A novel mechanism of Dimethyl ester of Alpha-ketoglutarate in suppressing Paraquat-induced BEAS-2B cell injury by alleviating GSDME dependent pyroptosis.

BACKGROUND: Acute lung injury (ALI) induced by paraquat (PQ) progresses rapidly, leading to high mortality; however, there is no specific antidote. Our limited knowledge of the pathogenic toxicological mechanisms of PQ has hindered the development of treatments against PQ exposure. PURPOSE: Pyroptosis is a form of programmed cell death recently identified as a novel molecular mechanism adopted by chemotherapeutic drugs for cancer therapy. However, the involvement of pyroptosis in PQ-induced lung injury has not been reported. Therefore, we investigated the effects of PQ on the lung tissues to elucidate the molecular mechanisms underlying its toxicity, especially its ability to induce pyroptosis. METHODS: To observe the morphological changes of BEAS-2B cells exposed to PQ, the plasma membrane damage of the cells was detected by LDH release assay, mitochondrial function and cell metabolism were detected by energy metabolism analysis. Western blotting was used to detect the protein levels of GSDMD, C-GSDMD, GSDME and N-GSDME in BEAS-2B cells. Metabolites of TCA cycle were detected by metabolomics, and the changes of TCA cycle metabolic enzymes in cells were detected by Western blotting. RESULTS: We observed that PQ induced proteolytic cleavage of gasdermin E (GSDME) with concomitant cleavage of caspase 3 in BEAS-2B cells. Knockout of GSDME attenuated PQ-induced cell death. Additionally, PQ induced ROS accumulation, mitochondrial depolarisation, and mitochondrial dysfunction in these cells. PQ activated the caspase 3/GSDME pathway and damaged the cytoplasmic membrane in cells, leading to pyroptosis. We demonstrated that DMK suppressed PQ-induced pyroptosis by blocking PQ-induced caspase 3/GSDME pathway activation, reducing cellular ROS levels, and improving mitochondrial function. CONCLUSION: These findings provide novel insights into the previously unrecognized mechanism of GSDME-dependent pyroptosis in PQ poisoning.

Emodin alleviates LPS-induced myocardial injury through inhibition of NLRP3 inflammasome activation.

Myocardial injury and cardiovascular dysfunction are serious consequences of sepsis and contribute to high mortality. Currently, the pathogenesis of myocardial injury in sepsis is still unclear, and therapeutic approaches are limited. In this study, we investigated the protective effect of emodin on septic myocardial injury and the underlying mechanism. Lipopolysaccharide (LPS)-induced C57BL/6 mice and cardiomyocytes were used as models of sepsis in vivo and in vitro, respectively. The results showed that emodin alleviated cardiac dysfunction, myocardial injury and improved survival rate in LPS-induced septic mice. Emodin attenuated the levels of inflammatory cytokines and cardiac inflammation induced by LPS. Emodin reduced NOD-like receptor protein 3 (NLRP3) and Gasdermin D (GSDMD) expression in the heart tissue of LPS-induced septic mice. In vitro, emodin alleviated LPS-induced cell injury and inflammation in cardiomyocytes by inhibiting NLRP3 inflammasome activation. In addition, an NLRP3 inhibitor was used to further confirm the function of the NLRP3 inflammasome in LPS-induced myocardial injury. Taken together, our findings suggest that emodin improves LPS-induced myocardial injury and cardiac dysfunction by alleviating the inflammatory response and cardiomyocyte pyroptosis by inhibiting NLRP3 inflammasome activation, which provides a feasible strategy for preventing and treating myocardial injury in sepsis.

Administration of nicotinamide riboside prevents oxidative stress and organ injury in sepsis.

AIMS: Sepsis-caused multiple organ failure remains the major cause of morbidity and mortality in intensive care units. Nicotinamide riboside (NR) is a precursor of nicotinamide adenine dinucleotide (NAD+), which is important in regulating oxidative stress. This study investigated whether administration of NR prevented oxidative stress and organ injury in sepsis. METHODS: Mouse sepsis models were induced by injection of lipopolysaccharides (LPS) or feces-injection-in-peritoneum. NR was given before sepsis onset. Cultured macrophages and endothelial cells were incubated with various agents. RESULTS: Administration of NR elevated the NAD+ levels, and elicited a reduction of oxidative stress, inflammation and caspase-3 activity in lung and heart tissues, which correlated with attenuation of pulmonary microvascular permeability and myocardial dysfunction, leading to less mortality in sepsis models. These protective effects of NR were associated with decreased levels of plasma high mobility group box-1 (HMGB1) in septic mice. Consistently, pre-treatment of macrophages with NR increased NAD+ content and reduced HMGB1 release upon LPS stimulation. NR also prevented reactive oxygen species (ROS) production and apoptosis in endothelial cells induced by a conditioned-medium collected from LPS-treated macrophages. Furthermore, inhibition of SIRT1 by EX527 offset the negative effects of NR on HMGB1 release in macrophages, and ROS and apoptosis in endothelial cells. CONCLUSIONS: Administration of NR prevents lung and heart injury, and improves the survival in sepsis, likely by inhibiting HMGB1 release and oxidative stress via the NAD+/SIRT1 signaling. Given NR has been used as a health supplement, it may be a useful agent to prevent organ injury in sepsis.

Curcumin attenuates sepsis-induced acute organ dysfunction by preventing inflammation and enhancing the suppressive function of Tregs.

Sepsis is characterized by the extensive release of cytokines and other mediators. It results in a dysregulated immune response and can lead to organ damage and death. Curcumin has anti-inflammatory properties and immunoregulation functions in various disorders such as sepsis, cancer, rheumatoid arthritis, cardiovascular diseases, lung fibrosis, gallstone formation, and diabetes. This paper investigates the effects of curcumin on immune status and inflammatory response in mice subjected to cecal ligation and puncture (CLP). Inflammatory tissue injury was evaluated by histological observation. Magnetic microbeads were used to isolate splenic CD4+CD25+regulatory T cells (Tregs), and phenotypes were then analyzed by flow cytometry. The levels of Foxp3 were detected by Western blot and real-time PCR and cytokine levels were determined by enzyme-linked immunosorbent assay. We found that the administration of curcumin significantly alleviated inflammatory injury of the lung and kidney in septic mice. The suppressive function of Treg cells was enhanced and the plasma levels of IL-10 increased after treatment with curcumin. Furthermore, the secretion of plasma TNF-α and IL-6 was notably inhibited in septic mice treated with curcumin and administration with curcumin could improve survival after CLP. These data suggest that curcumin could be used as a potential therapeutic agent for sepsis.

[The efficacy of traditional Chinese medicin in animal model of lung injury induced by paraquat: a meta-analysis].

OBJECTIVE: To systematically review the effect of traditional Chinese medicine (TCM) in an animal model of lung injury induced by paraquat (PQ), and to provide a theoretical basis for future clinical trials. METHODS: The Wanfang, CNKI, VIP, PubMed/MEDLINE, EMBASE database (from January 1979 to September 2012) were searched. All papers concerning TCM in animal model of lung injury induced by PQ were retrieved. Study selection and data extraction were performed on the basis of Cochrane systematic review methods. Weighted mean difference (WMD) and 95% confidence interval (95%CI) with random effects model was adopted to investigate the effect of TCM on lung injury induced by PQ. RESULTS: Eighteen papers involving 1 188 rats met our criteria. Meta-analysis showed that TCM could improve the lung coefficiency (WMD -0.07, 95%CI -0.14 to -0.01, P=0.03), reduce lung wet/dry weight ratio (WMD -1.15, 95%CI -2.03 to -0.27, P=0.01), increase the serum superoxide dismutase (SOD) activity (WMD 56.08, 95%CI 23.46 to 88.70, P=0.000 8), improve plasma glutathione peroxidase (GSH-Px) level (WMD 26.64, 95%CI 18.95 to 34.33, P<0.000 01), and lower serum malondialdehyde(MDA) level (WMD -0.65, 95%CI -1.00 to -0.30, P=0.000 2), however there was no significant difference in the level of serum tumor necrosis factor-α (TNF-α) and hydroxyproline(HYP) level between TCM and controls (TNF-α: WMD -25.15, 95%CI -54.87 to 4.57, P=0.10; HYP: WMD -0.11, 95%CI -2.71 to 0.48, P=0.17). CONCLUSIONS: These findings demonstrate the efficacy of TCM in animal models of lung injury induced by PQ. However taking account of heterogeneity, the efficacy should be interpreted with caution.

Up-regulation of mitofusin-2 protects CD4+ T cells from HMGB1-mediated immune dysfunction partly through Ca(2+)-NFAT signaling pathway.

High mobility group box 1 protein (HMGB1) was recently discovered to be a critical late-acting cytokine and innate immune-modulating factor in sepsis, but the potential role and mechanism of HMGB1 in adaptive immunity remains elusive. The present study demonstrated that HMGB1 had a dual influence on immune function of CD4(+) T lymphocytes. Low dose of HMGB1 had no effect on the proliferation activity of CD4(+) T lymphocytes, but the Th1 cytokines production was increased. In contrast, treatment with high amount of HMGB1 suppressed the proliferative response and induced Th2 polarization of CD4(+) T lymphocytes. We found that the expression of mitofusin-2 (Mfn2; also named hyperplasia suppressor gene), a member of the mitofusin family, was decreased in CD4(+) T lymphocytes when stimulated with high dose of HMGB1. Up-regulation of Mfn2 attenuated the suppressive effect of HMGB1 on CD4(+) T lymphocytes, which was associated with profound elevation of intracellular calcium concentration ([Ca(2+)](i)) and nuclear factor of activated T cells (NFAT) activity. These results indicate that HMGB1 have a direct role on adaptive immunity, and the decrease of Mfn2 expression may be a major cause of HMGB1-mediated immune dysfunction and Ca(2+)-NFAT signaling defect of CD4(+) T lymphocytes.