RESUMO
5-Methyluridine (5-MU) is a prominent intermediate for industrial synthesis of several antiviral-drugs, however, its availability over the past decades has overwhelmingly relied on chemical and enzymatic strategies. Here, we have realized efficient production of 5-MU in E. coli, for the first time, via a designer artificial pathway consisting of a two-enzyme cascade (UMP 5-methylase and phosphatase). More importantly, we have engineered the E. coli cell factory to boost 5-MU production by systematic evaluation of multiple strategies, and as a proof of concept, we have further developed an antibiotic-free fermentation strategy to realize 5-MU production (10.71 g/L) in E. coli MB229 (a ΔthyA strain). Remarkably, we have also established a versatile and robust platform with exploitation of the engineered E. coli for efficient production of diversified UMP-derived chemicals. This study paves the way for future engineering of E. coli as a synthetic biology platform for acceleratively accessing UMP-derived chemical diversities.
Assuntos
Escherichia coli , Engenharia Metabólica , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
BACKGROUND AND PURPOSE: Bispectral Index (BIS) and University of Michigan Sedation Scale (UMSS) were two commonly used methods of monitoring the sedation depth, but their correlation was not clear. The purpose of this study is to ascertain if BIS correlates with UMSS in determining the sedation level during pediatric drug-induced sleep endoscopy (DISE). METHODS: One-hundred children, aged 36-143 months, with ASA I~II grade, were enrolled. They were subject to general anesthesia for an elective adenotonsillectomy. Two drug regimens were used. After UMSS ≥ 3, the sites of airway obstructions were located by checking the supraglottic airway structures with a fibrous laryngoscope. UMSS scores, BIS values, electromyography (EMG), and signal quality indices (SQIs) were recorded at the pre-medication and pre-DISE baseline (T0), 5 min subsequent to medication administration but prior to DISE initiation (T1), 1 min after DISE was initiated (T2), 1 min after DISE was completed (T3), 1 min subsequent to tracheal intubation (T4), 1 min following extubation (T5), and 30 min past extubation (T6). RESULTS: There were strong correlations between BIS monitor readings and UMSS scores for total and two regimens. Kappa values revealed moderate agreement between BIS and UMSS for total and two regimens. The agreement rates were 67.47% for the total, 61.43% for Regimen 1, and 73.42% for Regimen 2, respectively. CONCLUSION: BIS correlates with UMSS in determining the sedation level during pediatric DISE for two regimens. BIS might serve as an appropriate indicator of sedation intensity when UMSS could not be used.
Assuntos
Sedação Consciente , Endoscopia , Tonsilectomia , Humanos , Masculino , Feminino , Criança , Pré-Escolar , Adenoidectomia , Hipnóticos e Sedativos/administração & dosagem , Monitores de Consciência , Anestesia Geral , EletromiografiaRESUMO
Mounting attention has been focused on defects of the autophagy-lysosomal pathway in sepsis, however, the precise mechanisms governing the autophagy-lysosomal process in sepsis are poorly known. We have previously reported that Erbin deficiency aggravated the inflammatory response and organ injuries caused by sepsis. In the present study, we found that Erbin knockout impaired the autophagy process in both muramyl dipeptide (MDP)-induced bone marrow-derived macrophages (BMDMs) and sepsis mouse liver and lung, as detected by the accumulation of LC3-II and SQSTM1/p62, and autophagosomes. Pretreatment with autophagy inhibitor chloroquine (CQ) further aggravated inflammatory response and organ injuries in vivo and in vitro sepsis model. We also observed that the impaired lysosomal function mediated autophagic blockade, as detected by the decreased expression of ATP6V, cathepsin B (CTSB) and LAMP2 protein. Immunoprecipitation revealed that the C-terminal of Erbin (aa 391-964) interacts with the N-terminal of transcription factor EB (TFEB) (aa 1-247), and affects the stability of TFEB-14-3-3 and TFEB-PPP3CB complexes and the phosphorylation status of TFEB, thereby promote the nucleus translocation of TFEB and the TFEB target genes transcription. Thus, our study suggested that Erbin alleviated sepsis-induced inflammatory responses and organ injuries by rescuing dysfunction of the autophagy-lysosomal pathway through TFEB-14-3-3 and TFEB-PPP3CB pathway.
Assuntos
Núcleo Celular , Peptídeos e Proteínas de Sinalização Intracelular , Sepse , Animais , Camundongos , Autofagossomos/metabolismo , Autofagia/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Núcleo Celular/metabolismo , Inflamação/etiologia , Inflamação/genética , Inflamação/metabolismo , Lisossomos/genética , Lisossomos/metabolismo , Sepse/complicações , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
Sepsis-associated encephalopathy (SAE) is a serious and common complication of sepsis. To study the ferroptosis in the pathogenesis of SAE and demonstrate the protection effect of ferroptosis resistance, cognitive function, neurological deficits, blood-brain barrier integrity and neuroinflammation were detected. SAE model was established by cecal ligation and puncture (CLP) in mice and an in vitro model was created by introducing LPS to HT22 cells. Ferroptosis inducer Fe-citrate (Fe) and ferroptosis inhibitor ferrostatin-1 (Fer-1) was post-treated in the models, respectively. SAE caused ferroptosis, as evidenced by an increase in reactive oxygen species (ROS), iron content and malondialdehyde (MDA) and a decrease in glutathione (GSH) level, as well as changes in the expression of ferroptosis-related proteins as acyl-CoA synthetase long-chain family member 4 (ACSL4), glutathione peroxidase 4 (GPX4), and cystine-glutamate antiporter (SLC7A11), and harmed mitochondrial function. In contrast, inhibiting ferroptosis with Fer-1 attenuated ferroptosis. Meanwhile, Fer-1 attenuated neurologic severity score, learning and memory impairment, Fluoro-Jade C (FJC) staining, and decreased Evans Blue (EB) extravasation, microglia activation and TNF-α and IL-1ß production following SAE. The benefit of Fer-1 was diminished by ferroptosis inducer Fe. In addition, Fer-1 up-regulated the nuclear factor erythroid-2-related factor 2 (Nrf2)/ heme oxygenase-1(HO-1) signaling axis both in vivo and in vitro. In conclusion, our study revealed that Fer-1 might inhibit feroptosis in neurons by triggering the Nrf2/OH-1 pathway, thereby providing a therapeutic solution for SAE.
Assuntos
Ferroptose , Encefalopatia Associada a Sepse , Animais , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Cicloexilaminas/farmacologiaRESUMO
BACKGROUND: This study aims to investigate the correlation between Erbin and sepsis, and the role of Erbin on the pyroptosis pathway in acute kidney injury caused by sepsis and NLRP3/caspase-1/Gasdermin D pathway. METHODS: In the study, lipopolysaccharide (LPS) treatment or cecal ligation and puncture (CLP) surgery on mice were used to stimulate the in vitro and in vivo sepsis-induced renal injury model. The male C57BL/6 of wild-type mice (WT) and Erbin-knockout mice (Erbin-/-, EKO) were randomly divided into four groups (WT + Sham, WT + CLP, EKO + Sham, EKO + CLP). Inflammatory cytokine, renal function, pyroptotic cell numbers and the levels of protein and mRNA expression of pyroptosis, including the NLRP3 (all P < 0.05), were analyzed and found increase in Erbin-/- mice with CLP and LPS-induced HK-2 cells. RESULTS: The inhibited of Erbin shows a renal damaged effect by promoting NLRP3 inflammasome-mediated pyroptosis in SI-AKI. CONCLUSION: This study demonstrated a novel mechanism by which Erbin regulates NLRP3 inflammasome-mediated pyroptosis in SI-AKI.
Assuntos
Injúria Renal Aguda , Sepse , Animais , Masculino , Camundongos , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , Caspase 1/metabolismo , Caspase 1/farmacologia , Gasderminas , Inflamassomos/metabolismo , Inflamassomos/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose , Sepse/complicaçõesRESUMO
BACKGROUND: Microglia pyroptosis-mediated neuroinflammation is thought to be the crucial pathogenesis of sepsis-associated encephalopathy (SAE). Erbin has been reported to be associated with various inflammatory diseases. However, the role of Erbin in SAE and the relationship between Erbin and microglia pyroptosis are unknown. In this study, we investigated the promising role and underlying molecular mechanism of Erbin in the regulation of microglia pyroptosis. METHODS: WT and Erbin knockout mice underwent cecum ligation perforation (CLP) to induce SAE. Primary mouse microglia and BV2 cells were treated with LPS/nigericin in vitro. Behavioral tests were performed to evaluate cognitive function. Nissl staining and transmission electron microscopy were used to assess histological and structural lesions. ELISA and qPCR were carried out to detect neuroinflammation. Western blot and immunofluorescence were used to analyze protein expression. Flow cytometry and confocal microscopy were utilized to observe the Ca2+ changes in the cytoplasm and endoplasmic reticulum (ER). To further explore the underlying mechanism, STF083010 was administered to block the IRE1α/Xbp1s pathway. RESULTS: Erbin deletion resulted in more pronounced neuronal damage and cognitive impairment in mice that underwent CLP. Erbin knockout promoted microglial pyroptosis and inflammatory cytokines secretion in vivo and in vitro, which was mediated by activation of the IRE1α/Xbp1s. Treatment with the selective inhibitor STF083010 significantly inhibited IRE1α/Xbp1s pathway activity, decreased intracytoplasmic Ca2+, attenuated microglial pyroptosis, reduced pro-inflammatory cytokine secretion, lessened neuronal damage, and improved cognitive function. CONCLUSIONS: In SAE, Erbin inhibits IRE1/Xbp1s pathway activity and reduces the ER Ca2+ influx to the cytoplasm, reducing microglial pyroptosis.
Assuntos
Encefalopatia Associada a Sepse , Animais , Citocinas/metabolismo , Endorribonucleases , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Knockout , Microglia/metabolismo , Nigericina , Proteínas Serina-Treonina Quinases/genética , Piroptose/fisiologia , Encefalopatia Associada a Sepse/metabolismo , Sulfonamidas , TiofenosRESUMO
BACKGROUND: Oxycodone is a synthetic opioid receptor agonist that exerts antinociceptive activity via κ-, µ- and δ-opioid receptors (KOR, MOR and DOR, respectively). Activation of MOR has been reported to provide protection against acute lung injury (ALI). We hypothesized that pretreatment with oxycodone would attenuate lung injury at the level of alveolar tight junctions (TJs) and aquaporins (AQPs) and investigated this possibility in a two-hit model of ALI induced by lipopolysaccharide (LPS) and mechanical ventilation (MV). METHOD: Male Sprague Dawley rats and A59 cells were divided into 6 groups: the control group, ALI group, oxycodone-pretreated group, and oxycodone/κ-, µ-, or δ-opioid receptor antagonist-pretreated groups. The rats were pretreated with oxycodone 30 min before intravenous injection of LPS and then allowed to recover for 24 h prior to MV, establishing a two-hit model of ALI. The cells were similarly treated with oxycodone (with or without antagonists) 30 min after exposure to lipopolysaccharide. The cells were cyclically stretched 24 h later to mirror the in vivo MV protocol. RESULTS: Oxycodone alleviated the histological lung changes in the rats with ALI and decreased pulmonary microvascular permeability both in vivo and in vitro. Oxycodone upregulated the expression of claudin-5, ZO-1, AQP1, and AQP5 but downregulated the expression of TNF-α, IL-1ß, TLR4, NF-κB, MMP9, and caspase-3 and suppressed endothelial apoptosis in vivo and in vitro. These protective effects of oxycodone were partly eliminated by KOR and MOR antagonists but not by DOR antagonists. CONCLUSION: Oxycodone pretreatment appears to act via κ- and µ-opioid receptors to ameliorate LPS- and MV-induced lung injury by suppressing inflammation and apoptosis, and this protective effect might be mediated through the inhibition of the TLR4/NF-κB pathways.
Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Inflamação/tratamento farmacológico , Lipopolissacarídeos/efeitos adversos , Pulmão/efeitos dos fármacos , NF-kappa B/metabolismo , Oxicodona/farmacologia , Respiração Artificial/efeitos adversos , Receptor 4 Toll-Like/metabolismo , Analgésicos/farmacologia , Animais , Apoptose , Aquaporinas/metabolismo , Linhagem Celular , Células Endoteliais/metabolismo , Epitélio/metabolismo , Humanos , Técnicas In Vitro , Pulmão/metabolismo , Masculino , Microcirculação , Permeabilidade , Ratos , Ratos Sprague-Dawley , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , Transdução de Sinais , Junções Íntimas/metabolismoRESUMO
BACKGROUND: Previous studies showed that GTS-21, a selective alpha 7 nAchR agonist, can trigger anti-inflammatory effects and improve the survival of septic animals. However, whether GTS-21 affects autophagy responses remains unclear. Here, we tested the hypothesis that GTS-21 ameliorates sepsis-induced hepatic injury by modulating autophagy in mice. METHOD: C57BL/6 male mice were randomly separated and categorized into four groups: the sham group, and CLP group subjected to caecal ligation and puncture (CLP, a model of polymicrobial sepsis). The CLP + GTS-21 group was administered GTS-21 immediately after CLP challenge. α-Bungarotoxin (an alpha 7 nAchR antagonist) was injected before CLP was performed, and then, after CLP challenge, GTS-21 was administered to α-BGT + CLP + GTS-21 group. The hepatic tissue and blood samples were harvested 6 h after the operation. RESULTS: CLP challenge increased TNF-α and IL-6 production, and hepatic enzyme alanine aminotransferase and aspartate transaminase levels. CLP also elevated the expression of hepatic LC3-II, sequestosome-1/p62, Atg7 and Atg5. The administration of GTS-21 inhibited pro-inflammatory cytokine production and hepatic enzymatic marker expression, promoted the expression of LC3-II, Atg7, Atg5, and decreased the expression of p62, which could be reversed by α-BGT treatment. CONCLUSION: Our findings suggested that α7nAchR is involved in diminishing hepatic damage by inhibiting inflammatory responses and improving autophagy in mice with polymicrobial sepsis.
Assuntos
Autofagia/efeitos dos fármacos , Compostos de Benzilideno/farmacologia , Hepatopatias/tratamento farmacológico , Hepatopatias/metabolismo , Piridinas/farmacologia , Sepse/tratamento farmacológico , Sepse/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucina-6/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/metabolismoRESUMO
AIMS: The purpose of the present study was to investigate the possible role of TIPE2 on acute lung injury (ALI) induced by myocardial ischemia/reperfusion (MIR) in diabetic rats. METHODS: Sprague-Dawley (SD) rats were randomly separated into four groups: control+sham (C + sham); control+MIR (C + MIR); diabetes+sham (D + sham); diabetes+MIR (D + MIR). Diabetes was induced using streptozotocin. Eight weeks after diabetes induction, MIR was conducted. At 2 h after MIR, myocardial injury indices were assessed; arterial blood, bronchoalveolar lavage fluid (BALF) and lung tissues were collected for corresponding detection. RESULTS: Rats subjected to MIR showed serious ALI (estimated via pathological changes, lung injury score and Wet/Dry weight ratio), lung inflammation and pulmonary cell apoptosis compared with sham groups, especially in D + MIR group. Evaluation of protein expression in lung tissues showed that p-JNK and nuclear NF-κB p65 protein levels were higher in D + MIR group as compared with C + MIR group. Besides, either hyperglycemia or MIR can significantly upregulate TIPE2 protein levels. CONCLUSIONS: In conclusion, diabetic lungs are more susceptible to MIR. TIPE2 may involve in this pathological process, possibly through regulation of inflammation, oxidative stress and apoptosis.
Assuntos
Lesão Pulmonar Aguda/etiologia , Diabetes Mellitus Experimental/complicações , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/metabolismo , Traumatismo por Reperfusão Miocárdica/complicações , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Animais , Apoptose , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Mediadores da Inflamação/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Pulmão/patologia , Masculino , Traumatismo por Reperfusão Miocárdica/metabolismo , Estresse Oxidativo , Fosforilação , Ratos Sprague-Dawley , Transdução de Sinais , Estreptozocina , Fator de Transcrição RelA/metabolismoRESUMO
BACKGROUND: DnaJ/Hsp40 homolog, subfamily B, member 6 (DNAJB6) is significantly down-regulated in esophageal squamous cell carcinoma (ESCC), while its complicated molecular mechanisms are still unknown. AIMS: To investigate the relationship between DNAJB6 and ESCC. METHODS: The expression of DNAJB6 was detected in ESCC patient by Western blot and immunohistochemistry. To overexpress DNAJB6a by lentivirus infection, colony-forming, CCK-8, transwell, mouse xenograft assays were utilized to verify the proliferous, invasive, and migratory role of DNAJB6a in ESCC cells. The MDA and GSH assays determine whether DNAJB6a participates in cell redox reaction. The variation of AKT and GPX4 was detected by Western blot. RESULTS: The correlation between DNAJB6 level and lymph node metastasis in ESCC patient was negative. Overexpressing DNAJB6a shows tumor-suppressive effects in vitro and in vivo. In addition, DNAJB6a overexpression was accompanied together with a remarkable reduction in the protein levels of GPX4 and phosphorylated AKT (p-AKT). CONCLUSION: DNAJB6 plays an important anti-oncogenic role in ESCC evolvement via ferroptosis.
Assuntos
Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Proteínas de Choque Térmico HSP40/genética , Chaperonas Moleculares/genética , Proteínas do Tecido Nervoso/genética , Animais , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Ferroptose/genética , Glutationa/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Mitocôndrias/ultraestrutura , Chaperonas Moleculares/metabolismo , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Proteínas do Tecido Nervoso/metabolismo , Oxirredução , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: Tumour necrosis factor-α-induced protein 8-like 2 (TIPE2) has strong anti-inflammatory properties. However, it is unknown whether increased TIPE2 is protective against lipopolysaccharide (LPS)-induced ALI. In the current study, we aimed to investigate whether increased TIPE2 can exert protective effects in a mouse model of ALI induced by LPS. METHODS: We administered TIPE2 adeno-associated virus (AAV-TIPE2) intratracheally into the lungs of mice. Three weeks later, ALI was induced by intratracheal injection of LPS into BALB/c mice. Twenty-four hours later, lung bronchoalveolar lavage fluid (BALF) was acquired to analyse cells and protein, arterial blood was collected for arterial blood gas analysis and the determination of pro-inflammatory factor levels, and lung issues were collected for histologic examination, transmission electron microscopy (TEM), TUNEL staining, wet/dry (W/D) weight ratio analysis, myeloperoxidase (MPO) activity analysis and blot analysis of protein expression. RESULTS: We found that TIPE2 overexpression markedly mitigated LPS-induced lung injury, which was evaluated by the deterioration of histopathology, histologic scores, the W/D weight ratio, and total protein expression in the BALF. Moreover, TIPE2 overexpression markedly attenuated lung inflammation, as evidenced by the downregulation of polymorphonuclear neutrophils (PMNs) in the BALF, lung MPO activity, and pro-inflammatory cytokine levels in the serum. Moreover, TIPE2 overexpression not only dramatically prevented LPS-induced pulmonary cell apoptosis in mice but also blocked LPS-activated JNK phosphorylation and NF-κB p65 nuclear translocation. CONCLUSIONS: Our study shows that the increased expression of AAV-mediated TIPE2 in the lungs of mice inhibits acute inflammation and apoptosis and suppresses the activation of NF-κB and JNK in a murine model of ALI.
Assuntos
Lesão Pulmonar Aguda/terapia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/patologia , Animais , Apoptose , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/sangue , Dependovirus/genética , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Inflamação/terapia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno/imunologia , Contagem de Leucócitos , Lipopolissacarídeos , Pulmão/patologia , Pulmão/ultraestrutura , Masculino , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , NF-kappa B/imunologia , Transdução GenéticaRESUMO
This study aimed to investigate the protective effects of ketamine against hepatic ischemia-reperfusion (I/R) injury by suppressing activation of Kupffer cells (KCs) in rat liver autotransplantation. Male Sprague-Dawley rats were randomized into 3 groups (n = 10 each). Group I, the sham group, received saline. Group II received saline and underwent orthotopic liver autotransplantation (OLAT). Group III received 10 mg/kg ketamine and underwent OLAT. Blood samples were obtained at 3, 6, 12, and 24 h after I/R, and following ALT, AST, LDH, IL-6, TNF-α, IL-1ß, and IL-10 in serum were detected. Model rats were sacrificed at the indicated time points and the graft liver tissues were evaluated histologically. KCs were isolated from rat liver tissues, and inflammatory products and proteins of NF-κB signaling pathway were detected using quantitative RT-PCR and Western blotting. Our results showed that ketamine significantly decreased ALT, AST, LDH, IL-6, TNF-α, and IL-1ß levels and increased IL-10 level. Furthermore, ketamine alleviated the histopathology changes, by less KC infiltration and lower hepatocyte apoptosis. Moreover, activity of NF-κB signaling pathway in KCs was suppressed. In addition, production of pro- and anti-inflammatory factors is consistent with the results in tissues. Ketamine ameliorated I/R injury after liver transplantation by suppressing activation of KCs in rats.
Assuntos
Ketamina/farmacologia , Células de Kupffer/efeitos dos fármacos , Transplante de Fígado/efeitos adversos , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/patologia , Animais , Apoptose/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Ketamina/uso terapêutico , Células de Kupffer/metabolismo , Masculino , NF-kappa B/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transplante Autólogo/efeitos adversosRESUMO
Sepsis-induced acute lung injury has been deemed to be an life-threatening pulmonary dysfunction caused by a dysregulated host response to infection. The modification of N6-Methyladenosine (m6A) is implicated in several biological processes, including mitochondrial transcription and ferroptosis. Ferroptosis is an iron-dependent type of programed cell death, which plays a role in sepsis-induced acute lung injury (ALI). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial regulator of intracellular oxidative homeostasis, linked to ferroptosis resistance. This research aims to explore the effect of m6A in ferroptosis in sepsis-induced ALI. First, we found a time-dependent dynamic alteration on pulmonary methylation level during sepsis-induced ALI. We identified METTL4 as a differentially expressed gene in ALI mice using m6A sequencing and RNA-sequencing, and revealed the methylation of several ferroptosis related genes (Nrf2). Thus, we generated METTL4 deficiency mice and found that METTL4 knockdown alleviated ferroptosis, as evidenced by lipid ROS, MDA, Fe2+, as well as alterations in GPX4 and SLC7A11 protein expression. Consistently, we found that METTL4 silencing could decrease ferroptosis sensitivity in LPS-induced TC-1 cells. Furthermore, both the dual-luciferase reporter assay and rescue experiments indicated that METTL4 mediated the N6-methyladenosine of Nrf2 3'UTR, then YTHDF2 binded with the m6A site, promoting the degradation of Nrf2. In conclusion, we revealed that METTL4 promoted alveolar epithelial cells ferroptosis in sepsis-induced lung injury via N6-methyladenosine of Nrf2, which might provide a novel approach to therapeutic strategies for sepsis-induced ALI.
Assuntos
Lesão Pulmonar Aguda , Ferroptose , Sepse , Animais , Camundongos , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Células Epiteliais Alveolares , Ferroptose/genética , Fator 2 Relacionado a NF-E2/genética , Sepse/complicações , Sepse/genética , Metiltransferases/metabolismoRESUMO
Metabolic disorder has been found to be an important factor in the pathogenesis and progression of sepsis. However, the causation of such an association between serum metabolites and sepsis has not been established. We conducted a two-sample Mendelian randomization (MR) study. A genome-wide association study of 486 human serum metabolites was used as the exposure, whereas sepsis and sepsis mortality within 28 days were set as the outcomes. In MR analysis, 6 serum metabolites were identified to be associated with an increased risk of sepsis, and 6 serum metabolites were found to be related to a reduced risk of sepsis. Furthermore, there were 9 metabolites positively associated with sepsis-related mortality, and 8 metabolites were negatively correlated with sepsis mortality. In addition, "glycolysis/gluconeogenesis" (p = 0.001), and "pyruvate metabolism" (p = 0.042) two metabolic pathways were associated with the incidence of sepsis. This MR study suggested that serum metabolites played significant roles in the pathogenesis of sepsis, which may provide helpful biomarkers for early disease diagnosis, therapeutic interventions, and prognostic assessments for sepsis.
Assuntos
Biomarcadores , Estudo de Associação Genômica Ampla , Análise da Randomização Mendeliana , Sepse , Humanos , Sepse/sangue , Sepse/mortalidade , Sepse/genética , Biomarcadores/sangue , Masculino , Polimorfismo de Nucleotídeo Único , Feminino , Pessoa de Meia-Idade , MetabolomaRESUMO
BACKGROUND: Sepsis refers to a systemic inflammatory response caused by infection, involving multiple organs. Sepsis-associated encephalopathy (SAE), as one of the most common complications in patients with severe sepsis, refers to the diffuse brain dysfunction caused by sepsis without central nervous system infection. However, there is no clear diagnostic criteria and lack of specific diagnostic markers. METHODS: The main active ingredients of coptidis rhizoma(CR) were identified from TCMSP and SwissADME databases. SwissTargetPrediction and PharmMapper databases were used to obtain targets of CR. OMIM, DisGeNET and Genecards databases were used to explore targets of SAE. Limma differential analysis was used to identify the differential expressed genes(DEGs) in GSE167610 and GSE198861 datasets. WGCNA was used to identify feature module. GO and KEGG enrichment analysis were performed using Metascape, DAVID and STRING databases. The PPI network was constructed by STRING database and analyzed by Cytoscape software. AutoDock and PyMOL software were used for molecular docking and visualization. Cecal ligation and puncture(CLP) was used to construct a mouse model of SAE, and the core targets were verified in vivo experiments. RESULTS: 277 common targets were identified by taking the intersection of 4730 targets related to SAE and 509 targets of 9 main active ingredients of CR. 52 common DEGs were mined from GSE167610 and GSE198861 datasets. Among the 25,864 DEGs in GSE198861, LCN2 showed the most significant difference (logFC = 6.9). GO and KEGG enrichment analysis showed that these 52 DEGs were closely related to "inflammatory response" and "innate immunity". A network containing 38 genes was obtained by PPI analysis, among which LCN2 ranked the first in Degree value. Molecular docking results showed that berberine had a well binding affinity with LCN2. Animal experiments results showed that berberine could inhibit the high expression of LCN2,S100A9 and TGM2 induced by CLP in the hippocampus of mice, as well as the high expression of inflammatory factors (TNFα, IL-6 and IL-1ß). In addition, berberine might reduce inflammation and neuronal cell death by partially inhibiting NFκB/LCN2 pathway in the hippocampus of CLP models, thereby alleviating SAE. CONCLUSION: Overall, Berberine may exert anti-inflammatory effects through multi-ingredients, multi-targets and multi-pathways to partially rescue neuronal death and alleviate SAE.
Assuntos
Berberina , Biologia Computacional , Lipocalina-2 , NF-kappa B , Farmacologia em Rede , Encefalopatia Associada a Sepse , Animais , Humanos , Masculino , Camundongos , Anti-Inflamatórios/uso terapêutico , Anti-Inflamatórios/farmacologia , Berberina/farmacologia , Berberina/uso terapêutico , Modelos Animais de Doenças , Regulação para Baixo , Medicamentos de Ervas Chinesas/uso terapêutico , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/química , Lipocalina-2/genética , Lipocalina-2/metabolismo , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Doenças Neuroinflamatórias/tratamento farmacológico , NF-kappa B/metabolismo , Mapas de Interação de Proteínas , Sepse/tratamento farmacológico , Encefalopatia Associada a Sepse/tratamento farmacológico , Encefalopatia Associada a Sepse/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Ovarian tumor domain-containing protease 1 (OTUD1) is a critical negative regulator that promotes innate immune homeostasis and is extensively involved in the pathogenesis of sepsis. In this study, we performed a powerful integration of multiomics analysis and an experimental mechanistic investigation to elucidate the immunoregulatory role of OTUD1 in sepsis at the clinical, animal and cellular levels. Our study revealed the upregulation of OTUD1 expression and the related distinctive alterations observed via multiomics profiling in clinical and experimental sepsis. Importantly, in vivo and in vitro, OTUD1 was shown to negatively regulate inflammatory responses and play a protective role in sepsis-induced pathological lung injury by mechanistically inhibiting the activation of the transforming growth factor-beta-activated kinase 1 (TAK1)-mediated mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling pathways in the present study. Subsequently, we probed the molecular mechanisms underlying OTUD1's regulation of NF-κB and MAPK pathways by pinpointing the target proteins that OTUD1 can deubiquitinate. Drawing upon prior research conducted in our laboratory, it has been demonstrated that tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) performs a protective function in septic lung injury and septic encephalopathy by suppressing the NF-κB and MAPK pathways. Hence, we hypothesized that TIPE2 might be a target protein of OTUD1. Additional experiments, including Co-IP, immunofluorescence co-localization, and Western blotting, revealed that OTUD1 indeed has the ability to deubiquitinate TIPE2. In summary, OTUD1 holds potential as an immunoregulatory and inflammatory checkpoint agent, and could serve as a promising therapeutic target for sepsis-induced lung injury.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , MAP Quinase Quinase Quinases , Camundongos Endogâmicos C57BL , NF-kappa B , Sepse , Proteases Específicas de Ubiquitina , Animais , Sepse/metabolismo , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/genética , NF-kappa B/metabolismo , Camundongos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Humanos , Proteases Específicas de Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/genética , Transdução de Sinais/fisiologia , Ubiquitinação , Lesão Pulmonar/metabolismo , Lesão Pulmonar/etiologia , Lesão Pulmonar/prevenção & controle , Sistema de Sinalização das MAP Quinases/fisiologiaRESUMO
Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that certain of the cell migration assay data shown in Fig. 2C were strikingly similar to data that had appeared in different form in other articles by different authors. Owing to the fact that the contentious data in the above article had already been published elsewhere, or were already under consideration for publication, prior to its submission to Molecular Medicine Reports, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 19: 19261934, 2019; DOI: 10.3892/mmr.2019.9830].
RESUMO
Anastomotic leak is still a severe complication in esophageal surgery due to high mortality. This article reviews the updates on the treatment of anastomotic leak after esophagectomy in order to provide reference for clinical treatment and research. The relevant studies published in the Chinese Zhiwang, Wanfang, and MEDLINE databases to December 21, 2021 were retrieved, and esophageal carcinoma, esophagectomy, anastomotic leakage, and fistula selected as the keywords. A total of 78 studies were finally included. The treatments include traditional surgical drainage, new reverse drainage trans-fistula, stent plugging, endoscopic clamping, biological protein glue injection plugging, endoluminal vacuum therapy (EVT), and reoperation, etc. Early diagnosis, accurate classification and optimal treatment can promote the rapid healing of anastomotic leaks. EVT may be the most valuable approach, simultaneously with good commercial prospects. Reoperation should be considered in patients with complex fistula in which conservative treatment is insufficient or has failed.
Assuntos
Fístula Anastomótica , Neoplasias Esofágicas , Humanos , Fístula Anastomótica/diagnóstico , Fístula Anastomótica/etiologia , Fístula Anastomótica/cirurgia , Neoplasias Esofágicas/cirurgia , Neoplasias Esofágicas/complicações , Esofagectomia/efeitos adversos , Anastomose Cirúrgica/efeitos adversos , Estudos Retrospectivos , Resultado do TratamentoRESUMO
The cholinergic anti-inflammatory pathway has been found to exert a protective role in myocardial ischemia-reperfusion injury (MIRI). Alpha7 nicotinic acetylcholine receptor (α7nAChR) is a regulator of cholinergic anti-inflammatory pathway; however, little information is available on the effect of α7nAChR on MIRI. In the present study, we hypothesized that 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxanol-3-yl)-urea (PNU-120596), a potent positive allosteric modulator of α7nAChR, could play a protective role on MIRI. Fifty-five rats were randomly assigned into 4 groups: Sham group, ischemia-reperfusion group, PNU-120596 group, α-bungarotoxin group. Compared with ischemia-reperfusion group, PNU-120596 treatment markedly decreased infarct size, ultrastructural damage, serum creatine kinase, and lactate dehydrogenase. Serum proinflammatory cytokine production, myocardium endothelial activation and neutrophil infiltration, myocardium malondialdehyde were also significantly decreased, accompanied by increased myocardium superoxide dismutase production, in the PNU-120596 group compared with the ischemia-reperfusion group. Meanwhile, we observed a significant inhibition of nuclear factor kappa B activation in PNU-120596 group compared with ischemia-reperfusion group. Pretreatment of α7nAChR-selective antagonist, α-bungarotoxin, abolished all the protective effects of PNU-120596 on MIRI. In conclusion, PNU might have a protective effect against MIRI. Its action mechanisms might be involved in the inhibition of inflammatory responses, attenuation of lipid peroxidation, and suppression of nuclear factor kappa B activity.
Assuntos
Isoxazóis/farmacologia , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Compostos de Fenilureia/farmacologia , Receptores Nicotínicos/metabolismo , Regulação Alostérica , Animais , Bungarotoxinas/farmacologia , Citocinas/metabolismo , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Inflamação/fisiopatologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Traumatismo por Reperfusão Miocárdica/fisiopatologia , NF-kappa B/metabolismo , Agonistas Nicotínicos/farmacologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Delayed neurocognitive recovery (dNCR), a postoperative complication that occurs in elderly patients, still lacks effective treatment. Interleukin-33 (IL-33) has been proved to modulate neuroinflammation and synaptic plasticity, among other effects, but the role of IL-33 in dNCR is not clear. We established a dNCR model in aged mice by laparotomy under sevoflurane anesthesia. Cognition was evaluated by Morris water maze (MWM) and fear conditioning test (FCT). Immunofluorescence was used to detect the density of IL-33 and glial fibrillary acidic protein (GFAP) co-localization, ionized calcium-binding adapter molecule 1, vesicular glutamate transporter 1 (vGlut1) and postsynaptic density protein-95 (PSD95) co-localization in the hippocampus. IL-33, GFAP, vGlut1 and PSD95 were tested by Western blotting. Enzyme-linked immunosorbent assay was used to detect the levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß) and IL-10. Surgery/anesthesia reduced the level of IL-33 in the hippocampus. Intraperitoneal injection of 200 ng IL-33 per mouse significantly decreased the latency to the platform and increased the number of platform crossings and the target quadrant dwell time in MWM, while increasing the freezing time in the context test of FCT. Furthermore, IL-33 inhibited microglial activation and the release of TNF-α and IL-1ß while upregulating the markers of excitatory synapses vGlut1 and PSD95. Our findings indicated that IL-33 improved cognition by inhibiting the hippocampal inflammatory response and upregulating the number of excitatory synapses. Therefore, IL-33 is a potential drug for the treatment of dNCR.