Serum HSP70 and VEGF levels are effective predictive factors of chemoradiosensitivity and prognosis of pancreatic cancer patients.

AIM: To evaluate the value of serum HSP70 and VEGF levels for predicting the chemoradiosensitivity of the pancreatic cancer patients. METHODS: A total of 255 patients with pancreatic cancer and 60 healthy subjects were enrolled in this study. Serum levels of HSP70 and VEGF were measured using enzyme-linked immunosorbent assay (ELISA) for the pre-treatment, during-treatment and post- chemoradiotherapy time-points. The results were analyzed to evaluate the potential of serum HSP70 and VEGF levels for predicting the chemoradiosensitivity of pancreatic cancer patients. RESULTS: The serum levels of both HSP70 and VEGF were found to be elevated in pancreatic cancer patients as compared to healthy subjects. After chemoradiotherapy treatment, 179 patients showed effective clinical response [complete response (CR) + partial response (PR)] while 76 patients showed ineffective clinical response [stable disease (SD) + progressive disease (PD)]. Compared with the pre-treatment levels, serum levels of HSP70 and VEGF were higher during chemoradiotherapy, and lower post-treatment in the effective group. However, serum levels of HSP70 and VEGF were higher during and after treatment in the ineffective group. At any given timepoint, serum levels of HSP70 and VEGF were higher in the ineffective group as compared to those of the effective group. The overall survival (OS) and progression free survival (PFS) trends were: HSP70 High/VEGFHigh < HSP70High/VEGFLow or HSP70Low/VEGFHigh < HSP70Low/VEGFLow. Serum levels of HSP70 and VEGF were individually effective, and their combination was even more effective in predicting the chemoradiosensitivity of pancreatic cancer patients. HSP70 and VEGF expression were independent risk factors for OS and PFS of pancreatic cancer patients. CONCLUSION: Higher levels of serum HSP70 and VEGF were associated with lower radiosensitivity and worse prognosis, while lower levels of serum HSP70 and VEGF were associated with improved radiosensitivity and better prognosis of pancreatic cancer patients.

Pyroptosis in sepsis induced organ dysfunction.

As an uncontrolled inflammatory response to infection, sepsis and sepsis induced organ dysfunction are great threats to the lives of septic patients. Unfortunately, the pathogenesis of sepsis is complex and multifactorial, which still needs to be elucidated. Pyroptosis is a newly discovered atypical form of inflammatory programmed cell death, which depends on the Caspase-1 dependent classical pathway or the non-classical Caspase-11 (mouse) or Caspase-4/5 (human) dependent pathway. Many studies have shown that pyroptosis is related to sepsis. The Gasdermin proteins are the key molecules in the membrane pores formation in pyroptosis. After cut by inflammatory caspase, the Gasdermin N-terminal fragments with perforation activity are released to cause pyroptosis. Pyroptosis is closely related to the occurrence and development of sepsis induced organ dysfunction. In this review, we summarized the molecular mechanism of pyroptosis, the key role of pyroptosis in sepsis and sepsis induced organ dysfunction, with the aim to bring new diagnostic biomarkers and potential therapeutic targets to improve sepsis clinical treatments.

The alterations of innate immunity and enhanced severity of infections in diabetes mellitus.

Diabetes mellitus (DM) is a metabolic inflammatory disease with a high incidence worldwide. Patients with DM are at a high risk for all types of infections. Type 1 DM is characterised with immune destruction of pancreatic ß cells, while type 2 diabetes is characterised with insulin resistance and ß cell dysfunction, both of which result in disorders of glucose and lipid metabolism. This metabolic disorder causes functional defects of immune cells, aberrant production of inflammatory cytokines, dysregulated immune responses, advanced pathophysiological injury of the body, and increased mortality in populations with DM upon infections. Starting with the change of natural immune system in patients with DM, this paper focused on the enhanced severity of infections in DM and the underlying innate immune alterations in preclinical and clinical studies, aiming to better understand the influence of DM on the susceptibility, pathophysiology, and clinical outcomes in infections.

Effects of Somatostatin and Indomethacin Mono or Combination Therapy on High-risk Hyperamylasemia and Post-pancreatitis Endoscopic Retrograde Cholangiopancreatography Patients: A Randomized Study.

BACKGROUND: Endoscopic retrograde cholangiopancreatography (ERCP) is a minimally invasive technique widely used to diagnose and treat pancreatic and biliary diseases; however, it is linked with imminent hyperamylasemia and post-ERCP pancreatitis (PEP). Somatostatin and indomethacin are the classic recommended drugs used for PEP prevention. OBJECTIVE: To elucidate the effects of somatostatin and indomethacin mono or in combination to prevent hyperamylasemia and PEP in high-risk individuals. METHODS: Altogether 1458 patients who underwent ERCP in our hospital from January 2016 to May 2022 were included in this investigation and categorized into 4 groups based on the treatment regimen: placebo, indomethacin, somatostatin, and indomethacin + somatostatin. The pre operation and post operation (at 6, 12, and 24 h) hospitalization cost, length of stay, the occurrence of hyperamylasemia and PEP, levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-8, and VAS pain score were determined in the 4 groups. In all the groups, VAS and IL-6, TNF-α, and IL-8 levels substantially increased in the pretreatment and decreased sequentially from 6 to 24 h post operation. The individuals in the indomethacin revealed substantially reduced hyperamylasemia, VAS, and levels of IL-6, TNF-α, and IL-8, 6 h post operation, whereas the hospitalization fee, length of stay, PEP incidence, VAS, levels of IL-6, TNF-α, and IL-8, 12 and 24 h post operation were not statistically important in comparison with the individuals who received placebo therapy. The somatostatin and the indomethacin + somatostatin groups indicated markedly alleviated hospitalization fee, length of stay, the occurrence of hyperamylasemia and PEP, VAS, and the levels of IL-6, TNF-α, and IL-8 at 6, 12, and 24 h post operation compared with the placebo cohort. Furthermore, compared with the indomethacin group, the above-determined factors notably reduced at 6, 12, and 24 h post operation in somatostatin and indomethacin + somatostatin groups. It was also observed that the indomethacin + somatostatin group has substantially decreased the occurrence of hyperamylasemia, VAS score, and levels of IL-6, TNF-α, and IL-8, 6 hours post operation, while at 12 and 24 h post operation, the hospitalization fee, length of stay and incidence of PEP, VAS, levels of IL-6, TNF-α, and IL-8 were not statistically important compared with the somatostatin group. It is also worth noting that the side effects of both drugs are rare and mild. RESULTS: For high-risk PEP patients, indomethacin and somatostatin can efficiently alleviate post-operative hyperamylasemia and improve their life standard within 6 hours and 24 hours, respectively. Indomethacin is suitable for individuals who underwent simple, short-duration ERCP with expected mild post-operative abdominal pain, whereas somatostatin is given to patients with complicated, long-duration ERCP and expected severe post-operative abdominal pain. Their combinational therapy produces a synergistic effect and can reduce the incidence of hyperamylasemia, thereby improving patients' quality of life within 6 h and is also effective against individuals who received a more complicated, longer-duration ERCP and were expected to have severer and longer post-operative abdominal pain.

A pilot study on pyroptosis related genes in peripheral blood mononuclear cells of non-small cell lung cancer patients.

OBJECTIVE: To investigate the GSDMD, CASP1, CASP4 and CASP5 expression in peripheral blood mononuclear cells of non-small cell lung cancer patients and analyze their clinical significance. METHODS: 71 non-small cell lung cancer patients were selected as the study group and 50 healthy individuals as the control group. The GSDMD, CASP1, CASP4 and CASP5 expression in peripheral blood mononuclear cells of the two groups were detected by real-time fluorescence quantitative PCR. The GSDMD, CASP1, CASP4, CASP5 expression and their relationship with the clinical characteristics of the patients were analyzed. RESULTS: Compared with the control group, the GSDMD, CASP4 and CASP5 expression in PBMCs of lung cancer patients was significantly higher(P < 0.05). Lymph node metastasis had significant difference with the CASP4 and GSDMD expression (P < 0.05); tumor volume had significant difference with CASP1 and CASP5 expression (P < 0.05). The areas under predictive ROC curve of the GSDMD, CASP1, CASP4, and CASP5 mRNA expression were 0.629(P < 0.05), 0.574(p > 0.05), 0.701(P < 0.05) and 0.628(P < 0.05), the sensitivity values were 84.5%, 67.6% 43.7%, and 84.3%;the specificity values were 42%, 52%, 84% and 64%, respectively. CONCLUSION: GSDMD, CASP1, CASP4 and CASP5 gene expression are highly increased in PBMCs of non-small cell lung cancer patients and their expression are closely related to the clinical characteristics of patients. The early enhanced pyroptosis-related gene expression may be potential molecular markers for early diagnosis of non-small cell lung cancer.

Validation of reference genes for the normalization of the RT-qPCR in peripheral blood mononuclear cells of septic patients.

Objective: To screen and validate reference genes suitable for gene mRNA expression study in peripheral blood mononuclear cells (PBMCs) between septic patients and healthy controls (HC). Methods: Total RNA in PBMCs was extracted and RT-qPCR was used to determine the mRNA expression profiles of 9 candidate genes, including ACTB, B2M, GAPDH, GUSB, HPRT1, PGK1, RPL13A, SDHA and YWHAZ. The genes expression stabilities were assessed by both geNorm and NormFinder software. Results: YWHAZ was the most stable gene among the 9 candidate genes evaluated by both geNorm and NormFinder in mixed and sepsis groups. The most stable gene combination in mixed group analyzed by geNorm was the combination of GAPDH, PKG1 and YWHAZ, while that in sepsis group was the combination of ACTB, PKG1 and YWHAZ. Conclusion: Our first systematic analysis of the reference genes in PBMC of septic patients suggested YWHAZ was the best candidate. The combination of ACTB, PKG1 and YWHAZ could improve RT-qPCR accuracy in septic patients. Our results identified the most stable reference genes to standardize RT-qPCR of sepsis patients, which can serve as a useful tool for gene function exploration in the future.

HSF1 Alleviates Brain Injury by Inhibiting NLRP3-Induced Pyroptosis in a Sepsis Model.

Background: Sepsis, which could cause a systemic inflammatory response, is a life-threatening disease with a high morbidity and mortality rate. There is evidence that brain injury may be related to severe systemic infection induced by sepsis. The brain injury caused by sepsis could increase the risk of mortality in septic patients, which seriously affects the septic patient's prognosis of survival. Although there remains a focus on sepsis research, clinical measures to prevent and treat brain injury in sepsis are not yet available, and the high mortality rate is still a big health burden. Therefore, it is necessary to investigate the new molecules or regulated pathways that can effectively inhibit the progress of sepsis. Objective: NLR family pyrin domain-containing 3 (NLRP3) increased in the procession of sepsis and functioned as the key regulator of pyroptosis. Heat shock factor 1 (HSF1) can protect organs from multiorgan dysfunction syndrome induced by lipopolysaccharides in mice, and NLRP3 could be inhibited by HSF1 in many organs. However, whether HSF1 regulated NLRP3 in sepsis-induced brain injury, as well as the detailed mechanism of HSF1 in brain injury, remains unknown in the sepsis model. In this research, we try to explore the relationship between HSF1 and NLRP3 in a sepsis model and try to reveal the mechanism of HSF1 inhibiting the process of brain injury. Methods: In this study, we used wild-type mice and hsf1 -/- mice for in vivo research and PC12 cells for in vitro research. Real-time PCR and Western blot were used to analyze the expression of HSF1, NLRP3, cytokines, and pyrolytic proteins. EthD-III staining was chosen to detect the pyroptosis of the hippocampus and PC12 cells. Results: The results showed that HSF1 is negatively related to pyroptosis. The pyroptosis in cells of brain tissue was significantly increased in the hsf1 -/- mouse model compared to hsf1 +/+ mice. In PC12 cells, hsf1 siRNA can upregulate pyroptosis while HSF1-transfected plasmid could inhibit the pyroptosis. HSF1 could negatively regulate the NLRP3 pathway in PC12 cells, while hsf1 siRNA enhanced the pyroptosis in PC12 cells, which could be reversed by nlrp3 siRNA. Conclusion: These results imply that HSF1 could alleviate sepsis-induced brain injury by inhibiting pyroptosis through the NLRP3-dependent pathway in brain tissue and PC12 cells, suggesting HSF1 as a potential molecular target for treating brain injury in sepsis clinical studies.

Increased Dp71 in ischemia-reperfusion injured rat heart exerts anti-apoptotic role via enhancing Bcl-2.

For the first time, increased Dp71 in ischemia-reperfusion injured rat heart were identified, both Dp71 mRNA and protein reached its peak expression 8 h after reperfusion. In H2O2 stimulated H9c2 cells, Dp71 mRNA and protein gradually increased and reached a peak at 16 h. Enhanced Dp71 in H9c2 could resist H2O2-induced cell apoptosis, while Dp71 depletion accelerated the apoptosis induced by H2O2. Enhanced Bcl-2 expression and Bcl-2∕Bax protein expression ratio was identified in Dp71 overexpressed H9c2 cells, while knocking down Dp71 significantly decreased the Bcl-2 and Bcl-2∕Bax protein expression ratio. Increased Dp71 can accelerate FAK and p65 phosphorylation, which finally resulted in enhanced Bcl-2 expression and explains the highly possible cardiac protection role of Dp71.

IL-1R1 blockade attenuates liver injury through inhibiting the recruitment of myeloid-derived suppressor cells in sepsis.

Myeloid-derived suppressor cells (MDSCs) mobilize and migrate from bone marrow to peripheral tissues or immune organs, which is associated with poor prognosis in sepsis. Intervention of MDSCs might be a potential target for the effective treatment of sepsis. In the present study, we demonstrated that IL-1R1 blockade with either recombinant human IL-1R antagonist Anakinra or IL-1R1 deficiency had a protective effect on the liver injury in septic mice. The possible mechanism was that Anakinra treatment and IL-1R1 knockout inhibited the migration of MDSCs to the liver in sepsis, thus attenuating the immune suppression of MDSCs on effector T cells characterized with the decrease in proportion of CD4+ and CD8+ T cells. Furthermore, the switch from pro-inflammatory M1 macrophage to anti-inflammatory M2 phenotype and the ability of bacterial clearance in the liver of septic mice were enhanced obviously by Anakinra and IL-1R1 deficiency, which contributes to the attenuated liver injury. Taken together, these findings provide new ideas for revealing the relationship between IL-1R1 and MDSCs in sepsis, thereby providing a potentially effective target for ameliorating septic liver injury.

HSF1 Protects Sepsis-Induced Acute Lung Injury by Inhibiting NLRP3 Inflammasome Activation.

As an important transcription factor, heat shock factor 1 (HSF1) plays an endogenous anti-inflammation role in the body and can alleviate multiple organ dysfunction caused by sepsis, which contributes to an uncontrolled inflammatory response. The NLRP3 inflammasome is a supramolecular complex that plays key roles in immune surveillance. Inflammation is accomplished by NLRP3 inflammasome activation, which leads to the proteolytic maturation of IL-1ß and pyroptosis. However, whether HSF1 is involved in the activation of the NLRP3 inflammasome in septic acute lung injury (ALI) has not been reported. Here, we show that HSF1 suppresses NLRP3 inflammasome activation in transcriptional and post-translational modification levels. HSF1 can repress NLRP3 expression via inhibiting NF-κB phosphorylation. HSF1 can inhibit caspase-1 activation and IL-1ß maturation via promoting NLRP3 ubiquitination. Our finding not only elucidates a novel mechanism for HSF1-mediated protection of septic ALI but also identifies new therapeutic targets for septic ALI and related diseases.

The Emerging Role of Ferroptosis in Sepsis.

Ferroptosis is a novel form of cell death characterized by the iron-dependent accumulation of lipid peroxides and is different from other types of cell death. The mechanisms of ferroptosis are discussed in the review, including System Xc-, Glutathione Peroxidase 4 pathway, Ferroptosis Suppressor Protein 1 and Dihydroorotate Dehydrogenase pathway. Ferroptosis is associated with the occurrence of various diseases, including sepsis. Research in recent years has displayed that ferroptosis is involved in sepsis occurrence and development. Iron chelators can inhibit the development of sepsis and improve the survival rate of septic mice. The ferroptotic cells can release damage-associated molecular patterns and lipid peroxidation, which further mediate inflammatory responses. Ferroptosis inhibitors can resist sepsis-induced multiple organ dysfunction and inflammation. Finally, we reviewed ferroptosis, an iron-dependent form of cell death that is different from other types of cell death in biochemistry, morphology, and major regulatory mechanisms, which is involved in multiple organ injuries caused by sepsis. Exploring the relationship between sepsis and ferroptosis may yield new treatment targets for sepsis.

NLRP3 inflammasome in sepsis (Review).

Sepsis is an imbalanced response to infection that leads to life-threatening organ dysfunction. Although an increasing number of anti-inflammatory drugs are available, the options for treating sepsis remain limited. Therefore, it is imperative to understand the pathogenesis and pathophysiology of sepsis and develop novel therapeutic targets to treat this state. The Nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is a cytoplasmic high-molecular weight protein complex composed of the sensor NLRP3, adapter protein apoptosis-related speck-like protein and pro-caspase-1. It functions by cleaving pro-caspase-1 to become active caspase-1, resulting in the maturation and release of IL-1ß and IL-18. Activation of the NLRP3 inflammasome is necessary for innate immune defense and also serves an important role in adaptive immune responses. Studies have shown that the NLRP3 inflammasome is involved in the occurrence and evolution of sepsis and other immune inflammatory diseases. The present paper reviews the activation pathways and biological function of the NLRP3 inflammasome in sepsis, with the aim to provide a basis for further research.

Bushen Huoxue Acupuncture Inhibits NLRP1 Inflammasome-Mediated Neuronal Pyroptosis in SAMP8 Mouse Model of Alzheimer's Disease.

BACKGROUND: It was indicated that nucleotide-binding oligomerisation domain­like receptor protein 1 (NLRP1) inflammasome-mediated pyroptosis is involveg in the progression of Alzheimer's disease (AD). This study was designed to explore the effect of Bushen Huoxue Acupuncture on cognitive defect and NLRP1 inflammasome-mediated pyroptosis in AD mouse. METHODS: Senescence-accelerated mouse prone 8 (SAMP8) mice were used as a model of AD. Bushen Huoxue Acupuncture was performed in four acupoints: "Baihui acupoint" (GV20), "Shenshu acupoint" (BL23), "Xuehai acupoint" (SP10), and "Geshu acupoint" (BL17). Morris water maze test was performed to evaluate the cognitive function of the mouse. The levels of Aß1-40, Aß1-42, IL-1ß, and IL-18 were examined by ELISA assay. Neuronal apoptosis and damage in hippocampal tissues were measured using TUNEL and Nissl staining, respectively. The expression of NLRP1, ASC, cleaved caspase-1, IL-1ß, and IL-18 was examined using Western blot. RESULTS: Bushen Huoxue Acupuncture improved the learning and memory deficits of AD mouse. Meanwhile, Bushen Huoxue Acupuncture decreased the production of Aß in hippocampal tissues of SAMP8 mice and attenuated the neuronal apoptosis and damage. Furthermore, Bushen Huoxue Acupuncture inhibited NLRP1 inflammasome activation in SAMP8 mice. CONCLUSION: Bushen Huoxue Acupuncture could notably attenuate the cognitive defect of mouse AD model and inhibit NLRP1 inflammasome-mediated pyroptosis.

HSF1 Alleviates Microthrombosis and Multiple Organ Dysfunction in Mice with Sepsis by Upregulating the Transcription of Tissue-Type Plasminogen Activator.

Sepsis is a life-threatening complication of infection closely associated with coagulation abnormalities. Heat shock factor 1 (HSF1) is an important transcription factor involved in many biological processes, but its regulatory role in blood coagulation remained unclear. We generated a sepsis model in HSF1-knockout mice to evaluate the role of HSF1 in microthrombosis and multiple organ dysfunction. Compared with septic wild-type mice, septic HSF1-knockout mice exhibited a greater degree of lung, liver, and kidney tissue damage, increased fibrin/: fibrinogen deposition in the lungs and kidneys, and increased coagulation activity. RNA-seq analysis revealed that tissue-type plasminogen activator (t-PA) was upregulated in the lung tissues of septic mice, and the level of t-PA was significantly lower in HSF1-knockout mice than in wild-type mice in sepsis. The effects of HSF1 on t-PA expression were further validated in HSF1-knockout mice with sepsis and in vitro in mouse brain microvascular endothelial cells using HSF1 RNA interference or overexpression under lipopolysaccharide stimulation. Bioinformatics analysis, combined with electromobility shift and luciferase reporter assays, indicated that HSF1 directly upregulated t-PA at the transcriptional level. Our results reveal, for the first time, that HSF1 suppresses coagulation activity and microthrombosis by directly upregulating t-PA, thereby exerting protective effects against multiple organ dysfunction in sepsis.

HSF1 Attenuates LPS-Induced Acute Lung Injury in Mice by Suppressing Macrophage Infiltration.

Heat shock factor 1 (HSF1) is a transcription factor involved in the heat shock response and other biological processes. We have unveiled here an important role of HSF1 in acute lung injury (ALI). HSF1 knockout mice were used as a model of lipopolysaccharide- (LPS-) induced ALI. Lung damage was aggravated, and macrophage infiltration increased significantly in the bronchoalveolar lavage fluid (BALF) and lung tissue of HSF-/- mice compared with the damage observed in HSF1+/+ mice. Upon LPS stimulation, HSF-/- mice showed higher levels of monocyte chemoattractant protein-1 (MCP-1) in the serum, BALF, and lung tissue and increased the expression of MCP-1 and chemokine (C-C motif) receptor 2 (CCR2) on the surface of macrophages compared with those in HSF1+/+. Electrophoretic mobility shift assays (EMSA) and dual luciferase reporter assays revealed that HSF1 could directly bind to heat shock elements (HSE) in the promoter regions of MCP-1 and its receptor CCR2, thereby inhibiting the expression of both genes. We concluded that HSF1 attenuated LPS-induced ALI in mice by directly suppressing the transcription of MCP-1/CCR2, which in turn reduced macrophage infiltration.

Heat Shock Factor 1 in Relation to Tumor Angiogenesis and Disease Progression in Patients With Pancreatic Cancer.

OBJECTIVE: In this article, the aims were to study the expression of heat shock factor 1 (HSF1) in patients with pancreatic cancer and to elucidate the relevance between HSF1, angiogenesis, clinicopathological factors, and prognosis. METHODS: Pancreatic cancer, paracancerous, and normal pancreatic tissues were collected. The HSF1 RNA and protein expressions were identified using quantitative real-time reverse transcription polymerase chain reaction and immunohistochemical staining. Associations of HSF1 and cluster of differentiation 34 with clinical variables and disease outcomes were investigated. RESULTS: Compared with the normal pancreatic and paracancerous tissue, HSF1 RNA and protein significantly showed higher expression in the pancreatic cancer tissue and was significantly associated with microvessel density. The high expression of HSF1 did not correspond to the patients' sex, age, carcinoembryonic antigen level, diameter of tumors, and locations; however, it corresponded significantly with carbohydrate antigen 19-9 level, lymph node metastasis, tumor node metastasis stage, differentiation degree, vascular invasion, and distant metastasis. The expression levels of HSF1 and cluster of differentiation 34 were significantly correlated with prognosis, disease specificity, and survival. The high expression of HSF1 would lead to worse prognosis and decrease in survival time and disease-free survival time. CONCLUSIONS: HSF1 expression level in pancreatic cancer tissue could be an ideal prognostic biomarker for risk stratification and a potential therapeutic target for patients with pancreatic cancer.

Metformin protects against ischaemic myocardial injury by alleviating autophagy-ROS-NLRP3-mediated inflammatory response in macrophages.

Myocardial ischaemia is usually accompanied by inflammatory response which plays a critical role in the myocardial healing and scar formation, while persistent inflammatory response contributes greatly to the myocardial remodeling and consequent heart failure. Metformin (Met), a widely used hypoglycemic drug, has increasingly been shown to exert remarkable cardioprotective effect on ischaemic myocardial injury such as acute myocardial infarction (AMI). However, the underlying mechanisms are still far from being fully understood. In this study, a mouse model of AMI was established through ligating the left anterior descending coronary artery (LAD), 100 mg/kg Met was given immediately after operation once daily for 3 days. It was demonstrated that Met effectively improved the cardiac haemodynamics (LVSP, LVEDP, +dp/dt, -dp/dt), diminished the infarct size, alleviated the disarrangement of myocardial cells and reduced the infiltration of inflammatory cells (macrophages, neutrophils and lymphocytes) in the heart of AMI mice. Mechanistically, Met decreased the expression of NLRP3 and enhanced the accumulation of LC3 puncta in F4/80-positive macrophages in the heart of AMI mice. Single cell suspension of cardiac macrophages was prepared from AMI mice and exhibited increased NLRP3 mRNA and protein expression. In contrast, Met decreased the expression of NLRP3 and p62, whereas increased the ratio of LC3II/LC3I. Additionally, both conditioned medium from H9c2 cardiomyocytes exposed to hydrogen peroxide (H9c2-H2O2-CM) and combination of mtDNA and ATP (mtDNA-ATP) increased the expression of NLRP3 and cleaved caspase-1 (p10) as well as intracellular ROS production in RAW264.7 macrophages, which were abrogated by Met treatment. Strikingly, chloroquine (CQ), 3-methyladenine (3-MA) and knockdown of autophagy-related gene (Atg5) abrogated the inhibitory effects of Met on H9c2-H2O2-CM and mtDNA-ATP-induced NLRP3 expression, release of IL-1ß and IL-18 as well as ROS production in RAW264.7 macrophages. Collectively, these findings suggest that Met protects against ischaemic myocardial injury through alleviating autophagy-ROS-NLRP3 axis-mediated inflammatory response in macrophages.

Inhibition of Aerobic Glycolysis Promotes Neutrophil to Influx to the Infectious Site Via CXCR2 in Sepsis.

Recent evidences suggest that metabolic reprogramming plays an important role in the regulation of innate inflammatory response; however, the specific mechanism is unclear. In this study, we found that glycolytic inhibitor 2-deoxyglucose (2-DG) significantly improved the survival rate in cecal ligation and puncture (CLP)-induced septic mice. 2-DG-treated mice developed increased neutrophil migration to the infectious site and more efficient bacterial clearance than untreated mice. 2-DG reversed the down-regulation of chemokine receptor 2 (CXCR2) and the impaired chemotaxis induced by CLP in mice or lipopolysaccharides (LPS) in human neutrophils. Furthermore, 2-DG reversed the down-regulation of CXCR2 in neutrophils by decreasing the expression of G protein-coupled receptor kinase-2 (GRK2), a serin-threonine protein kinase that mediated the internalization of chemokine receptors, which was induced via the inhibition of extracellular regulated protein kinases (ERK) phosphorylation and the promotion of P38 phosphorylation. Finally, SB225002, a CXCR2 antagonist, partially blocked the protective effects of 2-DG in sepsis. Together, we found a novel mechanism for the migration of neutrophils regulated by metabolism and suggested that aerobic glycolysis might be a potential target of intervention in sepsis.

LDK378 inhibits the recruitment of myeloid-derived suppressor cells to spleen via the p38-GRK2-CCR2 pathway in mice with sepsis.

Myeloid-derived suppressor cells (MDSCs) are functionally immunosuppressive cells that are persistently increased in abundance and associated with adverse clinical outcomes in sepsis. Here, we investigated the therapeutic potential of an anaplastic lymphoma kinase inhibitor, LDK378, in cecal ligation and puncture (CLP)-induced polymicrobial sepsis and examined its effects on the recruitment of MDSCs. LDK378 significantly improved the survival of CLP-induced polymicrobial septic mice, which was paralleled by reduced organ injury, decreased release of inflammatory cytokines and decreased recruitment of MDSCs to the spleen. Importantly, LDK378 inhibited the migration of MDSCs to the spleen by blocking the CLP-mediated upregulation of CC chemokine receptor 2 (CCR2), a chemokine receptor critical for the recruitment of MDSCs. Mechanistically, LDK378 treatment blocked the CLP-induced CCR2 upregulation of MDSCs via partially inhibiting the phosphorylation of p38 and G-protein-coupled receptor kinase-2 (GRK2) in bone marrow MDSCs of septic mice. Furthermore, in vitro experiments also showed that lipopolysaccharide (LPS)-induced migration of MDSCs was similarly owing to the activation of GRK2 and upregulation of CCR2 by LPS, whereas the treatment with LDK378 partially blocked the LPS-induced phosphorylation of p38 and GRK2 and decreased the expression of CCR2 on the cell surface, therefore leading to the suppression of MDSC migration. Together, these findings unravel a novel function of LDK378 in the host response to infection and suggest that LDK378 could be a potential therapeutic agent for sepsis.

Dp71 depleted HBE cells displayed increased DNA damage and apoptosis induced by H2O2.

Human bronchial epithelium (HBE)-Dp71 anti-sense(AS)cells with stably transfected Dp71 siRNA plasmids were prepared for further exploration of Dp71 biological traits in cells other than PC12. HBE-Dp71AS cells displayed increased DNA damage induced by H2O2. Apoptosis of HBE-Dp71AS cells induced by H2O2 was increased via enhancing caspase 3, caspase 8 and caspase 9. HBE-Dp71AS cells also displayed decreased proliferation and clonogenic formation. RAD51 was proved to be a new binding partner of Dp71 by co-immunoprecipitation (Ip) and immunofluorescence. Reduced RAD51 mRNA and protein levels were observed in HBE-Dp71AS cells. Decreased lamin B1, focal adhesion kinase (FAK), phosphorylated focal adhesion kinase (p-FAK) and phosphorylated protein kinase B (p-AKT) were detected in the HBE-Dp71AS cells, which functioned together with RAD51 as the molecular explanations for the character alterations of HBE-Dp71AS cells.