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1.
Int Immunopharmacol ; 139: 112710, 2024 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-39029229

RESUMO

PANoptosis is manifested with simultaneous activation of biomarkers for both pyroptotic, apoptotic and necroptotic signaling via the molecular platform PANoptosome and it is involved in pathologies of various inflammatory diseases including hemophagocytic lymphohistiocytosis (HLH). Scutellarin is a flavonoid isolated from herbal Erigeron breviscapus (Vant.) Hand.-Mazz. and has been shown to possess multiple pharmacological effects, but it is unknown whether scutellarin has any effects on PANoptosis and related inflammatory diseases. In this study, we found that scutellarin inhibited cell death in bone marrow-derived macrophages (BMDMs) and J774A.1 cells treated with TGF-ß-activated kinase 1 (TAK1) inhibitor 5Z-7-oxozeaenol (OXO) plus lipopolysaccharide (LPS), which has been commonly used to induce PANoptosis. Western blotting showed that scutellarin dose-dependently inhibited the activation biomarkers for pyroptotic (Caspase-1p10 and GSDMD-NT), apoptotic (cleaved Casp3/8/9 and GSDME-NT), and necroptotic (phosphorylated MLKL) signaling. The inhibitory effect of scutellarin was unaffected by NLRP3 or Caspase-1 deletion. Interestingly, scutellarin blocked the assembly of PANoptosome that encompasses ASC, RIPK3, Caspase-8 and ZBP1, suggesting its action on upstream signaling. Consistent with this, scutellarin inhibited mitochondrial damage and mitochondrial reactive oxygen species (mtROS) generation in cells treated with OXO+LPS. Further, mito-TEMPO that can scavenge mtROS significantly inhibited OXO+LPS-induced PANoptotic cell death. In line with the in vitro results, scutellarin markedly alleviated systemic inflammation, multiple organ injury, and activation of PANoptotic biomarkers in mice with HLH. Collectively, our data suggest that scutellarin can inhibit PANoptosis by suppressing mitochondrial damage and mtROS generation and thereby mitigating multiple organ injury in mice with inflammatory disorders.

2.
Int Immunopharmacol ; 130: 111680, 2024 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-38368772

RESUMO

Fulminant hepatitis (FH) is a severe clinical syndrome leading to hepatic failure and even mortality. D-galactosamine (D-GalN) plus lipopolysaccharide (LPS) challenge is commonly used to establish an FH mouse model, but the mechanism underlying D-GalN/LPS-induced liver injury is incompletely understood. Previously, it has been reported that extracellular ATP that can be released under cytotoxic and inflammatory stresses serves as a damage signal to induce potassium ion efflux and trigger the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome activation through binding to P2X7 receptor. In this study, we tried to investigate whether it contributed to the fulminant hepatitis (FH) induced by D-GalN plus LPS. In an in vitro cellular model, D-GalN plus extracellular ATP, instead of D-GalN alone, induced pyroptosis and apoptosis, accompanied by mitochondrial reactive oxygen species (ROS) burst, and the oligomerization of Drp1, Bcl-2, and Bak, as well as the loss of mitochondrial membrane potential in LPS-primed macrophages, well reproducing the events induced by D-GalN and LPS in vivo. Moreover, these events in the cellular model were markedly suppressed by both A-804598 (an ATP receptor P2X7R inhibitor) and glibenclamide (an ATP-sensitive potassium ion channel inhibitor); in the FH mouse model, administration of A-804598 significantly mitigated D-GalN/LPS-induced hepatic injury, mitochondrial damage, and the activation of apoptosis and pyroptosis signaling, corroborating the contribution of extracellular ATP to the cell death. Collectively, our data suggest that extracellular ATP acts as an autologous damage-associated molecular pattern to augment mitochondrial damage, hepatic cell death, and liver injury in D-GalN/LPS-induced FH mouse model.


Assuntos
Guanidinas , Lipopolissacarídeos , Necrose Hepática Massiva , Quinolinas , Camundongos , Animais , Espécies Reativas de Oxigênio/metabolismo , Lipopolissacarídeos/farmacologia , Galactosamina/farmacologia , Fígado/metabolismo , Apoptose , Trifosfato de Adenosina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
3.
Inflamm Res ; 73(4): 597-617, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38353723

RESUMO

OBJECTIVE: PANoptosis, a new form of regulated cell death, concomitantly manifests hallmarks for pyroptosis, apoptosis, and necroptosis. It has been usually observed in macrophages, a class of widely distributed innate immune cells in various tissues, upon pathogenic infections. The second-generation curaxin, CBL0137, can trigger necroptosis and apoptosis in cancer-associated fibroblasts. This study aimed to explore whether CBL0137 induces PANoptosis in macrophages in vitro and in mouse tissues in vivo. METHODS: Bone marrow-derived macrophages and J774A.1 cells were treated with CBL0137 or its combination with LPS for indicated time periods. Cell death was assayed by propidium iodide staining and immunoblotting. Immunofluorescence microscopy was used to detect cellular protein distribution. Mice were administered with CBL0137 plus LPS and their serum and tissues were collected for biochemical and histopathological analyses, respectively. RESULTS: The results showed that CBL0137 alone or in combination with LPS induced time- and dose-dependent cell death in macrophages, which was inhibited by a combination of multiple forms of cell death inhibitors but not each alone. This cell death was independent of NLRP3 expression. CBL0137 or CBL0137 + LPS-induced cell death was characterized by simultaneously increased hallmarks for pyroptosis, apoptosis and necroptosis, indicating that this is PANoptosis. Induction of PANoptosis was associated with Z-DNA formation in the nucleus and likely assembly of PANoptosome. ZBP1 was critical in mediating CBL0137 + LPS-induced cell death likely by sensing Z-DNA. Moreover, intraperitoneal administration of CBL0137 plus LPS induced systemic inflammatory responses and caused multi-organ (including the liver, kidney and lung) injury in mice due to induction of PANoptosis in these organs. CONCLUSIONS: CBL0137 alone or plus inflammatory stimulation induces PANoptosis both in vitro and in vivo, which is associated with systemic inflammatory responses in mice.


Assuntos
Carbazóis , DNA Forma Z , Neoplasias , Camundongos , Animais , Lipopolissacarídeos/farmacologia , Apoptose , Piroptose
4.
Acta Pharmacol Sin ; 45(3): 594-608, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37964019

RESUMO

PANoptosis is a new type of cell death featured with pyroptosis, apoptosis and necroptosis, and is implicated in organ injury and mortality in various inflammatory diseases, such as sepsis and hemophagocytic lymphohistiocytosis (HLH). Reverse electron transport (RET)-mediated mitochondrial reactive oxygen species (mtROS) has been shown to contribute to pyroptosis and necroptosis. In this study we investigated the roles of mtROS and RET in PANoptosis induced by TGF-ß-activated kinase 1 (TAK1) inhibitor 5Z-7-oxozeaenol (Oxo) plus lipopolysaccharide (LPS) as well as the effects of anti-RET reagents on PANoptosis. We showed that pretreatment with anti-RET reagents 1-methoxy PMS (MPMS) or dimethyl fumarate (DMF) dose-dependently inhibited PANoptosis in macrophages BMDMs and J774A.1 cells induced by Oxo/LPS treatment assayed by propidium iodide (PI) staining. The three arms of the PANoptosis signaling pathway, namely pyroptosis, apoptosis and necroptosis signaling, as well as the formation of PANoptosomes were all inhibited by MPMS or DMF. We demonstrated that Oxo/LPS treatment induced RET and mtROS in BMDMs, which were reversed by MPMS or DMF pretreatment. Interestingly, the PANoptosome was co-located with mitochondria, in which the mitochondrial DNA was oxidized. MPMS and DMF fully blocked the mtROS production and the formation of PANoptosome induced by Oxo plus LPS treatment. An HLH mouse model was established by poly(I:C)/LPS challenge. Pretreatment with DMF (50 mg·kg-1·d-1, i.g. for 3 days) or MPMS (10 mg·kg-1·d-1, i.p. for 2 days) (DMF i.g. MPMS i.p.) effectively alleviated HLH lesions accompanied by decreased hallmarks of PANoptosis in the liver and kidney. Collectively, RET and mtDNA play crucial roles in PANoptosis induction and anti-RET reagents represent a novel class of PANoptosis inhibitors by blocking oxidation of mtDNA, highlighting their potential application in treating PANoptosis-related inflammatory diseases. PANoptotic stimulation induces reverse electron transport (RET) and reactive oxygen species (ROS) in mitochondia, while 1-methoxy PMS and dimethyl fumarate can inhibit PANoptosis by suppressing RETmediated oxidation of mitochondrial DNA.


Assuntos
DNA Mitocondrial , Fumarato de Dimetilo , Animais , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Transporte de Elétrons , Fumarato de Dimetilo/metabolismo , Fumarato de Dimetilo/farmacologia , DNA Mitocondrial/metabolismo , Lipopolissacarídeos/farmacologia , Elétrons , Mitocôndrias , Apoptose
5.
Inflammation ; 47(1): 285-306, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37759136

RESUMO

Itaconate is an unsaturated dicarboxylic acid that is derived from the decarboxylation of the Krebs cycle intermediate cis-aconitate and has been shown to exhibit anti-inflammatory and anti-bacterial/viral properties. But the mechanisms underlying itaconate's anti-inflammatory activities are not fully understood. Necroptosis, a lytic form of regulated cell death (RCD), is mediated by receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL) signaling. It has been involved in the pathogenesis of organ injury in many inflammatory diseases. In this study, we aimed to explore whether itaconate and its derivatives can inhibit necroptosis in murine macrophages, a mouse MPC-5 cell line and a human HT-29 cell line in response to different necroptotic activators. Our results showed that itaconate and its derivatives dose-dependently inhibited necroptosis, among which dimethyl itaconate (DMI) was the most effective one. Mechanistically, itaconate and its derivatives inhibited necroptosis by suppressing the RIPK1/RIPK3/MLKL signaling and the oligomerization of MLKL. Furthermore, DMI promoted the nuclear translocation of Nrf2 that is a critical regulator of intracellular redox homeostasis, and reduced the levels of intracellular reactive oxygen species (ROS) and mitochondrial superoxide (mtROS) that were induced by necroptotic activators. Consistently, DMI prevented the loss of mitochondrial membrane potential induced by the necroptotic activators. In addition, DMI mitigated caerulein-induced acute pancreatitis in mice accompanied by reduced activation of the necroptotic signaling in vivo. Collectively, our study demonstrates that itaconate and its derivatives can inhibit necroptosis by suppressing the RIPK1/RIPK3/MLKL signaling, highlighting their potential applications for treating necroptosis-associated diseases.


Assuntos
Pancreatite , Proteínas Quinases , Succinatos , Camundongos , Humanos , Animais , Proteínas Quinases/metabolismo , Doença Aguda , Anti-Inflamatórios , Apoptose
6.
Free Radic Biol Med ; 212: 117-132, 2024 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-38151213

RESUMO

Damage-associated molecular patterns (DAMPs) such as extracellular ATP and nigericin (a bacterial toxin) not only act as potassium ion (K+) efflux inducers to activate NLRP3 inflammasome, leading to pyroptosis, but also induce cell death independently of NLRP3 expression. However, the roles of energy metabolism in determining NLRP3-dependent pyroptosis and -independent necrosis upon K+ efflux are incompletely understood. Here we established cellular models by pharmacological blockade of energy metabolism, followed by stimulation with a K+ efflux inducer (ATP or nigericin). Two energy metabolic inhibitors, namely CPI-613 that targets α-ketoglutarate dehydrogenase and pyruvate dehydrogenase (a rate-limiting enzyme) and 2-deoxy-d-glucose (2-DG) that targets hexokinase, are recruited in this study, and Nlrp3 gene knockout macrophages were used. Our data showed that CPI-613 and 2-DG dose-dependently inhibited NLRP3 inflammasome activation, but profoundly increased cell death in the presence of ATP or nigericin. The cell death was K+ efflux-induced but NLRP3-independent, which was associated with abrupt reactive oxygen species (ROS) production, reduction of mitochondrial membrane potential, and oligomerization of mitochondrial proteins, all indicating mitochondrial damage. Notably, the cell death induced by K+ efflux and blockade of energy metabolism was distinct from pyroptosis, apoptosis, necroptosis or ferroptosis. Furthermore, fructose 1,6-bisphosphate, a high-energy intermediate of glycolysis, significantly suppressed CPI-613+nigericin-induced mitochondrial damage and cell death. Collectively, our data show that energy deficiency diverts NLRP3 inflammasome activation-dependent pyroptosis to Nlrp3-independent necrosis upon K+ efflux inducers, which can be dampened by high-energy intermediate, highlighting a critical role of energy metabolism in cell survival and death under inflammatory conditions.


Assuntos
Caprilatos , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Sulfetos , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Inflamassomos/genética , Inflamassomos/metabolismo , Nigericina/farmacologia , Potássio/metabolismo , Necrose/genética , Metabolismo Energético/genética , Trifosfato de Adenosina/metabolismo , Interleucina-1beta/metabolismo , Espécies Reativas de Oxigênio/metabolismo
7.
Apoptosis ; 28(11-12): 1646-1665, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37702860

RESUMO

Macrophages represent the first lines of innate defense against pathogenic infections and are poised to undergo multiple forms of regulated cell death (RCD) upon infections or toxic stimuli, leading to multiple organ injury. Triptolide, an active compound isolated from Tripterygium wilfordii Hook F., possesses various pharmacological activities including anti-tumor and anti-inflammatory effects, but its applications have been hampered by toxic adverse effects. It remains unknown whether and how triptolide induces different forms of RCD in macrophages. In this study, we showed that triptolide exhibited significant cytotoxicity on cultured macrophages in vitro, which was associated with multiple forms of lytic cell death that could not be fully suppressed by any one specific inhibitor for a single form of RCD. Consistently, triptolide induced the simultaneous activation of pyroptotic, apoptotic and necroptotic hallmarks, which was accompanied by the co-localization of ASC specks respectively with RIPK3 or caspase-8 as well as their interaction with each other, indicating the formation of PANoptosome and thus the induction of PANoptosis. Triptolide-induced PANoptosis was associated with mitochondrial dysfunction and ROS production. PANoptosis was also induced by triptolide in mouse peritoneal macrophages in vivo. Furthermore, triptolide caused kidney and liver injury, which was associated with systemic inflammatory responses and the activation of hallmarks for PANoptosis in vivo. Collectively, our data reveal that triptolide induces PANoptosis in macrophages in vitro and exhibits nephrotoxicity and hepatotoxicity associated with induction of PANoptosis in vivo, suggesting a new avenue to alleviate triptolide's toxicity by harnessing PANoptosis.


Assuntos
Diterpenos , Fenantrenos , Camundongos , Animais , Apoptose , Macrófagos/metabolismo , Diterpenos/efeitos adversos , Diterpenos/metabolismo , Fenantrenos/toxicidade , Fenantrenos/metabolismo , Compostos de Epóxi/toxicidade , Compostos de Epóxi/metabolismo
8.
Acta Pharmacol Sin ; 44(10): 2019-2036, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37221235

RESUMO

Activation of NLR family pyrin domain-containing 3 (NLRP3) inflammasome plays important role in defending against infections, but its aberrant activation is causally linked to many inflammatory diseases, thus being a therapeutic target for these diseases. Theaflavin, one major ingredient of black tea, exhibits potent anti-inflammatory and anti-oxidative activities. In this study, we investigated the therapeutic effects of theaflavin against NLRP3 inflammasome activation in macrophages in vitro and in animal models of related diseases. We showed that theaflavin (50, 100, 200 µM) dose-dependently inhibited NLRP3 inflammasome activation in LPS-primed macrophages stimulated with ATP, nigericin or monosodium urate crystals (MSU), evidenced by reduced release of caspase-1p10 and mature interleukin-1ß (IL-1ß). Theaflavin treatment also inhibited pyroptosis as shown by decreased generation of N-terminal fragment of gasdermin D (GSDMD-NT) and propidium iodide incorporation. Consistent with these, theaflavin treatment suppressed ASC speck formation and oligomerization in macrophages stimulated with ATP or nigericin, suggesting reduced inflammasome assembly. We revealed that theaflavin-induced inhibition on NLRP3 inflammasome assembly and pyroptosis resulted from ameliorated mitochondrial dysfunction and reduced mitochondrial ROS production, thereby suppressing interaction between NLRP3 and NEK7 downstream of ROS. Moreover, we showed that oral administration of theaflavin significantly attenuated MSU-induced mouse peritonitis and improved the survival of mice with bacterial sepsis. Consistently, theaflavin administration significantly reduced serum levels of inflammatory cytokines including IL-1ß and attenuated liver inflammation and renal injury of mice with sepsis, concomitant with reduced generation of caspase-1p10 and GSDMD-NT in the liver and kidney. Together, we demonstrate that theaflavin suppresses NLRP3 inflammasome activation and pyroptosis by protecting mitochondrial function, thus mitigating acute gouty peritonitis and bacterial sepsis in mice, highlighting a potential application in treating NLRP3 inflammasome-related diseases.


Assuntos
Gota , Peritonite , Sepse , Camundongos , Animais , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Espécies Reativas de Oxigênio , Nigericina/uso terapêutico , Peritonite/tratamento farmacológico , Antioxidantes/uso terapêutico , Sepse/complicações , Sepse/tratamento farmacológico , Caspases , Trifosfato de Adenosina , Interleucina-1beta/metabolismo
9.
Int Immunopharmacol ; 117: 109974, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-37012867

RESUMO

Necroptosis is a necrotic form of regulated cell death, which is primarily mediated by the receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL) pathway in a caspase-independent manner. Necroptosis has been found to occur in virtually all tissues and diseases evaluated, including pancreatitis. Celastrol, a pentacyclic triterpene extracted from the roots of Tripterygium wilfordii (thunder god vine), possesses potent anti-inflammatory and anti-oxidative activities. Yet, it is unclear whether celastrol has any effects on necroptosis and necroptotic-related diseases. Here we showed that celastrol significantly suppressed necroptosis induced by lipopolysaccharide (LPS) plus pan-caspase inhibitor (IDN-6556) or by tumor-necrosis factor-α in combination with LCL-161 (Smac mimetic) and IDN-6556 (TSI). In these in vitro cellular models, celastrol inhibited the phosphorylation of RIPK1, RIPK3, and MLKL and the formation of necrosome during necroptotic induction, suggesting its possible action on upstream signaling of the necroptotic pathway. Consistent with the known role of mitochondrial dysfunction in necroptosis, we found that celastrol significantly rescued TSI-induced loss of mitochondrial membrane potential. TSI-induced intracellular and mitochondrial reactive oxygen species (mtROS), which are involved in the autophosphorylation of RIPK1 and recruitment of RIPK3, were significantly attenuated by celastrol. Moreover, in a mouse model of acute pancreatitis that is associated with necroptosis, celastrol administration significantly reduced the severity of caerulein-induced acute pancreatitis accompanied by decreased phosphorylation of MLKL in pancreatic tissues. Collectively, celastrol can attenuate the activation of RIPK1/RIPK3/MLKL signaling likely by attenuating mtROS production, thereby inhibiting necroptosis and conferring protection against caerulein-induced pancreatitis in mice.


Assuntos
Pancreatite , Camundongos , Animais , Pancreatite/induzido quimicamente , Pancreatite/tratamento farmacológico , Proteínas Quinases/metabolismo , Necroptose , Ceruletídeo , Doença Aguda , Triterpenos Pentacíclicos , Caspases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Apoptose
10.
Viruses ; 15(3)2023 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-36992326

RESUMO

Ranavirus is a large nucleocytoplasmic DNA virus. Chinese giant salamander iridovirus (CGSIV) belongs to the ranavirus genus, and its replication involves a series of essential viral genes. Viral PCNA is a gene closely associated with viral replication. CGSIV-025L also encodes PCNA-like genes. We have described the function of CGSIV-025L in virus replication. The promoter of CGSIV-025L is activated during viral infection, and it is an early (E) gene that can be effectively transcribed after viral infection. CGSIV-025L overexpression promoted viral replication and viral DNA replication. siRNA interfered with CGSIV-025L expression and attenuated viral replication and viral DNA replication. The Δ025L-CGSIV strain with the deletion of CGSIV-025L could not replicate normally and could be rescued by the replenishment of 025L. CGSIV-025L was proven to be an essential gene for CGSIV by overexpression, interference, and deletion mutation experiments. CGSIV-025L was found to interact with CGSIV-062L by yeast two-hybrid, CoIP, and GST pulldown. Thus, the current study demonstrated that CGSIV-025L is an essential gene of CGSIV, which may be involved in viral infection by participating in viral DNA replication and interacting with replication-related proteins.


Assuntos
Infecções por Vírus de DNA , Iridovirus , Ranavirus , Animais , Iridovirus/genética , Genes Essenciais , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/genética , DNA Viral/genética , Infecções por Vírus de DNA/veterinária , Replicação Viral , Ranavirus/genética , Genes Virais , Urodelos/genética
11.
Pharmacol Res ; 189: 106697, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36796462

RESUMO

Necroptosis has been implicated in various inflammatory diseases including tumor-necrosis factor-α (TNF-α)-induced systemic inflammatory response syndrome (SIRS). Dimethyl fumarate (DMF), a first-line drug for treating relapsing-remitting multiple sclerosis (RRMS), has been shown to be effective against various inflammatory diseases. However, it is still unclear whether DMF can inhibit necroptosis and confer protection against SIRS. In this study, we found that DMF significantly inhibited necroptotic cell death in macrophages induced by different necroptotic stimulations. Both the autophosphorylation of receptor-interacting serine/threonine kinase 1 (RIPK1) and RIPK3 and the downstream phosphorylation and oligomerization of MLKL were robustly suppressed by DMF. Accompanying the suppression of necroptotic signaling, DMF blocked the mitochondrial reverse electron transport (RET) induced by necroptotic stimulation, which was associated with its electrophilic property. Several well-known anti-RET reagents also markedly inhibited the activation of the RIPK1-RIPK3-MLKL axis accompanied by decreased necrotic cell death, indicating a critical role of RET in necroptotic signaling. DMF and other anti-RET reagents suppressed the ubiquitination of RIPK1 and RIPK3, and they attenuated the formation of necrosome. Moreover, oral administration of DMF significantly alleviated the severity of TNF-α-induced SIRS in mice. Consistent with this, DMF mitigated TNF-α-induced cecal, uterine, and lung damage accompanied by diminished RIPK3-MLKL signaling. Collectively, DMF represents a new necroptosis inhibitor that suppresses the RIPK1-RIPK3-MLKL axis through blocking mitochondrial RET. Our study highlights DMF's potential therapeutic applications for treating SIRS-associated diseases.


Assuntos
Proteínas Quinases , Fator de Necrose Tumoral alfa , Camundongos , Animais , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases/metabolismo , Fumarato de Dimetilo , Necroptose , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Síndrome de Resposta Inflamatória Sistêmica , Fosforilação Oxidativa , Apoptose
12.
Cell Mol Immunol ; 19(8): 925-943, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35799057

RESUMO

Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, has increased in incidence and prevalence in recent decades. Both clinical and animal studies are critical for understanding the pathogenesis of this disease. Dextran sodium sulfate (DSS)-induced colitis is a frequently used animal model of IBD, but the underlying mechanism of the model remains incompletely understood. In this study, we found that NOD-like receptor family pyrin containing 3 (NLRP3) depletion markedly mitigated DSS-induced colitis and was accompanied by decreased activation of the inflammasome in the colons of mice. However, in vitro assays showed that DSS did not directly trigger but instead potentiated NLRP3 inflammasome assembly in macrophages in response to suboptimal ATP or nigericin stimulation. Mechanistically, DSS potentiated NLRP3 inflammasome activation in macrophages by augmenting KCa3.1-mediated potassium ion (K+) efflux. Furthermore, we found that pharmacologic blockade of the K+ channel KCa3.1 with TRAM-34 or genetic depletion of the Kcnn4 gene (encoding KCa3.1) not only ameliorated the severity of DSS-induced colitis but also attenuated in vivo inflammasome assembly in the colonic tissues of mice, suggesting a causal link between KCa3.1-mediated augmentation of the NLRP3 inflammasome and DSS-induced inflammatory injuries. Collectively, these results indicate that KCa3.1 plays a critical role in mediating DSS-induced colitis in mice by potentiating NLRP3 inflammasome activation. Our data provide a previously unknown mechanism by which DSS induces colitis in mice and suggests that KCa3.1 is an alternative therapeutic target for treating IBD.


Assuntos
Colite , Doenças Inflamatórias Intestinais , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Animais , Colite/induzido quimicamente , Colite/patologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Inflamassomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Canais de Potássio , Sulfatos
13.
Int Immunopharmacol ; 108: 108885, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35623294

RESUMO

Necroptosis is a form of regulated necrosis mainly controlled by receptor-interacting protein kinases 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Necroptosis has important roles in defensing against pathogenic infections, but it is also implicated in various inflammatory diseases including pancreatitis. Baicalin, a flavonoid from Scutellaria baicalensis Georgi, has been shown to possess anti-inflammatory and anti-pyroptosis properties, yet it is unclear whether baicalin can inhibit necroptosis and confer protection against necroptosis-related diseases. Here we reported that baicalin significantly inhibited necroptosis in macrophages induced by lipopolysaccharide plus pan-caspase inhibitor (IDN-6556), or by tumor-necrosis factor-α in combination with LCL-161 (Smac mimetic) and IDN-6556 (TSI). Mechanistically, baicalin did not inhibit the phosphorylation of RIPK1, RIPK3 and MLKL, nor membrane translocation of p-MLKL, during necroptotic induction, but instead inhibited p-MLKL oligomerization that is required for executing necroptosis. As intracellular reactive oxygen species (ROS) has been reported to be involved in p-MLKL oligomerization, we assessed the effects of N-acetyl-L-cysteine (NAC), an ROS scavenger, on necroptosis and found that NAC significantly attenuated TSI-induced necroptosis and intracellular ROS production concomitantly with reduced levels of oligomerized p-MLKL, mirroring the effect of baicalin. Indeed, inhibitory effect of baicalin was associated with reduced TSI-induced superoxide (indicating mitochondrial ROS) production and increased mitochondrial membrane potential within cells during necroptosis. Besides, oral administration of baicalin significantly reduced the severity of caerulein-induced acute pancreatitis in mice, an animal model of necroptosis-related disease. Collectively, baicalin can inhibit necroptosis through attenuating p-MLKL oligomerization and confers protection against caerulein-induced pancreatitis in mice.


Assuntos
Necroptose , Pancreatite , Doença Aguda , Animais , Apoptose , Ceruletídeo/farmacologia , Flavonoides/farmacologia , Flavonoides/uso terapêutico , Camundongos , Necrose/tratamento farmacológico , Pancreatite/induzido quimicamente , Pancreatite/tratamento farmacológico , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo
14.
Int Immunopharmacol ; 108: 108867, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35605433

RESUMO

Dimethyl fumarate (DMF) is a fumaric acid derivative clinically approved for the treatment of some inflammatory diseases, but the underlying mechanism for its therapeutic effects remains incompletely understood. NLR family pyrin domain containing 3 (NLRP3) inflammasome activation has critical roles in innate immune responses to various infections and sterile inflammations. In this study, we aimed to explore whether DMF affects auto-immune hepatitis (AIH) in mice induced by concanavalin A (Con A) by modulating NLRP3 inflammasome activation. The results showed that DMF suppressed the activation of NLRP3 inflammasome activation in lipopolysaccharide-primed murine bone marrow-derived macrophages upon ATP or nigericin treatment, as evidenced by reduced cleavage of pro-caspase-1, release of mature interleukin-1ß (IL-1ß) and generation of gasdermin D N-terminal fragment (GSDMD-NT). DMF also greatly reduced ASC speck formation upon the stimulation of nigericin or ATP, indicating its inhibitory effect on NLRP3 inflammasome assembly. Consistent with reduced generation of GSDMD-NT, ATP or nigericin-induced pyroptosis was markedly suppressed by DMF. Moreover, DMF treatment alleviated mitochondrial damage induced by ATP or nigericin. Interestingly, all these effects were reversed by the protein kinase A (PKA) pathway inhibitors (H89 and MDL-12330A). Mechanistically, DMF enhanced PKA signaling and thus increased NLRP3 phosphorylation at PKA-specific sites to attenuate its activation. Importantly, DMF decreased serum levels of inflammatory cytokines and ameliorated liver injury in Con A-induced AIH of mice, concomitant with reduced the generation of caspase-1p10 and GSDMD-NT and alleviating mitochondrial aggregation in the liver. Collectively, DMF displayed anti-inflammatory effects by inhibiting NLRP3 inflammasome activation likely through regulating PKA signaling, highlighting its potential application in treating AIH.


Assuntos
Hepatite Autoimune , Inflamassomos , Trifosfato de Adenosina/farmacologia , Animais , Caspase 1/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico , Fumarato de Dimetilo/farmacologia , Fumarato de Dimetilo/uso terapêutico , Hepatite Autoimune/tratamento farmacológico , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nigericina/farmacologia , Nigericina/uso terapêutico
15.
Acta Biochim Biophys Sin (Shanghai) ; 54(1): 64-76, 2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35130622

RESUMO

Macrophages are critical sentinel cells armed with multiple regulated necrosis pathways, including pyroptosis, apoptosis followed by secondary necrosis, and necroptosis, and are poised to undergo distinct form(s) of necrosis for tackling dangers of pathogenic infection or toxic exposure. The natural BH3-mimetic gossypol is a toxic phytochemical that can induce apoptosis and/or pyroptotic-like cell death, but what exact forms of regulated necrosis are induced remains largely unknown. Here we demonstrated that gossypol induces pyroptotic-like cell death in both unprimed and lipopolysaccharide-primed mouse bone marrow-derived macrophages (BMDMs), as evidenced by membrane swelling and ballooning accompanied by propidium iodide incorporation and lactic acid dehydrogenase release. Notably, gossypol simultaneously induces the activation of both pyroptotic and apoptotic (followed by secondary necrosis) pathways but only weakly activates the necroptosis pathway. Unexpectedly, gossypol-induced necrosis is independent of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, as neither inhibitor for the NLRP3 pathway nor NLRP3 deficiency protects the macrophages from the necrosis. Furthermore, necrotic inhibitors or even pan-caspase inhibitor alone does not or only partly inhibit such necrosis. Instead, a combination of inhibitors composed of pan-caspase inhibitor IDN-6556, RIPK3 inhibitor GSK'872 and NADPH oxidase inhibitor GKT137831 not only markedly inhibits the necrosis, with all apoptotic and pyroptotic pathways being blocked, but also attenuates gossypol-induced peritonitis in mice. Lastly, the activation of the NLRP3 pathway and apoptotic caspase-3 appears to be independent of each other. Collectively, gossypol simultaneously induces the activation of multiple subroutines of regulated necrosis in macrophages depending on both apoptotic and inflammatory caspases.


Assuntos
Gossipol , Animais , Apoptose , Caspase 1/metabolismo , Gossipol/metabolismo , Gossipol/farmacologia , Inflamassomos/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Necrose/induzido quimicamente , Necrose/metabolismo
16.
Int Immunopharmacol ; 102: 108380, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34848154

RESUMO

Discovery of anti-inflammatory drugs that can suppress T lymphocyte activation and proliferation by inhibiting TCR/CD3 and IL-2/IL-2R signaling is still needed in clinic, though rapamycin and other related reagents have made great success. Taraxasterol (TAS) is an active ingredient of dandelion, an anti-inflammatory medicinal herb with low in vivo toxicity that has long been used in China. Yet the action mechanism of TAS on lymphocytes remains elusive. The anti-inflammatory effects of TAS were evaluated in C57BL/6 mouse primary lymphocytes stimulated with concanavalin A (Con A) in vitro and in mouse model of Con A-induced acute hepatitis in vivo. Our results showed that TAS significantly suppressed Con A-induced acute hepatitis in a mouse model, reducing the hepatic necrosis areas, the release of aminotransferases, and the production of IL-2 and other inflammatory cytokines. Supporting this, in vitro study also showed that TAS reduced the production of IL-2 and the expression of IL-2 receptor subunit α (CD25) upon the stimulation of Con A, which was likely mediated by suppressing NF-κB activation. The downstream pathways of IL-2/IL-2R signaling, including the activation of PI3K/PDK1/mTOR, STAT3 and STAT5, were also suppressed by TAS. Consistently, Con A-induced T cell proliferation was also inhibited by TAS in vitro. Our data indicate that TAS can suppress both T lymphocyte activation and cell proliferation by down-regulating IL-2 expression and its signaling pathway thereby ameliorating Con A-induced acute hepatitis, highlighting TAS as a potential drug candidate for treating inflammatory diseases including autoimmune hepatitis.


Assuntos
Anti-Inflamatórios/uso terapêutico , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Interleucina-2/imunologia , Esteróis/uso terapêutico , Linfócitos T/efeitos dos fármacos , Triterpenos/uso terapêutico , Animais , Anti-Inflamatórios/farmacologia , Proliferação de Células/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Concanavalina A , Citocinas/sangue , Feminino , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/patologia , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Esteróis/farmacologia , Linfócitos T/imunologia , Triterpenos/farmacologia
17.
Acta Pharmacol Sin ; 43(5): 1324-1336, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-34376811

RESUMO

Monosodium urate (MSU) crystals, the etiological agent of gout, are formed in joints and periarticular tissues due to long-lasting hyperuricemia. Although MSU crystal-triggered NLRP3 inflammasome activation and interleukin 1ß (IL-1ß) release are known to have key roles in gouty arthritis, recent studies revealed that MSU crystal-induced necrosis also plays a critical role in this process. However, it remains unknown what forms of necrosis have been induced and whether combined cell death inhibitors can block such necrosis. Here, we showed that MSU crystal-induced necrosis in murine macrophages was not dependent on NLRP3 inflammasome activation, as neither genetic deletion nor pharmacological blockade of the NLRP3 pathway inhibited the necrosis. Although many cell death pathways (such as ferroptosis and pyroptosis) inhibitors or reactive oxygen species inhibitors did not have any suppressive effects, necroptosis pathway inhibitors GSK'872 (RIPK3 inhibitor), and GW806742X (MLKL inhibitor) dose-dependently inhibited MSU crystal-induced necrosis. Moreover, a triple combination of GSK'872, GW806742X, and IDN-6556 (pan-caspase inhibitor) displayed enhanced inhibition of the necrosis, which was further fortified by the addition of MCC950 (NLRP3 inhibitor), suggesting that multiple cell death pathways might have been triggered by MSU crystals. Baicalin, a previously identified inhibitor of NLRP3, inhibited MSU crystal-induced inflammasome activation and suppressed the necrosis in macrophages. Besides, baicalin gavage significantly ameliorated MSU crystal-induced peritonitis in mice. Altogether, our data indicate that MSU crystals induce NLRP3-independent necrosis, which can be inhibited by combined inhibitors for multiple signaling pathways, highlighting a new avenue for the treatment of gouty arthritis.


Assuntos
Artrite Gotosa , Gota , Animais , Artrite Gotosa/induzido quimicamente , Artrite Gotosa/tratamento farmacológico , Artrite Gotosa/metabolismo , Gota/tratamento farmacológico , Gota/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Necrose/induzido quimicamente , Necrose/tratamento farmacológico , Transdução de Sinais , Ácido Úrico
18.
Genes (Basel) ; 14(1)2022 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-36672799

RESUMO

The DNAJ family, a class of chaperone proteins involved in protein folding, assembly, and transport, plays an essential role in viral infections. However, the role of DNAJA4 (DnaJ Heat Shock Protein Family (Hsp40) Member A4) in the ranavirus infection has not been reported. This study demonstrates the function of the epithelial papilloma of carp (EPC) DNAJA4 in Chinese giant salamander (Andrias davidianus) iridovirus (CGSIV) replication. DNAJA4 consists of 1479 base pairs and encodes a 492 amino acid polypeptide. Sequence analysis has shown that EPC DNAJA4 contains a conserved J domain and shares 84% homology with Danio rerio DNAJA4 and 68% homology with Homo sapiens DNAJA4. EPC DNAJA4 was localized in the cytoplasm, and its expression was significantly upregulated after CGSIV infection. Overexpression of EPC DNAJA4 promotes CGSIV replication and CGSIV DNA replication. siRNA knockdown of DNAJA4 expression attenuates CGSIV replication and viral DNA replication. Overexpression and interference experiments have proved that EPC DNAJA4 is a pro-viral factor. Co-IP, GST-pulldown, and immunofluorescence confirmed the interaction between EPC DNAJA4 and CGSIV proliferating cell nuclear antigen (PCNA). Our results demonstrate for the first time that EPC DNAJA4 is involved in viral infection by promoting viral DNA replication and interacting with proteins associated with viral replication.


Assuntos
Iridovirus , Humanos , Animais , Iridovirus/genética , Replicação do DNA/genética , Replicação Viral/genética , DNA Viral/genética , Urodelos/genética , Proteínas de Choque Térmico HSP40
19.
Methods Mol Biol ; 2321: 43-51, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34048006

RESUMO

Mouse models of bacterial sepsis are widely used in research to investigate the underlying molecular mechanisms of sepsis and to develop clinically useful therapeutic regimens. Three commonly used mouse sepsis models include (a) injection of bacterial endotoxin, (b) infusion of cultured bacteria, and (c) cecal ligation and puncture. Here we describe the induction of bacterial sepsis in mice by intraperitoneal injection of cultured live Escherichia coli cells. The severity of the sepsis can be regulated by the number of E. coli cells injected into the peritoneal cavity of mice.


Assuntos
Escherichia coli/crescimento & desenvolvimento , Sepse/microbiologia , Animais , Ceco/microbiologia , Modelos Animais de Doenças , Endotoxinas/administração & dosagem , Injeções Intraperitoneais/métodos , Ligadura/métodos , Camundongos , Camundongos Endogâmicos C57BL , Cavidade Peritoneal/microbiologia , Punções/métodos
20.
Inflammation ; 44(4): 1229-1245, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34009550

RESUMO

The NLRP3 inflammasome is a multi-molecular complex that acts as a molecular platform to mediate caspase-1 activation, leading to IL-1ß/IL-18 maturation and release in cells stimulated by various pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). This inflammasome plays an important role in the innate immunity as its activation can further promote the occurrence of inflammation, enhance the ability of host to remove pathogens, and thus facilitate the repair of injured tissues. But if the inflammasome activation is dysregulated, it will cause the development of various inflammatory diseases and metabolic disorders. Therefore, under normal conditions, the activation of inflammasome is tightly regulated by various positive and negative signaling pathways to respond to the stimuli without damaging the host itself while maintaining homeostasis. In this review, we summarize recent advances in the major signaling pathways (including TLRs, MAPK, mTOR, autophagy, PKA, AMPK, and IFNR) that regulate NLRP3 inflammasome activation, providing a brief view of the molecular network that regulates this inflammasome as a theoretical basis for therapeutic intervention of NLRP3 dysregulation-related diseases.


Assuntos
Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Transdução de Sinais/fisiologia , Quinases Proteína-Quinases Ativadas por AMP/metabolismo , Animais , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Serina-Treonina Quinases TOR/metabolismo
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