Lactobacillus crispatus reverses Escherichia coli-induced inflammatory injury and migration of cervical cancer cells by inhibiting ferroptosis

Lactobacillus crispatus reverses Escherichia coli-induced inflammatory injury and migration of cervical cancer cells by inhibiting ferroptosis | 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 Research Article Lactobacillus crispatus reverses Escherichia coli-induced inflammatory injury and migration of cervical cancer cells by inhibiting ferroptosis Dongli Tian, Lei Deng, Enting Lu, Huayang Li, Fanyi Meng, Xinyang Chen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6435097/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background In the post-antibiotic era, there is growing recognition of the critical role that microecological balance plays in human health. Extensive research has demonstrated that Lactobacillus, as the predominant bacterial genus in the female reproductive system, effectively inhibits harmful microorganisms and contributes to maintaining overall health. In this study, Lactobacillus crispatus, a species within the Lactobacillus genus, was selected as the subject of investigation to elucidate its antibacterial mechanisms against Escherichia coli and the molecular pathways involved in reversing cervical cancer complicated by Escherichia coli infection. Results Lactobacillus crispatus exhibited inhibitory effects on the growth of Escherichia coli, with an average inhibitory zone diameter of 24.31 ± 34 mm. The active components responsible for this inhibition were identified as live bacterial cells of Lactobacillus crispatus. The antibacterial activity of Lactobacillus crispatus was stable for up to 7 days at 4°C and 42 days at 25°C. Furthermore, Lactobacillus crispatus effectively suppressed the biofilm formation and cellular adhesion of Escherichia coli while limiting the release of extracellular soluble proteins. It has been demonstrated that Escherichia coli induces inflammatory damage in cervical cancer cells via the ferroptosis pathway, thereby promoting tumor cell migration. Following intervention with Lactobacillus crispatus, the bacterium was able to reverse the ferroptosis-induced and inflammatory damage caused by Escherichia coli in cervical cancer cells, inhibit tumor cell migration, and reduce tumor malignancy. Conclusion Lactobacillus crispatus as a microecological balance regulator can not only inhibit the growth of Escherichia coli, but also improve the migration ability of cervical cancer complicated with Escherichia coli infection and maintain the health of the female reproductive system. Lactobacillus crispatus Escherichia coli ferroptosis Cervical cancer Inflammatory injury inflammation Migration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background There exists a diverse array of microbial communities within the female vagina, collectively maintaining the balance and stability of vaginal microecology [ 1 , 2 ] . However, any disruption to this equilibrium may lead to gynecological diseases, including inflammation and tumors. Escherichia coli exhibits specific distribution patterns in the female reproductive system, frequently serving as a common pathogen in gynecological infections. Research has demonstrated that among women of childbearing age, factors such as sexual activity, pregnancy, childbirth, and others may impair vaginal microecology, thereby increasing the susceptibility to Escherichia coli infection [ 3 – 5 ] . Furthermore, postmenopausal women are at heightened risk for Escherichia coli infection due to decreased estrogen levels, vaginal mucosal atrophy, and alterations in the microecological environment. Escherichia coli infection can elicit an inflammatory response, releasing various cytokines and inflammatory mediators, such as interleukin and tumor necrosis factor. These substances may promote cell proliferation, inhibit apoptosis, and create favorable conditions for the growth and survival of tumor cells. As a critical anatomical structure connecting the vagina and uterus, the cervix is also a region prone to colonization by Escherichia coli, potentially leading to cervical epithelial damage, inflammation, and even carcinogenesis [ 6 ] . Lactobacillus crispatus serves as the dominant strain in the female vagina, playing a pivotal role in preserving the balance of vaginal microecology [ 7 ] . It maintains an acidic environment in the vagina, inhibits the proliferation of harmful bacteria, and produces hydrogen peroxide to suppress heterobacteria, which is associated with reduced levels of inflammation [ 8 ] . Currently, the mechanism by which Lactobacillus crispatus mitigates cervical cancer through the inhibition of Escherichia coli remains unclear. The objective of this study is to further investigate the role of Escherichia coli in the onset of cervical cancer, which holds significant implications for enhancing early prevention, improving treatment efficacy, and reducing mortality rates. Additionally, the application of Lactobacillus crispatus-based microecological therapy to control the occurrence and progression of cervical cancer carries substantial practical significance. Materials and methods Strains and cell lines and their cultures The freeze-dried powder of Lactobacillus crispatus CGMCC1.2743 was obtained from the China General Microbiological Culture Collection Center (CGMCC). An appropriate volume of sterile water was added to reconstitute the lyophilized powder, which was subsequently inoculated into MRS liquid medium(Solarbio, Beijing, China). The culture was maintained under controlled conditions at 37 ℃ with a shaking speed of 150 rpm for 18 hours. Following successful growth of the strain, an inoculation loop was used to transfer the culture onto MRS solid medium, which was then incubated at 37 ℃ for 18 to 24 hours. Escherichia coli ATCC25922 originates from the Laboratory of Shenyang Medical University Affiliated Central Hospital and is cultured in broth medium (Solarbio, Beijing, China) under conditions at 37 ℃, 180 r/min, for 18 hours. Human cervical cancer cell lines Hela and C-33A were obtained from Beijing Dingguo Changsheng Biotechnology Co., Ltd. Both Hela and C-33A cells were cultured in DMEM (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), along with 100 units/mL of penicillin (Genview, Australia) and 100 units/mL of streptomycin solution (Genview, Australia). The cultures were maintained at 37°C in a humidified atmosphere containing 5% CO 2 . Antibodies The primary antibodies include mouse monoclonal anti-NF-κB p65 (1:3000, Cat No. ab288751), rabbit polyclonal anti-LOX (1:5000, Cat No. ab174316), rabbit monoclonal anti-COX2 (1:3000, Cat No. ab179800), rabbit monoclonal anti-E-cadherin (1:6000, Cat No. ab314063), rabbit monoclonal anti-N-cadherin (1:6000, Cat No. ab76011), rabbit monoclonal anti-Vimentin (1:3000, Cat No. ab92547), rabbit monoclonal anti-Snail (1:1000, Cat No. ab216347), rabbit monoclonal anti-Slug (1:1000, Cat No. ab314086) were obtained from Abcam Biomedical Company (Cambridge, UK). The primary antibodies include mouse monoclonal anti-GPX4 (1:3000, Cat No. 67763-1-Ig), rabbit polyclonal anti-SLC7A11 (1:1000, Cat No. 26864-1-AP), rabbit polyclonal anti-SLC3A2 (1:20000, Cat No. 15193-1-AP) were obtained from Proteintech Company (Wuhan, Hubei, China). The primary antibodies include rabbit polyclonal anti-ompA (1:2000, Cat No. PA5-117575), rabbit polyclonal anti-ompC (1:2000, Cat No. PA5-117701), rabbit polyclonal anti-Lipoprotein p27 (1:1000, Cat No. 200-401-C30), were obtained from Thermo Fisher Scientific Company (Massachusetts, USA). HRP-Goat anti-rabbit recombinant secondary antibody (H + L) and HRP-Goat anti-mouse recombinant secondary antibody (H + L) were obtained from Proteintech Company (Wuhan, Hubei, China). Ferroptosis determination Ferroptosis was detected using fluorescence microscopy. The procedure involved adding 200 µL of Hela and C-33A cells (at a concentration of 2×10 4 cells/mL) to each well of a 24-well plate, followed by incubation for 18 hours at 37°C with 5% CO 2 . Subsequently, the culture medium was removed, and the cells were treated for 4 hours with 200 µL of MDEM medium containing Escherichia coli or MDEM medium containing Escherichia coli plus Lactobacillus crispatus. Afterward, the medium was replaced with 500 µL of DMEM supplemented with ferric ammonium sulfate (final concentration of 100 µmol/L), and the cells were incubated for 30 minutes in an incubator. Following this step, the cells were washed with PBS, fixed with 4% paraformaldehyde for 30 minutes at room temperature, and permeabilized with 1% Triton X-100 for 20 minutes. After washing again with PBS, the cells were incubated with 500 µL of Iron Green solution for 30 minutes. Finally, the intensity of the fluorescence signals was quantified using GFP and BF filters under a fluorescence microscope. Cell scratch assay Briefly, 2×10 5 cells/well Hela cells and C-33A were seeded in 6-well plates. After 18 h of adherence, they were scratched with a 200-µL pipette tip. Subsequently, the cells were treated with 200 µL of MDEM medium containing Escherichia coli or MDEM medium containing Escherichia coli plus Lactobacillus crispatus, cultured them for 24 h. The experiment was repeated three times, and the cell scratch area was calculated using ImageJ software. Bacteriostatic active ingredient detection The concentration of Escherichia coli during the logarithmic growth phase was adjusted to 1×10 6 CFU/mL. A volume of 100 µL was then taken and evenly spread onto MRS solid medium. The Lactobacillus crispatus bacterial solution cultured for logarithmic period was divided into 4 groups, whole bacterial solution, supernatant liquid, live bacteria, and inactivated bacteria. Using the disk diffusion method, 200 µL of each tested sample was added to a disk and incubated at 37°C for 18 hours. The inhibition diameter was measured using a vernier caliper. Minimum inhibitory concentration (MIC) determination The concentration of Lactobacillus crispatus was adjusted to 1×10 2 CFU/mL, 1×10 3 CFU/mL, 1×10 4 CFU/mL, 1×10 5 CFU/mL, 1×10 6 CFU/mL, and 1×10 7 CFU/mL using MRS medium. Subsequently, 100 µL of Lactobacillus crispatus bacterial suspensions at varying concentrations were added to a 96-well plate, followed by the addition of 10 µL of Escherichia coli suspension (1×10 6 CFU/mL) into each well. The plates were then incubated for 18 hours at 37°C in a bacterial incubator. After incubation, the minimum drug concentration that inhibited visible bacterial growth was determined using the plate viable counting method, which corresponds to the MIC of the bacteria against the tested drugs. Extracellular soluble protein detection 200 µL of Escherichia coli suspension (1×10 6 CFU/mL) was added to a 48-well plate. Subsequently, 200 µL of Lactobacillus crispatus suspension at final concentrations of 0 MIC, 1/4 MIC,1/2 MIC, and MIC were added to the respective wells. A negative control consisting of broth medium without bacteria was included. The Escherichia coli suspension from each group was centrifuged at 12,000 rpm for 20 minutes at 4°C to collect the bacterial pellet. The supernatant was discarded, and the cells were washed twice with pre-cooled phosphate-buffered saline (PBS). Following centrifugation at the same conditions, the supernatant was removed, and the bacteria were re-suspended in PBS. Subsequently, the bacterial samples were treated in a metal bath at 100°C for 15 minutes, with inversion every 2 minutes to ensure uniform release of soluble proteins from lysed cells. Protein samples (20 µL) were subjected to SDS-PAGE electrophoresis, followed by staining with Coomassie Brilliant Blue (Beyotime Biotechnology, Shanghai, China) for 40 minutes. The gel was then decolorized using distilled water and photographed for analysis of protein expression. Biofilm formation determination 200 µL of Escherichia coli suspension (1×10 6 CFU/mL) was added to a 48-well plate. Subsequently, 200 µL of Lactobacillus crispatus suspension at final concentrations of 0 MIC, 1/4 MIC,1/2 MIC, and MIC were added to the respective wells. A negative control consisting of broth medium without bacteria was included. The samples were incubated for 18 hours at 37°C. After incubation, the medium and non-adherent bacteria were removed, and the wells were washed with PBS buffer. The adherent bacteria were fixed with methanol for 20 minutes, stained with a 2% crystal violet solution for 15 minutes, and decolorized with 33% glacial acetic acid. Finally, the absorbance was measured at 630 nm. Adhesion ability determination 500 µL of Hela and C-33A cells, suspended at a concentration of 2×10 5 cells/mL, were seeded into 6-well plates and incubated overnight under standard cell culture conditions (37°C, 5% CO₂). Subsequently, the cells were cultured in DMEM supplemented with serum for 3 days, followed by serum-free DMEM for 12 hours. After removing the culture medium and washing the wells twice with PBS, the following treatments were added to each well: 1 mL of Escherichia coli suspension, 1 mL of Escherichia coli suspension combined with 1/4 MIC Lactobacillus crispatus mixture,1 mL of Escherichia coli suspension combined with 1/2 MIC Lactobacillus crispatus mixture, 1 mL of Escherichia coli suspension combined with MIC Lactobacillus crispatus mixture, and 1 mL of DMEM culture medium as a control. The contents were mixed thoroughly and co-incubated for 4 hours. Following incubation, the cells were washed with PBS and fixed with 4% paraformaldehyde for 1 hour. The number of Escherichia coli adhering to the cell surface was observed and quantified under a microscope. Adhesion levels were categorized based on the following criteria: non-adherent ( 100 bacteria). Fermentation broth conditions determination A single colony of Lactobacillus crispatus was inoculated into MRS Liquid medium. The pH value ranged from 1 to 12, the temperature ranged from 28 to 43℃, and the culture time ranged from 12 to 42 hours to optimize the culture condition with the best antibacterial effect. In addition, live Lactobacillus crispatus was placed at 4℃ and 25℃ to detect the maintenance days of antibacterial activity of Lactobacillus crispatus. Inflammatory cytokines detection The levels of inflammatory factors in cells were quantified using the Enzyme-Linked Immunosorbent Assay (ELISA). Hela and C-33A cells, treated with Escherichia coli or a combination of Escherichia coli and Lactobacillus crispatus, were centrifuged at 1000g for 20 minutes at 4°C to collect the supernatant. According to the manufacturer's instructions (Mibio, Shanghai, China), standard wells and sample wells were prepared for TNF-α, IL-6, and IL-8 detection. Fifty microliters (50 µL) of standards at varying concentrations were added to the standard wells, while 50 µL of samples were added to the sample wells. Each well was subsequently incubated with 100 µL of horseradish peroxidase (HRP)-conjugated detection antibody for 60 minutes at 37°C. Following this, each well was washed five times with 300 µL of washing buffer. Substrate A and substrate B (50 µL each) were then added to each well and incubated for 15 minutes at 37°C in the dark. Finally, 50 µL of stop solution was added to each well, and the absorbance was measured at a wavelength of 450 nm. Western Blot Hela and C-33A cells were treated with Escherichia coli or a combination of Escherichia coli and Lactobacillus crispatus. The cells were subsequently lysed using RIPA lysate(Beyotime Biotechnology, Shanghai, China) containing 1mM PMSF(Beyotime Biotechnology, Shanghai, China), and total protein extracts were prepared. Equal quantities of protein samples from each experimental group were resolved by SDS-PAGE at 80 V for 25 min and then at 120 V for an additional 50 min. The separated proteins were transferred onto PVDF membranes (Millipore, Boston, MA, USA) under constant conditions of 15 V and 1.0 A for 18 min. The membranes were extensively washed with TBST, blocked overnight at 4°C with 5% skim milk (Sigma, Louis, MO, USA) in TBST, and incubated with the primary antibody for 2 h at room temperature. This was followed by incubation with the secondary antibody for 1 h under the same conditions. Protein bands were visualized using ECL reagent (Vazyme, Shanghai, China), and the gray values of the protein bands were calculated by Image J software. Statistical Analysis Each experiment was conducted a minimum of three times, with the results expressed as mean ± SD. Statistical analysis of all data was performed using SPSS 22.0 software. To compare the cell ferroptosis level, inflammatory factors, migration level with or without Escherichia coli intervention, a two-tailed Student's t-test was applied. The effects of Escherichia coli in combination with Lactobacillus crispatus at various concentrations on ferroptosis, inflammatory factors, and migration of human ovarian cancer cells were assessed using one-way ANOVA. Statistical significance was defined as a P value below 0.05. Results Escherichia coli induces inflammatory injury of cervical cancer cells through ferroptosis Human cervical cancer cells Hela and C-33A were infected with Escherichia coli. As shown in Figure 1-A, using fluorescence microscopy, the intracellular ferroptosis fluorescence signal was significantly increased in Hela and C-33A cells infected with Escherichia coli compared with those not infected with Escherichia coli. In addition, detection of ferroptosis-related regulatory proteins showed that the expression of GPX4, SLC7A11 and SLC3A2 was significantly decreased in Hela and C-33A cells after Escherichia coli infection ( p <0.05) (Figure 1-B). Studies have confirmed that ferroptosis is closely related to inflammatory injury. The results showed that the expression of TNF-α, IL-6 and IL-8 in Hela and C-33A cells after Escherichia coli infection was significantly increased ( p <0.001) (Figure 1-C). Escherichia coli promotes the migration of cervical cancer cells We investigated the impact of Escherichia coli on the migratory capacity of cervical cancer Hela and C-33A cells. As shown in Figure 2-A, the migratory ability of cervical cancer Hela and C-33A cells infected with Escherichia coli was significantly enhanced compared to that of uninfected cells ( p <0.05). Western blot analysis revealed that the expression levels of adhesion-related proteins N-cadherin, Vimentin, Snail, and Slug were significantly upregulated in Escherichia coli-infected cervical cancer Hela and C-33A cells relative to uninfected cells ( p <0.05) (Figure 2-B). Conversely, the expression level of E-cadherin was significantly decreased ( p <0.05) (Figure 2-B). Lactobacillus crispatus inhibited the growth of Escherichia coli We divided the cultured Lactobacillus crispatus bacterial solution into four groups: whole bacterial solution, sterile supernatant, active bacterial and inactivated bacterial. As shown in Figure 3-A, the whole solution of Lactobacillus crispatus and the active strain could inhibit the growth of Escherichia coli, but neither the supernatant nor the inactivated strain could inhibit the growth of Escherichia coli. The inhibitory diameters of the whole fermentation solution and the active strain against Escherichia coli were 17.32±13mm and 17.87±21mm, respectively. We optimized the culture conditions, and the results showed that the best antibacterial effect was obtained at pH3.0-5.0 and 37℃ for 18 hours, and the inhibition diameter was 24.31±34 mm (Figure 3-B to D). The antibacterial effect of Lactobacillus crispatus could be maintained for 42 days at 4℃. The antibacterial effect of Lactobacillus crispatus could be maintained for 7 days at 25℃ (Figure 3-E). The minimum inhibitory concentration (MIC) of Lactobacillus crispatus against Escherichia coli was 1×10 4 CFU/mL (Figure 3-F). Inhibitory mechanism analysis of Lactobacillus crispatus against Escherichia coli We selected 4 concentrations of Lactobacillus crispatus live bacteria to analyze the bacteriostatic mechanism, and the 4 concentrations were divided into 0 MIC, 1/4 MIC, 1/2 MIC, and MIC. As shown in Figures 4-A and 4-B, biofilm formation and adhesion of Escherichia coli were significantly reduced when the Lactobacillus crispatus concentration was ≥1/2MIC (p<0.05). The protein gel imaging system showed that when the concentration of Lactobacillus crispatus was ≥1/2MIC, the expression level of soluble protein in Escherichia coli cells decreased (Figure 4-C). Subsequently, we conducted a more in-depth analysis of the expression of biofilm-related proteins and key regulatory proteins involved in adhesion. The results demonstrated that the expression levels of Escherichia coli Outer membrane protein A (OmpA), Outer membrane protein C (OmpC), and Lipoprotein p27 could be significantly suppressed when the Lactobacillus crispatus concentration was ≥1/2MIC (p<0.05) (Figure 4-D) . Lactobacillus crispatus reversed the inflammatory injury induced by Escherichia coli in cervical cancer cells We investigated the impact of Lactobacillus crispatus on the inflammatory response triggered by ferroptosis induced by Escherichia coli in Hela and C-33A cells through the addition of Lactobacillus crispatus. As shown in Figure 5-A, fluorescence microscopy results demonstrated that when the concentration of Lactobacillus crispatus was ≥1/2MIC, the intensity of the ferroptosis-related fluorescence signal in Escherichia coli-infected Hela and C-33A cells was significantly reduced. Additionally, at a concentration of Lactobacillus crispatus ≥1/2MIC, the expression levels of ferroptosis-associated regulatory proteins GPX4, SLC7A11, and SLC3A2 were significantly decreased (p<0.05). Furthermore, the expression levels of TNF-α, IL-6, and IL-8 were significantly inhibited under the same conditions (p<0.05). Moreover, when the concentration of Lactobacillus crispatus was ≥1/2MIC, the expression levels of inflammatory regulatory proteins NF-κB, COX2, and LOX in Hela and C-33A cells were significantly decreased (p<0.05). Lactobacillus crispatus inhibits Escherichia coli-induced migration of cervical cancer cells As shown in Figure 6-A, Escherichia coli infected Hela and C-33A cells were able to significantly inhibit the migration of Hela and C-33A cells when Lactobacillus crispatus was added at a concentration ≥1/2MIC (p<0.05). In addition, Western blot results showed that the expression levels of migration-related proteins N-cadherin, vimentin, Snail, Slug were significantly decreased (p<0.05), and the expression level of E-cadherin was significantly increased at the concentration of Lactobacillus crispatus ≥1/2MIC (p<0.05). Discussion Currently, the global incidence of gynecological diseases among women has surpassed 93%, with approximately 9 million individuals succumbing to these conditions annually. This figure is escalating at an annual rate of 8% [9] . In China, gynecological diseases affect 80% of women, with the prevalence reaching as high as 87% among married women. The incidence of gynecological diseases among Chinese women is rising annually by 20% to 30%, indicating a significant upward trend. Common gynecological conditions in Chinese women include cervicitis, pelvic inflammatory disease, and vaginitis. Approximately 60% of women will experience at least one gynecological infection during their lifetime, while the prevalence of such infections among women aged 55 and above reaches 42%, with a recurrence rate as high as 50% [10-12] . Escherichia coli, a prevalent bacterium in the intestinal and vaginal microbial communities, typically maintains a balanced population and species diversity within the vaginal microbiota of healthy women. However, an abnormal increase in bacterial load or alterations in the composition and structure of the vaginal microecosystem may disrupt this balance, potentially leading to gynecological inflammation and even tumor development [13,14] . Recent evidence suggests that Escherichia coli may serve as a potential risk factor for the onset and progression of various cancers. Colibactin, a genotoxin produced by Escherichia coli harboring the polyketide synthase (PKS) island, has been shown to induce DNA double-strand breaks, contributing to colon cancer progression [15] . Studies have demonstrated that cathepsin K (CTSK), a protein secreted by colon cancer cells stimulated by Escherichia coli-derived lipopolysaccharide (LPS), can bind to Toll-like receptor 4 (TLR4) via the mTOR signaling pathway, thereby promoting M2 polarization of tumor-associated macrophages (TAMs) and enhancing the migration and motility of colon cancer cells [16] . Additionally, reports indicate a significantly higher detection rate of Escherichia coli in patients with HPV16-positive cervical cancer. Escherichia coli induces upregulation of Foxp3 and promotes IL-10 expression in cervical cancer, exacerbating cervical intraepithelial neoplasia and enhancing the adhesion and invasion capabilities of cervical cancer cells [17] . In this study, we investigated the effects of Escherichia coli on cervical cancer cells in vitro. Our findings revealed significant increases in the expression levels of GPX4, SLC7A11, and SLC3A2 in Hela and C-33A cells following Escherichia coli treatment, accompanied by enhanced intracellular ferroptosis fluorescence signals. We also assessed the expression of TNF-α, IL-6, and IL-8 in Hela and C-33A cervical cancer cells post-treatment with Escherichia coli . Our results are similar to those of other research results. Yang et al. demonstrated that Escherichia coli could increase the iron content in teleost erythrocytes, induce the Fenton reaction to release reactive oxygen species (ROS), increase the expression of inflammatory factors, and activate the ferroptosis signaling pathway [18] . Extensive research has established that inflammatory injury can facilitate tumor cell migration. Following Escherichia coli treatment, we observed increased expression of N-cadherin, Vimentin, Snail, and Slug, along with decreased E-cadherin expression, which promoted the migration of Hela and C-33A cells. Lactobacillus crispatus is the predominant microflora in the genital tract of healthy women and serves as a key indicator reflecting the vaginal microenvironment's health status [19] . Research has demonstrated that Lactobacillus crispatus maintains the vagina's mildly acidic environment through lactic acid secretion and produces antimicrobial substances such as hydrogen peroxide and bacteriocins, effectively inhibiting pathogenic bacterial growth and preventing genital infections [20,21] . In this study, we provide the first evidence that Lactobacillus crispatus can inhibit Escherichia coli growth, with its active component being live bacteria. At temperatures of 4°C and 25°C, the bacteriostatic activity of the live strain persists for up to 42 days and 7 days, respectively. Our results indicate that concentrations of Lactobacillus crispatus ≥1/2MIC inhibit Escherichia coli biofilm formation, cellular adhesion, and intracellular soluble protein expression. We further analyzed the bacteriostasis mechanism and found that Lactobacillus crispatus could inhibit the expression levels of Escherichia coli Outer membrane protein A (OmpA), Outer membrane protein C (OmpC) and Lipoprotein p27. In earlier phases of this study, we established that Escherichia coli induces ferroptosis pathways, promoting cervical cancer cell migration. Consequently, we investigated Lactobacillus crispatus's impact on Escherichia coli 's tumorigenic function. We found that Lactobacillus crispatus reverses Escherichia coli -induced ferroptosis in Hela and C-33A cells, significantly reducing GPX4, SLC7A11, and SLC3A2 expression levels while markedly diminishing intracellular ferroptosis fluorescence signals. Furthermore, we discovered that Lactobacillus crispatus targets NF-κB to suppress inflammatory regulatory proteins COX2 and LOX expression, reduce pro-inflammatory cytokines TNF-α, IL-6, and IL-8 levels, and ultimately inhibit Hela and C-33A cell migration. However, this study also has some limitations. All experimental results are based on relevant studies conducted in vitro cell models. In the future, further animal and human experiments are needed to fully verify the migration mechanism of L. crispatus against E. coli-induced cervical cancer. Conclusions Escherichia coli is among the most prevalent bacteria associated with gynecological infections in women. Escherichia coli triggers inflammatory injury in cervical cancer cells via the ferroptosis pathway and enhances cell migration. Lactobacillus crispatus, as the dominant strain of colonization in the female reproductive system, can inhibit the growth of Escherichia coli, reduce cell inflammatory damage and inhibit the migration of cervical cancer cells in vitro. The results of this study provide theoretical basis for the future application of Lactobacillus crispatus as a microecological agent for anti-infection and anti-tumor in clinical practice. Abbreviations glutathione peroxidase 4 (GPX4), solute carrier family 7, member 11 (SLC7A11), solute carrier family 3, member 3 (SLC3A2), tumor necrosis factor-α (TNF-α), Interleukin (IL), potential of hydrogen (pH), minimum inhibitory concentration (MIC), colony forming units (CFU), outer membrane protein A (OmpA), outer membrane protein C (OmpC), nuclear factor kappa-B (NF-κB), cyclooxygenase-2 (COX2), lectin-type oxidized LDL receptor 1(LOX), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), phosphate-buffered saline (PBS). Declarations Ethics approval and consent to participate This study was conducted at the School of Life Sciences and Health at Northeastern University in accordance with the Declaration of Helsinki and approved by the local Council of Governance (no. NEU-EC-2023A079S). Consent for publication Not applicable. Availability of data and materials All data included in this study are available on request from the corresponding author. Clinical trial number Not applicable Competing interests The authors declare that they have no competing interests. Funding This work was financially supported by Key Research and Development Project of Liaoning Province (Grant No.2024JH2/102500019), Shenyang Science and Technology Plan (Grant No.22-321-33-08), and Shenyang Bureau of Science and Technology (2023 Special Project to Assist China Medical University in high-quality development，Grant No.23-506-3-01-10). Corresponding Author Ye Sun , Email: [email protected] ； Yi Zhang , Email: [email protected] Authors' contributions YZ: Experimental design (lead); funding support (lead). YS: Experimental operation (lead); data analysis (lead); article writing (lead). DT: Experimental design. EL: Experimental operation. HL: Experimental design. FM and XC: Data analysis. D ata availablity All data included in this study are available on request from the corresponding author Acknowledgements The author thanks the Science and Technology Department of Liaoning Province and Science and Technology Bureau of Shenyang City for funding this study. Authors' information Teacher of Shenyang Medical College Doctor of the First Affiliated Hospital of China Medical University Informed consent —Written informed consent was obtained from individual or guardian participants. References de Sire A, de Sire R, Curci C, et al. Role of Dietary Supplements and Probiotics in Modulating Microbiota and Bone Health: The Gut-Bone Axis. Cells. 2022 Feb 21;11(4):743. Shen L, Zhang W, Yuan Y, et al. Vaginal microecological characteristics of women in different physiological and pathological period. Front Cell Infect Microbiol. 2022 Jul 22;12:959793. Salmanov AG, Kostikov VV, Lytvak O, et al. POSTOPERATIVE INFECTIONS AFTER GYNECOLOGICAL SURGERIES IN UKRAINE. 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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-6435097","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":448121431,"identity":"f7d4cb7f-ae18-44f5-9fc6-624d73f8622a","order_by":0,"name":"Dongli Tian","email":"","orcid":"","institution":"the First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dongli","middleName":"","lastName":"Tian","suffix":""},{"id":448121432,"identity":"83063d38-b870-43ef-be6e-6afd832da698","order_by":1,"name":"Lei Deng","email":"","orcid":"","institution":"the First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Deng","suffix":""},{"id":448121438,"identity":"104889b6-71d0-4d40-a108-d0e9cbe8e1fe","order_by":2,"name":"Enting Lu","email":"","orcid":"","institution":"the First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Enting","middleName":"","lastName":"Lu","suffix":""},{"id":448121439,"identity":"0ba863af-81c2-49de-84bf-94db4343e298","order_by":3,"name":"Huayang Li","email":"","orcid":"","institution":"the First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Huayang","middleName":"","lastName":"Li","suffix":""},{"id":448121441,"identity":"a222e99b-7fba-454f-a407-570fa989e273","order_by":4,"name":"Fanyi Meng","email":"","orcid":"","institution":"the First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fanyi","middleName":"","lastName":"Meng","suffix":""},{"id":448121442,"identity":"0038d195-92ed-4a6d-8166-49a918222500","order_by":5,"name":"Xinyang Chen","email":"","orcid":"","institution":"the First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinyang","middleName":"","lastName":"Chen","suffix":""},{"id":448121443,"identity":"a8c232f8-a562-459e-898e-62f7cf7b19d0","order_by":6,"name":"Yi Zhang","email":"","orcid":"","institution":"the First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Zhang","suffix":""},{"id":448121444,"identity":"6747880e-4f7d-4f5e-9e6d-462a3e728792","order_by":7,"name":"Ye Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYNCCCijNQ7yWMyRrYWwjRYv8jNxjEh/n1SWubT/A+OBtG4O8OSEtBjfy0iRnbjucuO1MArPh3DYGw50NhLRI5JhJ8247kLjtBgObNG8bQ4LBAYIOA2r5O6cOpIX9N1FaGG4AtTA2MINtYSZKi8GZN8aWPccOG287k9gsOeechOEGgg5rzzG88aOmTnbb8cMHP7wps5En7DAGBhYJIOHYwMDYAKQlCKsHAuYPQMKeKKWjYBSMglEwMgEA52pAhTH27lwAAAAASUVORK5CYII=","orcid":"","institution":"Shenyang Medical College","correspondingAuthor":true,"prefix":"","firstName":"Ye","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2025-04-12 14:23:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6435097/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6435097/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81549829,"identity":"6d7bd7ae-f2a6-4ac4-ab31-fab5df3bc70e","added_by":"auto","created_at":"2025-04-28 12:37:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":249742,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEscherichia\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e coli induces inflammatory damage through ferroptosis pathway in cervical cancer cells.\u003c/strong\u003e (A) Detection of ferroptosis in Hela and C-33A cells by \u003cem\u003eEscherichia\u003c/em\u003e coli by fluorescence assay. (B) The expression of ferroptosis related protein in Hela and C-33A cells was detected by Western blot. (C) The effects of \u003cem\u003eEscherichia\u003c/em\u003e coli on inflammatory factors of Hela and C-33A cells of cervical cancer were detected by Elisa. ns, \u003cem\u003ep\u003c/em\u003e\u0026gt;0.05; * # ^, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; ** ## ^^, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; *** ### ^^^, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001; Full-length gels are presented in supplementary figure S1.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6435097/v1/a2b6b8250496f4e542027061.png"},{"id":81549825,"identity":"6777636e-ac4a-46f5-b7b5-7b7b9b039a80","added_by":"auto","created_at":"2025-04-28 12:37:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":165817,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEscherichia\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e coli promotes the migration of cervical cancer Hela cells and C-33A cells.\u003c/strong\u003e (A) The effect of \u003cem\u003eEscherichia\u003c/em\u003ecoli on the migration of cervical cancer Hela cells and C-33A cells was detected by scratch assay. (B) The effect of \u003cem\u003eEscherichia\u003c/em\u003e coli on the expression of migration-related proteins in Hela cells and C-33A cells of cervical cancer was detected by Western blot. * #, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; ** ##, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01. Full-length gels are presented in supplementary figure S2.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6435097/v1/3fc4fb54b7210bd63c8c4019.png"},{"id":81549826,"identity":"fbfad0dd-68c9-442e-a58e-50f8de5d2c9a","added_by":"auto","created_at":"2025-04-28 12:37:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":192947,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLive \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLactobacillus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e crispatus could inhibit the growth of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEscherichia\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ecoli.\u003c/strong\u003e (A) The active components of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus inhibiting the growth of \u003cem\u003eEscherichia\u003c/em\u003ecoli were detected by disk dispersion method. (B) Effect of culture medium pH on inhibition of \u003cem\u003eEscherichia\u003c/em\u003e coli growth by \u003cem\u003eLactobacillus\u003c/em\u003ecrispatus. (C) Effect of incubation temperature on the inhibition of \u003cem\u003eEscherichia\u003c/em\u003ecoli growth by \u003cem\u003eLactobacillus\u003c/em\u003e crispatus. (D) Effect of incubation time on the inhibition of \u003cem\u003eEscherichia\u003c/em\u003e coli growth by \u003cem\u003eLactobacillus\u003c/em\u003ecrispatus. (E) Effect of storage conditions on inhibition of \u003cem\u003eEscherichia\u003c/em\u003ecoli growth by \u003cem\u003eLactobacillus\u003c/em\u003e crispatus. (F) The minimum inhibitory concentration (MIC) of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus to inhibit the growth of \u003cem\u003eEscherichia\u003c/em\u003ecoli was determined by double dilution method. a, whole culture medium; b, culture medium supernatant; c, living bacteria; d, inactivate bacteria; e, DMSO.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6435097/v1/1502177c9477c2e84c9d4ad6.png"},{"id":81549827,"identity":"81fed0cd-0a08-4ac0-af17-32a711fe2d00","added_by":"auto","created_at":"2025-04-28 12:37:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":195511,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibitory mechanism analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLactobacillus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e crispatus against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEscherichia\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ecoli. \u003c/strong\u003e(A) Effects of different concentrations of \u003cem\u003eLactobacillus\u003c/em\u003ecrispatus on \u003cem\u003eEscherichia\u003c/em\u003e coli biofilms. (B) Effect of different concentrations of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus on the adhesion of \u003cem\u003eEscherichia\u003c/em\u003ecoli. (C) Effects of different concentrations of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus on the levels of extracellular soluble protein secreted by \u003cem\u003eEscherichia\u003c/em\u003ecoli. (D) The effect of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus on the expression of key regulatory proteins in \u003cem\u003eEscherichia\u003c/em\u003e coli biofilm and adhesion was detected by western blot. ns, \u003cem\u003ep\u003c/em\u003e\u0026gt;0.05; * #, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; ** ##, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01. Full-length gels are presented in supplementary figure S3.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6435097/v1/0b6607145242a4fb3b3e9db6.png"},{"id":81549845,"identity":"8402181c-4bd0-4f06-9e07-54292c617d61","added_by":"auto","created_at":"2025-04-28 12:37:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":341938,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eLactobacillus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e reverses the inflammatory injury of cervical cancer cells induced by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEscherichia\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e coli through ferroptosis pathway.\u003c/strong\u003e (A) The effect of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus at different concentrations on the ferroptosis of cervical cancer cells induced by \u003cem\u003eEscherichia\u003c/em\u003e coli was detected by fluorescence method. (B) The effect of \u003cem\u003eLactobacillus\u003c/em\u003ecrispatus at different concentrations on the expression of ferroptosis related proteins in cervical cancer cells induced by \u003cem\u003eEscherichia\u003c/em\u003e coli were detected by Western blot. (C) The effect of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus at different concentrations on the expression of proteins related to the regulation of inflammation in cervical cancer cells induced by \u003cem\u003eEscherichia\u003c/em\u003ecoli was detected by Western blot. The effect of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus at different concentrations on the expression of inflammatory factors TNF-α (D)、IL-6 (E)、 IL-6 (F) in cervical cancer cells induced by \u003cem\u003eEscherichia\u003c/em\u003e coli was detected by Elisa. ns, \u003cem\u003ep\u003c/em\u003e\u0026gt;0.05; * #, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; ** ##, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01. Full-length gels are presented in supplementary figure S4 and figure S5.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6435097/v1/5902d0f415937a8e55eb4705.png"},{"id":81549841,"identity":"ba9a1000-46d4-4cad-a82d-58283a5ede54","added_by":"auto","created_at":"2025-04-28 12:37:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":258997,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eLactobacillus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ecrispatus inhibits the migration of cervical cancer cells induced by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEscherichia\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ecoli.\u003c/strong\u003e (A) The effect of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus at different concentrations on the migration of cervical cancer cells induced by \u003cem\u003eEscherichia\u003c/em\u003e coli was detected by scratch method. (B) The effect of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus at different concentrations on the expression of migration-related proteins in cervical cancer cells induced by \u003cem\u003eEscherichia\u003c/em\u003e coli was detected by Western blot. ns, \u003cem\u003ep\u003c/em\u003e\u0026gt;0.05; * #, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; ** ##, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01. Full-length gels are presented in supplementary figure S6.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6435097/v1/49a2f56801066d4634064cff.png"},{"id":87882241,"identity":"9e92a4dc-f702-4c93-acbe-3e8fb24bbc6b","added_by":"auto","created_at":"2025-07-30 04:31:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2653735,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6435097/v1/bb66b73f-da17-4f19-bf9f-19252bb6f649.pdf"},{"id":81549832,"identity":"f341a133-869d-4d0d-af4b-49b4b5fdf074","added_by":"auto","created_at":"2025-04-28 12:37:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1964139,"visible":true,"origin":"","legend":"","description":"","filename":"fulluncroppedGelsandBlotsimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6435097/v1/ccdceedc663f23669c3abd37.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lactobacillus crispatus reverses Escherichia coli-induced inflammatory injury and migration of cervical cancer cells by inhibiting ferroptosis","fulltext":[{"header":"Background","content":"\u003cp\u003eThere exists a diverse array of microbial communities within the female vagina, collectively maintaining the balance and stability of vaginal microecology\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. However, any disruption to this equilibrium may lead to gynecological diseases, including inflammation and tumors. \u003cem\u003eEscherichia\u003c/em\u003e coli exhibits specific distribution patterns in the female reproductive system, frequently serving as a common pathogen in gynecological infections. Research has demonstrated that among women of childbearing age, factors such as sexual activity, pregnancy, childbirth, and others may impair vaginal microecology, thereby increasing the susceptibility to \u003cem\u003eEscherichia\u003c/em\u003e coli infection\u003csup\u003e[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Furthermore, postmenopausal women are at heightened risk for \u003cem\u003eEscherichia\u003c/em\u003e coli infection due to decreased estrogen levels, vaginal mucosal atrophy, and alterations in the microecological environment. \u003cem\u003eEscherichia\u003c/em\u003e coli infection can elicit an inflammatory response, releasing various cytokines and inflammatory mediators, such as interleukin and tumor necrosis factor. These substances may promote cell proliferation, inhibit apoptosis, and create favorable conditions for the growth and survival of tumor cells. As a critical anatomical structure connecting the vagina and uterus, the cervix is also a region prone to colonization by \u003cem\u003eEscherichia\u003c/em\u003e coli, potentially leading to cervical epithelial damage, inflammation, and even carcinogenesis\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLactobacillus\u003c/em\u003e crispatus serves as the dominant strain in the female vagina, playing a pivotal role in preserving the balance of vaginal microecology\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. It maintains an acidic environment in the vagina, inhibits the proliferation of harmful bacteria, and produces hydrogen peroxide to suppress heterobacteria, which is associated with reduced levels of inflammation\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Currently, the mechanism by which \u003cem\u003eLactobacillus\u003c/em\u003e crispatus mitigates cervical cancer through the inhibition of \u003cem\u003eEscherichia\u003c/em\u003e coli remains unclear. The objective of this study is to further investigate the role of \u003cem\u003eEscherichia\u003c/em\u003e coli in the onset of cervical cancer, which holds significant implications for enhancing early prevention, improving treatment efficacy, and reducing mortality rates. Additionally, the application of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus-based microecological therapy to control the occurrence and progression of cervical cancer carries substantial practical significance.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains and cell lines and their cultures\u003c/h2\u003e \u003cp\u003eThe freeze-dried powder of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus CGMCC1.2743 was obtained from the China General Microbiological Culture Collection Center (CGMCC). An appropriate volume of sterile water was added to reconstitute the lyophilized powder, which was subsequently inoculated into MRS liquid medium(Solarbio, Beijing, China). The culture was maintained under controlled conditions at 37 ℃ with a shaking speed of 150 rpm for 18 hours. Following successful growth of the strain, an inoculation loop was used to transfer the culture onto MRS solid medium, which was then incubated at 37 ℃ for 18 to 24 hours. \u003cem\u003eEscherichia\u003c/em\u003e coli ATCC25922 originates from the Laboratory of Shenyang Medical University Affiliated Central Hospital and is cultured in broth medium (Solarbio, Beijing, China) under conditions at 37 ℃, 180 r/min, for 18 hours.\u003c/p\u003e \u003cp\u003eHuman cervical cancer cell lines Hela and C-33A were obtained from Beijing Dingguo Changsheng Biotechnology Co., Ltd. Both Hela and C-33A cells were cultured in DMEM (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), along with 100 units/mL of penicillin (Genview, Australia) and 100 units/mL of streptomycin solution (Genview, Australia). The cultures were maintained at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAntibodies\u003c/h3\u003e\n\u003cp\u003eThe primary antibodies include mouse monoclonal anti-NF-κB p65 (1:3000, Cat No. ab288751), rabbit polyclonal anti-LOX (1:5000, Cat No. ab174316), rabbit monoclonal anti-COX2 (1:3000, Cat No. ab179800), rabbit monoclonal anti-E-cadherin (1:6000, Cat No. ab314063), rabbit monoclonal anti-N-cadherin (1:6000, Cat No. ab76011), rabbit monoclonal anti-Vimentin (1:3000, Cat No. ab92547), rabbit monoclonal anti-Snail (1:1000, Cat No. ab216347), rabbit monoclonal anti-Slug (1:1000, Cat No. ab314086) were obtained from Abcam Biomedical Company (Cambridge, UK). The primary antibodies include mouse monoclonal anti-GPX4 (1:3000, Cat No. 67763-1-Ig), rabbit polyclonal anti-SLC7A11 (1:1000, Cat No. 26864-1-AP), rabbit polyclonal anti-SLC3A2 (1:20000, Cat No. 15193-1-AP) were obtained from Proteintech Company (Wuhan, Hubei, China). The primary antibodies include rabbit polyclonal anti-ompA (1:2000, Cat No. PA5-117575), rabbit polyclonal anti-ompC (1:2000, Cat No. PA5-117701), rabbit polyclonal anti-Lipoprotein p27 (1:1000, Cat No. 200-401-C30), were obtained from Thermo Fisher Scientific Company (Massachusetts, USA). HRP-Goat anti-rabbit recombinant secondary antibody (H\u0026thinsp;+\u0026thinsp;L) and HRP-Goat anti-mouse recombinant secondary antibody (H\u0026thinsp;+\u0026thinsp;L) were obtained from Proteintech Company (Wuhan, Hubei, China).\u003c/p\u003e\n\u003ch3\u003eFerroptosis determination\u003c/h3\u003e\n\u003cp\u003eFerroptosis was detected using fluorescence microscopy. The procedure involved adding 200 \u0026micro;L of Hela and C-33A cells (at a concentration of 2\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/mL) to each well of a 24-well plate, followed by incubation for 18 hours at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Subsequently, the culture medium was removed, and the cells were treated for 4 hours with 200 \u0026micro;L of MDEM medium containing \u003cem\u003eEscherichia\u003c/em\u003e coli or MDEM medium containing \u003cem\u003eEscherichia\u003c/em\u003e coli plus \u003cem\u003eLactobacillus\u003c/em\u003e crispatus. Afterward, the medium was replaced with 500 \u0026micro;L of DMEM supplemented with ferric ammonium sulfate (final concentration of 100 \u0026micro;mol/L), and the cells were incubated for 30 minutes in an incubator. Following this step, the cells were washed with PBS, fixed with 4% paraformaldehyde for 30 minutes at room temperature, and permeabilized with 1% Triton X-100 for 20 minutes. After washing again with PBS, the cells were incubated with 500 \u0026micro;L of Iron Green solution for 30 minutes. Finally, the intensity of the fluorescence signals was quantified using GFP and BF filters under a fluorescence microscope.\u003c/p\u003e\n\u003ch3\u003eCell scratch assay\u003c/h3\u003e\n\u003cp\u003eBriefly, 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well Hela cells and C-33A were seeded in 6-well plates. After 18 h of adherence, they were scratched with a 200-\u0026micro;L pipette tip. Subsequently, the cells were treated with 200 \u0026micro;L of MDEM medium containing \u003cem\u003eEscherichia\u003c/em\u003e coli or MDEM medium containing \u003cem\u003eEscherichia\u003c/em\u003e coli plus \u003cem\u003eLactobacillus\u003c/em\u003e crispatus, cultured them for 24 h. The experiment was repeated three times, and the cell scratch area was calculated using ImageJ software.\u003c/p\u003e\n\u003ch3\u003eBacteriostatic active ingredient detection\u003c/h3\u003e\n\u003cp\u003eThe concentration of \u003cem\u003eEscherichia\u003c/em\u003e coli during the logarithmic growth phase was adjusted to 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mL. A volume of 100 \u0026micro;L was then taken and evenly spread onto MRS solid medium. The \u003cem\u003eLactobacillus\u003c/em\u003e crispatus bacterial solution cultured for logarithmic period was divided into 4 groups, whole bacterial solution, supernatant liquid, live bacteria, and inactivated bacteria. Using the disk diffusion method, 200 \u0026micro;L of each tested sample was added to a disk and incubated at 37\u0026deg;C for 18 hours. The inhibition diameter was measured using a vernier caliper.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMinimum inhibitory concentration (MIC) determination\u003c/h2\u003e \u003cp\u003eThe concentration of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus was adjusted to 1\u0026times;10\u003csup\u003e2\u003c/sup\u003e CFU/mL, 1\u0026times;10\u003csup\u003e3\u003c/sup\u003e CFU/mL, 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e CFU/mL, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e CFU/mL, 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mL, and 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e CFU/mL using MRS medium. Subsequently, 100 \u0026micro;L of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus bacterial suspensions at varying concentrations were added to a 96-well plate, followed by the addition of 10 \u0026micro;L of \u003cem\u003eEscherichia\u003c/em\u003e coli suspension (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mL) into each well. The plates were then incubated for 18 hours at 37\u0026deg;C in a bacterial incubator. After incubation, the minimum drug concentration that inhibited visible bacterial growth was determined using the plate viable counting method, which corresponds to the MIC of the bacteria against the tested drugs.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExtracellular soluble protein detection\u003c/h3\u003e\n\u003cp\u003e200 \u0026micro;L of \u003cem\u003eEscherichia\u003c/em\u003e coli suspension (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mL) was added to a 48-well plate. Subsequently, 200 \u0026micro;L of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus suspension at final concentrations of 0 MIC, 1/4 MIC,1/2 MIC, and MIC were added to the respective wells. A negative control consisting of broth medium without bacteria was included. The \u003cem\u003eEscherichia\u003c/em\u003e coli suspension from each group was centrifuged at 12,000 rpm for 20 minutes at 4\u0026deg;C to collect the bacterial pellet. The supernatant was discarded, and the cells were washed twice with pre-cooled phosphate-buffered saline (PBS). Following centrifugation at the same conditions, the supernatant was removed, and the bacteria were re-suspended in PBS. Subsequently, the bacterial samples were treated in a metal bath at 100\u0026deg;C for 15 minutes, with inversion every 2 minutes to ensure uniform release of soluble proteins from lysed cells. Protein samples (20 \u0026micro;L) were subjected to SDS-PAGE electrophoresis, followed by staining with Coomassie Brilliant Blue (Beyotime Biotechnology, Shanghai, China) for 40 minutes. The gel was then decolorized using distilled water and photographed for analysis of protein expression.\u003c/p\u003e\n\u003ch3\u003eBiofilm formation determination\u003c/h3\u003e\n\u003cp\u003e200 \u0026micro;L of \u003cem\u003eEscherichia\u003c/em\u003e coli suspension (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mL) was added to a 48-well plate. Subsequently, 200 \u0026micro;L of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus suspension at final concentrations of 0 MIC, 1/4 MIC,1/2 MIC, and MIC were added to the respective wells. A negative control consisting of broth medium without bacteria was included. The samples were incubated for 18 hours at 37\u0026deg;C. After incubation, the medium and non-adherent bacteria were removed, and the wells were washed with PBS buffer. The adherent bacteria were fixed with methanol for 20 minutes, stained with a 2% crystal violet solution for 15 minutes, and decolorized with 33% glacial acetic acid. Finally, the absorbance was measured at 630 nm.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAdhesion ability determination\u003c/h2\u003e \u003cp\u003e500 \u0026micro;L of Hela and C-33A cells, suspended at a concentration of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL, were seeded into 6-well plates and incubated overnight under standard cell culture conditions (37\u0026deg;C, 5% CO₂). Subsequently, the cells were cultured in DMEM supplemented with serum for 3 days, followed by serum-free DMEM for 12 hours. After removing the culture medium and washing the wells twice with PBS, the following treatments were added to each well: 1 mL of \u003cem\u003eEscherichia\u003c/em\u003e coli suspension, 1 mL of \u003cem\u003eEscherichia\u003c/em\u003e coli suspension combined with 1/4 MIC \u003cem\u003eLactobacillus\u003c/em\u003e crispatus mixture,1 mL of \u003cem\u003eEscherichia\u003c/em\u003e coli suspension combined with 1/2 MIC \u003cem\u003eLactobacillus\u003c/em\u003e crispatus mixture, 1 mL of \u003cem\u003eEscherichia\u003c/em\u003e coli suspension combined with MIC \u003cem\u003eLactobacillus\u003c/em\u003e crispatus mixture, and 1 mL of DMEM culture medium as a control. The contents were mixed thoroughly and co-incubated for 4 hours. Following incubation, the cells were washed with PBS and fixed with 4% paraformaldehyde for 1 hour. The number of \u003cem\u003eEscherichia\u003c/em\u003e coli adhering to the cell surface was observed and quantified under a microscope. Adhesion levels were categorized based on the following criteria: non-adherent (\u0026lt;\u0026thinsp;40 bacteria), weakly adherent (41\u0026ndash;100 bacteria), and strongly adherent (\u0026gt;\u0026thinsp;100 bacteria).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFermentation broth conditions determination\u003c/h2\u003e \u003cp\u003eA single colony of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus was inoculated into MRS Liquid medium. The pH value ranged from 1 to 12, the temperature ranged from 28 to 43℃, and the culture time ranged from 12 to 42 hours to optimize the culture condition with the best antibacterial effect. In addition, live \u003cem\u003eLactobacillus\u003c/em\u003e crispatus was placed at 4℃ and 25℃ to detect the maintenance days of antibacterial activity of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eInflammatory cytokines detection\u003c/h2\u003e \u003cp\u003eThe levels of inflammatory factors in cells were quantified using the Enzyme-Linked Immunosorbent Assay (ELISA). Hela and C-33A cells, treated with \u003cem\u003eEscherichia\u003c/em\u003e coli or a combination of \u003cem\u003eEscherichia\u003c/em\u003e coli and \u003cem\u003eLactobacillus\u003c/em\u003e crispatus, were centrifuged at 1000g for 20 minutes at 4\u0026deg;C to collect the supernatant. According to the manufacturer's instructions (Mibio, Shanghai, China), standard wells and sample wells were prepared for TNF-α, IL-6, and IL-8 detection. Fifty microliters (50 \u0026micro;L) of standards at varying concentrations were added to the standard wells, while 50 \u0026micro;L of samples were added to the sample wells. Each well was subsequently incubated with 100 \u0026micro;L of horseradish peroxidase (HRP)-conjugated detection antibody for 60 minutes at 37\u0026deg;C. Following this, each well was washed five times with 300 \u0026micro;L of washing buffer. Substrate A and substrate B (50 \u0026micro;L each) were then added to each well and incubated for 15 minutes at 37\u0026deg;C in the dark. Finally, 50 \u0026micro;L of stop solution was added to each well, and the absorbance was measured at a wavelength of 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot\u003c/h2\u003e \u003cp\u003eHela and C-33A cells were treated with \u003cem\u003eEscherichia\u003c/em\u003e coli or a combination of \u003cem\u003eEscherichia\u003c/em\u003e coli and \u003cem\u003eLactobacillus\u003c/em\u003e crispatus. The cells were subsequently lysed using RIPA lysate(Beyotime Biotechnology, Shanghai, China) containing 1mM PMSF(Beyotime Biotechnology, Shanghai, China), and total protein extracts were prepared. Equal quantities of protein samples from each experimental group were resolved by SDS-PAGE at 80 V for 25 min and then at 120 V for an additional 50 min. The separated proteins were transferred onto PVDF membranes (Millipore, Boston, MA, USA) under constant conditions of 15 V and 1.0 A for 18 min. The membranes were extensively washed with TBST, blocked overnight at 4\u0026deg;C with 5% skim milk (Sigma, Louis, MO, USA) in TBST, and incubated with the primary antibody for 2 h at room temperature. This was followed by incubation with the secondary antibody for 1 h under the same conditions. Protein bands were visualized using ECL reagent (Vazyme, Shanghai, China), and the gray values of the protein bands were calculated by Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eEach experiment was conducted a minimum of three times, with the results expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical analysis of all data was performed using SPSS 22.0 software. To compare the cell ferroptosis level, inflammatory factors, migration level with or without \u003cem\u003eEscherichia\u003c/em\u003e coli intervention, a two-tailed Student's t-test was applied. The effects of \u003cem\u003eEscherichia\u003c/em\u003e coli in combination with \u003cem\u003eLactobacillus\u003c/em\u003e crispatus at various concentrations on ferroptosis, inflammatory factors, and migration of human ovarian cancer cells were assessed using one-way ANOVA. Statistical significance was defined as a P value below 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEscherichia\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;coli induces inflammatory injury of cervical cancer cells through ferroptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman cervical cancer cells Hela and C-33A were infected with \u003cem\u003eEscherichia\u003c/em\u003e coli. As shown in Figure 1-A, using fluorescence microscopy, the intracellular ferroptosis fluorescence signal was significantly increased in Hela and C-33A cells infected with \u003cem\u003eEscherichia\u003c/em\u003e coli compared with those not infected with \u003cem\u003eEscherichia\u003c/em\u003e coli. In addition, detection of ferroptosis-related regulatory proteins showed that the expression of GPX4, SLC7A11 and SLC3A2 was significantly decreased in Hela and C-33A cells after \u003cem\u003eEscherichia\u003c/em\u003e coli infection (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) (Figure 1-B). Studies have confirmed that ferroptosis is closely related to inflammatory injury. The results showed that the expression of TNF-\u0026alpha;, IL-6 and IL-8 in Hela and C-33A cells after \u003cem\u003eEscherichia\u003c/em\u003e coli infection was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001) (Figure 1-C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEscherichia\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;coli promotes the migration of cervical cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated the impact of \u003cem\u003eEscherichia\u003c/em\u003e coli on the migratory capacity of cervical cancer Hela and C-33A cells. As shown in Figure 2-A, the migratory ability of cervical cancer Hela and C-33A cells infected with \u003cem\u003eEscherichia\u003c/em\u003e coli was significantly enhanced compared to that of uninfected cells (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). Western blot analysis revealed that the expression levels of adhesion-related proteins N-cadherin, Vimentin, Snail, and Slug were significantly upregulated in \u003cem\u003eEscherichia\u003c/em\u003e coli-infected cervical cancer Hela and C-33A cells relative to uninfected cells (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) (Figure 2-B). Conversely, the expression level of E-cadherin was significantly decreased (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) (Figure 2-B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLactobacillus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;crispatus\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;inhibited the growth of \u003cem\u003eEscherichia\u003c/em\u003e coli\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe divided the cultured \u003cem\u003eLactobacillus\u003c/em\u003e crispatus bacterial solution into four groups: whole bacterial solution, sterile supernatant, active bacterial and inactivated bacterial. As shown in Figure 3-A, the whole solution of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus and the active strain could inhibit the growth of \u003cem\u003eEscherichia\u003c/em\u003e coli, but neither the supernatant nor the inactivated strain could inhibit the growth of \u003cem\u003eEscherichia\u003c/em\u003e coli. The inhibitory diameters of the whole fermentation solution and the active strain against \u003cem\u003eEscherichia\u003c/em\u003e coli were 17.32\u0026plusmn;13mm and 17.87\u0026plusmn;21mm, respectively. We optimized the culture conditions, and the results showed that the best antibacterial effect was obtained at pH3.0-5.0 and 37℃ for 18 hours, and the inhibition diameter was 24.31\u0026plusmn;34 mm (Figure 3-B to D). The antibacterial effect of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus could be maintained for 42 days at 4℃. The antibacterial effect of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus could be maintained for 7 days at 25℃ (Figure 3-E). The minimum inhibitory concentration (MIC) of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus against \u003cem\u003eEscherichia\u003c/em\u003e coli was 1\u0026times;10\u003csup\u003e4\u0026nbsp;\u003c/sup\u003eCFU/mL (Figure 3-F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibitory mechanism analysis of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus against \u003cem\u003eEscherichia\u003c/em\u003e coli\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe selected 4 concentrations of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus live bacteria to analyze the bacteriostatic mechanism, and the 4 concentrations were divided into 0 MIC, 1/4 MIC, 1/2 MIC, and MIC. As shown in Figures 4-A and 4-B, biofilm formation and adhesion of \u003cem\u003eEscherichia\u003c/em\u003e coli were significantly reduced when the \u003cem\u003eLactobacillus\u003c/em\u003e crispatus concentration was \u0026ge;1/2MIC (p\u0026lt;0.05). The protein gel imaging system showed that when the concentration of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus was \u0026ge;1/2MIC, the expression level of soluble protein in \u003cem\u003eEscherichia\u003c/em\u003e coli cells decreased (Figure 4-C). Subsequently, we conducted a more in-depth analysis of the expression of biofilm-related proteins and key regulatory proteins involved in adhesion. The results demonstrated that the expression levels of \u003cem\u003eEscherichia\u003c/em\u003e coli Outer membrane protein A (OmpA), Outer membrane protein C (OmpC), and Lipoprotein p27 could be significantly suppressed when the \u003cem\u003eLactobacillus\u003c/em\u003e crispatus concentration was \u0026ge;1/2MIC (p\u0026lt;0.05) (Figure 4-D) .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLactobacillus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;crispatus reversed the inflammatory injury induced by \u003cem\u003eEscherichia\u003c/em\u003e coli in cervical cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated the impact of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus on the inflammatory response triggered by ferroptosis induced by \u003cem\u003eEscherichia\u003c/em\u003e coli in Hela and C-33A cells through the addition of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus. As shown in Figure 5-A, fluorescence microscopy results demonstrated that when the concentration of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus was \u0026ge;1/2MIC, the intensity of the ferroptosis-related fluorescence signal in \u003cem\u003eEscherichia\u003c/em\u003e coli-infected Hela and C-33A cells was significantly reduced. Additionally, at a concentration of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus \u0026ge;1/2MIC, the expression levels of ferroptosis-associated regulatory proteins GPX4, SLC7A11, and SLC3A2 were significantly decreased (p\u0026lt;0.05). Furthermore, the expression levels of TNF-\u0026alpha;, IL-6, and IL-8 were significantly inhibited under the same conditions (p\u0026lt;0.05). Moreover, when the concentration of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus was \u0026ge;1/2MIC, the expression levels of inflammatory regulatory proteins NF-\u0026kappa;B, COX2, and LOX in Hela and C-33A cells were significantly decreased (p\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLactobacillus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;crispatus inhibits \u003cem\u003eEscherichia\u003c/em\u003e coli-induced migration of cervical cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 6-A, \u003cem\u003eEscherichia\u003c/em\u003e coli infected Hela and C-33A cells were able to significantly inhibit the migration of Hela and C-33A cells when \u003cem\u003eLactobacillus\u003c/em\u003e crispatus was added at a concentration \u0026ge;1/2MIC (p\u0026lt;0.05). In addition, Western blot results showed that the expression levels of migration-related proteins N-cadherin, vimentin, Snail, Slug were significantly decreased (p\u0026lt;0.05), and the expression level of E-cadherin was significantly increased at the concentration of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus \u0026ge;1/2MIC (p\u0026lt;0.05).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCurrently, the global incidence of gynecological diseases among women has surpassed 93%, with approximately 9 million individuals succumbing to these conditions annually. This figure is escalating at an annual rate of 8%\u003csup\u003e[9]\u003c/sup\u003e. In China, gynecological diseases affect 80% of women, with the prevalence reaching as high as 87% among married women. The incidence of gynecological diseases among Chinese women is rising annually by 20% to 30%, indicating a significant upward trend. Common gynecological conditions in Chinese women include cervicitis, pelvic inflammatory disease, and vaginitis. Approximately 60% of women will experience at least one gynecological infection during their lifetime, while the prevalence of such infections among women aged 55 and above reaches 42%, with a recurrence rate as high as 50%\u003csup\u003e[10-12]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEscherichia\u003c/em\u003e coli, a prevalent bacterium in the intestinal and vaginal microbial communities, typically maintains a balanced population and species diversity within the vaginal microbiota of healthy women. However, an abnormal increase in bacterial load or alterations in the composition and structure of the vaginal microecosystem may disrupt this balance, potentially leading to gynecological inflammation and even tumor development\u003csup\u003e[13,14]\u003c/sup\u003e. Recent evidence suggests that \u003cem\u003eEscherichia\u003c/em\u003e coli may serve as a potential risk factor for the onset and progression of various cancers. Colibactin, a genotoxin produced by \u003cem\u003eEscherichia\u003c/em\u003e coli harboring the polyketide synthase (PKS) island, has been shown to induce DNA double-strand breaks, contributing to colon cancer progression\u003csup\u003e[15]\u003c/sup\u003e. Studies have demonstrated that cathepsin K (CTSK), a protein secreted by colon cancer cells stimulated by \u003cem\u003eEscherichia\u003c/em\u003e coli-derived lipopolysaccharide (LPS), can bind to Toll-like receptor 4 (TLR4) via the mTOR signaling pathway, thereby promoting M2 polarization of tumor-associated macrophages (TAMs) and enhancing the migration and motility of colon cancer cells\u003csup\u003e[16]\u003c/sup\u003e. Additionally, reports indicate a significantly higher detection rate of \u003cem\u003eEscherichia\u003c/em\u003e coli in patients with HPV16-positive cervical cancer. \u003cem\u003eEscherichia\u003c/em\u003e coli induces upregulation of Foxp3 and promotes IL-10 expression in cervical cancer, exacerbating cervical intraepithelial neoplasia and enhancing the adhesion and invasion capabilities of cervical cancer cells\u003csup\u003e[17]\u003c/sup\u003e. In this study, we investigated the effects of \u003cem\u003eEscherichia coli\u003c/em\u003e on cervical cancer cells in vitro. Our findings revealed significant increases in the expression levels of GPX4, SLC7A11, and SLC3A2 in Hela and C-33A cells following \u003cem\u003eEscherichia coli\u003c/em\u003e treatment, accompanied by enhanced intracellular ferroptosis fluorescence signals. We also assessed the expression of TNF-\u0026alpha;, IL-6, and IL-8 in Hela and C-33A cervical cancer cells post-treatment with \u003cem\u003eEscherichia coli\u003c/em\u003e. Our results are similar to those of other research results. Yang et al. demonstrated that \u003cem\u003eEscherichia coli\u003c/em\u003e could increase the iron content in teleost erythrocytes, induce the Fenton reaction to release reactive oxygen species (ROS), increase the expression of inflammatory factors, and activate the ferroptosis signaling pathway\u003csup\u003e[18]\u003c/sup\u003e. Extensive research has established that inflammatory injury can facilitate tumor cell migration. Following \u003cem\u003eEscherichia coli\u003c/em\u003e treatment, we observed increased expression of N-cadherin, Vimentin, Snail, and Slug, along with decreased E-cadherin expression, which promoted the migration of Hela and C-33A cells.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLactobacillus\u003c/em\u003e crispatus is the predominant microflora in the genital tract of healthy women and serves as a key indicator reflecting the vaginal microenvironment\u0026apos;s health status\u003csup\u003e[19]\u003c/sup\u003e. Research has demonstrated that \u003cem\u003eLactobacillus\u003c/em\u003e crispatus maintains the vagina\u0026apos;s mildly acidic environment through lactic acid secretion and produces antimicrobial substances such as hydrogen peroxide and bacteriocins, effectively inhibiting pathogenic bacterial growth and preventing genital infections\u003csup\u003e[20,21]\u003c/sup\u003e. In this study, we provide the first evidence that \u003cem\u003eLactobacillus\u003c/em\u003e crispatus can inhibit \u003cem\u003eEscherichia\u003c/em\u003e coli growth, with its active component being live bacteria. At temperatures of 4\u0026deg;C and 25\u0026deg;C, the bacteriostatic activity of the live strain persists for up to 42 days and 7 days, respectively. Our results indicate that concentrations of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus \u0026ge;1/2MIC inhibit \u003cem\u003eEscherichia coli\u003c/em\u003e biofilm formation, cellular adhesion, and intracellular soluble protein expression. We further analyzed the bacteriostasis mechanism and found that \u003cem\u003eLactobacillus\u003c/em\u003e crispatus could inhibit the expression levels of \u003cem\u003eEscherichia coli\u003c/em\u003e Outer membrane protein A (OmpA), Outer membrane protein C (OmpC) and Lipoprotein p27. In earlier phases of this study, we established that \u003cem\u003eEscherichia coli\u003c/em\u003e induces ferroptosis pathways, promoting cervical cancer cell migration. Consequently, we investigated \u003cem\u003eLactobacillus\u003c/em\u003e crispatus\u0026apos;s impact on \u003cem\u003eEscherichia coli\u003c/em\u003e\u0026apos;s tumorigenic function. We found that \u003cem\u003eLactobacillus\u003c/em\u003e crispatus reverses \u003cem\u003eEscherichia coli\u003c/em\u003e-induced ferroptosis in Hela and C-33A cells, significantly reducing GPX4, SLC7A11, and SLC3A2 expression levels while markedly diminishing intracellular ferroptosis fluorescence signals. Furthermore, we discovered that \u003cem\u003eLactobacillus\u003c/em\u003e crispatus targets NF-\u0026kappa;B to suppress inflammatory regulatory proteins COX2 and LOX expression, reduce pro-inflammatory cytokines TNF-\u0026alpha;, IL-6, and IL-8 levels, and ultimately inhibit Hela and C-33A cell migration. However, this study also has some limitations. All experimental results are based on relevant studies conducted in vitro cell models. In the future, further animal and human experiments are needed to fully verify the migration mechanism of L. crispatus against E. coli-induced cervical cancer.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e\u003cem\u003eEscherichia\u003c/em\u003e coli is among the most prevalent bacteria associated with gynecological infections in women. \u003cem\u003eEscherichia\u003c/em\u003e coli triggers inflammatory injury in cervical cancer cells via the ferroptosis pathway and enhances cell migration. \u003cem\u003eLactobacillus\u003c/em\u003e crispatus, as the dominant strain of colonization in the female reproductive system, can inhibit the growth of \u003cem\u003eEscherichia\u003c/em\u003e coli, reduce cell inflammatory damage and inhibit the migration of cervical cancer cells in vitro. The results of this study provide theoretical basis for the future application of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus as a microecological agent for anti-infection and anti-tumor in clinical practice.\u003c/p\u003e\n"},{"header":"Abbreviations","content":"\u003cp\u003eglutathione peroxidase 4 (GPX4), solute carrier family 7, member 11 (SLC7A11), solute carrier family 3, member 3 (SLC3A2), tumor necrosis factor-\u0026alpha; (TNF-\u0026alpha;), Interleukin (IL), potential of hydrogen (pH), minimum inhibitory concentration (MIC), colony forming units (CFU), outer membrane protein A (OmpA), outer membrane protein C (OmpC), nuclear factor kappa-B (NF-\u0026kappa;B), cyclooxygenase-2 (COX2), lectin-type oxidized LDL receptor 1(LOX), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), phosphate-buffered saline (PBS).\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted at the School of Life Sciences and Health at Northeastern University in accordance with the Declaration of Helsinki and approved by the local Council of Governance (no. NEU-EC-2023A079S).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data included in this study are available on request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by Key Research and Development Project of Liaoning Province (Grant No.2024JH2/102500019), Shenyang Science and Technology Plan (Grant No.22-321-33-08), and Shenyang Bureau of Science and Technology (2023 Special Project to Assist China Medical University in high-quality development，Grant No.23-506-3-01-10).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding Author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYe Sun\u003c/strong\u003e, Email: [email protected]；\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYi Zhang\u003c/strong\u003e, Email: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYZ: Experimental design (lead); funding support (lead).\u003c/p\u003e\n\u003cp\u003eYS: Experimental operation (lead); data analysis (lead); article writing (lead).\u003c/p\u003e\n\u003cp\u003eDT: Experimental design.\u003c/p\u003e\n\u003cp\u003eEL: Experimental operation.\u003c/p\u003e\n\u003cp\u003eHL: Experimental design.\u003c/p\u003e\n\u003cp\u003eFM and XC: Data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cstrong\u003eata availablity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data included in this study are available on request from the corresponding author\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author thanks the Science and Technology Department of Liaoning Province and Science and Technology Bureau of Shenyang City for funding this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTeacher of Shenyang Medical College\u003c/p\u003e\n\u003cp\u003eDoctor of the First Affiliated Hospital of China Medical University\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u003c/strong\u003e\u0026mdash;Written informed consent was obtained from individual or guardian participants.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ede Sire A, de Sire R, Curci C, et al. Role of Dietary Supplements and Probiotics in Modulating Microbiota and Bone Health: The Gut-Bone Axis. Cells. 2022 Feb 21;11(4):743. \u003c/li\u003e\n\u003cli\u003eShen L, Zhang W, Yuan Y, et al. Vaginal microecological characteristics of women in different physiological and pathological period. Front Cell Infect Microbiol. 2022 Jul 22;12:959793. \u003c/li\u003e\n\u003cli\u003eSalmanov AG, Kostikov VV, Lytvak O, et al. POSTOPERATIVE INFECTIONS AFTER GYNECOLOGICAL SURGERIES IN UKRAINE. Pol Merkur Lekarski. 2023;51(4):299-305. \u003c/li\u003e\n\u003cli\u003eXiao R, Li Y, Liu X, et al. Antibiotic susceptibility of Escherichia coli isolated from neonates admitted to neonatal intensive care units across China from 2015 to 2020. Front Cell Infect Microbiol. 2023 May 22;13:1183736. \u003c/li\u003e\n\u003cli\u003eJang S, Jeon M, Mun SJ, et al. Clinical characteristics and risk factors for septic shock in patients with pyometra: A retrospective multicenter cohort study. J Infect Public Health. 2024 May;17(5):862-867. \u003c/li\u003e\n\u003cli\u003eLopez LR, Bleich RM, Arthur JC. Microbiota Effects on Carcinogenesis: Initiation, Promotion, and Progression. Annu Rev Med. 2021 Jan 27;72:243-261.\u003c/li\u003e\n\u003cli\u003eGarza J, Gandhi K, Choi S, et al. Cytokine profiles and Lactobacillus species presence in pre-menopausal subjects with genital Mycoplasma genitalium or Ureaplasma urealyticum colonization. Womens Health (Lond). 2021 Jan-Dec;17:17455065211009181.\u003c/li\u003e\n\u003cli\u003eDricot CEMK, Erreygers I, Cauwenberghs E, et al. Riboflavin for women\u0026apos;s health and emerging microbiome strategies. NPJ Biofilms Microbiomes. 2024 Oct 18;10(1):107.\u003c/li\u003e\n\u003cli\u003eCao Y, Guo Y, Long Z, et al. The Global Burden of Gynecological Diseases from 1990 to 2019. Am J Prev Med. 2024 Nov;67(5):698-704. \u003c/li\u003e\n\u003cli\u003eSun P, Yu C, Yin L,et al. Global, regional, and national burden of female cancers in women of child-bearing age, 1990-2021: analysis of data from the global burden of disease study 2021. EClinicalMedicine. 2024 Jul 2;74:102713. \u003c/li\u003e\n\u003cli\u003eKocarnik JM, Compton K, Dean FE, et al . Cancer Incidence, Mortality, Years of Life Lost, Years Lived With Disability, and Disability-Adjusted Life Years for 29 Cancer Groups From 2010 to 2019: A Systematic Analysis for the Global Burden of Disease Study 2019. JAMA Oncol. 2022 Mar 1;8(3):420-444.\u003c/li\u003e\n\u003cli\u003eFarid Mojtahedi M, Sepidarkish M, Almukhtar M, et al. Global incidence of surgical site infections following caesarean section: a systematic review and meta-analysis. J Hosp Infect. 2023 Sep;139:82-92.\u003c/li\u003e\n\u003cli\u003eTimm MR, Russell SK, Hultgren SJ. Urinary tract infections: pathogenesis, host susceptibility and emerging therapeutics. Nat Rev Microbiol. 2025 Feb;23(2):72-86. \u003c/li\u003e\n\u003cli\u003eFarid Mojtahedi M, Sepidarkish M, Almukhtar M, et al. Global incidence of surgical site infections following caesarean section: a systematic review and meta-analysis. J Hosp Infect. 2023 Sep;139:82-92. \u003c/li\u003e\n\u003cli\u003eOliero M, Calv\u0026eacute; A, Fragoso G, et al. Oligosaccharides increase the genotoxic effect of colibactin produced by pks+ \u003cem\u003eEscherichia\u003c/em\u003e coli strains. BMC Cancer. 2021 Feb 17;21(1):172. \u003c/li\u003e\n\u003cli\u003eLi R, Zhou R, Wang H, et al. Gut microbiota-stimulated cathepsin K secretion mediates TLR4-dependent M2 macrophage polarization and promotes tumor metastasis in colorectal cancer. Cell Death Differ. 2019 Nov;26(11):2447-2463. \u003c/li\u003e\n\u003cli\u003eZou Q, Wu Y, Zhang S, et al. \u003cem\u003eEscherichia\u003c/em\u003e coli and HPV16 coinfection may contribute to the development of cervical cancer. Virulence. 2024 Dec;15(1):2319962.\u003c/li\u003e\n\u003cli\u003eYang M, Lu Z, Li F, et al. \u003cem\u003eEscherichia\u003c/em\u003e coli induced ferroptosis in red blood cells of grass carp (Ctenopharyngodon idella). Fish Shellfish Immunol. 2021 May;112:159-167.\u003c/li\u003e\n\u003cli\u003eVitale SG, Ferrari F, Ciebiera M, et al. The Role of Genital Tract Microbiome in Fertility: A Systematic Review. Int J Mol Sci. 2021 Dec 24;23(1):180. \u003c/li\u003e\n\u003cli\u003eBreshears LM, Edwards VL, Ravel J, et al. Lactobacillus crispatus inhibits growth of Gardnerella vaginalis and Neisseria gonorrhoeae on a porcine vaginal mucosa model. BMC Microbiol. 2015 Dec 9;15:276.\u003c/li\u003e\n\u003cli\u003eDing C, Wu H, Cao X, et al. Lactobacillus crispatus-derived exopolysaccharides with antibacterial activity limit Salmonella typhimurium invasion by inhibiting inflammasome-mediated pyroptosis. Food Funct. 2022 Oct 17;13(20):10501-10515. \u003c/li\u003e\n\u003c/ol\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lactobacillus crispatus, Escherichia coli, ferroptosis, Cervical cancer, Inflammatory injury, inflammation, Migration","lastPublishedDoi":"10.21203/rs.3.rs-6435097/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6435097/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIn the post-antibiotic era, there is growing recognition of the critical role that microecological balance plays in human health. Extensive research has demonstrated that Lactobacillus, as the predominant bacterial genus in the female reproductive system, effectively inhibits harmful microorganisms and contributes to maintaining overall health. In this study, \u003cem\u003eLactobacillus\u003c/em\u003e crispatus, a species within the Lactobacillus genus, was selected as the subject of investigation to elucidate its antibacterial mechanisms against \u003cem\u003eEscherichia\u003c/em\u003e coli and the molecular pathways involved in reversing cervical cancer complicated by \u003cem\u003eEscherichia\u003c/em\u003e coli infection.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLactobacillus\u003c/em\u003e crispatus exhibited inhibitory effects on the growth of \u003cem\u003eEscherichia\u003c/em\u003e coli, with an average inhibitory zone diameter of 24.31\u0026thinsp;\u0026plusmn;\u0026thinsp;34 mm. The active components responsible for this inhibition were identified as live bacterial cells of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus. The antibacterial activity of \u003cem\u003eLactobacillus\u003c/em\u003e crispatus was stable for up to 7 days at 4\u0026deg;C and 42 days at 25\u0026deg;C. Furthermore, \u003cem\u003eLactobacillus\u003c/em\u003e crispatus effectively suppressed the biofilm formation and cellular adhesion of \u003cem\u003eEscherichia\u003c/em\u003e coli while limiting the release of extracellular soluble proteins. It has been demonstrated that \u003cem\u003eEscherichia\u003c/em\u003e coli induces inflammatory damage in cervical cancer cells via the ferroptosis pathway, thereby promoting tumor cell migration. Following intervention with \u003cem\u003eLactobacillus\u003c/em\u003e crispatus, the bacterium was able to reverse the ferroptosis-induced and inflammatory damage caused by \u003cem\u003eEscherichia\u003c/em\u003e coli in cervical cancer cells, inhibit tumor cell migration, and reduce tumor malignancy.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLactobacillus\u003c/em\u003e crispatus as a microecological balance regulator can not only inhibit the growth of \u003cem\u003eEscherichia\u003c/em\u003e coli, but also improve the migration ability of cervical cancer complicated with \u003cem\u003eEscherichia\u003c/em\u003e coli infection and maintain the health of the female reproductive system.\u003c/p\u003e","manuscriptTitle":"Lactobacillus crispatus reverses Escherichia coli-induced inflammatory injury and migration of cervical cancer cells by inhibiting ferroptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-28 12:37:33","doi":"10.21203/rs.3.rs-6435097/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1da35015-5320-4b5c-a7b9-d48b3cdb6311","owner":[],"postedDate":"April 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-30T04:23:34+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-28 12:37:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6435097","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6435097","identity":"rs-6435097","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}