Article Type: Apigenin alleviated peritoneal dialysis-related peritoneal fibrosis by regulating pyroptosis

Article Type: Apigenin alleviated peritoneal dialysis-related peritoneal fibrosis by regulating pyroptosis | 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 Article Type: Apigenin alleviated peritoneal dialysis-related peritoneal fibrosis by regulating pyroptosis Dandan Xue, Nana Luo, Xiang Li, Zhanfeng Jiao, Yiming Zhang, Xiaofen Ma This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4196099/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 Prolonged exposure of the peritoneum to peritoneal dialysis fluid, peritonitis, and peritoneal blood accumulation can cause peritoneal inflammation and injury, followed by progressive fibrosis, ultimately leading to the cessation of peritoneal dialysis. Apigenin is an herbal medicine with anti-inflammatory, antioxidant, and anti-fibrotic properties. Our previous study found that apigenin could inhibit the process of peritoneal fibrosis, but whether apigenin could treat fibrosis by inhibiting pyroptosis is not known. This study aimed to investigate the protective effects of apigenin against peritoneal mesothelial cell pyroptosis and fibrosis. First, we observed changes in peritoneal mesothelial cells using VX-765, an inhibitor of cellular pyroptosis. Western blotting, immunoblotting, and electron microscopy showed that cellular pyroptosis was significantly inhibited, and peritoneal mesothelial cell fibrosis was reduced accordingly. Secondly, when the cells were co-treated with high glucose medium and apigenin, apigenin attenuated the activation of cellular pyroptosis by decreasing the levels of Gasdermin D, NOD-like receptor protein 3 inflammatory niche activation, caspase-1 and IL-1β and also inhibited the development of peritoneal fibrosis. Thus, our results suggest that apigenin plays a protective role against peritoneal fibrosis by inhibiting pyroptosis. Peritoneal fibrosis pyroptosis apigenin vx-765 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Approximately 2 million people worldwide are estimated to have end-stage renal disease (ESRD), and this number is increasing annually. 1 Peritoneal dialysis (PD) is a recognized renal replacement therapy. 2 , 3 Prolonged exposure to PD fluid activates a series of pathological events, including changes in peritoneal vascular solutes (neovascularization), exacerbation of interstitial microchanges (fibrosis), reduction of osmotic conductivity, recruitment of inflammatory cells, and increased production of inflammatory cytokines, which worsens and exacerbates peritoneal fibrosis (PF), leading to ultrafiltration failure, and for which there is currently no effective treatment. 456 . Pyroptosis is a type of hemolytic programmed cell death characterized by cell swelling, rupture, secretion of cell contents, and significant pro-inflammatory effects. 7 It is typically mediated by the assembly of inflammatory vesicles. 8 The assembly of inflammatory vesicles begins with cytosolic pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) and are formed by the assembly of NOD-like receptors (NLRs) (as sensor proteins) together with caspase-1 precursors, apoptosis-associated speck-like proteins (ASC). 9 – 11 Caspase-1 efficiently cleaves Gasdermin D (GSDMD) to become the GSDMD-N structural domain mediating the formation of pores in the plasma membrane that release pro-inflammatory factors such as interleukin (IL)-1β, IL-18, and high mobility group protein B1 (HMGB1), leading to an inflammatory response. 12 , 13 Caspase-1, as a key component of inflammatory vesicles, is considered an important target for inhibiting inflammasome activation. VX-765 is a potent, bioavailable, and non-toxic small-molecule inhibitor of caspase1, 14 and it effectively inhibits inflammatory cell infiltration and pyroptosis-associated protein expression, thereby reducing renal inflammation and fibrosis. 15 , 16 We hypothesized that VX-765 has anti-inflammatory or anti-extracellular matrix accumulation effects. However, whether VX-765 protects against PF by regulating pyroptosis remains unclear. Apigenin (API) is a 4′,5,7-trihydroxyflavone that has been shown to exert various pharmacological effects, including anti-inflammatory, antioxidant, and anti-fibrotic effects. 17 , 18 Meng et al. reported that API inhibits NOD-like receptor protein 3 (NLRP3) inflammasome activation by activating autophagy, thereby attenuating hepatocyte pyroptosis. 19 Previous studies have demonstrated that API can inhibit miR34a expression by downregulating PF in mice; nevertheless, the exact mechanism remains unclear. 20 Moreover, no evidence suggesting that API protects against peritoneal mesothelial cell fibrosis by inhibiting pyroptosis exists. Therefore, this study aimed to investigate the molecular-level effects and mechanism of action of API on PF. 2. Materials and methods 2.1 Cell culture and treatment Isolated mouse mesothelial peritoneal cells (MPMCs) were cultured in RPMI 1640 medium supplemented with 15% fetal calf serum, L-glutamine, hydrocortisone, insulin, transferrin, selenium, 20 mM HEPES, 4 mg/L gentamicin, 120 mg/L benzylpenicillin, and 2.5 mg/L amphotericin at 37°C in a humidified culture chamber supplied with 5% CO 2 . The culture medium was changed every 3 days until the cells were confluent. Subsequently, cells were washed with phosphate-buffered saline and digested with 0.007% ethylenediaminetetraacetic acid solution containing 0.02% trypsin at 37°C for 2 min, followed by the addition of fresh medium for a new round of culture or subsequent analysis. The API used for cell treatment was purchased from Merck with a purity of over 99%, as determined using high-performance liquid chromatography (HPLC). VX-765 was purchased from MedChemexpress. To examine the effects of API on MPMCs in high-glucose fluids, MPMCs were divided into four groups: i) vehicle control group, cells were cultured in regular media and treated with dimethyl sulfoxide (DMSO); ii) high glycemic model group, cells were cultured in high glucose culture solution; iii) high glycemic model + vehicle group, cells were cultured in high glucose culture solution and treated with DMSO; and iv) high glycemic model + API group, cells were cultured in high glucose culture solution and treated with API. 2.2 Western blot analysis The primary mouse peritoneal mesothelial cells were inoculated onto 6-well plates and allowed to grow to 70–80% at 37°C. Subsequently, MPMCs were treated with API in a 75 mM high-sugar medium. Each sample was collected from six parallel wells. The protein solutions of MPMCs were extracted with a lysis buffer comprising radioimmunoprecipitation assay buffer, protease inhibitor, phosphatase inhibitor, and phenylmethylsulfonyl fluoride (50:1:1:1) and left to stand on ice for 40 min. The cell lysate was centrifuged at 12,000 rpm for 20 min, and the protein concentration in the supernatant was determined using the bicinchoninic acid assay (Beyotime, China). Equal amounts of protein per sample were loaded into each well, separated on a 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% bovine serum albumin (BSA) for 2 h and then incubated overnight with the following rabbit-derived polyclonal antibodies: NLRP3 (Affinity, China), caspase 1 (Affinity, China), IL-1β (Affinity, China), GSDMD (CST, USA), N-cadherin (Abclonal, China), and E-cadherin (Abclonal, China), and GAPDH (Abclonal, China) was used as an internal control. Membranes were washed with tris-buffered saline with Tween-20 and incubated with a secondary antibody (Abclonal, China) for 1 h at room temperature. The bound antibodies were visualized using enhanced chemiluminescence Plus (Proteintech, China). 2.3 Immunofluorescence staining The MPMCs were seeded in a glass-bottom cell culture dish (1×10⁵ cells per well) and allowed to adhere overnight at 37℃. After various treatments, MPMCs were treated with 4% buffered paraformaldehyde for 20 min at room temperature and then treated with 0.5% Triton X-100 for 20 min. Subsequently, 5% BSA was used to block the membrane for 1 h at room temperature and then treated with the primary antibodies against NLRP3, GSDND-N and Collagen III at 4°C overnight before treatment with the secondary antibody for 1 h at room temperature. Nuclei were stained with 4',6-diamidino-2-phenylindole (Beyotime, China). Images were captured using a fluorescence microscope. The immunostaining results were quantified and statistically analyzed using Image-Pro Plus 6.0. 2.4 Enzyme-linked immunosorbent assays (ELISA) The cell culture media were collected and centrifuged at 3,000 rpm for 20 min at 4°C. The cell supernatant was assayed for lactate dehydrogenase (LDH) using an ELISA kit according to the manufacturer’s instructions, using a microplate reader. The development of color intensity in each well was visualized by measuring the 450-nm absorbance. 2.5 Scanning electron microscopy Slides containing cells treated with and without hydrogen peroxide were dehydrated using increasing concentrations of ethanol (30, 40, 50, 60, 70, 80, 90, and 100%). After drying in a CO 2 critical point dryer, the sample was adhered to the sample stage with a double-sided conductive tape, sprayed using ion sputtering, and then observed and photographed using an electron microscope (Hitachi, SU8010). 2.6 Statistical analysis All results data was expressed as mean ± standard deviation (SD). GraphPad Prism software version 8.0 was used for all statistical analyses. Each experiment was conducted in triplicates. P < 0.05 was considered statistically significant. 3. Results 3.1 High glucose treatment induces pyroptosis and epithelial-mesenchymal transition (EMT) in MPMCs Primary mouse peritoneal mesothelial cells were used to investigate the role of peritoneal mesothelial cell pyroptosis in PF. According to our previous research, 1 we treated MPMCs with 75 mM D-glucose and observed that NLRP3, cleaved caspase-1, GSDMD p30 fragment, and cleaved IL-1β expression was increased in high glucose medium as shown in Fig. 1 . Simultaneously, we observed that high glucose levels decreased E-cadherin expression and increased N-cadherin expression in peritoneal mesothelial cells (Fig. 3 ). The results showed that high glucose levels promoted the expression of the above-mentioned proteins and activated the pyroptotic pathway and EMT. 3.2 VX-765 suppresses high glucose-induced pyroptosis and fibrosis in MPMCs To determine whether caspase1 inhibitor VX-765 could attenuate indicators related to pyroptosis in mouse peritoneal mesothelial cells, the expression of pyroptosis-related proteins IL-1β, NLRP3, caspase-1, and GSDMD was assessed using western blotting and immunofluorescence methods. We found that the expression of cleaved-IL-1β, NLRP3, cleaved-caspase-1, and GSDMD-N, markers related to cellular pyroptosis, was significantly decreased by the administration of VX-765 and induced by high glucose for 48 h, indicating that VX-765 successfully inhibited cellular pyroptosis. Consistent with the western blotting results, immunofluorescence analysis demonstrated that VX-765 significantly inhibited the expression of NLRP3 and GSDMD-N. The effect of VX-765 on cell death was detected using electron microscopy, and it was found that under the influence of high glucose, MPMCs continued to swell until the cell membrane was ruptured, the nucleus remained intact, and the cell structure was obviously damaged, with many bubble-like protrusions and release of contents. However, in the VX-765 group, the cell structure was intact, and cell death was significantly reduced compared to that in the high glucose group (Fig. 2 ). These results confirmed the inhibitory effect of VX-765 on high glucose-induced pyroptosis in MPMCs (Fig. 1 ). After VX-765 treatment of MPMCs, the protein expression level of N-cadherin, a mesenchymal phenotype related to PF, was significantly reduced, and that of E-cadherin, an epithelial phenotype, was significantly increased compared to that of the high glucose group. The protein expression level of the epithelial phenotype E-cadherin was significantly higher than that in the high glucose group (Fig. 3 ). Collectively, these results indicated that VX-765 alleviated PF in MPMCs. PF in MPMCs and MPMCs pyroptosis are closely associated with fibrosis. 3.3 API inhibits high glucose-induced pyroptosis and fibrosis in MPMCs ELISA was used to detect the expression level of LDH in cell culture supernatants, and the level of LDH was found to be lower in the API group than in the high glucose group (Fig. 4 ). Protein expression levels of pyroptosis markers (IL-1β, NLRP3, Caspase-1, and GSDMD) were detected USING western blotting after treatment of MPMCs with 75 mM high glucose and API. As shown in Fig. 5 , the expression level of NLRP3, cleaved caspase-1, GSDMD-N, and cleaved IL-1β was substantially increased in the high glucose model group relative to the control group, and the increase of NLRP3, cleaved caspase-1, GSDMD-N, and cleaved IL-1β expression mediated by high glucose was attenuated by API in MPMCs. Moreover, API acted as both an inhibitor and stimulator of EMT-related indices (N-cadherin and E-cadherin) (Fig. 6 ). The immunofluorescence results were consistent with the western blotting results (Fig. 7 ). These results showed that API attenuated the effects of high glucose-mediated MPMCs EMT and pyroptosis. 4. Discussion PD is an effective renal replacement method for patients with ESRD. Peritonitis and prolonged PD can cause PF. Peritonitis induces angiogenesis and fibrosis, and the development of PF is usually the primary cause of PD interruption. 21, 22 In our study, API pretreatment attenuated peritoneal mesothelial cell pyroptosis, decreased protein expression of caspase-1, GSDMD, and NLRP3, and reduced IL-1β leakage. Additionally, API delayed the onset of PF by attenuating cellular pyroptosis. Indeed, our previous studies have demonstrated that API significantly promotes the proliferation of mouse peritoneal mesothelial cells, inhibits high glucose-induced apoptosis, and effectively inhibits high glucose-induced fibrosis in mouse peritoneal mesothelial cells. 20 API, a flavonoid, is a low-toxicity free radical scavenger and an antioxidant phenolic compound that exhibits anti-inflammatory, antiviral, and anti-fibrotic effects, possibly reducing the risk of chronic diseases. API effectively mitigates pyroptosis and plays a critical role in cardiovascular disease, particularly in ischemia/hypoxia-induced cardiomyocyte injury. Additionally, API significantly reduces the level of pro-inflammatory factors in hypoxia-induced cardiomyocytes, and the mechanism may include effective attenuation of cardiomyocyte pyroptosis and apoptosis, inhibition of the level of pro-inflammatory factors (IL-1β and IL-18) levels, effectively attenuating myocardial injury. 23 Moreover, API attenuated palmitic acid (PA)-induced NLRP3 inflammasome activation and cellular charring in primary mouse hepatocytes, which regulated NLRP3 inflammasomes through two pathways, thereby attenuating PA-induced cellular charring. 24 In our present study, we used API to treat high glucose-stimulated mouse MPMCs, and the results demonstrated that API attenuated the onset of MPMCs pyroptosis and fibrosis. The pyroptosis mechanism, leading to cell death, is triggered by the production and initiation of inflammasome-associated components, often referred to as intracellular protein complexes. 25 In the innate immune response, at the initiation step, specific PRRs, including Toll-like receptors, NLRs, and cytokine receptors, recognize PAMP, DAMP, and cytokines, which activate the NF-κB signaling pathway and increase the levels of NLRP3, pro-IL-1β, and pro-IL-18. 26,27 During the activation phase, NLRP3 aggregates via the Nacht structural domain and interacts with pyrin domain (PYD)-PYD to recruit PYD-caspase activation and recruitment domain (CARD)-containing ASC proteins, and CARD then binds to caspase-1 to initiate downstream signaling. 28 In cells, NLRP3 forms inflammatory vesicle complexes by interacting with ASC, caspase-1, and other proteins.29 On the one hand, activated caspase-1 cleaves pro-IL-1β and pro-IL-18 to generate activated IL-1β and IL-18, promoting the release of pro-inflammatory cytokines and triggering an inflammatory response 30 ; on the other hand, activated caspase-1 cleaves GSDMD into amino-terminal (GSDMD-CT) and carboxy-terminal (GSDMD-NT) fragments and induces pyroptosis. GSDMD-NT can bind to lipids and insert into the cell membrane, forming pores that secrete small-diameter substrates, including IL-1β and IL-18, 31,32 and as the number of molecular pores increases, the cell membrane eventually becomes relaxed and ruptured, and intracellular contents, such as IL-1α and HMGB1, are also released. 33 However, only a few studies have examined the pathogenic role of cellular pyroptosis in PF. We observed the characteristic morphological and biochemical features of cell pyroptosis, including balloon-like cell membrane swelling, caspase-1 activation, GSDMD shearing, pro-inflammatory factor release, and exudation of cytoplasmic contents in MPMCs modeled with high glucose. VX-765 is an orally bioavailable small-molecule inhibitor that increases caspase-1 with no signs of toxicity and has been used to treat inflammatory and autoimmune diseases. 14 Oral administration of VX-765 was found to be safe in humans in a 6-week phase II clinical trial on epilepsy, making VX-765 a viable drug. 34 Previous studies have demonstrated that VX-765 improves renal function in diabetic animals, inhibits high glucose-induced inflammatory cell infiltration and cellular pyroptosis-related protein expression, and attenuates tubulointerstitial fibrosis. 19 Whether VX-765 protects against high glucose-induced PF by regulating cellular pyroptosis remains unclear. We used VX-765 for the first time to treat high glucose-stimulated MPMCs and observed that VX-765 effectively reduced pyroptosis rate, inflammatory factors release, and fibrosis occurrence, suggesting that cellular pyroptosis inhibitors effectively regulate the inflammation and fibrosis of MPMCs in a high glucose environment. Conclusively, API effectively attenuated peritoneal mesothelial cell fibrosis in mice, and its mechanism of action may be related to the inhibition of peritoneal mesothelial cell pyroptosis and a cascade of inflammatory responses. The findings of the present study may provide important information for the prevention and treatment of PF. However the specific mechanism by which API attenuates PF and pyroptosis needs to be further explored. We still need to improve animal experiments to verify that apigenin attenuates PF. This is our next experimental focus. Declarations Author Contribution XFM and YMZ dsign experiments and develop experimental plans， DDX and NNL complete the experiment， XL and ZFJ analyzed the data. Declaration of interest statement The authors declare that there are no conflicts of interest. Funding details This work was funded by Shandong Traditional Chinese Medicine Technology Project (2019-0484). References Howell M, Walker RC, Howard K. Cost effectiveness of dialysis modalities: A systematic review of economic evaluations. Appl Health Econ Health Policy . 2019;17(3):315-330. doi.org/10.1007/s40258-018-00455-2 Masola V, Bonomini M, Borrelli S, et al. Fibrosis of peritoneal membrane as target of new therapies in peritoneal dialysis. Int J Mol Sci . 2022;23(9): 4831. doi.org/10.3390/ijms23094831 Mehrotra R, Devuyst O, Davies SJ, Johnson DW. The current state of peritoneal dialysis. J Am Soc Nephrol . 2016;27(11): 3238-3252. doi.org/10.1681/ASN.2016010112 Wang Y, Shi Y, Tao M, Zhuang S, Liu N. 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Antioxid Redox Signal . 2019;30(12):1503-1520. doi.org/10.1089/ars.2017.7366 Gaidt MM, Hornung V. Pore formation by GSDMD is the effector mechanism of pyroptosis. EMBO J . 2016;35(20):2167-2169. doi.org/10.15252/embj.201695415 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. 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-4196099","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":286183332,"identity":"004885c1-ae08-41f1-938b-5d53da7a223b","order_by":0,"name":"Dandan Xue","email":"","orcid":"","institution":"Affiliated Hospital of Jining Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dandan","middleName":"","lastName":"Xue","suffix":""},{"id":286183334,"identity":"2ccff068-b66a-49c5-bbc4-0e3076cd9651","order_by":1,"name":"Nana Luo","email":"","orcid":"","institution":"Affiliated Hospital of Jining Medical University","correspondingAuthor":false,"prefix":"","firstName":"Nana","middleName":"","lastName":"Luo","suffix":""},{"id":286183336,"identity":"197bb5b3-6e57-4c11-8bed-6b50f57f7c47","order_by":2,"name":"Xiang Li","email":"","orcid":"","institution":"Affiliated Hospital of Jining Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Li","suffix":""},{"id":286183338,"identity":"e786ffb6-6d1d-4260-9ab9-073624a7ff37","order_by":3,"name":"Zhanfeng Jiao","email":"","orcid":"","institution":"Affiliated Hospital of Jining Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhanfeng","middleName":"","lastName":"Jiao","suffix":""},{"id":286183339,"identity":"c5c7a1d6-5f18-4c78-980e-2ed0727c9145","order_by":4,"name":"Yiming Zhang","email":"","orcid":"","institution":"Affiliated Hospital of Jining Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yiming","middleName":"","lastName":"Zhang","suffix":""},{"id":286183340,"identity":"11113ead-c2d7-4818-8fbb-e7754c3a5814","order_by":5,"name":"Xiaofen Ma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYJCCAwwMcmDqA0OFhBw/kVqMQRTjDIYzFsaSDcRZBNLCwDiDsa0icQMhLbrtZwwP/KgwkDNnPGPYzDtPgnEDA/PDRzfwaDE7k5ZwsOeMgbFlA0jLNglmcwY2Y+McfFoOJB84wNv2J3HDgTPmj4Fa2CwbeNik8Wo5/7Dh4N82g3qgFqAtcyR4DA4Q0nIj+cBh3jaDBAOwlgYJCSK0PEs4LHPGwHDDgWOFjXOOSRhINhPyy/kc449vKgzkDW4c3tjwpqauvp+9+eFjfFoQQOKEARMPiMFMlHIQ4G9/wPiDaNWjYBSMglEwkgAAXD5WDX8pXs8AAAAASUVORK5CYII=","orcid":"","institution":"Affiliated Hospital of Jining Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xiaofen","middleName":"","lastName":"Ma","suffix":""}],"badges":[],"createdAt":"2024-03-31 14:44:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4196099/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4196099/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54082038,"identity":"044e0768-94d1-4f37-a9b3-0e7f22a1df77","added_by":"auto","created_at":"2024-04-04 10:01:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":375400,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of VX-765 on the pyroptosis and fibrosis of high glucose-induced MPMCs. Western blot analysis of the effects of VX-765 on caspase 1, NLRP3, GSDME-N, and IL-1β expression of MPMCs induced by high glucose medium.\u003c/p\u003e","description":"","filename":"Binder11.png","url":"https://assets-eu.researchsquare.com/files/rs-4196099/v1/14a23fac7cc51941c0f96b3c.png"},{"id":54082039,"identity":"803b5604-bfb1-4b64-86a3-2f806e22bbba","added_by":"auto","created_at":"2024-04-04 10:01:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":219447,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of VX-765 on pyroptosis and fibrosis in high glucose-treated MPMCs. Representative transmission electron micrographs showing MPMCs. Electron microscopy revealed that in the high glucose group, MPMCs continued to swell until the cell membrane ruptured, the nucleus was still intact, and the cell structure was obviously damaged, with many bubble-like protrusions and the release of contents; however, it was significantly reduced in the VX-765 group.\u003c/p\u003e","description":"","filename":"Binder12.png","url":"https://assets-eu.researchsquare.com/files/rs-4196099/v1/1ea834ffa457c8ceaa5a050b.png"},{"id":54082037,"identity":"5f8a848e-e978-4fe8-bc47-c1d8dd12cd61","added_by":"auto","created_at":"2024-04-04 10:01:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1634060,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of VX-765 on N-cadherin and E-cadherin expression were determined using western blot analysis of MPMCs induced by high glucose medium. *P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Binder13.png","url":"https://assets-eu.researchsquare.com/files/rs-4196099/v1/6379bb481d24f61573a54306.png"},{"id":54082041,"identity":"c5579a32-df54-4833-91be-6f0bad558925","added_by":"auto","created_at":"2024-04-04 10:01:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":12856,"visible":true,"origin":"","legend":"\u003cp\u003eELISA analysis of LDH in high glucose-induced MPMCs treated with high glucose and apigenin. The concentration of LDH significantly decreased in high glucose-induced MPMCs after apigenin treatment.\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4196099/v1/584fa1d44ef95c8161551278.png"},{"id":54082040,"identity":"0febca81-b751-4a3a-b79f-3ede19f533f4","added_by":"auto","created_at":"2024-04-04 10:01:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":356090,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of apigenin on pyroptosis and fibrosis in high glucose-treated MPMCs. The effects of apigenin on caspase 1, NLRP3, GSDME-N, and IL-1β expression were determined using Western blot analysis of MPMCs induced by high glucose medium.\u003c/p\u003e","description":"","filename":"Binder21.png","url":"https://assets-eu.researchsquare.com/files/rs-4196099/v1/3562a284b2f3794eb4a55ce6.png"},{"id":54082044,"identity":"64db5132-acc8-43dc-a130-e019bf50b60a","added_by":"auto","created_at":"2024-04-04 10:01:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":243053,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of apigenin on N-cadherin and E-cadherin expression were determined using western blot analysis of MPMCs induced by a high glucose medium. *P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Binder22.png","url":"https://assets-eu.researchsquare.com/files/rs-4196099/v1/7173aa609ec977eba18b1987.png"},{"id":54082042,"identity":"5a03d04a-a726-4fbf-a014-dc53da321ea0","added_by":"auto","created_at":"2024-04-04 10:01:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1210467,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of apigenin on pyroptosis and fibrosis in high glucose-treated MPMCs. Immunofluorescence and quantitative analyses of Collagen III, GSDMD-N, and NLRP3. *P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Binder23.png","url":"https://assets-eu.researchsquare.com/files/rs-4196099/v1/f8958f76dc9a828f6e9fbf4f.png"},{"id":55799137,"identity":"f796cb5f-b43c-4df8-ae65-a947884fdf9b","added_by":"auto","created_at":"2024-05-03 12:03:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4623140,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4196099/v1/d8c3dc50-d8c7-4771-99a0-b7c1dad18461.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Article Type: Apigenin alleviated peritoneal dialysis-related peritoneal fibrosis by regulating pyroptosis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eApproximately 2\u0026nbsp;million people worldwide are estimated to have end-stage renal disease (ESRD), and this number is increasing annually.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Peritoneal dialysis (PD) is a recognized renal replacement therapy.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Prolonged exposure to PD fluid activates a series of pathological events, including changes in peritoneal vascular solutes (neovascularization), exacerbation of interstitial microchanges (fibrosis), reduction of osmotic conductivity, recruitment of inflammatory cells, and increased production of inflammatory cytokines, which worsens and exacerbates peritoneal fibrosis (PF), leading to ultrafiltration failure, and for which there is currently no effective treatment.\u003csup\u003e456\u003c/sup\u003e .\u003c/p\u003e \u003cp\u003ePyroptosis is a type of hemolytic programmed cell death characterized by cell swelling, rupture, secretion of cell contents, and significant pro-inflammatory effects.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e It is typically mediated by the assembly of inflammatory vesicles.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e The assembly of inflammatory vesicles begins with cytosolic pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) and are formed by the assembly of NOD-like receptors (NLRs) (as sensor proteins) together with caspase-1 precursors, apoptosis-associated speck-like proteins (ASC).\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Caspase-1 efficiently cleaves Gasdermin D (GSDMD) to become the GSDMD-N structural domain mediating the formation of pores in the plasma membrane that release pro-inflammatory factors such as interleukin (IL)-1β, IL-18, and high mobility group protein B1 (HMGB1), leading to an inflammatory response.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eCaspase-1, as a key component of inflammatory vesicles, is considered an important target for inhibiting inflammasome activation. VX-765 is a potent, bioavailable, and non-toxic small-molecule inhibitor of caspase1,\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e and it effectively inhibits inflammatory cell infiltration and pyroptosis-associated protein expression, thereby reducing renal inflammation and fibrosis.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e We hypothesized that VX-765 has anti-inflammatory or anti-extracellular matrix accumulation effects. However, whether VX-765 protects against PF by regulating pyroptosis remains unclear.\u003c/p\u003e \u003cp\u003eApigenin (API) is a 4\u0026prime;,5,7-trihydroxyflavone that has been shown to exert various pharmacological effects, including anti-inflammatory, antioxidant, and anti-fibrotic effects.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Meng et al. reported that API inhibits NOD-like receptor protein 3 (NLRP3) inflammasome activation by activating autophagy, thereby attenuating hepatocyte pyroptosis.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Previous studies have demonstrated that API can inhibit miR34a expression by downregulating PF in mice; nevertheless, the exact mechanism remains unclear.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Moreover, no evidence suggesting that API protects against peritoneal mesothelial cell fibrosis by inhibiting pyroptosis exists. Therefore, this study aimed to investigate the molecular-level effects and mechanism of action of API on PF.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell culture and treatment\u003c/h2\u003e \u003cp\u003eIsolated mouse mesothelial peritoneal cells (MPMCs) were cultured in RPMI 1640 medium supplemented with 15% fetal calf serum, L-glutamine, hydrocortisone, insulin, transferrin, selenium, 20 mM HEPES, 4 mg/L gentamicin, 120 mg/L benzylpenicillin, and 2.5 mg/L amphotericin at 37\u0026deg;C in a humidified culture chamber supplied with 5% CO\u003csub\u003e2\u003c/sub\u003e. The culture medium was changed every 3 days until the cells were confluent. Subsequently, cells were washed with phosphate-buffered saline and digested with 0.007% ethylenediaminetetraacetic acid solution containing 0.02% trypsin at 37\u0026deg;C for 2 min, followed by the addition of fresh medium for a new round of culture or subsequent analysis. The API used for cell treatment was purchased from Merck with a purity of over 99%, as determined using high-performance liquid chromatography (HPLC). VX-765 was purchased from MedChemexpress. To examine the effects of API on MPMCs in high-glucose fluids, MPMCs were divided into four groups: i) vehicle control group, cells were cultured in regular media and treated with dimethyl sulfoxide (DMSO); ii) high glycemic model group, cells were cultured in high glucose culture solution; iii) high glycemic model\u0026thinsp;+\u0026thinsp;vehicle group, cells were cultured in high glucose culture solution and treated with DMSO; and iv) high glycemic model\u0026thinsp;+\u0026thinsp;API group, cells were cultured in high glucose culture solution and treated with API.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Western blot analysis\u003c/h2\u003e \u003cp\u003eThe primary mouse peritoneal mesothelial cells were inoculated onto 6-well plates and allowed to grow to 70\u0026ndash;80% at 37\u0026deg;C. Subsequently, MPMCs were treated with API in a 75 mM high-sugar medium. Each sample was collected from six parallel wells. The protein solutions of MPMCs were extracted with a lysis buffer comprising radioimmunoprecipitation assay buffer, protease inhibitor, phosphatase inhibitor, and phenylmethylsulfonyl fluoride (50:1:1:1) and left to stand on ice for 40 min. The cell lysate was centrifuged at 12,000 rpm for 20 min, and the protein concentration in the supernatant was determined using the bicinchoninic acid assay (Beyotime, China). Equal amounts of protein per sample were loaded into each well, separated on a 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% bovine serum albumin (BSA) for 2 h and then incubated overnight with the following rabbit-derived polyclonal antibodies: NLRP3 (Affinity, China), caspase 1 (Affinity, China), IL-1β (Affinity, China), GSDMD (CST, USA), N-cadherin (Abclonal, China), and E-cadherin (Abclonal, China), and GAPDH (Abclonal, China) was used as an internal control. Membranes were washed with tris-buffered saline with Tween-20 and incubated with a secondary antibody (Abclonal, China) for 1 h at room temperature. The bound antibodies were visualized using enhanced chemiluminescence Plus (Proteintech, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Immunofluorescence staining\u003c/h2\u003e \u003cp\u003eThe MPMCs were seeded in a glass-bottom cell culture dish (1\u0026times;10⁵ cells per well) and allowed to adhere overnight at 37℃. After various treatments, MPMCs were treated with 4% buffered paraformaldehyde for 20 min at room temperature and then treated with 0.5% Triton X-100 for 20 min. Subsequently, 5% BSA was used to block the membrane for 1 h at room temperature and then treated with the primary antibodies against NLRP3, GSDND-N and Collagen III at 4\u0026deg;C overnight before treatment with the secondary antibody for 1 h at room temperature. Nuclei were stained with 4',6-diamidino-2-phenylindole (Beyotime, China). Images were captured using a fluorescence microscope. The immunostaining results were quantified and statistically analyzed using Image-Pro Plus 6.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Enzyme-linked immunosorbent assays (ELISA)\u003c/h2\u003e \u003cp\u003eThe cell culture media were collected and centrifuged at 3,000 rpm for 20 min at 4\u0026deg;C. The cell supernatant was assayed for lactate dehydrogenase (LDH) using an ELISA kit according to the manufacturer\u0026rsquo;s instructions, using a microplate reader. The development of color intensity in each well was visualized by measuring the 450-nm absorbance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Scanning electron microscopy\u003c/h2\u003e \u003cp\u003eSlides containing cells treated with and without hydrogen peroxide were dehydrated using increasing concentrations of ethanol (30, 40, 50, 60, 70, 80, 90, and 100%). After drying in a CO\u003csub\u003e2\u003c/sub\u003e critical point dryer, the sample was adhered to the sample stage with a double-sided conductive tape, sprayed using ion sputtering, and then observed and photographed using an electron microscope (Hitachi, SU8010).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll results data was expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). GraphPad Prism software version 8.0 was used for all statistical analyses. Each experiment was conducted in triplicates. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 High glucose treatment induces pyroptosis and epithelial-mesenchymal transition (EMT) in MPMCs\u003c/h2\u003e \u003cp\u003ePrimary mouse peritoneal mesothelial cells were used to investigate the role of peritoneal mesothelial cell pyroptosis in PF. According to our previous research,\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e we treated MPMCs with 75 mM D-glucose and observed that NLRP3, cleaved caspase-1, GSDMD p30 fragment, and cleaved IL-1β expression was increased in high glucose medium as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Simultaneously, we observed that high glucose levels decreased E-cadherin expression and increased N-cadherin expression in peritoneal mesothelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The results showed that high glucose levels promoted the expression of the above-mentioned proteins and activated the pyroptotic pathway and EMT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 VX-765 suppresses high glucose-induced pyroptosis and fibrosis in MPMCs\u003c/h2\u003e \u003cp\u003eTo determine whether caspase1 inhibitor VX-765 could attenuate indicators related to pyroptosis in mouse peritoneal mesothelial cells, the expression of pyroptosis-related proteins IL-1β, NLRP3, caspase-1, and GSDMD was assessed using western blotting and immunofluorescence methods. We found that the expression of cleaved-IL-1β, NLRP3, cleaved-caspase-1, and GSDMD-N, markers related to cellular pyroptosis, was significantly decreased by the administration of VX-765 and induced by high glucose for 48 h, indicating that VX-765 successfully inhibited cellular pyroptosis. Consistent with the western blotting results, immunofluorescence analysis demonstrated that VX-765 significantly inhibited the expression of NLRP3 and GSDMD-N. The effect of VX-765 on cell death was detected using electron microscopy, and it was found that under the influence of high glucose, MPMCs continued to swell until the cell membrane was ruptured, the nucleus remained intact, and the cell structure was obviously damaged, with many bubble-like protrusions and release of contents. However, in the VX-765 group, the cell structure was intact, and cell death was significantly reduced compared to that in the high glucose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results confirmed the inhibitory effect of VX-765 on high glucose-induced pyroptosis in MPMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). After VX-765 treatment of MPMCs, the protein expression level of N-cadherin, a mesenchymal phenotype related to PF, was significantly reduced, and that of E-cadherin, an epithelial phenotype, was significantly increased compared to that of the high glucose group. The protein expression level of the epithelial phenotype E-cadherin was significantly higher than that in the high glucose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Collectively, these results indicated that VX-765 alleviated PF in MPMCs. PF in MPMCs and MPMCs pyroptosis are closely associated with fibrosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 API inhibits high glucose-induced pyroptosis and fibrosis in MPMCs\u003c/h2\u003e \u003cp\u003eELISA was used to detect the expression level of LDH in cell culture supernatants, and the level of LDH was found to be lower in the API group than in the high glucose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Protein expression levels of pyroptosis markers (IL-1β, NLRP3, Caspase-1, and GSDMD) were detected USING western blotting after treatment of MPMCs with 75 mM high glucose and API. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the expression level of NLRP3, cleaved caspase-1, GSDMD-N, and cleaved IL-1β was substantially increased in the high glucose model group relative to the control group, and the increase of NLRP3, cleaved caspase-1, GSDMD-N, and cleaved IL-1β expression mediated by high glucose was attenuated by API in MPMCs. Moreover, API acted as both an inhibitor and stimulator of EMT-related indices (N-cadherin and E-cadherin) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The immunofluorescence results were consistent with the western blotting results (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results showed that API attenuated the effects of high glucose-mediated MPMCs EMT and pyroptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePD is an effective renal replacement method for patients with ESRD. Peritonitis and prolonged PD can cause PF. Peritonitis induces angiogenesis and fibrosis, and the development of PF is usually the primary cause of PD interruption.\u003csup\u003e21,\u003c/sup\u003e\u003csup\u003e22\u003c/sup\u003e In our study, API pretreatment attenuated peritoneal mesothelial cell pyroptosis, decreased protein expression of caspase-1, GSDMD, and NLRP3, and reduced IL-1β leakage. Additionally, API delayed the onset of PF by attenuating cellular pyroptosis.\u003c/p\u003e\n\u003cp\u003eIndeed, our previous studies have demonstrated that API significantly promotes the proliferation of mouse peritoneal mesothelial cells, inhibits high glucose-induced apoptosis, and effectively inhibits high glucose-induced fibrosis in mouse peritoneal mesothelial cells.\u003csup\u003e20\u003c/sup\u003e API, a flavonoid, is a low-toxicity free radical scavenger and an antioxidant phenolic compound that exhibits anti-inflammatory, antiviral, and anti-fibrotic effects, possibly reducing the risk of chronic diseases. API effectively mitigates pyroptosis and plays a critical role in cardiovascular disease, particularly in ischemia/hypoxia-induced cardiomyocyte injury. Additionally, API significantly reduces the level of pro-inflammatory factors in hypoxia-induced cardiomyocytes, and the mechanism may include effective attenuation of cardiomyocyte pyroptosis and apoptosis, inhibition of the level of pro-inflammatory factors (IL-1β and IL-18) levels, effectively attenuating myocardial injury.\u003csup\u003e23\u0026nbsp;\u003c/sup\u003eMoreover, API attenuated palmitic acid (PA)-induced NLRP3 inflammasome activation and cellular charring in primary mouse hepatocytes, which regulated NLRP3 inflammasomes through two pathways, thereby attenuating PA-induced cellular charring.\u003csup\u003e24\u003c/sup\u003e In our present study, we used API to treat high glucose-stimulated mouse MPMCs, and the results demonstrated that API attenuated the onset of MPMCs pyroptosis and fibrosis.\u003c/p\u003e\n\u003cp\u003eThe pyroptosis mechanism, leading to cell death, is triggered by the production and initiation of inflammasome-associated components, often referred to as intracellular protein complexes.\u003csup\u003e25\u003c/sup\u003e In the innate immune response, at the initiation step, specific PRRs, including Toll-like receptors, NLRs, and cytokine receptors, recognize PAMP, DAMP, and cytokines, which activate the NF-κB signaling pathway and increase the levels of NLRP3, pro-IL-1β, and pro-IL-18.\u003csup\u003e26,27\u003c/sup\u003e During the activation phase, NLRP3 aggregates via the Nacht structural domain and interacts with pyrin domain (PYD)-PYD to recruit PYD-caspase activation and recruitment domain (CARD)-containing ASC proteins, and CARD then binds to caspase-1 to initiate downstream signaling.\u003csup\u003e28\u003c/sup\u003e In cells, NLRP3 forms inflammatory vesicle complexes by interacting with ASC, caspase-1, and other proteins.29 On the one hand, activated caspase-1 cleaves pro-IL-1β and pro-IL-18 to generate activated IL-1β and IL-18, promoting the release of pro-inflammatory cytokines and triggering an inflammatory response\u003csup\u003e30\u003c/sup\u003e; on the other hand, activated caspase-1 cleaves GSDMD into amino-terminal (GSDMD-CT) and carboxy-terminal (GSDMD-NT) fragments and induces pyroptosis. GSDMD-NT can bind to lipids and insert into the cell membrane, forming pores that secrete small-diameter substrates, including IL-1β and IL-18,\u003csup\u003e31,32\u003c/sup\u003e and as the number of molecular pores increases, the cell membrane eventually becomes relaxed and ruptured, and intracellular contents, such as IL-1α and HMGB1, are also released.\u003csup\u003e33\u003c/sup\u003e However, only a few studies have examined the pathogenic role of cellular pyroptosis in PF. We observed the characteristic morphological and biochemical features of cell pyroptosis, including balloon-like cell membrane swelling, caspase-1 activation, GSDMD shearing, pro-inflammatory factor release, and exudation of cytoplasmic contents in MPMCs modeled with high glucose.\u003c/p\u003e\n\u003cp\u003eVX-765 is an orally bioavailable small-molecule inhibitor that increases caspase-1 with no signs of toxicity and has been used to treat inflammatory and autoimmune diseases.\u003csup\u003e14\u003c/sup\u003e Oral administration of VX-765 was found to be safe in humans in a 6-week phase II clinical trial on epilepsy, making VX-765 a viable drug.\u003csup\u003e34\u003c/sup\u003e Previous studies have demonstrated that VX-765 improves renal function in diabetic animals, inhibits high glucose-induced inflammatory cell infiltration and cellular pyroptosis-related protein expression, and attenuates tubulointerstitial fibrosis.\u003csup\u003e19\u003c/sup\u003e Whether VX-765 protects against high glucose-induced PF by regulating cellular pyroptosis remains unclear. We used VX-765 for the first time to treat high glucose-stimulated MPMCs and observed that VX-765 effectively reduced pyroptosis rate, inflammatory factors release, and fibrosis occurrence, suggesting that cellular pyroptosis inhibitors effectively regulate the inflammation and fibrosis of MPMCs in a high glucose environment.\u003c/p\u003e\n\u003cp\u003eConclusively, API effectively attenuated peritoneal mesothelial cell fibrosis in mice, and its mechanism of action may be related to the inhibition of peritoneal mesothelial cell pyroptosis and a cascade of inflammatory responses. The findings of the present study may provide important information for the prevention and treatment of PF. However the specific mechanism by which API attenuates PF and pyroptosis needs to be further explored. We still need to improve animal experiments to verify that apigenin attenuates PF. This is our next experimental focus.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXFM and YMZ dsign experiments and develop experimental plans， DDX and NNL complete the experiment， XL and ZFJ analyzed the data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by Shandong Traditional Chinese Medicine Technology Project (2019-0484).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHowell M, Walker RC, Howard K. 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Pore formation by GSDMD is the effector mechanism of pyroptosis. \u003cem\u003eEMBO J\u003c/em\u003e. 2016;35(20):2167-2169. doi.org/10.15252/embj.201695415\u003c/li\u003e\n\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":"Peritoneal fibrosis, pyroptosis, apigenin, vx-765","lastPublishedDoi":"10.21203/rs.3.rs-4196099/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4196099/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProlonged exposure of the peritoneum to peritoneal dialysis fluid, peritonitis, and peritoneal blood accumulation can cause peritoneal inflammation and injury, followed by progressive fibrosis, ultimately leading to the cessation of peritoneal dialysis. Apigenin is an herbal medicine with anti-inflammatory, antioxidant, and anti-fibrotic properties. Our previous study found that apigenin could inhibit the process of peritoneal fibrosis, but whether apigenin could treat fibrosis by inhibiting pyroptosis is not known. This study aimed to investigate the protective effects of apigenin against peritoneal mesothelial cell pyroptosis and fibrosis. First, we observed changes in peritoneal mesothelial cells using VX-765, an inhibitor of cellular pyroptosis. Western blotting, immunoblotting, and electron microscopy showed that cellular pyroptosis was significantly inhibited, and peritoneal mesothelial cell fibrosis was reduced accordingly. Secondly, when the cells were co-treated with high glucose medium and apigenin, apigenin attenuated the activation of cellular pyroptosis by decreasing the levels of Gasdermin D, NOD-like receptor protein 3 inflammatory niche activation, caspase-1 and IL-1β and also inhibited the development of peritoneal fibrosis. Thus, our results suggest that apigenin plays a protective role against peritoneal fibrosis by inhibiting pyroptosis.\u003c/p\u003e","manuscriptTitle":"Article Type: Apigenin alleviated peritoneal dialysis-related peritoneal fibrosis by regulating pyroptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-04 10:01:38","doi":"10.21203/rs.3.rs-4196099/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":"f31e2da7-84c4-4388-a291-a749253b1240","owner":[],"postedDate":"April 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-03T11:55:04+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-04 10:01:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4196099","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4196099","identity":"rs-4196099","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}