Hydrogen-rich water reduces cell damage by reducing excessive autophagy in mouse neuronal cells after oxygen glucose deprivation/reoxygenation / 中华危重病急救医学

OBJECTIVE@#To investigate whether hydrogen-rich water exerts a protective effect against cellular injury by affecting the level of autophagy after oxygen glucose deprivation/reoxygenation (OGD/R) in a mouse hippocampal neuronal cell line (HT22 cells).@*METHODS@#HT22 cells in logarithmic growth phase were cultured in vitro. Cell viability was detected by cell counting kit-8 (CCK-8) assay to find the optimal concentration of Na2S2O4. HT22 cells were divided into control group (NC group), OGD/R group (sugar-free medium+10 mmol/L Na2S2O4 treated for 90 minutes and then changed to normal medium for 4 hours) and hydrogen-rich water treatment group (HW group, sugar-free medium+10 mmol/L Na2S2O4 treated for 90 minutes and then changed to medium containing hydrogen-rich water for 4 hours). The morphology of HT22 cells was observed by inverted microscopy; cell activity was detected by CCK-8 method; cell ultrastructure was observed by transmission electron microscopy; the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 was detected by immunofluorescence; the protein expression of LC3II/I and Beclin-1, markers of cellular autophagy, was detected by Western blotting.@*RESULTS@#Inverted microscopy showed that compared with the NC group, the OGD/R group had poor cell status, swollen cytosol, visible cell lysis fragments and significantly lower cell activity [(49.1±2.7)% vs. (100.0±9.7)%, P < 0.01]; compared with the OGD/R group, the HW group had improved cell status and remarkably higher cell activity [(63.3±1.8)% vs. (49.1±2.7)%, P < 0.01]. Transmission electron microscopy showed that the neuronal nuclear membrane of cells in the OGD/R group was lysed and a higher number of autophagic lysosomes were visible compared with the NC group; compared with the OGD/R group, the neuronal damage of cells in the HW group was reduced and the number of autophagic lysosomes was notably decreased. The results of immunofluorescence assay showed that the expressions of LC3 and Beclin-1 were outstandingly enhanced in the OGD/R group compared with the NC group, and the expressions of LC3 and Beclin-1 were markedly weakened in the HW group compared with the OGD/R group. Western blotting assay showed that the expressions were prominently higher in both LC3II/I and Beclin-1 in the OGD/R group compared with the NC group (LC3II/I: 1.44±0.05 vs. 0.37±0.03, Beclin-1/β-actin: 1.00±0.02 vs. 0.64±0.01, both P < 0.01); compared with the OGD/R group, the protein expression of both LC3II/I and Beclin-1 in the HW group cells were notably lower (LC3II/I: 0.54±0.02 vs. 1.44±0.05, Beclin-1/β-actin: 0.83±0.07 vs. 1.00±0.02, both P < 0.01).@*CONCLUSIONS@#Hydrogen-rich water has a significant protective effect on OGD/R-causing HT22 cell injury, and the mechanism may be related to the inhibition of autophagy.

Differential gene expression and bioinformatics analysis in sepsis secondary to pneumonia / 中华危重病急救医学

Objective:To analyze and screen the key genes of sepsis secondary to pulmonary infection by bioinformatics, and to provide theoretical basis for the basic research of the disease and find an ideal animal model program.Methods:Experiment 1 (bioinformatics analysis): gene expression data sets of pulmonary infection secondary sepsis patients and multiple sepsis animal models were screened by Gene Expression Omnibus (GEO) Database, and gene differences were analyzed by R software. Differential genes were analyzed by gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Correlation analysis was conducted between differential genes and clinical symptoms in the data set of pulmonary infection secondary sepsis, and the correlation heat map between differential genes and clinical symptoms was drawn. Key genes were screened by weighted gene co-expression network analysis (WGCNA) and protein-protein interaction network analysis (PPIN) clustering. Experiment 2 (sepsis animal model preparation): male mice weighing 21-25 g were randomly divided into the key genes group and the control (Sham) group. And cecal ligation and puncture (CLP) was used to establish mouse sepsis model, while the mice in sham group were performed by exposure of cecum. And all the mice were scarified 24 hours after surgery to extract the total RNA from lung tissue, real time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used to detect mRNA expression of key genes.Results:Experiment 1 (bioinformatics analysis): 319 differential genes were showed by GSE 134364 and GSE 65682 data set analysis of pulmonary infection secondary sepsis. And there was no genetic difference between community acquired pneumonia (CAP) and hospital acquired pneumonia (HAP) in patients with pulmonary infection secondary to sepsis. Obvious differences existed between differential genes in animal models, and there was no common differential gene. Differential genes in patients and animal models were similarly enriched in GO function, mainly in cell differentiation, regulation of cell process, and regulation of cellular response to stimuli, there were significant differences in pathway enrichment, among which, CLP animal models showed higher consistency with patients. The key genes obtained by WGCNA and PPIN analysis were MAPK14, NLRC4 and LCN2. Experiment 2 (sepsis animal model preparation): animal experiment results showed that the mRNA expressions of MAPK14, NLRC4 and LCN2 in lung tissue of CLP model mice were significantly up-regulated compared with the sham group.Conclusions:MAPK14, NLRC4 and LCN2 are key genes involved in the regulation of biological processes of pulmonary sepsis secondary to infection, and are potential research directions of this disease. What's more, CLP animal model can better reflect the biological characteristics of patients with pulmonary infection secondary sepsis, and is one of the ideal animal model schemes for pulmonary infection secondary sepsis.