A model of Helicobacter infection using an artificial liver constructed by a radial-flow bioreactor

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This study aimed to investigate the mechanism of Helicobacter invasion into cells and the structural and functional responses of cells to this invasion. Using the radial-flow bioreactor methodology, a novel artificial liver model was used to reconstruct cell cultures in three dimensions and to explore how Helicobacter pylori infects human hepatocytes. H. pylori was found to attach to the hepatocyte surface and to enter hepatocytes, existing within intercellular spaces. H. pylori infection was also associated with increased apoptosis as well as NF-kB and TNF-α activation in the artificial liver. Therefore, H. pylori exhibits a potential carcinogenic role in artificial liver environments. Further studies are warranted to clarify whether these effects occur in the human liver in vivo . artificial liver Helicobacter pylori hepatocellular carcinoma liver diseases cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Over the 30 years since the discovery of Helicobacter pylori , at least 42 new Helicobacter species have been identified [ 1 ]. Several of them have been detected in the liver of mice and dogs and reported to induce inflammatory cell infiltration in liver tissue and tumor formation [ 2 – 4 ]. In humans, Helicobacter DNA has been detected in the liver tissue of patients with hepatocellular carcinoma (HCC) and other liver diseases, primarily through cytomolecular methods, suggesting a potential role for Helicobacter infection in the etiology of liver diseases [ 5 – 7 ]. Previously, we detected Helicobacter DNA in liver tissues of patients with HCC, which was higher in patients than in controls, indicating its potential involvement in HCC pathogenesis [ 8 ]. DNA sequencing and immunostaining results suggested that Helicobacter pylori , a known stomach pathogen, or a Helicobacter very close to it, may be involved in hepatocellular carcinogenesis. Because detailed studies on how Helicobacter affects hepatocytes are lacking, we used hepatocytes and H. pylori , a well-studied representative of the Helicobacter group, to examine their interrelationship [ 9 ]. The interaction was significant: H. pylori adhered to hepatocytes, invaded cells, and grew intracellularly over an extended period; it also increased hepatocyte apoptosis and promoted DNA synthesis [ 10 ], suggesting that H. pylori contributes to carcinogenesis by increasing cell turnover. Here, we investigated the mechanism of Helicobacter invasion into cells and the structural and functional cellular responses that have not been fully elucidated in previous experiments. A radial flow bioreactor (RFB) enabled us to realize an in vitro state similar to that of in vivo cells in tissues by constructing cells in three dimensions [ 11 ]. Cell polarity can also be reproduced, making it possible to study the routes and conditions of Helicobacter infection in vivo and its effects on cells more detailly. Helicobacter intracellular invasion affects cells and allows bacteria to evade the host immune system, enabling long-lasting infection in vivo . Helicobacter may be critical as a virulence factor in the liver and stomach. Additionally, unpublished data have shown differences between hepatocytes and gastric epithelial cells in Helicobacter adhesion, cellular response to intracellular invasion, and intracellular infection duration. Nonetheless, the causes for these differences remain unclear and warrant further investigation. Moreover, Helicobacter may adhere to and invade cells from the sinusoidal side (unpublished data) in hepatocytes and from the luminal side of the glandular tissue in gastric epithelial cells. Three-dimensionally constructed cells are the ideal experimental system for testing this hypothesis, and the present study was designed to investigate it. This experimental system elucidated the mechanism of persistent intracellular infection of Helicobacter in both cell types. A unique feature of this study was that the experiments were conducted using an RFB, a new method enabling 3D construction of cell cultures and dramatically improving the quality of in vitro studies of pathological conditions. By utilizing the characteristics of this experimental system to mimic Helicobacter infection in actual in vivo tissues, we believe that we can elucidate the infection route and method of persistent infection in the liver, which has been difficult to identify to date. HCC is among the most common types of cancer worldwide, and the incidence of both hepatocellular and gastric cancers is particularly high in Japan compared to that in other countries. Specifically, HCC exhibits a high recurrence risk, even when detected and treated at an early stage, making carcinogenesis prevention a major issue. If this study further clarifies the influence of Helicobacter on hepatocarcinogenesis, identifies the infection route, and elucidates the mechanism of persistent infection, it could greatly contribute to preventing HCC in the future. Several virulent factors, such as CagA, VacA, and activation-induced cytidine deaminase (AID), are associated with Helicobacter pathogenesis [ 12 – 15 ]. CagA and VacA may be closely related to H. pylori intracellularly; nevertheless, this remains to be proven. Additionally, AID may promote multistep carcinogenesis in the stomach and in the liver. Therefore, Helicobacter infection may become a more important pathogenic factor in the stomach and liver in the future. This study will provide further insight into Helicobacter infection pathogenesis and advance research on liver carcinogenesis. Methods Ethical Approval In accordance with the ethical guidelines of the Japanese Ministry of Health, Labour, and Welfare, this experiment using non-human tissues did not require ethical approval. 3D Cell Culture in RFB A 3D cell culture system was constructed using 5 mL RFB (RA-5; ABLE, Tokyo, Japan) filled with cellulose beads (Asahi Kasei, Tokyo, Japan), a mass flow controller (RAD925, ABLE), and a closed-circuit reservoir, as described previously [16]. The system was filled with minimal essential medium (Invitrogen, Carlsbad, CA, USA) and 5% fetal bovine serum. Huh7 cells (1 × 10 7 ), cultured cells of HCC origin, were infused into the reservoir to facilitate attachment to the cellulose beads, and a 3D cell culture was constructed for 2 weeks until the cell culture reached approximate confluence. The culture medium was changed manually to maintain optimal glucose, lactate, and pH levels [16]. Bacterial Infection of 3D-Constructed Cells Helicobacter pylori NCTC11637 strain (1 × 10 12 colony forming units [CFUs]), a putative and virulent strain that contains the toxic factors CagA and VacA, was cultured on blood agar plates consisting of brain-heart infusion agar (Difco, Sparks, MD, USA) and 7% horse blood for 2 days before infection in a CO 2 incubator with 12% CO 2 at 37℃. The bacteria were suspended in cell culture medium as described above and adjusted to an appropriate concentration before use. The bacterial concentration was standardized by measuring the optical density at 600 nm. Overall, 15 mL of 1.1 x 10 9 /mL bacterial suspension with culture medium was inserted into the reserve. CFUs were measured as described previously [9] and found to be 7.5 x 10 9 , confirming that the multiplicity of infection was approximately 100. Moreover, 15 mL culture medium without bacteria was used as the control. After 24 and 48 h of incubation, 3D cell culture was collected, stored at –80°C, frozen in optimal cutting temperature compound for cryo-sections, and fixed in 2.5% glutaraldehyde for electron microscopy and 10% buffered formalin. Histology Morphology was assessed using hematoxylin and eosin (H&E) staining. Paraffin-embedded and formalin-fixed 3D cell culture sections (4-μm thick) were stained with H&E. Immunohistochemistry (IHC) for H. pylori was performed to assess the infection status. After de-embedding and blocking with 3% MtOH in H 2 O 2 for 10 min, antigen retrieval was performed using Liberate Antibody Binding solution (Polysciences Inc., Warrington, PA, USA) for 5 min at room temperature. After washing three times with phosphate-buffered saline (PBS) for 5 min, 1:50 diluted rabbit polyclonal anti- H. pylori antibody (DAKO, Kyoto, Japan) in 5% bovine serum albumin with PBS was applied to the slides and incubated for 60 min at room temperature. Secondary antibody and enhancement steps were performed as follows. Electron Microscopy Electron microscopy was performed using standard techniques at the Molecular Cell Biology Facility at the institute [16]. Measurement of Interleukin-8 (IL-8) Expression H. pylori induces IL-8 expression in stomach tissues and is critical in pathogenesis within the stomach. Here, we evaluated whether H. pylori induces IL-8 expression in an artificial liver using reverse transcription-polymerase chain reaction (RT-PCR) methods described below. Cell Cycle Balance Assessment Cell proliferation and apoptosis assessments are important for assessing the cell cycle balance. Increased cell proliferation and apoptosis accelerate the cell cycle [17]. Cell cycle acceleration promotes DNA misreading, leading to oncogene activation and inhibition of tumor suppressor genes. Here, we measured Ki-67 expression in the nucleus to assess cell proliferation using IHC with an anti-Ki-67 antibody. Ki-67 is a widely recognized cell proliferation marker. The Ki-67 nuclear antigen is expressed in all proliferating cells during the active phases of the cell cycle, namely late G1, S, M, and G2. IHC was performed to detect Ki-67 expression using a rabbit polyclonal anti-Ki-67 antibody (Novocastra, Newcastle, UK). Sections were de-embedded and processed according to the instructions of the N-Histofine Simple Stain MAX-PO(R) kit (NICHIREI BIOSCIENCES Inc., Tokyo, Japan). Antigen retrieval was performed as follows. Before incubation with the blocking reagent, the sections were soaked in antigen retrieval reagent (DAKO) for 40 min at 95°C and then cooled at room temperature for 15 min. The sections were then washed three times with PBS for 5 min. Data were analyzed and compared using the labelling index (LI), which is expressed as the percentage of nuclei with signals among 500 nuclei in a randomly chosen area. Additionally, we analyzed the expression of the proliferating cell nuclear antigen (PCNA), typical of proliferating cell nuclei, using RT-PCR (described below) to evaluate the status of cell proliferation of hepatocytes infected by H. pylori . Furthermore, we evaluated apoptosis using the TUNEL assay, which is excellent for detecting apoptosis in tissues [18]. The TUNEL method is highly efficient and specifically labels the free 3'-OH ends of fragmented DNA in apoptotic cells with fluorescein-dUTP using terminal transferase in situ or in cells. The TUNEL assay was performed in 4 µm of formalin-fixed and paraffin-embedded sections using the In Situ Cell Death Detection Kit, POD (Roche Applied Science, Tokyo, Japan). Data were analyzed and compared using LI, as described above. Carcinogenesis Factor Assessment We assessed whether H. pylori would affect the activation of several factors, such as tumor necrosis factor (TNF)-α, NF-kB, Akt, Met, β-catenin, IL-8, and Ets-1, which are involved in carcinogenesis, using an artificial liver model. Akt is crucial in controlling survival and apoptosis [19–21]. AKT promotes cell survival by inhibiting apoptosis. Met is a high-affinity tyrosine kinase receptor for hepatocyte growth factor [22, 23]. Altered Met levels and/or tyrosine kinase activity are found in several types of tumors, including renal, colon, and breast cancers [24]. β-catenin is a protein that binds to the cytoplasmic tail of E-Cadherin and is critical in cell adhesion; it has a recognized association with some cancer types, including colon cancer [25]. H. pylori activates β-catenin on the surface of human cells [26]; moreover, this protein is important in human cancer cells [27]. Expression of phospho-Akt, phospho-Met, and beta-catenin in 3D formation hepatocytes was detected using immunofluorescence with rabbit monoclonal anti-phospho-Akt antibody (Cell Signaling Technology Cat. no. 4060; Danvers, MA, USA), rabbit monoclonal anti-phospho-Met (Cell Signaling Technology, Cat. No. 3077, Danvers, MA, USA), and mouse monoclonal anti-beta-catenin antibody (BD Transduction Laboratories Cat. No. 610153, Franklin Lakes, NJ, USA). Immunofluorescence was performed using the same methods for Ki67 detection described above, except for the secondary antibody incubation step. The following secondary antibodies were used for IHC to detect β-catenin instead of secondary antibodies in the HISTOFINE kit: fluorescein-conjugated AffiniPure goat anti-mouse IgG or anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). In all immunofluorescence assays, the nuclei were stained with DAPI to facilitate cell identification. TNF-α and NF-kB are involved in the cell cycle [28]. H. pylori infection induces TNF-α and NF-kB activation in vitro and in vivo [29], with TNF-α being critical in carcinogenesis through apoptosis induction or inhibition [30], specifically inducing apoptosis through the cell death signaling pathway through TNF receptor 1 [31]. Moreover, TNF-α induces cell proliferation and cell survival by inhibiting apoptosis through the NF-kB activation pathway [32]. As described below, TNF-α expression was measured by quantitative RT-PCR, and NF-kB activation was assessed and quantified by chemiluminescence assay. IL-8, a member of the Cys-X-Cys chemokine family, is an activator and chemoattractant for neutrophils and lymphocytes [33]. Gastric mucosal IL-8 levels increase significantly after H. pylori infection and induce stomach inflammation [34], resulting in atrophic gastritis, the most important cause of gastric carcinogenesis [34]. We measured IL-8 expression in artificial livers infected with H. pylori by RT-PCR. Ets-1 may be a transcriptional regulator involved in tumorigenesis; it is believed to be upregulated in chronic hepatitis, slightly decreased in the presence of HCC relative to chronic hepatitis, and upregulated in the normal liver [35, 36]. In HCC, higher Ets-1 expression is associated with smaller tumors, whereas poorly differentiated carcinomas exhibit lower Ets-1 expression [36]. Therefore, Ets-1 may be a marker of early-stage HCC or a pre-cancerous state. We assessed Ets-1 expression levels by quantitative RT-PCR. RT-PCR Quantitative RT-PCR measured TNF-α, PCNA, IL-8, and Ets-1 levels. Total RNA was extracted from 5–10–µm–thick specimens of frozen cell culture using RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Ambion, Austin, TX, USA), following the provided instructions and stored at -80ºC. cDNA was prepared from the total RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). qRT-PCR was performed with TaqMan Gene Expression Assays (Applied Biosystems, Waltham, MA, USA) with TaqMan Gene Expression Master Mix (Applied Biosystems, Waltham, MA, USA), following the instructions provided. The probe for GAPDH was used as an internal control. The PCR cycling program consisted of 40 cycles of 95ºC for 1 s and 60ºC for 20 s using the StepOne RT-PCR systems (Applied Biosystems, Waltham, MA, USA) following the holding step with 95ºC for 20 s. The data were analyzed using comparative ΔΔCt methods, comparing the H. pylori -infected and uninfected control groups. NF-kB Chemiluminescence Assay NF-kB activation of 3D formation hepatocytes incubated with or without H. pylori was measured and quantified by Trans AM TM NF-kB p50Chemi/NF-kB p52 Chemi/NF-kB p65 Chemi (Active Motif, Cat. No.40097, Carlsbad, CA, USA). Nuclear extract was extracted from a 0.05 g frozen sample of 3D formation hepatocytes with cellulose beads using a Nuclear Extract Kit (version C4; Active Motif Cat. No.40010; Carlsbad, CA, USA) according to a standard protocol. The positive control cell extract included in the kit was used to confirm the assay accuracy. Statistical Analysis Data are reported as mean ± standard error and were evaluated using one-way analysis of variance or Student’s t -test. Statistical analyses were performed using SigmaPlot version 10 (SYSTAT Software Inc., San Jose, CA, USA). Statistical significance was set at P < 0.05. Results Immunohistological Findings of 3D Hepatocyte Culture with H. pylori H&E staining demonstrated that the hepatocytes were attached to each other with cellulose beads, forming a hepatocyte mass (Fig. 1). Apoptotic cells were visible, with pyknosis evidence, particularly in cells incubated with H. pylori compared to control cells incubated without it. IHC with an anti-HP antibody showed numerous H. pylori -attached hepatocytes on the surface of the hepatocyte mass (Fig. 1). Some H. pylori was internalized within hepatocytes. Nonetheless, H. pylori infection is rarely observed inside a mass. Electron Microscopic Findings Transmission electron microscopy exhibited large amounts of H. pylori attached to the surface of the hepatocytes with infiltration of the intercellular space (Fig. 2A). The cell surface rose around H. pylori attached to the cell surface, appearing to wrap around the head of H. pylori ; scanning electron microscopy confirmed that hepatocytes wrapped around H. pylori (Fig. 2B). H. pylori invades and remains in the intercellular spaces and on the surface of the sterically constructed cell mass. In these locations, it remains in the bacillus form rather than converting to the coccoid form, the defensive morphology adopted to survive when environmental conditions are unfavorable. Therefore, H. pylori might not only attach to the surface of the liver to invade the intracellular space, but it may also loosen intercellular junctions and invade intercellular spaces, thereby escaping from an environment that is difficult to survive in and facilitating survival. Loosening the intercellular space destabilizes intercellular connections, making cells more susceptible to cancer [37, 38]. Therefore, the entry of H. pylori into the intercellular space may also favor cancer development due to infection. Cell Cycle Balance Assessment We assessed the balance of the cell cycle by performing a TUNEL assay to evaluate the status of apoptosis and an IHC for Ki-67 expression to evaluate the status of cell proliferation. The TUNEL assay demonstrated that apoptotic cells were mostly inside the artificial liver rather than on the artificial liver surface (Fig. 3A). H. pylori incubation increased artificial liver apoptosis compared to that of control cells without such incubation. Additionally, quantification results (Fig. 3C) revealed an apoptosis increase in H. pylori -infected artificial liver (LI = 30.7±19.6) compared to that of controls (LI = 5.2±3.3) (P = 0.00147). IHC with an anti-Ki-67 antibody demonstrated that Ki-67 expression in the nuclei was detected in cells close to the surface of the artificial liver (Fig. 3B). The pattern and number of cells expressing Ki-67 (Fig. 3D) were similar between cells incubated with H. pylori (LI =28.1±10.9) and control cells without such incubation (LI = 25.7±4.5) (P = 0.426), suggesting that cell proliferation in the artificial liver was not augmented by H. pylori infection. Phosphorylated Akt was detected in the cytoplasm of 3D hepatocytes incubated with H. pylori (Fig. 4), indicating H. pylori activated Akt, which inhibited apoptosis and promoted cell survival in 3D hepatocytes. Incubation of 3D formation hepatocytes with H. pylori did not increase phosphorylated Met compared with the control, but it activated β-catenin (Fig. 4). Factors Activated by Helicobacter pylori Infection RT-PCR results confirmed significantly higher levels of TNF-α in H. pylori -infected cells (mean 1.16 × 10 -5 [SE 6.31 × 10 -7 ]) than they did in non-infected controls (mean 7.65 × 10 -6 [SE 4.77 × 10 -7 ]; t = -5.065, P = 0.007), suggesting that H. pylori induced TNF-α expression, thereby upregulating apoptosis critical in hepatocarcinogenesis. Nevertheless, IL-8 levels (P = 0.419) were similar between the two groups (Fig. 5), suggesting that H. pylori does not induce the expression of the most representative inflammatory cytokines in the artificial liver, unlike in the stomach. H. pylori did not change Ets-1 levels (P = 0.704), and PCNA levels were significantly lower in the H. pylori -infected cells (mean 0.000267 [SE 1.14 x 10 -5 ]) than in non-infected controls (mean 0.00047 [SE 1.28 x 10 -7 ]; t = 11.840, P < 0.001). Significantly higher levels of NF-kB accumulation were detected in H. pylori -incubated cells than in controls (Fig. 6). Discussion H. pylori incubation with 3D-formed hepatocytes clarifies how it infects human hepatocytes. Electron microscopy demonstrated that H. pylori attaches to the hepatocyte surface and enters hepatocytes. Our previous in vitro study demonstrated H. pylori inoculation into hepatocytes [9], and the present findings support these results. Here, H. pylori occupied intercellular spaces, suggesting loss of cell-to-cell connections and facilitating entry via this intercellular route. IHC and anti-β-catenin antibody findings suggest that β-catenin activation at cell surfaces during H. pylori- hepatocyte incubation contributed to disrupted cell–cell contacts and promoted intracellular entry through gaps between cells. H. pylori usually inhabits human stomachs containing gastric juice. One of the reasons H. pylori can survive in an acidic environment is that it produces urease, which neutralizes acids. Based on our findings, another reason may be its ability to move through the gaps between cells and internalize them to avoid a difficult extracellular environment. β-catenin activation may be crucial for H. pylori survival in a difficult environment. In our artificial liver model, H. pylori also activated β-catenin and potentially inhabited inter- and intra-cellular spaces for protracted periods. We previously reported that H. pylori induces both cell proliferation and apoptosis [10], inferring that H. pylori -driven cell-cycle acceleration can lead to carcinogenesis. Here, H. pylori infection increased apoptosis and decreased cell proliferation in an artificial liver model. Thus, H. pylori may contribute to liver carcinogenesis by increasing apoptosis in vitro. Further studies are needed to elucidate the mechanisms underlying hepatocarcinogenesis. H. pylori infection induces several factors related to carcinogenesis. The transcription factor NF-kB is a critical mediator of immune and inflammatory responses and may play key roles in several diseases, including cancer; H. pylori induces NF-kB activation [29]. Here, H. pylori was associated with activation of NF-kB, Akt, and TNF-α, suggesting a role in carcinogenesis within the artificial liver environment. However, IL-8 was not increased by H. pylori , indicating that chronic inflammation with chemokine production is not involved in H. pylori -associated hepatocarcinogenesis, unlike gastric carcinogenesis. IL-8 is a chemokine produced by macrophages, epithelial cells, airway smooth muscle cells, and vascular endothelial cells, and, unsurprisingly, IL-8 production did not increase in artificial livers. H. pylori infection did not affect Ets-1 levels in this study; therefore, the involvement of Ets-1 in hepatocarcinogenesis was not clarified in our artificial liver model, and further studies are required. The main limitation of this study is the use of an artificial liver model, potentially limiting real-life applicability. Nevertheless, compared with other in vitro methods for studying pathological conditions, RFB is advantageous because it facilitates cell culture 3D construction. Additional studies involving clinical populations are warranted to confirm the clinical validity of our findings. Conclusions H. pylori attached to the hepatocyte surface and entered hepatocytes, existing within intercellular spaces, and was associated with NF-kB, Akt, and TNF-α activation, indicating a potential carcinogenic role within the artificial liver environment. Further studies clarifying whether these effects occur in the human liver in vivo are warranted. Declarations Acknowledgments We greatly appreciate Professor Sae Ochi’s kind support of this study. We also thank the staff of the Core Research Facility, Research Center for Medical Sciences, and The Jikei University School of Medicine for their excellent electron microscopy techniques. Editorial support, in the form of medical writing, assembling tables and creating high-resolution images based on authors’ detailed directions, collating author comments, copyediting, fact checking, and referencing, was provided by Editage, Cactus Communications, and funded by Grant-in-Aid for Scientific Research (C) 2008 (grant number: 20590457). Authors’ references: 8. Ito K, Nakamura M, Toda G, Negishi M, Torii A, Ohno T. Potential role of Helicobacter pylori in hepatocarcinogenesis. Int J Mol Med. 2004;13:221–7. https://doi.org/10.3892/ijmm.13.2.221 9. Ito K, Yamaoka Y, Ota H, El-Zimaity H, Graham DY. Adherence, internalization, and persistence of Helicobacter pylori in hepatocytes. Dig Dis Sci. 2008;53:2541–9. https://doi.org/10.1007/s10620-007-0164-z 10. Ito K, Yamaoka Y, Yoffe B, Graham DY. Disturbance of apoptosis and DNA synthesis by Helicobacter pylori infection of hepatocytes. Dig Dis Sci. 2008;53:2532–40. https://doi.org/10.1007/s10620-007-0163-0 Funding : This study was funded by a Grant-in-Aid for Scientific Research (C) 2008 (grant number: 20590457). Competing interests : The authors have no relevant financial or nonfinancial interests to disclose. Availability of data and material : The datasets generated and/or analyzed in the current study are available from the corresponding author upon reasonable request. Code availability : Not applicable. Authors' contributions : All authors contributed to the conception and design of this study. The material preparation, data collection, and analyses were performed by Ito, Maehashi, and Matsuura. Kyoko Ito wrote the first draft of the manuscript, and all authors commented on the previous versions of the manuscript. All the authors have read and approved the final version of the manuscript. Ethics approval : In line with the ethical guidelines issued by the Japanese Ministry of Health, Labor, and Welfare, this experiment on non-human tissues did not require ethical approval. Consent to participate : Not applicable. Consent for publication : Not applicable. References Yamanaka H, Non. - H. pylori Helicobacter species detected in laboratory animals. Shinshu Med. 2019;J2019:81–9. (in Japanese). Yamanaka H, Arita M, Oi R, et al. Prevalence of an unidentified Helicobacter species in laboratory mice and its distribution in the hepatobiliary system and gastrointestinal tract. Exp Anim. 2013;62:109–16. https://doi.org/10.1538/expanim.62.109 . Cao S, Miao J, Qian M, et al. 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Expression and possible role of ets-1 in hepatocellular carcinoma. Am J Clin Pathol. 2000;114:719–25. https://doi.org/10.1309/RAVV-8NM1-CJB7-GJFR . Miyazaki K, Ashida Y, Kihira Y, Mashima K, Yamashita J, Horio T. Transformation of rat liver cell line by rous sarcoma virus causes loss of cell surface fibronectin, accompanied with secretion of metallo-proteinase that preferentially digests the fibronectin. J Biochem. 1987;102:569–82. https://doi.org/10.1093/oxfordjournals.jbchem.a122090 . Behrens J, Vakaet L, Friis R, et al. Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/β-catenin complex in cells transformed with a temperature-sensitive v-SRC gene. J Cell Biol. 1993;120:757–66. https://doi.org/10.1083/jcb.120.3.757 . Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 10 Oct, 2025 Reviewers invited by journal 09 Oct, 2025 Editor assigned by journal 07 Oct, 2025 First submitted to journal 06 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7797022","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":527391005,"identity":"24ca7f58-00d0-4b34-be8c-c963ee1b9b85","order_by":0,"name":"Kyoko Ito","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIie2RsQrCMBCGLwjpIrp2Mq/Q4Fp8lhMhTq4iOFgJpEvBVR/DN6gEdNE+Q/sIbh0cPCs4moyC+SCQhHzc3R+AQOAH4bRYBumIadpid1c6FN4pauyvwFux06wr6MNA6JIdjJrrnE/rGiYC2O17Gc45sqNJF1vNbYIwkxlU6FD6CWuMIiUyMUKPnl8TH8XOmY7yFmHjqRyNRUrsTIlZH0XhaV8pSbPMYkwu0rhmEdqemmKZCrk7y3u7Woth7EjsRcnoSygpglri8dVpEA8q9zkMCx8lEAgE/ognL1lB19AxCewAAAAASUVORK5CYII=","orcid":"","institution":"The Jikei University Hospital: Tokyo Jikeikai Ika Daigaku Fuzoku Byoin","correspondingAuthor":true,"prefix":"","firstName":"Kyoko","middleName":"","lastName":"Ito","suffix":""},{"id":527391006,"identity":"6f1bbdf7-eb36-45ad-83dd-5061a0319b19","order_by":1,"name":"Haruka Maehashi","email":"","orcid":"","institution":"Jikei University School of Medicine: Tokyo Jikeikai Ika 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19:11:19","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114955,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7797022/v1/3b2ee163e148e4c1204ccb35.html"},{"id":94224332,"identity":"086ccf10-6e72-43e6-ade1-f882514cf08e","added_by":"auto","created_at":"2025-10-23 19:19:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":679726,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemistry results\u003c/p\u003e\n\u003cp\u003eThe top row shows cells incubated with \u003cem\u003eH. pylori\u003c/em\u003e (HP), and the bottom row shows control (non-infected) cells. (A) Hematoxylin and eosin staining showing hepatocytes attached to each other, forming a mass of hepatocytes with cellulose beads. Apoptotic cells were visible (arrowheads). (B) Immunohistochemistry with an anti-HP antibody demonstrating numerous \u003cem\u003eH. pylori\u003c/em\u003e(brown)-attached hepatocytes on the surface of the hepatocyte mass, with some \u003cem\u003eH. pylori\u003c/em\u003e internalized within the hepatocytes. Magnification x 400\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7797022/v1/ce72c29947edb3198506528b.png"},{"id":94223862,"identity":"13a70e1a-68b9-4f0a-aef9-ee0185eb71f0","added_by":"auto","created_at":"2025-10-23 19:11:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":810281,"visible":true,"origin":"","legend":"\u003cp\u003eElectron microscopy images of \u003cem\u003eH. pylori \u003c/em\u003eattaching to or infiltrating hepatocytes of an artificial liver.\u003c/p\u003e\n\u003cp\u003e(A) Transmission electron microscopy showing \u003cem\u003eH. pylori\u003c/em\u003e attached (arrows) to the surface of hepatocytes with infiltration (arrowheads) of the intercellular space. The scale bar represents 100 nm in the left-most image and 500 nm in the middle and right-most images. (B) Scanning electron microscopy images demonstrating the hepatocytes wrapped around \u003cem\u003eH. pylori\u003c/em\u003e.\u003cem\u003e H. pylori\u003c/em\u003e was found to invade and stay in the intercellular spaces and on the surface of the sterically constructed cell mass. Scale bars represent 1 µm\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7797022/v1/9148ad394bb35ec2d99367c5.png"},{"id":94225124,"identity":"0b75dc48-d940-4b66-af71-dab5c0d232e3","added_by":"auto","created_at":"2025-10-23 19:27:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":584539,"visible":true,"origin":"","legend":"\u003cp\u003eTUNEL assay and Ki67 staining results\u003c/p\u003e\n\u003cp\u003eThe top row shows cells incubated with \u003cem\u003eH. pylori\u003c/em\u003e (HP), and the bottom row shows control (non-infected) cells (CTR). (A) TUNEL assay results demonstrate that apoptotic cells (brown) have a propensity to localize inside the artificial liver. Magnification x 400. (B) Ki67 staining with anti-Ki-67 antibody demonstrating that Ki-67 expression (brown) within the nuclei was detected in cells close to the surface of the artificial liver. Magnification x100. (C) Quantification of TUNEL assay results. The y-axis represents the labelling index. (D) Ki67 assay results demonstrating the number of cells expressing Ki-67 in the CTR and HP groups. The y-axis represents the labelling index.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7797022/v1/f4e87d874a1d7d982185d169.png"},{"id":94224334,"identity":"7790a9cb-819b-4bd6-b0d8-531f8fd547f5","added_by":"auto","created_at":"2025-10-23 19:19:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1038407,"visible":true,"origin":"","legend":"\u003cp\u003eExpression (green) of (A) phospho-Akt (200 x magnification), (B) beta-catenin (x 400 magnification), and (C) phospho-Met (x 200 magnification) in 3D formation hepatocytes\u003c/p\u003e\n\u003cp\u003eNuclei are stained blue by DAPI\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7797022/v1/5296b77f608027fe8c2e53df.png"},{"id":94223869,"identity":"343b755a-aa6f-4a33-a288-47f8cc9d283b","added_by":"auto","created_at":"2025-10-23 19:11:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":138087,"visible":true,"origin":"","legend":"\u003cp\u003eReal-time quantitative PCR results for various markers\u003c/p\u003e\n\u003cp\u003eGAPDH was used as a reference gene. The y-axis represents the ratio of expression levels in cells incubated with \u003cem\u003eH. pylori\u003c/em\u003eto those in control (non-infected) cells, where a value of 1 indicates identical levels of expression in cells incubated with \u003cem\u003eH. pylori\u003c/em\u003e and (non-infected) controls. *P \u0026lt;0.05\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7797022/v1/775c89a912449cd0753262fe.png"},{"id":94223865,"identity":"772eeebf-7f61-4bd9-90be-7350568d9446","added_by":"auto","created_at":"2025-10-23 19:11:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":105957,"visible":true,"origin":"","legend":"\u003cp\u003eLevels of NF-kB activation in cells incubated with \u003cem\u003eH. pylori\u003c/em\u003e compared to control (non-infected) cells\u003c/p\u003e\n\u003cp\u003eThe positive control showed high NF-kB activation, indicating that this assay was appropriate. The y-axis represents absorbance at 450 nm.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7797022/v1/719e82ed10693333be6e94fd.png"},{"id":94225785,"identity":"264f0bea-8c4f-4009-8b05-afe9f2443bda","added_by":"auto","created_at":"2025-10-23 19:35:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4255863,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7797022/v1/74aa9ec9-1051-4340-93e5-3a22653e960e.pdf"}],"financialInterests":"","formattedTitle":"A model of Helicobacter infection using an artificial liver constructed by a radial-flow bioreactor","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOver the 30 years since the discovery of \u003cem\u003eHelicobacter pylori\u003c/em\u003e, at least 42 new \u003cem\u003eHelicobacter\u003c/em\u003e species have been identified [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Several of them have been detected in the liver of mice and dogs and reported to induce inflammatory cell infiltration in liver tissue and tumor formation [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In humans, \u003cem\u003eHelicobacter\u003c/em\u003e DNA has been detected in the liver tissue of patients with hepatocellular carcinoma (HCC) and other liver diseases, primarily through cytomolecular methods, suggesting a potential role for \u003cem\u003eHelicobacter\u003c/em\u003e infection in the etiology of liver diseases [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Previously, we detected \u003cem\u003eHelicobacter\u003c/em\u003e DNA in liver tissues of patients with HCC, which was higher in patients than in controls, indicating its potential involvement in HCC pathogenesis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. DNA sequencing and immunostaining results suggested that \u003cem\u003eHelicobacter pylori\u003c/em\u003e, a known stomach pathogen, or a \u003cem\u003eHelicobacter\u003c/em\u003e very close to it, may be involved in hepatocellular carcinogenesis. Because detailed studies on how \u003cem\u003eHelicobacter\u003c/em\u003e affects hepatocytes are lacking, we used hepatocytes and \u003cem\u003eH. pylori\u003c/em\u003e, a well-studied representative of the \u003cem\u003eHelicobacter\u003c/em\u003e group, to examine their interrelationship [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The interaction was significant: \u003cem\u003eH. pylori\u003c/em\u003e adhered to hepatocytes, invaded cells, and grew intracellularly over an extended period; it also increased hepatocyte apoptosis and promoted DNA synthesis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], suggesting that \u003cem\u003eH. pylori\u003c/em\u003e contributes to carcinogenesis by increasing cell turnover.\u003c/p\u003e\u003cp\u003eHere, we investigated the mechanism of \u003cem\u003eHelicobacter\u003c/em\u003e invasion into cells and the structural and functional cellular responses that have not been fully elucidated in previous experiments. A radial flow bioreactor (RFB) enabled us to realize an \u003cem\u003ein vitro\u003c/em\u003e state similar to that of \u003cem\u003ein vivo\u003c/em\u003e cells in tissues by constructing cells in three dimensions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Cell polarity can also be reproduced, making it possible to study the routes and conditions of \u003cem\u003eHelicobacter\u003c/em\u003e infection \u003cem\u003ein vivo\u003c/em\u003e and its effects on cells more detailly. \u003cem\u003eHelicobacter\u003c/em\u003e intracellular invasion affects cells and allows bacteria to evade the host immune system, enabling long-lasting infection \u003cem\u003ein vivo\u003c/em\u003e. \u003cem\u003eHelicobacter\u003c/em\u003e may be critical as a virulence factor in the liver and stomach. Additionally, unpublished data have shown differences between hepatocytes and gastric epithelial cells in \u003cem\u003eHelicobacter\u003c/em\u003e adhesion, cellular response to intracellular invasion, and intracellular infection duration. Nonetheless, the causes for these differences remain unclear and warrant further investigation. Moreover, \u003cem\u003eHelicobacter\u003c/em\u003e may adhere to and invade cells from the sinusoidal side (unpublished data) in hepatocytes and from the luminal side of the glandular tissue in gastric epithelial cells. Three-dimensionally constructed cells are the ideal experimental system for testing this hypothesis, and the present study was designed to investigate it. This experimental system elucidated the mechanism of persistent intracellular infection of \u003cem\u003eHelicobacter\u003c/em\u003e in both cell types.\u003c/p\u003e\u003cp\u003eA unique feature of this study was that the experiments were conducted using an RFB, a new method enabling 3D construction of cell cultures and dramatically improving the quality of \u003cem\u003ein vitro\u003c/em\u003e studies of pathological conditions. By utilizing the characteristics of this experimental system to mimic \u003cem\u003eHelicobacter\u003c/em\u003e infection in actual \u003cem\u003ein vivo\u003c/em\u003e tissues, we believe that we can elucidate the infection route and method of persistent infection in the liver, which has been difficult to identify to date.\u003c/p\u003e\u003cp\u003eHCC is among the most common types of cancer worldwide, and the incidence of both hepatocellular and gastric cancers is particularly high in Japan compared to that in other countries. Specifically, HCC exhibits a high recurrence risk, even when detected and treated at an early stage, making carcinogenesis prevention a major issue. If this study further clarifies the influence of \u003cem\u003eHelicobacter\u003c/em\u003e on hepatocarcinogenesis, identifies the infection route, and elucidates the mechanism of persistent infection, it could greatly contribute to preventing HCC in the future. Several virulent factors, such as CagA, VacA, and activation-induced cytidine deaminase (AID), are associated with \u003cem\u003eHelicobacter\u003c/em\u003e pathogenesis [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. CagA and VacA may be closely related to \u003cem\u003eH. pylori\u003c/em\u003e intracellularly; nevertheless, this remains to be proven. Additionally, AID may promote multistep carcinogenesis in the stomach and in the liver. Therefore, \u003cem\u003eHelicobacter\u003c/em\u003e infection may become a more important pathogenic factor in the stomach and liver in the future. This study will provide further insight into \u003cem\u003eHelicobacter\u003c/em\u003e infection pathogenesis and advance research on liver carcinogenesis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthical Approval\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn accordance with the ethical guidelines of the Japanese Ministry of Health, Labour, and Welfare, this experiment using non-human tissues did not require ethical approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3D Cell Culture in RFB\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 3D cell culture system was constructed using 5 mL RFB (RA-5; ABLE, Tokyo, Japan) filled with cellulose beads (Asahi Kasei, Tokyo, Japan), a mass flow controller (RAD925, ABLE), and a closed-circuit reservoir, as described previously [16]. The system was filled with minimal essential medium (Invitrogen, Carlsbad, CA, USA) and 5% fetal bovine serum. Huh7 cells (1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e), cultured cells of HCC origin, were infused into the reservoir to facilitate attachment to the cellulose beads, and a 3D cell culture was constructed for 2 weeks until the cell culture reached approximate confluence. The culture medium was changed manually to maintain optimal glucose, lactate, and pH levels [16].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBacterial Infection of 3D-Constructed Cells\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHelicobacter pylori\u0026nbsp;\u003c/em\u003eNCTC11637 strain (1 \u0026times; 10\u003csup\u003e12\u0026nbsp;\u003c/sup\u003ecolony forming units [CFUs]), a putative and virulent strain that contains the toxic factors CagA and VacA, was cultured on blood agar plates consisting of brain-heart infusion agar (Difco, Sparks, MD, USA) and 7% horse blood for 2 days before infection in a CO\u003csub\u003e2\u003c/sub\u003e incubator with 12% CO\u003csub\u003e2\u003c/sub\u003e at 37℃. The bacteria were suspended in cell culture medium as described above and adjusted to an appropriate concentration before use.\u003c/p\u003e\n\u003cp\u003eThe bacterial concentration was standardized by measuring the optical density at 600 nm. Overall, 15 mL of 1.1 x 10\u003csup\u003e9\u0026nbsp;\u003c/sup\u003e/mL bacterial suspension with culture medium was inserted into the reserve. CFUs were measured as described previously [9] and found to be 7.5 x 10\u003csup\u003e9\u003c/sup\u003e, confirming that the multiplicity of infection was approximately 100. Moreover, 15 mL culture medium without bacteria was used as the control. After 24 and 48 h of incubation, 3D cell culture was collected, stored at \u0026ndash;80\u0026deg;C, frozen in optimal cutting temperature compound for cryo-sections, and fixed in 2.5% glutaraldehyde for electron microscopy and 10% buffered formalin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHistology\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMorphology was assessed using hematoxylin and eosin (H\u0026amp;E) staining. Paraffin-embedded and formalin-fixed 3D cell culture sections (4-\u0026mu;m thick) were stained with H\u0026amp;E. Immunohistochemistry (IHC) for \u003cem\u003eH. pylori\u003c/em\u003e was performed to assess the infection status. After de-embedding and blocking with 3% MtOH in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 10 min, antigen retrieval was performed using Liberate Antibody Binding solution (Polysciences Inc., Warrington, PA, USA) for 5 min at room temperature. After washing three times with phosphate-buffered saline (PBS) for 5 min, 1:50 diluted rabbit polyclonal anti-\u003cem\u003eH. pylori\u003c/em\u003e antibody (DAKO, Kyoto, Japan) in 5% bovine serum albumin with PBS was applied to the slides and incubated for 60 min at room temperature. Secondary antibody and enhancement steps were performed as follows.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eElectron Microscopy\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eElectron microscopy was performed using standard techniques at the Molecular Cell Biology Facility at the institute [16].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMeasurement of Interleukin-8 (IL-8) Expression\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eH. pylori\u003c/em\u003e induces IL-8 expression in stomach tissues and is critical in pathogenesis within the stomach. Here, we evaluated whether \u003cem\u003eH. pylori\u003c/em\u003e induces IL-8 expression in an artificial liver using reverse transcription-polymerase chain reaction (RT-PCR) methods described below.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell Cycle Balance Assessment\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell proliferation and apoptosis assessments are important for assessing the cell cycle balance. Increased cell proliferation and apoptosis accelerate the cell cycle [17]. Cell cycle acceleration promotes DNA misreading, leading to oncogene activation and inhibition of tumor suppressor genes. Here, we measured Ki-67 expression in the nucleus to assess cell proliferation using IHC with an anti-Ki-67 antibody. Ki-67 is a widely recognized cell proliferation marker. The Ki-67 nuclear antigen is expressed in all proliferating cells during the active phases of the cell cycle, namely late G1, S, M, and G2.\u003c/p\u003e\n\u003cp\u003eIHC was performed to detect Ki-67 expression using a rabbit polyclonal anti-Ki-67 antibody (Novocastra, Newcastle, UK). Sections were de-embedded and processed according to the instructions of the N-Histofine Simple Stain MAX-PO(R) kit (NICHIREI BIOSCIENCES Inc., Tokyo, Japan). Antigen retrieval was performed as follows. Before incubation with the blocking reagent, the sections were soaked in antigen retrieval reagent (DAKO) for 40 min at 95\u0026deg;C and then cooled at room temperature for 15 min. The sections were then washed three times with PBS for 5 min. Data were analyzed and compared using the labelling index (LI), which is expressed as the percentage of nuclei with signals among 500 nuclei in a randomly chosen area. Additionally, we analyzed the expression of the proliferating cell nuclear antigen (PCNA), typical of proliferating cell nuclei, using RT-PCR (described below) to evaluate the status of cell proliferation of hepatocytes infected by \u003cem\u003eH. pylori\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eFurthermore, we evaluated apoptosis using the TUNEL assay, which is excellent for detecting apoptosis in tissues [18]. The TUNEL method is highly efficient and specifically labels the free 3\u0026apos;-OH ends of fragmented DNA in apoptotic cells with fluorescein-dUTP using terminal transferase \u003cem\u003ein situ\u003c/em\u003e or in cells. The TUNEL assay was performed in 4 \u0026micro;m of formalin-fixed and paraffin-embedded sections using the In Situ Cell Death Detection Kit, POD (Roche Applied Science, Tokyo, Japan). Data were analyzed and compared using LI, as described above.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCarcinogenesis Factor Assessment\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe assessed whether \u003cem\u003eH. pylori\u003c/em\u003e would affect the activation of several factors, such as tumor necrosis factor (TNF)-\u0026alpha;, NF-kB, Akt, Met, \u0026beta;-catenin, IL-8, and Ets-1, which are involved in carcinogenesis, using an artificial liver model. Akt is crucial in controlling survival and apoptosis [19\u0026ndash;21]. AKT promotes cell survival by inhibiting apoptosis. Met is a high-affinity tyrosine kinase receptor for hepatocyte growth factor [22, 23]. Altered Met levels and/or tyrosine kinase activity are found in several types of tumors, including renal, colon, and breast cancers [24]. \u0026beta;-catenin is a protein that binds to the cytoplasmic tail of E-Cadherin and is critical in cell adhesion; it has a recognized association with some cancer types, including colon cancer [25]. \u003cem\u003eH. pylori\u003c/em\u003e activates \u0026beta;-catenin on the surface of human cells [26]; moreover, this protein is important in human cancer cells [27].\u003c/p\u003e\n\u003cp\u003eExpression of phospho-Akt, phospho-Met, and beta-catenin in 3D formation hepatocytes was detected using immunofluorescence with rabbit monoclonal anti-phospho-Akt antibody (Cell Signaling Technology Cat. no. 4060; Danvers, MA, USA), rabbit monoclonal anti-phospho-Met (Cell Signaling Technology, Cat. No. 3077, Danvers, MA, USA), and mouse monoclonal anti-beta-catenin antibody (BD Transduction Laboratories Cat. No. 610153, Franklin Lakes, NJ, USA). Immunofluorescence was performed using the same methods for Ki67 detection described above, except for the secondary antibody incubation step. The following secondary antibodies were used for IHC to detect \u0026beta;-catenin instead of secondary antibodies in the HISTOFINE kit: fluorescein-conjugated AffiniPure goat anti-mouse IgG or anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). In all immunofluorescence assays, the nuclei were stained with DAPI to facilitate cell identification.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTNF-\u0026alpha; and NF-kB are involved in the cell cycle [28]. \u003cem\u003eH. pylori\u003c/em\u003e infection induces TNF-\u0026alpha; and NF-kB activation \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eand \u003cem\u003ein vivo\u003c/em\u003e [29], with TNF-\u0026alpha; being critical in carcinogenesis through apoptosis induction or inhibition [30], specifically inducing apoptosis through the cell death signaling pathway through TNF receptor 1 [31]. Moreover, TNF-\u0026alpha; induces cell proliferation and cell survival by inhibiting apoptosis through the NF-kB activation pathway [32]. As described below, TNF-\u0026alpha; expression was measured by quantitative RT-PCR, and NF-kB activation was assessed and quantified by chemiluminescence assay.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIL-8, a member of the Cys-X-Cys chemokine family, is an activator and chemoattractant for neutrophils and lymphocytes [33]. Gastric mucosal IL-8 levels increase significantly after \u003cem\u003eH. pylori\u003c/em\u003e infection and induce stomach inflammation [34], resulting in atrophic gastritis, the most important cause of gastric carcinogenesis [34]. We measured IL-8 expression in artificial livers infected with \u003cem\u003eH. pylori\u003c/em\u003e by RT-PCR.\u003c/p\u003e\n\u003cp\u003eEts-1 may be a transcriptional regulator involved in tumorigenesis; it is believed to be upregulated in chronic hepatitis, slightly decreased in the presence of HCC relative to chronic hepatitis, and upregulated in the normal liver [35, 36]. In HCC, higher Ets-1 expression is associated with smaller tumors, whereas poorly differentiated carcinomas exhibit lower Ets-1 expression [36]. Therefore, Ets-1 may be a marker of early-stage HCC or a pre-cancerous state. We assessed Ets-1 expression levels by quantitative RT-PCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRT-PCR\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantitative RT-PCR measured TNF-\u0026alpha;, PCNA, IL-8, and Ets-1 levels. Total RNA was extracted from 5\u0026ndash;10\u0026ndash;\u0026micro;m\u0026ndash;thick specimens of frozen cell culture using RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Ambion, Austin, TX, USA), following the provided instructions and stored at -80\u0026ordm;C. cDNA was prepared from the total RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). qRT-PCR was performed with TaqMan Gene Expression Assays (Applied Biosystems, Waltham, MA, USA) with TaqMan Gene Expression Master Mix (Applied Biosystems, Waltham, MA, USA), following the instructions provided. The probe for GAPDH was used as an internal control. The PCR cycling program consisted of 40 cycles of 95\u0026ordm;C for 1 s and 60\u0026ordm;C for 20 s using the StepOne RT-PCR systems (Applied Biosystems, Waltham, MA, USA) following the holding step with 95\u0026ordm;C for 20 s. The data were analyzed using comparative \u0026Delta;\u0026Delta;Ct methods, comparing the \u003cem\u003eH. pylori\u003c/em\u003e-infected and uninfected control groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNF-kB Chemiluminescence Assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNF-kB activation of 3D formation hepatocytes incubated with or without \u003cem\u003eH. pylori\u0026nbsp;\u003c/em\u003ewas measured and quantified by Trans AM\u003csup\u003eTM\u003c/sup\u003eNF-kB p50Chemi/NF-kB p52 Chemi/NF-kB p65 Chemi (Active Motif, Cat. No.40097, Carlsbad, CA, USA). Nuclear extract was extracted from a 0.05 g frozen sample of 3D formation hepatocytes with cellulose beads using a Nuclear Extract Kit (version C4; Active Motif Cat. No.40010; Carlsbad, CA, USA) according to a standard protocol. The positive control cell extract included in the kit was used to confirm the assay accuracy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical Analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are reported as mean \u0026plusmn; standard error and were evaluated using one-way analysis of variance or Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. Statistical analyses were performed using SigmaPlot version 10 (SYSTAT Software Inc., San Jose, CA, USA). Statistical significance was set at P \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunohistological Findings of 3D Hepatocyte Culture with H. pylori\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E staining demonstrated that the hepatocytes were attached to each other with cellulose beads, forming a hepatocyte mass (Fig. 1). Apoptotic cells were visible, with pyknosis evidence, particularly in cells incubated with \u003cem\u003eH. pylori\u003c/em\u003e compared to control cells incubated without it. IHC with an anti-HP antibody showed numerous \u003cem\u003eH. pylori\u003c/em\u003e-attached hepatocytes on the surface of the hepatocyte mass (Fig. 1). Some \u003cem\u003eH. pylori\u003c/em\u003e was internalized within hepatocytes. Nonetheless, \u003cem\u003eH. pylori\u003c/em\u003e infection is rarely observed inside a mass.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eElectron Microscopic Findings\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransmission electron microscopy exhibited large amounts of \u003cem\u003eH. pylori\u003c/em\u003e attached to the surface of the hepatocytes with infiltration of the intercellular space (Fig. 2A). The cell surface rose around \u003cem\u003eH. pylori\u003c/em\u003e attached to the cell surface, appearing to wrap around the head of \u003cem\u003eH. pylori\u003c/em\u003e; scanning electron microscopy confirmed that hepatocytes wrapped around H. pylori (Fig. 2B). \u003cem\u003eH. pylori\u003c/em\u003e invades and remains in the intercellular spaces and on the surface of the sterically constructed cell mass. In these locations, it remains in the bacillus form rather than converting to the coccoid form, the defensive morphology adopted to survive when environmental conditions are unfavorable. Therefore, \u003cem\u003eH. pylori\u003c/em\u003e might not only attach to the surface of the liver to invade the intracellular space, but it may also loosen intercellular junctions and invade intercellular spaces, thereby escaping from an environment that is difficult to survive in and facilitating survival. Loosening the intercellular space destabilizes intercellular connections, making cells more susceptible to cancer [37, 38]. Therefore, the entry of \u003cem\u003eH. pylori\u003c/em\u003e into the intercellular space may also favor cancer development due to infection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell Cycle Balance Assessment\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe assessed the balance of the cell cycle by performing a TUNEL assay to evaluate the status of apoptosis and an IHC for Ki-67 expression to evaluate the status of cell proliferation. The TUNEL assay demonstrated that apoptotic cells were mostly inside the artificial liver rather than on the artificial liver surface (Fig. 3A). \u003cem\u003eH. pylori\u003c/em\u003e incubation increased artificial liver apoptosis compared to that of control cells without such incubation. Additionally, quantification results (Fig. 3C) revealed an apoptosis increase in \u003cem\u003eH. pylori\u003c/em\u003e-infected artificial liver (LI = 30.7\u0026plusmn;19.6) compared to that of controls (LI = 5.2\u0026plusmn;3.3) (P = 0.00147).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIHC with an anti-Ki-67 antibody demonstrated that Ki-67 expression in the nuclei was detected in cells close to the surface of the artificial liver (Fig. 3B). The pattern and number of cells expressing Ki-67 (Fig. 3D) were similar between cells incubated with \u003cem\u003eH. pylori\u0026nbsp;\u003c/em\u003e(LI =28.1\u0026plusmn;10.9) and control cells without such incubation (LI = 25.7\u0026plusmn;4.5) (P = 0.426), suggesting that cell proliferation in the artificial liver was not augmented by \u003cem\u003eH. pylori\u003c/em\u003e infection.\u003c/p\u003e\n\u003cp\u003ePhosphorylated Akt was detected in the cytoplasm of 3D hepatocytes incubated with \u003cem\u003eH. pylori\u003c/em\u003e (Fig. 4), indicating \u003cem\u003eH. pylori\u0026nbsp;\u003c/em\u003eactivated Akt, which inhibited apoptosis and promoted cell survival in 3D hepatocytes. Incubation of 3D formation hepatocytes with \u003cem\u003eH. pylori\u003c/em\u003e did not increase phosphorylated Met compared with the control, but it activated \u0026beta;-catenin (Fig. 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFactors Activated by Helicobacter pylori Infection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRT-PCR results confirmed significantly higher levels of TNF-\u0026alpha; in \u003cem\u003eH. pylori\u003c/em\u003e-infected cells (mean 1.16 \u0026times; 10\u003csup\u003e-5\u003c/sup\u003e [SE 6.31 \u0026times; 10\u003csup\u003e-7\u003c/sup\u003e]) than they did in non-infected controls (mean 7.65 \u0026times; 10\u003csup\u003e-6\u003c/sup\u003e [SE 4.77 \u0026times; 10\u003csup\u003e-7\u003c/sup\u003e]; t = -5.065, P = 0.007), suggesting that \u003cem\u003eH. pylori\u003c/em\u003e induced TNF-\u0026alpha; expression, thereby upregulating apoptosis critical in hepatocarcinogenesis. Nevertheless, IL-8 levels (P = 0.419) were similar between the two groups (Fig. 5), suggesting that \u003cem\u003eH. pylori\u003c/em\u003e does not induce the expression of the most representative inflammatory cytokines in the artificial liver, unlike in the stomach. \u003cem\u003eH. pylori\u003c/em\u003e did not change Ets-1 levels (P = 0.704), and PCNA levels were significantly lower in the\u003cem\u003e\u0026nbsp;H. pylori\u003c/em\u003e-infected cells (mean 0.000267 [SE 1.14 x 10\u003csup\u003e-5\u003c/sup\u003e]) than in non-infected controls (mean 0.00047 [SE 1.28 x 10\u003csup\u003e-7\u003c/sup\u003e]; t = 11.840, P \u0026lt; 0.001). Significantly higher levels of NF-kB accumulation were detected in \u003cem\u003eH. pylori\u003c/em\u003e-incubated cells than in controls (Fig. 6).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eH. pylori\u003c/em\u003e incubation with 3D-formed hepatocytes clarifies how it infects human hepatocytes. Electron microscopy demonstrated that \u003cem\u003eH. pylori\u003c/em\u003e attaches to the hepatocyte surface and enters hepatocytes. Our previous \u003cem\u003ein vitro\u003c/em\u003e study demonstrated \u003cem\u003eH. pylori\u003c/em\u003e inoculation into hepatocytes [9], and the present findings support these results. Here, \u003cem\u003eH. pylori\u003c/em\u003e occupied intercellular spaces, suggesting loss of cell-to-cell connections and facilitating entry via this intercellular route. IHC and anti-\u0026beta;-catenin antibody findings suggest that \u0026beta;-catenin activation at cell surfaces during \u003cem\u003eH. pylori-\u003c/em\u003ehepatocyte incubation contributed to disrupted cell\u0026ndash;cell contacts and promoted intracellular entry through gaps between cells. \u003cem\u003eH. pylori\u003c/em\u003e usually inhabits human stomachs containing gastric juice. One of the reasons \u003cem\u003eH. pylori\u003c/em\u003e can survive in an acidic environment is that it produces urease, which neutralizes acids. Based on our findings, another reason may be its ability to move through the gaps between cells and internalize them to avoid a difficult extracellular environment. \u0026beta;-catenin activation may be crucial for \u003cem\u003eH. pylori\u003c/em\u003e survival in a difficult environment. In our artificial liver model, \u003cem\u003eH. pylori\u003c/em\u003e also activated \u0026beta;-catenin and potentially inhabited inter- and intra-cellular spaces for protracted periods.\u003c/p\u003e\n\u003cp\u003eWe previously reported that \u003cem\u003eH. pylori\u003c/em\u003e induces both cell proliferation and apoptosis [10], inferring that \u003cem\u003eH. pylori\u003c/em\u003e-driven cell-cycle acceleration can lead to carcinogenesis. Here, \u003cem\u003eH. pylori\u003c/em\u003e infection increased apoptosis and decreased cell proliferation in an artificial liver model. Thus, \u003cem\u003eH. pylori\u003c/em\u003e may contribute to liver carcinogenesis by increasing apoptosis in vitro. Further studies are needed to elucidate the mechanisms underlying hepatocarcinogenesis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eH. pylori\u003c/em\u003e infection induces several factors related to carcinogenesis. The transcription factor NF-kB is a critical mediator of immune and inflammatory responses and may play key roles in several diseases, including cancer; \u003cem\u003eH. pylori\u003c/em\u003e induces NF-kB activation [29]. Here,\u0026nbsp;\u003cem\u003eH. pylori\u003c/em\u003e was associated with activation of NF-kB, Akt, and TNF-\u0026alpha;, suggesting a role in carcinogenesis within the artificial liver environment. However, IL-8 was not increased by \u003cem\u003eH. pylori\u003c/em\u003e,\u0026nbsp;indicating that chronic inflammation with chemokine production is not involved in \u003cem\u003eH. pylori\u003c/em\u003e-associated hepatocarcinogenesis, unlike gastric carcinogenesis. IL-8 is a chemokine produced by macrophages, epithelial cells, airway smooth muscle cells, and vascular endothelial cells, and, unsurprisingly, IL-8 production did not increase in artificial livers. \u003cem\u003eH. pylori\u003c/em\u003e infection did not affect Ets-1 levels in this study; therefore, the involvement of Ets-1 in hepatocarcinogenesis was not clarified in our artificial liver model, and further studies are required.\u003c/p\u003e\n\u003cp\u003eThe main limitation of this study is the use of an artificial liver model, potentially limiting real-life applicability. Nevertheless, compared with other \u003cem\u003ein vitro\u003c/em\u003e methods for studying pathological conditions, RFB is advantageous because it facilitates cell culture 3D construction. Additional studies involving clinical populations are warranted to confirm the clinical validity of our findings.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e\u003cem\u003eH. pylori\u003c/em\u003e attached to the hepatocyte surface and entered hepatocytes, existing within intercellular spaces, and was associated with NF-kB, Akt, and TNF-\u0026alpha; activation, indicating a potential carcinogenic role within the artificial liver environment. Further studies clarifying whether these effects occur in the human liver \u003cem\u003ein vivo\u003c/em\u003e are warranted.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe greatly appreciate Professor Sae Ochi\u0026rsquo;s kind support of this study. We also thank the staff of the Core Research Facility, Research Center for Medical Sciences, and The Jikei University School of Medicine for their excellent electron microscopy techniques. Editorial support, in the form of medical writing, assembling tables and creating high-resolution images based on authors\u0026rsquo; detailed directions, collating author comments, copyediting, fact checking, and referencing, was provided by Editage, Cactus Communications, and funded by Grant-in-Aid for Scientific Research (C) 2008 (grant number: 20590457).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; references:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e8. \u0026nbsp; \u0026nbsp;Ito K, Nakamura M, Toda G, Negishi M, Torii A, Ohno T. Potential role of \u003cem\u003eHelicobacter pylori\u003c/em\u003e in hepatocarcinogenesis. Int J Mol Med. 2004;13:221\u0026ndash;7. https://doi.org/10.3892/ijmm.13.2.221\u003c/p\u003e\n\u003cp\u003e9. \u0026nbsp; \u0026nbsp;Ito K, Yamaoka Y, Ota H, El-Zimaity H, Graham DY. Adherence, internalization, and persistence of \u003cem\u003eHelicobacter pylori\u003c/em\u003e in hepatocytes. Dig Dis Sci. 2008;53:2541\u0026ndash;9. https://doi.org/10.1007/s10620-007-0164-z\u003c/p\u003e\n\u003cp\u003e10. \u0026nbsp;Ito K, Yamaoka Y, Yoffe B, Graham DY. Disturbance of apoptosis and DNA synthesis by \u003cem\u003eHelicobacter pylori\u003c/em\u003e infection of hepatocytes. Dig Dis Sci. 2008;53:2532\u0026ndash;40. https://doi.org/10.1007/s10620-007-0163-0\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This study was funded by a Grant-in-Aid for Scientific Research (C) 2008 (grant number: 20590457).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e: The authors have no relevant financial or nonfinancial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e: The datasets generated and/or analyzed in the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e: All authors contributed to the conception and design of this study. The material preparation, data collection, and analyses were performed by Ito, Maehashi, and Matsuura. Kyoko Ito wrote the first draft of the manuscript, and all authors commented on the previous versions of the manuscript. All the authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e: In line with the ethical guidelines issued by the Japanese Ministry of Health, Labor, and Welfare, this experiment on non-human tissues did not require ethical approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYamanaka H, Non. -\u003cem\u003eH. pylori Helicobacter\u003c/em\u003e species detected in laboratory animals. 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J Cell Biol. 1993;120:757\u0026ndash;66. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1083/jcb.120.3.757\u003c/span\u003e\u003cspan address=\"10.1083/jcb.120.3.757\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"artificial liver, Helicobacter pylori, hepatocellular carcinoma, liver diseases, cancer","lastPublishedDoi":"10.21203/rs.3.rs-7797022/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7797022/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eHelicobacter\u003c/em\u003e DNA has been detected in the liver tissues of patients with hepatocellular carcinoma and other liver diseases, suggesting that \u003cem\u003eHelicobacter\u003c/em\u003e infection may be involved in the etiology of liver diseases. This study aimed to investigate the mechanism of \u003cem\u003eHelicobacter\u003c/em\u003e invasion into cells and the structural and functional responses of cells to this invasion. Using the radial-flow bioreactor methodology, a novel artificial liver model was used to reconstruct cell cultures in three dimensions and to explore how \u003cem\u003eHelicobacter pylori\u003c/em\u003e infects human hepatocytes. \u003cem\u003eH. pylori\u003c/em\u003e was found to attach to the hepatocyte surface and to enter hepatocytes, existing within intercellular spaces. \u003cem\u003eH. pylori\u003c/em\u003e infection was also associated with increased apoptosis as well as NF-kB and TNF-α activation in the artificial liver. Therefore, \u003cem\u003eH. pylori\u003c/em\u003e exhibits a potential carcinogenic role in artificial liver environments. Further studies are warranted to clarify whether these effects occur in the human liver \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"A model of Helicobacter infection using an artificial liver constructed by a radial-flow bioreactor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-23 19:11:14","doi":"10.21203/rs.3.rs-7797022/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-10-10T08:10:26+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-10T03:08:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-07T13:46:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Human Cell","date":"2025-10-07T03:45:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9ce13f4d-2898-496a-8f26-545728ee054e","owner":[],"postedDate":"October 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-08T03:34:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-23 19:11:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7797022","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7797022","identity":"rs-7797022","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}