Blockage of NLRP3 inflammasome by MCC950 can reverse the effect of pyroptosis in HUVECs and thrombosis of pregnancy rats

Blockage of NLRP3 inflammasome by MCC950 can reverse the effect of pyroptosis in HUVECs and thrombosis of pregnancy rats | 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 Blockage of NLRP3 inflammasome by MCC950 can reverse the effect of pyroptosis in HUVECs and thrombosis of pregnancy rats Yanhua Xu, Qinghua Wang, Yue Lu, Qin Shi, Linjie Xu, Siyi Chen, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2935166/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 Venous thromboembolism (VTE) is a common disease, the morbidity and mortality during pregnancy and the puerperium are significantly increased. However, the current understanding of its pathogenesis and pathophysiology is not fully adequate, which limits the choice of therapeutic approaches to some extent. In this study, we speculate that the massive accumulation of ROS in vascular endothelial cells after ischemia and hypoxia activates the NLRP3 inflammasome, which in turn releases a large amount of inflammatory factors that contribute to venous thrombosis. To verify the hypothesis, we established a DVT model in pregnant rats by the stenosis method and an H2O2-induced HUVECs model. The study hypothesis was fully confirmed by HE, IHC, Masson, Western blot, qRT-PCR, IF, ELISA, CCK8, LDH, invasion, migration, scratching, tube formation, TUNEL, JC-1 and flow cytometry experiments. We have reached the following results: 1.NLRP3 inflammasome was activated during the development of DVT (P < 0.0001). 2.MCC950 promoted the lysis, mechanization and recanalization of DVT in pregnant rats by inhibiting the activation of NLRP3 inflammasome (P< 0.05). 3.MCC950 reduced embryo loss and improved pregnancy outcome. 4.MCC950 inhibited NLRP3 inflammasome activation after H2O2-induced injury in HUVECs by enhancing ROS sensitivity (P < 0.05). To sum up, the study provides a new basic theoretical basis and potential therapeutic reference for the prevention and treatment of pregnancy-associated venous thromboembolism (PA-VTE). oxidative stress PA-VTE NLRP3 inflammasome pyroptosis ROS MCC950 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Venous thromboembolism (VTE) is a common disease including deep vein thrombosis (DVT) and pulmonary thromboembolism (PE)(Essien and Rali et al., 2019 ). The risk of VTE increases significantly during pregnancy(Soma-Pillay and Nelson-Piercy et al., 2016 ), and it is approximately five times higher in pregnant women and 30 to 60 times higher in the puerperium(Park and Park et al., 2021 ). Simultaneously, PE caused by DVT is a leading cause of maternal death(Konstantinides and Meyer et al., 2019 ). In addition, VTE is associated with adverse pregnancy outcomes. Symptoms associated with VTE, such as obstructed blood flow(Ibeh and Okocha et al., 2015 ), placental microthrombosis(Meng and Hu et al., 2015 ), and placental ischemia and hypoxia(Barut and Barut et al., 2010 ), can also lead to recurrent miscarriage, fetal growth restriction, pre-eclampsia, and placental abruption. Therefore, VTE during pregnancy and puerperium not only threatens the life of the mother, but may also affect fetal growth(Peeters, 2001 ). Once diagnosed, DVT in pregnancy and puerperium should be treated as soon as possible with a comprehensive approach based on anti-coagulation. This mainly includes physiotherapy, anti-coagulation, thrombolytic therapy and inferior vena cava filters (IVCF)(Ernst and Oporto et al., 2021 ). Low molecular weight heparin (LMWH) does not cross the placental barrier(Duffett and Rodger, 2015 ), has a relatively low incidence of bleeding, and is a clinically recognized antithrombotic agent(ACOG, 2018) However, all these treatments do not completely alleviate thrombotic disease due to local endothelial damage caused by inflammation and oxidative stress. Therefore, further studies on the pathogenesis and pathophysiology are needed to identify new therapeutic targets. Increasing evidence suggests that the pathogenesis of DVT is closely related to elevated levels of procoagulant factors/proteins, endothelial cell injury and apoptosis (Marik and Plante, 2008 ), platelet activation and aggregation(Budnik and Brill, 2018 ), overproduction of reactive oxygen species (ROS)(Herkert and Diebold et al., 2002 ), and inflammatory cell infiltration. Among these, the inflammatory response plays a key role. Inflammation activates the coagulation system and also inactivates the anticoagulation system, thereby inducing DVT, a process also known as immunothrombosis(Rider and Kaplanov et al., 2012 ). The intrinsic immune response is the body's first line of defense against foreign bodies and invasion by exogenous pathogens. The target signals of the intrinsic immune response can be classified into pathogen-associated molecular patterns (PAMPS) and damage-associated molecular patterns (DAMPs) for two different sources(Gong and Liu et al., 2020 ). NOD-like receptor thermal protein domain associated protein 3 (NLRP3) is the most studied and important pattern recognition receptor in the cytosol(Prochnicki and Latz, 2017 ). It is composed of apoptosis-associated speck-like protein containing a CARD (ASC), cysteine aspartate-specific proteinase-1 (caspase-1), and a CARD-associated speck-like protein (CARD), which assembles into inflammasome complexes to perform its functions(Broz and Dixit, 2016 ). Activated inflammasomes promote the maturation of interleukin-1β (IL-1β) and interleukin-18 (IL-18), induce the synthesis and release of other inflammatory cytokines, and amplify the inflammatory response(Godo and Shimokawa, 2017 ). It also cleaves perforin gasdermin D (GSDMD) and the cleaved product (N-terminal fragment) disrupts the cell membrane by forming open pores in the cell membrane and causing pyroptosis(Shi and Gao et al., 2017 ). Studies have shown that NLRP3 inflammasome activation is associated with the vascular inflammatory response(Xiao and Lu et al., 2013 ), which drives the development and progression of DVT. However, the role of NLRP3 inflammasome-mediated pyroptosis in the development of DVT in pregnant rats has been less studied both domestically and internationally. Moreover, the upstream regulatory mechanisms of NLRP3 inflammasome-mediated pyroptosis are complex. ROS production is one of the first identified triggers of NLRP3 inflammasome activation and a natural byproduct of aerobic metabolism produced in response to cellular stimulation by cytokines, invasion by foreign organisms and bacteria. There is growing evidence that ROS plays a key role in DVT after causing NLRP3 inflammasome activation(Gutmann and Siow et al., 2020 ). MCC950, a small molecule inhibitor that specifically inhibits NLRP3 activity, is a potential therapeutic approach for autoinflammatory and autoimmune diseases. For example, in the presence of excess palmitic acid (PA) in obese pregnant women, Sano et al.(Sano and Shimazaki et al., 2020 )found higher levels of PA exposure activated the NLRP3 inflammasome and induced placental inflammation leading to pregnancy complications after intravenous administration of palmitic acid solution to pregnant mice on day 12 of gestation, whereas MCC950 inhibited NLRP3 inflammasome activation and improved pregnancy outcome. The specific inhibition of MCC950 opens up the possibility of treating diseases caused by the NLRP3 inflammasome(Yang and Wang et al., 2019 ). Given the key role of inflammation and oxidative stress in thrombosis, and assuming that MCC950 is safe for the fetus, we propose the following research hypotheses: 1, the massive accumulation of ROS in ECs after ischemia and hypoxia activates the NLRP3 inflammasome, which in turn releases a large amount of inflammatory factors through pyroptosis to contribute to venous thrombosis; 2, MCC950 can inhibit pyroptosis through the ROS/NLRP3 pathway to achieve amelioration of pregnancy-related venous thromboembolism. To test the above hypothesis, we established a DVT model in pregnant rats and an H 2 O 2 -induced HUVECs (human umbilic vein endothelial cells, HUVECs) model. Through multiple biological approaches, we confirmed the hypothesis. In general, the study provided a new theoretical basis and potential therapeutic reference for the prevention and treatment of PA-VTE. 2. Materials and Methods 2.1 Animal Ethics and Design of Experiments Sixty 3-day pregnant Sprague-Dawley rats (6–8 weeks, 200-250g) were purchased from Nantong University Laboratory Animal Center (Jiangsu, China). The rats were housed in an SPF environment at 23–25°C, 12h/12h dark/light cycle, 50 ± 5% humidity, free access to food and water, and acclimatized for one week. Following the study of Cheng et al.(Cheng and Zhang et al., 2022 ), the tenth day of gestation (G10) was chosen for the subsequent experiments. General anesthesia was induced with 3% isoflurane, followed by maintenance anesthesia with 2.5% isoflurane until the end of the procedure (300–400 µL/100g body weight). Animals were euthanized at the end of the experiment. The care and use of all animal, experimental protocols, and procedures were performed in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments, ARRIVE) guidelines and the Guide for the Care and Use of Laboratory Animals proposed by the Ministry of Health of the People's Republic of China. The project was approved by the Animal Ethics Committee of Nantong University (S20221222-008). Firstly, G10 rats were randomly divided into two groups: an induced DVT model group ( n = 24) and a sham-operated control group ( n = 24). The model group was further divided into 4 subgroups of 6 hours, 1 day (1d, n = 6), 3 days (3d, n = 6), and 7 days (7d, n = 6). In the second part, pregnant rats were randomly divided into PBS ( n = 6) and MCC950 ( n = 6) group, and administered (20 mg/kg/d) three times by intraperitoneal injection(Bellut and Papp et al., 2021 ), 1 hour before isoflurane anesthesia, 1 day and 2 days after modeling, respectively. An equal dose of PBS was injected intraperitoneally as a negative control. The mortality rate of the experimental animals was 8.9% (3/60). The cause of death was hemorrhage from accidental puncture of the IVC (inferior vena cava, IVC) in two cases and incisional dehiscence after molding in one case. 2.2 Establishment of experimental model of DVT in pregnant rats The DVT model was created using the IVC "stenosis method" on day G10. Briefly, after successful anesthesia, the abdomen was opened along the midline and the IVC was gently separated from the aorta, 4 − 0 absorbable sutures are placed parallel to the IVC, and then the IVC and all visible side branches are ligated with non-reactive 5 − 0 absorbable sutures. The 4 − 0 sutures were then removed after confirming their tightness, resulting in 90% stenosis. In the control group, only sutures were passed and no vessels were ligated. Rats were anesthetized and euthanized at 6 h, 1 d, 3 d, and 7 d after DVT and 24 h after the third dose. Then the thrombotic tissues were excised, measured for length, and weighed. Blood samples were centrifuged at 3000×g for 10 min, and 200 µL of serum was collected. The serum and three thrombus tissues were fixed at -80°C, and the remaining three tissues were fixed at 4°C in 4% para-formaldehyde for 12 hours for subsequent experiments. 2.3 Histopathologic analysis and measurement of thrombotic tissue Tissues were removed from para-formaldehyde and washed thoroughly in PBS. After dehydrated through graded alcohol, the tissues were cleared through xylene, embedded in paraffin. Each group was sectioned separately with a paraffin microtome (thickness, 4µm). To observe the characteristics of thrombus formation, recanalization, and pathological changes after formation, at least three tissues from each group were randomly selected for the experiment. HE staining was performed with the kit (C0105M, Beyotime Biotechnology, China) according to the manufacturer's instructions. Processing of thrombus tissue sections was performed as described previously. Immunohistochemistry (IHC) staining was performed using a universal kit (PV6000, ZSGB-BIO, China). Briefly, sections were placed in Tris-EDTA antigen repair solution (1×) and heated in boiling water for 15 minutes to repair antigenic epitopes. To block endogenous peroxidase activity, endogenous peroxidase was added dropwise to each tissue and then incubated with 5% BSA for 30 minutes. After incubation with CD34 primary antibody (ICO-115, 1:200, Novus Biologicals, USA), sections were placed in a refrigerator at 4°C overnight. The next day, sections were incubated dropwise with reaction enhancer, washed with PBS, and incubated with secondary antibody for 30 minutes at 37°C. Color changes were observed microscopically during DAB incubation and finally incubated with hematoxylin for 15 minutes, fractionated with hydrochloric acid fractionation solution, dehydrated, transparent and sealed. Masson's trichrome stain is the definitive and classic technique, which stains collagen fibrils, extracellular matrix and vessel wall blue and cell nucleus black. It was performed with the kit (C0105, Beyotime Biotechnology, China) according to the manufacturer's instructions. Thrombus mechanization rate was calculated based on the percentage of thrombus area occupied by collagen fibrils and extracellular matrix-stained areas. At least three stained sections of each thrombus were observed sequentially under a microscope (Olympus, Tokyo, Japan). The cumulative optical density (IOD) values of positive expression sites for collagen fibrils (blue in Masson's stain) and CD34 (brown in thrombus tissue) were then calculated separately for each group of sections using FIJI Image 8.0 software. 2.4 Western blot Total protein was extracted from thrombus tissue or cells using a protein extraction reagent. Protein concentrations were determined using the BCA protein assay kit (P0010, Beyotime Biotechnology, China) according to the manufacturer's instructions. Solubilized proteins (30–50µg per lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (10%, 15% separation gels and 5% stacking gels) and transferred to PVDF membranes (IPVH00010, Merck Millipore, USA), blocked in tris-buffered saline (TBS) for one hour, and incubated with specific primary antibodies at 4°C overnight. The following primary antibodies were then used: rabbit anti-NLRP3 (T55651, 1:1000, Abmart, China), mouse anti-caspase-1 (22915-1-AP, 1:1000, Proteintech, USA), mouse anti-GSDMD (P57764, 1:1000, Abmart, China ), rabbit anti-IL-1β (P10749, 1:2000, Abmart, China), or mouse anti-GAPDH (60004-1-lg, 1:2000, Proteintech, USA). After washing with TBST, protein bands were visualized by incubation with HRP-conjugated affinipure goat anti-rabbit or anti-mouse IgG(H + L) (SA00001-1; SA00001-2, 1:5000, Proteintech, USA) for 2 h. With the BeyoECL Plus Kit (P0018S, Beyotime Biotechnology, China). protein bands were visualized with ECL luminescence solution (Thermo Fisher Scientific, Inc. USA) and then analyzed with FIJI ImageJ (v1.8.0; National Institutes of Health) and normalized to GAPDH levels. 2.5 Quantitative real-time polymerase chain reaction (qRT-PCR) To examine NLRP3 inflammasome mRNA expression, total RNA (ribonucleic acid, RNA) was extracted from frozen tissues or HUVECs using Trizol reagent (abs60154, absin, China) according to the manufacturer's instructions. Then, 2 µg of total RNA from each sample was reverse transcribed into complementary deoxyribonucleic acid (cDNA) using HiScript II Q RT SuperMix for qPCR (R223, Vazyme, China), with ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme, Nanjing, China) on the LightCycler® 96 system (Thermo Fisher, USA). The PCR reactions were performed as follows. First, 95℃ for 30s, pre-denaturation once;then 95℃ for10s and 60℃ for 30s, cycle forty times༛last, 95℃ for 5s, 60℃for 60s and 95℃for15s dissolution once. Quantitative RT-PCR was performed on all samples in triplicate. The mRNA expression levels of the target genes were normalized to GAPDH, and the results were expressed as 2 −ΔΔct . The main primer sequences are shown in Table 1 . Table 1 Sequences of primary genes Species Genes Leading and trailing chains Sequences Rat NLRP3 Forward 5' -GAG CIG GAC CTC AGT GAC AAT GC-3' Reverse 5' -AGA ACC AAT GCG AGA TCC TGA CAA C-3' Caspase-1 Forward 5' -GAA GGT GGC GCA TT CCT GG-3' Reverse 5' -AGG GCA AGA CGT GTA CGA GT-3' GSDMD Forward 5' -CAG CAG GCA GCA TCC TTG AGT G-3' Reverse 5' -CCT CCA GAG CCT TAG TAG CCA GTA G-3' IL-1β Forward 5' -AAT CTC ACA GCA GCA TCT CGA CAA G-3' Reverse 5' -TCC ACG GGC AAG ACA TAG GTA GC-3' Human NLRP3 Forward 5' -AGG GAT GAG AGT GTT GTG TGA AAC G-3' Reverse 5' -GCT TCT GGT TGC TGC TGA GGA C-3' Caspase-1 Forward 5' -GAA GAA ACA CTC TGA GCA AGT C-3' Reverse 5' -GAT GAT GAT CAC CLT CGG TTT G-3' GSDMD Forward 5' -TTG AAG AAR TGA GTG LGG ACA GAG C-3' Reverse 5' -TGG TGG TGT GTG CGT TGG AAT G-3' IL-1β Forward 5' -GCC AGT GAA ALG ATG GCT TAT T-3' Reverse 5' -AGG AGC ACT TCA TCT GTT TAG G-3' 2.6 Enzyme-linked Immunosorbent Assay (ELISA) for IL-1β and IL-18 in Serum Serum concentrations of IL-1β and IL-18 were determined using the appropriate ELISA kits (MM-0194R1, MM-0047R1, Jiangsu Meimian Industrial, China) according to the supplier's protocol. Absorbance at 450 nm was analyzed using an ELISA instrument (Thermo Fisher Scientific, Inc. USA). 2.7 Immunofluorescence Analysis of NLRP3 and Caspase-1 in Tissues and Cells The co-localization and differential expression of NLRP3, caspase-1 and CD34 in tissues and cells was evaluated by immunofluorescence (IF). IF of tissues was performed with some special treatments as follows. The thrombus tissue was dehydrated in different glucose gradients on alternate days, and finally embedded in optimal cutting temperature compound (4583, Sakura, USA) and stored at -80°C. Then, continuous coronal sections of 4 µM thickness were cut on a frozen section machine (Leica, Germany) and placed on 0.01% poly-L-lysine-coated coverslips. The frozen sections or cells were fixed with 4% paraformaldehyde for 25 min, followed by permeabilization with 0.1% Triton X-100 for 1 hour, blocked with 10% normal goat serum (SL038, Solarbio, China), frozen sections or cells were incubated with NLRP3 antibody (T55651, 1:100, Abmart, China), caspase-1(22915-1-AP, 1:100, Proteintech, USA) or CD34 (ICO-115, 1:200, Novus Biologicals, USA) were incubated overnight at 4°C. After washing, cells and tissues were incubated with goat anti-rabbit (Alexa Fluor® 568, 1:500, Thermo Fisher Scientific, for anti-NLRP3 antibody) and goat anti-mouse (Alexa Fluor® 488, 1:500, Thermo Fisher Scientific, for anti-caspase-1 or anti-CD34 antibody) secondary antibodies for 1 hour at 37°C, followed by staining with Hoechst33342 (4083, 1:1000, Cell Signaling Technology, USA) for five minutes at room temperature. Coverslips were mounted with antifade mounting medium (HY-K1042, MedChemExpress, USA). Visualization was then performed under a scanning fluorescence microscope (Leica, Germany). Finally, the average fluorescence intensity of frozen sections or cells was analyzed using Fiji Image. 2.8 Design of Cell Experiments HUVEC lines were generously donated by Dr. Xi Cheng (Nantong University, China) and cultured in DMEM/F-12 medium (A4192001, Gibco, USA;) supplemented with 10% fetal bovine serum (1925624, Biological Industries, Israel) and antibiotics (100 units/mL penicillin, 100 mg/mL streptomycin) at 37°C in a 5%CO 2 , 95% air incubator. HUVECs were plated in 6-, 24-, and 96-well plates for various experiments. In the co-culture system, HUVECs were cultured in the above mentioned chambers. Meanwhile, all cultures were grown in DMEM/F12 medium. Based on the results of CCK-8 as well as LDH experiments, the optimal concentration of H 2 O 2 was determined to be 400 µM with an action time of 24 h (Magenta and Cencioni et al., 2011 ; Ugusman and Zakaria et al., 2011 ; Han and Tang et al., 2017 ). In addition, the optimal concentration of MCC950 (HY-12815, MedChemExpress, USA) was determined to be 10 µM with an action time of 2 h according to the reference(Franke and Bieber et al., 2021 ). Cells were divided into the following groups: control group (cells were cultured in normal environment without any stimulation); H 2 O 2 group (cells were pretreated with 400 µM H 2 O 2 for 24 hours); H 2 O 2 + MCC950 group (cells were pretreated with 10 µM MCC950 for 2 hours and then co-cultured with 400 µM H 2 O 2 for 24 hours. The experiments were performed in triplicate. 2.9 In vitro cytotoxicity of MC950 and different concentrations of H 2 O 2 2.9.1 CCK-8 assay The cytotoxicity of different concentrations of H 2 O 2 (H13022648, HebeiHengjian, China) in HUVECs was evaluated by CCK-8 assay and LDH assays. The Cell Counting Kit-8 (CK04, Dojindo, Japan) was performed to assess the viability of HUVECs according to the manufacturer's protocol. Specifically, HUVECs were seeded in 96-well plates (1×10 4 cells/well) in 100 µL DMEM/F-12 medium containing FBS, penicillin, and streptomycin as previously mentioned. After overnight incubation, the medium was removed. Next, HUVECs were treated with 10 µM MCC950 (MCC950 group) or no treatment (control group) for 2 hours and then treated with different concentrations of H 2 O 2 (0, 100, 200, 400, 600 µM) for 24 hours. The medium was then removed and replaced with 100 µL fresh DMEM/F-12 medium, and 10 µL CCK-8 solution was added to each well. After 2 hours of incubation, absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, USA). Cell viability (%) was expressed as a percentage of the control condition. 2.9.2 LDH assay HUVECs were treated with H 2 O 2 , DMEM/F-12, or MCC950 as described above. Cytotoxicity was also determined by measuring LDH (lactate dehydrogenase, LDH) released from the cells using a LDH assay kit (C0016, Beyotime Biotechnology, China) according to the manufacturer's protocol. Absorbance and method of calculating are the same as those used in the CCK-8 experiment. 2.10 Invasion, Migration and Tube formation assay Invasion, migration and tube formation assays were used to observe the impairment of cell invasion, migration and angiogenesis ability after treatment of HUVECs with H 2 O 2 or MCC950. For these three assays, the upper surface of transwell cell culture inserts (353097-1, 8 µm pore size, BD Biosciences, USA) in 24-well plates were coated with 20 µL/well Matrigel (8246003, 1.25mg/ml, Corning, USA) for 2 hours. After treatment with H 2 O 2 or MCC950 as described above, cells were collected in the same manner, and then cells were resuspended separately with serum-free DMEM/F-12 medium. In detail, a total of 3×10 4 HUVECs were seeded in the upper chamber of the transwell. Then 500 µl of complete medium was added to the lower part of the transwell. For invasion and migration assays, cells were incubated in the incubator for 18–24 hours and then fixed with 4% paraformaldehyde, stained with crystal violet, and observed under a microscope (Olympus, Japan). The average number of invading or migrating cells was calculated from five randomly selected areas. For tube formation assay, after 4–6 hours of incubation, images of the blood vessels formed in each transwell were observed under an inverted microscope and photographed. In three independent experiments, vessel branching was measured using FIJI ImageJ software to determine total vessel length and branching images. 2.11 Scratch Healing Assay HUVECs were seeded in 6-well plates (5×10 5 cells per well, 2 mL DMEM/F-12). After washing with PBS, DMEM/F-12 containing 5% FBS with or without MCC950 (10 µM) and H 2 O 2 (400 µM) was added. The cells were placed in an incubator for incubation. The wells were then photographed immediately after 0, 12, and 24 hours, and the distance and area of each scratch was measured via FIJI image. 2.12 Flow Cytometric Assessment of ROS in HUVECs DCHF-DA is a reliable fluorescent marker of ROS that can be oxidized by intracellular ROS to 2', 7'-dichlorofluorescein (DCF). The level of intracellular ROS can be known by detecting the fluorescence of DCF. Therefore, followed the manufacturer's instructions, we used a reactive oxygen species detection kit (S0033S, Beyotime Biotechnology, China) to detect intracellular ROS production in HUVECs. Briefly, different groups of HUVECs were incubated with 10 µM DCFH-DA at 37°C for 20 minutes in the dark. After washing to remove extracellular residues of DCFH-DA, the mean fluorescence intensity of ROS was analyzed and averaged using a flow cytometer (Becton-Dickinson, San Jose, USA) with FlowJo 7.6 software (Tree Star, Inc.) 2.13 Mitochondrial membrane potential assay JC-1 is an ideal fluorescent probe and is widely used to detect mitochondrial membrane potential as an indicator of early apoptosis detection. After treatment, cells were washed twice with PBS, 1 ml of JC-1 staining solution (C2006, Beyotime Biotechnology, China) was added, mixed thoroughly, and incubated for 20 min at 37ºC. After incubation, the supernatant was aspirated, and the cells were washed 4 times with JC-1 staining buffer in a shaking side swing bed to reduce the background, 3 µL Hoechst 33342 for 5 minutes at room temperature in the dark, and immediately observed under a fluorescence microscope and photographed. 2.14 Hoechst33342/TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) fluorescence staining To detect DNA fragments of HUVECs, cells were double stained with Hoechst33342 and TUNEL. Briefly, HUVECs (3×10 4 cells/well) were cultured in 24-well plates. After the indicated treatments, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 30 minutes at room temperature. Then cells from each group were stained with 3 µl TUNEL (A113, 100 µg/ml, Vazyme, China) for 1 hour at 37°C in the dark and with 3 µl Hoechst33342 for 5 minutes at room temperature in the dark. After that, photographed under an inverted fluorescence microscope (Nikon, TE-2000 U) at ×40 magnification. 2.14 Statistical Analysis Vector images were statistically analyzed using FIJI ImageJ(Schindelin and Arganda-Carreras et al., 2012 ) followed by GraphPad Prism software (version8.0, GraphPad Software Inc., CA, USA). All data followed a normal distribution, and all continuous values were expressed as mean ± standard error (SEM). Student's t-test was used to compare the two experimental groups, and differences between groups were analyzed by one-way ANOVA. All relevant experiments were repeated three times with similar results, showing a representative series of images, and values less than 0.05 were considered statistically significant. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, # p < 0.05, ## p < 0.01, ### p < 0.001, ### p < 0.0001). 3. Results 3.1 Lysis, mechanization, and recanalization of DVT in pregnant rats are accompanied by changes in inflammatory factors, neovascularization, and fibrotic area. 3.1.1 Pathological results To observe the process of thrombus formation and evolution, we performed HE, CD34 immunohistochemical and Masson’s staining at different time intervals, with the following results. As shown in Fig. 1A and Table 2 , 6 hours after modeling, the blood vessels showed bruise-like changes with a small number of inflammatory cells (Fig. 1A); the relative expression of CD34 was 0.128 ± 0.0237 (Fig. 1B,1C). Masson's trichrome staining showed that the relative fibrotic area was 1.009 ± 0.007, with a light blue color around the vessel wall (Fig. 1D,1E). One day after modeling, the thrombus was laminar and filled the entire lumen, and inflammatory cells were scattered throughout the vein wall, with neutrophils predominant (Fig. 1A). The relative expression of CD34 increased to 1.279 ± 0.0877 ( P < 0.01, vs. 6h group) (Fig. 1B,1C), the blue area representing collagen fibers, extracellular matrix and vessel wall was dominated by thrombus edges, a little fibrin-like composition in the middle of thrombus tissue was analyzed, and the area of fibrosis increased to 1.303 ± 0.069 ( P < 0.01, vs. 6h group) (Fig. 1D,1E). Three days after modeling, inflammatory cells were significantly increased in the thrombus tissue (Fig. 1A), endothelial cells were proliferated, the relative expression of CD34 was upregulated to 2.294 ± 0.107 ( P < 0.0001, vs. 6h and sham groups) (Fig. 1B,1C), fibrin-like formation was clearly visible in the center of the thrombus in addition to the thrombus edge, and the fibrosis area increased to 1.428 ± 0.010, indicating a significant proliferation of collagen fibers ( P < 0.0001, vs. 6h and sham groups) (Fig. 1D,1E). Seven days after modeling, the number of inflammatory cells in the venous wall continued to increase (Fig. 1A), there were relatively larger neovascularizations, granulation tissue was seen at the junction of thrombotic vessels to the thrombus, there was recanalized small vessel formation in the thrombus, and the relative expression of CD34 was 3.926 ± 0.48 ( P < 0.0001, vs. the 6h and sham groups) (Fig. 1B,1C). The increase in collagen fiber content was more significant, the venous thrombus was mechanized, most of the thrombus was occupied by collagen fibers, and the relative fibrosis area was 1.519 ± 0.015 ( P < 0.0001, vs. the 6h and sham groups) (Fig. 1D,1E). Table 2 Statistical values and P -value of the pathological results of DVT in different groups Group Ratio of venous thrombus weight/length(mg/mm) Relative expression of CD34 (%) Relative fibrosis area (%) sham 1.667 ± 0.333 0.483 ± 0.034 1 ± 0.002 6h 11.67 ± 0.881 0.128 ± 0.023 1.009 ± 0.007 1d 15.75 ± 0.629 1.279 ± 0.087 1.303 ± 0.069 3d 12.33 ± 0.333 2.294 ± 0.107 1.428 ± 0.010 7d 11.33 ± 0.881 3.926 ± 0.48 1.519 ± 0.015 P -Value ༜0.0001 ༜0.0001 ༜0.0001 Table 6 NLRP3 inflammasome protein relative expression results statistical values and P -values Group NLRP3 Caspase-1 GSDMD IL-1β PBS 0.303 ± 0.057 0.556 ± 0.076 0.547 ± 0.028 0.237 ± 0.033 MCC950 0.099 ± 0.021 0.229 ± 0.086 0.158 ± 0.009 0.077 ± 0.001 P -Value 0.029 0.047 0.0002 0.008 3.1.2 Ratio 0f Venous Thrombus Weight/Length in Pregnant Rats Due to certain anatomical differences in the length and thickness of the IVC in each pregnant rat and certain differences in the manipulation by each surgeon, the overall condition of the thrombus was first assessed by the ratio of weight/length. No significant thrombus formation was observed in the sham group. As shown in Fig. 1F and Table 2 , the weight/length ratio (mg/mm) increased rapidly from 6 h, peaked at 1 d, and then decreased slowly to near 6 h at 7 d ( P < 0.0001, vs. the 6h and sham groups) (Fig. 1F). 3.2 Upregulation of NLRP3 inflammasome expression in the development of DVT in pregnant rats. To evaluate whether the expression of NLRP3 inflammasome was upregulated and the trend of change, we detected it by Western blot, qRT-PCR and IF. As shown in Fig. 2A-2B and Table 3 , the protein levels of NLRP3 were not significantly in the sham and 6h groups, and started to increase and peaked at 1d and slowly decreased from day 3. It decreased at day 7, but was still higher than the sham and 6h groups. The protein expression of caspase-1 was not significant in the sham group and gradually increased after 6 hours, peaked at day 7 ( P < 0.0001) (Fig. 2C). GSDMD protein levels gradually increased from 6h, reached a maximum at day 3, and began to decrease at day 7, but were still higher than at day 1 (P < 0.0001, P < 0.05, vs. sham and 6h groups) (Fig. 2D). IL-1β began to show significant changes at 6 h ( P < 0.001, vs. sham group), peaked at day 1 ( P < 0.0001), and then gradually decreased, didn’t return to normal levels at day 7 ( P < 0.01, vs. 6h group) (Fig. 2E). Table 3 Statistical values and P- values of NLRP3 inflammasome protein expression in DVT tissue of pregnant rats Group NLRP3 Caspase-1 GSDMD IL-1β sham 0.041 ± 0.007 0.101 ± 0.023 0.061 ± 0.016 0.049 ± 0.007 6h 0.064 ± 0.053 0.216 ± 0.030 0.153 ± 0.006 0.392 ± 0.044 1d 0.519 ± 0.053 0.375 ± 0.045 0.331 ± 0.034 0.635 ± 0.062 3d 0.328 ± 0.035 0.715 ± 0.022 0.883 ± 0.102 0.201 ± 0.030 7d 0.163 ± 0.012 0.981 ± 0.088 0.532 ± 0.081 0.095 ± 0.024 P- Value < 0.0001 < 0.0001 < 0.0001 < 0.0001 Consistent with protein expression, NLRP3, caspase-1, GSDMD, and IL-1β mRNA expression levels were increased to varying degrees at different time intervals with statistically significant differences ( P < 0.0001, P = 0.0003, P < 0.0001, P = 0.0067) (Fig. 2F-2I, Table 4 ). Table 4 Statistical values and P- values of NLRP3 inflammasome mRNA expression in DVT tissue of pregnant rats Group NLRP3 Caspase-1 GSDMD IL-1β sham 0.349 ± 0.065 0.249 ± 0.009 0.100 ± 0.009 0.040 ± 0.002 6h 0.429 ± 0.033 0.567 ± 0.034 0.31 ± 0.034 2.337 ± 0.016 1d 1.954 ± 0.037 0.829 ± 0.096 2.242 ± 0.064 2.774 ± 0.098 3d 1.44 ± 0.061 1.287 ± 0.064 2.714 ± 0.031 2.24 ± 0.183 7d 1.312 ± 0.055 1.92 ± 0.103 2.557 ± 0.114 0.351 ± 0.027 P -Value < 0.0001 0.0003 < 0.0001 0.0067 As shown in Fig. 2J-2K, compared with the sham group, the relative expression of NLRP3 was 1.056 ± 0.013 at 6 hours; it increased to a maximum of 1.837 ± 0.086 at day 1 ( P < 0.001); gradually decreased to 1.539 ± 0.042 at day 3 ( P < 0.001), and then decreased to 1.368 ± 0.015 at day 7 ( P < 0.05), in agreement with the Western blot and qRT-PCR results. A more significant finding was that NLRP3 was not significantly expressed in the sham and 6h groups, and predominantly expressed in the vascular endothelium around the thrombus from day 1, gradually shifting to predominantly expressed in the middle of the thrombus tissue after day 3. To detect the basal levels of IL-1β and IL-18, serum samples were collected before each modeling session as the before group. As shown in Fig. 3 and Table 5 , IL-1β was elevated from 6h, reached its highest level at 1 day, then slowly decreased and remained above the 6h level at 7 days ( P < 0.001, v.s) (Fig. 2L). IL-18 reached its highest level at 6h ( P < 0.0001) then gradually decreased and remained above the 6h level at 7 days ( P < 0.05) (Fig. 2M, Table 5 ). Table 5 Serum IL-1β and IL-18 concentrations and P- value of DVT in pregnancy rats Group IL-1β(pg/mL) IL-18(pg/mL) Before 2.99 ± 0.060 29.67 ± 2.523 sham 4.488 ± 0.091 29.14 ± 1.816 6h 6.095 ± 0.176 62.53 ± 4.245 1d 9.579 ± 0.267 52.83 ± 3.374 3d 7.839 ± 0.204 48.25 ± 2.357 7d 6.892 ± 0.446 46.06 ± 3.829 P -Value < 0.0001 < 0.0001 3.3 MCC950 Promotes Thrombus Lysis, Mechanization and Recanalization Based on the results obtained from the above experiments, MCC950 was chosen as an inhibitor to see whether it could reduce the inflammatory response and thus promote thrombus lysis absorption and recanalization. As shown in Fig. 3A-3B, the number of inflammatory cells in the venous wall was reduced and thrombus lysis was faster in the MCC950 group. The relative expression of CD34 was from 1.0 ± 0.123 to 1.536 ± 0.187 ( P = 0.0314) (Fig. 3C,3D), indicating that the number of neovascularization was less in the PBS group. The relative fibrosis area after PBS treatment was from 1,000 ± 0.025 to 0.914 ± 0.017 ( P = 0.0165) (Fig. 3E,3F,3G), indicating that MCC950 treatment could promote thrombus mechanization. Simultaneously, the thrombus weight/length ratio was 0.111 ± 0.002 in the PBS group and decreased to 0.082 ± 0.000 in the MCC group ( P < 0.0001) (Fig. 3H). In conclusion, MCC950 can reduce inflammatory cell infiltration in thrombus and promote thrombus lysis, mechanization and recanalization. 3.4 MCC950 down-regulates the expression of the NLRP3 inflammasome in the model of DVT in pregnant rats To evaluate the inhibitory function of MCC950 on NLRP3 inflammasome in DVT of pregnant rats, the protein expression levels were first analyzed by Western blot. Next, the mRNA expression levels were evaluated by qRT-PCR assay. As shown in Fig. 4A-4I, Table 6 and Table 7 , NLRP3 inflammasome mRNA levels were all downregulated in the MCC950 group (p < 0.05), consistent with the trend of NLRP3 inflammasome protein expression. The expression and localization of NLRP3 were also detected by IF assay. As shown in Fig. 3–9, the relative expression of NLRP3 was from 1 ± 0.074 to 0.565 ± 0.025, which mean that it was significantly decreased after MCC950 treatment ( P = 0.0002). Moreover, it was predominantly expressed in the middle of thrombus, which is consistent with the results of the first part (Fig. 4J-4K). Table 7 NLRP3 inflammasome mRNA relative expression results statistical values and P -values Group NLRP3 Caspase-1 GSDMD IL-1β PBS 1.254 ± 0.064 1.959 ± 0.200 2.625 ± 0.150 3.246 ± 0.199 MCC950 0.181 ± 0.037 0.950 ± 0.0.069 0.906 ± 0.110 1.999 ± 0.160 P -Value < 0.0001 0.0003 < 0.0001 0.0067 IL-1β and IL-18 levels (pg/mL) were then measured by ELISA. As shown in Fig. 3-10A, serum IL-1β concentration was 7.234 ± 0.274 before modeling and 10.170 ± 0.452 in the PBS-treated group, while it decreased to 8.010 ± 0.274 after MCC950 treatment ( P < 0.0001) (Fig. 4L). The serum IL-18 concentration was 29.41 ± 1.484 and increased to 55.13 ± 1.529, while it decreased to 43.68 ± 1.544 ( P < 0.0001) (Fig. 4M). This indicates MCC950 treatment inhibits the expression of IL-1β and IL-18. 3.5 MCC950 improves pregnancy outcome As shown in Fig. 5, by carefully observing the vaginal bleeding of pregnant rats before collecting the material, we found that rats in the PBS group showed different degrees of vaginal bleeding (A), while the vaginal opening of rats in the MCC950 group was dry without any blood (B). After opening the abdominal cavity, we found that the oocytes were reduced to different degrees in the PBS group, while the reduction of oocytes was not obvious in the MCC950 group (C). During the retrieval process, we found that there were very few peri-thrombus adhesions in the MCC950 group of pregnant rats, which were easier to isolate. The thrombus in the PBS group was dark red in color and hard in texture after harvesting, whereas the thrombus tissue in the MCC950 group was bright red in color, soft in texture, and extremely easy to separate from the vascular endothelium (D). 3.6 MCC950 attenuates toxic effect of H 2 O 2 on HUVECs and improves cell proliferation, angiogenesis To investigate the effects of H 2 O 2 and MCC950 on HUVECs and the optimal concentrations, HUVECs were incubated with different concentrations of H 2 O 2 (0, 100, 200, 400, 600 µM) for 24 h. Cell viability (%) gradually decreased at 100–600 µM H 2 O 2 compared to control cells (0 µM H 2 O 2 ), and decreased significantly at H 2 O 2 concentrations > 400 µM ( P 0.05), and gradually increased after more than 100 µM, showing a dose-dependent effect (Fig. 6B). Therefore, based on the results of CCK8 and LDH analysis, 400 µM was selected as the injury model for the following experiments (Fig. 6B). Next, whether MCC950 could protect against H 2 O 2 -induced damage was tested. As shown in Fig. 5A and Table 8 , cell viability increased to different degrees in each group after pre-treatment with MCC950 compared to the same concentration without pre-treatment. It was most interesting at a concentration of 400 µM. Significant differences can be seen between this group and the 400 µM group without pretreatment with MCC950, and also with the group without H 2 O 2 co-incubation, further confirming the correct choice of 400 µM. The results showed that the release of LDH still increased with increasing concentration after MCC950 pretreatment, but the increase was significantly lower in each group than without MCC950 pretreatment. The results of both experiments confirmed MCC950 protects against H 2 O 2 -induced damage and attenuates LDH release (Fig. 6B). Treatment with 10 µM MCC950 for 2 h could be selected as the optimal concentration and time and used in the following experiments. Table 8 Statistical values and p -values for CCK-8 and LDH results Group Cell ability(%) P -Value (VS.Control) P -Value Control 1 ± 0.032 100 µM H 2 O 2 0.685 ± 0.027 < 0.0001 200 µM H 2 O 2 0.592 ± 0.008 < 0.0001 400 µM H 2 O 2 0.495 ± 0.020 < 0.0001 600 µM H 2 O 2 0.168 ± 0.003 0.9999 0.046 (VS. 100 µM H 2 O 2 ) MCC950 10 µM + 200 µM H 2 O 2 0.769 ± 0.0328 0.9137 0.019 (VS. 200 µM H 2 O 2 ) MCC950 10 µM + 400 µM H 2 O 2 0.608 ± 0.026 0.0008 0.046 (VS. 400 µM H 2 O 2 ) MCC950 10 µM + 600 µM H 2 O 2 0.205 ± 0.006 < 0.0001 0.998 (VS. 600 µM H 2 O 2 ) Group Relative release of LDH(%) P -Value (VS.Control) P -Value Control 0.183 ± 0.004 100 µM H 2 O 2 0.221 ± 0.010 0.66 200 µM H 2 O 2 0.346 ± 0.020 < 0.0001 400 µM H 2 O 2 0.563 ± 0.022 < 0.0001 600 µM H 2 O 2 0.749 ± 0.016 0.9999 MCC950 10 µM + 100µM H 2 O 2 0.295 ± 0.016 0.0006 0.0285 (VS. 100 µM H 2 O 2 ) MCC950 10 µM + 200 µM H 2 O 2 0.384 ± 0.001 < 0.0001 0.6183 (VS. 200 µM H 2 O 2 ) MCC950 10 µM + 200 µM H 2 O 2 0.384 ± 0.001 < 0.0001 0.6183 (VS. 200 µM H 2 O 2 ) MCC950 10 µM + 400 µM H 2 O 2 0.456 ± 0.012 < 0.0001 0.0007 (VS. 400 µM H 2 O 2 ) MCC950 10 µM + 600 µM H 2 O 2 0.597 ± 0.011 < 0.0001 < 0.0001 (VS. 600 µM H 2 O 2 ) Numerous studies have shown functional impairment occurs after H 2 O 2 induction in HUVECs, and we next investigated whether MCC950 could ameliorate the functional impairment caused by H 2 O 2 . Cell migration refers to the movement of cells after receiving migration signals. Cell invasion refers to the ability of cells to migrate from one area to another through the extracellular matrix. From the invasion and migration experiments in Fig. 6C-6F and Table 9 , H 2 O 2 significantly reduced the invasion and migration ability of HUVECs compared with the control group, while it was gradually restored after pretreatment with MCC950. Table 9 Statistical values and p -values of the experimental results for invasion, migration, and formation of tubules Group Invading cells Migrating cells Branches Total branches length Control 170.1 ± 2.283 144.2 ± 1.376 141.2 ± 4.234 7291 ± 207.6 H 2 O 2 65.14 ± 2.623 55.5 ± 1.478 108.1 ± 2.142 6397 ± 170 MCC950 + H 2 O 2 118.4 ± 1.925 73.33 ± 1.563 125.5 ± 4.479 7274 ± 152.2 P -Value < 0.0001 < 0.0001 < 0.0001 0.0012 The tube formation assay is a rapid and quantifiable method to measure angiogenesis in vitro. By assessing the number of branches and total branch length, we found H 2 O 2 reduced the angiogenic capacity of HUVECs, while MCC950 restored angiogenic function ( p < 0.0001) (Fig. 6G-6I). The scratch experiment can analyze the cell migration function from another perspective. As shown in Fig. 6J-6K, the percentage of wound healing distance between cell scratches in the H 2 O 2 group was 0.522 ± 0.038, which was significantly lower than that in the control group ( P < 0. 0001); whereas, after pre-incubation with MCC950, it rose to 0.745 ± 0.057, which was lower than that in the control group and higher in the H 2 O 2 group ( P < 0.0001). In conclusion, MCC950 significantly reduced pyroptosis of HUVECs and partially restored cell function of proliferation and angiogenesis. 3.7 MCC950 Inhibits NLRP3 Inflammasome Activity in H 2 O 2 -stimulated HUVECs In the H 2 O 2 -stimulated HUVECs, to investigate whether MCC950 acts by inhibiting the NLRP3 inflammasome, we also performed Western blot, qRT-PCR and IF experiments. As shown in Fig. 7A-7G, Fig. 7I-7J, Table 10 and Table 11 , the protein and mRNA expression of NLRP3, caspase-1, GSDMD, and IL-1β were significantly upregulated in the H 2 O 2 group compared with the control group, and decreased after 2 h pretreatment with MCC950. Table 10 NLRP3 inflammasome protein expression statistics and p -values Group NLRP3 Caspase-1 GSDMD IL-1β Control 0.386 ± 0.0142 0.451 ± 0.032 0.748 ± 0.048 0.773 ± 0.098 H 2 O 2 1.508 ± 0.124 1.665 ± 0.149 2.248 ± 0.134 1.784 ± 0.097 H 2 O 2 + MCC950 1.119 ± 0.021 0.861 ± 0.040 1.289 ± 0.048 1.237 ± 0.088 P -Value < 0.0001 < 0.0001 < 0.0001 0.0001 Table 11 NLRP3 inflammasome mRNA expression statistics and p -values Group NLRP3 Caspase-1 GSDMD IL-1β Control 0.635 ± 0.037 0.843 ± 0.020 0.748 ± 0.048 0.436 ± 0.086 H 2 O 2 1.583 ± 0.022 1.298 ± 0.057 2.248 ± 0.134 3.368 ± 0.138 MCC950 + H 2 O 2 1.143 ± 0.019 1.081 ± 0.004 1.289 ± 0.048 1.455 ± 0.047 P -Value < 0.0001 0.0004 < 0.0001 < 0.0001 NLRP3 inflammasome can be activated through caspase-1-mediated classical pathway and caspase-4, -5, and − 11-mediated non-classical pathway. Therefore, caspase-1 and CD34 were detected simultaneously during IF experiments in addition to co-localization of NLRP3 and CD34. As shown in Fig. 7H, 7K, the relative expression of caspase-1 was 1.000 + 0.058 in the control group, increased to 25.35 ± 1.209, and then decreased to 18.42 + 1.548 ( P < 0.01). The relative NLRP3 expression in the control group was 0.903 + 0.114, which was significantly upregulated to 2.914 ± 0.077 in the H 2 O 2 group, while decreased to 2.241 + 0.105 after MCC950 treatment ( P < 0.01) (Fig. 7M,7L). Consistent with the Western blot and qRT-PCR results, again indicating that MCC950 inhibits NLRP3 inflammasome activity in H 2 O 2 -stimulated HUVECs. 3.8 MCC950 inhibits NLRP3 inflammasome activity by increasing sensitivity to ROS In this study, flow cytometry was selected to detect intracellular ROS levels. As shown in Fig. 8A-8F, the DCF fluorescence intensity was 0.233 ± 0.012 in the control group and increased to 0.52 ± 0.055 in the H 2 O 2 group ( P < 0.01). It decreased to 0.2 ± 0.040 after pretreatment with MCC950, which was significantly lower than that in the H 2 O 2 group ( P < 0.01). At high mitochondrial membrane potential, JC-1 aggregates in the mitochondrial matrix to produce red fluorescence; conversely, it can produce green fluorescence. As shown in Fig. 8G, the green fluorescence intensity was increased in the H 2 O 2 group compared with the control group, and the green fluorescence intensity was significantly decreased and the red fluorescence intensity was increased after MCC950 pretreatment. The results suggested that H 2 O 2 causes damage to the mitochondrial membrane potential, while MCC950 can restore the membrane potential. TUNEL staining can detect nuclear DNA breakage during focal cell death. Therefore, cell damage could be further detected by TUNEL. As shown in Fig. 8H, DNA damage was significantly increased in the H 2 O 2 group compared with the control group, whereas it was reduced after pre-incubation with MCC950. Taken together, the ROS, JC-1 and TUNEL results indicated MCC950 increased intracellular antioxidant capacity and enhanced the sensitivity of H 2 O 2 -induced HUVECs to ROS. 4 Discussion Venous thrombus lysis, mechanization, and recanalization is a complex and dynamic evolutionary process involving multiple cells and cytokines(Nosaka and Ishida et al., 2017 ). Since previous studies have demonstrated the feasibility of a venous stasis thrombosis model(Henke and Pearce et al., 2006 ), we used the "stenosis method" to construct a model of IVC in pregnant rats. The development of DVT was dynamically observed by HE, IHC and Masson's trichrome staining. These results showed mechanization and recanalization, neocapillary formation and fibrosis processes play an important role in the process of DVT in pregnancy rats(Ishida and Kimura et al., 2015 ; Nosaka and Ishida et al., 2017 ), which provides the basis for later evaluation of the efficacy of MCC950 treatment from a pathological point of view. This study elucidated the involvement of NLRP3 inflammasome activation in DVT of pregnancy rats. Inflammation and hemostasis are two highly interrelated processes, with inflammation inducing coagulation and coagulation amplifying inflammation. The two coordinates with each other in a positive feedback loop, and when dysregulated, they lead to disease. Thrombosis is a confirmation of the interdependent interaction of this pathological process(Gutmann and Siow et al., 2020 ). Qiao et al.(Qiao and Wu et al., 2018 )identified a role for NLRP3 and IL-1β in platelet function and provided a novel potential link between thrombosis and inflammation, suggesting that therapies targeting NLRP3 or interleukin-1β may be beneficial in the treatment of inflammation-associated thrombosis. Moraes et al.(Moraes and Hottz et al., 2023 ) confirmed the dependence of IL-1β secretion on intracellular ROS after platelet NLRP3 activation and suggested an IL-1β-dependent relationship between inflammasome IL-1β activation and release and increased vascular permeability. In conclusion, the NLRP3 inflammasome plays an important role in thrombosis by activating the coagulation system. The present study suggested the development of DVT may begin in ECs. In IF experiments, we found a very interesting phenomenon. Over time, the location of NLRP3 expression changed significantly, beginning with a gradual shift from the endothelial tissue at the periphery of the thrombus to the tissue in the middle of the thrombus. Previous studies have found that the NLRP3 inflammasome can be activated in blood cells through one or more receptors under conditions of tissue injury or cell necrosis that produce endogenous DAMPs(Jiang and Jiang et al., 2018 ). So far, studies on NLRP3 inflammasomes and DVT have mainly focused on platelets. Therefore, HUVECs were selected as experimental subjects to further investigate how this process actually occurs in this study. The endothelium is a single layer of epithelium covering the surface of blood vessels and is highly active. It plays an important role in maintaining vasodilation, coagulation and anticoagulation systems, immune regulation, vascular smooth muscle proliferation and migration(Takeuchi and Akira, 2010 ). Under physiological conditions, ROS are essential for physiological cellular functions such as host defense, post-translational processing of proteins, cell signaling, regulation of gene expression and cell differentiation(Bedard and Krause, 2007 ). However, certain risk factors, such as hypoxia, can lead to excessive ROS production, resulting in endothelial dysfunction and cell death(Forstermann and Xia et al., 2017 ). Endothelial dysfunction or endothelial cell death is closely associated with the development of many diseases in obstetrics and gynecology. Maynard et al.(Maynard and Min et al., 2003 ) found that excess placental soluble fms-like tyrosine kinase 1 (sFlt1) can lead to endothelial dysfunction, hypertension, and proteinuria in pre-eclampsia. In ECs, ROS act as a bridge between pathological stimuli and NLRP3 inflammasome activation. ROS has been shown to activate the NLRP3 inflammasome and plays an important role in various diseases(Li and Zhou et al., 2019 ; Zhao and Wang et al., 2019 ). Gupta(Gupta and Sahu et al., 2017 )et al. found that hypoxia induces the production of hypoxia-inducible factor (HIF)-1a, and HIF-1a has been shown to be formed by ROS activation of NLRP3. Therefore, ROS are considered to be a common upstream cause of blood disorders and pathologies (e.g., clot formation). Other studies have shown that the antioxidant capacity within the vascular endothelium of DVT patients is reduced and that oxidative stress may play an important role in the pathophysiology of DVT(Takeuchi and Akira, 2010 ; Yang and Tu et al., 2012 ). In conclusion, it can be hypothesized that ROS increase after endothelial cell hypoxia, leading to NLRP3 inflammasome activation(Roberto and Micucci et al., 2010 )and activation of the coagulation system and consequently thrombosis after endothelial dysfunction. MCC950 was shown to promote lysis, mechanization and recanalization of DVT in SD pregnant rats. Li et al. (Li and Qin et al., 2022 )used hirudin to cause middle cerebral artery occlusion in mice. MCC950 inhibited ischemia-induced overexpression of NLRP3 and its downstream caspase-1, ASC and IL-1β, and protected BV-2 microglia from viability and death. However, Lemarchand et al.(Lemarchand and Barrington et al., 2019 ) used a mouse model of middle cerebral artery thrombosis with increased expression of pro-inflammatory cytokines and NLRP3 after stroke, and targeting NLRP3 with the inhibitor MCC950 or using NLRP3 knockout mice had no effect on the extent of stroke-induced injury. This differs from the results of the present study and may be due to different defense mechanisms in the brain barrier. Altogether, cellular experiments demonstrated increased oxidative stress in endothelial cells promotes the activation and assembly of the NLRP3 inflammasome, which contributes to the pathogenesis and pathophysiology of DVT. The present study, the first therapeutic study of MCC950 in a pregnant animal model, is innovative but has some limitations. First, treatment with only 20 mg/kg of MCC950 did not allow evaluation of whether different doses of MCC950 have different effects on thrombosis in rats. Second, the effect of NLRP3 inflammasome inactivation on the coagulation system was not confirmed by further experiments due to time constraints. Third, the effect of MCC950 on placental function was not further investigated and its efficacy, especially safety, remains to be verified. The present study fully utilized multiple molecular biology methods, from whole-tissue-cell-molecule, confirmed the research hypothesis at multiple levels and directions, and came to the following conclusions: the massive accumulation of ROS in ECs after ischemia and hypoxia activates the NLRP3 inflammasome, which in turn releases inflammatory factors through pyroptosis and may be a mechanism contributing to DVT. MCC950 may inhibit vascular endothelial cell pyroptosis through the ROS/NLRP3 signaling axis to ameliorate pregnancy-related venous thromboembolism. This mechanism provides new insights into the pathophysiology of DVT in pregnant rats and may open new avenues for the development of techniques to diagnose, evaluate, and treat maternal DVT. In addition, there are some prospects for future studies. During the experiment, we also observed the mental status, vaginal bleeding, and pregnancy loss rate of pregnant rats in the MCC950 treatment group were significantly better than those in the control group. In addition, concurrent thrombosis in pregnant women can lead to placental ischemia and hypoxia, resulting in intrauterine growth restriction and delayed fetal development; can MCC950 improve these adverse outcomes? Finally, the possibility of further clinical translational studies of MCC950 treatment for DVT or obstetric and gynecologic disorders during pregnancy will be the focus of our future studies. Declarations Ethical Approval Ethics of the study was approved by the Laboratory Animal Ce nter of Nantong University (S20221222-008). Competing interests All authors declare that there are no conflicts of interest. Authors’ contributions YHX, QHW, YL and YQZ contributed to the conception and design of the project. QS, LJX, SYC, YZH and MRH performed all the figures. 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Basic Clin Pharmacol Toxicol 106 (1): 38–44. Sano, M. and S. Shimazaki, et al. (2020). "Palmitic acid activates NLRP3 inflammasome and induces placental inflammation during pregnancy in mice." J Reprod Dev 66 (3): 241–248. Schindelin, J. and I. Arganda-Carreras, et al. (2012). "Fiji: an open-source platform for biological-image analysis." Nat Methods 9 (7): 676–82. Shi, J. and W. Gao, et al. (2017). "Pyroptosis: Gasdermin-Mediated Programmed Necrotic Cell Death." Trends Biochem Sci 42 (4): 245–254. Soma-Pillay, P. and C. Nelson-Piercy, et al. (2016). "Physiological changes in pregnancy." Cardiovasc J Afr 27 (2): 89–94. Takeuchi, O. and S. Akira (2010). "Pattern recognition receptors and inflammation." Cell 140 (6): 805–20. Ugusman, A. and Z. Zakaria, et al. (2011). "Piper sarmentosum inhibits ICAM-1 and Nox4 gene expression in oxidative stress-induced human umbilical vein endothelial cells." BMC Complement Altern Med 11: 31. Xiao, H. and M. Lu, et al. (2013). "Sterol regulatory element binding protein 2 activation of NLRP3 inflammasome in endothelium mediates hemodynamic-induced atherosclerosis susceptibility." Circulation 128 (6): 632–42. Yang, Y. and H. Wang, et al. (2019). "Recent advances in the mechanisms of NLRP3 inflammasome activation and its inhibitors." Cell Death Dis 10 (2): 128. Yang, Z. and Q. Tu, et al. (2012). "The role of heparin binding surfaces in the direction of endothelial and smooth muscle cell fate and re-endothelialization." Biomaterials 33 (28): 6615–25. Zhao, Y. and Z. Wang, et al. (2019). "p66Shc Contributes to Liver Fibrosis through the Regulation of Mitochondrial Reactive Oxygen Species." Theranostics 9 (5): 1510–1522. Additional Declarations No competing interests reported. Supplementary Files S20221222008.pdf 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-2935166","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":202909376,"identity":"8cd91ba6-4579-4c05-ac28-77f70ebcf2cc","order_by":0,"name":"Yanhua Xu","email":"","orcid":"","institution":"Nantong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yanhua","middleName":"","lastName":"Xu","suffix":""},{"id":202909377,"identity":"3eb59387-d565-4b58-92af-c4e884bf8072","order_by":1,"name":"Qinghua Wang","email":"","orcid":"","institution":"Nantong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qinghua","middleName":"","lastName":"Wang","suffix":""},{"id":202909378,"identity":"0e4cb5e1-8c7d-427c-a0e8-dd4f41fa5a7c","order_by":2,"name":"Yue Lu","email":"","orcid":"","institution":"Nantong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Lu","suffix":""},{"id":202909379,"identity":"a558e039-9686-4b12-889c-d089edfff8a1","order_by":3,"name":"Qin Shi","email":"","orcid":"","institution":"Center For Reproductive Medicine, Afliated Hospital of Nantong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qin","middleName":"","lastName":"Shi","suffix":""},{"id":202909380,"identity":"37805295-7a7c-4206-8156-8b1f75e52747","order_by":4,"name":"Linjie Xu","email":"","orcid":"","institution":"Nantong 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University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yuquan","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2023-05-15 03:44:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2935166/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2935166/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":37454579,"identity":"25fa1289-8b65-4c64-927d-ad0d4c0b3c99","added_by":"auto","created_at":"2023-05-24 18:50:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1025632,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"518Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2935166/v1/06496c6eab96f0585a3bf893.jpg"},{"id":37455522,"identity":"13696692-ebb9-4e8a-b48b-b3fb014f4bcb","added_by":"auto","created_at":"2023-05-24 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6","display":"","copyAsset":false,"role":"figure","size":925393,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"518Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2935166/v1/ea16202f23067a3a038ae853.jpg"},{"id":37455707,"identity":"c421300d-d3a0-4b17-a378-8b93b9dd535d","added_by":"auto","created_at":"2023-05-24 19:22:47","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1176972,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"518Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2935166/v1/ab6648546bd3fe0a985eac3e.jpg"},{"id":37454580,"identity":"5eb37fb3-9b7b-422d-9947-07d0f2a9bd00","added_by":"auto","created_at":"2023-05-24 18:50:47","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":716829,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"518Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2935166/v1/74f6a0be71d8a3eeeb3e96d6.jpg"},{"id":38201481,"identity":"8f1f4fda-a07d-4191-b1a8-1c1b1b40e56d","added_by":"auto","created_at":"2023-06-08 05:29:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2161353,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2935166/v1/8e0f39dd-db95-4848-9045-86a0cccdf36b.pdf"},{"id":37454582,"identity":"75ef5c08-1720-4514-82d5-8fcf4010a61b","added_by":"auto","created_at":"2023-05-24 18:50:47","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":784467,"visible":true,"origin":"","legend":"","description":"","filename":"S20221222008.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2935166/v1/4a00d4c9695872d51f456eaa.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Blockage of NLRP3 inflammasome by MCC950 can reverse the effect of pyroptosis in HUVECs and thrombosis of pregnancy rats","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eVenous thromboembolism (VTE) is a common disease including deep vein thrombosis (DVT) and pulmonary thromboembolism (PE)(Essien and Rali et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The risk of VTE increases significantly during pregnancy(Soma-Pillay and Nelson-Piercy et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and it is approximately five times higher in pregnant women and 30 to 60 times higher in the puerperium(Park and Park et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Simultaneously, PE caused by DVT is a leading cause of maternal death(Konstantinides and Meyer et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In addition, VTE is associated with adverse pregnancy outcomes. Symptoms associated with VTE, such as obstructed blood flow(Ibeh and Okocha et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), placental microthrombosis(Meng and Hu et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and placental ischemia and hypoxia(Barut and Barut et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), can also lead to recurrent miscarriage, fetal growth restriction, pre-eclampsia, and placental abruption. Therefore, VTE during pregnancy and puerperium not only threatens the life of the mother, but may also affect fetal growth(Peeters, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOnce diagnosed, DVT in pregnancy and puerperium should be treated as soon as possible with a comprehensive approach based on anti-coagulation. This mainly includes physiotherapy, anti-coagulation, thrombolytic therapy and inferior vena cava filters (IVCF)(Ernst and Oporto et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Low molecular weight heparin (LMWH) does not cross the placental barrier(Duffett and Rodger, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), has a relatively low incidence of bleeding, and is a clinically recognized antithrombotic agent(ACOG, 2018) However, all these treatments do not completely alleviate thrombotic disease due to local endothelial damage caused by inflammation and oxidative stress. Therefore, further studies on the pathogenesis and pathophysiology are needed to identify new therapeutic targets.\u003c/p\u003e \u003cp\u003eIncreasing evidence suggests that the pathogenesis of DVT is closely related to elevated levels of procoagulant factors/proteins, endothelial cell injury and apoptosis (Marik and Plante, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), platelet activation and aggregation(Budnik and Brill, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), overproduction of reactive oxygen species (ROS)(Herkert and Diebold et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), and inflammatory cell infiltration. Among these, the inflammatory response plays a key role. Inflammation activates the coagulation system and also inactivates the anticoagulation system, thereby inducing DVT, a process also known as immunothrombosis(Rider and Kaplanov et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe intrinsic immune response is the body's first line of defense against foreign bodies and invasion by exogenous pathogens. The target signals of the intrinsic immune response can be classified into pathogen-associated molecular patterns (PAMPS) and damage-associated molecular patterns (DAMPs) for two different sources(Gong and Liu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). NOD-like receptor thermal protein domain associated protein 3 (NLRP3) is the most studied and important pattern recognition receptor in the cytosol(Prochnicki and Latz, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It is composed of apoptosis-associated speck-like protein containing a CARD (ASC), cysteine aspartate-specific proteinase-1 (caspase-1), and a CARD-associated speck-like protein (CARD), which assembles into inflammasome complexes to perform its functions(Broz and Dixit, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Activated inflammasomes promote the maturation of interleukin-1β (IL-1β) and interleukin-18 (IL-18), induce the synthesis and release of other inflammatory cytokines, and amplify the inflammatory response(Godo and Shimokawa, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It also cleaves perforin gasdermin D (GSDMD) and the cleaved product (N-terminal fragment) disrupts the cell membrane by forming open pores in the cell membrane and causing pyroptosis(Shi and Gao et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Studies have shown that NLRP3 inflammasome activation is associated with the vascular inflammatory response(Xiao and Lu et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), which drives the development and progression of DVT. However, the role of NLRP3 inflammasome-mediated pyroptosis in the development of DVT in pregnant rats has been less studied both domestically and internationally. Moreover, the upstream regulatory mechanisms of NLRP3 inflammasome-mediated pyroptosis are complex. ROS production is one of the first identified triggers of NLRP3 inflammasome activation and a natural byproduct of aerobic metabolism produced in response to cellular stimulation by cytokines, invasion by foreign organisms and bacteria. There is growing evidence that ROS plays a key role in DVT after causing NLRP3 inflammasome activation(Gutmann and Siow et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMCC950, a small molecule inhibitor that specifically inhibits NLRP3 activity, is a potential therapeutic approach for autoinflammatory and autoimmune diseases. For example, in the presence of excess palmitic acid (PA) in obese pregnant women, Sano et al.(Sano and Shimazaki et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)found higher levels of PA exposure activated the NLRP3 inflammasome and induced placental inflammation leading to pregnancy complications after intravenous administration of palmitic acid solution to pregnant mice on day 12 of gestation, whereas MCC950 inhibited NLRP3 inflammasome activation and improved pregnancy outcome. The specific inhibition of MCC950 opens up the possibility of treating diseases caused by the NLRP3 inflammasome(Yang and Wang et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven the key role of inflammation and oxidative stress in thrombosis, and assuming that MCC950 is safe for the fetus, we propose the following research hypotheses: 1, the massive accumulation of ROS in ECs after ischemia and hypoxia activates the NLRP3 inflammasome, which in turn releases a large amount of inflammatory factors through pyroptosis to contribute to venous thrombosis; 2, MCC950 can inhibit pyroptosis through the ROS/NLRP3 pathway to achieve amelioration of pregnancy-related venous thromboembolism.\u003c/p\u003e \u003cp\u003eTo test the above hypothesis, we established a DVT model in pregnant rats and an H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HUVECs (human umbilic vein endothelial cells, HUVECs) model. Through multiple biological approaches, we confirmed the hypothesis. In general, the study provided a new theoretical basis and potential therapeutic reference for the prevention and treatment of PA-VTE.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animal Ethics and Design of Experiments\u003c/h2\u003e \u003cp\u003eSixty 3-day pregnant Sprague-Dawley rats (6\u0026ndash;8 weeks, 200-250g) were purchased from Nantong University Laboratory Animal Center (Jiangsu, China). The rats were housed in an SPF environment at 23\u0026ndash;25\u0026deg;C, 12h/12h dark/light cycle, 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5% humidity, free access to food and water, and acclimatized for one week. Following the study of Cheng et al.(Cheng and Zhang et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), the tenth day of gestation (G10) was chosen for the subsequent experiments. General anesthesia was induced with 3% isoflurane, followed by maintenance anesthesia with 2.5% isoflurane until the end of the procedure (300\u0026ndash;400 \u0026micro;L/100g body weight). Animals were euthanized at the end of the experiment. The care and use of all animal, experimental protocols, and procedures were performed in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments, ARRIVE) guidelines and the Guide for the Care and Use of Laboratory Animals proposed by the Ministry of Health of the People's Republic of China. The project was approved by the Animal Ethics Committee of Nantong University (S20221222-008).\u003c/p\u003e \u003cp\u003eFirstly, G10 rats were randomly divided into two groups: an induced DVT model group (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;24) and a sham-operated control group (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;24). The model group was further divided into 4 subgroups of 6 hours, 1 day (1d, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6), 3 days (3d, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6), and 7 days (7d, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6). In the second part, pregnant rats were randomly divided into PBS (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6) and MCC950 (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6) group, and administered (20 mg/kg/d) three times by intraperitoneal injection(Bellut and Papp et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), 1 hour before isoflurane anesthesia, 1 day and 2 days after modeling, respectively. An equal dose of PBS was injected intraperitoneally as a negative control. The mortality rate of the experimental animals was 8.9% (3/60). The cause of death was hemorrhage from accidental puncture of the IVC (inferior vena cava, IVC) in two cases and incisional dehiscence after molding in one case.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Establishment of experimental model of DVT in pregnant rats\u003c/h2\u003e \u003cp\u003eThe DVT model was created using the IVC \"stenosis method\" on day G10. Briefly, after successful anesthesia, the abdomen was opened along the midline and the IVC was gently separated from the aorta, 4\u0026thinsp;\u0026minus;\u0026thinsp;0 absorbable sutures are placed parallel to the IVC, and then the IVC and all visible side branches are ligated with non-reactive 5\u0026thinsp;\u0026minus;\u0026thinsp;0 absorbable sutures. The 4\u0026thinsp;\u0026minus;\u0026thinsp;0 sutures were then removed after confirming their tightness, resulting in 90% stenosis. In the control group, only sutures were passed and no vessels were ligated.\u003c/p\u003e \u003cp\u003eRats were anesthetized and euthanized at 6 h, 1 d, 3 d, and 7 d after DVT and 24 h after the third dose. Then the thrombotic tissues were excised, measured for length, and weighed. Blood samples were centrifuged at 3000\u0026times;g for 10 min, and 200 \u0026micro;L of serum was collected. The serum and three thrombus tissues were fixed at -80\u0026deg;C, and the remaining three tissues were fixed at 4\u0026deg;C in 4% para-formaldehyde for 12 hours for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Histopathologic analysis and measurement of thrombotic tissue\u003c/h2\u003e \u003cp\u003eTissues were removed from para-formaldehyde and washed thoroughly in PBS. After dehydrated through graded alcohol, the tissues were cleared through xylene, embedded in paraffin. Each group was sectioned separately with a paraffin microtome (thickness, 4\u0026micro;m). To observe the characteristics of thrombus formation, recanalization, and pathological changes after formation, at least three tissues from each group were randomly selected for the experiment. HE staining was performed with the kit (C0105M, Beyotime Biotechnology, China) according to the manufacturer's instructions.\u003c/p\u003e \u003cp\u003eProcessing of thrombus tissue sections was performed as described previously. Immunohistochemistry (IHC) staining was performed using a universal kit (PV6000, ZSGB-BIO, China). Briefly, sections were placed in Tris-EDTA antigen repair solution (1\u0026times;) and heated in boiling water for 15 minutes to repair antigenic epitopes. To block endogenous peroxidase activity, endogenous peroxidase was added dropwise to each tissue and then incubated with 5% BSA for 30 minutes. After incubation with CD34 primary antibody (ICO-115, 1:200, Novus Biologicals, USA), sections were placed in a refrigerator at 4\u0026deg;C overnight. The next day, sections were incubated dropwise with reaction enhancer, washed with PBS, and incubated with secondary antibody for 30 minutes at 37\u0026deg;C. Color changes were observed microscopically during DAB incubation and finally incubated with hematoxylin for 15 minutes, fractionated with hydrochloric acid fractionation solution, dehydrated, transparent and sealed.\u003c/p\u003e \u003cp\u003eMasson's trichrome stain is the definitive and classic technique, which stains collagen fibrils, extracellular matrix and vessel wall blue and cell nucleus black. It was performed with the kit (C0105, Beyotime Biotechnology, China) according to the manufacturer's instructions. Thrombus mechanization rate was calculated based on the percentage of thrombus area occupied by collagen fibrils and extracellular matrix-stained areas.\u003c/p\u003e \u003cp\u003eAt least three stained sections of each thrombus were observed sequentially under a microscope (Olympus, Tokyo, Japan). The cumulative optical density (IOD) values of positive expression sites for collagen fibrils (blue in Masson's stain) and CD34 (brown in thrombus tissue) were then calculated separately for each group of sections using FIJI Image 8.0 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Western blot\u003c/h2\u003e \u003cp\u003eTotal protein was extracted from thrombus tissue or cells using a protein extraction reagent. Protein concentrations were determined using the BCA protein assay kit (P0010, Beyotime Biotechnology, China) according to the manufacturer's instructions. Solubilized proteins (30\u0026ndash;50\u0026micro;g per lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (10%, 15% separation gels and 5% stacking gels) and transferred to PVDF membranes (IPVH00010, Merck Millipore, USA), blocked in tris-buffered saline (TBS) for one hour, and incubated with specific primary antibodies at 4\u0026deg;C overnight. The following primary antibodies were then used: rabbit anti-NLRP3 (T55651, 1:1000, Abmart, China), mouse anti-caspase-1 (22915-1-AP, 1:1000, Proteintech, USA), mouse anti-GSDMD (P57764, 1:1000, Abmart, China ), rabbit anti-IL-1β (P10749, 1:2000, Abmart, China), or mouse anti-GAPDH (60004-1-lg, 1:2000, Proteintech, USA). After washing with TBST, protein bands were visualized by incubation with HRP-conjugated affinipure goat anti-rabbit or anti-mouse IgG(H\u0026thinsp;+\u0026thinsp;L) (SA00001-1; SA00001-2, 1:5000, Proteintech, USA) for 2 h. With the BeyoECL Plus Kit (P0018S, Beyotime Biotechnology, China). protein bands were visualized with ECL luminescence solution (Thermo Fisher Scientific, Inc. USA) and then analyzed with FIJI ImageJ (v1.8.0; National Institutes of Health) and normalized to GAPDH levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Quantitative real-time polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTo examine NLRP3 inflammasome mRNA expression, total RNA (ribonucleic acid, RNA) was extracted from frozen tissues or HUVECs using Trizol reagent (abs60154, absin, China) according to the manufacturer's instructions. Then, 2 \u0026micro;g of total RNA from each sample was reverse transcribed into complementary deoxyribonucleic acid (cDNA) using HiScript II Q RT SuperMix for qPCR (R223, Vazyme, China), with ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme, Nanjing, China) on the LightCycler\u0026reg; 96 system (Thermo Fisher, USA). The PCR reactions were performed as follows. First, 95℃ for 30s, pre-denaturation once;then 95℃ for10s and 60℃ for 30s, cycle forty times༛last, 95℃ for 5s, 60℃for 60s and 95℃for15s dissolution once. Quantitative RT-PCR was performed on all samples in triplicate. The mRNA expression levels of the target genes were normalized to GAPDH, and the results were expressed as 2\u003csup\u003e\u0026minus;ΔΔct\u003c/sup\u003e. The main primer sequences are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSequences of primary genes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLeading and trailing chains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSequences\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNLRP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5' -GAG CIG GAC CTC AGT GAC AAT GC-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5' -AGA ACC AAT GCG AGA TCC TGA CAA C-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCaspase-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5' -GAA GGT GGC GCA TT CCT GG-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5' -AGG GCA AGA CGT GTA CGA GT-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGSDMD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5' -CAG CAG GCA GCA TCC TTG AGT G-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5' -CCT CCA GAG CCT TAG TAG CCA GTA G-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIL-1β\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5' -AAT CTC ACA GCA GCA TCT CGA CAA G-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5' -TCC ACG GGC AAG ACA TAG GTA GC-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNLRP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5' -AGG GAT GAG AGT GTT GTG TGA AAC G-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5' -GCT TCT GGT TGC TGC TGA GGA C-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCaspase-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5' -GAA GAA ACA CTC TGA GCA AGT C-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5' -GAT GAT GAT CAC CLT CGG TTT G-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGSDMD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5' -TTG AAG AAR TGA GTG LGG ACA GAG C-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5' -TGG TGG TGT GTG CGT TGG AAT G-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIL-1β\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5' -GCC AGT GAA ALG ATG GCT TAT T-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5' -AGG AGC ACT TCA TCT GTT TAG G-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Enzyme-linked Immunosorbent Assay (ELISA) for IL-1β and IL-18 in Serum\u003c/h2\u003e \u003cp\u003e Serum concentrations of IL-1β and IL-18 were determined using the appropriate ELISA kits (MM-0194R1, MM-0047R1, Jiangsu Meimian Industrial, China) according to the supplier's protocol. Absorbance at 450 nm was analyzed using an ELISA instrument (Thermo Fisher Scientific, Inc. USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Immunofluorescence Analysis of NLRP3 and Caspase-1 in Tissues and Cells\u003c/h2\u003e \u003cp\u003eThe co-localization and differential expression of NLRP3, caspase-1 and CD34 in tissues and cells was evaluated by immunofluorescence (IF). IF of tissues was performed with some special treatments as follows. The thrombus tissue was dehydrated in different glucose gradients on alternate days, and finally embedded in optimal cutting temperature compound (4583, Sakura, USA) and stored at -80\u0026deg;C. Then, continuous coronal sections of 4 \u0026micro;M thickness were cut on a frozen section machine (Leica, Germany) and placed on 0.01% poly-L-lysine-coated coverslips. The frozen sections or cells were fixed with 4% paraformaldehyde for 25 min, followed by permeabilization with 0.1% Triton X-100 for 1 hour, blocked with 10% normal goat serum (SL038, Solarbio, China), frozen sections or cells were incubated with NLRP3 antibody (T55651, 1:100, Abmart, China), caspase-1(22915-1-AP, 1:100, Proteintech, USA) or CD34 (ICO-115, 1:200, Novus Biologicals, USA) were incubated overnight at 4\u0026deg;C. After washing, cells and tissues were incubated with goat anti-rabbit (Alexa Fluor\u0026reg; 568, 1:500, Thermo Fisher Scientific, for anti-NLRP3 antibody) and goat anti-mouse (Alexa Fluor\u0026reg; 488, 1:500, Thermo Fisher Scientific, for anti-caspase-1 or anti-CD34 antibody) secondary antibodies for 1 hour at 37\u0026deg;C, followed by staining with Hoechst33342 (4083, 1:1000, Cell Signaling Technology, USA) for five minutes at room temperature. Coverslips were mounted with antifade mounting medium (HY-K1042, MedChemExpress, USA). Visualization was then performed under a scanning fluorescence microscope (Leica, Germany). Finally, the average fluorescence intensity of frozen sections or cells was analyzed using Fiji Image.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Design of Cell Experiments\u003c/h2\u003e \u003cp\u003eHUVEC lines were generously donated by Dr. Xi Cheng (Nantong University, China) and cultured in DMEM/F-12 medium (A4192001, Gibco, USA;) supplemented with 10% fetal bovine serum (1925624, Biological Industries, Israel) and antibiotics (100 units/mL penicillin, 100 mg/mL streptomycin) at 37\u0026deg;C in a 5%CO\u003csub\u003e2\u003c/sub\u003e, 95% air incubator. HUVECs were plated in 6-, 24-, and 96-well plates for various experiments. In the co-culture system, HUVECs were cultured in the above mentioned chambers. Meanwhile, all cultures were grown in DMEM/F12 medium. Based on the results of CCK-8 as well as LDH experiments, the optimal concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was determined to be 400 \u0026micro;M with an action time of 24 h (Magenta and Cencioni et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Ugusman and Zakaria et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Han and Tang et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In addition, the optimal concentration of MCC950 (HY-12815, MedChemExpress, USA) was determined to be 10 \u0026micro;M with an action time of 2 h according to the reference(Franke and Bieber et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Cells were divided into the following groups: control group (cells were cultured in normal environment without any stimulation); H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group (cells were pretreated with 400 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 24 hours); H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;MCC950 group (cells were pretreated with 10 \u0026micro;M MCC950 for 2 hours and then co-cultured with 400 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 24 hours. The experiments were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003e2.9 In vitro cytotoxicity of MC950 and different concentrations of\u003c/span\u003e H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.9.1 CCK-8 assay\u003c/h2\u003e \u003cp\u003eThe cytotoxicity of different concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (H13022648, HebeiHengjian, China) in HUVECs was evaluated by CCK-8 assay and LDH assays. The Cell Counting Kit-8 (CK04, Dojindo, Japan) was performed to assess the viability of HUVECs according to the manufacturer's protocol. Specifically, HUVECs were seeded in 96-well plates (1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well) in 100 \u0026micro;L DMEM/F-12 medium containing FBS, penicillin, and streptomycin as previously mentioned. After overnight incubation, the medium was removed. Next, HUVECs were treated with 10 \u0026micro;M MCC950 (MCC950 group) or no treatment (control group) for 2 hours and then treated with different concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (0, 100, 200, 400, 600 \u0026micro;M) for 24 hours. The medium was then removed and replaced with 100 \u0026micro;L fresh DMEM/F-12 medium, and 10 \u0026micro;L CCK-8 solution was added to each well. After 2 hours of incubation, absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, USA). Cell viability (%) was expressed as a percentage of the control condition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.9.2 LDH assay\u003c/h2\u003e \u003cp\u003eHUVECs were treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, DMEM/F-12, or MCC950 as described above. Cytotoxicity was also determined by measuring LDH (lactate dehydrogenase, LDH) released from the cells using a LDH assay kit (C0016, Beyotime Biotechnology, China) according to the manufacturer's protocol. Absorbance and method of calculating are the same as those used in the CCK-8 experiment.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Invasion, Migration and Tube formation assay\u003c/h2\u003e \u003cp\u003eInvasion, migration and tube formation assays were used to observe the impairment of cell invasion, migration and angiogenesis ability after treatment of HUVECs with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e or MCC950. For these three assays, the upper surface of transwell cell culture inserts (353097-1, 8 \u0026micro;m pore size, BD Biosciences, USA) in 24-well plates were coated with 20 \u0026micro;L/well Matrigel (8246003, 1.25mg/ml, Corning, USA) for 2 hours. After treatment with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e or MCC950 as described above, cells were collected in the same manner, and then cells were resuspended separately with serum-free DMEM/F-12 medium. In detail, a total of 3\u0026times;10\u003csup\u003e4\u003c/sup\u003e HUVECs were seeded in the upper chamber of the transwell. Then 500 \u0026micro;l of complete medium was added to the lower part of the transwell. For invasion and migration assays, cells were incubated in the incubator for 18\u0026ndash;24 hours and then fixed with 4% paraformaldehyde, stained with crystal violet, and observed under a microscope (Olympus, Japan). The average number of invading or migrating cells was calculated from five randomly selected areas. For tube formation assay, after 4\u0026ndash;6 hours of incubation, images of the blood vessels formed in each transwell were observed under an inverted microscope and photographed. In three independent experiments, vessel branching was measured using FIJI ImageJ software to determine total vessel length and branching images.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Scratch Healing Assay\u003c/h2\u003e \u003cp\u003eHUVECs were seeded in 6-well plates (5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well, 2 mL DMEM/F-12). After washing with PBS, DMEM/F-12 containing 5% FBS with or without MCC950 (10 \u0026micro;M) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (400 \u0026micro;M) was added. The cells were placed in an incubator for incubation. The wells were then photographed immediately after 0, 12, and 24 hours, and the distance and area of each scratch was measured via FIJI image.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Flow Cytometric Assessment of ROS in HUVECs\u003c/h2\u003e \u003cp\u003eDCHF-DA is a reliable fluorescent marker of ROS that can be oxidized by intracellular ROS to 2', 7'-dichlorofluorescein (DCF). The level of intracellular ROS can be known by detecting the fluorescence of DCF. Therefore, followed the manufacturer's instructions, we used a reactive oxygen species detection kit (S0033S, Beyotime Biotechnology, China) to detect intracellular ROS production in HUVECs. Briefly, different groups of HUVECs were incubated with 10 \u0026micro;M DCFH-DA at 37\u0026deg;C for 20 minutes in the dark. After washing to remove extracellular residues of DCFH-DA, the mean fluorescence intensity of ROS was analyzed and averaged using a flow cytometer (Becton-Dickinson, San Jose, USA) with FlowJo 7.6 software (Tree Star, Inc.)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Mitochondrial membrane potential assay\u003c/h2\u003e \u003cp\u003eJC-1 is an ideal fluorescent probe and is widely used to detect mitochondrial membrane potential as an indicator of early apoptosis detection. After treatment, cells were washed twice with PBS, 1 ml of JC-1 staining solution (C2006, Beyotime Biotechnology, China) was added, mixed thoroughly, and incubated for 20 min at 37\u0026ordm;C. After incubation, the supernatant was aspirated, and the cells were washed 4 times with JC-1 staining buffer in a shaking side swing bed to reduce the background, 3 \u0026micro;L Hoechst 33342 for 5 minutes at room temperature in the dark, and immediately observed under a fluorescence microscope and photographed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Hoechst33342/TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) fluorescence staining\u003c/h2\u003e \u003cp\u003eTo detect DNA fragments of HUVECs, cells were double stained with Hoechst33342 and TUNEL. Briefly, HUVECs (3\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well) were cultured in 24-well plates. After the indicated treatments, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 30 minutes at room temperature. Then cells from each group were stained with 3 \u0026micro;l TUNEL (A113, 100 \u0026micro;g/ml, Vazyme, China) for 1 hour at 37\u0026deg;C in the dark and with 3 \u0026micro;l Hoechst33342 for 5 minutes at room temperature in the dark. After that, photographed under an inverted fluorescence microscope (Nikon, TE-2000 U) at \u0026times;40 magnification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Statistical Analysis\u003c/h2\u003e \u003cp\u003eVector images were statistically analyzed using FIJI ImageJ(Schindelin and Arganda-Carreras et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) followed by GraphPad Prism software (version8.0, GraphPad Software Inc., CA, USA). All data followed a normal distribution, and all continuous values were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SEM). Student's t-test was used to compare the two experimental groups, and differences between groups were analyzed by one-way ANOVA. All relevant experiments were repeated three times with similar results, showing a representative series of images, and values less than 0.05 were considered statistically significant. (*\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003e3.1 Lysis, mechanization, and recanalization of DVT in pregnant rats are accompanied by changes in inflammatory factors, neovascularization, and fibrotic area.\u003c/span\u003e \u003c/p\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.1.1 Pathological results\u003c/h2\u003e \u003cp\u003eTo observe the process of thrombus formation and evolution, we performed HE, CD34 immunohistochemical and Masson\u0026rsquo;s staining at different time intervals, with the following results.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;1A and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e6\u003c/span\u003e hours after modeling, the blood vessels showed bruise-like changes with a small number of inflammatory cells (Fig.\u0026nbsp;1A); the relative expression of CD34 was 0.128\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0237 (Fig.\u0026nbsp;1B,1C). Masson's trichrome staining showed that the relative fibrotic area was 1.009\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007, with a light blue color around the vessel wall (Fig.\u0026nbsp;1D,1E). One day after modeling, the thrombus was laminar and filled the entire lumen, and inflammatory cells were scattered throughout the vein wall, with neutrophils predominant (Fig.\u0026nbsp;1A). The relative expression of CD34 increased to 1.279\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0877 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, vs. 6h group) (Fig.\u0026nbsp;1B,1C), the blue area representing collagen fibers, extracellular matrix and vessel wall was dominated by thrombus edges, a little fibrin-like composition in the middle of thrombus tissue was analyzed, and the area of fibrosis increased to 1.303\u0026thinsp;\u0026plusmn;\u0026thinsp;0.069 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, vs. 6h group) (Fig.\u0026nbsp;1D,1E). Three days after modeling, inflammatory cells were significantly increased in the thrombus tissue (Fig.\u0026nbsp;1A), endothelial cells were proliferated, the relative expression of CD34 was upregulated to 2.294\u0026thinsp;\u0026plusmn;\u0026thinsp;0.107 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, vs. 6h and sham groups) (Fig.\u0026nbsp;1B,1C), fibrin-like formation was clearly visible in the center of the thrombus in addition to the thrombus edge, and the fibrosis area increased to 1.428\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010, indicating a significant proliferation of collagen fibers (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, vs. 6h and sham groups) (Fig.\u0026nbsp;1D,1E). Seven days after modeling, the number of inflammatory cells in the venous wall continued to increase (Fig.\u0026nbsp;1A), there were relatively larger neovascularizations, granulation tissue was seen at the junction of thrombotic vessels to the thrombus, there was recanalized small vessel formation in the thrombus, and the relative expression of CD34 was 3.926\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, vs. the 6h and sham groups) (Fig.\u0026nbsp;1B,1C). The increase in collagen fiber content was more significant, the venous thrombus was mechanized, most of the thrombus was occupied by collagen fibers, and the relative fibrosis area was 1.519\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, vs. the 6h and sham groups) (Fig.\u0026nbsp;1D,1E).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistical values and \u003cem\u003eP\u003c/em\u003e-value of the pathological results of DVT in different groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRatio of venous thrombus weight/length(mg/mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRelative expression of CD34 (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRelative fibrosis area (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esham\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.667\u0026thinsp;\u0026plusmn;\u0026thinsp;0.333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.483\u0026thinsp;\u0026plusmn;\u0026thinsp;0.034\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.881\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.128\u0026thinsp;\u0026plusmn;\u0026thinsp;0.023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.009\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.629\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.279\u0026thinsp;\u0026plusmn;\u0026thinsp;0.087\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.303\u0026thinsp;\u0026plusmn;\u0026thinsp;0.069\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.294\u0026thinsp;\u0026plusmn;\u0026thinsp;0.107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.428\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.881\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.926\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.519\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-Value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e༜0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e༜0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e༜0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNLRP3 inflammasome protein relative expression results statistical values and \u003cem\u003eP\u003c/em\u003e-values\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNLRP3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGSDMD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIL-1β\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.303\u0026thinsp;\u0026plusmn;\u0026thinsp;0.057\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.556\u0026thinsp;\u0026plusmn;\u0026thinsp;0.076\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.547\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.237\u0026thinsp;\u0026plusmn;\u0026thinsp;0.033\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCC950\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.099\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.229\u0026thinsp;\u0026plusmn;\u0026thinsp;0.086\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.158\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.077\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-Value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.047\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2 Ratio 0f Venous Thrombus Weight/Length in Pregnant Rats\u003c/h2\u003e \u003cp\u003eDue to certain anatomical differences in the length and thickness of the IVC in each pregnant rat and certain differences in the manipulation by each surgeon, the overall condition of the thrombus was first assessed by the ratio of weight/length. No significant thrombus formation was observed in the sham group. As shown in Fig.\u0026nbsp;1F and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the weight/length ratio (mg/mm) increased rapidly from 6 h, peaked at 1 d, and then decreased slowly to near 6 h at 7 d (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, vs. the 6h and sham groups) (Fig.\u0026nbsp;1F).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Upregulation of NLRP3 inflammasome expression in the development of DVT in pregnant rats.\u003c/h2\u003e \u003cp\u003eTo evaluate whether the expression of NLRP3 inflammasome was upregulated and the trend of change, we detected it by Western blot, qRT-PCR and IF.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;2A-2B and Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the protein levels of NLRP3 were not significantly in the sham and 6h groups, and started to increase and peaked at 1d and slowly decreased from day 3. It decreased at day 7, but was still higher than the sham and 6h groups. The protein expression of caspase-1 was not significant in the sham group and gradually increased after 6 hours, peaked at day 7 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;2C). GSDMD protein levels gradually increased from 6h, reached a maximum at day 3, and began to decrease at day 7, but were still higher than at day 1 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, vs. sham and 6h groups) (Fig.\u0026nbsp;2D). IL-1β began to show significant changes at 6 h (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, vs. sham group), peaked at day 1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and then gradually decreased, didn\u0026rsquo;t return to normal levels at day 7 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, vs. 6h group) (Fig.\u0026nbsp;2E).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistical values and \u003cem\u003eP-\u003c/em\u003evalues of NLRP3 inflammasome protein expression in DVT tissue of pregnant rats\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNLRP3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGSDMD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIL-1β\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esham\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.041\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.101\u0026thinsp;\u0026plusmn;\u0026thinsp;0.023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.061\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.049\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.064\u0026thinsp;\u0026plusmn;\u0026thinsp;0.053\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.216\u0026thinsp;\u0026plusmn;\u0026thinsp;0.030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.153\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.392\u0026thinsp;\u0026plusmn;\u0026thinsp;0.044\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.519\u0026thinsp;\u0026plusmn;\u0026thinsp;0.053\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.375\u0026thinsp;\u0026plusmn;\u0026thinsp;0.045\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.331\u0026thinsp;\u0026plusmn;\u0026thinsp;0.034\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.635\u0026thinsp;\u0026plusmn;\u0026thinsp;0.062\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.328\u0026thinsp;\u0026plusmn;\u0026thinsp;0.035\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.715\u0026thinsp;\u0026plusmn;\u0026thinsp;0.022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.883\u0026thinsp;\u0026plusmn;\u0026thinsp;0.102\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.201\u0026thinsp;\u0026plusmn;\u0026thinsp;0.030\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.163\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.981\u0026thinsp;\u0026plusmn;\u0026thinsp;0.088\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.532\u0026thinsp;\u0026plusmn;\u0026thinsp;0.081\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.095\u0026thinsp;\u0026plusmn;\u0026thinsp;0.024\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP-\u003c/em\u003e Value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eConsistent with protein expression, NLRP3, caspase-1, GSDMD, and IL-1β mRNA expression levels were increased to varying degrees at different time intervals with statistically significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0003, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0067) (Fig.\u0026nbsp;2F-2I, Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistical values and \u003cem\u003eP-\u003c/em\u003evalues of NLRP3 inflammasome mRNA expression in DVT tissue of pregnant rats\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNLRP3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGSDMD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIL-1β\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esham\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.349\u0026thinsp;\u0026plusmn;\u0026thinsp;0.065\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.249\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.040\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.429\u0026thinsp;\u0026plusmn;\u0026thinsp;0.033\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.567\u0026thinsp;\u0026plusmn;\u0026thinsp;0.034\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.034\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.337\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.954\u0026thinsp;\u0026plusmn;\u0026thinsp;0.037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.829\u0026thinsp;\u0026plusmn;\u0026thinsp;0.096\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.242\u0026thinsp;\u0026plusmn;\u0026thinsp;0.064\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.774\u0026thinsp;\u0026plusmn;\u0026thinsp;0.098\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.061\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.287\u0026thinsp;\u0026plusmn;\u0026thinsp;0.064\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.714\u0026thinsp;\u0026plusmn;\u0026thinsp;0.031\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.183\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.312\u0026thinsp;\u0026plusmn;\u0026thinsp;0.055\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.557\u0026thinsp;\u0026plusmn;\u0026thinsp;0.114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.351\u0026thinsp;\u0026plusmn;\u0026thinsp;0.027\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-Value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0067\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;2J-2K, compared with the sham group, the relative expression of NLRP3 was 1.056\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013 at 6 hours; it increased to a maximum of 1.837\u0026thinsp;\u0026plusmn;\u0026thinsp;0.086 at day 1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001); gradually decreased to 1.539\u0026thinsp;\u0026plusmn;\u0026thinsp;0.042 at day 3 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and then decreased to 1.368\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015 at day 7 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), in agreement with the Western blot and qRT-PCR results. A more significant finding was that NLRP3 was not significantly expressed in the sham and 6h groups, and predominantly expressed in the vascular endothelium around the thrombus from day 1, gradually shifting to predominantly expressed in the middle of the thrombus tissue after day 3.\u003c/p\u003e \u003cp\u003eTo detect the basal levels of IL-1β and IL-18, serum samples were collected before each modeling session as the before group. As shown in Fig.\u0026nbsp;3 and Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e5\u003c/span\u003e, IL-1β was elevated from 6h, reached its highest level at 1 day, then slowly decreased and remained above the 6h level at 7 days (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, v.s) (Fig.\u0026nbsp;2L). IL-18 reached its highest level at 6h (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) then gradually decreased and remained above the 6h level at 7 days (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;2M, Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSerum IL-1β and IL-18 concentrations and \u003cem\u003eP-\u003c/em\u003evalue of DVT in pregnancy rats\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIL-1β(pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIL-18(pg/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBefore\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.060\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.523\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esham\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.488\u0026thinsp;\u0026plusmn;\u0026thinsp;0.091\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.816\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.095\u0026thinsp;\u0026plusmn;\u0026thinsp;0.176\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.53\u0026thinsp;\u0026plusmn;\u0026thinsp;4.245\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.579\u0026thinsp;\u0026plusmn;\u0026thinsp;0.267\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52.83\u0026thinsp;\u0026plusmn;\u0026thinsp;3.374\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.839\u0026thinsp;\u0026plusmn;\u0026thinsp;0.204\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.357\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.892\u0026thinsp;\u0026plusmn;\u0026thinsp;0.446\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.06\u0026thinsp;\u0026plusmn;\u0026thinsp;3.829\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-Value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.3 MCC950 Promotes Thrombus Lysis, Mechanization and Recanalization\u003c/h2\u003e \u003cp\u003eBased on the results obtained from the above experiments, MCC950 was chosen as an inhibitor to see whether it could reduce the inflammatory response and thus promote thrombus lysis absorption and recanalization.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;3A-3B, the number of inflammatory cells in the venous wall was reduced and thrombus lysis was faster in the MCC950 group. The relative expression of CD34 was from 1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.123 to 1.536\u0026thinsp;\u0026plusmn;\u0026thinsp;0.187 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0314) (Fig.\u0026nbsp;3C,3D), indicating that the number of neovascularization was less in the PBS group. The relative fibrosis area after PBS treatment was from 1,000\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025 to 0.914\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0165) (Fig.\u0026nbsp;3E,3F,3G), indicating that MCC950 treatment could promote thrombus mechanization. Simultaneously, the thrombus weight/length ratio was 0.111\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002 in the PBS group and decreased to 0.082\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000 in the MCC group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;3H). In conclusion, MCC950 can reduce inflammatory cell infiltration in thrombus and promote thrombus lysis, mechanization and recanalization.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003e3.4 MCC950 down-regulates the expression of the NLRP3 inflammasome in the model of DVT in pregnant rats\u003c/span\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the inhibitory function of MCC950 on NLRP3 inflammasome in DVT of pregnant rats, the protein expression levels were first analyzed by Western blot. Next, the mRNA expression levels were evaluated by qRT-PCR assay. As shown in Fig.\u0026nbsp;4A-4I, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, NLRP3 inflammasome mRNA levels were all downregulated in the MCC950 group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), consistent with the trend of NLRP3 inflammasome protein expression. The expression and localization of NLRP3 were also detected by IF assay. As shown in Fig.\u0026nbsp;3\u0026ndash;9, the relative expression of NLRP3 was from 1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.074 to 0.565\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025, which mean that it was significantly decreased after MCC950 treatment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0002). Moreover, it was predominantly expressed in the middle of thrombus, which is consistent with the results of the first part (Fig.\u0026nbsp;4J-4K).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNLRP3 inflammasome mRNA relative expression results statistical values and \u003cem\u003eP\u003c/em\u003e-values\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNLRP3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGSDMD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIL-1β\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.254\u0026thinsp;\u0026plusmn;\u0026thinsp;0.064\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.959\u0026thinsp;\u0026plusmn;\u0026thinsp;0.200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.625\u0026thinsp;\u0026plusmn;\u0026thinsp;0.150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.246\u0026thinsp;\u0026plusmn;\u0026thinsp;0.199\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCC950\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.181\u0026thinsp;\u0026plusmn;\u0026thinsp;0.037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.950\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0.069\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.906\u0026thinsp;\u0026plusmn;\u0026thinsp;0.110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.999\u0026thinsp;\u0026plusmn;\u0026thinsp;0.160\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-Value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0067\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIL-1β and IL-18 levels (pg/mL) were then measured by ELISA. As shown in Fig.\u0026nbsp;3-10A, serum IL-1β concentration was 7.234\u0026thinsp;\u0026plusmn;\u0026thinsp;0.274 before modeling and 10.170\u0026thinsp;\u0026plusmn;\u0026thinsp;0.452 in the PBS-treated group, while it decreased to 8.010\u0026thinsp;\u0026plusmn;\u0026thinsp;0.274 after MCC950 treatment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;4L). The serum IL-18 concentration was 29.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.484 and increased to 55.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.529, while it decreased to 43.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.544 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;4M). This indicates MCC950 treatment inhibits the expression of IL-1β and IL-18.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.5 MCC950 improves pregnancy outcome\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;5, by carefully observing the vaginal bleeding of pregnant rats before collecting the material, we found that rats in the PBS group showed different degrees of vaginal bleeding (A), while the vaginal opening of rats in the MCC950 group was dry without any blood (B). After opening the abdominal cavity, we found that the oocytes were reduced to different degrees in the PBS group, while the reduction of oocytes was not obvious in the MCC950 group (C). During the retrieval process, we found that there were very few peri-thrombus adhesions in the MCC950 group of pregnant rats, which were easier to isolate. The thrombus in the PBS group was dark red in color and hard in texture after harvesting, whereas the thrombus tissue in the MCC950 group was bright red in color, soft in texture, and extremely easy to separate from the vascular endothelium (D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003e3.6 MCC950 attenuates toxic effect of\u003c/span\u003e H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eon HUVECs and improves cell proliferation, angiogenesis\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eTo investigate the effects of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and MCC950 on HUVECs and the optimal concentrations, HUVECs were incubated with different concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (0, 100, 200, 400, 600 \u0026micro;M) for 24 h. Cell viability (%) gradually decreased at 100\u0026ndash;600 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e compared to control cells (0 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and decreased significantly at H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations\u0026thinsp;\u0026gt;\u0026thinsp;400 \u0026micro;M (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The higher the concentration is, the greater the toxicity of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to cells (Fig.\u0026nbsp;6A). Meanwhile, LDH release was not significantly increased at H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations less than 100 \u0026micro;M (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), and gradually increased after more than 100 \u0026micro;M, showing a dose-dependent effect (Fig.\u0026nbsp;6B). Therefore, based on the results of CCK8 and LDH analysis, 400 \u0026micro;M was selected as the injury model for the following experiments (Fig.\u0026nbsp;6B). Next, whether MCC950 could protect against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced damage was tested. As shown in Fig.\u0026nbsp;5A and Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, cell viability increased to different degrees in each group after pre-treatment with MCC950 compared to the same concentration without pre-treatment. It was most interesting at a concentration of 400 \u0026micro;M. Significant differences can be seen between this group and the 400 \u0026micro;M group without pretreatment with MCC950, and also with the group without H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e co-incubation, further confirming the correct choice of 400 \u0026micro;M. The results showed that the release of LDH still increased with increasing concentration after MCC950 pretreatment, but the increase was significantly lower in each group than without MCC950 pretreatment. The results of both experiments confirmed MCC950 protects against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced damage and attenuates LDH release (Fig.\u0026nbsp;6B). Treatment with 10 \u0026micro;M MCC950 for 2 h could be selected as the optimal concentration and time and used in the following experiments.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistical values and \u003cem\u003ep\u003c/em\u003e-values for CCK-8 and LDH results\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell ability(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-Value\u003c/p\u003e \u003cp\u003e(VS.Control)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-Value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.685\u0026thinsp;\u0026plusmn;\u0026thinsp;0.027\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e200 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.592\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e400 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.495\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e600 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.168\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCC950 10 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.837\u0026thinsp;\u0026plusmn;\u0026thinsp;0.062\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCC950 10 \u0026micro;M \u003c/p\u003e \u003cp\u003e+\u0026thinsp;100 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.847\u0026thinsp;\u0026plusmn;\u0026thinsp;0.048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.046\u003c/p\u003e \u003cp\u003e (VS. 100 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCC950 10 \u0026micro;M \u003c/p\u003e \u003cp\u003e+\u0026thinsp;200 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.769\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0328\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.019\u003c/p\u003e \u003cp\u003e(VS. 200 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCC950 10 \u0026micro;M\u003c/p\u003e \u003cp\u003e\u0026thinsp;+\u0026thinsp;400 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.608\u0026thinsp;\u0026plusmn;\u0026thinsp;0.026\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.046\u003c/p\u003e \u003cp\u003e (VS. 400 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCC950 10 \u0026micro;M \u003c/p\u003e \u003cp\u003e+\u0026thinsp;600 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.205\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.998\u003c/p\u003e \u003cp\u003e (VS. 600 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRelative release of LDH(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-Value\u003c/p\u003e \u003cp\u003e(VS.Control)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-Value\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.183\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.221\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e200 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.346\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e400 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.563\u0026thinsp;\u0026plusmn;\u0026thinsp;0.022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e600 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.749\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCC950 10 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.186\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCC950 10 \u0026micro;M\u003c/p\u003e \u003cp\u003e\u0026thinsp;+\u0026thinsp;100\u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.295\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0285 \u003c/p\u003e \u003cp\u003e(VS. 100 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCC950 10 \u0026micro;M\u003c/p\u003e \u003cp\u003e\u0026thinsp;+\u0026thinsp;200 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.384\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6183 \u003c/p\u003e \u003cp\u003e(VS. 200 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCC950 10 \u0026micro;M\u003c/p\u003e \u003cp\u003e\u0026thinsp;+\u0026thinsp;200 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.384\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6183 \u003c/p\u003e \u003cp\u003e(VS. 200 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCC950 10 \u0026micro;M\u003c/p\u003e \u003cp\u003e\u0026thinsp;+\u0026thinsp;400 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.456\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0007\u003c/p\u003e \u003cp\u003e(VS. 400 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCC950 10 \u0026micro;M\u003c/p\u003e \u003cp\u003e\u0026thinsp;+\u0026thinsp;600 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.597\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001 \u003c/p\u003e \u003cp\u003e(VS. 600 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eNumerous studies have shown functional impairment occurs after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e induction in HUVECs, and we next investigated whether MCC950 could ameliorate the functional impairment caused by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Cell migration refers to the movement of cells after receiving migration signals. Cell invasion refers to the ability of cells to migrate from one area to another through the extracellular matrix. From the invasion and migration experiments in Fig.\u0026nbsp;6C-6F and Table\u0026nbsp;\u003cspan refid=\"Tab9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e significantly reduced the invasion and migration ability of HUVECs compared with the control group, while it was gradually restored after pretreatment with MCC950.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab9\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 9\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistical values and \u003cem\u003ep\u003c/em\u003e-values of the experimental results for invasion, migration, and formation of tubules\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInvading cells\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMigrating cells\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBranches\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal branches length\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e170.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.283\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e144.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.376\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e141.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7291\u0026thinsp;\u0026plusmn;\u0026thinsp;207.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e65.14\u0026thinsp;\u0026plusmn;\u0026thinsp;2.623\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.478\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e108.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6397\u0026thinsp;\u0026plusmn;\u0026thinsp;170\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCC950\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e118.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.925\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e73.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.563\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e125.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.479\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7274\u0026thinsp;\u0026plusmn;\u0026thinsp;152.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-Value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe tube formation assay is a rapid and quantifiable method to measure angiogenesis in vitro. By assessing the number of branches and total branch length, we found H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e reduced the angiogenic capacity of HUVECs, while MCC950 restored angiogenic function (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;6G-6I).\u003c/p\u003e \u003cp\u003eThe scratch experiment can analyze the cell migration function from another perspective. As shown in Fig.\u0026nbsp;6J-6K, the percentage of wound healing distance between cell scratches in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group was 0.522\u0026thinsp;\u0026plusmn;\u0026thinsp;0.038, which was significantly lower than that in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0. 0001); whereas, after pre-incubation with MCC950, it rose to 0.745\u0026thinsp;\u0026plusmn;\u0026thinsp;0.057, which was lower than that in the control group and higher in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In conclusion, MCC950 significantly reduced pyroptosis of HUVECs and partially restored cell function of proliferation and angiogenesis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003e3.7 MCC950 Inhibits NLRP3 Inflammasome Activity in\u003c/span\u003e H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003e-stimulated HUVECs\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eIn the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated HUVECs, to investigate whether MCC950 acts by inhibiting the NLRP3 inflammasome, we also performed Western blot, qRT-PCR and IF experiments. As shown in Fig.\u0026nbsp;7A-7G, Fig.\u0026nbsp;7I-7J, Table\u0026nbsp;\u003cspan refid=\"Tab10\" class=\"InternalRef\"\u003e10\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab11\" class=\"InternalRef\"\u003e11\u003c/span\u003e, the protein and mRNA expression of NLRP3, caspase-1, GSDMD, and IL-1β were significantly upregulated in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group compared with the control group, and decreased after 2 h pretreatment with MCC950.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab10\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 10\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNLRP3 inflammasome protein expression statistics and \u003cem\u003ep\u003c/em\u003e-values\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNLRP3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGSDMD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIL-1β\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.386\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.451\u0026thinsp;\u0026plusmn;\u0026thinsp;0.032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.748\u0026thinsp;\u0026plusmn;\u0026thinsp;0.048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.773\u0026thinsp;\u0026plusmn;\u0026thinsp;0.098\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.508\u0026thinsp;\u0026plusmn;\u0026thinsp;0.124\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.665\u0026thinsp;\u0026plusmn;\u0026thinsp;0.149\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.248\u0026thinsp;\u0026plusmn;\u0026thinsp;0.134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.784\u0026thinsp;\u0026plusmn;\u0026thinsp;0.097\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;MCC950\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.119\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.861\u0026thinsp;\u0026plusmn;\u0026thinsp;0.040\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.289\u0026thinsp;\u0026plusmn;\u0026thinsp;0.048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.237\u0026thinsp;\u0026plusmn;\u0026thinsp;0.088\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-Value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab11\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 11\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNLRP3 inflammasome mRNA expression statistics and \u003cem\u003ep\u003c/em\u003e-values\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNLRP3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGSDMD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIL-1β\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.635\u0026thinsp;\u0026plusmn;\u0026thinsp;0.037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.843\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.748\u0026thinsp;\u0026plusmn;\u0026thinsp;0.048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.436\u0026thinsp;\u0026plusmn;\u0026thinsp;0.086\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.583\u0026thinsp;\u0026plusmn;\u0026thinsp;0.022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.298\u0026thinsp;\u0026plusmn;\u0026thinsp;0.057\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.248\u0026thinsp;\u0026plusmn;\u0026thinsp;0.134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.368\u0026thinsp;\u0026plusmn;\u0026thinsp;0.138\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCC950\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.143\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.081\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.289\u0026thinsp;\u0026plusmn;\u0026thinsp;0.048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.455\u0026thinsp;\u0026plusmn;\u0026thinsp;0.047\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-Value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eNLRP3 inflammasome can be activated through caspase-1-mediated classical pathway and caspase-4, -5, and \u0026minus;\u0026thinsp;11-mediated non-classical pathway. Therefore, caspase-1 and CD34 were detected simultaneously during IF experiments in addition to co-localization of NLRP3 and CD34. As shown in Fig.\u0026nbsp;7H, 7K, the relative expression of caspase-1 was 1.000\u0026thinsp;+\u0026thinsp;0.058 in the control group, increased to 25.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.209, and then decreased to 18.42\u0026thinsp;+\u0026thinsp;1.548 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The relative NLRP3 expression in the control group was 0.903\u0026thinsp;+\u0026thinsp;0.114, which was significantly upregulated to 2.914\u0026thinsp;\u0026plusmn;\u0026thinsp;0.077 in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group, while decreased to 2.241\u0026thinsp;+\u0026thinsp;0.105 after MCC950 treatment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;7M,7L). Consistent with the Western blot and qRT-PCR results, again indicating that MCC950 inhibits NLRP3 inflammasome activity in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated HUVECs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.8 MCC950 inhibits NLRP3 inflammasome activity by increasing sensitivity to ROS\u003c/h2\u003e \u003cp\u003eIn this study, flow cytometry was selected to detect intracellular ROS levels. As shown in Fig.\u0026nbsp;8A-8F, the DCF fluorescence intensity was 0.233\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012 in the control group and increased to 0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.055 in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). It decreased to 0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.040 after pretreatment with MCC950, which was significantly lower than that in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eAt high mitochondrial membrane potential, JC-1 aggregates in the mitochondrial matrix to produce red fluorescence; conversely, it can produce green fluorescence. As shown in Fig.\u0026nbsp;8G, the green fluorescence intensity was increased in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group compared with the control group, and the green fluorescence intensity was significantly decreased and the red fluorescence intensity was increased after MCC950 pretreatment. The results suggested that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e causes damage to the mitochondrial membrane potential, while MCC950 can restore the membrane potential.\u003c/p\u003e \u003cp\u003eTUNEL staining can detect nuclear DNA breakage during focal cell death. Therefore, cell damage could be further detected by TUNEL. As shown in Fig.\u0026nbsp;8H, DNA damage was significantly increased in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group compared with the control group, whereas it was reduced after pre-incubation with MCC950.\u003c/p\u003e \u003cp\u003eTaken together, the ROS, JC-1 and TUNEL results indicated MCC950 increased intracellular antioxidant capacity and enhanced the sensitivity of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HUVECs to ROS.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eVenous thrombus lysis, mechanization, and recanalization is a complex and dynamic evolutionary process involving multiple cells and cytokines(Nosaka and Ishida et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Since previous studies have demonstrated the feasibility of a venous stasis thrombosis model(Henke and Pearce et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), we used the \"stenosis method\" to construct a model of IVC in pregnant rats. The development of DVT was dynamically observed by HE, IHC and Masson's trichrome staining. These results showed mechanization and recanalization, neocapillary formation and fibrosis processes play an important role in the process of DVT in pregnancy rats(Ishida and Kimura et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Nosaka and Ishida et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), which provides the basis for later evaluation of the efficacy of MCC950 treatment from a pathological point of view.\u003c/p\u003e \u003cp\u003eThis study elucidated the involvement of NLRP3 inflammasome activation in DVT of pregnancy rats. Inflammation and hemostasis are two highly interrelated processes, with inflammation inducing coagulation and coagulation amplifying inflammation. The two coordinates with each other in a positive feedback loop, and when dysregulated, they lead to disease. Thrombosis is a confirmation of the interdependent interaction of this pathological process(Gutmann and Siow et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Qiao et al.(Qiao and Wu et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)identified a role for NLRP3 and IL-1β in platelet function and provided a novel potential link between thrombosis and inflammation, suggesting that therapies targeting NLRP3 or interleukin-1β may be beneficial in the treatment of inflammation-associated thrombosis. Moraes et al.(Moraes and Hottz et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) confirmed the dependence of IL-1β secretion on intracellular ROS after platelet NLRP3 activation and suggested an IL-1β-dependent relationship between inflammasome IL-1β activation and release and increased vascular permeability. In conclusion, the NLRP3 inflammasome plays an important role in thrombosis by activating the coagulation system.\u003c/p\u003e \u003cp\u003eThe present study suggested the development of DVT may begin in ECs. In IF experiments, we found a very interesting phenomenon. Over time, the location of NLRP3 expression changed significantly, beginning with a gradual shift from the endothelial tissue at the periphery of the thrombus to the tissue in the middle of the thrombus. Previous studies have found that the NLRP3 inflammasome can be activated in blood cells through one or more receptors under conditions of tissue injury or cell necrosis that produce endogenous DAMPs(Jiang and Jiang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). So far, studies on NLRP3 inflammasomes and DVT have mainly focused on platelets. Therefore, HUVECs were selected as experimental subjects to further investigate how this process actually occurs in this study.\u003c/p\u003e \u003cp\u003eThe endothelium is a single layer of epithelium covering the surface of blood vessels and is highly active. It plays an important role in maintaining vasodilation, coagulation and anticoagulation systems, immune regulation, vascular smooth muscle proliferation and migration(Takeuchi and Akira, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Under physiological conditions, ROS are essential for physiological cellular functions such as host defense, post-translational processing of proteins, cell signaling, regulation of gene expression and cell differentiation(Bedard and Krause, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). However, certain risk factors, such as hypoxia, can lead to excessive ROS production, resulting in endothelial dysfunction and cell death(Forstermann and Xia et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Endothelial dysfunction or endothelial cell death is closely associated with the development of many diseases in obstetrics and gynecology. Maynard et al.(Maynard and Min et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) found that excess placental soluble fms-like tyrosine kinase 1 (sFlt1) can lead to endothelial dysfunction, hypertension, and proteinuria in pre-eclampsia. In ECs, ROS act as a bridge between pathological stimuli and NLRP3 inflammasome activation. ROS has been shown to activate the NLRP3 inflammasome and plays an important role in various diseases(Li and Zhou et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhao and Wang et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Gupta(Gupta and Sahu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)et al. found that hypoxia induces the production of hypoxia-inducible factor (HIF)-1a, and HIF-1a has been shown to be formed by ROS activation of NLRP3. Therefore, ROS are considered to be a common upstream cause of blood disorders and pathologies (e.g., clot formation). Other studies have shown that the antioxidant capacity within the vascular endothelium of DVT patients is reduced and that oxidative stress may play an important role in the pathophysiology of DVT(Takeuchi and Akira, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Yang and Tu et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In conclusion, it can be hypothesized that ROS increase after endothelial cell hypoxia, leading to NLRP3 inflammasome activation(Roberto and Micucci et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2010\u003c/span\u003e)and activation of the coagulation system and consequently thrombosis after endothelial dysfunction.\u003c/p\u003e \u003cp\u003eMCC950 was shown to promote lysis, mechanization and recanalization of DVT in SD pregnant rats. Li et al. (Li and Qin et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)used hirudin to cause middle cerebral artery occlusion in mice. MCC950 inhibited ischemia-induced overexpression of NLRP3 and its downstream caspase-1, ASC and IL-1β, and protected BV-2 microglia from viability and death. However, Lemarchand et al.(Lemarchand and Barrington et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) used a mouse model of middle cerebral artery thrombosis with increased expression of pro-inflammatory cytokines and NLRP3 after stroke, and targeting NLRP3 with the inhibitor MCC950 or using NLRP3 knockout mice had no effect on the extent of stroke-induced injury. This differs from the results of the present study and may be due to different defense mechanisms in the brain barrier. Altogether, cellular experiments demonstrated increased oxidative stress in endothelial cells promotes the activation and assembly of the NLRP3 inflammasome, which contributes to the pathogenesis and pathophysiology of DVT.\u003c/p\u003e \u003cp\u003eThe present study, the first therapeutic study of MCC950 in a pregnant animal model, is innovative but has some limitations. First, treatment with only 20 mg/kg of MCC950 did not allow evaluation of whether different doses of MCC950 have different effects on thrombosis in rats. Second, the effect of NLRP3 inflammasome inactivation on the coagulation system was not confirmed by further experiments due to time constraints. Third, the effect of MCC950 on placental function was not further investigated and its efficacy, especially safety, remains to be verified.\u003c/p\u003e \u003cp\u003eThe present study fully utilized multiple molecular biology methods, from whole-tissue-cell-molecule, confirmed the research hypothesis at multiple levels and directions, and came to the following conclusions: the massive accumulation of ROS in ECs after ischemia and hypoxia activates the NLRP3 inflammasome, which in turn releases inflammatory factors through pyroptosis and may be a mechanism contributing to DVT. MCC950 may inhibit vascular endothelial cell pyroptosis through the ROS/NLRP3 signaling axis to ameliorate pregnancy-related venous thromboembolism. This mechanism provides new insights into the pathophysiology of DVT in pregnant rats and may open new avenues for the development of techniques to diagnose, evaluate, and treat maternal DVT.\u003c/p\u003e \u003cp\u003eIn addition, there are some prospects for future studies. During the experiment, we also observed the mental status, vaginal bleeding, and pregnancy loss rate of pregnant rats in the MCC950 treatment group were significantly better than those in the control group. In addition, concurrent thrombosis in pregnant women can lead to placental ischemia and hypoxia, resulting in intrauterine growth restriction and delayed fetal development; can MCC950 improve these adverse outcomes? Finally, the possibility of further clinical translational studies of MCC950 treatment for DVT or obstetric and gynecologic disorders during pregnancy will be the focus of our future studies.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics of the study was approved by the\u0026nbsp;Laboratory Animal Ce \u0026nbsp; nter\u0026nbsp;of Nantong University (S20221222-008).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYHX, QHW, YL and YQZ contributed to the conception and design of the project. QS, LJX, SYC, YZH and MRH performed all the figures. YHX drafted the manuscript. YQZ supervised the findings of this work. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanks to Dr. Cheng Xi of Nantong University for the gift of HUVECs. We also would like to thank Editage (www.editage.cn) for editing this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEssien EO, Rali P, et al. (2018). \".Pulmonary Embolism\". Med Clin North Am;103(3):549\u0026ndash;564. Soma-Pillay P, Nelson-Piercy C, Tolppanen H, Mebazaa A. Physiological changes in pregnancy. 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However, the current understanding of its pathogenesis and pathophysiology is not fully adequate, which limits the choice of therapeutic approaches to some extent. In this study, we speculate that the massive accumulation of ROS in vascular endothelial cells after ischemia and hypoxia activates the NLRP3 inflammasome, which in turn releases a large amount of inflammatory factors that contribute to venous thrombosis. To verify the hypothesis, we established a DVT model in pregnant rats by the stenosis method and an H2O2-induced HUVECs model. The study hypothesis was fully confirmed by HE, IHC, Masson, Western blot, qRT-PCR, IF, ELISA, CCK8, LDH, invasion, migration, scratching, tube formation, TUNEL, JC-1 and flow cytometry experiments. We have reached the following results: 1.NLRP3 inflammasome was activated during the development of DVT (P \u003c 0.0001). 2.MCC950 promoted the lysis, mechanization and recanalization of DVT in pregnant rats by inhibiting the activation of NLRP3 inflammasome (P\u003c 0.05). 3.MCC950 reduced embryo loss and improved pregnancy outcome. 4.MCC950 inhibited NLRP3 inflammasome activation after H2O2-induced injury in HUVECs by enhancing ROS sensitivity (P \u003c 0.05). To sum up, the study provides a new basic theoretical basis and potential therapeutic reference for the prevention and treatment of pregnancy-associated venous thromboembolism (PA-VTE).","manuscriptTitle":"Blockage of NLRP3 inflammasome by MCC950 can reverse the effect of pyroptosis in HUVECs and thrombosis of pregnancy rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-24 18:50:42","doi":"10.21203/rs.3.rs-2935166/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":"1ca2c807-d331-4dc3-84b7-2df0fcc6ce51","owner":[],"postedDate":"May 24th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-06-08T05:29:42+00:00","versionOfRecord":[],"versionCreatedAt":"2023-05-24 18:50:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2935166","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2935166","identity":"rs-2935166","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}