Monoclonal Antibody to Podoplanin SZ-168 Attenuates Microglia Pyroptosis and Neuroinflammation in Experimental Cerebral Haemorrhage

Monoclonal Antibody to Podoplanin SZ-168 Attenuates Microglia Pyroptosis and Neuroinflammation in Experimental Cerebral Haemorrhage | 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 Monoclonal Antibody to Podoplanin SZ-168 Attenuates Microglia Pyroptosis and Neuroinflammation in Experimental Cerebral Haemorrhage Shiyao Liu, Jie Cui, Ruyi Liang, xia Wang, Yiming Zhao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5120185/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 Neuroinflammation is an important factor in the development of secondary brain injury after intracerebral haemorrhage (ICH), involving the interaction of haemorrhage formation and inflammatory pathways. Podoplanin is a small transmembrane mucin-like glycoprotein, and it has been demonstrated that the PDPN/CLEC-2 axis is involved in inflammatory diseases. This study aims to investigate the therapeutic effect of a monoclonal antibody against podoplanin, SZ168, on neuroinflammation in a mouse model of cerebral haemorrhage. Methods Male C57BL/6 mice (weight 22–25 g, 6–8 weeks old, n = 108) were randomly divided into sham-operated, ICH-operated and SZ168-treated groups. Brain haemorrhage was induced by collagenase injection into the striatum, and SZ168 IgG was administered intraperitoneally 6 h after ICH. Neurological regression was assessed at the molecular, cellular, histological and functional levels by short-term neurobehavioural tests, cerebral oedema, Evans blue staining, Western blotting, ELISA and immunofluorescence staining. In vitro, human-derived microglial HMC3 cells were pretreated with SZ168 IgG and washed platelets. Then hemin treatment was added to construct an in vitro model of cerebral haemorrhage, which was detected by wound healing assay, transwell migration assay, cell counting kit-8 reagent, colony formation on solid media and western blot to detect the functional changes of microglia after PDPN inhibition. Results PDPN expression in mouse brains was elevated 48 h after ICH, and inhibition of PDPN function with SZ168 improved short-term neurological function after ICH and reduced cerebral oedema and cerebral haemorrhage. In vitro, SZ168 treatment inhibited microglial invasion, migration and proliferation functions. Mechanistically, inhibition of PDPN function with SZ168 in vivo and in vitro was able to attenuate ICH-induced expression of nlrp3, caspase-1 and gasdermin D as well as inflammatory factors IL-1β, TNF-α and MPO. Conclusions Blocking PDPN can reduce microglial invasion, migration and proliferation functions by inhibiting the microglial NLRP3/Caspase-1/GSDMD inflammatory pathway, which may play a role in reducing the inflammatory situation at the site of cerebral haemorrhage and ameliorating secondary brain injury. podoplanin cerebral haemorrhage microglia neuroinflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Intracerebral haemorrhage (ICH) is a severe, fatal subtype of stroke that accounts for approximately 10-15% of all stroke patients and has a high mortality and disability rate 1 ,2 . After cerebral haemorrhage, disease progression is mainly influenced by primary brain injury and secondary brain injury (SBI) 3 , which refers to a series of pathological processes triggered by erythrocyte debris and degradation products, such as inflammatory incubation, mitochondrial dysfunction, oxidative stress and neuroendocrine 4 . Neuroinflammation is an important contributor to brain injury following cerebral haemorrhage through the mechanism of microglial activation and neutrophil infiltration, resulting in the release of pro-inflammatory cytokines, including tumour necrosis factor (TNF-α), interleukin (IL-1β), myeloperoxidase (MPO) and other inflammation-related factors. Microglia pyroptosis is closely associated with the inflammatory response and is characterised by rapid plasma membrane rupture and release of cellular contents 5 . In addition, microglia pyroptosis is involved in the pathogenesis of secondary brain injury after ICH. Therefore, inhibiting microglia pyroptosis and the inflammatory response is a potential therapeutic approach to attenuate secondary brain injury after ICH 6 ,7 . Cell pyroptosis is a form of cell death in which caspase-1 regulates the hydrolytic maturation of IL-1β and IL-18 proteins, leading to rapid and damaging inflammatory cell death by the specific mechanism of activated caspase-1 cleavage of the precursors of gasdermin D (GSDMD), IL-1β (pro-IL-1β) and IL-18 (pro-IL-18) 8 . Gasdermin-N structural domain fragments are translocated to the membrane to form a stable circular pore. The pore can allow the passage of inflammatory cell contents, leading to microglia pyroptosis, which results in excessive inflammation 9 . It has been shown that in central nervous system disorders such as Alzheimer's disease and cerebral ischaemia, inhibition of caspase-1 activation and cleavage of GSDMD can effectively ameliorate neuroinflammation and disease progression and attenuate brain damage 10 . In particular, microglia pyroptosis executive GSDMD has been shown in various studies to play a key role in influencing neuroinflammation in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE) 11 ,12 . Therefore, modulation of GSDMD-mediated microglia pyroptosis may help to attenuate neuroinflammation after ICH 13 . Podoplanin (PDPN) is a small transmembrane mucin-like glycoprotein that is an endogenous ligand for platelet C-type lectin-like receptor-2 (CLEC-2). PDPN interaction with platelet CLEC-2 has been shown to induce platelet aggregation and thrombosis, and the PDPN/CLEC-2 axis has been implicated in inflammatory diseases. A mAb SZ168 against the extracellular region of human PDPN was developed at Jiangsu Institute of Hematology, Jiangsu province, China and SZ168 effectively attenuates the secretion of pro-inflammatory cytokines in lung-injured mice 14 and that blocking PDPN in tumours with high levels of PDPN expression inhibits tumour growth and metastasis 15 . In a mouse model of ischaemic stroke, treatment with α-PDPN significantly reduced the levels of caspase-1 and GSDMD in microglia and inhibited the secretion of IL-18 and IL-1β, thereby attenuating the disruption of the blood-brain barrier and the level of inflammation 16,17 . In the present study, we hypothesised that inhibition of PDPN might attenuate secondary brain injury after ICH in mice by inhibiting caspase-1/GSDMD-mediated microglia pyroptosis and activation of neuroinflammation. Materials and Methods Animals C57BL/6 mice (male, weighing approximately 22-25 g, n=108) were purchased and bred at the Animal Centre of Soochow University. All mice were maintained at room temperature (22±1°C) with a 12-hour day/night cycle (humidity: 60±5%) and food ad libitum. All animal procedures were under the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Guidelines Committee of Soochow University. Cell culture The human HMC3 cell line was purchased from Wuhan Punosai (Wuhan, Hubei province, China). Cells were cultured in MEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a 5% CO2 incubator. When cells reached approximately 80% fusion, they were digested with trypsin and passaged for further experiments. Cells were treated with haemoglobin (hemin, 100 μmol/L) dissolved in DMSO for 48 h. At the end of stimulation, cells were lysed with lysis solution and finally centrifuged, and cells were collected and assayed as previously described. Data collection The RNA-seq transcriptome data of GSE200575 in this study were obtained from the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) database. The GSE200575 dataset was divided into an older and a younger group, from each group containing 3 models of cerebral haemorrhage (ICH) and 3 models of sham surgery (SHAM). The transcriptome sequencing data were generated using the GPL24247 platform and derived from C57BL/6 mice. ICH model ICH was induced by stereotactically guided injection of collagenase into the right basal ganglia region. Briefly, mice were anaesthetised with 1% sodium pentobarbital intraperitoneally during the procedure and body temperature was maintained at 37.0 ± 0.5°C with a heated blanket. Mice were placed in the prone position on a stereotactic head frame. A coronal incision was made to expose the skull until the fontanel was visible 18 . A 1 mm cranial hole was then drilled into the skull and a syringe was inserted under stereotactic guidance into the right basal ganglia region (coordinates: 0.2 mm anterior to bregma, 2.0 mm lateral to bregma). A total volume of 0.5 μL collagenase was then infused at a rate of 0.25 μL/min using a micro infusion pump (injection started 3.5 mm inferior to the dura mater). To prevent leakage, the needle was left in place for 10 min after completion of the 0.5 μL infusion and slowly withdrawn at a rate of 1 mm/min, after which the burr hole was closed with sterilised medical bone wax and the scalp incision was sutured. The mice were allowed to fully recover on a 37°C heating pad and neurological deficits were closely monitored. The sham-operated group underwent the same procedure without collagenase injection. Animal groups and the Administration of drug Male C57BL/6 mice (n=108) were randomLy divided into sham-operated (n=32), ICH-operated (n=32) and SZ168-treated groups (n=32) .The experimental design was shown in Part 1 of the Additional file 1: Fig. S1. In the in vivo model, the anti-human PDPN extracellular region mAb (SZ168) was administered intraperitoneally at 100 μg/mL 6 hours after the ICH model was established. In the in vitro model, cells were treated with hemin 100 μmol/L for 2h and then re-treated by adding different concentrations of platelets and SZ168 simultaneously. Neurobehavioural function tests The neurobehavioural function of mice was assessed 24 hours after ICH using the modified Garcia test, administered by a blinded investigator. The modified Garcia test consisted of seven items: spontaneous activity, axial sensation, tentacles, limb symmetry, lateral rotation, forelimb walking and climbing. Each test is scored from 0 to 3 or 1 to 3, with a total score of 3 to 21. Higher scores indicate better neurological function. Brain Tissue Water Content Brain water content (BWC) was measured by the wet/dry method as reported 19 . Overall, mice were killed by decapitation under deep anaesthesia 48 hours after ICH. Brains were immediately removed and divided into five sections: ipsilateral and contralateral cortex, ipsilateral and contralateral basal ganglia, and cerebellum. Each brain section was immediately measured on an analytical microbalance to obtain wet weight (WW) and then dried at 100°C for 24 h to obtain dry weight (DW). Brain tissue water content was calculated using the following formula: brain tissue water content (%) = [(WW-DW)/WW] * 100%. Blood-brain barrier permeability To assess blood-brain barrier permeability, 200 μL of 2% Evans Blue (Genye, China) was injected via a tail vein as reported 20 . After 24 h of circulation, mice were perfused via the heart under deep anaesthesia with cold phosphate buffer (0.1 M, PBS, p H 7.4). The brains were then removed, divided into left and right hemispheres and immediately stored at -80°C. The right hemisphere was homogenised in 1100 mL PBS, disrupted by ultrasound and centrifuged (12 000 g, 4 °C, 30 min). The supernatant was collected, added to acetone in a 3:7 ratio and incubated overnight at 4°C. After centrifugation (12 000 g, 4 °C, 30 min), the Evans blue staining was measured at 600 nm using a spectrophotometer. Immunofluorescence Staining Two-colour immunofluorescence staining was performed as previously described 21 . Mice were deeply anaesthetised and transcardially perfused with 30 mL ice-cold PBS and 30 mL 4% paraformaldehyde 48 h after ICH. Whole brains were collected and fixed in 4% paraformaldehyde for 24 hours. The brains were then dehydrated in 75%, 80%, 95%, 100% (I) and 100% (II) ethanol for half an hour each. The brains were then removed and placed in ethanol + xylene (1:1) for 2 hours; xylene (I) and xylene (II) for 10 minutes each, after which they were immersed in wax and embedded in melted pure wax. After cooling and confirmation of the embedding, the sections were cut into 5 mm thick coronal sections using a paraffin slicer. For double immunohistochemical staining, brain sections were incubated overnight at 4°C with an antibody: anti-calcium binding protein (Iba-1; 1:1000, Abcam, UK), anti-podoplanin (5 μg/mL, ThermoFisher Scientific, USA), anti-myeloperoxidase (MPO, 1:250, Santa Cruz Biotechnology, USA) and anti-myeloperoxidase (MPO.) Santa Cruz Biotechnology, USA), anti-gasdermin D (1:250, Santa Cruz Biotechnology, USA) were incubated overnight at 4°C and then observed and photographed by fluorescence microscopy after incubation with the corresponding secondary antibody (1:100, Biyuntian, China) for 1 h at 37°C. Images were analysed using Fiji/ImageJ software. Immunohistochemistry Immunohistochemical staining was performed as previously described 22,23 , and mice used for immunohistochemistry were anaesthetised with sodium pentobarbital and perfused transcardially with 30 mL ice-cold PBS and 30 mL 4% paraformaldehyde. Whole brains were collected, fixed in 4% paraformaldehyde for 24 h and transferred to 70% ethanol for paraffin embedding and 5 μm sectioning. Heat-induced antigen retrieval was performed on tissue sections in citrate or EDTA buffer. Sections were oxidase treated, blocked with serum-free proteins, stained with anti-podoplanin (5 μg/mL, ThermoFisher Scientific, USA) and, after overnight incubation at 4°C, immunoreactivity was detected with HRP-conjugated secondary antibodies and stained with diaminobenzidine and haematoxylin. The density of podoplanin immunostaining was measured in five sections using Image J software. Western Blot Western blotting was performed as previously described 24 . Briefly, after mice were perfused with ice-cold PBS (0.1 M, pH 7.4), the tissue surrounding the haematoma was removed 24 hours post-operatively and stored at -80°C in a refrigerator for spare parts. Brain tissue haematoma sites were thoroughly crushed in a glass homogeniser with pre-cooled RIPA lysis buffer containing protease and phosphatase inhibitors to obtain a 10% tissue homogenate, which was then lysed on ice for 30 min, followed by centrifugation at 12,000 rpm for 20 min at 4°C. Macrophages and microglia were lysed on ice for 30 min using RIPA lysis buffer containing protease inhibitors. The cells were then centrifuged at 12,000g for 20 minutes at 4°C. After collecting the supernatant, the concentration of the supernatant was determined using the BCA technique. Equal amounts of proteins (30 μg of cellular proteins or 90 μg of tissue proteins were placed in each well channel) were separated by 10% SDS polyacrylamide gel electrophoresis (treated at 80 V for 30 min, then 120 V for 40 min) and transferred to nitrocellulose (NC) membranes, which were sealed with 5% nonfat dry milk. After primary antibody incubation at 4 °C overnight, the membranes were treated with anti-rabbit or anti-mouse IgG coupled secondary antibodies. The antibodies used were anti-NLRP3 (1:1,000, Proteintech, Wuhan, China); anti-caspase 1 (1:10,000, Proteintech, Wuhan, China); anti-GSDMD/GSDMD-n (1:1,000, Abcam, USA); anti-Gapdh ( 1: 10,000, Proteintech, Wuhan, China); anti-GSDMD/N (1:10,000, Abcam, USA); anti-Gapdh ( 1:10,000, Proteintech, Wuhan, China); and anti-mouse IgG coupling. Proteintech, Wuhan, China); SZ168 (anti-podoplanin, 2 μg/mL, Suzhou, China); anti-mouse IgG ( 1 : 5,000, Biyun Tian, China); anti-rabbit IgG ( 1 : 10,000, Proteintech, Wuhan, China). At the end of the secondary antibody incubation, the membranes were rinsed with PBS and the proteins were then visualised three times using an enhanced chemiluminescence (ECL) system. Quantitative analysis of each protein band was performed using Image J software. Enzyme-linked immunosorbent assay Enzyme-linked immunosorbent assay (ELISA) was performed as previously described 25 . Histone proteins from ICH experimental animals were obtained and assayed using an ELISA kit (Multi Sciences, China) according to the manufacturer's instructions. Mice were anaesthetised 24 h after ICH and then perfused with 0.1 M PBS. Tissue proteins from ICH experimental animals were collected and homogenised in lysis buffer containing protease inhibitors (100 mL lysate per 10 mg tissue); undiluted supernatants were used for the determination of IL-1b, TNF-α and MPO, respectively. The mouse double antibody sandwich ELISA kits for IL-1b, TNF-α and MPO were purchased from Multi Sciences and performed according to the manufacturer's instructions. Platelet isolation Fresh whole blood from healthy donors was collected in anticoagulated tubes containing 0.38% sodium citrate and centrifuged at 1100 rpm for 10 minutes to isolate platelet-rich plasma (PRP). The PRP was aspirated into a new centrifuge tube, centrifuged at 3500 rpm for 2 min, the supernatant discarded and the precipitate resuspended in ETDA-PBS, centrifuged at 2000 rpm for 2 min and repeated three times to obtain washed platelets. Cell counting kit (CCK-8) assay Human microglia were inoculated into 96-well plates (5*10 3 cells per well), the hemin group then added 100 μmol of hemin and cultured for 24 h; the platelet group added different concentrations of washed platelets for 2 h and then added 100 μmol of hemin and cultured for 24 h; the SZ168 and mIgG groups added 5*10 6 platelets and different concentrations of SZ168 or 60 μg/mL mIgG were incubated for 2 h before adding 100 μmol of hemin for 24 h. 10μL CCK-8 solution from the CCK-8 kit (BiyunTian, Shanghai, China) was added to each well and incubated at 37°C for 4 hours. Optical density (OD) values at 450 nm were measured in each well using an enzyme marker to assess cell viability. The OD values were normalised to the 24-hour control time point to show changes more clearly 26 . Wound-scratch assay Human microglial cells were inoculated into 24-well plates (1*10 5 /well) and cultured to 90% fusion at 37°C in a 5% CO2 incubator, and after 12 hours of starvation in serum-free medium, the tip of a sterile 200 μL pipette was used to scratch along the cells. The hemin group was then cultured with 100 μmol of hemin for 24 hours; the platelet group was cultured with different concentrations of washed platelets for 2 hours followed by 100 μmol of hemin for 24 hours; the SZ168-treated group and the mIgG group were cultured with 1*10 6 platelets and different concentrations of SZ168 or 25 μg/mL mIgG for 2 hours; after 2 hours, 100 μmol of hemin was added and incubated for 24 hours. Cells were photographed with a light microscope (Leica, Wetzlar, Germany; magnification: ×100) in 2 random fields of view at 0 h and 24 h after scratching. Cell migration distances were calculated using Image Pro-Plus 6.0 software (NIH, USA). Transwell experiments Human microglia were inoculated using 24-well Transwell chambers (Corning, Sigma-Aldrich, USA) (upper layer) at a cell density of 2*10 4 cells per well and cultured in serum-free medium, the hemin group was incubated for 24 h with the addition of 100 μmol of hemin; the platelet group was incubated for 24 hours with the addition of washed platelets at different concentrations for 2 hours followed by the addition of 100 μmol of hemin; SZ168 and mIgG groups were incubated by the addition of 1*10 7 platelets and different concentrations of SZ168 or 25 μg/mL mIgG for 2 hours followed by 100 μmol of hemin for 24 hours. FBS (20%) was prepared in the lower chamber. The incubation time was 24 hours. The invasive cells attached to the lower surface were then stripped and treated with methanol ( 4%, 0.5 h) and crystal violet ( 0.1%, 15 min). Final data were calculated based on five randomly selected fields of view under a light microscope (Leica; magnification: × 100). Plate cloning experiment The human microglial suspension was adjusted to 1000 cells/mL and 1000 cells were inoculated into each well of a 6-well plate; the hemin group was incubated with another 100 μmol hemin for 1 week; the platelet group was incubated with washed platelets of different concentrations for 2 h and then 100 μmol hemin for 1 week; the SZ168-treated group and the mIgG group were incubated with 1*10 7 platelets, and different SZ168 and mIgG groups were cultured with 1*10 7 platelets and different concentrations of SZ168 or 25μg/mL mIgG for 2 hours before adding 100µmol hemin for 1 week, during which the solution was changed every 3-4 days, At the end of the culture, the cells were washed with PBS and 1 mL of 4% paraformaldehyde was added to each well to fix the cells for 30-60 minutes, then washed again and stained by adding 1 mL of crystal violet staining solution to each well for 10 minutes. Cells were stained under a microscope. The cells were photographed under a microscope, the number of clone formations (>50 cells) was counted and the cell images were processed using Image software to obtain the number of clone formations in different groups. Results Mortality and rejection A total of 108 male C57BL/6 mice were used in this study and the overall mortality rate of the ICH mice was 9.26% (10/108), none of the sham-operated group died and 2 mice were excluded from this study because they were found to have no haematoma in the brain after euthanasia(Part 2 of the Additional file 1: Table S1). Differential analysis of PDPN genes in brain tissue of ICH-operated and Sham-operated groups Brain tissue PDPN genes of ICH-operated and SHAM-operated groups were obtained from the GSE200575 dataset, and the two groups were directly divided into old and young groups based on age, respectively, and each group contained information from three ICH samples and three SHAM samples, and the data were obtained from the brain tissues of mice at 24 h after ICH. Differential analysis of the ICH-operated and SHAM-operated groups in the old and young groups showed that the expression of the PDPN gene was increased in both the old and young groups in the ICH-operated group compared with the SHAM-operated group( p <0.05, Fig. 1A-B). Podoplanin expression in ICH and cells over time To observe the expression of PDPN, tissues were harvested from the brain haematoma site after establishing the ICH model in mice and subjected to Western blot analysis, immunohistochemistry and immunofluorescence staining. Endogenous expression of PDPN in the ipsilateral/right cerebral hemisphere was observed by Western blot analysis, immunohistochemistry and immunofluorescence staining 48 hours after ICH creation.WB results showed that the expression of PDPN in mouse brain tissue was significantly increased at 48 hours after ICH compared to the sham-operated group ( p <0.05, Fig.1C). Immunohistochemical results were consistent with the above, PDPN expression was increased at 48h after ICH compared with the sham-operated group ( p <0.001, Fig. 1D), whereas PDPN expression was decreased in the SZ168-treated group ( p <0.05, Fig. 1D). Immunofluorescence double-label staining showed that PDPN was predominantly expressed in microglia/macrophages (Iba-1 + ) in the perihematomal tissue at 48h after ICH, and both PDPN and microglia/macrophage markers were reduced in the SZ168-treated group. ( p <0.001, Fig. 1E). An in vitro cerebral haemorrhage model was established using human microglia treated with hemin, and cellular proteins were extracted and subjected to Western blot analysis separately as the concentration of hemin treatment gradually increased. Compared with the DMSO-treated group, the expression of PDPN gradually increased with hemin concentrations of 40, 60, 80 and 100 μmol/L, reaching the peak at 100 μmol/L( p <0.05, Fig. 1G). SZ168 treatment reduces neurobehavioural deficits, BBB permeability, Brain Edema and Hematoma Size after ICH The ICH model was established using C57BL/6 mice and SZ168 was injected intraperitoneally after 6 hours (Fig. 2A). Two days later, the brains of the mice were removed to observe the cerebral haemorrhage, which showed that the cerebral haematoma area of the mice in the control group was significantly enlarged compared with that of the SZ168-treated group, and it was speculated that the treatment with SZ168 was able to reduce the size of the cerebral haematoma of the mice after ICH (Fig. 2B); In the short-term neurological function observation, by observing the neurological function of mice at 1, 3 and 7 days after modelling, the results showed that the neurological deficits in the ICH+mIgG group were higher than those in the SZ168 treatment group at 1, 3 and 7 days after ICH and that the treatment with SZ168 was able to effectively reduce the degree of neurological impairment( p <0.05, Fig. 2C). The results of brain tissue water content experiments showed that treatment with SZ168 effectively reduced the cerebral oedema phenomenon of bleeding in the brain tissue of mice, and the cerebral oedema in the ipsilateral/right basal ganglia and cortical areas was effectively reduced compared with that in the ICH+mIgG group ( p <0.05, Figure 2E). Evans blue staining experiments showed that SZ168 treatment was able to reduce the permeability of the blood-brain barrier ( p <0.05, Fig. 2D). Overall, the SZ168-treated ICH-operated group significantly improved neurological deficits, reduced cerebral oedema, and reduced BB permeability and haematoma size. SZ168 treatment reduces the expressiodeath microglia pyroptosis molecules and inflammatory cytokines after ICH The therapeutic effect of SZ168 in reducing cerebral haemorrhage after ICH in mice has been observed in previous experiments. It has been demonstrated that the upregulation of PDPN can regulate the inflammatory response and prognosis of diseases through the involvement of inflammatory pathways, especially the Nlrp3/Capase-1/GSDMD pyroptosis pathway, so it observed the expression of PDPN and related inflammatory factors in ICH after SZ168 treatment. Western blot analysis showed that PDPN expression was increased after ICH, accompanied by upregulation of the expression of microglia pyroptosis molecules such as Nlrp3, caspase-1 and GSDMD. However, treatment with SZ168 downregulated the expression of both PDPN and pyroptosis molecules( p <0.05, Fig. 3A). Immunofluorescence staining results were consistent with the above, the number of GSDMD-positive and MPO-positive microglia was increased in the perihematoma tissues after ICH, whereas treatment with SZ168 decreased the number of GSDMD- and MPO-positive microglia in the perihematoma tissues after 48 hours of ICH( p <0.05, Fig. 3B-C). In addition, ELISA results showed that the expression of inflammatory cytokines IL-1β, TNF-α and MPO was increased after ICH, whereas treatment with SZ168 decreased the expression of inflammatory cytokines( p <0.01, Fig. 3D-F). The results of the in vitro experiments were consistent with the above (Fig. 4A), HMC3 cells were treated with hemin to simulate the environment of cerebral haemorrhage in vitro, PLT and SZ168 were added for treatment after two hours of modelling. Western blot showed that the expression of pyroptosis molecules caspase-1 and GSDMD-N was increased in the mIgG group, while the expression of pyroptosis molecules was decreased in the SZ168-treated group as a dose dependent of SZ168.( p <0.05, Fig. 4B,4D). SZ168 treatment reduces microglial invasion and migration function From the results of the above experiments, it was observed that PDPN was able to influence microglia to participate in the development of disease inflammation. Immunofluorescence co-localisation also showed a decrease in microglia co-expressing PDPN and inflammatory pathway proteins, so we speculated whether this was because PDPN reduces the number of PDPN + microglia that have been transferred to the site of haemorrhage by modulating the invasive, migratory and other motility functions of microglia. Therefore, an in vitro ICH model was constructed using human HMC3 cells, a certain concentration of platelets was added to co-incubate with the cells to achieve a better effect in restoring the environment of cerebral haemorrhage in vivo, and the effect of PDPN up-regulation on microglia motility function was observed by cell scratch assay and transwell invasion assay. HMC3 cells were treated with 100 μmol/L hemin and different concentrations of PLT to simulate the environment of cerebral haemorrhage in vitro, and the migration and invasion functions of human microglia were observed by cell scratch assay and transwell invasion assay. As shown in the cell scratch assay, microglia migration gradually increased with the platelet concentration from 1*10 5 , 5*10 5 to 1*10 6 (/mL), with 1*10 6 (/mL) as the peak( p <0.05, Fig. 5A). The cells were then co-incubated with 100 μmol hemin and 1*10 6 (/mL) platelets before being treated with a low (5 μg/mL) or high (25μg/mL) concentration of SZ168. The results showed that SZ168 IgG gradually inhibited microglial migration in a dose dependent ( p <0.05, Fig. 5B). The transwell invasion assay showed that microglial cells invasion was gradually increased with the platelet concentration from 5*10 5 , 5*10 6 to 1*10 7 (/mL) ( p <0.05, Fig. 5D). The cells were then co-incubated with 100 μmol hemin and 1*10 7 (/mL) platelets before being treated with a low ((5 μg/mL) or high (25μg/mL) concentration of SZ168,and the results showed that the invasive ability of microglia was gradually suppressed with the increase of SZ168 concentration( p <0.05, Fig. 5E). SZ168 treatment reduces the proliferative capacity of microglia HMC3 cells were treated with 100 μmol/L hemin with different concentrations of PLT to simulate the environment of cerebral haemorrhage in vitro, and the proliferation function of microglia was observed by cell viability assay and plate cloning experiment. In the cell viability assay, the number of proliferating cells gradually increased with the concentration of platelets from 1*10 3 , 1*10 4 , 1*10 5 , 1*10 6 to 5*10 6 (/mL) ( p <0.01, Fig. 6A). In contrast, SZ168 effectively inhibited cell proliferation, and the inhibitory effect became stronger as the concentration of SZ168 increased ( p <0.05, Fig. 6B). The results of plate cloning experiment were consistent with the above, in brief, when the platelet concentration from 5*10 3 , 1*10 4 to 5*10 4 (/mL), the number of microglia proliferation gradually increased, and 5*10 4 (/mL) reached the peak ( p <0.05, Fig. 6C). The inhibitory results showed that the proliferative ability of microglia was gradually inhibited with the increase of SZ168 concentration from 5, to 50 μg/mL p <0.05, Fig. 6D). From the migration, invasion and proliferation experiments, it can be seen that SZ168 can effectively reduce the invasion, migration and proliferation of microglia after inhibiting the function of PDPN. This result, combined with the results of immunofluorescence staining, suggests that the increased expression of PDPN on microglia enhances their invasion and migration functions, promotes the transfer of microglia to the haematoma site to induce cell death, releases inflammatory factors, and causes aggravation of the haematoma. SZ168 antibody treatment reduces microglial proliferation, migration and invasion into the haematoma site, reduces brain inflammation and decreases the production of inflammatory cytokines, thereby improving neurological function and cerebral haematoma. Discussion In this study, firstly, we found the upregulation of PDPN expression in the cerebral haemorrhage group in the gene set GSE200575, and the experimental results were consistent with the genetic analysis, which showed that PDPN expression was upregulated in the cerebral haemorrhage model in mouse brain tissue, and that inhibition of its function by using SZ168 reduced the neurological impairments, cerebral oedema as well as the impairment of the blood-brain barrier, and, secondly, that this effect can be presumed to be due to the release of large amounts of inflammatory factors mediated by the Nlrp3/Caspase-1/GSDMD signalling pathway via the release of a large number of inflammatory factors, that this effect can be speculated to be due to the release of large amounts of inflammatory factors mediated by the Nlrp3/Caspase-1/GSDMD signalling pathway and the downregulation of inflammatory pathway proteins such as Nlrp3, Caspase-1 and GSDMD and inflammatory factors such as IL-1β, TNF-α and MPO after treatment with SZ168, thus serving to ameliorate the therapeutic effects of secondary brain injury. Finally, by observing that the expression of PDPN and microglial cell markers were reduced in the brain tissue of mice after treatment with SZ168, and by reducing the migration, invasion and proliferation of microglial cells after using SZ168 in vitro experiments, it was speculated that SZ168 was able to inhibit the proliferation and migration and invasion of microglial cells into the cerebral haematoma site, thereby reducing the occurrence of cerebral haematoma inflammation, which was an anti-microglia pyroptosis and anti-inflammatory factor. SZ168 can inhibit the proliferation and migration of microglia to the haematoma site, thereby reducing the inflammation in the haematoma site and exerting anti-microglia pyroptosis and anti-inflammatory effects. A large number of studies have demonstrated that inhibition of neuroinflammation can reduce secondary brain injury and improve neurological function after ICH, as neuroinflammation is an important pathogenetic mechanism of secondary brain injury after ICH 27,28 . The neuroinflammatory response in the CNS is a complex process influenced by the synergistic interaction of different groups of neuroglia 29 , and microglia play an important activating role in activating the initial steps of the inflammatory response, promoting the infiltration of neutrophils, amplifying the inflammatory response and leading to the disruption of the extracellular matrix, the integrity of cell membranes and the Impaired blood-brain barrier 30,31,32 . However, in the present study, PDPN expression was found to be up-regulated in microglia, a transmembrane mucin that has been implicated in lymphatic endothelium and lymphangiogenesis 33 and is more widely expressed in a variety of human tumours and inflammatory diseases, where PDPN is implicated in platelet aggregation and metastasis formation through binding to CLEC-2, and where it can regulate the cytoskeleton, thereby increasing cell migration and invasion. Cytoskeleton, thereby increasing cell migration and invasion 34 . There is evidence that the knockdown of PDPN can affect microglial motility and phagocytosis, and the intracellular structural domain of PDPN binds directly to ERM proteins and also regulates cell motility and morphology through the activation of various cellular signalling pathways such as RhoGTPases 35,36 . It has been reported that in neuro-malignant gliomas and melanomas, PDPN can increase the migratory capacity of tumour cells and enhance the tube-forming activity of endothelial cells and that PDPN expression is an important step in tumour progression 37 , and the role of PDPN in rheumatoid arthritis (RA), sepsis and the wound healing process 38,39 , and that the CLEC-2-PDPN pathway was able to promote infiltration of PDPN-positive immune cells and macrophage recruitment at the site of infection. There is also evidence that PDPN may influence the prognosis of diseases such as cerebral ischaemia-reperfusion and traumatic brain injury by promoting the pyroptosis pathway and releasing inflammatory factors, leading to more severe neurological impairment, cerebral oedema and BBB destruction 40,41 . In conclusion, we also observed elevated protein expression of the pyroptosis pathway in microglia, which correlated with increased PDPN expression following ICH. Treatment with SZ168 resulted in decreased pyroptosis protein expression and PDPN expression, as well as a reduction in neurological impairments, cerebral oedema, and blood-brain barrier disruption. This suggests a correlation between PDPN expression and prognosis in patients with cerebral haemorrhage, indicating a potential therapeutic strategy for ICH. Nevertheless, further elucidation is required regarding the mechanism of PDPN's role in microglia inflammation, as well as the specific mechanisms and pathways through which PDPN affects microglia motor function. Conclusions In summary, we have first demonstrated that inhibition of PDPN function using the monoclonal antibody SZ168 can improve impaired neurological function, reduce cerebral haematomas and ameliorate neuroinflammation and cerebral oedema after ICH via the Nlrp3/Caspsae-1/GSDMD pathway. Therefore, podoplanin protein may be a promising therapeutic idea for early treatment of ICH to improve prognosis. Abbreviations ICH Intracerebral haemorrhage PDPN Podoplanin NLRP3 Pyrin Domain Containing Protein 3 GSDMD Gasdermin D MPO Myeloperoxidase IL-1β Interleukin-1β TNF-α Tumor necrosis factorα DMSO Dimethylsulfoxide Iba-1 Ionized calcium-binding adaptor molecule-1 PBS Phosphate-buffered saline BWC Brain water content WW Wet weight DW Dry weight BBB Blood-brain barrier. Declarations Author contributions SYLworked on the experimental design, conducted the experiments, analysed the data, and drafted the manuscript. JC, RYL, XW and YMZ participated in the experimental design, data analysis and interpretation, and manuscript preparation. All authors read and approved the final manuscript. Funding This project was supported by the National Natural Science Foundation of China-funded projects(Grant81873431) Availability of data and materials Other researchers who wish to reproduce the results or replicate the procedures will have access to the data, analysis methods, and study materials. The data supporting the results of this study are available from the corresponding author upon reasonable request. The authors are responsible for maintaining availability. Ethics approval and consent to participate All animal experiments were approved by the Animal Care and Use Committee of Soochow University First Affiliated Hospital. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Keep RF, Hua Y, Xi G. Intracerebral haemorrhage: mechanisms of injury and therapeutic targets. Lancet Neurol. 2012;11(8):720–31. Lapchak PA, Zhang JH. The high cost of stroke and stroke cytoprotection research. Transl Stroke Res. 2017;8(4):307–17. Tschoe C, Bushnell CD, Duncan PW, Alexander-Miller MA, Wolfe SQ. Neuroinflammation after intracerebral hemorrhage and potential therapeutic targets. J Stroke. 2020;22:29–46. Wu X, Luo J, Liu H, Cui W, Guo K, Zhao L, Bai H, Guo W, Guo H, Feng D, Qu Y. 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Luo X, Li L, Zheng W, Gu L, Zhang X, Li Y, et al. HLY78 Protects Blood-Brain Barrier Integrity Through Wnt/Beta-Catenin Signaling Pathway Following Subarachnoid Hemorrhage in Rats. Brain Res Bull (2020) 162:107–14. Paradowska-Gorycka A, Wajda A, Romanowska-Prochnicka K, Walczuk E, Kuca-Warnawin E, Kmiolek T, et al. Th17/Treg-Related Transcriptional Factor Expression and Cytokine Profile in Patients With Rheumatoid Arthritis. Front Immunol (2020) 11:572858. Feng, C.; Yu, A.; Wang, Z.; Wang, K.; Chen, J.; Wu, Y.; Deng, T.; Chen, H.; Hou, Y.; Ma, S.; Dai, X.; Huang, L. A Novel PDPN Antagonist Peptide CY12-RP2 Inhibits Melanoma Growth via Wnt/β-Catenin and Modulates the Immune Cells. J Exp Clin Cancer Res 2024, 43 (1), 9. Wang J. Preclinical and Clinical Research on Inflammation After Intracerebral Hemorrhage. Prog Neurobiol (2010) 92(4):463–77. Xu P, Hong Y, Xie Y, Yuan K, Li J, Sun R, et al. 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Podoplanin increases migration and angiogenesis in malignant glioma. Int J Clin Exp Pathol. 2015;8(7):8663–8670. J. Sikorska, D. Gawel, H. Domek, M. Rudzinska, B. Czarnocka, Podoplanin (PDPN) affects the invasiveness of thyroid carcinoma cells by inducing ezrin, radixin and moesin (E/R/M) phosphorylation in association with matrix metalloproteinases, BMC Canc. 19 (2019) 85. B. Fernandez-Munoz, M.M. Yurrita, E. Martin-Villar, P. Carrasco-Ramirez, D. Megias, J. Renart, M. Quintanilla, The transmembrane domain of podoplanin is required for its association with lipid rafts and the induction of epithelial-mesenchymal transition, Int. J. Biochem. Cell Biol. 43 (2011) 886e896. Feng C, Yu A, Wang Z, et al. A novel PDPN antagonist peptide CY12-RP2 inhibits melanoma growth via Wnt/β-catenin and modulates the immune cells. J Exp Clin Cancer Res. 2024;43(1):9. Published 2024 Jan 2. M. Honma, T. Shibuya, K. Hayashi, S. Iinuma, M. Fujii, A. Ishida-Yamamoto, Suppression of podoplanin expression during differentiation of epidermal keratinocytes, J. Dermatol. 46 (2019) 922e924. J. Rayes, S. Lax, S. Wichaiyo, S.K. Watson, Y. Di, S. Lombard, B. Grygielska, S.W. Smith, K. Skordilis, S.P. Watson, The podoplanin-CLEC-2 axis inhibits inflammation in sepsis, Nat. Commun. 8 (2017) 2239. Fei M, Wang H, Zhou M, Deng C, Zhang L, Han Y. Podoplanin influences the inflammatory phenotypes and mobility of microglia in traumatic brain injury. Biochem Biophys Res Commun. 2020;523(2):361–367. Meng D, Ma X, Li H, et al. A Role of the PodoplaninCLEC-2 Axis in Promoting Inflammatory Response After Ischemic Stroke in Mice. Neurotox Res 2021;39:477 − 88. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials.docx Additional file 1. Supplementary Fig. S1. Experimental design and animal groups. ICH, intracerebral hemorrhage; WB, western blot; ELISA, enzyme-linked immunosorbent assay; Supplementary Table S1. Summary of experimental groups and mortality rate in the study. 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-5120185","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":371255226,"identity":"6238fb56-f2ba-4fdf-942f-5dbde74856d6","order_by":0,"name":"Shiyao Liu","email":"","orcid":"","institution":"National Clinical Research Center for Hematologic Diseases, The First Affiliated Hospital of Soochow University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shiyao","middleName":"","lastName":"Liu","suffix":""},{"id":371255227,"identity":"7f367297-1b9d-4c0c-a632-6867f7b68599","order_by":1,"name":"Jie Cui","email":"","orcid":"","institution":"Changning Maternity and Infant Health Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Cui","suffix":""},{"id":371255228,"identity":"64c02541-0a7d-40fe-8092-bb72bd03c7f2","order_by":2,"name":"Ruyi Liang","email":"","orcid":"","institution":"National Clinical Research Center for Hematologic Diseases, The First Affiliated Hospital of Soochow University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ruyi","middleName":"","lastName":"Liang","suffix":""},{"id":371255229,"identity":"8e37dff3-02f1-42c0-bbe5-6bdc96c4a342","order_by":3,"name":"xia Wang","email":"","orcid":"","institution":"The Affiliated Suzhou Hospital of Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"xia","middleName":"","lastName":"Wang","suffix":""},{"id":371255230,"identity":"8361b263-e98e-4f05-866d-2d07e6279413","order_by":4,"name":"Yiming Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYDACCRBhwMDDAEIMFRJy8iRqOWNhbNhAlBYwAGphbKtIZDhAQIf87OaHj3kKtsmY8689+Jh3nkQCYwPzw0c38GhhnHPM2JjH4DaP5Yx3yYYzt0nksTOwGRvn4NHCLJFgJg3SYnDjjJnEx20SxYwNPGzS+LSwSaR/Q2hJnCOR2HCAgBYeiRyoLed7gLY0EKFFQiKn2HAO2Ba+ZMMZxySMDZsJ+EV+RvrGB2/+3LY3OH/24GOemjo5eXZgGOLTAgJMPGD7EqBcZgLKQYDxB4jkP0CE0lEwCkbBKBiRAABi3Uc9e75WmwAAAABJRU5ErkJggg==","orcid":"","institution":"National Clinical Research Center for Hematologic Diseases, The First Affiliated Hospital of Soochow University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yiming","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2024-09-20 02:56:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5120185/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5120185/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70474861,"identity":"84996020-ba8d-492d-9ba8-dcecad8be269","added_by":"auto","created_at":"2024-12-03 14:05:46","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":270111,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of PDPN on microglia after ICH in vivo or in vitro. (A-B) PDPN genes were obtained from the GSE200575 dataset, and the differences in gene expression of PDPN in the normal and ICH-operated groups were analysed in the older and younger age groups, respectively. * p \u0026lt; 0.05, * * p \u0026lt; 0.01, * * * p \u0026lt; 0.001 vs. sham-operated group. Error bars are expressed as mean ± SD, n = 3 for each group. (C) Representative Western blot bands for quantitative analysis of PDPN expression in the experimental group and sham-operated groups after ICH. * p \u0026lt; 0.05, * * p \u0026lt; 0.01, * * * p \u0026lt; 0.001 versus sham-operated group. Error bars are expressed as mean ± SD, n = 3 for each group. (D) The area around the haematoma on the ipsilateral cerebral hemisphere after ICH was harvested from mice in the sham-operated, control and SZ168-treated groups, respectively and the area was stained immunohistochemically. Brown areas are indicated as PDPN positive and blue areas are indicated as PDPN negative, scale bar = 200 μm, n = 3/group. (E) Immunofluorescence staining images of podoplanin (green) co-localised with microglia (Iba-1, red) in the haematoma area of the sham-operated, control and SZ168-treated groups as shown above. Nuclei were stained with DAPI (blue). Scale bar = 200 μm, n = 3/group.F. Flowchart of in vitro experimental design of hemin, i.e. haemoglobin. (G)After the construction of brain haemorrhage environment for human HMC3 cells in vitro, representative western blot bands of cellular proteins treated with different concentrations of hemin were extracted and analysed for quantitative analysis of PDPN expression, respectively, * p \u0026lt; 0.05, * * p \u0026lt; 0.01, * * * p \u0026lt; 0.001 vs. dmso group. Error bars are expressed as mean ± SD, n = 3 per group.\u003c/p\u003e","description":"","filename":"FIGURE1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5120185/v1/fc5ef2a8b47b0750f98afa67.jpg"},{"id":70474560,"identity":"618df5f5-3c9f-44a1-bef3-0aa703ce7398","added_by":"auto","created_at":"2024-12-03 13:57:46","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":89911,"visible":true,"origin":"","legend":"\u003cp\u003eSZ168 treatment attenuates neurobehavioural deficits, BBB permeability, cerebral oedema and hematoma size after ICH (A). Flowchart of the experimental design of ICH in mice; Elisa, enzyme-linked immunosorbent assay. Ich, i.e. intracerebral haemorrhage. IF staining, immunofluorescence staining. wb, western blot. (B-E) Mice were examined 48 hours after ICH for ipsilateral cerebral haematoma size, neurological deficits, blood-brain barrier permeability and brain tissue water content. * p \u0026lt; 0.05, * * p \u0026lt; 0.01, * * * p \u0026lt; 0.001 vs. sham/vehicle-operated group. Error bars are expressed as mean ± SD, n = 5 for each group.\u003c/p\u003e","description":"","filename":"FIGURE2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5120185/v1/225859985179880f0dae27e9.jpg"},{"id":70474558,"identity":"30895ed4-a113-4407-bc48-043d24bc98cf","added_by":"auto","created_at":"2024-12-03 13:57:46","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":122155,"visible":true,"origin":"","legend":"\u003cp\u003eSZ168 treatment reduces the expression of microglial cell pyroptosis molecules and inflammatory cytokines after ICH.(A)Representative Western blot bands for quantitative analysis of Nlrp3, Caspsae-1, GSDMD and GSDMD-N expression in sham-operated, control and SZ168-treated groups in mice with ipsilateral cerebral hematoma tissues harvested 48 hours after ICH. * p \u0026lt; 0.05, * * p \u0026lt; 0.01, * * * p \u0026lt; 0.001 versus vehicle-operated group. Error bars are expressed as mean ± SD, n = 3 per group. (B-C) As shown above, the figure shows immunofluorescence staining images of GSDMD (green) or MPO (green) co-localised with microglia (Iba-1, red) in the haematoma region in the sham-operated, control and SZ168-treated groups. Nuclei were stained with DAPI (blue). Scale bar = 200 μm, n = 3/group. (D-F). ELISA kit for IL - 1b, TNF - a and MPO production, * p \u0026lt; 0.05, * * p \u0026lt; 0.01, * * * p \u0026lt; 0.001 vs. sham/vehicle-operated group. Error bars are expressed as mean ± SD, n = 6 per group.\u003c/p\u003e","description":"","filename":"FIGURE3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5120185/v1/e4c87a86979c8a05cd38ea38.jpg"},{"id":70474565,"identity":"835bee56-094e-4ae4-83cb-8616ae95fe2a","added_by":"auto","created_at":"2024-12-03 13:57:46","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":73396,"visible":true,"origin":"","legend":"\u003cp\u003eSZ168 treatment reduces the expression of microglia pyroptosis molecules on human HMC cell lines. (A) Flowchart of the in vitro experimental design. (B-E) Representative western blots for quantitative analysis of the expression of cellular proteins Capsae-1, and GSDMD-N extracted from different concentrations of PLT or SZ168 treatment, respectively, after in vitro construction of brain haemorrhagic environments on human HMC3 cells.Blot bands, * p \u0026lt; 0.05, * * p \u0026lt; 0.01, * * * p \u0026lt; 0.001 vs. sham/vehicle-operated group. Error bars are expressed as mean ± SD, n = 3 per group.\u003c/p\u003e","description":"","filename":"FIGURE4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5120185/v1/42391fe302c54d4ff571e471.jpg"},{"id":70474859,"identity":"af7c8268-b002-4aca-8841-bfd796ed92fa","added_by":"auto","created_at":"2024-12-03 14:05:46","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":133160,"visible":true,"origin":"","legend":"\u003cp\u003eSZ168 treatment reduces microglial invasion and migration functions. (A-B) The effect of PDPN on the migration ability of the human HMC3 cell line was analysed by scratch assay, power field scale bar, 100 μm.C. Statistical analysis was performed to determine the migration distance. * p \u0026lt; 0.05, * * p \u0026lt; 0.01, * * * p \u0026lt; 0.001 vs. vehicle-operated group. Error bars are expressed as mean ± SD, n = 3 for each group. (D-E) The effect of PDPN on the migratory ability of the human HMC3 cell line was analysed by Transwell assay. The power field scale bar is 100 μm. Statistical analysis was performed to determine the number of migrated cells. * p \u0026lt; 0.05, * * p \u0026lt; 0.01, * * * p \u0026lt; 0.001 vs. vehicle-operated group. Error bars are mean ± SD, n = 3 per group.\u003c/p\u003e","description":"","filename":"FIGURE5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5120185/v1/970f885f3f312759d3fab40e.jpg"},{"id":70474562,"identity":"89b35e02-ce17-4d9f-abfc-8ff46ea1343d","added_by":"auto","created_at":"2024-12-03 13:57:46","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":88432,"visible":true,"origin":"","legend":"\u003cp\u003eSZ168 treatment reduces the proliferative capacity of microglia. (A-D) The effect of PDPN on the proliferative capacity of the human HMC3 cell line was analysed by CCK-8 assay and plate cloning assay. Colony formation statistics were analysed to determine the number of cell proliferation colonies. * p \u0026lt; 0.05, * * p \u0026lt; 0.01, * * * p \u0026lt; 0.001 vs. vehicle-operated group. Error bars are mean ± SD, n = 3 per group.\u003c/p\u003e","description":"","filename":"FIGURE6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5120185/v1/68e1fd4fb2d93b137ab6a63f.jpg"},{"id":70786347,"identity":"bdbb2500-1d55-4809-82a6-d68fa222b214","added_by":"auto","created_at":"2024-12-06 17:02:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1213418,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5120185/v1/d2525756-bdcf-47a5-a8be-4bb0f506b4dc.pdf"},{"id":70474862,"identity":"136ae4c0-8088-46d0-bef1-333e03a8fe7b","added_by":"auto","created_at":"2024-12-03 14:05:46","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":154382,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 1. Supplementary Fig. S1. Experimental design and animal groups. ICH, intracerebral hemorrhage; WB, western blot; ELISA, enzyme-linked immunosorbent assay; Supplementary Table S1. Summary of experimental groups and mortality rate in the study.\u003c/p\u003e","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-5120185/v1/8f056f3bfdc1e7670ab7d6c8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Monoclonal Antibody to Podoplanin SZ-168 Attenuates Microglia Pyroptosis and Neuroinflammation in Experimental Cerebral Haemorrhage","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIntracerebral haemorrhage (ICH) is a severe, fatal subtype of stroke that accounts for approximately 10-15% of all stroke patients and has a high mortality and disability rate\u003csup\u003e1\u003c/sup\u003e\u003csup\u003e,2\u003c/sup\u003e. After cerebral haemorrhage, disease progression is mainly influenced by primary brain injury and secondary brain injury (SBI)\u003csup\u003e3\u003c/sup\u003e, which refers to a series of pathological processes triggered by erythrocyte debris and degradation products, such as inflammatory incubation, mitochondrial dysfunction, oxidative stress and neuroendocrine\u003csup\u003e4\u003c/sup\u003e. Neuroinflammation is an important contributor to brain injury following cerebral haemorrhage through the mechanism of microglial activation and neutrophil infiltration, resulting in the release of pro-inflammatory cytokines, including tumour necrosis factor (TNF-\u0026alpha;), interleukin (IL-1\u0026beta;), myeloperoxidase (MPO) and other inflammation-related factors. Microglia pyroptosis is closely associated with the inflammatory response and is characterised by rapid plasma membrane rupture and release of cellular contents\u003csup\u003e5\u003c/sup\u003e. In addition, microglia pyroptosis is involved in the pathogenesis of secondary brain injury after ICH. Therefore, inhibiting microglia pyroptosis and the inflammatory response is a potential therapeutic approach to attenuate secondary brain injury after ICH\u003csup\u003e6\u003c/sup\u003e\u003csup\u003e,7\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eCell pyroptosis is a form of cell death in which caspase-1 regulates the hydrolytic maturation of IL-1\u0026beta; and IL-18 proteins, leading to rapid and damaging inflammatory cell death by the specific mechanism of activated caspase-1 cleavage of the precursors of gasdermin D (GSDMD), IL-1\u0026beta; (pro-IL-1\u0026beta;) and IL-18 (pro-IL-18)\u003csup\u003e8\u003c/sup\u003e. Gasdermin-N structural domain fragments are translocated to the membrane to form a stable circular pore. The pore can allow the passage of inflammatory cell contents, leading to microglia pyroptosis, which results in excessive inflammation\u003csup\u003e9\u003c/sup\u003e. It has been shown that in central nervous system disorders such as Alzheimer\u0026apos;s disease and cerebral ischaemia, inhibition of caspase-1 activation and cleavage of GSDMD can effectively ameliorate neuroinflammation and disease progression and attenuate brain damage\u003csup\u003e10\u003c/sup\u003e. In particular, microglia pyroptosis executive GSDMD has been shown in various studies to play a key role in influencing neuroinflammation in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE)\u003csup\u003e11\u003c/sup\u003e\u003csup\u003e,12\u003c/sup\u003e. Therefore, modulation of GSDMD-mediated microglia pyroptosis may help to attenuate neuroinflammation after ICH\u003csup\u003e13\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003ePodoplanin (PDPN) is a small transmembrane mucin-like glycoprotein that is an endogenous ligand for platelet C-type lectin-like receptor-2 (CLEC-2). PDPN interaction with platelet CLEC-2 has been shown to induce platelet aggregation and thrombosis, and the PDPN/CLEC-2 axis has been implicated in inflammatory diseases. A mAb SZ168 against the extracellular region of human PDPN was developed at Jiangsu Institute of Hematology, Jiangsu province, China\u0026nbsp;and SZ168 effectively attenuates the secretion of pro-inflammatory cytokines in lung-injured mice\u003csup\u003e14\u003c/sup\u003e and that blocking PDPN in tumours with high levels of PDPN expression inhibits tumour growth and metastasis\u003csup\u003e15\u003c/sup\u003e. In a mouse model of ischaemic stroke, treatment with \u0026alpha;-PDPN significantly reduced the levels of caspase-1 and GSDMD in microglia and inhibited the secretion of IL-18 and IL-1\u0026beta;, thereby attenuating the disruption of the blood-brain barrier and the level of inflammation\u003csup\u003e16,17\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn the present study, we hypothesised that inhibition of PDPN might attenuate secondary brain injury after ICH in mice by inhibiting caspase-1/GSDMD-mediated microglia pyroptosis and activation of neuroinflammation.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eAnimals\u003c/p\u003e\n\u003cp\u003eC57BL/6 mice (male, weighing approximately 22-25 g, n=108) were purchased and bred at the Animal Centre of Soochow University. All mice were maintained at room temperature (22\u0026plusmn;1\u0026deg;C) with a 12-hour day/night cycle (humidity: 60\u0026plusmn;5%) and food ad libitum. All animal procedures were under the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Guidelines Committee of Soochow University.\u003c/p\u003e\n\u003cp\u003eCell culture\u003c/p\u003e\n\u003cp\u003eThe human HMC3 cell line was purchased from Wuhan Punosai (Wuhan, Hubei province, China). Cells were cultured in MEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37\u0026deg;C in a 5% CO2 incubator. When cells reached approximately 80% fusion, they were digested with trypsin and passaged for further experiments. Cells were treated with haemoglobin (hemin, 100 \u0026mu;mol/L) dissolved in DMSO for 48 h. At the end of stimulation, cells were lysed with lysis solution and finally centrifuged, and cells were collected and assayed as previously described.\u003c/p\u003e\n\u003cp\u003eData collection\u003c/p\u003e\n\u003cp\u003eThe RNA-seq transcriptome data of GSE200575 in this study were obtained from the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) database. The GSE200575 dataset was divided into an older and a younger group, from each group containing 3 models of cerebral haemorrhage (ICH) and 3 models of sham surgery (SHAM). The transcriptome sequencing data were generated using the GPL24247 platform and derived from C57BL/6 mice.\u003c/p\u003e\n\u003cp\u003eICH model\u003c/p\u003e\n\u003cp\u003eICH was induced by stereotactically guided injection of collagenase into the right basal ganglia region. Briefly, mice were anaesthetised with 1% sodium pentobarbital intraperitoneally during the procedure and body temperature was maintained at 37.0 \u0026plusmn; 0.5\u0026deg;C with a heated blanket. Mice were placed in the prone position on a stereotactic head frame. A coronal incision was made to expose the skull until the fontanel was visible\u003csup\u003e18\u003c/sup\u003e. A 1 mm cranial hole was then drilled into the skull and a syringe was inserted under stereotactic guidance into the right basal ganglia region (coordinates: 0.2 mm anterior to bregma, 2.0 mm lateral to bregma). A total volume of 0.5 \u0026mu;L collagenase was then infused at a rate of 0.25 \u0026mu;L/min using a micro infusion pump (injection started 3.5 mm inferior to the dura mater). To prevent leakage, the needle was left in place for 10 min after completion of the 0.5 \u0026mu;L infusion and slowly withdrawn at a rate of 1 mm/min, after which the burr hole was closed with sterilised medical bone wax and the scalp incision was sutured. The mice were allowed to fully recover on a 37\u0026deg;C heating pad and neurological deficits were closely monitored. The sham-operated group underwent the same procedure without collagenase injection.\u003c/p\u003e\n\u003cp\u003eAnimal groups and the Administration of drug\u003c/p\u003e\n\u003cp\u003eMale C57BL/6 mice (n=108) were randomLy divided into sham-operated\u0026nbsp;(n=32), ICH-operated\u0026nbsp;(n=32) and SZ168-treated groups\u0026nbsp;(n=32) .The experimental design was shown in Part 1 of the Additional file 1: Fig. S1.\u003c/p\u003e\n\u003cp\u003eIn the \u003cem\u003ein\u0026nbsp;\u003c/em\u003e\u003cem\u003evivo\u003c/em\u003e model, the anti-human PDPN extracellular region mAb (SZ168) was administered intraperitoneally at 100 \u0026mu;g/mL 6 hours after the ICH model was established. In the \u003cem\u003ein vitro\u003c/em\u003e model, cells were treated with hemin 100 \u0026mu;mol/L for 2h and then re-treated by adding different concentrations of platelets and SZ168 simultaneously.\u003c/p\u003e\n\u003cp\u003eNeurobehavioural function tests\u003c/p\u003e\n\u003cp\u003eThe neurobehavioural function of mice was assessed 24 hours after ICH using the modified Garcia test, administered by a blinded investigator. The modified Garcia test consisted of seven items: spontaneous activity, axial sensation, tentacles, limb symmetry, lateral rotation, forelimb walking and climbing. Each test is scored from 0 to 3 or 1 to 3, with a total score of 3 to 21. Higher scores indicate better neurological function.\u003c/p\u003e\n\u003cp\u003eBrain Tissue Water Content\u003c/p\u003e\n\u003cp\u003eBrain water content (BWC) was measured by the wet/dry method as reported\u003csup\u003e19\u003c/sup\u003e. Overall, mice were killed by decapitation under deep anaesthesia 48 hours after ICH. Brains were immediately removed and divided into five sections: ipsilateral and contralateral cortex, ipsilateral and contralateral basal ganglia, and cerebellum. Each brain section was immediately measured on an analytical microbalance to obtain wet weight (WW) and then dried at 100\u0026deg;C for 24 h to obtain dry weight (DW). Brain tissue water content was calculated using the following formula: brain tissue water content (%) = [(WW-DW)/WW] * 100%.\u003c/p\u003e\n\u003cp\u003eBlood-brain barrier permeability\u003c/p\u003e\n\u003cp\u003eTo assess blood-brain barrier permeability, 200 \u0026mu;L of 2% Evans Blue (Genye, China) was injected via a tail vein as reported\u003csup\u003e20\u003c/sup\u003e. After 24 h of circulation, mice were perfused via the heart under deep anaesthesia with cold phosphate buffer (0.1 M, PBS, p H 7.4). The brains were then removed, divided into left and right hemispheres and immediately stored at -80\u0026deg;C. The right hemisphere was homogenised in 1100 mL PBS, disrupted by ultrasound and centrifuged (12 000 g, 4 \u0026deg;C, 30 min). The supernatant was collected, added to acetone in a 3:7 ratio and incubated overnight at 4\u0026deg;C. After centrifugation (12 000 g, 4 \u0026deg;C, 30 min), the Evans blue staining was measured at 600 nm using a spectrophotometer.\u003c/p\u003e\n\u003cp\u003eImmunofluorescence Staining\u003c/p\u003e\n\u003cp\u003eTwo-colour immunofluorescence staining was performed as previously described\u003csup\u003e21\u003c/sup\u003e. Mice were deeply anaesthetised and transcardially perfused with 30 mL ice-cold PBS and 30 mL 4% paraformaldehyde 48 h after ICH. Whole brains were collected and fixed in 4% paraformaldehyde for 24 hours. The brains were then dehydrated in 75%, 80%, 95%, 100% (I) and 100% (II) ethanol for half an hour each. The brains were then removed and placed in ethanol + xylene (1:1) for 2 hours; xylene (I) and xylene (II) for 10 minutes each, after which they were immersed in wax and embedded in melted pure wax. After cooling and confirmation of the embedding, the sections were cut into 5 mm thick coronal sections using a paraffin slicer. For double immunohistochemical staining, brain sections were incubated overnight at 4\u0026deg;C with an antibody: anti-calcium binding protein (Iba-1; 1:1000, Abcam, UK), anti-podoplanin (5 \u0026mu;g/mL, ThermoFisher Scientific, USA), anti-myeloperoxidase (MPO, 1:250, Santa Cruz Biotechnology, USA) and anti-myeloperoxidase (MPO.) Santa Cruz Biotechnology, USA), anti-gasdermin D (1:250, Santa Cruz Biotechnology, USA) were incubated overnight at 4\u0026deg;C and then observed and photographed by fluorescence microscopy after incubation with the corresponding secondary antibody (1:100, Biyuntian, China) for 1 h at 37\u0026deg;C. Images were analysed using Fiji/ImageJ software.\u003c/p\u003e\n\u003cp\u003eImmunohistochemistry\u003c/p\u003e\n\u003cp\u003eImmunohistochemical staining was performed as previously described\u003csup\u003e22,23\u003c/sup\u003e, and mice used for immunohistochemistry were anaesthetised with sodium pentobarbital and perfused transcardially with 30 mL ice-cold PBS and 30 mL 4% paraformaldehyde. Whole brains were collected, fixed in 4% paraformaldehyde for 24 h and transferred to 70% ethanol for paraffin embedding and 5 \u0026mu;m sectioning. Heat-induced antigen retrieval was performed on tissue sections in citrate or EDTA buffer. Sections were oxidase treated, blocked with serum-free proteins, stained with anti-podoplanin (5 \u0026mu;g/mL, ThermoFisher Scientific, USA) and, after overnight incubation at 4\u0026deg;C, immunoreactivity was detected with HRP-conjugated secondary antibodies and stained with diaminobenzidine and haematoxylin. The density of podoplanin immunostaining was measured in five sections using Image J software.\u003c/p\u003e\n\u003cp\u003eWestern Blot\u003c/p\u003e\n\u003cp\u003eWestern blotting was performed as previously described\u003csup\u003e24\u003c/sup\u003e. Briefly, after mice were perfused with ice-cold PBS (0.1 M, pH 7.4), the tissue surrounding the haematoma was removed 24 hours post-operatively and stored at -80\u0026deg;C in a refrigerator for spare parts. Brain tissue haematoma sites were thoroughly crushed in a glass homogeniser with pre-cooled RIPA lysis buffer containing protease and phosphatase inhibitors to obtain a 10% tissue homogenate, which was then lysed on ice for 30 min, followed by centrifugation at 12,000 rpm for 20 min at 4\u0026deg;C. Macrophages and microglia were lysed on ice for 30 min using RIPA lysis buffer containing protease inhibitors. The cells were then centrifuged at 12,000g for 20 minutes at 4\u0026deg;C. After collecting the supernatant, the concentration of the supernatant was determined using the BCA technique.\u003c/p\u003e\n\u003cp\u003eEqual amounts of proteins (30 \u0026mu;g of cellular proteins or 90 \u0026mu;g of tissue proteins were placed in each well channel) were separated by 10% SDS polyacrylamide gel electrophoresis (treated at 80 V for 30 min, then 120 V for 40 min) and transferred to nitrocellulose (NC) membranes, which were sealed with 5% nonfat dry milk. After primary antibody incubation at 4 \u0026deg;C overnight, the membranes were treated with anti-rabbit or anti-mouse IgG coupled secondary antibodies. The antibodies used were anti-NLRP3 (1:1,000, Proteintech, Wuhan, China); anti-caspase 1 (1:10,000, Proteintech, Wuhan, China); anti-GSDMD/GSDMD-n (1:1,000, Abcam, USA); anti-Gapdh ( 1: 10,000, Proteintech, Wuhan, China); anti-GSDMD/N (1:10,000, Abcam, USA); anti-Gapdh ( 1:10,000, Proteintech, Wuhan, China); and anti-mouse IgG coupling. Proteintech, Wuhan, China); SZ168 (anti-podoplanin, 2 \u0026mu;g/mL, Suzhou, China); anti-mouse IgG ( 1 : 5,000, Biyun Tian, China); anti-rabbit IgG ( 1 : 10,000, Proteintech, Wuhan, China). At the end of the secondary antibody incubation, the membranes were rinsed with PBS and the proteins were then visualised three times using an enhanced chemiluminescence (ECL) system. Quantitative analysis of each protein band was performed using Image J software.\u003c/p\u003e\n\u003cp\u003eEnzyme-linked immunosorbent assay\u003c/p\u003e\n\u003cp\u003eEnzyme-linked immunosorbent assay (ELISA) was performed as previously described\u003csup\u003e25\u003c/sup\u003e. Histone proteins from ICH experimental animals were obtained and assayed using an ELISA kit (Multi Sciences, China) according to the manufacturer\u0026apos;s instructions. Mice were anaesthetised 24 h after ICH and then perfused with 0.1 M PBS. Tissue proteins from ICH experimental animals were collected and homogenised in lysis buffer containing protease inhibitors (100 mL lysate per 10 mg tissue); undiluted supernatants were used for the determination of IL-1b, TNF-\u0026alpha; and MPO, respectively. The mouse double antibody sandwich ELISA kits for IL-1b, TNF-\u0026alpha; and MPO were purchased from Multi Sciences and performed according to the manufacturer\u0026apos;s instructions.\u003c/p\u003e\n\u003cp\u003ePlatelet isolation\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFresh whole blood from healthy donors was collected in anticoagulated tubes containing 0.38% sodium citrate and centrifuged at 1100 rpm for 10 minutes to isolate platelet-rich plasma (PRP). The PRP was aspirated into a new centrifuge tube, centrifuged at 3500 rpm for 2 min, the supernatant discarded and the precipitate resuspended in ETDA-PBS, centrifuged at 2000 rpm for 2 min and repeated three times to obtain washed platelets.\u003c/p\u003e\n\u003cp\u003eCell counting kit (CCK-8) assay\u003c/p\u003e\n\u003cp\u003eHuman microglia were inoculated into 96-well plates (5*10\u003csup\u003e3\u003c/sup\u003e cells per well), the hemin group then added 100 \u0026mu;mol of hemin and cultured for 24 h; the platelet group added different concentrations of washed platelets for 2 h and then added 100 \u0026mu;mol of hemin and cultured for 24 h; the SZ168 and mIgG groups added 5*10\u003csup\u003e6\u003c/sup\u003e platelets and different concentrations of SZ168 or 60 \u0026mu;g/mL mIgG were incubated for 2 h before adding 100 \u0026mu;mol of hemin for 24 h. 10\u0026mu;L CCK-8 solution from the CCK-8 kit (BiyunTian, Shanghai, China) was added to each well and incubated at 37\u0026deg;C for 4 hours. Optical density (OD) values at 450 nm were measured in each well using an enzyme marker to assess cell viability. The OD values were normalised to the 24-hour control time point to show changes more clearly\u003csup\u003e26\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWound-scratch assay\u003c/p\u003e\n\u003cp\u003eHuman microglial cells were inoculated into 24-well plates (1*10\u003csup\u003e5\u003c/sup\u003e/well) and cultured to 90% fusion at 37\u0026deg;C in a 5% CO2 incubator, and after 12 hours of starvation in serum-free medium, the tip of a sterile 200 \u0026mu;L pipette was used to scratch along the cells. The hemin group was then cultured with 100 \u0026mu;mol of hemin for 24 hours; the platelet group was cultured with different concentrations of washed platelets for 2 hours followed by 100 \u0026mu;mol of hemin for 24 hours; the SZ168-treated group and the mIgG group were cultured with 1*10\u003csup\u003e6\u003c/sup\u003e platelets and different concentrations of SZ168 or 25 \u0026mu;g/mL mIgG for 2 hours; after 2 hours, 100 \u0026mu;mol of hemin was added and incubated for 24 hours. Cells were photographed with a light microscope (Leica, Wetzlar, Germany; magnification: \u0026times;100) in 2 random fields of view at 0 h and 24 h after scratching. Cell migration distances were calculated using Image Pro-Plus 6.0 software (NIH, USA).\u003c/p\u003e\n\u003cp\u003eTranswell experiments\u003c/p\u003e\n\u003cp\u003eHuman microglia were inoculated using 24-well Transwell chambers (Corning, Sigma-Aldrich, USA) (upper layer) at a cell density of 2*10\u003csup\u003e4\u003c/sup\u003e cells per well and cultured in serum-free medium, the hemin group was incubated for 24 h with the addition of 100 \u0026mu;mol of hemin; the platelet group was incubated for 24 hours with the addition of washed platelets at different concentrations for 2 hours followed by the addition of 100 \u0026mu;mol of hemin; SZ168 and mIgG groups were incubated by the addition of 1*10\u003csup\u003e7\u003c/sup\u003e platelets and different concentrations of SZ168 or 25 \u0026mu;g/mL mIgG for 2 hours followed by 100 \u0026mu;mol of hemin for 24 hours. FBS (20%) was prepared in the lower chamber. The incubation time was 24 hours. The invasive cells attached to the lower surface were then stripped and treated with methanol ( 4%, 0.5 h) and crystal violet ( 0.1%, 15 min). Final data were calculated based on five randomly selected fields of view under a light microscope (Leica; magnification: \u0026times; 100).\u003c/p\u003e\n\u003cp\u003ePlate cloning experiment\u003c/p\u003e\n\u003cp\u003eThe human microglial suspension was adjusted to 1000 cells/mL and 1000 cells were inoculated into each well of a 6-well plate; the hemin group was incubated with another 100 \u0026mu;mol hemin for 1 week; the platelet group was incubated with washed platelets of different concentrations for 2 h and then 100 \u0026mu;mol hemin for 1 week; the SZ168-treated group and the mIgG group were incubated with 1*10\u003csup\u003e7\u003c/sup\u003e platelets, and different SZ168 and mIgG groups were cultured with 1*10\u003csup\u003e7\u003c/sup\u003e platelets and different concentrations of SZ168 or 25\u0026mu;g/mL mIgG for 2 hours before adding 100\u0026micro;mol hemin for 1 week, during which the solution was changed every 3-4 days, At the end of the culture, the cells were washed with PBS and 1 mL of 4% paraformaldehyde was added to each well to fix the cells for 30-60 minutes, then washed again and stained by adding 1 mL of crystal violet staining solution to each well for 10 minutes. Cells were stained under a microscope. The cells were photographed under a microscope, the number of clone formations (\u0026gt;50 cells) was counted and the cell images were processed using Image software to obtain the number of clone formations in different groups.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eMortality and rejection\u003c/p\u003e\n\u003cp\u003eA total of 108 male C57BL/6 mice were used in this study and the overall mortality rate of the ICH mice was 9.26% (10/108), none of the sham-operated group died and 2 mice were excluded from this study because they were found to have no haematoma in the brain after euthanasia(Part 2 of the Additional file 1: Table S1).\u003c/p\u003e\n\u003cp\u003eDifferential analysis of PDPN genes in brain tissue of ICH-operated and Sham-operated groups\u003c/p\u003e\n\u003cp\u003eBrain tissue PDPN genes of ICH-operated and SHAM-operated groups were obtained from the GSE200575 dataset, and the two groups were directly divided into old and young groups based on age, respectively, and each group contained information from three ICH samples and three SHAM samples, and the data were obtained from the brain tissues of mice at 24 h after ICH. Differential analysis of the ICH-operated and SHAM-operated groups in the old and young groups showed that the expression of the PDPN gene was increased in both the old and young groups in the ICH-operated group compared with the SHAM-operated group(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Fig. 1A-B).\u003c/p\u003e\n\u003cp\u003ePodoplanin expression in ICH and cells over time\u003c/p\u003e\n\u003cp\u003eTo observe the expression of PDPN, tissues were harvested from the brain haematoma site after establishing the ICH model in mice and subjected to Western blot analysis, immunohistochemistry and immunofluorescence staining. Endogenous expression of PDPN in the ipsilateral/right cerebral hemisphere was observed by Western blot analysis, immunohistochemistry and immunofluorescence staining 48 hours after ICH creation.WB results showed that the expression of PDPN in mouse brain tissue was significantly increased at 48 hours after ICH compared to the sham-operated group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Fig.1C). Immunohistochemical results were consistent with the above, PDPN expression was increased at 48h after ICH compared with the sham-operated group\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, Fig. 1D), whereas PDPN expression was decreased in the SZ168-treated group\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Fig. 1D). Immunofluorescence double-label staining showed that PDPN was predominantly expressed in microglia/macrophages (Iba-1\u003csup\u003e+\u003c/sup\u003e) in the perihematomal tissue at 48h after ICH, and both PDPN and microglia/macrophage markers were reduced in the SZ168-treated group.\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, Fig. 1E).\u003c/p\u003e\n\u003cp\u003eAn in vitro cerebral haemorrhage model was established using human microglia treated with hemin, and cellular proteins were extracted and subjected to Western blot analysis separately as the concentration of hemin treatment gradually increased. Compared with the DMSO-treated group, the expression of PDPN gradually increased with hemin concentrations of 40, 60, 80 and 100 \u0026mu;mol/L, reaching the peak at 100 \u0026mu;mol/L(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Fig. 1G).\u003c/p\u003e\n\u003cp\u003eSZ168 treatment reduces neurobehavioural deficits, BBB permeability, Brain Edema and Hematoma Size after ICH\u003c/p\u003e\n\u003cp\u003eThe ICH model was established using C57BL/6 mice and SZ168 was injected intraperitoneally after 6 hours\u0026nbsp;(Fig. 2A). Two days later, the brains of the mice were removed to observe the cerebral haemorrhage, which showed that the cerebral haematoma area of the mice in the control group was significantly enlarged compared with that of the SZ168-treated group, and it was speculated that the treatment with SZ168 was able to reduce the size of the cerebral haematoma of the mice after ICH\u0026nbsp;(Fig. 2B); In the short-term neurological function observation, by observing the neurological function of mice at 1, 3 and 7 days after modelling, the results showed that the neurological deficits in the ICH+mIgG group were higher than those in the SZ168 treatment group at 1, 3 and 7 days after ICH and that the treatment with SZ168 was able to effectively reduce the degree of neurological impairment(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Fig. 2C). The results of brain tissue water content experiments showed that treatment with SZ168 effectively reduced the cerebral oedema phenomenon of bleeding in the brain tissue of mice, and the cerebral oedema in the ipsilateral/right basal ganglia and cortical areas was effectively reduced compared with that in the ICH+mIgG group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Figure 2E). Evans blue staining experiments showed that SZ168 treatment was able to reduce the permeability of the blood-brain barrier\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Fig. 2D). Overall, the SZ168-treated ICH-operated group significantly improved neurological deficits, reduced cerebral oedema, and reduced BB permeability and haematoma size.\u003c/p\u003e\n\u003cp\u003eSZ168 treatment reduces the expressiodeath microglia pyroptosis molecules and inflammatory cytokines after ICH\u003c/p\u003e\n\u003cp\u003eThe therapeutic effect of SZ168 in reducing cerebral haemorrhage after ICH in mice has been observed in previous experiments. It has been demonstrated that the upregulation of PDPN can regulate the inflammatory response and prognosis of diseases through the involvement of inflammatory pathways, especially the Nlrp3/Capase-1/GSDMD pyroptosis pathway, so it observed the expression of PDPN and related inflammatory factors in ICH after SZ168 treatment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWestern blot analysis showed that PDPN expression was increased after ICH, accompanied by upregulation of the expression of microglia pyroptosis molecules such as Nlrp3, caspase-1 and GSDMD. However, treatment with SZ168 downregulated the expression of both PDPN and pyroptosis molecules(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Fig. 3A). Immunofluorescence staining results were consistent with the above, the number of GSDMD-positive and MPO-positive microglia was increased in the perihematoma tissues after ICH, whereas treatment with SZ168 decreased the number of GSDMD- and MPO-positive microglia in the perihematoma tissues after 48 hours of ICH(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Fig. 3B-C). In addition, ELISA results showed that the expression of inflammatory cytokines IL-1\u0026beta;, TNF-\u0026alpha; and MPO was increased after ICH, whereas treatment with SZ168 decreased the expression of inflammatory cytokines(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, Fig. 3D-F).\u003c/p\u003e\n\u003cp\u003eThe results of the in vitro experiments were consistent with the above (Fig. 4A), HMC3 cells were treated with hemin to simulate the environment of cerebral haemorrhage in vitro, \u0026nbsp;PLT and SZ168 were added for treatment after two hours of modelling. Western blot showed that the expression of pyroptosis molecules caspase-1 and GSDMD-N was increased in the mIgG group, while the expression of pyroptosis molecules was decreased in the SZ168-treated group as a dose dependent of SZ168.(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Fig. 4B,4D).\u003c/p\u003e\n\u003cp\u003eSZ168 treatment reduces microglial invasion and migration function\u003c/p\u003e\n\u003cp\u003eFrom the results of the above experiments, it was observed that PDPN was able to influence microglia to participate in the development of disease inflammation. Immunofluorescence co-localisation also showed a decrease in microglia co-expressing PDPN and inflammatory pathway proteins, so we speculated whether this was because PDPN reduces the number of PDPN\u003csup\u003e+\u003c/sup\u003e microglia that have been transferred to the site of haemorrhage by modulating the invasive, migratory and other motility functions of microglia. Therefore, an in vitro ICH model was constructed using human HMC3 cells, a certain concentration of platelets was added to co-incubate with the cells to achieve a better effect in restoring the environment of cerebral haemorrhage in vivo, and the effect of PDPN up-regulation on microglia motility function was observed by cell scratch assay and transwell invasion assay.\u003c/p\u003e\n\u003cp\u003eHMC3 cells were treated with 100 \u0026mu;mol/L hemin and different concentrations of PLT to simulate the environment of cerebral haemorrhage in vitro, and the migration and invasion functions of human microglia were observed by cell scratch assay and transwell invasion assay. As shown in the cell scratch assay, microglia migration gradually increased with the platelet concentration from 1*10\u003csup\u003e5\u003c/sup\u003e, 5*10\u003csup\u003e5\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eto 1*10\u003csup\u003e6\u003c/sup\u003e (/mL), with 1*10\u003csup\u003e6\u003c/sup\u003e(/mL)\u0026nbsp;as the peak(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Fig. 5A). The cells were then co-incubated with 100 \u0026mu;mol hemin and 1*10\u003csup\u003e6\u003c/sup\u003e (/mL) platelets before being treated with a low (5 \u0026mu;g/mL) or high (25\u0026mu;g/mL) concentration of SZ168. The results showed that SZ168 IgG gradually inhibited microglial migration in a dose dependent (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Fig. 5B).\u003c/p\u003e\n\u003cp\u003eThe transwell invasion assay showed that\u0026nbsp;microglial cells\u0026nbsp;invasion\u0026nbsp;was gradually increased\u0026nbsp;with the platelet concentration from 5*10\u003csup\u003e5\u003c/sup\u003e, 5*10\u003csup\u003e6\u003c/sup\u003e to 1*10\u003csup\u003e7\u003c/sup\u003e (/mL) \u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Fig. 5D).\u0026nbsp;The cells were then co-incubated with 100 \u0026mu;mol hemin and 1*10\u003csup\u003e7\u003c/sup\u003e (/mL) platelets before being treated with a low ((5 \u0026mu;g/mL) or high (25\u0026mu;g/mL) concentration of SZ168,and the results showed that the invasive ability of microglia was gradually suppressed with the increase of SZ168 concentration(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Fig. 5E).\u003c/p\u003e\n\u003cp\u003eSZ168 treatment reduces the proliferative capacity of microglia\u003c/p\u003e\n\u003cp\u003eHMC3 cells were treated with 100 \u0026mu;mol/L hemin with different concentrations of PLT to simulate the environment of cerebral haemorrhage in vitro, and the proliferation function of microglia was observed by cell viability assay and plate cloning experiment.\u0026nbsp;In the cell viability assay, the number of proliferating cells gradually increased with the concentration of platelets from 1*10\u003csup\u003e3\u003c/sup\u003e, 1*10\u003csup\u003e4\u003c/sup\u003e, 1*10\u003csup\u003e5\u003c/sup\u003e, 1*10\u003csup\u003e6\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eto 5*10\u003csup\u003e6\u003c/sup\u003e (/mL) (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, Fig. 6A). In contrast, SZ168 effectively inhibited cell proliferation, and the inhibitory effect became stronger as the concentration of SZ168 increased (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Fig. 6B). The results of plate cloning experiment were consistent with the above, in brief, when the platelet concentration from 5*10\u003csup\u003e3\u003c/sup\u003e, 1*10\u003csup\u003e4\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eto 5*10\u003csup\u003e4\u003c/sup\u003e (/mL), the number of microglia proliferation gradually increased, and 5*10\u003csup\u003e4\u003c/sup\u003e (/mL) reached the peak (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Fig. 6C). The inhibitory results showed that the proliferative ability of microglia was gradually inhibited with the increase of SZ168 concentration\u0026nbsp;from\u0026nbsp;5,\u0026nbsp;to\u0026nbsp;50 \u0026mu;g/mL\u0026nbsp;\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Fig. 6D).\u003c/p\u003e\n\u003cp\u003eFrom the migration, invasion and proliferation experiments, it can be seen that SZ168 can effectively reduce the invasion, migration and proliferation of microglia after inhibiting the function of PDPN. This result, combined with the results of immunofluorescence staining, suggests that the increased expression of PDPN on microglia enhances their invasion and migration functions, promotes the transfer of microglia to the haematoma site to induce cell death, releases inflammatory factors, and causes aggravation of the haematoma. SZ168 antibody treatment reduces microglial proliferation, migration and invasion into the haematoma site, reduces brain inflammation and decreases the production of inflammatory cytokines, thereby improving neurological function and cerebral haematoma.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, firstly, we found the upregulation of PDPN expression in the cerebral haemorrhage group in the gene set GSE200575, and the experimental results were consistent with the genetic analysis, which showed that PDPN expression was upregulated in the cerebral haemorrhage model in mouse brain tissue, and that inhibition of its function by using SZ168 reduced the neurological impairments, cerebral oedema as well as the impairment of the blood-brain barrier, and, secondly, that this effect can be presumed to be due to the release of large amounts of inflammatory factors mediated by the Nlrp3/Caspase-1/GSDMD signalling pathway via the release of a large number of inflammatory factors, that this effect can be speculated to be due to the release of large amounts of inflammatory factors mediated by the Nlrp3/Caspase-1/GSDMD signalling pathway and the downregulation of inflammatory pathway proteins such as Nlrp3, Caspase-1 and GSDMD and inflammatory factors such as IL-1\u0026beta;, TNF-\u0026alpha; and MPO after treatment with SZ168, thus serving to ameliorate the therapeutic effects of secondary brain injury. Finally, by observing that the expression of PDPN and microglial cell markers were reduced in the brain tissue of mice after treatment with SZ168, and by reducing the migration, invasion and proliferation of microglial cells after using SZ168 in vitro experiments, it was speculated that SZ168 was able to inhibit the proliferation and migration and invasion of microglial cells into the cerebral haematoma site, thereby reducing the occurrence of cerebral haematoma inflammation, which was an anti-microglia pyroptosis and anti-inflammatory factor. SZ168 can inhibit the proliferation and migration of microglia to the haematoma site, thereby reducing the inflammation in the haematoma site and exerting anti-microglia pyroptosis and anti-inflammatory effects.\u003c/p\u003e\n\u003cp\u003eA large number of studies have demonstrated that inhibition of neuroinflammation can reduce secondary brain injury and improve neurological function after ICH, as neuroinflammation is an important pathogenetic mechanism of secondary brain injury after ICH\u003csup\u003e27,28\u003c/sup\u003e. The neuroinflammatory response in the CNS is a complex process influenced by the synergistic interaction of different groups of neuroglia\u003csup\u003e29\u003c/sup\u003e, and microglia play an important activating role in activating the initial steps of the inflammatory response, promoting the infiltration of neutrophils, amplifying the inflammatory response and leading to the disruption of the extracellular matrix, the integrity of cell membranes and the Impaired blood-brain barrier\u003csup\u003e30,31,32\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eHowever, in the present study, PDPN expression was found to be up-regulated in microglia, a transmembrane mucin that has been implicated in lymphatic endothelium and lymphangiogenesis\u003csup\u003e33\u003c/sup\u003e and is more widely expressed in a variety of human tumours and inflammatory diseases, where PDPN is implicated in platelet aggregation and metastasis formation through binding to CLEC-2, and where it can regulate the cytoskeleton, thereby increasing cell migration and invasion. Cytoskeleton, thereby increasing cell migration and invasion\u003csup\u003e34\u003c/sup\u003e. There is evidence that the knockdown of PDPN can affect microglial motility and phagocytosis, and the intracellular structural domain of PDPN binds directly to ERM proteins and also regulates cell motility and morphology through the activation of various cellular signalling pathways such as RhoGTPases\u003csup\u003e35,36\u003c/sup\u003e. It has been reported that in neuro-malignant gliomas and melanomas, PDPN can increase the migratory capacity of tumour cells and enhance the tube-forming activity of endothelial cells and that PDPN expression is an important step in tumour progression\u003csup\u003e37\u003c/sup\u003e, and the role of PDPN in rheumatoid arthritis (RA), sepsis and the wound healing process\u003csup\u003e38,39\u003c/sup\u003e, and that the CLEC-2-PDPN pathway was able to promote infiltration of PDPN-positive immune cells and macrophage recruitment at the site of infection. There is also evidence that PDPN may influence the prognosis of diseases such as cerebral ischaemia-reperfusion and traumatic brain injury by promoting the pyroptosis pathway and releasing inflammatory factors, leading to more severe neurological impairment, cerebral oedema and BBB destruction\u003csup\u003e40,41\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn conclusion, we also observed elevated protein expression of the pyroptosis pathway in microglia, which correlated with increased PDPN expression following ICH. Treatment with SZ168 resulted in decreased pyroptosis protein expression and PDPN expression, as well as a reduction in neurological impairments, cerebral oedema, and blood-brain barrier disruption. This suggests a correlation between PDPN expression and prognosis in patients with cerebral haemorrhage, indicating a potential therapeutic strategy for ICH. Nevertheless, further elucidation is required regarding the mechanism of PDPN\u0026apos;s role in microglia inflammation, as well as the specific mechanisms and pathways through which PDPN affects microglia motor function.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, we have first demonstrated that inhibition of PDPN function using the monoclonal antibody SZ168 can improve impaired neurological function, reduce cerebral haematomas and ameliorate neuroinflammation and cerebral oedema after ICH via the Nlrp3/Caspsae-1/GSDMD pathway. Therefore, podoplanin protein may be a promising therapeutic idea for early treatment of ICH to improve prognosis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eICH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIntracerebral haemorrhage\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDPN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePodoplanin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNLRP3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePyrin Domain Containing Protein 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGSDMD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGasdermin D\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMPO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMyeloperoxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIL-1β\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterleukin-1β\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNF-α\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor necrosis factorα\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMSO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDimethylsulfoxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIba-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIonized calcium-binding adaptor molecule-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBWC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBrain water content\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWet weight\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDry weight\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBBB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBlood-brain barrier.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSYLworked on the experimental design, conducted the experiments, analysed the data, and drafted the manuscript. JC, RYL, XW and YMZ participated in the experimental design, data analysis and interpretation, and manuscript preparation. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was supported by the National Natural Science Foundation of China-funded projects(Grant81873431)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOther researchers who wish to reproduce the results or replicate the procedures will have access to the data, analysis methods, and study materials. The data supporting the results of this study are available from the corresponding author upon reasonable request. The authors are responsible for maintaining availability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Animal Care and Use Committee of Soochow University First Affiliated Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Keep RF, Hua Y, Xi G. Intracerebral haemorrhage: mechanisms of injury and therapeutic targets. Lancet Neurol. 2012;11(8):720\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Lapchak PA, Zhang JH. 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Nature (2015) 526(7575):660\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e de Vasconcelos NM, Van Opdenbosch N, Van Gorp H, Parthoens E, Lamkanfi M. Single-Cell Analysis of Pyroptosis Dynamics Reveals Conserved GSDMD-Mediated Subcellular Events That Precede Plasma Membrane Rupture. Cell Death Differ (2019) 26(1):146\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Flores J, Noel A, Foveau B, Beauchet O, LeBlanc AC. Pre-Symptomatic Caspase-1 Inhibitor Delays CognitiveDeclineinaMouseModelof Alzheimer Disease and Aging. Nat Commun (2020) 11(1):4571.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e McKenzie BA, Dixit VM, Power C. Fiery Cell Death: Pyroptosis in the Central Nervous System. Trends Neurosci (2020) 43(1):55\u0026ndash;73. doi: 10.1016/ j.tins.2019.11.005.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Li S, Wu Y, Yang D, Wu C, Ma C, Liu X, et al. 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CNS Neurosci Ther 2024, 30 (3), e14643.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Flores, J.; No\u0026euml;l, A.; Foveau, B.; Beauchet, O.; LeBlanc, A. C. Pre-Symptomatic Caspase-1 Inhibitor Delays Cognitive Decline in a Mouse Model of Alzheimer Disease and Aging. Nat Commun 2020, 11 (1), 4571.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Luo X, Li L, Zheng W, Gu L, Zhang X, Li Y, et al. HLY78 Protects Blood-Brain Barrier Integrity Through Wnt/Beta-Catenin Signaling Pathway Following Subarachnoid Hemorrhage in Rats. Brain Res Bull (2020) 162:107\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Paradowska-Gorycka A, Wajda A, Romanowska-Prochnicka K, Walczuk E, Kuca-Warnawin E, Kmiolek T, et al. Th17/Treg-Related Transcriptional Factor Expression and Cytokine Profile in Patients With Rheumatoid Arthritis. 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Cell Biol. 43 (2011) 886e896.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFeng C, Yu A, Wang Z, et al. A novel PDPN antagonist peptide CY12-RP2 inhibits melanoma growth via Wnt/β-catenin and modulates the immune cells. J Exp Clin Cancer Res. 2024;43(1):9. Published 2024 Jan 2.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eM. Honma, T. Shibuya, K. Hayashi, S. Iinuma, M. Fujii, A. Ishida-Yamamoto, Suppression of podoplanin expression during differentiation of epidermal keratinocytes, J. Dermatol. 46 (2019) 922e924.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJ. Rayes, S. Lax, S. Wichaiyo, S.K. Watson, Y. Di, S. Lombard, B. Grygielska, S.W. Smith, K. Skordilis, S.P. Watson, The podoplanin-CLEC-2 axis inhibits inflammation in sepsis, Nat. Commun. 8 (2017) 2239.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFei M, Wang H, Zhou M, Deng C, Zhang L, Han Y. Podoplanin influences the inflammatory phenotypes and mobility of microglia in traumatic brain injury. Biochem Biophys Res Commun. 2020;523(2):361\u0026ndash;367.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeng D, Ma X, Li H, et al. A Role of the PodoplaninCLEC-2 Axis in Promoting Inflammatory Response After Ischemic Stroke in Mice. Neurotox Res 2021;39:477\u0026thinsp;\u0026minus;\u0026thinsp;88.\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":"podoplanin, cerebral haemorrhage, microglia, neuroinflammation","lastPublishedDoi":"10.21203/rs.3.rs-5120185/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5120185/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eNeuroinflammation is an important factor in the development of secondary brain injury after intracerebral haemorrhage (ICH), involving the interaction of haemorrhage formation and inflammatory pathways. Podoplanin is a small transmembrane mucin-like glycoprotein, and it has been demonstrated that the PDPN/CLEC-2 axis is involved in inflammatory diseases. This study aims to investigate the therapeutic effect of a monoclonal antibody against podoplanin, SZ168, on neuroinflammation in a mouse model of cerebral haemorrhage.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eMale C57BL/6 mice (weight 22\u0026ndash;25 g, 6\u0026ndash;8 weeks old, n\u0026thinsp;=\u0026thinsp;108) were randomly divided into sham-operated, ICH-operated and SZ168-treated groups. Brain haemorrhage was induced by collagenase injection into the striatum, and SZ168 IgG was administered intraperitoneally 6 h after ICH. Neurological regression was assessed at the molecular, cellular, histological and functional levels by short-term neurobehavioural tests, cerebral oedema, Evans blue staining, Western blotting, ELISA and immunofluorescence staining. In vitro, human-derived microglial HMC3 cells were pretreated with SZ168 IgG and washed platelets. Then hemin treatment was added to construct an in vitro model of cerebral haemorrhage, which was detected by wound healing assay, transwell migration assay, cell counting kit-8 reagent, colony formation on solid media and western blot to detect the functional changes of microglia after PDPN inhibition.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ePDPN expression in mouse brains was elevated 48 h after ICH, and inhibition of PDPN function with SZ168 improved short-term neurological function after ICH and reduced cerebral oedema and cerebral haemorrhage. In vitro, SZ168 treatment inhibited microglial invasion, migration and proliferation functions. Mechanistically, inhibition of PDPN function with SZ168 in vivo and in vitro was able to attenuate ICH-induced expression of nlrp3, caspase-1 and gasdermin D as well as inflammatory factors IL-1β, TNF-α and MPO.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eBlocking PDPN can reduce microglial invasion, migration and proliferation functions by inhibiting the microglial NLRP3/Caspase-1/GSDMD inflammatory pathway, which may play a role in reducing the inflammatory situation at the site of cerebral haemorrhage and ameliorating secondary brain injury.\u003c/p\u003e","manuscriptTitle":"Monoclonal Antibody to Podoplanin SZ-168 Attenuates Microglia Pyroptosis and Neuroinflammation in Experimental Cerebral Haemorrhage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-03 13:57:41","doi":"10.21203/rs.3.rs-5120185/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":"7f3588d3-470c-4f53-8e46-f7763cd98f47","owner":[],"postedDate":"December 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-06T16:53:54+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-03 13:57:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5120185","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5120185","identity":"rs-5120185","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}