/SVneo cells as a model. Our results showed that TSA decreased cell viability, upregulated FDX1, and downregulated lipoic acid synthetase (LIAS), suggesting that TSA may enhance the sensiti vity of HTR-8/SVneo cells to cuproptosis by regulating FDX1. Further experiments revealed that TSA exacerbated cuproptosis. Specifically, TSA treatment significantly reduced mitochondrial membrane potential, indicating impaired mitochondrial function. Additionally, the expression of the pro-apoptotic protein Bax was upregulated, while the anti-apoptotic protein Bcl-2 was downregulated, indicating a shift toward apoptosis. The altered Bax/Bcl-2 ratio further exacerbated oxidative stress and cell deat. Tanshinone IIA may have a dual effect on cellular oxidative stress, potentially promoting or inhibiting it, which could depend on different disease states. Our data suggest that TSA may disrupt the adaptation of gestational trophoblast cells to hypoxic environments by exacerbating reactive oxygen species (ROS) accumulation (Figure 4a-c), leading to mitochondrial dysfunction (Figure 4f) and apoptosis. This is consistent with the observed downregulation of Bcl-2 and upregulation of Bax (Figure 5), which are known mediators of hypoxia adaptation. However, the precise mechanisms require further investigation, as some studies report that TSA can protect hypoxic trophoblast cells from endothelial cell injury [31], indicating its dual roles in regulating cell growth and its high safety profile. Future studies should further investigate the molecular mechanisms of TSA and its potential clinical applications, aiming to develop novel therapeutic strategies for tubal pregnancy and refractory trophoblastic diseases. Conclusion Our study highlights the critical role of oxidative stress and cuproptosis in trophoblast cell death during tubal pregnancy (TP). Tanshinone IIA (TSA) was shown to elevate reactive oxygen species (ROS) levels and induce apoptosis in trophoblast cells, potentially through modulating FDX1 expression and enhancing susceptibility to copper-induced cell death. The upregulation of Bax and downregulation of Bcl-2 following TSA treatment indicated a shift toward an apoptotic state, accompanied by exacerbated oxidative stress and mitochondrial damage. These findings suggest that TSA may disrupt trophoblast adaptation to hypoxic conditions, ultimately promoting apoptosis. Abbreviations TP Tubal Pregnancy NP Normal Pregnancy TSA Tanshinone IIA TTM Tetrathiomolybdate ROS Reactive Oxygen Species CFDA Chinese State Food and Drug Administration PDC Dehydrogenase Complex KGDC Ketoglutarate Dehydrogenase Complex PFA Paraformaldehyde LIAS Lipoic Acid Synthetase FDX1 Ferredoxin 1 DLAT Dihydrolipoamide Acetyltransferase DLST Dihydrolipoamide S-acetyltransferase HIF-1α Hypoxia-Inducible Factor 1α ISC Iron-Sulfur Cluster GSH Glutathione Declarations Ethics approval and consent to participate The study was conducted in accordance with the Declaration of Helsinki, and approved by the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine (Guangzhou, China) (JY2024-149 10/23/2024). Consent for publication All authors agree to publish this article. Funding This research was supported by the National Natural Science Foundation of China (82174417) and the Sanming Project of Medicine in Shenzhen（No. SZZYSM202106003）. Contribution statement Conceptualization, J.D. and G.D.; methodology, J.D., X.W. and Y.Z.; software, J.D., X.W. and Y.Z.; validation, J.F., L.Y. and Y.H.; formal analysis, J.D.; investigation, Y.H. and G.D.; resources, J.F. and Y.H.; data curation, J.D. and X.W.; writing—original draft preparation, J.D., X.W. and Y.Z.; writing—review and editing, J.F., L.Y., Y.H. and G.D.; visualization, J.D. and G.D.; supervision, X.W., Y.Z. and G.D.; project administration, Y.H. and G.D.; funding acquisition, Y.H. and G.D. All authors have read and agreed to the published version of the manuscript. Declaration of competing interest The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. 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Rev. 87 , 1011–1046. 10.1152/physrev.00004.2006 (2007). Bhatti, J. S., Bhatti, G. K. & Reddy, P. H. Mitochondrial Dysfunction and Oxidative Stress in Metabolic Disorders - A Step towards Mitochondria Based Therapeutic Strategies. Biochim. Biophys. Acta Mol. Basis Dis. 1863 , 1066–1077. 10.1016/j.bbadis.2016.11.010 (2017). Schulz, V. et al. Functional Spectrum and Structural Specificity of Mitochondrial Ferredoxins FDX1 and FDX2. Nat Chem Biol. 19, 206–217 . (2023). 10.1038/s41589-022-01159-4 Tsui, K. H. et al. The Cross-Communication of Cuproptosis and Regulated Cell Death in Human Pathophysiology. Int. J. Biol. Sci. 20 , 218. 10.7150/ijbs.84733 (2024). Guan, Z., Chen, J., Li, X., Dong, N. & Tanshinone IIA Induces Ferroptosis in Gastric Cancer Cells through P53-Mediated SLC7A11 down-Regulation. Biosci. Rep. 40 , BSR20201807. 10.1042/BSR20201807 (2020). Yun, S. M. et al. Activation of C-Jun N-Terminal Kinase Mediates Tanshinone IIA-Induced Apoptosis in KBM-5 Chronic Myeloid Leukemia Cells. Biol Pharm Bull. 36, 208–214 . (2013). 10.1248/bpb.b12-00537 Zhao, M. et al. Sodium Tanshinone IIA Sulfonate Prevents Hypoxic Trophoblast-Induced Endothelial Cell Dysfunction via Targeting HMGB1 Release. J. Biochem. Mol. Toxicol. 31 10.1002/jbt.21903 (2017). Additional Declarations No competing interests reported. Supplementary Files floatimage1.png Cite Share Download PDF Status: Published Journal Publication published 11 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 21 May, 2025 Reviews received at journal 20 May, 2025 Reviewers agreed at journal 30 Apr, 2025 Reviews received at journal 28 Apr, 2025 Reviewers agreed at journal 28 Apr, 2025 Reviewers invited by journal 28 Apr, 2025 Submission checks completed at journal 25 Apr, 2025 First submitted to journal 04 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5772434","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":448880693,"identity":"167de4f2-56e5-4b66-8bae-d6a06560f210","order_by":0,"name":"Juemeng Du","email":"","orcid":"","institution":"Guangzhou University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Juemeng","middleName":"","lastName":"Du","suffix":""},{"id":448880696,"identity":"a4c7e5c2-e7f3-43ee-8550-33557047ac24","order_by":1,"name":"Xuanyin Wang","email":"","orcid":"","institution":"Guangzhou University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xuanyin","middleName":"","lastName":"Wang","suffix":""},{"id":448880701,"identity":"4124161b-5a9e-4534-b603-19b71d966946","order_by":2,"name":"Yuerui Zeng","email":"","orcid":"","institution":"Guangzhou University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuerui","middleName":"","lastName":"Zeng","suffix":""},{"id":448880704,"identity":"0fae1e48-9afb-463c-8567-99878c2583c2","order_by":3,"name":"Jingjing Feng","email":"","orcid":"","institution":"Guangzhou University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"Feng","suffix":""},{"id":448880705,"identity":"ba6eaf59-275e-42f0-b6cf-7824692f7b86","order_by":4,"name":"Lisha Yang","email":"","orcid":"","institution":"Guangzhou University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Lisha","middleName":"","lastName":"Yang","suffix":""},{"id":448880706,"identity":"118108fd-f87a-497d-8199-d500108f1d8b","order_by":5,"name":"Yanxi Huang","email":"","orcid":"","institution":"Guangzhou University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yanxi","middleName":"","lastName":"Huang","suffix":""},{"id":448880707,"identity":"89e058ec-7f7f-48ff-8727-425c4e9b6c68","order_by":6,"name":"Gaopi Deng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYDCCA1Cajb2x8cEH0rTwHG42nEGSFgaJ9DZpDmJ08B3vPfzyS4VdHp/kwwZpBgY7Od0GAlokz5xLs5Y5k1zMJp3YYFzAkGxsdoCAFoMbOWbGkm3MiW1ALckzGA4kbiOo5f4bkJb6xDbJgw2HeYjScoPH+OHHtsOJbRKMjc1EaZE8k2PGzHDmeDEbT2Iz4wwDIvzCd/yM8ccfFdV58u3Hn//4UGEnR1ALELBJ8zAwJEDdSVg5CDB//AHXMgpGwSgYBaMACwAASRhGrCKRvtMAAAAASUVORK5CYII=","orcid":"","institution":"Guangzhou University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Gaopi","middleName":"","lastName":"Deng","suffix":""}],"badges":[],"createdAt":"2025-01-06 09:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5772434/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5772434/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-07238-5","type":"published","date":"2025-07-11T15:57:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82045021,"identity":"915bed6f-f36f-42a5-80f9-ad29c65cb0b1","added_by":"auto","created_at":"2025-05-06 09:32:38","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":254706,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of Cuproptosis-related Proteins in Chorionic Villi from NP (n=10) and TP (n=10) Patients. \u003cstrong\u003ea.\u003c/strong\u003e Western blotting analysis of DLAT, DLST, and FDX1 protein levels. \u003cstrong\u003eb.\u003c/strong\u003eImmunohistochemical staining of LIAS, FDX1, Bax, and Bcl2 in the chorionic villi. Data are presented as standard deviation±mean; \u003cem\u003ens p≥0.05，\u003c/em\u003e*\u003cem\u003ep＜0.05，*\u003c/em\u003e*\u003cem\u003ep＜0.01 \u003c/em\u003e,\u003cem\u003e \u003c/em\u003evs NP group.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5772434/v1/47f4255d8dafd3748671cef9.jpeg"},{"id":82048928,"identity":"328a4631-a101-413c-934b-00977cc4c3c5","added_by":"auto","created_at":"2025-05-06 09:48:38","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":93167,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eCell viability measured by the CCK8 assay after treatment with different concentrations of TSA for 24 hours (vs 0μM).\u003cstrong\u003e b-c.\u003c/strong\u003e Expression of LIAS and FDX1 proteins in cells treated with 30 μM TSA for 24 hours (vs NP group). Data are presented as standard deviation±mean; \u003cem\u003ens p≥0.05，\u003c/em\u003e*\u003cem\u003ep＜0.05，*\u003c/em\u003e*\u003cem\u003ep＜0.01\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5772434/v1/530ac607ec295157159aa0af.jpeg"},{"id":82045025,"identity":"ff5e8600-c54c-42fe-8758-d9c58f4f8c94","added_by":"auto","created_at":"2025-05-06 09:32:38","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":203280,"visible":true,"origin":"","legend":"\u003cp\u003eCell invasion and migration assays after different treatments for 24 hours. \u003cstrong\u003ea-b. \u003c/strong\u003eTranswell assay for cell invasion. \u003cstrong\u003ec-d. \u003c/strong\u003eScratch assay for cell migration. Data are presented as standard deviation±mean; \u003cem\u003ens p≥0.05，\u003c/em\u003e*\u003cem\u003ep＜0.05，*\u003c/em\u003e*\u003cem\u003ep＜0.01\u003c/em\u003e , vs MOD group.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5772434/v1/3574a37e3540524564df63c1.jpeg"},{"id":82051710,"identity":"2e123d8c-d549-4dda-8261-0b8b874e71b4","added_by":"auto","created_at":"2025-05-06 10:04:38","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":274615,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of oxidative stress markers. \u003cstrong\u003ea. \u003c/strong\u003eIntracellular ROS levels detected using DCFH-DA fluorescent probe. \u003cstrong\u003eb-c. \u003c/strong\u003eFlow cytometry analysis of ROS levels. \u003cstrong\u003ed-e.\u003c/strong\u003e Flow cytometry analysis of apoptosis levels. \u003cstrong\u003ef. \u003c/strong\u003eMitochondrial membrane potential detected with JC1 probe. Data are presented as standard deviation±mean; \u003cem\u003ens p≥0.05，\u003c/em\u003e*\u003cem\u003ep＜0.05，*\u003c/em\u003e*\u003cem\u003ep＜0.01\u003c/em\u003e , vs MOD group.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5772434/v1/64b576d43e6142bedc838226.jpeg"},{"id":82047140,"identity":"f0fad173-e085-497b-ac64-6bda24bdd330","added_by":"auto","created_at":"2025-05-06 09:40:38","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":111711,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea-b.\u003c/strong\u003e Western blotting analysis of cuproptosis-related protein expression after 24-hour treatments. \u003cstrong\u003ec. \u003c/strong\u003eImmunofluorescence staining of cuproptosis-related proteins after 24-hour treatments. Data are presented as standard deviation±mean; \u003cem\u003ens p≥0.05，\u003c/em\u003e*\u003cem\u003ep＜0.05，*\u003c/em\u003e*\u003cem\u003ep＜0.01\u003c/em\u003e , vs MOD group.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5772434/v1/26e20ebcb625291a3c5a930b.jpeg"},{"id":86699340,"identity":"1b310f8c-3865-44f6-bb84-d896e9f3b50a","added_by":"auto","created_at":"2025-07-14 16:07:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1855336,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5772434/v1/1fdcee4d-6bbe-45fd-9642-18643474e1ab.pdf"},{"id":82045022,"identity":"8b2710e4-f5b2-45b7-af33-41a18718d4cc","added_by":"auto","created_at":"2025-05-06 09:32:38","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":247315,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5772434/v1/07149a47a5225c0a01d6e5e4.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tanshinone IIA induces oxidative stress in trophoblast cells and enhances copper dependent death","fulltext":[{"header":"Highlights","content":"\u003cul start=\"50\"\u003e\n \u003cli\u003eTanshinone IIA (TSA) impacts cuproptosis pathways in trophoblast cells.\u003c/li\u003e\n \u003cli\u003eTSA increases the production of reactive oxygen species (ROS) and induces mitochondrial apoptosis，which contributes to cell damage and dysfunction.\u003c/li\u003e\n \u003cli\u003eThe oxidative stress and apoptosis in trophoblast cells may be involved in the development of tubal pregnancy.\u003c/li\u003e\n \u003cli\u003eThis process could potentially be targeted by copper-related inhibitors to prevent or mitigate the condition.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eEctopic pregnancy is a common condition in early pregnancy, most frequently occurring in the fallopian tube, known as TP. Untimely diagnosis and treatment can seriously jeopardize the health of pregnant women and lead to death. Currently, the main treatment involves surgical removal of the fallopian tube (salpingectomy) or removal of the ectopic pregnancy tissue while preserving the fallopian tube (salpingostomy). However, surgical treatment also increases the risk of pelvic inflammatory disease, recurrence of ectopic pregnancy, and may reduce the chances of natural conception[1]. The main alternative is to inhibit trophoblast proliferation with MTX[2]. However, MTX is reproductively toxic[3]\u0026nbsp;and may also impair liver function and cause gastrointestinal reaction.\u003c/p\u003e\n\u003cp\u003eEarly ectopic pregnancy occurs when trophoblast cells grow in an unsuitable environment, where insufficient blood supply and local hypoxia result in elevated ROS production[4]. Excess ROS damages cellular structures, proteins, and DNA, triggering inflammation and cell death, which subsequently disrupts normal embryo implantation and development. This oxidative stress exacerbates local tissue damage and further worsens the condition of ectopic pregnancy[5].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e, a traditional Chinese herbal medicine, has been used in Asian countries for centuries. TSA, a fat-soluble active ingredient extracted from \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e, is a terpenoid with diverse pharmacological properties, including antioxidant, antibacterial[6], anti-inflammatory[7], antitumor, and antithrombotic activities[8]. The water-soluble derivatives of\u003cem\u003e\u0026nbsp;Salvia miltiorrhiza\u0026nbsp;\u003c/em\u003ehave been approved by the National Medical Products Administration (NMPA, China) for the treatment of cardiovascular diseases. The main pharmacological effects are mediated through the regulation of inflammatory pathways, oxidative stress responses, and apoptotic signaling. In normal cells, TSA primarily exerts protective effects[9], whereas in tumor cells and other pathological cells, it mainly promotes ROS production, induces ferroptosis[10] to enhance cellular oxidative stress, or mediates apoptosis and inhibits cellular proliferation through the PI3K/AKT/mTOR pathway, JNK signaling, and other related pathways.\u003c/p\u003e\n\u003cp\u003eTsvetkov et al. identified a novel form of programmed cell death termed “cuproptosis”[11]. Intracellular Cu\u003csup\u003e2+\u003c/sup\u003e is reduced to the more toxic Cu\u003csup\u003e+\u003c/sup\u003e by FDX1 through an indirect mechanism. LIAS links the lipoyl group to DLAT and DLST, and Cu\u003csup\u003e+\u003c/sup\u003e binds to the lipoylated DLAT and DLST through disulfide bonds, inducing oligomerization. DLAT and DLST are core components of the Pyruvate Dehydrogenase Complex (PDC) and α-Ketoglutarate Dehydrogenase Complex (KGDC), respectively, which are essential for the TCA cycle. Therefore, alterations in DLAT and DLST ultimately lead to cell death, and their depletion also results in the accumulation of ROS[12]. Cuproptosis relies on mitochondrial respiration, and inhibitors of electron transport chain complexes I and II can attenuate it. Experimental results demonstrate that cuproptosis cannot be inhibited by known cell death inhibitors but can be reversed by copper chelators.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch2\u003eEthical Statement\u003c/h2\u003e\n\u003cp\u003eThe study was approved by the Research Medical Ethics Committee of the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine (Guangzhou, China) (JY2024-149). Written informed consent was obtained from all patients and healthy volunteers prior to their participation. The study was conducted in accordance with the 1964 Declaration of Helsinki and its later amendments.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eInclusion and Exclusion Criteria\u003c/h2\u003e\n\u003cp\u003ePatients in all groups were free from primary diseases, including cardiac, hepatic, and renal insufficiency, as well as hematologic, neoplastic, and immune system diseases. Additionally, patients with a history of drug therapy within the last 3 months, or those who were smokers and/or alcoholics, were excluded from the study.\u003c/p\u003e\n\u003cp\u003ePatients diagnosed with TP based on β-hCG levels and transvaginal ultrasound findings, showing a gestational sac within the fallopian tube, were included in the TP group. Pathologic examination of intrauterine tissues confirmed the absence of chorionic tissue[13]. Patients with ectopic pregnancies located outside the fallopian tube or those who were hemodynamically unstable on admission were excluded from the study.\u003c/p\u003e\n\u003cp\u003ePatients with NP, confirmed by transvaginal ultrasound (TVUS), were included in the control group. Pathologic examination of uterine tissues confirmed the presence of chorionic tissue in the sections.\u003c/p\u003e\n\u003ch2\u003eStudy Cohort\u003c/h2\u003e\n\u003cp\u003eThe study population included patients with tubal pregnancy (TP; n = 10) and women with normal intrauterine pregnancy (NP; n = 10). Clinical data of the participants are summarized in Table 1. Human chorionic villi were collected from the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine. Tissue samples were divided into two portions: one was stored at -80°C for protein expression analysis, and the other was fixed in 4% paraformaldehyde (PFA) and embedded in paraffin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Clinical data of the participants.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"566\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eParameter\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNormal Pregnancy (n=10)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003etubal pregnancy (n=10)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e27.1\u0026nbsp;±\u0026nbsp;3.4 (22–34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e28.7\u0026nbsp;±\u0026nbsp;3.4 (24–35)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBMI (kg/m²)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e23.8\u0026nbsp;±\u0026nbsp;3.3 (20–29)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e23.3\u0026nbsp;±\u0026nbsp;3.3 (18–28)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGestational age (days)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e47.1\u0026nbsp;±\u0026nbsp;4.6 (42–53)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e48.8\u0026nbsp;±\u0026nbsp;4.1 (44–56)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePrevious ectopic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0 [0–0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0 [0–0.25]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSerum hCG (IU/L)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10,076 [5,450–17,543]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1,286 [918–1,678]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eData presented as mean ± SD (range) for normally distributed variables (age, BMI, gestational age) or median [IQR] for non-normally distributed variables (hCG). Categorical data shown as n (%).\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBMI, body mass index.\u003c/em\u003e \u003cem\u003e*p \u0026lt; 0.05 considered statistically significant.\u003c/em\u003e\u003c/p\u003e\n\u003ch2\u003eCe ll Lines, Reagents, and Antibodies\u003c/h2\u003e\n\u003cp\u003eHTR-8/SVneo cells [14] were purchased from the American Type Culture Collection (CRL-3271). This cell line contains two cell populations: trophoblast and stromal/mesenchymal cells [15]. The cells were cultured in RPMI-1640 medium (Gibco, 11875093) supplemented with 10% fetal bovine serum (FBS; Gibco, A5670701) and antibiotics (penicillin, 100 IU/mL; streptomycin, 100 μg/mL; NCM Biotech, C100C5) at 37°C in a 5% CO2 atmosphere.\u003c/p\u003e\n\u003cp\u003eTanshinone IIA (568-72-9) and Elesclomol (STA-4783) were purchased from MedChemExpress. TTM (323446) was obtained from Sigma-Aldrich. Antibodies against DLAT (13426-1-AP), FDX1 (82957-2-RR), Bax (50599-2-Ig), Bcl2 (12789-1-AP), and β-actin (20536-1-AP) were purchased from Proteintech. Antibodies against DLST (ab177934), lipoic acid (ab58724), and LIAS (ab246917) were purchased from Abcam.\u003c/p\u003e\n\u003cp\u003eThe cells were divided into the following groups: NC (negative control), TSA (Tanshinone IIA treatment), MOD (model group treated with 5 μM CuCl\u003csub\u003e2\u003c/sub\u003e and 50 nM Elesclomol), MOD+TSA (MOD group with Tanshinone IIA treatment), and MOD+TSA+TTM (MOD+TSA group with 20 μM TTM treatment) [16–18].The concentrations of Elesclomol (50 nM) and CuCl₂ (5 μM) were selected based on previous studies demonstrating cuproptosis induction in various cell types at these ranges [11,19-20].\u003c/p\u003e\n\u003ch2\u003eCCK8 cell viability assay\u003c/h2\u003e\n\u003cp\u003eCell viability was determined using the Cell Counting Kit-8 (CCK-8; Solarbio, CA12120) according to the manufacturer’s instructions. Cells were seeded in 96-well plates at a density of 1×10^4 cells per well and treated according to the experimental groups, with three replicate wells per group. After treatment, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C for 1.5 hours. Absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, USA).\u003c/p\u003e\n\u003ch2\u003eImmunofluorescence Staining (IF)\u003c/h2\u003e\n\u003cp\u003eCell culture chamber slides were placed in 12-well plates, and cells were seeded at a density of 4×10^4 cells per well. After grouping, cells were fixed on the slides with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 20 minutes, and blocked with BSA. Subsequently, the cells were incubated overnight with a primary antibody (anti-FDX1 antibody, 1:500 dilution), followed by incubation with a FITC-conjugated secondary antibody (Proteintech, SA00003-2, 1:200 dilution) for 2 hours at room temperature. After washing, antifading mounting medium containing DAPI (Solarbio, S2110) was added dropwise to the slides. The slides were coverslipped, and images were captured using a fluorescence microscope (Olympus, Japan).\u003c/p\u003e\n\u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e\n\u003cp\u003eTissues were fixed in 4% paraformaldehyde for 24h, embedded in paraffin, and the wax blocks were made into paraffin sections, followed by dewaxing hydration operation, restore antigenic epitopes using Tris-EDTA antigen retrieval solution (Beyotime P0084) at 37℃, blocking non-specific binding sites with 20% goat serum (Solarbio SL038), and then primary antibody(anti-FDX1 antibody 1:200, anti-Bax antibody 1:200, anti-Bcl\u003csub\u003e2\u003c/sub\u003e antibody 1:200) was used at 4°C overnight incubation, reaction using HRP-labeled secondary antibody, color development with DAB color solution (proteintech PK10006), and scanning imaging using Digital Pathology Slice Scanning System (3DHistech Pannoramic 250).\u003c/p\u003e\n\u003ch2\u003eWestern blot\u003c/h2\u003e\n\u003cp\u003eCells or tissues were lysed using RIPA Lysis Buffer (Beyotime, P0013B) on ice for 30 minutes, followed by centrifugation to collect the supernatant. The supernatant was mixed with loading buffer (Beyotime, P0015L) and heated at 100°C for 5 minutes. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (MCE, IPVH00010). The membranes were blocked with 5% skimmed milk for 2 hours at room temperature to prevent non-specific binding. Subsequently, the membranes were incubated overnight at 4°C with primary antibodies, including anti-FDX1 (1:1000), anti-Bax (1:2000), anti-Bcl-2 (1:2000), anti-β-actin (1:10000), anti-Lipoic Acid (1:10000), and anti-LIAS (1:2500). After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG or anti-mouse IgG for 1 hour at room temperature. Chemiluminescent reactions were performed using an ECL substrate (Tanon, 180-5001), and images were captured using a multifunctional imager (Bio-Rad Laboratories, Hercules, USA). Protein band intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, USA).\u003c/p\u003e\n\u003ch2\u003eROS and Mitochondrial Membrane Potential (MMP) Detection\u003c/h2\u003e\n\u003cp\u003eCells were seeded in 12-well plates at a density of 4×10^4 cells per well and treated according to the experimental groups. A solution of dichloro-dihydro-fluorescein diacetate (DCFH-DA; Beyotime, S0035S) or JC-1 fluorescent probe (Solarbio, M8650) was prepared according to the manufacturer’s instructions. The cells were incubated with the probes at 37°C for the indicated time in the dark, followed by three washes with PBS. Images were captured using a fluorescence microscope (Olympus, Japan).\u003c/p\u003e\n\u003ch2\u003eFlow Cytometry for Apoptosis or ROS Detection\u003c/h2\u003e\n\u003cp\u003eCells were seeded into 6-well plates at a density of 5×10^5 cells per well and treated according to the experimental groups. Working solutions of Annexin V-FITC and PI (MULTI SCIENCES, AP101) or DCFH-DA were prepared according to the manufacturer’s instructions. After treatment, the cells were harvested and resuspended in the working solutions. For apoptosis detection, the cells were incubated with Annexin V-FITC and PI at 37°C in the dark for 5 minutes. For ROS detection, the cells were incubated with DCFH-DA at 37°C in the dark for 20 minutes. Following incubation, the cells were \u0026nbsp;analyzed using a FACSCanto II flow cytometer (BD Biosciences, USA).\u003c/p\u003e\n\u003ch2\u003eCell Invasion Assay\u003c/h2\u003e\n\u003cp\u003eMatrigel matrix gel (Corning, 356234) was added to the upper chambers of Transwell inserts (Corning, 3422) and allowed to solidify. HTR-8/SVneo cells were suspended in medium containing the drug and 1% fetal bovine serum (FBS), and 1×10^5 cells were seeded into the upper chambers. The lower chambers were filled with medium containing 10% FBS. After incubation according to the experimental groups, cells on the lower surface of the membrane were fixed with 4% paraformaldehyde (PFA) and stained with crystal violet. Images were captured using a microscope (Olympus, Japan) and analyzed with ImageJ software.\u003c/p\u003e\n\u003ch2\u003eWound-Healing Assay\u003c/h2\u003e\n\u003cp\u003eCells were seeded into 6-well plates at a density of 4×10^4 cells per well and cultured until they reached 80% confluence. A sterile pipette tip was used to create a wound in the cell monolayer. The cells were rinsed twice with RPMI-1640 medium, and images were captured at 0 hours using a microscope (Olympus, Japan). After wounding, the cells were incubated for 24 hours according to the experimental groups. Following the intervention, the cells were cultured for an additional 24 hours, and images were captured again for analysis using ImageJ software.\u003c/p\u003e\n\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n\u003cp\u003e.All statistical analyses were performed using SPSS 27.0 (IBM, USA) and GraphPad Prism 8.0 (GraphPad Software, USA). Normality was assessed by Shapiro-Wilk testing. Continuous variables with normal distribution were expressed as mean ± standard deviation and analyzed using independent t-tests (two-group comparisons) or one-way ANOVA with Tukey’s post-hoc test (multi-group comparisons), while non-normally distributed data were presented as median [interquartile range] and evaluated with Mann-Whitney U tests or Kruskal-Wallis tests with Bonferroni’s correction, respectively. P-value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003e\u003cstrong\u003eCuproptosis in Chorionic Villi of Patients with Tubal Pregnancy\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eWe collected samples from 20 patients aged between 22 and 35 years, including 10 cases of NP and 10 cases of TP (\u003cstrong\u003eTable 1\u003c/strong\u003e). The tissues were obtained from chorionic villi in the uterine cavity (NP group) or from TP. Protein expression and immunohistochemical analyses were performed (\u003cstrong\u003eFigure 1\u003c/strong\u003e). The results showed that the levels of FDX1, DLST, and DLAT were significantly higher in normal chorionic villi compared to those from TP, with only DLAT showing statistical significance. Immunohistochemical staining revealed that trophoblast cells expressed higher levels of LIAS, FDX1, Bax, and Bcl2 compared to stromal cells within the chorionic villi. Compared to the NP group, TP tissues exhibited increased Bax expression and decreased expression of LIAS, FDX1, and Bcl2. The reduced expression of FDX1 and Bcl2, combined with elevated Bax levels in TP tissues, suggests enhanced apoptosis in trophoblast cells, potentially associated with cuproptosis.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eEffect of Tanshinone IIA on HTR8-SVneo Cells\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eTo determine the optimal concentration of tanshinone IIA (TSA) for intervention, cells were treated with various concentrations of TSA for 12 or 24 hours, and cell viability was assessed using the CCK-8 assay. The results showed that concentrations of 30-40 \u0026mu;M reduced cell viability by 50% (\u003cstrong\u003eFigure 2a\u003c/strong\u003e). Based on these findings, 30-40 \u0026mu;M TSA was selected for subsequent experiments. After 24 hours of treatment, the levels of lipoic acid synthetase (LIAS) and ferredoxin 1 (FDX1) were measured (\u003cstrong\u003eFigure 2b, 2c\u003c/strong\u003e). Compared to the untreated group, TSA treatment decreased LIAS levels and increased FDX1 expression; however, these changes were not statistically significant.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eEffect of Tanshinone IIA on Cuproptosis and Cell Function in HTR8-SVneo Cells\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eTrophoblast cells in early pregnancy exhibit migratory and invasive capabilities, which contribute to the invasion of the fallopian tube and its eventual rupture in tubal pregnancies. To further investigate the effects of cuproptosis and tanshinone IIA (TSA) on cell function, cells were treated according to the same grouping method. The results showed that cuproptosis induced by Elesclomol and CuCl\u003csub\u003e2\u003c/sub\u003e significantly inhibited the invasion and migration abilities of the cells. However, the combination of TSA with cuproptosis further suppressed these cellular functions, and tetrathiomolybdate (TTM) partially reversed this effect (\u003cstrong\u003eFigure 3\u003c/strong\u003e).\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eEffect of Tanshinone IIA on Oxidative Stress in HTR8-SVneo Cells\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eTo further explore the effects of cuproptosis and tanshinone IIA (TSA) on oxidative stress, we used fluorescent probes to detect intracellular reactive oxygen species (ROS), apoptosis, and mitochondrial membrane potential (\u003cstrong\u003eFigure 4\u003c/strong\u003e). The results showed that cuproptosis induced by Elesclomol and CuCl\u003csub\u003e2\u003c/sub\u003e significantly increased intracellular ROS levels and promoted apoptosis. However, the combination of TSA with cuproptosis further enhanced these effects, and tetrathiomolybdate (TTM) partially reversed this phenomenon .\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eEffect of Tanshinone IIA on Cuproptosis-related Protein Expression in HTR8-SVneo Cells\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eWestern blotting and immunofluorescence were performed to detect the expression levels of acylated dihydrolipoamide acetyltransferase (lip-DLAT), acylated dihydrolipoamide S-acetyltransferase (lip-DLST), FDX1, Bax, and Bcl2 proteins in the cells. The results showed that cuproptosis induced by Elesclomol and CuCl\u003csub\u003e2\u003c/sub\u003e downregulated the expression of lip-DLAT, lip-DLST, FDX1, and Bcl\u003csub\u003e2\u003c/sub\u003e, while Bax expression was upregulated. Combined treatment with tanshinone IIA (TSA) further enhanced these changes, and tetrathiomolybdate (TTM) partially reversed the effects (\u003cstrong\u003eFigure 5\u003c/strong\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOxidative stress occurs during normal pregnancy, where increased energy demands and elevated oxygen consumption enhance mitochondrial activity, subsequently increasing ROS production. As crucial signaling molecules, ROS participate in cellular proliferation, migration, and angiogenesis[21]. A balanced ROS level, supported by an efficient antioxidant defense system, maintains redox homeostasis, ensuring normal fetal development and maternal health[22]. However, excessive ROS can cause cellular damage, lipid peroxidation, DNA oxidation, and protein modification, leading to cellular and tissue dysfunction[18]. Studies have shown that excessive ROS plays a significant role in the pathophysiology of pregnancy complications, including miscarriage, preeclampsia[22], intrauterine growth restriction, preterm premature rupture of membranes, and TP[23].\u003c/p\u003e\n\u003cp\u003eROS can alter the oxidation state of intracellular copper ions, increasing their reactivity. Oxidative stress increases mitochondrial membrane permeability, depletes antioxidants such as glutathione (GSH) and metallothionein (MT), and impairs the cell’s ability to detoxify copper ions[24]. This reduces copper-binding capacity, elevates free copper levels, and ultimately triggers cuproptosis. Additionally, oxidative stress activates transcription factors such as NF-κB or directly modifies copper transport proteins such as CTR1 and ATP7A/B[25], leading to increased copper uptake or reduced copper efflux, which promotes copper accumulation and cuproptosis. Moreover, cuproptosis disrupts the mitochondrial tricarboxylic acid (TCA) cycle, further exacerbating oxidative stress[26].\u003c/p\u003e\n\u003cp\u003eFDX1, an iron-sulfur cluster (ISC) protein, plays a crucial role in mitochondrial metabolism and the cuproptosis pathway. It facilitates electron transfer from NADPH to downstream enzymes via iron-sulfur clusters (2Fe-2S) [27]. Additionally, FDX1 promotes the deposition of copper ions into mitochondria and interacts with lipoylases to induce cytosolic lipoylation, disrupting their normal function and leading to cell death [28]. According to a study by Tsvetkov et al. published in Science, knockdown of FDX1 reduces lipoylation of tricarboxylic acid cycle components DLAT and DLST, partially reversing cuproptosis. However, FDX1 expression tends to decrease during cuproptosis [11]. Our findings indicate that, in the absence of drug treatment, trophoblast cells in chorionic villi tissues from patients with tubal pregnancy exhibit higher levels of apoptosis and cuproptosis-associated protein expression compared to those from patients with normal intrauterine pregnancy. This suggests that trophoblast cells in tubal pregnancy are more prone to apoptosis and cuproptosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe antitumor activity of tanshinone IIA (TSA) has been demonstrated in various cancer cell lines, with its role in mediating apoptosis through increased reactive oxygen species (ROS) levels being particularly significant. Previous studies have shown that TSA reduces glutathione (GSH) and cysteine levels while increasing ROS levels in BGC-823 and NCI-H87 cells [29], and alters mitochondrial membrane potential in chronic myeloid leukemia (CML) cells, thereby inducing intrinsic apoptosis [30]. It was also also suggested that Sodium Tanshinone IIA Sulfonate inhibits the release of High Mobility Group Box 1 protein (HMGB1) from hypoxic trophoblasts, thereby reducing inflammatory responses and oxidative stress, protecting endothelial cell function[31]. In the present study, we investigated the effects of TSA on tubal pregnancy (TP) trophoblast cells using HTR-8/SVneo cells as a model. Our results showed that TSA decreased cell viability, upregulated FDX1, and downregulated lipoic acid synthetase (LIAS), suggesting that TSA may enhance the sensiti vity of HTR-8/SVneo cells to cuproptosis by regulating FDX1. Further experiments revealed that TSA exacerbated cuproptosis. Specifically, TSA treatment significantly reduced mitochondrial membrane potential, indicating impaired mitochondrial function. Additionally, the expression of the pro-apoptotic protein Bax was upregulated, while the anti-apoptotic protein Bcl-2 was downregulated, indicating a shift toward apoptosis. The altered Bax/Bcl-2 ratio further exacerbated oxidative stress and cell deat. Tanshinone IIA may have a dual effect on cellular oxidative stress, potentially promoting or inhibiting it, which could depend on different disease states.\u003c/p\u003e\n\u003cp\u003eOur data suggest that TSA may disrupt the adaptation of gestational trophoblast cells to hypoxic environments by exacerbating reactive oxygen species (ROS) accumulation (Figure 4a-c), leading to mitochondrial dysfunction (Figure 4f) and apoptosis. This is consistent with the observed downregulation of Bcl-2 and upregulation of Bax (Figure 5), which are known mediators of hypoxia adaptation. However, the precise mechanisms require further investigation, as some studies report that TSA can protect hypoxic trophoblast cells from endothelial cell injury [31], indicating its dual roles in regulating cell growth and its high safety profile. Future studies should further investigate the molecular mechanisms of TSA and its potential clinical applications, aiming to develop novel therapeutic strategies for tubal pregnancy and refractory trophoblastic diseases.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study highlights the critical role of oxidative stress and cuproptosis in trophoblast cell death during tubal pregnancy (TP). Tanshinone IIA (TSA) was shown to elevate reactive oxygen species (ROS) levels and induce apoptosis in trophoblast cells, potentially through modulating FDX1 expression and enhancing susceptibility to copper-induced cell death. The upregulation of Bax and downregulation of Bcl-2 following TSA treatment indicated a shift toward an apoptotic state, accompanied by exacerbated oxidative stress and mitochondrial damage. These findings suggest that TSA may disrupt trophoblast adaptation to hypoxic conditions, ultimately promoting apoptosis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"524\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eTP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eTubal Pregnancy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eNP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eNormal Pregnancy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eTSA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eTanshinone IIA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eTTM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eTetrathiomolybdate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eROS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eReactive Oxygen Species\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eCFDA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eChinese State Food and Drug Administration\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003ePDC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eDehydrogenase Complex\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eKGDC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eKetoglutarate Dehydrogenase Complex\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003ePFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eParaformaldehyde\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eLIAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eLipoic Acid Synthetase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eFDX1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eFerredoxin 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eDLAT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eDihydrolipoamide Acetyltransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eDLST\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eDihydrolipoamide S-acetyltransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eHIF-1\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eHypoxia-Inducible Factor 1\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eISC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eIron-Sulfur Cluster\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eGSH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eGlutathione\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki, and approved by the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine (Guangzhou, China) (JY2024-149 10/23/2024).\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eAll authors agree to publish this article.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (82174417) and the Sanming Project of Medicine in Shenzhen（No. SZZYSM202106003）.\u003c/p\u003e\n\u003cp\u003eContribution statement\u003c/p\u003e\n\u003cp\u003eConceptualization, J.D. and G.D.; methodology, J.D., X.W. and Y.Z.; software, J.D., X.W. and Y.Z.; validation, J.F., L.Y. and Y.H.; formal analysis, J.D.; investigation, Y.H. and G.D.; resources, J.F. and Y.H.; data curation, J.D. and X.W.; writing\u0026mdash;original draft preparation, J.D., X.W. and Y.Z.; writing\u0026mdash;review and editing, J.F., L.Y., Y.H. and G.D.; visualization, J.D. and G.D.; supervision, X.W., Y.Z. and G.D.; project administration, Y.H. and G.D.; funding acquisition, Y.H. and G.D. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eDeclaration of competing interest\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eWe gratefully thank all participants of the study and members of the Department of Gynecology, First Affiliated Hospital of Guangzhou University of Chinese Medicine.\u003c/p\u003e\n\u003cp\u003eSupplementary Materials\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eData Availability Statement\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed in this study can be obtained from the corresponding author on reasonable request. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRamphal, S. R. Feasability and Clinical Outcome With Laparoscopic Management of Extrauterine Pregnancies. \u003cem\u003eJ. Minim. 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Sodium Tanshinone IIA Sulfonate Prevents Hypoxic Trophoblast-Induced Endothelial Cell Dysfunction via Targeting HMGB1 Release. \u003cem\u003eJ. Biochem. Mol. Toxicol.\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/jbt.21903\u003c/span\u003e\u003cspan address=\"10.1002/jbt.21903\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ectopic pregnancy, Tanshinone IIA, oxidative stress, copper dependent","lastPublishedDoi":"10.21203/rs.3.rs-5772434/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5772434/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOxidative stress occurs in trophoblast cells during the development of tubal pregnancy (TP), compared to normal pregnancy. It has been demonstrated that Previous studies have shown that Tanshinone IIA (TSA) can increase reactive oxygen species (ROS) levels and exacerbate oxidative stress in tumor cells, while its effects on trophoblast cells or affects cuproptosis pathways remain unclear.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe collected chorionic tissue from patients with normal intrauterine pregnancies (NP) or TP to detect the expression of related proteins. HTR-8/SVneo cells were cultured in vitro and treated with Elesclomol, CuCl2 and/or TSA, tetrathiomolybdate (TTM). The expressions of proteins such as DLAT, DLST, FDX1, Lipo-DLAT, Lipo-DLST, Bax, and Bcl-2 were measured. Mitochondrial membrane potential, cell apoptosis, and cell function were also assessed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResult\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe concentration of TSA added to HTR8-SVneo cells was 30 µM. The protein expression of DLAT, DLST, Lipo-DLAT, Lipo-DLST monomers, FDX1 and Bcl-2/ Bax was downregulated by the addition of Elesclomol and CuCl2 intervention in the cells. Meanwhile, the levels of reactive oxygen species (ROS) increased, mitochondrial membrane potential decreased, cell apoptosis increased, and cell invasion and migration were attenuated. The addition of TSA enhanced these effects, while the addition of TTM mitigated them.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTSA can promote oxidative stress in HTR-8/SVneo cells, leading to cell apoptosis. This process can be reversed by copper chelator.\u003c/p\u003e","manuscriptTitle":"Tanshinone IIA induces oxidative stress in trophoblast cells and enhances copper dependent death","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 09:32:33","doi":"10.21203/rs.3.rs-5772434/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-21T11:56:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-20T17:17:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"287728829820076424392736154728310966840","date":"2025-04-30T13:18:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-28T06:51:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6565598497924498204314972261364048083","date":"2025-04-28T06:23:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-28T06:18:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-25T12:26:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-04-04T07:22:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"822ffa2d-6d62-4e1a-b0f1-1661174c2757","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47763900,"name":"Biological sciences/Drug discovery"},{"id":47763901,"name":"Health sciences/Diseases"},{"id":47763902,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2025-07-14T16:00:09+00:00","versionOfRecord":{"articleIdentity":"rs-5772434","link":"https://doi.org/10.1038/s41598-025-07238-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-11 15:57:12","publishedOnDateReadable":"July 11th, 2025"},"versionCreatedAt":"2025-05-06 09:32:33","video":"","vorDoi":"10.1038/s41598-025-07238-5","vorDoiUrl":"https://doi.org/10.1038/s41598-025-07238-5","workflowStages":[]},"version":"v1","identity":"rs-5772434","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5772434","identity":"rs-5772434","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}