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ObjectiveTo investigate the effect of Glycyrrhizae Radix et Rhizoma （GR）-containing serum on lipopolysaccharide （LPS）-induced inflammation in human colon epithelial adenocarcinoma cells （Caco2） based on inhibition of ferroptosis by the nuclear factor erythroid 2-related factor 2 （Nrf2）/heme oxygenase-1 （HO-1） pathway. MethodCaco2 cells were divided into a normal group， a model group （LPS， 200 μg·L-1）， low-， medium-， and high-dose GR-containing serum groups （5%， 10%， 20%）， and a ferroptosis inhibitor group （3-amino-4-cyclohexylamino-benzoic acid ethyl ester， Fer-1， 10 μmol·L-1）. The cells in the normal group were cultured normally， while those in other groups underwent the induction of an inflammation model. The cells in the low-， medium-， and high-dose GR-containing serum groups were treated with 5%， 10%， and 20% GR-containing serum for 24 hours， respectively， and the cells in the ferroptosis inhibitor group were treated with Fer-1 for 24 hours. Transmission electron microscopy was used to observe mitochondrial morphology in each group. Flow cytometry was used to detect intracellular Fe2+ levels. Microplate assays were performed to measure superoxide dismutase （SOD） activity， malondialdehyde （MDA） and glutathione peroxidase （GSH-Px） levels. Enzyme-linked immunosorbent assay （ELISA） was used to measure interleukin-1β （IL-1β）， IL-6， IL-10， and tumor necrosis factor-α （TNF-α） levels. Western blot was used to measure the expression levels of Nrf2， HO-1， ferritin heavy chain 1 （FTH1）， and glutathione peroxidase 4 （GSH-Px4） proteins. Small interfering RNA （siRNA） was used to investigate the role of Nrf2 in ferroptosis regulation. The cells after interference were divided into a negative control （NC） group， a Si-Nrf2 group， a GR-containing serum （20%） + Si-Nrf2 group， and a GR-containing serum （20%） + NC group. Microplate assays were performed to measure MDA， SOD， and GSH-Px levels， and Western blot was used to measure the expression levels of Nrf2， HO-1， FTH1， and GSH-Px4 proteins. ResultCompared with the normal group， the model group showed mitochondrial contraction， increased mitochondrial membrane thickness， and smaller mitochondrial morphology， increased Fe2+ content （P<0.01）， blunted SOD activity （P<0.01）， decreased GSH-Px expression （P<0.01）， increased MDA content （P<0.01）， reduced expression levels of Nrf2 and HO-1 （P<0.05）， reduced FTH1 expression （P<0.01）， and down-regulated GSH-Px4 expression （P<0.01）. In the GR-containing serum groups， the medium- and high-dose groups showed a significant decrease in Fe2+ content （P<0.01）， potentiated SOD and GSH-Px activities （P<0.01）， and decreased MDA levels （P<0.01）. The high-dose group showed a significant increase in Nrf2 expression （P<0.05）， and the medium-dose group showed increased expression of HO-1 and GSH-Px4 proteins （P<0.05）. The expression levels of FTH1 significantly increased in the low-， medium-， and high-dose groups （P<0.01）. The study on mechanism revealed that compared with the NC group， the cells transfected with Nrf2 siRNA showed increased MDA content （P<0.01）， blunted SOD activity （P<0.01）， decreased GSH-Px activity （P<0.01）， decreased expression of Nrf2 and HO-1 （P<0.01）， and reduced levels of FTH1 and GSH-Px4 proteins （P<0.01）. Compared with the Si-Nrf2 group， the cells treated with GR-containing serum showed a decrease in MDA content （P<0.01）， an increase in SOD activity （P<0.01）， an increase in GSH-Px activity （P<0.01）， increased expression of Nrf2 and FTH1 proteins （P<0.05）， and higher expression levels of HO-1 and GSH-Px4 proteins （P<0.01）. ConclusionGR-containing serum can reduce the inflammatory cytokines and oxidative stress levels in LPS-induced Caco2 cells. Its mechanism is related to the promotion of Nrf2/HO-1 signaling pathway expression， alleviating intracellular lipid peroxidation and inhibiting ferroptosis.
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Objective:To identify the potential pathogenic genes for perioperative neurocognitive disorder (PND) in the patients with digestive system tumors.Methods:The gene expression data of esophageal cancer, gastric cancer, colon cancer, rectal cancer and liver cancer in The Cancer Genome Atlas database were analyzed by bioinformatics analysis method, and the differentially expressed genes in tumor tissues in above-mentioned disease samples were identified compared with para-carcinoma tissues.Secretory proteome differential genes with the same expression trend in digestive system tumors were obtained by comparing with human secretory proteome genes.The correlation between secretomics and PND was determined by comparing with the GeneCards database.Hub genes were identified through PPI network construction and calculation, and the functions and signaling pathways of the above-mentioned differential genes were identified through GO and KEGG enrichment analysis.Results:Compared with para-carcinoma tissues, the expression of 2 640 genes was significantly up-regulated and the expression of 1 423 genes was down-regulated in esophageal cancer tissues; the expression of 3 748 genes was up-regulated and the expression of 908 genes was down-regulated in gastric cancer samples; the expression of 2 684 genes was up-regulated and the expression of 2 678 genes was down-regulated in colon cancer samples; the expression of 2 876 genes was up-regulated and the expression of 2 945 genes was down-regulated in rectal cancer samples; the expression of 1 484 genes was up-regulated and the expression of 723 genes was down-regulated in hepatocellular carcinoma samples.Among them, the expression of the encoding genes of 53 secreted proteins was uniformly up-regulated and the expression of the encoding genes of 20 secreted proteins was uniformly down-regulated in the above tumors.Twenty up-regulated genes and 3 down-regulated genes were associated with PND.PPI network analysis showed that MMP9 was the hub gene.The results of GO and KEGG analysis suggested that differentially expressed genes were mainly related to receptor-ligand activity, cytokine activity and chemokine activity, and were mainly enriched in signaling pathways related to cell cycle and cellular senescence.Conclusions:About 23 differentially expressed genes in digestive system tumors are potentially related to PND, of which MMP9 and other genes may be the hub genes, mainly acting on receptor-ligand binding, regulation of cytokine and chemokine activity, cell cycle, cellular senescence and other related signaling pathways.
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Objective To evaluate the relationship between the failed mechanism of sevoflurane postconditioning-induced myocardial protection and the activity of dynamin-related protein 1(Drp1)in dia-betic rats. Methods Pathogen-free healthy adult male Sprague-Dawley rats, weighing 220-280 g, in which diabetes mellitus was induced by combination of high-fat and high-sucrose diet and intraperitoneal injection of streptozotoein 30 mg∕kg, were studied.Sixty rats with diabetes mellitus were divided into 5 groups(n=12 each)using a random number table: sham operation group(group Sham), myocardial ischemia∕reperfusion (I∕R)group(group I∕R), sevoflurane postconditioning group(group SP), Drp1 inhibitor mitochondrial division inhibitor-1(Mdivi-1)group(group M)and Mdivi-1 plus sevoflurane postconditioning group(group M-SP). Myocardial I∕R was induced by occluding the left anterior descending branch of the coronary artery for 30 min followed by 120 min reperfusion except for group Sham. Mdivi-1 1.2 mg∕kg was intraperito-neally injected at 15 min before ischemia in M and M-SP groups, and 2.5% sevoflurane was inhaled starting from 5 min of reperfusion in SP and M-SP groups. Blood samples were collected from the right internal jugular vein at 120 min of reperfusion for measurement of serum cardiac troponin I(cTnI)concentrations(by en-zyme-linked immunosorbent assay). Rats were then sacrificed and myocardial specimens were obtained for de-termination of the myocardial infarct size(by TTC), cell apoptosis(by TUNEL), expression of Bax, Bcl-2 and activated caspase-3(by Western blot)and nicotinamide adenine dinucleotide(NAD+)content(by spectrophotometry). Apoptosis index(AI)and Bax∕Bcl-2 ratio were calculated. Results Compared with group Sham, the percentage of myocardial infarct size, serum concentration of cTnI, AI and Bax∕Bcl-2 ratio were significantly increased, the expression of activated caspase-3 was up-regulated, and the NAD+content was decreased in the other four groups(P<0.05). Compared with group I∕R, the percentage of myocardial infarct size, serum concentration of cTnI, AI and Bax∕Bcl-2 ratio were significantly decreased, the expres-sion of activated caspase-3 was down-regulated, and the NAD+content was increased in group M-SP(P<0.05), and no significant change was found in the parameters mentioned above in SP and M groups(P>0.05). Compared with group SP, the percentage of myocardial infarct size, serum concentration of cTnI, AI and Bax∕Bcl-2 ratio were significantly decreased, the expression of activated caspase-3 was down-regulated, and the NAD+content was decreased in group M-SP(P<0.05), and no significant change was found in the parameters mentioned above in group M(P>0.05). ConclusionThe failed mechanism of sevoflurane postconditioning-induced myocardial protection may be related to the activity of Drp1 in diabetic rats.
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Objective To investigate the effect of inhibition of glycogen synthase kinase?3 beta ( GSK?3β) activity on sevoflurane postconditioning?induced cardioprotection in diabetic rats. Methods Healthy adult male Sprague?Dawley rats, weighing 250-300 g, in which diabetes mellitus was induced by intraperitoneal 1% streptozotocin 60 mg∕kg combined with high?fat and high?sucrose diet and confirmed by blood glucose level >16. 7 mmol∕L. Forty rats with diabetes mellitus were divided into 5 groups ( n=8 each) using a random number table: sham operation group ( S group ) , ischemia?reperfusion ( I∕R ) group, sevoflurane postconditioning group ( SP group) , GSK?3β inhibitor SB216763 group ( SB group) , and sevoflurane postconditioning plus SB216763 group ( SS group ) . Myocardial ischemia was induced by 30 min occlusion of the left anterior descending branch of the coronary artery followed by 120 min reperfu? sion. The rats inhaled sevoflurane with the end?tidal concentration of 2.5% for 5 min starting from 1 min be?fore reperfusion in group SP. SB216763 0.2 mg∕kg was injected via the caudal vein at 1 min before reperfu?sion in group SB. In group SS, the rats inhaled sevoflurane with the end?tidal concentration of 2.5% for 5 min starting from 1 min before reperfusion, and SB216763 0.2 mg∕kg was injected via the caudal vein at 1 min before reperfusion. At 120 min of reperfusion, blood samples were collected from the carotid artery for determination of serum creatine kinase?MB (CK?MB) activity and cardiac troponin I (cTnI) concentra?tions. Myocardial specimens were collected at 120 min of reperfusion for microscopic examination of the pathological changes and for determination of myocardial infarct size ( by 2,3,5?triphenyltetrazolium chlo?ride staining) and phosphorylated GSK?3β (p?GSK?3β) expression (by Western blot). Results Com?pared with group S, the myocardial infarct size and serum CK?MB activity and cTnI concentration were sig?nificantly increased, and the expression of p?GSK?3βwas significantly down?regulated in I∕R, SP, SB and SS groups (P0.05) . Compared with group SB, the myocardial infarct size and serum CK?MB activity and cTnI concentration were significantly decreased, and the expression of p?GSK?3β was sig?nificantly up?regulated in group SS (P<0.05). The pathological changes of myocardium were significantly attenuated in SB and SS groups as compared with group I∕R and group SP . Conclusion Inhibition of GSK?3β activity can improve sevoflurane postconditioning?induced cardioprotection in diabetic rats.