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This study investigated the protective effect of chrysin on hepatic fibrosis by regulating AMP-activated kinase (AMPK)-NOD-like receptor protein 3 (NLRP3) mediated pyroptosis pathway. The hepatic fibrosis model of mice was established by thioacetamide (TAA) in vivo. Except the control and chrysin alone groups, the mice were injected intraperitoneally with TAA at 100 mg·kg-1, three times per week for the first week. From the 2nd to 5th week, mice were injected intraperitoneally with TAA at 200 mg·kg-1, three times per week for the next 4 weeks. Chrysin groups were intragastrically administrated once per day to 5th week. The histopathological changes were detected by HE and Masson staining. The levels of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assessed by the kits. All animal experiments were approved by the Medical Ethics Committee of Affiliated Zhongshan Hospital of Dalian University (DWLL2019060). LX-2 cells were stimulated by (transforming growth factor-β, TGF-β) in vitro. The protein expressions of AMPKα, p-AMPKα, NLRP3, cysteinyl aspartate specific proteinase-1 (caspase-1), gasdermin D (GSDMD) were detected by Western blot, and the mRNA levels of collagen-Ι, α-smooth muscle actin (α-SMA), interleukin-1β (IL-1β), IL-18, caspase-1, GSDMD were analysis by reverse transcription-polymerase chain reaction (RT-PCR). Chrysin attenuated the increases in serum AST and ALT levels in the TAA group, while significantly improved the changes of liver morphology, reduced liver tissue inflammatory cell infiltration and inhibited collagens deposition. Compared with TAA group, chrysin effectively activated AMPKα phosphorylation and inhibited hepatic NLRP3 inflammasome activation. Additionally, the protein expressions and mRNA levels of IL-1β, IL-18, caspase-1 and GSDMD in chrysin groups were decreased. Chrysin inhibited the expressions of collagen-Ι and α-SMA, enhanced the phosphorylation of AMPKα, and decreased the expressions of NLRP3 and GSDMD. Therefore, chrysin may inhibit inflammatory injury and pyroptosis possibly by activating AMPK and inhibiting NLRP3 inflammasome to alleviate hepatic fibrosis.
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ObjectiveTo explore the anti-tumor effect and mechanism of Shenqi Yiliu prescription in the intervention of pyroptosis. MethodTen male BALB/c mice were randomly selected and assigned to the blank group. The remaining 40 mice underwent the induction of the liver cancer xenograft model. After 5 days of modeling， 40 surviving mice were randomly divided into model group， cisplatin group ［2.5×10-3 g·kg-1·（3 d）-1］， Shenqi Yiliu prescription group （27 g·kg-1·d-1）， and a combination group （Shenqi Yiliu prescription group + cisplatin）. The mice in the blank group and the model group were treated with an equal volume of normal saline for 10 days. The general conditions of mice in each group were observed. After the intervention， the tumor weight of the mice was weighed and the tumor inhibition rate was calculated. Hematoxylin-eosin （HE） staining was used to observe the pathological changes in tumor tissues. The levels of mouse liver function indicators， including alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were detected. The TdT-mediated dUTP-biotin nick end labeling （TUNEL） assay was used to detect DNA damage in mouse tumor tissue cells. Immunohistochemistry （IHC）， immunofluorescence （IF）， and Western blot were used to detect the protein expression levels of NOD-like receptor protein 3 （NLRP3）， cysteinyl aspartate-specific protease-1 （Caspase-1）， and gasdermin D （GSDMD） in tumor tissues. The levels of interleukin-1β （IL-1β） and interleukin-18 （IL-18） in tumor tissues were detected by enzyme-linked immunosorbent assay （ELISA）. ResultCompared with the mice in the blank group， those in the model group were in a poor mental state， sleepy， and lazy， and their fur color was dull， with increased levels of serum ALT and AST in liver function tests （P<0.01）. Compared with the model group， the groups with drug intervention showed improved mental state， inhibited tumor growth to varying degrees， and decreased tumor weight， and the tumor inhibition rate in the combination group was the highest （P<0.01）. HE staining showed that the pathological and morphological lesions of the tumor tissues in the model group were significant， while those in all groups with drug intervention were improved to a certain extent. The karyolysis and nuclear rupture in the Shenqi Yiliu prescription group and the combination group were more significant. In the liver function test， the serum ALT and AST levels of mice in the Shenqi Yiliu prescription group and the combination group decreased （P<0.01）， and the inflammatory factors IL-1β and IL-18 in each group with drug intervention decreased （P<0.05， P<0.01）. Among them， the declining trend of IL-1β and IL-18 in the Shenqi Yiliu prescription group was the most significant （P<0.01）. TUNEL staining showed that the positive TUNEL staining in each group with drug intervention decreased after intervention （P<0.05， P<0.01）， especially the cisplatin group and Shenqi Yiliu prescription group （P<0.01）. Western blot， IHC， and IF found that the protein expression levels of NLRP3， Caspase-1， and GSDMD in each group with drug intervention decreased （P<0.05， P<0.01）. Compared with the mice in the cisplatin group， those in the Shenqi Yiliu prescription group and the combination group had better mental state and regular tumor morphology， and the tumor weight of the mice in the combination group decreased （P<0.05）. The levels of ALT and AST in the Shenqi Yiliu prescription group decreased （P<0.05）， and the levels of IL-1β and IL-18 in the Shenqi Yiliu prescription group and the combination group decreased （P<0.05， P<0.01）， especially in the combination group （P<0.01）. The results of IHC showed that the expression of GSDMD protein in the tumor tissues of mice in the combination group was reduced （P<0.01）. IF detection showed that the expression of NLRP3 in the tumor tissues of the Shenqi Yiliu prescription group was reduced （P<0.01）. The results of Western blot showed that the expression level of NLRP3 protein in the Shenqi Yiliu prescription group and the combination group decreased （P<0.01）， and the expression level of Caspase-1 protein in the combination group decreased （P<0.01）. The decrease in GSDMD protein expression was not significant， and the difference was not statistically significant. ConclusionShenqi Yiliu prescription combined with cisplatin has an obvious anti-tumor effect， which may be achieved by down-regulating the NLRP3/Caspase-1/GSDMD inflammatory pyroptosis pathway to inhibit cell pyroptosis， and relieve the inflammatory response in mice with liver cancer.
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Pyroptosis， a new type of inflammatory programmed cell death， is different from apoptosis， necrosis， cytosis， ferroptosis， and autophagy. Pyroptosis is dependent on the activation of cysteine aspartate-specific protease （Caspase）， which cleaves key mediator proteins to form pores in the cell membrane and induces the maturation and release of the proinflammatory cytokines interleukin-1β and interleukin-18 into the extracellular environment， resulting in a cascade of inflammatory reactions. Gastric cancer as a malignant tumor of the digestive tract is refractory and has poor prognosis， and the chemoradiotherapy of this disease may lead to a variety of complications. At present， the pathogenesis of gastric cancer remains unclear. Studies have proved that pyroptosis is associated with the occurrence and development of gastric cancer， which has attracted wide attention. Pyroptosis is a double-edged sword for gastric cancer. On the one hand， it can release the contents of proinflammatory cells to amplify or maintain inflammation and induce the "inflammation-cancer" transformation of cells. On the other hand， pyroptosis can enhance the sensitivity of drugs for chemotherapy to improve the therapeutic effect and survival. In recent years， the anti-tumor mechanism of traditional Chinese medicine （TCM） has become a research hotspot as TCM has demonstrated significant effects in clinical application. Therefore， the regulation of pyroptosis by TCM may be a new direction for the treatment of gastric cancer in the future. Based on the available studies， this paper introduces the roles of pyroptosis-associated key proteins in the occurrence and development of gastric cancer. Furthermore， this paper summarizes the effects of TCM prescriptions and active ingredients on alleviating gastric mucosal damage， reducing the incidence of gastric cancer， and preventing tumor metastasis and recurrence by mediating pyroptosis pathways， aiming to provide new ideas for deciphering the mechanism of pyroptosis and exploring the TCM treatment of gastric cancer in the future.
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Pyroptosis， an atypical new cell death mode other than apoptosis and necrosis， has been discovered in recent years. Pyroptosis depends on the cleavage of gasdermins （GSDMs） by Caspases. The activated GSDMs act on the plasma membrane to form a perforation， which results in cell lysis and triggers inflammation and immune response. Pyroptosis can be induced by four distinct signaling pathways， including canonical and non-canonical inflammasome pathways， apoptosis-associated Caspases-mediated pathway， and granzyme pathway. In these signaling pathways， GSDMs are the executors of pyroptosis. Pyroptosis is associated with the death of tumor cells and the inflammatory damage of normal tissues. Recent studies have demonstrated that moderate pyroptosis can lead to tumor cell death to exert an anti-tumor effect， and meanwhile stimulate the tumor immune microenvironment， while it can promote tumor development. Despite the good performance， drug-based anti-tumor therapies such as tumor immunotherapy， chemotherapy， and targeted therapy have some shortcomings such as drug resistance， recurrence， and damage to normal tissues. The latest research shows that a variety of natural compounds have anti-tumor effects in the auxiliary treatment of tumors by mediating the pyroptosis pathways in a multi-target and multi-pathway manner， which provide new ideas for the study of anti-tumor therapy. We reviewed the molecular mechanism of pyroptosis and the regulatory role of pyroptosis in tumors and tumor immune microenvironment， and summarized the recent research progress in the natural medicinal components regulating pyroptosis in anti-tumor therapy， with a view to providing ideas for the research on the anti-tumor therapy based on pyroptosis.
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As a novel star molecule, gasdermin D (GSDMD) plays an important role in the amplification of immune inflammatory response and the process of pyroptosis. After being cleaved and activated by caspase-1, the N-terminal of GSDMD is rapidly released, which anchors on the cell membrane and forms pores, thereby leading to pyroptosis, accompanied by the release of a large amount of the strong proinflammatory factors IL-1β and IL-18. Acute/chronic liver inflammatory response and cell death are the common pathological features of liver diseases such as viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease, autoimmune liver disease, liver failure, and hepatocellular carcinoma. This article summarizes the basic structural characteristics of GSDMD and elaborates on its important role in the pathological progression of various liver diseases. In addition, it is proposed that prevention and treatment strategies with GSDMD as a potential therapeutic target can provide new ideas for further studies on the clinical prevention and treatment of liver diseases.
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Cardiomyocyte death is one of the major mechanisms contributing to the development of myocardial infarction (MI) and myocardial ischemia/reperfusion (MI/R) injury. Due to the limited regenerative ability of cardiomyocytes, understanding the mechanisms of cardiomyocyte death is necessary. Pyroptosis, one of the regulated programmed cell death pathways, has recently been shown to play important roles in MI and MI/R injury. Pyroptosis is activated by damage-associated molecular patterns (DAMPs) that are released from damaged myocardial cells and activate the formation of an apoptosis-associated speck-like protein containing a CARD (ASC) interacting with NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), resulting in caspase-1 cleavage which promotes the activation of Gasdermin D (GSDMD). This pathway is known as the canonical pathway. GSDMD has also been shown to be activated in a non-canonical pathway during MI and MI/R injury via caspase-4/5/11. Suppression of GSDMD has been shown to provide cardioprotection against MI and MI/R injury. Although the effects of MI or MI/R injury on pyroptosis have previously been discussed, knowledge concerning the roles of GSDMD in these settings remains limited. In this review, the evidence from in vitro, in vivo, and clinical studies focusing on cardiac GSDMD activation during MI and MI/R injury is comprehensively summarized and discussed. Implications from this review will help pave the way for a new therapeutic target in ischemic heart disease.
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Silicosis is a leading cause of occupational disease-related morbidity and mortality worldwide, but the molecular basis underlying its development remains unclear. An accumulating body of evidence supports gasdermin D (GSDMD)-mediated pyroptosis as a key component in the development of various pulmonary diseases. However, there is little experimental evidence connecting silicosis and GSDMD-driven pyroptosis. In this work, we investigated the role of GSDMD-mediated pyroptosis in silicosis. Single-cell RNA sequencing of healthy and silicosis human and murine lung tissues indicated that GSDMD-induced pyroptosis in macrophages was relevant to silicosis progression. Through microscopy we then observed morphological alterations of pyroptosis in macrophages treated with silica. Measurement of interleukin-1β release, lactic dehydrogenase activity, and real-time propidium iodide staining further revealed that silica induced pyroptosis of macrophages. Additionally, we verified that both canonical (caspase-1-mediated) and non-canonical (caspase-4/5/11-mediated) signaling pathways mediated silica-induced pyroptosis activation, in vivo and in vitro. Notably, Gsdmd knockout mice exhibited dramatically alleviated silicosis phenotypes, which highlighted the pivotal role of pyroptosis in this disease. Taken together, our results demonstrated that macrophages underwent GSDMD-dependent pyroptosis in silicosis and inhibition of this process could serve as a viable clinical strategy for mitigating silicosis.
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ABSTRACT Purpose: Diabetic retinopathy is currently considered a chronic inflammatory disease involving NOD-like receptor family pyrin domain containing 3 inflammasome activation and retinal microglial pyroptosis. In this study, we aimed to investigate whether NOD-like receptor family pyrin domain containing 3 inflammasome signaling induces pyroptotic death of retinal microglia under high-glucose conditions. Methods: Retinal microglia were stimulated by high glucose levels for 24 h. Cell viability, lactate dehydrogenase release, and caspase-1 activity were detected in vitro. The expression of pro-inflammatory cytokine (interleukin-1β, activated microglia marker ionized calcium-binding adapter molecule-1), NOD-like receptor family pyrin domain containing 3, cleaved caspase-1, and cleaved gasdermin D were examined. Subsequently, retinal microglia were pretreated with the inhibitors of NOD-like receptor family pyrin domain containing 3 inflammasome signaling prior to stimulation with high glucose, and their molecular and functional changes were evaluated. Results: High-glucose (25, 50, or 100 mM) stimulation decreased cell viability, but enhanced lactate dehydrogenase release and caspase-1 activity in a dose-dependent manner. Moreover, high glucose upregulated the protein expression of interleukin-1β, ionized calcium-binding adapter molecule-1, NOD-like receptor family pyrin domain containing 3, cleaved caspase-1, and cleaved gasdermin D. However, pretreatment with the inhibitors of NOD-like receptor family pyrin domain containing 3 inflammasome signaling inhibited high glucose (25 mM)-induced cytotoxicity, NOD-like receptor family pyrin domain containing 3 inflammasome activation, and pyroptosis of retinal microglia. Conclusions: NOD-like receptor family pyrin domain containing 3 inflammasome signaling may modulate retinal microglia-related inflammation and pyroptosis under high-glucose conditions.
RESUMO Objetivo: Atualmente, a retinopatia diabética é considerada uma doença inflamatória crônica envolvendo a ativação de inflamassomas NLRP3 e piroptose da micróglia da retina. Neste estudo, objetivamos investigar se a sinalização de inflamassomas NLRP3 induz a morte da micróglia da retina sob condições de alta glicose. Métodos: A micróglia da retina foi estimulada por altos níveis de glicose durante 24 horas. A viabilidade celular, a liberação de LDH e a atividade da caspase1 foram analisadas in vitro. Avaliou-se a expressão de citocina pró-inflamatória (IL1β), de marcador de micróglia ativado (Iba1), de NLRP3, de caspase1 clivada e de GSDMD clivada. Subsequentemente, a micróglia da retina foi pré-tratada com inibidores da sinalização de inflamassomas NLRP3 antes da estimulação com altos níveis de glicose e suas alterações moleculares e funcionais foram avaliadas. Resultados: A estimulação com altos níveis de glicose (25 mM, 50 mM ou 100 mM) diminuiu a viabilidade celular, mas aumentou a liberação de LDH e a atividade da caspase1 de forma dependente da dose. Além disso, os altos níveis de glicose aumentaram a expressão das proteínas IL1β, Iba1, NLRP3, caspase1 clivada e GSDMD clivada. No entanto, o pré-tratamento com inibidores da sinalização de inflamassomas NLRP3 e a posterior estimulação com altos níveis de glicose (25 mM) induziu citotoxicidade, a ativação de inflamassomas NLRP3 e a piroptose da micróglia da retina. Conclusão: A sinalização de inflamassomas NLRP3 pode modular a inflamação e a piroptose da micróglia da retina na presença de altos níveis de glicose.
Subject(s)
Humans , Inflammasomes , Pyroptosis , Microglia , Interleukin-1beta , NLR Family, Pyrin Domain-Containing 3 Protein , GlucoseABSTRACT
@#Inflammatory caspase-11 senses and is activated by intracellular lipopolysaccharide (LPS) leading to pyroptosis that has critical role in defensing against bacterial infection, whereas its excess activation under pathogenic circumstances may cause various inflammatory diseases. However, there are few known drugs that can control caspase-11 activation. We report here that scutellarin, a flavonoid from Erigeron breviscapus, acted as an inhibitor for caspase-11 activation in macrophages. Scutellarin dose-dependently inhibited intracellular LPS-induced release of caspase-11p26 (indicative of caspase-11 activation) and generation of N-terminal fragment of gasdermin D (GSDMD-NT), leading to reduced pyroptosis. It also suppressed the activation of non-canonical nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome as evidenced by reduced apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and decreased interleukin-1 beta (IL-1β) and caspase-1p10 secretion, whereas the NLRP3-specific inhibitor MCC950 only inhibited IL-1β and caspase-1p10 release and ASC speck formation but not pyroptosis. Scutellarin also suppressed LPS-induced caspase-11 activation and pyroptosis in RAW 264.7 cells lacking ASC expression. Moreover, scutellarin treatment increased Ser/Thr phosphorylation of caspase-11 at protein kinase A (PKA)-specific sites, and its inhibitory action on caspase-11 activation was largely abrogated by PKA inhibitor H89 or by adenylyl cyclase inhibitor MDL12330A. Collectively, our data indicate that scutellarin inhibited caspase-11 activation and pyroptosis in macrophages at least partly via regulating the PKA signaling pathway.
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Sepsis is a serious life-threatening organ dysfunction disease caused by the host′s unbalanced response to infection, and it′s a common cause of death in intensive care units.Pyroptosis is a kind of necrosis and inflammatory programmed cell death mediated by caspase.Its mechanism can be divided into typical inflammasome activation and atypical inflammasome activation.Caspase-1/11 and Gasdermin-D are the key substrates for pyroptosis.Mitochondrial DNA(mtDNA)as a damage-associated molecular pattern mediates pyroptosis and is associated with severity of sepsis.This review summarized the progress on the relationship between mtDNA-mediated pyroptosis and sepsis.
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ObjectiveA good invasion ability of extravilloustrophoblas (EVTs) is the prerequisite for successful placental colonization and effective remodeling of the uterine spiral artery. This article aims to simulate the pathophysiological process of oxidative stress inducing trophoblasts to pyroptosis in vitro, exploring the correlation between trophoblasts pyroptosis and the pathogenesis of preeclampsia.MethodsTwenty-five patients with preeclampsia were selected from the Department of Obstetrics and Gynecology, Zhongda Hospital affiliated to Southeast University from September 2017 to January 2019. Among them, early-onset preeclampsia (gestational weeks<34) was early-onset group (n=17), late-onset preeclampsia (gestational weeks≥34) was late-onset group (n=8), and full-term pregnant women with normal blood pressure (39＜gestational weeks＞42) were selected as normal group (n=10). Human trophoblasts were cultured with HTR-8/SVneo for 12 hours, and then treated with H2O2 (100, 150, 200, 250μmol/L) (2, 4, 6, 12 h), to induce human trophoblast HTR-8/SVneo pyrolysis model; the control group was normal cultured cells of 1640+10% fetal bovine serum + 1% antibiotics. Placental specimens from 7 patients with preeclampsia were randomly selected, including 3 cases in early onset group, 4 cases in late onset group and 1 case in normal group. The total proteins of cells and placenta were extracted respectively, and the expression of scorch death-related molecular proteins was detected. The mRNA levels of pyroptosis related molecules in cells was detected by RT-qPCR, and the morphological changes of cells were observed by inverted phase contrast microscope.ResultsThe Western blot results showed that the activation of the key molecular activation form of the cell pyrogenesis pathway, Cleaved caspase1, could be detected in the placenta. When H2O2 was 150 mol/L for 2h, the mRNA levels of NLRP3 and IL-1, the key molecules of the upstream activation signal, were significantly up-regulated (8.680±0.481, 14.136±0.244) compared with the control group (1.00±0.00) (P<0.000). At 4h, mRNA levels of key molecule GSDMD and downstream inflammatory factor IL-18 (1.639±0.354 and 1.794±0.043) in the pyrogenesis pathway were significantly higher than those in the control group (1.00±0.00), with statistically significant differences (P<0.05). By reverse validation of the mRNA levels of the molecules associated with pyroptosis, the optimal conditions of the model induced by H2O2 were 150 mol/L and 4h, and the typical changes, such as cell swelling, fragmentation and plasma membrane bubble formation, could be seen under the light microscope.ConclusionThe pyroptosis model of trophoblast cells was successfully established, and the physiological process of oxidative stress inducing trophoblasts to pyroptosis in vitro was successfully simulated, providing new ideas and directions for the diagnosis and treatment of preeclampsia and the development of new drugs.
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In recent years, researchers have shown great interest in the mechanism of induced programmed cell death (PCD) in tumor cells. Pyroptosis is a newly-discovered programmed cell death, which has a proinflammatory effect and is accompanied by cell pore formation, cytoplasmic swelling, cell membrane rupture, and release of cytoplasmic contents into the extracellular environment. A large number of studies have shown that pyroptosis is closely related to the occurrence and development of various diseases such as cancer. Currently, gasdermin family mediate cell pyroptosis, due to gasdermin D and gasdermin E are two molecules that are extensively studied in pyroptosis. This article mainly explores the relevant mechanism of these two proteins and their relationship with cancer, which will broaden our understanding of new insights to cancer and provide a new perspective on cancer prevention and treatment.
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To explore whether extracellular histones aggravate acute respiratory distress syndrome (ARDS) by inducing peripheral blood mononuclear cell (PBMC) pyroptosis. Methods Twenty patients with ARDS admitted to Shanghai Pulmonary Hospital, Tongji University School of Medicine from April to September in 2019 were enrolled, and 20 healthy volunteers were enrolled as controls. In vivo experiment: peripheral blood samples of patients with ARDS within 24 hours after diagnosis and healthy volunteers were collected, and the levels of plasma extracellular histone, interleukins (IL-1β and IL-18) and lactic dehydrogenase (LDH) were determined by enzyme-linked immunosorbent assay (ELISA). PBMC were harvested, the expression levels of the pyroptosis associated N terminal-gasdermin-D (GSDMD-N) protein were determined by Western Blot. In vitro experiment: PBMC isolated from healthy volunteers were divided into four groups. Blank control group without any treatment; lipopolysaccharide (LPS) group was treated with 1 mg/L LPS for 4 hours; LPS+histones group was treated with 100 mg/L exogenous histones for 24 hours after LPS treatment; LPS+histone+heparin group was treated with 200 U heparin for 24 hours after LPS and exogenous histones treatment. The GSDMD-N protein expression was determined by Western Blot, and the levels of IL-1β, IL-18 and LDH in cell supernatant were determined by ELISA. Spearman test was used to test the correlation among the parameters. Results In vivo experiment results: compared with healthy control group, the GSDMD-N protein expression in PBMC of patients with ARDS was significantly increased [GSDMD-N/GAPDH: 0.136 (0.062, 0.246) vs. 0.026 (0.018, 0.036), P < 0.01], as well as the plasma levels of IL-1β, IL-18, LDH and extracellular histones [IL-1β (ng/L): 120.0 (94.2, 213.0) vs. 88.5 (82.3, 105.3), IL-18 (ng/L): 164.5 (70.8, 236.3) vs. 60.5 (52.0, 89.0), LDH (U/L): 30.9 (24.7, 39.5) vs. 19.8 (17.2, 21.5), extracellular histones (mg/L): 73.0 (42.8, 112.9) vs. 12.2 (9.6, 16.9), all P < 0.01], indicating that the PBMC of ARDS patients had significant pyroptosis and release of a large number of inflammatory factors. The oxygenation index (PaO2/FiO2) of ARDS patients was 135.5 (94.5, 196.0) mmHg (1 mmHg = 0.133 kPa). Correlation analysis showed that the expression of GSDMD-N protein in patients with ARDS was negatively correlated with PaO2/FiO2 (r = -0.935, P <0.01) and positively correlated with IL-1β, IL-18, LDH and extracellular histones (r value was 0.844, 0.843, 0.887, 0.899, respectively, all P < 0.01). In vitro experiment results: compared with blank control group, the expression of GSDMD-N protein in PBMC and the levels of inflammatory mediators in the supernatant of the LPS group were significantly increased [GSDMD-N/GAPDH: 0.035±0.006 vs. 0.028±0.006, IL-1β (ng/L): 39.8±5.5 vs. 22.6±4.7, IL-18 (ng/L): 31.2±4.4 vs. 20.0±2.2, LDH (U/L): 51.2±7.3 vs. 36.6±7.6, all P < 0.05], indicating that LPS stimulation could increase PBMC pyroptosis and the release of inflammatory mediators. Compared with LPS group, the expression of GSDMD-N protein and the levels of inflammatory mediators of the LPS+histones group were further increased [GSDMD-N/GAPDH:0.114±0.009 vs. 0.035±0.006, IL-1β (ng/L): 119.0±18.7 vs. 39.8±5.5, IL-18 (ng/L): 49.2±8.5 vs. 31.2±4.4, LDH (U/L): 127.8±19.8 vs. 51.2±7.3, all P < 0.01], indicating that the stimulation of LPS on PBMC could be significantly amplified by exogenous histone treatment, GSDMD-N protein expression could be up-regulated and inflammatory factor release could be promoted to further induce PBMC pyroptosis. These adverse effects of exogenous histones on PBMC could be abrogated by heparin, the expression of GSDMD-N protein and the levels of inflammatory mediators were significantly lower than those of LPS+histones group [GSDMD-N/GAPDH: 0.063±0.004 vs. 0.114±0.009, IL-1β (ng/L): 46.8±8.6 vs. 119.0±18.7, IL-18 (ng/L): 33.0±5.1 vs. 49.2±8.5, LDH (U/L): 65.4±11.0 vs. 127.8±19.8, all P < 0.05]. Conclusion Extracellular histones in plasma may aggravate ARDS by mediating PBMC pyroptosis.
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Objective@#To explore whether extracellular histones aggravate acute respiratory distress syndrome (ARDS) by inducing peripheral blood mononuclear cell (PBMC) pyroptosis.@*Methods@#Twenty patients with ARDS admitted to Shanghai Pulmonary Hospital, Tongji University School of Medicine from April to September in 2019 were enrolled, and 20 healthy volunteers were enrolled as controls. In vivo experiment: peripheral blood samples of patients with ARDS within 24 hours after diagnosis and healthy volunteers were collected, and the levels of plasma extracellular histone, interleukins (IL-1β and IL-18) and lactic dehydrogenase (LDH) were determined by enzyme-linked immunosorbent assay (ELISA). PBMC were harvested, the expression levels of the pyroptosis associated N terminal-gasdermin-D (GSDMD-N) protein were determined by Western Blot. In vitro experiment: PBMC isolated from healthy volunteers were divided into four groups. Blank control group without any treatment; lipopolysaccharide (LPS) group was treated with 1 mg/L LPS for 4 hours; LPS+histones group was treated with 100 mg/L exogenous histones for 24 hours after LPS treatment; LPS+histone+heparin group was treated with 200 U heparin for 24 hours after LPS and exogenous histones treatment. The GSDMD-N protein expression was determined by Western Blot, and the levels of IL-1β, IL-18 and LDH in cell supernatant were determined by ELISA. Spearman test was used to test the correlation among the parameters.@*Results@#In vivo experiment results: compared with healthy control group, the GSDMD-N protein expression in PBMC of patients with ARDS was significantly increased [GSDMD-N/GAPDH: 0.136 (0.062, 0.246) vs. 0.026 (0.018, 0.036), P < 0.01], as well as the plasma levels of IL-1β, IL-18, LDH and extracellular histones [IL-1β (ng/L): 120.0 (94.2, 213.0) vs. 88.5 (82.3, 105.3), IL-18 (ng/L): 164.5 (70.8, 236.3) vs. 60.5 (52.0, 89.0), LDH (U/L): 30.9 (24.7, 39.5) vs. 19.8 (17.2, 21.5), extracellular histones (mg/L): 73.0 (42.8, 112.9) vs. 12.2 (9.6, 16.9), all P < 0.01], indicating that the PBMC of ARDS patients had significant pyroptosis and release of a large number of inflammatory factors. The oxygenation index (PaO2/FiO2) of ARDS patients was 135.5 (94.5, 196.0) mmHg (1 mmHg = 0.133 kPa). Correlation analysis showed that the expression of GSDMD-N protein in patients with ARDS was negatively correlated with PaO2/FiO2 (r = -0.935, P < 0.01) and positively correlated with IL-1β, IL-18, LDH and extracellular histones (r value was 0.844, 0.843, 0.887, 0.899, respectively, all P < 0.01). In vitro experiment results: compared with blank control group, the expression of GSDMD-N protein in PBMC and the levels of inflammatory mediators in the supernatant of the LPS group were significantly increased [GSDMD-N/GAPDH: 0.035±0.006 vs. 0.028±0.006, IL-1β (ng/L): 39.8±5.5 vs. 22.6±4.7, IL-18 (ng/L): 31.2±4.4 vs. 20.0±2.2, LDH (U/L): 51.2±7.3 vs. 36.6±7.6, all P < 0.05], indicating that LPS stimulation could increase PBMC pyroptosis and the release of inflammatory mediators. Compared with LPS group, the expression of GSDMD-N protein and the levels of inflammatory mediators of the LPS+histones group were further increased [GSDMD-N/GAPDH: 0.114±0.009 vs. 0.035±0.006, IL-1β (ng/L): 119.0±18.7 vs. 39.8±5.5, IL-18 (ng/L): 49.2±8.5 vs. 31.2±4.4, LDH (U/L): 127.8±19.8 vs. 51.2±7.3, all P < 0.01], indicating that the stimulation of LPS on PBMC could be significantly amplified by exogenous histone treatment, GSDMD-N protein expression could be up-regulated and inflammatory factor release could be promoted to further induce PBMC pyroptosis. These adverse effects of exogenous histones on PBMC could be abrogated by heparin, the expression of GSDMD-N protein and the levels of inflammatory mediators were significantly lower than those of LPS+histones group [GSDMD-N/GAPDH: 0.063±0.004 vs. 0.114±0.009, IL-1β (ng/L): 46.8±8.6 vs. 119.0±18.7, IL-18 (ng/L): 33.0±5.1 vs. 49.2±8.5, LDH (U/L): 65.4±11.0 vs. 127.8±19.8, all P < 0.05].@*Conclusion@#Extracellular histones in plasma may aggravate ARDS by mediating PBMC pyroptosis.
ABSTRACT
Gasdermin family (GSDMs), consisting of six proteins (GSDMA, GSDMB, GSDMC, GSDMD, GSDME and DFNB59) in humans and ten proteins (GSDMA1-3, GSDMC1-4, GSDMD, GSDME and DFNB59) in mice, might be involved in multiple physiological and pathological processes, including epithelial cell development, apoptosis, inflammation, carcinogenesis and immune-related diseases. Recent studies confirmed GSDMD, which containing an N-terminal domain with pore-forming activity and a C-terminal domain with structural autoinhibition, as a crucial substrate of inflammatory caspases in pyroptosis, pioneering a new area for structural and functional research on Gasdermin family proteins. This review will summarize the latest progress in the structures, functions and association with diseases of several Gasdermin family proteins.
ABSTRACT
Gasdermin family (GSDMs), consisting of six proteins (GSDMA, GSDMB, GSDMC, GSDMD, GSDME and DFNB59) in humans and ten proteins (GSDMA1-3, GSDMC1-4, GSDMD, GSDME and DFNB59) in mice, might be involved in multiple physiological and pathological processes, including ep-ithelial cell development, apoptosis, inflammation, carcinogenesis and immune-related diseases. Recent studies confirmed GSDMD, which containing an N-terminal domain with pore-forming activity and a C-termi-nal domain with structural autoinhibition, as a crucial substrate of inflammatory caspases in pyroptosis, pio-neering a new area for structural and functional research on Gasdermin family proteins. This review will sum-marize the latest progress in the structures, functions and association with diseases of several Gasdermin fam-ily proteins.
ABSTRACT
Pyroptosis is a newly discovered form of programmed cell death, and morphologically, it has the characteristics of both necrosis and apoptosis. Gasdermin-D is the executive protein in pyroptosis. Pyroptosis-related caspases were activated in cells after stimulation, and then caspases hydrolyzed to gasdermin-D.Subsequently, gasdermin-N domain combined to cytomembrane to form membrane pores. The pores disrupt the osmotic potential and cause cell swelling and eventual lysis. The process was also accompanied by releasing of inflammatory cytokines. This review focused on the morphology, molecular mechanism of pyroptosis and its role in viral infections.