Pilot study of the role of ferroptosis in abnormal biological behaviour of keratinocytes in psoriasis vulgaris

Pilot study of the role of ferroptosis in abnormal biological behaviour of keratinocytes in psoriasis vulgaris | 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 Pilot study of the role of ferroptosis in abnormal biological behaviour of keratinocytes in psoriasis vulgaris Ningling Wu, Qian Hu, Lu zhou, Zhibing Fu, Xiaoliang Tong, Lihua Gao, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4113873/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 Abnormal biological behaviour of keratinocytes (KCs) is a critical pathophysiological manifestation of psoriasis. Ferroptosis is programmed cell death induced by the accumulation of lipid reactive oxygen species (ROS) in the presence of increased intracellular iron ions or inhibition of GPX4. Objectives The purpose of this study was to investigate the effects of ferroptosis on the biological behaviour of KCs in psoriasis vulgaris and its possible regulatory mechanisms in clinical samples, cells, and mouse models. Methods We first examined the differences in the expression of GPX4 and 4-HNE between psoriasis and normal human lesions. And detected KRT6, FLG, and inflammatory cytokines after inducing ferroptosis in animal and cell models by RT-qPCR, Western blot, immunohistochemistry, and flow cytometry. Results We found that GPX4 was decreased and that the oxidation product 4-hydroxy-2-nonenal (HNE) was increased in the skin lesions of patients with psoriasis vulgaris. The expression level of GPX4 correlates with the severity of skin lesions. Moreover, inducing ferroptosis promoted the expression of FLG and reduced the expression of KRT6 and inflammatory cytokines in vitro, and alleviated the phenotype of skin lesions in vivo. Conclusions This study confirms the existence of ferroptosis in psoriatic lesions, which may be inversely correlated with disease severity. The ferroptosis inducer RSL3 ameliorated psoriatic symptoms by improving the abnormal biological behaviour of KCs. Psoriasis vulgaris ferroptosis Keratinocytes GPX4 abnormal biological behaviours Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Psoriasis is a common inflammatory skin disease characterised by the hyperproliferation of keratinocytes (KC) [ 1 , 2 ] . The abnormal biological behaviour of KCs in psoriasis, namely excessive proliferation, delayed differentiation, and release of inflammatory chemokines, plays a crucial role in the pathogenesis of psoriasis [ 3 – 5 ] . In the initial phase of psoriasis, antimicrobial peptides (AMPs) released by KCs bind to their DNA and trigger dendritic cells (DCs) to activate helper T (Th)17 inflammatory response [ 6 , 7 ] . In the maintenance and expansion stage of inflammation, over-proliferating KCs synthesise and release more IL-6, IL-1, TNF-α, and chemokines CXCL1, CXCL8, and CCL20 to further attract macrophages and neutrophils to produce an increasingly fierce inflammatory reaction [ 8 – 10 ] . Therefore, further clarification and improvement of specific mechanisms of the abnormal biological behaviour of KCs are necessary. Ferroptosis is programmed cell death induced by the accumulation of lipid reactive oxygen species (ROS) in the presence of increased intracellular iron ions or inhibition of glutathione peroxidase 4 (GPX4) [ 11 ] . GPX4 is a central inhibitor of ferroptosis that converts toxic lipid hydroperoxides into nontoxic alcohols [ 12 – 14 ] . In other words, inhibiting GPX4, such as RSL3, can directly induce ferroptosis in different cells [ 15 – 18 ] . The latest research on ferroptosis has been extended to the mechanisms of hepatitis C virus replication, neutrophils triggering systemic lupus erythematosus, asthma, rheumatoid arthritis, and other infectious immune-inflammatory diseases [ 19 – 22 ] . Notably, Arbiser found significant differences in ferroptosis-related genes, including GPX4, transferrin (TFRC or TFR1), and Nrf2, in the lesions of patients with psoriasis by analysing biological information in the GEO database [ 23 ] . The authors speculated that increased ferroptosis plays a protective role in the disease process, which is called physiological ferroptosis [ 23 ] . Shou et al. also demonstrated the presence of ferroptosis in psoriasis lesions [ 24 ] . However, the correlation between ferroptosis and psoriasis and the effect of ferroptosis on the biological behaviour of KCs remains unclear. In this study, we confirmed that ferroptosis-related molecules change in psoriasis lesions and that the occurrence of ferroptosis may be negatively correlated with disease severity. Furthermore, the ferroptosis inducer RSL3 might regulate the abnormal biological behaviour of HaCaT cells and alleviate the phenotype of IMQ-induced psoriasis-like mouse models, providing a new potential target for the prevention and treatment of psoriasis vulgaris. 2. Materials and Methods 2.1 Human subjects. Forty-nine skin biopsy specimens were obtained: 23 from patients with psoriasis vulgaris and 26 from healthy individuals. Patient information, PASI scores, and staging criteria were recorded. This study was approved by the Ethics Committee of Third Xiangya Hospital, Central South University (Ethics No. 2021-S247) and consent was obtained from all subjects. 2.2 M5-induced psoriasis-like cell model. Human immortalised keratinocyte HaCaT cells were purchased from the China Typical Culture Preservation Center, Wuhan University (item number: GDC0106). Medium 1640 (Pricella, China) supplemented with 10% foetal bovine serum (Pricella, China) was cultured in a 37°C cell incubator containing 5% CO 2 . A psoriasis-like keratinocyte model was established by adding an M5 cytokine combination (IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α, each at a final concentration of 5ng/ml) to the medium of HaCaT keratinocytes [ 25 ] . 2.3 IMQ-induced psoriasis mice model establishment, RSL3 intervention, and PASI score BALB/c mice were purchased from the Hunan Slyke Jingda Company. BALB/c mice were used to establish an IMQ-induced psoriasis-like mouse model [ 26 ] . Female BALB/C mice aged 6–8 weeks were adaptatively fed for 1 week and depilated at approximately 2 cm×2 cm on their backs. Sixteen mice were randomly divided into four groups: blank control group (Control, Ctrl), IMQ model group (IMQ), IMQ model + ferroptosis inducer group (IMQ + RSL 3), and IMQ model + solvent control group (IMQ + Vehicle). The investigator was not blinded to the group allocation. Five percent IMQ (Sichuan Mingxin Pharmaceutical, China) (62.5 mg) was applied to the back hair removal area once daily for 8 days. An equal volume of RSL3 (2.0 mg/kg/d, MCE, China) or 10% DMSO (100 µl) was painted on the back of the mouse 30 min before IMQ application. The intervention was performed once daily, and the mice were weighed, photographed, and scored according to a previous mouse PASI scoring standard [ 27 ] . Details are as follows: PASI scoring criteria were scored based on erythema and scaliness, respectively, and each item was rated as 0–4 points according to severity. 0, none ; 1, mild ; 2, moderate ; 3, severe ; and 4, very severe . On day 9, significant differences were observed among the groups. The experimental Animal Welfare Ethics Committee of Central South University has approved this part of the experimental protocol (Ethics No. 2021sydw0063). 2.4 Cell viability was detected by the CCK8 kit HaCaT cells were inoculated in 10% complete medium and treated with different concentrations of RSL3 for 24, 48, and 72 h. Cell viability was determined using a CCK8 kit (Beyotime, China). 2.5 ROS detection Intracellular ROS levels were observed by flow cytometry and fluorescence microscopy, according to the instructions of the reactive oxygen species detection kit (Beyotime, China). 2.6 Detection of apoptosis Flow cytometry and fluorescence microscopy were performed according to the procedures of the Annexin-V-FITC apoptosis detection kit (Beyotime, China). 2.7 Transmission electron mMicroscope (TEM) TEM was used to examine the mitochondrial morphological changes in HaCaT cells treated with 5 µM RSL3 for 24 h. The cells were digested with trypsin, harvested, and fixed in 2.5% glutaraldehyde (EM grade) overnight at 4°C. After fixation in 1% osmic acid, the cell samples were dehydrated and placed in standard embedding moulds. Suitable areas were selected, and 0.08 µm ultrathin sections were stained with lead citrate and uranyl acetate. The sections were examined using a TEM (JEM-1400Plus, JEOL, Ltd., Tokyo, Japan). 2.8 RT-qPCR Total RNA was extracted from tissues and cells using Trizol (AG, China). Next, 700 µg RNA was reversely transcribed into cDNA according to the manufacturer’s instructions (Vazyme, China). The amplification solution was prepared according to the instructions for SYBR Green fluorescent dye reagent (Novizan, China), and a real-time PCR system (Bio-Rad, USA) was used for the amplification reaction. The primer sequences used are listed in Supplementary Table S1. 2.9 Western blot Protein concentrations were measured in a lysis buffer solution (Beyotime, China) using a BCA assay kit (Beyotime, China). A 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) was prepared and transferred to a polyvinylidene fluoride membrane (Millipore, USA) after electrophoresis. After sealing in a 5% skim milk solution, GPX4 (AiFang, AF301385, 1:500), 4HNE (Abcam, ab46545, 1:1000), GAPDH (Affinity, AF7021, 1:300), KRT6 (Abcam, ab93279, 1:2000), and FLG (Affinity, DF13653, 1:1000) were diluted with 1 × PBST. The goat was incubated on a low-speed shaker at 4 ℃ overnight, and the secondary antibody was diluted with 1 PBST: HRP goat anti-mouse IgG (Proteintech, SA00001-1, 1:5000) and HRP goat anti-rabbit IgG (Proteintech, SA00001-2, 1:6000). 2.10 HE and immunohistochemical staining analysis Human and mouse skin tissues were fixed with formalin, and paraffin-embedded sections (6µm) were treated with haematoxylin and eosin (H&E) stain and stored at room temperature. The immunohistochemical procedure was described in the literature [ 28 ] . The following antibodies were used: GPX4 (AF301385, 1:80; AiFang), 4-HNE (ab46545, 1:100; Abcam), KRT6 (10590-1-AP, 1:400; Proteintech), and FLG (AF10489, 1:100; AiFang). The immune response intensity of each section was divided into four grades [ 29 ] :0 (no staining), 1+ (weak staining), 2+ (medium staining), and 3+ (strong staining). The percentage of stained cells was divided into two grades:1 (≤ 30%) and 2 (> 30%). Final immunohistochemical staining score = staining score×percentage of stained cells. 2.11 Statistical methods All charts of the data in this study were generated using GraphPad Prism software (version 8.0), and the data were represented by mean ± standard deviation (SD). Two independent sample t tests were used to compare the measurement data of the two groups. Comparison of measurement data of multiple groups was conducted using one-way ANOVA. Non-normally distributed data were analysed using a non-parametric test (Mann-Whitney U test). Statistical significance was set at P < 0.05. 3. Results 3.1 Ferroptosis was increased in psoriatic lesions. GPX4 is a critical gene involved in ferroptosis. The expression of GPX4 in psoriasis vulgaris lesions and normal specimens was detected using RT-qPCR, immunohistochemistry, and Western Blot. The results of RT-qPCR showed that the expression level of GPX4 mRNA in psoriasis lesions (N = 23, 0.48 ± 0.48) was significantly lower than that in healthy controls (N = 26, 1.72 ± 1.54. Figure 1 A). Immunohistochemical and Western blot results confirmed this finding (Figs. 1 B and 1 D). Western blot and immunohistochemistry revealed an increase in the oxidation product 4-hydroxy-2-nonenal (HNE) in psoriasis lesions (Figs. 1 C and 1 E). These results are consistent with the characteristics of ferroptosis. We then conducted a correlation analysis between the GPX4 mRNA relative expression level and PASI score. The results showed that the higher the PASI score, the higher the GPX4 expression level and that the two were positively correlated. (Fig. 1 F). 3.2 RSL3 induced ferroptosis in HaCaT cells. Cell viability at different concentrations of RSL3 and at three treatment times was assessed using a Cell Counting Kit-8 (CCK-8) (Fig. 2 A). At a concentration of 10 µM RSL3, cell viability was close to 0 at all three treatment times. Under a light microscope, the number of cells decreased with an increase in the concentration after 24 h of treatment (Fig. 2 B). The IC50 of RSL3 was 6.04 µM after 24 h of treatment. RSL3 seldom showed cytotoxicity at a concentration below 6.04 µM. Western blot showed that 5 µM and 7.5 µM RSL3 inhibited the expression of GPX4 protein (Fig. 2 C). Consequently, we used 5 µM RSL3 in HaCaT cells for 24 h. The cells displayed increased intracellular ROS levels, as assessed by fluorescence microscopy and flow cytometry (Figs. 2 D and 2 E). We also found that HaCaT cells displayed an extensive percentage of dead cells after RSL3 treatment (Figs. 2 F and 2 G). Electron microscopy revealed increased mitochondrial membrane density and decreased crista levels (Fig. 2 H). Therefore, 5 µM was the optimal concentration of RSL3 for inducing ferroptosis in HaCaT cells. 3.3 RSL3 regulated biological behaviours of cells in vitro. HaCaT cells were induced with M5 to establish a psoriatic cell model, and the cells were treated with 5 µM RSL3 for 24 h [ 25 ] . KRT6 is an excessive proliferation marker of psoriatic KCs; filaggrin (FLG) is a late differentiation marker of normal KCs [ 30 , 31 ] . RT-qPCR results showed an increased expression of IL-17a, IL-6, IL-1β, CXCL1, and KRT6 and a decreased expression of FLG, prompting the successful establishment of the M5-induced psoriasis cell model. The RSL3 groups showed decreased KRT6 (Fig. 3 A and 3 B); increased FLG (Figs. 3 C and 3 D); and decreased IL-17a, IL-6, IL-1β, CXCL1, CXCL2, and CCL20 (Fig. 3 E). The results of this study suggest that the ferroptosis inducer RSL3 inhibits proliferation, promotes differentiation, and inhibits the expression of chemokines and inflammation in HaCaT cells induced by M5. 3.4 RSL3 alleviated the phenotype of skin lesions in vivo Five-percent Imiquimod (IMQ) of 62.5 mg was applied to the depilated area of the back once daily for 8 d. An equal volume of RSL3 (2.0 mg/kg/d) or 10% DMSO (100 µl) was painted on the back of the mouse 30 min before IMQ application. On day 9, we observed that erythema and scales in the back lesions of mice in the RSL3 group were significantly reduced, suggesting that RSL3 could improve psoriatic dermatitis induced by IMQ (Figs. 4 A and 4 B). HE results showed that the IMQ group was characterised by thickening of the epidermis, hyperkeratosis, and parakeratosis (Fig. 4 C). These results indicate that RSL3 improved the local inflammatory response and reduced epidermal thickness in IMQ-induced psoriatic mouse models. 3.5 RSL3 regulated the KCs’ biological behaviours in IMQ- treated mouse models. RT-qPCR and immunohistochemical results revealed that RSL3 decreased KRT6 expression and promoted FLG expression in IMQ-induced psoriasis lesions in mice (Figs. 5 A, 5 B, 5 C, and 5 D). The expression of IL-17a and CXCL1 in the IMQ + RSL3 group was decreased, and that in the model and solvent control groups was increased, with a statistical difference, and IL-6, IL-1β, CXCL2, and CCL20 showed a downward trend, with no significant statistical significance (Fig. 5 E). These results suggest that RSL3 inhibits proliferation, promotes differentiation, and inhibits inflammation during psoriasis treatment. 4. Discussion Ferroptosis, a form of regulated cell death discovered recently, is characterized by iron-dependent cell demise triggered by excessive lipid peroxidation in the cell membrane [ 32 ] . It has been verified that KCs in psoriasis vulgaris exhibit anti-death properties. This impediment in the cell death and differentiation process leads to rapid cell proliferation, mirroring the behavior of tumor cells [ 33 ] . However, the relationship between psoriasis vulgaris and Ferroptosis remains unclear. GPX4, a negative regulator that decreases in the process of ferroptosis, is notably diminished in psoriasis vulgaris, aligning with findings from Shou et al. [ 23 , 24 ] . The association between GPX4 levels and the PASI score indicates a negative correlation between ferroptosis occurrence and the severity of psoriasis. In an effort to delve deeper into the specific specific interaction between ferroptosis and psoriasis vulgaris, we employed a ferroptosis inducer, RSL3, which inhibit GPX4 protein in HaCaT cells. Our findings reveal that HaCaT cells are highly susceptible to RSL3, indicating that RSL3 curtails HaCaT cell proliferation in a dose- and time-dependent manner. Concurrently, intracellular ROS and apoptosis levels rise following treatment with RSL3. Furthermore, in the M5-induced psoriasis model, RSL3 overexpression leads to a reduction in the proliferation marker KRT6 and an increase in the expression of barrier protection factors and late differentiation gene FLG. Animal experiment corroborate these findings. Those outcomes align with the tumor-like characteristics of KCs in psoriasis, suggesting that ferroptosis mitigates psoriasis progression by inhibiting KC proliferation and promoting KC differentiation. Considering the activation of inflammatory pathways in Keratinocytes (KCs) and the correlation between ferroptosis and psoriasis, we next examined whether using ferroptosis inducers would exacerbate inflammation. Surprisingly, we observed a significant reduction in most inflammatory chemokines, including IL-17, IL-22, CXCL1, IL-6. Specifically, IL-17 and CXCL1 levels decreased significantly at both cellular and animal levels, indicating that RSL3 may supress certain inflammatory chemokines’ expression. The lack of statistical difference in the animal model may stem from an incomplete understanding of physical ferroptosis’s optimal range. The whole mouse may retain a self-compensatory ability and exhibit individual differences, increasing the exploration of physiological ferroptosis in mice. Recent studies reveal the intricate relationship between programmed cell death and inflammation [ 34 , 35 ] . Ferroptosis, a type of programmed cell death, can either exacerbate inflammation through damage-associated molecular patterns (DAMPs) [ 36 ] or attract immune cells, complicating the inflammatory response. Conversely, pathogen-associated molecular patterns (PAMPs) induce the specific antibacterial immune response to control infection [ 37 ] . Research on ferroptosis’s role in skin inflammation is still in its infancy. Shou et al. demonstrated that ferroptosis inducers do not promote the inflammation during psoriasis, suggesting that the ferroptosis inhibitors may alleviate inflammation by decreasing the intracellular oxidative stress levels [ 24 ] . Additionally, studies have indicated that the RSL3 may suppress the LPS-induced cellular inflammation by increasing ROS, which activates the Nrf2 anti-oxidation system, thereby reducing the secretion of inflammatory factors [ 38 ] . Therefore, the inhibitors and inducers of ferroptosis indirectly affect the inflammation by regulating the level of intracellular oxidative stress. It's important to note that ferroptosis can act as a double-edged sword in the organism. Among tumors, inducing ferroptosis in cancer cells may decrease the number of cells and inhibit cell growth. However, ferroptotic cells appeal immune cells by DAMPs, potentially promoting the development of tumors [ 39 ] . In the context of inflammation, ferroptosis influences the quantity and function of immune cells, weakening immune response; while also allowing immune cells to identify the ferroptotic cells, causing a series of inflammatory and specific reactions [ 40 ] . This leads us to hypothesize the existence of a balance in organism ferroptosis, which we term “physiological ferroptosis”. This balance maximizes ferroptosis’s beneficial effects while also decreases the harmful impacts. In conclusion, our study elucidates the differential expression of GPX4 in skin lesions between healthy controls and patients with psoriasis vulgaris. Additionally, it has been confirmed that the HaCaT cell are highly sensitive to RSL3. The application of RSL3 could inhibit cell proliferation and promote cell differentiation by downregulating the GPX4 protein, thereby alleviating the symptoms of psoriatic dermatitis through inflammation control. This may become the significant target for psoriasis vulgaris treatment. Our study has limitations, notably small sample size. Larger clinical trials are necessary to investigate the association between ferroptosis and disease progression further. More research is necessary to explore how the ferroptosis inducer RSL3 regulates the abnormal biological behaviour of KCs at both cellular and animal levels and establish ferroptosis inhibitors as controls. 5. Conclusions In conclusion, this study confirms the existence of ferroptosis in psoriatic lesions, which may be inversely correlated with disease severity. The ferroptosis inducer RSL3 ameliorated psoriatic symptoms by improving the abnormal biological behaviour of KCs. Abbreviations Abbreviations Full name KCs keratinocytes GPX4 glutathione peroxidase 4 IMQ imiquimod Th T helper cells TNF tumour necrosis factor IL interleukin CXCL C-X-C motif chemokine ligand CCL C-C motif chemokine ligand GSH glutathione Xc ― cystine/glutamate transporter ROS reactive oxygen species 4HNE 4-Hydroxynonenal KRT keratin FLG filaggrin PI propidium iodide HE hematoxylin and eosin stain PASI psoriasis area and severity index Ctrl control Declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of Third Xiangya Hospital, Central South University (Ethics No. 2021-S247) and the experimental Animal Welfare Ethics Committee of Central South University (Ethics No. 2021sydw0063). Authors' contributions Jinrong Zeng and Jianyun Lu: Conceptualization and Supervision. Jinrong Zeng, Lu zhou, and Zhibing Fu: Writing- Original draft preparation. Qian Hu, Ningling Wu: Investigation, Methodology and Formal analysis. Ningling Wu: Visualization. Xiaoliang Tong: Validation. Lina Tan, Siyu Yan, Lihua Gao, and Dan Wang: Data Curation. Jianyun Lu: Writing - Review & Editing. Jinrong Zeng All authors read and approved the final manuscript. Consent for publication Not applicable Availability of data and materials All relevant data are included in the present manuscript or in the supplements. Raw data are available upon reasonable request by the first author R.M. Acknowledgements Not applicable. Contents of the manuscript have not been previously published and are not currently submitted elsewhere. I accept responsibility for the scientific integrity of the work described in this manuscript. All listed authors have seen and approved of the manuscript and will sign off on any subsequent manuscript revisions References Ding X, Wang T, Shen Y et al. Prevalence of psoriasis in China: a population-based study in six cities. 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Microglia and macrophage exhibit attenuated inflammatory response and ferroptosis resistance after RSL3 stimulation via increasing Nrf2 expression. J Neuroinflammation. 2021; 18: 249. doi: 10.1186/s12974-021-02231-x Tang D, Chen X, Kang R et al. Ferroptosis: molecular mechanisms and health implications. Cell Res. 2021; 31: 107–25. doi: 10.1038/s41422-020-00441-1 Chen X, Kang R, Kroemer G et al. Ferroptosis in infection, inflammation, and immunity. J Exp Med. 2021; 218. doi: 10.1084/jem.20210518 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-4113873","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":281188176,"identity":"47c8f2a3-8ad5-458c-a00e-8e1794a27a9c","order_by":0,"name":"Ningling Wu","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Ningling","middleName":"","lastName":"Wu","suffix":""},{"id":281188177,"identity":"baa01997-383d-4a92-b5bd-851a32f05e3d","order_by":1,"name":"Qian Hu","email":"","orcid":"","institution":"Changsha Fourth Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Hu","suffix":""},{"id":281188178,"identity":"1c77c476-40e7-4d8e-9180-5cdcb5f4e3da","order_by":2,"name":"Lu zhou","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"zhou","suffix":""},{"id":281188179,"identity":"fe116168-c18c-49ae-b54a-5c0a01dc390d","order_by":3,"name":"Zhibing Fu","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Zhibing","middleName":"","lastName":"Fu","suffix":""},{"id":281188180,"identity":"9f60a008-8876-4dd8-a600-56ddcfb072f7","order_by":4,"name":"Xiaoliang Tong","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoliang","middleName":"","lastName":"Tong","suffix":""},{"id":281188181,"identity":"65911314-956a-4be3-bd53-d1b6a0f58154","order_by":5,"name":"Lihua Gao","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Lihua","middleName":"","lastName":"Gao","suffix":""},{"id":281188182,"identity":"921bacac-4c16-4c8c-8668-260a99e3e073","order_by":6,"name":"Lina Tan","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Lina","middleName":"","lastName":"Tan","suffix":""},{"id":281188183,"identity":"337296a5-262a-4362-8069-4b3b6f68e79e","order_by":7,"name":"Siyu Yan","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Siyu","middleName":"","lastName":"Yan","suffix":""},{"id":281188184,"identity":"c94fe782-bcf6-4274-a33d-2021ae09036c","order_by":8,"name":"Dan Wang","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Wang","suffix":""},{"id":281188185,"identity":"52d89efb-b0d8-4bb5-b87d-48adbb10538e","order_by":9,"name":"Jinrong Zeng","email":"data:image/png;base64,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","orcid":"","institution":"Central South University","correspondingAuthor":true,"prefix":"","firstName":"Jinrong","middleName":"","lastName":"Zeng","suffix":""},{"id":281188186,"identity":"064e330b-1f2b-450f-8daa-740f85dcf423","order_by":10,"name":"Jianyun Lu","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Jianyun","middleName":"","lastName":"Lu","suffix":""}],"badges":[],"createdAt":"2024-03-16 15:44:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4113873/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4113873/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53183722,"identity":"da083669-2bb0-4c64-aab0-127b13691fef","added_by":"auto","created_at":"2024-03-21 16:03:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1222535,"visible":true,"origin":"","legend":"\u003cp\u003eFerroptosis was increased in psoriatic lesions.\u003c/p\u003e\n\u003cp\u003e(A) RT-qPCR was performed to measure GPX4 expression. (B) Protein levels of GPX4 were determined by Western blot. (C) Protein levels of 4-HNE were detected by Western blot. (D) Immunohistochemical staining of GPX4 (Scale bars: 50 μm) was observed by immunohistochemistry staining. (E) 4-HNE (Scale bars: 50 μm) was observed by immunohistochemistry staining. (F) Correlational analysis of GPX4 expression and PASI score. **= P\u0026lt;0.01.***=P\u0026lt;0.001. Figure 2. RSL3 induced ferroptosis in HaCaT cells.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4113873/v1/2764d04fe13286c900fa544a.png"},{"id":53183723,"identity":"361ba028-969e-481b-a91f-1eb9ae4b3e64","added_by":"auto","created_at":"2024-03-21 16:03:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":977852,"visible":true,"origin":"","legend":"\u003cp\u003eRSL3 induced ferroptosis in HaCaT cells.\u003c/p\u003e\n\u003cp\u003e(A) HaCaT cells were treated with RSL3 at the concentration of 0, 0.5, 1, 1.5, 2, 5, and 10 μM for 24, 48, and 72 h, respectively. Proliferation of HaCaT cells was detected by a CCK8 kit. (B) HaCaT cells were treated with RSL3 at the concentration of 0, 2.5, 5, and 10 μM for 24 h, and the number and morphology of HaCaT cells were observed under the light microscope. (C) HaCaT cells were treated with RSL3 at the concentration of 0, 2.5, 5, and 7.5 μM for 24 h, and the expression of GPX4 protein was detected by Western blot. (D) HaCaT cells were treated with 5 μM RSL3 for 24 h, and the ROS content was detected under fluorescence microscope. (E) Cellular ROS was analysed with flow cytometry. (F) Representative results of annexin V-FITC/PI staining by flow cytometry and fluorescence microscope (G). Annexin V-FITC staining positive cells showed green fluorescence, PI staining positive cells showed red fluorescence, apoptotic cells were only marked with green fluorescence, necrotic cells were marked with both green and red fluorescence, and normal cells were without fluorescence staining. (H) Ultrastructure of HaCaT cells was determined by TEM after 24 h treatment with 5 μΜ RSL3. Yellow arrows: typical mitochondria morphology. Red arrows: shrunken mitochondria morphology. Scale bar is 1 μm. ns=nonsignificant. *= P\u0026lt;0.05. **= P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4113873/v1/bf680d9e4f35ac4f60e54928.png"},{"id":53183724,"identity":"a65732fc-87be-4ff7-9f0e-9f7ffa46aa4c","added_by":"auto","created_at":"2024-03-21 16:03:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":243088,"visible":true,"origin":"","legend":"\u003cp\u003eRSL3 regulated biological behaviours of cells in vitro.\u003c/p\u003e\n\u003cp\u003eHaCaT cells were stimulated with M5 (5ng/ml) consisting of IL-17A, IL-22, IL-1α, TNF-α, and oncostatin M cytokines for 24 h to establish a psoriatic cell model that mimics characteristics of psoriatic inflammation and hyperproliferation in vitro, followed by treatment with 5 μM RSL3 for 24 h. (A) Cells were harvested, and RNA was extracted for RT-qPCR analysis of KRT6 mRNA levels. (B) Protein levels of KRT6 were determined by Western blot analysis. (C) FLG mRNA expression in cells was measured. (D) Protein levels of FLG in the cells were measured. (E) Cells were harvested, and RNA was extracted for RT-qPCR analysis of IL-17A, IL-6, IL-1β, CXCL1, CXCL2, and CCL20. ns=nonsignificant. *= P\u0026lt;0.05. **=P\u0026lt;0.01. ***=P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4113873/v1/ac56c998dcf9fc235a02b247.png"},{"id":53183725,"identity":"7b86050f-af77-4796-ba05-2fe89b8a9148","added_by":"auto","created_at":"2024-03-21 16:03:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1898985,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic changes in skin lesions on backs of mice.\u003c/p\u003e\n\u003cp\u003eNo intervention was performed in the control group. IMQ was applied daily to each experimental group, and RSL3 corresponding to the control solvent was applied externally in the IMQ + RSL 3 IMQ + Vehicle group 30 min before IMQ was applied daily. (A) Macroscopic appearance of the mouse back on day 9; (B) erythema and scaling were scored on days 0, 2, 4, 6, and 8 based on the PASI. (C) H\u0026amp;E staining of mouse skin (scale bars:100 μm, 50 μm). (D) Immunohistochemical staining of KRT6 in mouse skin lesions (Scale bars: 50 μm); (E) Immunohistochemical staining score of KRT6. (F) Immunohistochemical staining of FLG in mouse skin lesions. (Scale bars: 100 μm). ns=nonsignificant. *= P\u0026lt;0.05. **=P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4113873/v1/d102b6f16dade4afd43128a3.png"},{"id":53185626,"identity":"9cdd630e-5fbd-4f88-8ba2-4791f79aee0a","added_by":"auto","created_at":"2024-03-21 16:11:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1308486,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of RSL3 on IMQ-induced psoriasis-like skin lesion.\u003c/p\u003e\n\u003cp\u003eRT-qPCR was performed to measure the expression of KRT6 (A), FLG (C), and inflammatory cytokines and chemokines in mouse skin lesions (E). Protein levels of KRT6 (B) and FLG (D) were measured by Western blot. GAPDH served as an internal reference. ns=nonsignificant. *= P\u0026lt;0.05. **=P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4113873/v1/fa6a654fdd8fdd68faa339af.png"},{"id":53418197,"identity":"825aa304-73e3-418f-bb32-471cbfc2a281","added_by":"auto","created_at":"2024-03-25 18:06:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2992156,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4113873/v1/61331419-6023-4d00-a277-9895321f812f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pilot study of the role of ferroptosis in abnormal biological behaviour of keratinocytes in psoriasis vulgaris","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePsoriasis is a common inflammatory skin disease characterised by the hyperproliferation of keratinocytes (KC) \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The abnormal biological behaviour of KCs in psoriasis, namely excessive proliferation, delayed differentiation, and release of inflammatory chemokines, plays a crucial role in the pathogenesis of psoriasis \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. In the initial phase of psoriasis, antimicrobial peptides (AMPs) released by KCs bind to their DNA and trigger dendritic cells (DCs) to activate helper T (Th)17 inflammatory response \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. In the maintenance and expansion stage of inflammation, over-proliferating KCs synthesise and release more IL-6, IL-1, TNF-α, and chemokines CXCL1, CXCL8, and CCL20 to further attract macrophages and neutrophils to produce an increasingly fierce inflammatory reaction \u003csup\u003e[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Therefore, further clarification and improvement of specific mechanisms of the abnormal biological behaviour of KCs are necessary.\u003c/p\u003e \u003cp\u003eFerroptosis is programmed cell death induced by the accumulation of lipid reactive oxygen species (ROS) in the presence of increased intracellular iron ions or inhibition of glutathione peroxidase 4 (GPX4) \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. GPX4 is a central inhibitor of ferroptosis that converts toxic lipid hydroperoxides into nontoxic alcohols \u003csup\u003e[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. In other words, inhibiting GPX4, such as RSL3, can directly induce ferroptosis in different cells \u003csup\u003e[\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. The latest research on ferroptosis has been extended to the mechanisms of hepatitis C virus replication, neutrophils triggering systemic lupus erythematosus, asthma, rheumatoid arthritis, and other infectious immune-inflammatory diseases \u003csup\u003e[\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Notably, Arbiser found significant differences in ferroptosis-related genes, including GPX4, transferrin (TFRC or TFR1), and Nrf2, in the lesions of patients with psoriasis by analysing biological information in the GEO database \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. The authors speculated that increased ferroptosis plays a protective role in the disease process, which is called physiological ferroptosis \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Shou et al. also demonstrated the presence of ferroptosis in psoriasis lesions \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. However, the correlation between ferroptosis and psoriasis and the effect of ferroptosis on the biological behaviour of KCs remains unclear.\u003c/p\u003e \u003cp\u003eIn this study, we confirmed that ferroptosis-related molecules change in psoriasis lesions and that the occurrence of ferroptosis may be negatively correlated with disease severity. Furthermore, the ferroptosis inducer RSL3 might regulate the abnormal biological behaviour of HaCaT cells and alleviate the phenotype of IMQ-induced psoriasis-like mouse models, providing a new potential target for the prevention and treatment of psoriasis vulgaris.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Human subjects.\u003c/h2\u003e \u003cp\u003eForty-nine skin biopsy specimens were obtained: 23 from patients with psoriasis vulgaris and 26 from healthy individuals. Patient information, PASI scores, and staging criteria were recorded. This study was approved by the Ethics Committee of Third Xiangya Hospital, Central South University (Ethics No. 2021-S247) and consent was obtained from all subjects.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 M5-induced psoriasis-like cell model.\u003c/h2\u003e \u003cp\u003eHuman immortalised keratinocyte HaCaT cells were purchased from the China Typical Culture Preservation Center, Wuhan University (item number: GDC0106). Medium 1640 (Pricella, China) supplemented with 10% foetal bovine serum (Pricella, China) was cultured in a 37\u0026deg;C cell incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e. A psoriasis-like keratinocyte model was established by adding an M5 cytokine combination (IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α, each at a final concentration of 5ng/ml) to the medium of HaCaT keratinocytes \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 IMQ-induced psoriasis mice model establishment, RSL3 intervention, and PASI score\u003c/h2\u003e \u003cp\u003eBALB/c mice were purchased from the Hunan Slyke Jingda Company. BALB/c mice were used to establish an IMQ-induced psoriasis-like mouse model \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Female BALB/C mice aged 6\u0026ndash;8 weeks were adaptatively fed for 1 week and depilated at approximately 2 cm\u0026times;2 cm on their backs. Sixteen mice were randomly divided into four groups: blank control group (Control, Ctrl), IMQ model group (IMQ), IMQ model\u0026thinsp;+\u0026thinsp;ferroptosis inducer group (IMQ\u0026thinsp;+\u0026thinsp;RSL 3), and IMQ model\u0026thinsp;+\u0026thinsp;solvent control group (IMQ\u0026thinsp;+\u0026thinsp;Vehicle). The investigator was not blinded to the group allocation. Five percent IMQ (Sichuan Mingxin Pharmaceutical, China) (62.5 mg) was applied to the back hair removal area once daily for 8 days. An equal volume of RSL3 (2.0 mg/kg/d, MCE, China) or 10% DMSO (100 \u0026micro;l) was painted on the back of the mouse 30 min before IMQ application. The intervention was performed once daily, and the mice were weighed, photographed, and scored according to a previous mouse PASI scoring standard \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Details are as follows: PASI scoring criteria were scored based on erythema and scaliness, respectively, and each item was rated as 0\u0026ndash;4 points according to severity. 0, \u003cem\u003enone\u003c/em\u003e; 1, \u003cem\u003emild\u003c/em\u003e; 2, \u003cem\u003emoderate\u003c/em\u003e; 3, \u003cem\u003esevere\u003c/em\u003e; and 4, \u003cem\u003every severe\u003c/em\u003e. On day 9, significant differences were observed among the groups. The experimental Animal Welfare Ethics Committee of Central South University has approved this part of the experimental protocol (Ethics No. 2021sydw0063).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cell viability was detected by the CCK8 kit\u003c/h2\u003e \u003cp\u003eHaCaT cells were inoculated in 10% complete medium and treated with different concentrations of RSL3 for 24, 48, and 72 h. Cell viability was determined using a CCK8 kit (Beyotime, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 ROS detection\u003c/h2\u003e \u003cp\u003eIntracellular ROS levels were observed by flow cytometry and fluorescence microscopy, according to the instructions of the reactive oxygen species detection kit (Beyotime, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Detection of apoptosis\u003c/h2\u003e \u003cp\u003eFlow cytometry and fluorescence microscopy were performed according to the procedures of the Annexin-V-FITC apoptosis detection kit (Beyotime, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Transmission electron mMicroscope (TEM)\u003c/h2\u003e \u003cp\u003eTEM was used to examine the mitochondrial morphological changes in HaCaT cells treated with 5 \u0026micro;M RSL3 for 24 h. The cells were digested with trypsin, harvested, and fixed in 2.5% glutaraldehyde (EM grade) overnight at 4\u0026deg;C. After fixation in 1% osmic acid, the cell samples were dehydrated and placed in standard embedding moulds. Suitable areas were selected, and 0.08 \u0026micro;m ultrathin sections were stained with lead citrate and uranyl acetate. The sections were examined using a TEM (JEM-1400Plus, JEOL, Ltd., Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 RT-qPCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from tissues and cells using Trizol (AG, China). Next, 700 \u0026micro;g RNA was reversely transcribed into cDNA according to the manufacturer\u0026rsquo;s instructions (Vazyme, China). The amplification solution was prepared according to the instructions for SYBR Green fluorescent dye reagent (Novizan, China), and a real-time PCR system (Bio-Rad, USA) was used for the amplification reaction. The primer sequences used are listed in Supplementary Table S1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Western blot\u003c/h2\u003e \u003cp\u003eProtein concentrations were measured in a lysis buffer solution (Beyotime, China) using a BCA assay kit (Beyotime, China). A 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) was prepared and transferred to a polyvinylidene fluoride membrane (Millipore, USA) after electrophoresis. After sealing in a 5% skim milk solution, GPX4 (AiFang, AF301385, 1:500), 4HNE (Abcam, ab46545, 1:1000), GAPDH (Affinity, AF7021, 1:300), KRT6 (Abcam, ab93279, 1:2000), and FLG (Affinity, DF13653, 1:1000) were diluted with 1 \u0026times; PBST. The goat was incubated on a low-speed shaker at 4 ℃ overnight, and the secondary antibody was diluted with 1 PBST: HRP goat anti-mouse IgG (Proteintech, SA00001-1, 1:5000) and HRP goat anti-rabbit IgG (Proteintech, SA00001-2, 1:6000).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 HE and immunohistochemical staining analysis\u003c/h2\u003e \u003cp\u003eHuman and mouse skin tissues were fixed with formalin, and paraffin-embedded sections (6\u0026micro;m) were treated with haematoxylin and eosin (H\u0026amp;E) stain and stored at room temperature. The immunohistochemical procedure was described in the literature \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. The following antibodies were used: GPX4 (AF301385, 1:80; AiFang), 4-HNE (ab46545, 1:100; Abcam), KRT6 (10590-1-AP, 1:400; Proteintech), and FLG (AF10489, 1:100; AiFang). The immune response intensity of each section was divided into four grades \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e:0 (no staining), 1+ (weak staining), 2+ (medium staining), and 3+ (strong staining). The percentage of stained cells was divided into two grades:1 (\u0026le;\u0026thinsp;30%) and 2 (\u0026gt;\u0026thinsp;30%). Final immunohistochemical staining score\u0026thinsp;=\u0026thinsp;staining score\u0026times;percentage of stained cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical methods\u003c/h2\u003e \u003cp\u003eAll charts of the data in this study were generated using GraphPad Prism software (version 8.0), and the data were represented by mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Two independent sample t tests were used to compare the measurement data of the two groups. Comparison of measurement data of multiple groups was conducted using one-way ANOVA. Non-normally distributed data were analysed using a non-parametric test (Mann-Whitney U test). Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Ferroptosis was increased in psoriatic lesions.\u003c/h2\u003e \u003cp\u003eGPX4 is a critical gene involved in ferroptosis. The expression of GPX4 in psoriasis vulgaris lesions and normal specimens was detected using RT-qPCR, immunohistochemistry, and Western Blot. The results of RT-qPCR showed that the expression level of GPX4 mRNA in psoriasis lesions (N\u0026thinsp;=\u0026thinsp;23, 0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48) was significantly lower than that in healthy controls (N\u0026thinsp;=\u0026thinsp;26, 1.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Immunohistochemical and Western blot results confirmed this finding (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Western blot and immunohistochemistry revealed an increase in the oxidation product 4-hydroxy-2-nonenal (HNE) in psoriasis lesions (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). These results are consistent with the characteristics of ferroptosis. We then conducted a correlation analysis between the GPX4 mRNA relative expression level and PASI score. The results showed that the higher the PASI score, the higher the GPX4 expression level and that the two were positively correlated. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2 RSL3 induced ferroptosis in HaCaT cells.\u003c/h2\u003e \u003cp\u003eCell viability at different concentrations of RSL3 and at three treatment times was assessed using a Cell Counting Kit-8 (CCK-8) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). At a concentration of 10 \u0026micro;M RSL3, cell viability was close to 0 at all three treatment times. Under a light microscope, the number of cells decreased with an increase in the concentration after 24 h of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The IC50 of RSL3 was 6.04 \u0026micro;M after 24 h of treatment. RSL3 seldom showed cytotoxicity at a concentration below 6.04 \u0026micro;M. Western blot showed that 5 \u0026micro;M and 7.5 \u0026micro;M RSL3 inhibited the expression of GPX4 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Consequently, we used 5 \u0026micro;M RSL3 in HaCaT cells for 24 h. The cells displayed increased intracellular ROS levels, as assessed by fluorescence microscopy and flow cytometry (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). We also found that HaCaT cells displayed an extensive percentage of dead cells after RSL3 treatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Electron microscopy revealed increased mitochondrial membrane density and decreased crista levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Therefore, 5 \u0026micro;M was the optimal concentration of RSL3 for inducing ferroptosis in HaCaT cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3 RSL3 regulated biological behaviours of cells in vitro.\u003c/h2\u003e \u003cp\u003eHaCaT cells were induced with M5 to establish a psoriatic cell model, and the cells were treated with 5 \u0026micro;M RSL3 for 24 h \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. KRT6 is an excessive proliferation marker of psoriatic KCs; filaggrin (FLG) is a late differentiation marker of normal KCs \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. RT-qPCR results showed an increased expression of IL-17a, IL-6, IL-1β, CXCL1, and KRT6 and a decreased expression of FLG, prompting the successful establishment of the M5-induced psoriasis cell model. The RSL3 groups showed decreased KRT6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB); increased FLG (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD); and decreased IL-17a, IL-6, IL-1β, CXCL1, CXCL2, and CCL20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). The results of this study suggest that the ferroptosis inducer RSL3 inhibits proliferation, promotes differentiation, and inhibits the expression of chemokines and inflammation in HaCaT cells induced by M5.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 RSL3 alleviated the phenotype of skin lesions in vivo\u003c/h2\u003e \u003cp\u003eFive-percent Imiquimod (IMQ) of 62.5 mg was applied to the depilated area of the back once daily for 8 d. An equal volume of RSL3 (2.0 mg/kg/d) or 10% DMSO (100 \u0026micro;l) was painted on the back of the mouse 30 min before IMQ application. On day 9, we observed that erythema and scales in the back lesions of mice in the RSL3 group were significantly reduced, suggesting that RSL3 could improve psoriatic dermatitis induced by IMQ (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). HE results showed that the IMQ group was characterised by thickening of the epidermis, hyperkeratosis, and parakeratosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These results indicate that RSL3 improved the local inflammatory response and reduced epidermal thickness in IMQ-induced psoriatic mouse models.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5 RSL3 regulated the KCs\u0026rsquo; biological behaviours in IMQ- treated mouse models.\u003c/h2\u003e \u003cp\u003eRT-qPCR and immunohistochemical results revealed that RSL3 decreased KRT6 expression and promoted FLG expression in IMQ-induced psoriasis lesions in mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The expression of IL-17a and CXCL1 in the IMQ\u0026thinsp;+\u0026thinsp;RSL3 group was decreased, and that in the model and solvent control groups was increased, with a statistical difference, and IL-6, IL-1β, CXCL2, and CCL20 showed a downward trend, with no significant statistical significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). These results suggest that RSL3 inhibits proliferation, promotes differentiation, and inhibits inflammation during psoriasis treatment.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eFerroptosis, a form of regulated cell death discovered recently, is characterized by iron-dependent cell demise triggered by excessive lipid peroxidation in the cell membrane\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. It has been verified that KCs in psoriasis vulgaris exhibit anti-death properties. This impediment in the cell death and differentiation process leads to rapid cell proliferation, mirroring the behavior of tumor cells\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. However, the relationship between psoriasis vulgaris and Ferroptosis remains unclear. GPX4, a negative regulator that decreases in the process of ferroptosis, is notably diminished in psoriasis vulgaris, aligning with findings from Shou et al. \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. The association between GPX4 levels and the PASI score indicates a negative correlation between ferroptosis occurrence and the severity of psoriasis.\u003c/p\u003e \u003cp\u003eIn an effort to delve deeper into the specific specific interaction between ferroptosis and psoriasis vulgaris, we employed a ferroptosis inducer, RSL3, which inhibit GPX4 protein in HaCaT cells. Our findings reveal that HaCaT cells are highly susceptible to RSL3, indicating that RSL3 curtails HaCaT cell proliferation in a dose- and time-dependent manner. Concurrently, intracellular ROS and apoptosis levels rise following treatment with RSL3. Furthermore, in the M5-induced psoriasis model, RSL3 overexpression leads to a reduction in the proliferation marker KRT6 and an increase in the expression of barrier protection factors and late differentiation gene FLG. Animal experiment corroborate these findings. Those outcomes align with the tumor-like characteristics of KCs in psoriasis, suggesting that ferroptosis mitigates psoriasis progression by inhibiting KC proliferation and promoting KC differentiation.\u003c/p\u003e \u003cp\u003eConsidering the activation of inflammatory pathways in Keratinocytes (KCs) and the correlation between ferroptosis and psoriasis, we next examined whether using ferroptosis inducers would exacerbate inflammation. Surprisingly, we observed a significant reduction in most inflammatory chemokines, including IL-17, IL-22, CXCL1, IL-6. Specifically, IL-17 and CXCL1 levels decreased significantly at both cellular and animal levels, indicating that RSL3 may supress certain inflammatory chemokines\u0026rsquo; expression. The lack of statistical difference in the animal model may stem from an incomplete understanding of physical ferroptosis\u0026rsquo;s optimal range. The whole mouse may retain a self-compensatory ability and exhibit individual differences, increasing the exploration of physiological ferroptosis in mice.\u003c/p\u003e \u003cp\u003eRecent studies reveal the intricate relationship between programmed cell death and inflammation\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Ferroptosis, a type of programmed cell death, can either exacerbate inflammation through damage-associated molecular patterns (DAMPs)\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e or attract immune cells, complicating the inflammatory response. Conversely, pathogen-associated molecular patterns (PAMPs) induce the specific antibacterial immune response to control infection \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Research on ferroptosis\u0026rsquo;s role in skin inflammation is still in its infancy. Shou et al. demonstrated that ferroptosis inducers do not promote the inflammation during psoriasis, suggesting that the ferroptosis inhibitors may alleviate inflammation by decreasing the intracellular oxidative stress levels\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Additionally, studies have indicated that the RSL3 may suppress the LPS-induced cellular inflammation by increasing ROS, which activates the Nrf2 anti-oxidation system, thereby reducing the secretion of inflammatory factors\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Therefore, the inhibitors and inducers of ferroptosis indirectly affect the inflammation by regulating the level of intracellular oxidative stress.\u003c/p\u003e \u003cp\u003eIt's important to note that ferroptosis can act as a double-edged sword in the organism. Among tumors, inducing ferroptosis in cancer cells may decrease the number of cells and inhibit cell growth. However, ferroptotic cells appeal immune cells by DAMPs, potentially promoting the development of tumors\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. In the context of inflammation, ferroptosis influences the quantity and function of immune cells, weakening immune response; while also allowing immune cells to identify the ferroptotic cells, causing a series of inflammatory and specific reactions\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. This leads us to hypothesize the existence of a balance in organism ferroptosis, which we term \u0026ldquo;physiological ferroptosis\u0026rdquo;. This balance maximizes ferroptosis\u0026rsquo;s beneficial effects while also decreases the harmful impacts.\u003c/p\u003e \u003cp\u003eIn conclusion, our study elucidates the differential expression of GPX4 in skin lesions between healthy controls and patients with psoriasis vulgaris. Additionally, it has been confirmed that the HaCaT cell are highly sensitive to RSL3. The application of RSL3 could inhibit cell proliferation and promote cell differentiation by downregulating the GPX4 protein, thereby alleviating the symptoms of psoriatic dermatitis through inflammation control. This may become the significant target for psoriasis vulgaris treatment.\u003c/p\u003e \u003cp\u003eOur study has limitations, notably small sample size. Larger clinical trials are necessary to investigate the association between ferroptosis and disease progression further. More research is necessary to explore how the ferroptosis inducer RSL3 regulates the abnormal biological behaviour of KCs at both cellular and animal levels and establish ferroptosis inhibitors as controls.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn conclusion, this study confirms the existence of ferroptosis in psoriatic lesions, which may be inversely correlated with disease severity. The ferroptosis inducer RSL3 ameliorated psoriatic symptoms by improving the abnormal biological behaviour of KCs.\u003c/p\u003e"},{"header":"Abbreviations","content":" \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"453\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.69757174392936%\" valign=\"top\"\u003e\n \u003cp\u003eAbbreviations\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.30242825607064%\" valign=\"top\"\u003e\n \u003cp\u003eFull name\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.69757174392936%\" valign=\"top\"\u003e\n \u003cp\u003eKCs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.30242825607064%\" valign=\"top\"\u003e\n \u003cp\u003ekeratinocytes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.69757174392936%\" valign=\"top\"\u003e\n \u003cp\u003eGPX4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.30242825607064%\" valign=\"top\"\u003e\n \u003cp\u003eglutathione peroxidase 4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.69757174392936%\" valign=\"top\"\u003e\n \u003cp\u003eIMQ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.30242825607064%\" valign=\"top\"\u003e\n \u003cp\u003eimiquimod\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.69757174392936%\" valign=\"top\"\u003e\n \u003cp\u003eTh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.30242825607064%\" valign=\"top\"\u003e\n \u003cp\u003eT helper cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.69757174392936%\" valign=\"top\"\u003e\n \u003cp\u003eTNF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.30242825607064%\" valign=\"top\"\u003e\n \u003cp\u003etumour necrosis factor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.69757174392936%\" valign=\"top\"\u003e\n \u003cp\u003eIL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.30242825607064%\" valign=\"top\"\u003e\n \u003cp\u003einterleukin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.69757174392936%\" valign=\"top\"\u003e\n \u003cp\u003eCXCL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.30242825607064%\" valign=\"top\"\u003e\n \u003cp\u003eC-X-C motif chemokine ligand\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.69757174392936%\" valign=\"top\"\u003e\n \u003cp\u003eCCL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.30242825607064%\" valign=\"top\"\u003e\n \u003cp\u003eC-C motif chemokine ligand\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.69757174392936%\" valign=\"top\"\u003e\n \u003cp\u003eGSH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.30242825607064%\" valign=\"top\"\u003e\n \u003cp\u003eglutathione\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.69757174392936%\" valign=\"top\"\u003e\n \u003cp\u003eXc\u003csup\u003e―\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.30242825607064%\" valign=\"top\"\u003e\n \u003cp\u003ecystine/glutamate transporter\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.69757174392936%\" valign=\"top\"\u003e\n \u003cp\u003eROS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.30242825607064%\" valign=\"top\"\u003e\n \u003cp\u003ereactive oxygen species\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.69757174392936%\" valign=\"top\"\u003e\n \u003cp\u003e4HNE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.30242825607064%\" valign=\"top\"\u003e\n \u003cp\u003e4-Hydroxynonenal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.69757174392936%\" valign=\"top\"\u003e\n \u003cp\u003eKRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.30242825607064%\" valign=\"top\"\u003e\n \u003cp\u003ekeratin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.69757174392936%\" valign=\"top\"\u003e\n \u003cp\u003eFLG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.30242825607064%\" valign=\"top\"\u003e\n \u003cp\u003efilaggrin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.69757174392936%\" valign=\"top\"\u003e\n \u003cp\u003ePI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.30242825607064%\" valign=\"top\"\u003e\n \u003cp\u003epropidium iodide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.69757174392936%\" valign=\"top\"\u003e\n \u003cp\u003eHE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.30242825607064%\" valign=\"top\"\u003e\n \u003cp\u003ehematoxylin and eosin stain\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.69757174392936%\" valign=\"top\"\u003e\n \u003cp\u003ePASI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.30242825607064%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;psoriasis area and severity index\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.69757174392936%\" valign=\"top\"\u003e\n \u003cp\u003eCtrl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.30242825607064%\" valign=\"top\"\u003e\n \u003cp\u003econtrol\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of Third Xiangya Hospital, Central South University (Ethics No. 2021-S247) and the experimental Animal Welfare Ethics Committee of Central South University (Ethics No. 2021sydw0063).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJinrong Zeng and Jianyun Lu: Conceptualization and Supervision. Jinrong Zeng, Lu zhou, and Zhibing Fu: Writing- Original draft preparation. Qian Hu, Ningling Wu: Investigation, Methodology and Formal analysis. Ningling Wu: Visualization. Xiaoliang Tong: Validation. Lina Tan, Siyu Yan, Lihua Gao, and Dan Wang: Data Curation. \u0026nbsp;Jianyun Lu: Writing - Review \u0026amp; Editing. Jinrong Zeng All authors read and approved the final manuscript.\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 relevant data are included in the present manuscript or in the supplements. Raw data are available upon reasonable request by the first author R.M.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eContents of the manuscript have not been previously published and are not currently submitted elsewhere. I accept responsibility for the scientific integrity of the work described in this manuscript. All listed authors have seen and approved of the manuscript and will sign off on any subsequent manuscript revisions\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDing X, Wang T, Shen Y et al. Prevalence of psoriasis in China: a population-based study in six cities. 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J Exp Med. 2021; 218. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1084/jem.20210518\u003c/span\u003e\u003cspan address=\"10.1084/jem.20210518\" 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":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":"Psoriasis vulgaris, ferroptosis, Keratinocytes, GPX4, abnormal biological behaviours","lastPublishedDoi":"10.21203/rs.3.rs-4113873/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4113873/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAbnormal biological behaviour of keratinocytes (KCs) is a critical pathophysiological manifestation of psoriasis. Ferroptosis is programmed cell death induced by the accumulation of lipid reactive oxygen species (ROS) in the presence of increased intracellular iron ions or inhibition of GPX4.\u003c/p\u003e\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eThe purpose of this study was to investigate the effects of ferroptosis on the biological behaviour of KCs in psoriasis vulgaris and its possible regulatory mechanisms in clinical samples, cells, and mouse models.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe first examined the differences in the expression of GPX4 and 4-HNE between psoriasis and normal human lesions. And detected KRT6, FLG, and inflammatory cytokines after inducing ferroptosis in animal and cell models by RT-qPCR, Western blot, immunohistochemistry, and flow cytometry.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found that GPX4 was decreased and that the oxidation product 4-hydroxy-2-nonenal (HNE) was increased in the skin lesions of patients with psoriasis vulgaris. The expression level of GPX4 correlates with the severity of skin lesions. Moreover, inducing ferroptosis promoted the expression of FLG and reduced the expression of KRT6 and inflammatory cytokines in vitro, and alleviated the phenotype of skin lesions in vivo.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study confirms the existence of ferroptosis in psoriatic lesions, which may be inversely correlated with disease severity. The ferroptosis inducer RSL3 ameliorated psoriatic symptoms by improving the abnormal biological behaviour of KCs.\u003c/p\u003e","manuscriptTitle":"Pilot study of the role of ferroptosis in abnormal biological behaviour of keratinocytes in psoriasis vulgaris","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-21 16:03:19","doi":"10.21203/rs.3.rs-4113873/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":"de57b8a6-97aa-4a06-aeeb-2109db0d5cdd","owner":[],"postedDate":"March 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-25T18:06:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-21 16:03:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4113873","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4113873","identity":"rs-4113873","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}