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ObjectiveTo explore the mechanism of Qigesan （QGS） in intervening in the migration and invasion of esophageal carcinoma TE-1 cells. MethodMicroarray technology was used to screen differentially expressed genes （DEGs） in the normal group and the QGS group， and the ontological functions and signaling pathways of DEGs were analyzed. The thiazolyl tetrazolium （MTT） assay was used to detect the effect of QGS on the viability of TE-1 cells. In the subsequent experiments for verification， a blank group， a transforming growth factor-β1 （TGF-β1） group， a TGF-β1 + QGS group， and a TGF-β1 + SB431542 group were set up. The cell morphology in each experimental group was observed by microscopy. The migration and invasion abilities of cells were detected by wound healing assay， and the mRNA expression levels of E-Cadherin， vimentin， Smad2， and Smad7 were detected by Real-time quantitative polymerase chain reaction （Real-time PCR）. The protein expression of E-Cadherin， vimentin， p-Smad2/3， Smad2/3， and Smad7 was detected by Western blot. ResultThere were 1 487 DEGs between the QGS group and the blank group， including 1 080 down-regulated ones （accounting for 72.63%） and 407 up-regulated ones. The down-regulated genes were mainly involved in biological processes such as cytoskeletal protein binding， ATP binding， adenylate nucleotide binding， and adenylate ribonucleotide binding， and the involved Kyoto Encyclopedia of Genes and Genomes （KEGG） pathways included TGF-β signaling pathway， cell cycle， extracellular matrix-receptor interaction protein， tumor pathways， and oocyte meiosis. The up-regulated genes were mainly involved in RNA binding， DNA binding， transcriptional regulator activity， transcriptional activator activity， and nucleotide binding， and the KEGG pathways involved mainly included mitogen-activated protein kinase （MAPK） signaling pathway， bladder cancer， renal cell carcinoma， cancer pathways， and p53 signaling pathway. Compared with the blank group， the inhibition rate of cell viability of TE-1 cells increased after QGS （20， 30， 40， 60， 80 mg·L-1） intervention for 12， 24， 36， 48， 60 h （P<0.05）， and the inhibition rate was time- and dose-dependent. Compared with the blank group， the TGF-β1 group showed lengthened cells with fibroblast phenotype. Compared with the TGF-β1 group， the TGF-β1 + QGS group showed shortened cells with normal morphology and epithelial phenotype. The cell morphology in the TGF-β1 + SB431542 group was similar to that of the TGF-β1 + QGS group. Compared with the blank group， the TGF-β1 group showed potentiated ability of cell migration and invasion （P<0.05）. Compared with the TGF-β1 group， the TGF-β1 + QGS group and the TGF-β1 + SB431542 group showed inhibited and weakened migration and invasion abilities of cells （P<0.05）. However， there was no significant difference in migration and invasion abilities between the TGF-β1 + QGS group and the TGF-β1 + SB431542 group. The mRNA expression levels of vimentin and Smad2 in the TGF-β1 group were higher （P<0.05）， and the mRNA expression levels of E-Cadherin and Smad7 were lower （P<0.05） than those in the blank group. Compared with the TGF-β1 group， the TGF-β1 + QGS group and the TGF-β1+ SB431542 group exhibited decreased expression levels of vimentin and Smad2 mRNA （P<0.05）， and elevated expression levels of E-Cadherin and Smad7 mRNA （P<0.05）. Compared with the blank group， the TGF-β1 group showed up-regulated protein expression levels of vimentin， p-Smad2/3， and Smad2/3 （P<0.05）， and reduced protein expression levels of E-Cadherin and Smad7 （P<0.05）. Compared with the TGF-β1 group， the TGF-β1 + QGS group and the TGF-β1 + SB431542 group displayed decreased protein expression levels of vimentin， p-Smad2/3， and Smad2/3 （P<0.05）， and increased protein expression levels of E-Cadherin and Smad7 （P<0.05）. ConclusionThe ethyl acetate extract of QGS inhibits the epithelial-mesenchymal transition （EMT） of TE-1 cells through the TGF-β1 pathway to reduce the migration and invasion of TE-1 cells.
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ObjectiveTo investigate the mechanism of icariin in ameliorating efferocytosis dysfunction and inflammatory response of alveolar macrophages induced by cigarette smoke extract via the peroxisome proliferator-activated receptor gamma （PPARγ） signaling pathway. MethodThe untreated rat alveolar macrophages （NR8383） were taken as the blank group. The NR8383 cells treated with 10% cigarette smoke extract were divided into model， low-， medium-， and high-dose （10， 20， 40 μmol·L-1） icariin， PPARγ inhibitor， and PPARγ inhibitor + low-， medium-， and high-dose icariin groups. Alamar blue colorimetry was employed to examine the proliferation and toxicity of icariin on NR8383 cells. The efferocytosis rate of NR8383 cells was detected by flow cytometry. Enzyme-linked immunosorbent assay was employed to measure the levels of tumor necrosis factor-alpha （TNF-α）， transforming growth factor-β1 （TGF-β1）， and milk fat globule-epidermal growth factor 8 （MFG-E8）. Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） were employed to determine the protein and mRNA levels， respectively， of PPARγ， CD36， and RAS-related C3 botulinum toxin substrate 1 （Rac1）. ResultThe efferocytosis dysfunction model of NR8383 was established with the cigarette smoke extract. Compared with the blank control group， the model group showed decreased efferocytosis rate （P<0.05）， elevated TNF-α level （P<0.05）， lowered TGF-β1 and MFG-E8 levels （P<0.01）， and down-regulated mRNA and protein levels of PPARγ， CD36， and Rac1 （P<0.05， P<0.01）. Compared with the model group， the treatment with icariin increased the efferocytosis rate （P<0.05， P<0.01）， lowered the TNF-α level （P<0.01）， elevated TGF-β1 and MFG-E8 levels （P<0.05）， and up-regulated the protein and mRNA levels of PPARγ， CD36， and Rac1 （P<0.05， P<0.01）. Compared with icariin alone， PPARγ inhibitor + icariin decreased the efferocytosis rate （P<0.05） and down-regulated the protein and mRNA levels of PPARγ （P<0.05， P<0.01）. In addition， PPARγ inhibitor + low-dose icariin down-regulated the protein level of CD36 （P<0.01） and PPARγ inhibitor + low-/medium-dose icariin up-regulated the protein level of Rac1 （P<0.05）. ConclusionIcariin ameliorates the cigarette smoke extract-induced efferocytosis dysfunction of alveolar macrophage by regulating the PPARγ signaling pathway and cytoskeletal structure rearrangement.
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Objective:To observe the effects of Xiaoqinglong Decoction and Qingqi Huatan Pills on interleukin-1β(IL-1β)-induced mucushypersecretion model of human airway epithelial H292 cellsand related molecules of nuclear factor-κB/microRNA-494 (NF-κB/miR-494) signaling pathway, and to explore the mechanism of the two medicines in improving pathological airway mucus.Methods:Methyl thiazolyl tetrazolium (MTT) colorimetric method was used to detect the effects of different concentrations of Xiaoqinglong Decoction and Qingqi Huatan Pills on the activity of H292 cellsinduced by IL-1β, and the appropriate concentration was selected for subsequent experiments. Cells were randomly divided into blank group, IL-1β model group (5 μg/L IL-1β), NF-κB inhibitor pyrrolidinedithiocarbamate (PDTC) group (5 μg/L IL-1β+100 μmol/L PDTC), Xiaoqinglong Decoction (5 μg/L IL-1β+1 000 mg/L Xiaoqinglong Decoction) and Qingqi Huatan Pill group (5 μg/L IL-1β+1 000 mg/L Qingqi Huatan Pills). 5 μg/L IL-1β was used to induce H292 cells for 24 hours to establish a model of airway epithelial mucus hypersecretion. Enzyme linked immunosorbent assay (ELISA) method was used to detect the levels of mucin 5AC (MUC5AC), tumor necrosis factor-α (TNF-α) and IL-8 and the synthesis of intracellular MUC5AC and cystic fibrosis transmembrane conductance regulator (CFTR). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression levels of MUC5AC mRNA, CFTR mRNA, miR-494. Western blotting was used to detect protein expression of key proteins (p65) and NF-κB inhibitors (IκB) in NF-κB signaling pathway.Results:Xiaoqinglong Decoction and Qingqi Huatan Pills with the concentration of 1 000 mg/L were selected for the follow-up experiment. Compared with the blank group, the levels of MUC5AC, TNF-α and IL-8 were significantly increased in the model group, intracellular MUC5AC protein content and mRNA expression were also significantly increased, intracellular CFTR protein content and mRNA expression were significantly decreased, and intracellular p65 protein expression was significantly up-regulated, the expression of IκB protein was significantly down-regulated, and the expression of miR-494 was significantly increased. Compared with the model group, the levels of MUC5AC, TNF-α and IL-8 were significantly reduced in PDTC group, Xiaoqinglong Decoction group and Qingqi Huatan Pill group, intracellular MUC5AC protein content and mRNA expression were also significantly decreased, and intracellular p65 protein expression was significantly down-regulated, and IκB protein expression was significantly up-regulated, miR-494 expression was significantly reduced. Intracellular CFTR protein content and mRNA expression were significantly increased in both PDTC group and Qingqi Huatan Pill group. Compared with the PDTC group, the level of TNF-α in the Xiaoqinglong Decoction group was significantly increased (ng/L: 22.77±3.14 vs. 11.09±3.37, P < 0.05), the content and mRNA expression of CFTR and IκB protein expression was significantly decreased [CFTR protein (ng/L): 97.38±6.62 vs. 227.04±19.48, CFTR mRNA (2 -ΔΔCt): 0.99±0.08 vs. 1.21±0.08, IκB/β-actin: 1.69±0.11 vs. 2.00±0.18, all P < 0.05], the level of TNF-α in Qingqi Huatan Pill group was significantly higher (ng/L: 19.08±3.71 vs. 11.09±3.37, P < 0.05). Compared with Xiaoqinglong Decoction group, the protein content and mRNA expression of CFTR and IκB protein expression in Qingqi Huatan Pill group were significantly increased [CFTR protein (ng/L) : 235.01±22.71 vs. 97.38±6.62, CFTR mRNA(2 -ΔΔCt): 1.32±0.15 vs. 0.99±0.08, IκB/β-actin: 1.94±0.16 vs. 1.69±0.11, all P < 0.05]. Conclusions:The effect of Xiaoqinglong Decoctionin improving the hypersecretion of mucus in the airway epithelium may be related to the inhibition of NF-κB/miR-494 inflammatory signal-mediated MUC5AC hypersecretion, while the effect of Qingqi Huatan Pills may be related to the inhibition of NF-κB/miR-494 inflammatory signal-mediated MUC5AC hypersecretion and CFTR dysfunction. Therefore, the difference in the mechanism of the two treatments of airway pathological mucus is mainly in the regulation of CFTR mRNA and protein.
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Objective:To investigate the influence of hemoglobin glycation index (HGI) on the risk of incident chronic kidney disease (CDK) among nondiabetic patients.Methods:Prospective cohort study. At baseline, a total of 7 407 nondiabetic patients without a history of CKD from Pingguoyuan Community of the Shijingshan District in Beijing were included from December 2011 to August 2012, who were then divided into three groups according to the tertiles of their baseline HGI levels. The CKD incidence rate was compared among the different HGI groups at last follow-up. Cox multivariable regression was applied to evaluate whether HGI measures predicted CKD risk. Test for trend across tertiles were examined using ordinal values in separate models.Results:The mean age of the subjects was (56.4±7.5) years, and 4 933 (66.6%) were female. At mean follow-up of 3.23 years, 107 (1.4%) individuals developed CKD. The incidence of CKD was gradually increasing from the low to high HGI groups [1.1% (28/2 473) vs. 1.2% (31/2 564) vs. 2.0% (48/2 370), P=0.016]. In the multivariate Cox regression analysis, after adjustment for potential confounders, the high HGI group had a 68.5% increased risk of CKD compared with the low HGI group ( HR=1.685, 95% CI 1.023 to 2.774). CKD risk increased with increasing HGI tertiles ( P for trend=0.028). Conclusion:High HGI is associated with an increased risk for CKD in the nondiabetic population, indicating that HGI may help identify individuals at high risk for CKD.
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Objective:To investigate the effects and its mechanism of chronic unpredictable stress on intestine and liver injuries in rats, and explore the possibility of the existence of brain-gut-liver axis.Methods:Twenty male SD rats were randomly divided into control group and stress group (with 10 in each group). The rats in the stress group were stimulated by chronic unpredictable stress for 4 weeks to prepare the chronic stress model. The rats in the control group were fed normally without stress stimulation. After modeling, ten rats in the control group and seven rats in the stress group were included. The depressive behavior of the two groups was evaluated by sugar water preference experiment. Then the rats were sacrificed. The diversity of gut flora in intestinal feces was analyzed by 16S rRNA sequencing analysis. The pathological injuries of ileum and liver were detected by HE staining. The expressions of occludin in ileum and Toll-like receptor 4 (TLR4) in liver were detected by immunohistochemistry. The expression of TLR4 protein in liver tissue was detected by Western blot. The level of lipopolysaccharide (LPS) in rat portal vein serum was detected by AZO chromogenic limulus test and blood biochemical method was used to detect liver function.Statistical analysis was performed using SPSS 25.0 software, and t-test or Mann-Whitney U test was used for comparison between the two groups. Using STAMP software, Wilcoxon rank sum test was used to analyze the difference in bacterial abundance between the two groups. Results:The consumption of sugar water ((7.86±0.90)ml) and the preference rate of sugar water ((43.06±5.65)%) in the stress group were lower than those in the control group ((15.10±1.51)ml, (76.81±6.44)%), and the difference were statistically significant ( t=11.33, 11.16, both P<0.01). Chronic stress caused pathological damage to rat ileum tissue. Compared with the control group, the ileum villi of rats in the chronic stress group were longer ((448.93±12.71)μm, (497.12±16.72)μm, t=-5.88, P<0.01) and thicker ((81.99±16.54)μm, (133.93±6.78)μm, t=-7.12, P<0.01), and the expression of occludin was significantly down-regulated ((0.236±0.011), (0.130±0.026), t=9.12 , P<0.01), the LPS level increased significantly ((18.83±2.62)EU/L, (38.64±2.51)EU/L, t=-5.79, P<0.01). The Beta diversity of rat intestinal flora changed under chronic stress, and the abundance of WPS-2 phylum in intestinal tract of rats in stress group was higher than that in control group ( t=2.76, P<0.05). Chronic stress caused pathological damage to the liver tissue of rats. Compared with the control group, the expression of TLR4 protein in the liver tissue of the chronic stress group increased ((0.169±0.014), (0.475±0.034), Z=-2.37, P<0.05). Compared with the control group, the ALT ((39.7±6.2)U/L, (82.9±43.1)U/L, Z=-2.35, P<0.05) and AST((130.9±28.9)U/L, (472.7±263.3)U/L, Z=-2.64, P<0.05) levels of the chronic stress group increased, especially in AST. Conclusion:Chronic stress cause synchronous damage to the intestine and liver in rats. The mechanism may be related to the results caused by chronic stress such as the changes of the diversity of intestinal flora, the increasing of intestinal permeability, the action of LPS translocated through portal vein blood on TLR4 in liver.
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Objective:To screen the time points of high survival rate and efferocytosis dysfunction of rat alveolar macrophages stimulated by cigarette smoke extract (CSE), establish an in vitro model of alveolar macrophage efferocytosis function, and study chronic respiratory diseases with chronic inflammatory reaction as the main pathological changes. Methods:① Time point screening experiment: rat alveolar macrophages (NR8383 cells) were cultured in vitro, and the cells in logarithmic growth phase were divided into blank control group (100 μL complete medium) and 5% CSE group (90 μL complete medium + 10 μL 100% CSE). Alma blue method was used to detect the effect of 5% CSE on the activity of NR8383 cells at 6, 12, 24 and 48 hours. ② Apoptosis induction experiment: rat type Ⅱ alveolar epithelial cells (RLE-6TN cells) were cultured in vitro as phagocytic target cells of NR8383 cells, and the cells in logarithmic growth phase were divided into blank control group and 10, 30 and 60 minutes groups after ultraviolet exposure (apoptosis was induced by 30 000 μJ/cm 2 ultraviolet irradiation for 15 minutes). Flow cytometry was used to detect the apoptosis rate of RLE-6TN cells cultured for 10, 30 and 60 minutes after ultraviolet exposure. ③ Cell efferocytosis experiment: NR8383 cells in logarithmic phase were divided into blank control group and 5% CSE group. Two hours before NR8383 cells were stimulated by CSE for 6, 12 and 24 hours, RLE-6TN cells were exposed to ultraviolet to induce apoptosis, and the RLE-6TN cell suspension was added to NR8383 cells (the ratio of RLE-6TN cells to NR8383 cells was 5∶1). Flow cytometry was used to detect the efferocytosis rate of NR8383 cells to RLE-6TN cells at different time points treated with 5% CSE. Results:① Compared with the blank control group, the activity of NR8383 cells significantly decreased after treatment with 5% CSE for 48 hours [cell reduction rate: (68.5±4.1)% vs. (73.6±2.3)%, P < 0.05]. However, there were no significant differences when the activities of NR8383 cells treated with 5% CSE for 6, 12 and 24 hours were compared with the blank control group, so these three time points were selected for the subsequent establishment of alveolar macrophage cell efferocytosis dysfunction in vitro model experiment. ② Compared with the blank control group, the apoptosis rate of RLE-6TN cells significantly increased at 10, 30 and 60 minutes after ultraviolet exposure [(66.87±8.63)%, (85.51±2.39)%, (96.13±2.74)% vs. (9.13±3.17)%, all P < 0.01] in a time-dependent manner. Considering that it taked about 50 minutes for RLE-6TN cells to be labeled with PKH26 membrane labeling probe, 10 minutes after ultraviolet exposure was selected to label RLE-6TN cells. ③ Compared with the blank control group, the efferocytosis function of NR8383 cells was significantly decreased after treatment with 5% CSE for 12 hours [cell efferocytosis rate: (33.64±1.30)% vs. (44.02±2.71)%, P < 0.01], but there was no significant effect on the efferocytosis function of NR8383 cells at 6 hours and 24 hours. Conclusions:CSE can induce alveolar macrophage cell efferocytosis dysfunction. Based on the test results of the effect of 5% CSE on NR8383 cell activity and cell efferocytosis function, 12 hours with high survival rate and weak efferocytosis effect of NR8383 cells can be selected as the in vitro model condition of alveolar macrophage cell efferocytosis dysfunction.
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OBJECTIVE@#To investigate the protective effect of melatonin against myocardial ischemia reperfusion (IR) injury in isolated rat hearts and explore the underlying mechanisms.@*METHODS@#The isolated hearts from 40 male SD rats were randomly divided into 4 groups (=10): the control group, where the hearts were perfused with KH solution for 175 min; IR group, where the hearts were subjected to global ischemia for 45 min followed by reperfusion for 120 min; IR+melatonin (Mel+IR) group, where melatonin (5 μmol/L) was administered to the hearts 1 min before ischemia and during the first 5 min of reperfusion, followed by 115 min of reperfusion; and IR+2, 3-butanedione monoxime (IR+BDM) group, where the hearts were treated with BDM (20 mmol/L) in the same manner as melatonin treatment. Myocardial injury in the isolated hearts was assessed based on myocardial injury area, caspase-3 activity, and expressions of cytochrome C and cleaved caspase-3 proteins. Cardiac contracture was assessed using HE staining and by detecting lactate dehydrogenase (LDH) activity and the content of cardiac troponin I (cTnI) in the coronary outflow, measurement of left ventricular end-diastolic pressure (LVEDP) and electron microscopy. The content of ATP in the cardiac tissue was also determined.@*RESULTS@#Compared with those in the control group, the isolated hearts in IR group showed significantly larger myocardial injury area and higher caspase-3 activity and the protein expressions of cytochrome C and cleaved caspase-3 with significantly increased LDH activity and cTnI content in the coronary outflow and elevated LVEDP at the end of reperfusion; HE staining showed obvious fractures of the myocardial fibers and the content of ATP was significantly decreased in the cardiac tissue; electron microscopy revealed the development of contraction bands. In the isolated hearts with IR, treatment with Mel or BDM significantly reduced the myocardial injury area, caspase-3 activity, and protein expressions of cytochrome C and cleaved caspase-3, obviously inhibited LDH activity, lowered the content of cTnI and LVEDP, reduced myocardial fiber fracture, and increased ATP content in the cardiac tissue. Both Mel and BDM inhibited the formation of contraction bands in the isolated hearts with IR injury.@*CONCLUSIONS@#Mel can alleviate myocardial IR injury in isolated rat hearts by inhibiting cardiac contracture, the mechanism of which may involve the upregulation of ATP in the cardiac myocytes to lessen the tear of membrane and reduce cell content leakage.
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Animals , Male , Rats , Contracture , Melatonin , Myocardial Ischemia , Myocardial Reperfusion Injury , Myocardium , Myocytes, Cardiac , Rats, Sprague-DawleyABSTRACT
OBJECTIVE@#To investigate the protective effect of melatonin against myocardial ischemia reperfusion (IR) injury in isolated rat hearts and explore the underlying mechanisms.@*METHODS@#The isolated hearts from 40 male SD rats were randomly divided into 4 groups (=10): the control group, where the hearts were perfused with KH solution for 175 min; IR group, where the hearts were subjected to global ischemia for 45 min followed by reperfusion for 120 min; IR+melatonin (Mel+IR) group, where melatonin (5 μmol/L) was administered to the hearts 1 min before ischemia and during the first 5 min of reperfusion, followed by 115 min of reperfusion; and IR+2, 3-butanedione monoxime (IR+BDM) group, where the hearts were treated with BDM (20 mmol/L) in the same manner as melatonin treatment. Myocardial injury in the isolated hearts was assessed based on myocardial injury area, caspase-3 activity, and expressions of cytochrome C and cleaved caspase-3 proteins. Cardiac contracture was assessed using HE staining and by detecting lactate dehydrogenase (LDH) activity and the content of cardiac troponin I (cTnI) in the coronary outflow, measurement of left ventricular end-diastolic pressure (LVEDP) and electron microscopy. The content of ATP in the cardiac tissue was also determined.@*RESULTS@#Compared with those in the control group, the isolated hearts in IR group showed significantly larger myocardial injury area and higher caspase-3 activity and the protein expressions of cytochrome C and cleaved caspase-3 with significantly increased LDH activity and cTnI content in the coronary outflow and elevated LVEDP at the end of reperfusion; HE staining showed obvious fractures of the myocardial fibers and the content of ATP was significantly decreased in the cardiac tissue; electron microscopy revealed the development of contraction bands. In the isolated hearts with IR, treatment with Mel or BDM significantly reduced the myocardial injury area, caspase-3 activity, and protein expressions of cytochrome C and cleaved caspase-3, obviously inhibited LDH activity, lowered the content of cTnI and LVEDP, reduced myocardial fiber fracture, and increased ATP content in the cardiac tissue. Both Mel and BDM inhibited the formation of contraction bands in the isolated hearts with IR injury.@*CONCLUSIONS@#Mel can alleviate myocardial IR injury in isolated rat hearts by inhibiting cardiac contracture, the mechanism of which may involve the upregulation of ATP in the cardiac myocytes to lessen the tear of membrane and reduce cell content leakage.
Subject(s)
Animals , Male , Rats , Heart , Melatonin , Pharmacology , Therapeutic Uses , Muscle Contraction , Myocardial Reperfusion Injury , Drug Therapy , Myocytes, Cardiac , Rats, Sprague-DawleyABSTRACT
As one of the top three causes of death in the world, chronic obstructive pulmonary disease (COPD) is a serious hazard to human health. Macrophages play an important role in COPD, and their efferocytosis function is essential for ending chronic inflammation of COPD. Efferocytosis damage of alveolar macrophages (AM) in patients with COPD causes the rising of bacterial infection and airway bacterial colonization risk in lungs, which is the main reason for the acute exacerbation and the rising of incidence rate and mortality rate in COPD. In recent years, the regulation of macrophage efferocytosis function in COPD has becoming a research hotspot. Progress on the role of macrophage efferocytosis function on COPD, and the breakthrough points of improving AM efferocytosis dysfunction by traditional Chinese medicine is reviewed, so as to provide new ideas for the prevention and treatment of COPD.
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Objective:To investigate the role of Na+/Ca2+ exchanger (NCX) in myocardial ischemiareperfusion injury and the underlying mechanisms.Methods:Forty Sprague-Dawley rats were divided into 4 groups randomly:a control group,a KBR7943 group,an ischemia-reperfusion group (IR group),and an IR plus KB-R7943 group (KB-R7943+IR group).Isolated Sprague Dawley male rat hearts underwent Langendorffperfusion.The ratio of left ventricular developed pressure (LVDP),left ventricular end-diastolic pressure (LVEDP),the infarct size of myocardium,and the lactate dehydrogenase (LDH) activity in the coronary flow was determined.HE staining was used to assess the change of myocardial morphology.Western blot was used to determine the levels of cleaved caspase-3,cytochrome c and the phosphorylation of Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) and the Thr17 site ofphospholamban.Results:Compared with the control group,IR group significantly induced an enlarged infarct size,reduction of the ratio of LVDP,up-regulation of cytochrome c,cleaved caspase-3,p-CaMKⅡ and p-phospholamban,and increased in the activity of LDH,the level of LVEDP (P＜0.01) and the disordered myocardial morphology.These effects were significantly attenuated in the presence of KB-R7943 treatment (10 μmol/L).Conclusion:NCX mediates myocardial ischemia-reperfusion-induced cell apoptosis and necrosis through activation of CaMKⅡ.
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Objective To observe the effects of nourishing lung and kidney formulas on inflammatory response of alveolar epithelial cells stimulated by monocytes conditioned medium and study the anti-inflammatory mechanism of the formulas for the treatment of chronic obstructive pulmonary diseases (COPD).Methods The reproduction of inflammation models of A549 cells were stimulated by monocyte THP-1 cell strain conditioned medium.A549 cells were randomly divided into blank control group (20％ blank rabbits serum),model group (20％ blank rabbits serum+25.0％ THP-1 cell conditioned medium),nuclear transcription factor activator protein-1 (AP-1) pathway inhibitor T-5224group (20％ blank rabbits serum+25.0％ THP-1 cells conditioned medium+100 μmol/L T-5224),nourishing lung and kidney group (20％ rabbits serum with nourishing lung and kidney formulas+25.0％ THP-1 cells conditioned medium).The contents of interleukins (IL-6,IL-8),tumor necrosis factor-α (TNF-αα),matrix metalloprotein-9 (MMP-9) in cell culture supernatant were detected with enzyme linked immunosorbent assay (ELISA),the supernatant content of malondialdehyde (MDA) was detected with thibabituric acid (TBA) method,the total activity of superoxide dismutase (T-SOD) was detected with hydroxylamine method,and the activity of AP-1 pathway was detected with electrophoretic mobility shift assay (EMSA) method.Results Compared with the blank control group,the A549 cell proliferation were significantly increased at 24 hours,48 hours stimulation by 25.0％ cell conditioned medium (A value:24 hours was 0.41 ± 0.02 vs.0.37 ± 0.04,48 hours was 1.30 ± 0.09 vs.1.15 ± 0.19).Compared with the blank control group,the contents of IL-6,IL-8,TNF-αα,MMP-9,MDA,AP-1 expression were significantly increased in model group [IL-6 (ng/L):35.00±3.63 vs.23.15±1.72,IL-8 (ng/L):273.09± 164.36 vs.231.45±33.90,TNF-α(ng/L):51.61 ± 9.51 vs.28.87 ± 3.34,MMP-9 (ng/L):442.85 ± 78.86 vs.235.60 ± 14.62,MDA (μmol/L):6.90 ± 0.11 vs.6.01 ± 0.12,AP-1 expression (A value):2.260 ± 0.062 vs.1.000 ± 0.000],MDA/T-SOD ratio was increased (4.43 ± 0.05vs.3.96 ± 0.06).Compared with model group,the levels of IL-8 (ng/L:100.29 ± 17.03),TNF-α (ng/L:25.13 ± 0.46),AP-1 expression (A value:1.38 ± 0.02),and the MDA/T-SOD ratio (4.23 ± 0.23) in T-5224 group,and MMP-9 (ng/L:195.44±9.80),MDA (μmol/L:5.86±0.30),MDA/T-SOD ratio (3.56±0.41),AP-1 expression (A value:0.76 ± 0.01) in nourishing lung and kidney group were all reduced significantly (all P ＜ 0.05).Conclusion Nourishing lung and kidney formulas can suppress the inflammatory response through regulating the alveolar epithelial cells AP-1 signaling pathways.
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Objective To investigate the influence of three different processing methods on contents and speciation of heavy metals Pb,Cd,Hg and Cu and a harmful nonmetallic element As in Cortex Eucommiae. Methods The five elements were measured by atomic absorption spectrometry. The determination wavelengthes of Pb and Cd was 283.3 and 228.8 nm using graphite furnace atom?ic absorption spectrometry;the determination wavelength of Cu was 324.8 nm using flame atomic absorption spectrometry;the determi?nation wavelengthes of As and Hg were 193.7 and 253.6 nm using flow injection-hydride generation method. Results Heavy metal′s contents in Cortex Eucommiae stir-fried with salt were increased. Contents of heavy metals in Cortex Eucommiae stir-fried and baked were decreased. Heavy metal morphology had no differences between processed products and raw materials. The experimental results showed that main existing form of Pb was in organic matter-bound,and mercury existed in exchange condition and organic matter-bound. Copper existed in organic matter-bound and carbonate bound and arsenic mainly existed with residual forms. Cadmium mainly exited in carbonate exchange condition. Conclusion There is a certain effect on the heavy metal content but less effect on the form of heavy metals by different processing methods.
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Cell culture technology is the most commonly used method in the in vitro experiments at present. However, monolayer cell culture technology has been unable to meet the demand of the researchers. This is because that monolayer cell culture cannot mimic the cellular environment in which multiple cells interact with each other in the body. We cannot discuss the relationship of many cells, because we do not know the relationship between cells through a single kind of cell. So cell co-culture medicine arises at the historic moment for the demand. With the development of research method in recent years, cell co-culture method also has been improved in practice: from direct contact co-cultures to indirect contact co-cultures, from two-dimensional co-cultures to three-dimensional co-cultures. Cell co-culture method is closer to the human body. It is also more advantageous to study the interaction among cells. Nowadays, there are more researchers tend to select this method to study the physiological and pathological in vitro model, tissue engineering, and cell differentiation research. At the same time, it has become the focus of drug research and development, drug analysis, mechanism of drug action, and drug targets. This article will review the studies of cell co-culture method, summarize advantages and disadvantages of various methods, so as to promote improvement of cell culture methods, to build cells co-culture system that more close to human body, and build the in vitro model that simulate internal circulation of human body further.
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Objective:To study the related factors of severe acute radiation-induced lung injury (SAR) caused by IMRT and concurrent chemotherapy for non-small cell lung cancer. Methods:We retrospectively analyzed the data of 2 323 non-small cell lung cancer pa-tients who underwent IMRT radiotherapy and concurrent chemotherapy at the Department of Radiotherapy of Tianjin Medical Univer-sity Cancer Institute and Hospital from January 2010 to January 2014. We analyzed the clinical factors and parameters that affect dose by univariate and multivariate analysis. Results:A total of 2 323 patients enrolled and 1 241 cases suffering from acute radiation-in-duced lung injury with the rate of 53.4%. Only 185 cases suffered from SARP with a rate of 7.96%. Univariate analysis showed that the gender, histopathological type, total radiation dose, V5 (%), and average dose rate are not related to SARP (P>0.05). By contrast an age of>60 years, 1%predicted FEV, docetaxel+carboplatin/cisplatin chemotherapy, V20 (%), V30 (%), and mean lung dose (MLD) are sig-nificantly related to SARP (P60 years, docetaxel+carboplatin/cisplatin che-motherapy, V20 (%), and V30 (%) are the independent risk factors of SARP. Conclusion:Among the non-small cell lung cancer patients undergoing IMRT radiotherapy and concurrent chemotherapy, further attention should be given to elderly patients, patients receiving docetaxel and platinum chemotherapy, as well as V20 and V30 with high doses. The necessary preventive treatment should be given to reduce the incidence of SARP, improve the quality of life of patients, and reduce the incidence of respiratory failure and mortality.
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ABSTRACT:Objective To analyze the correlation between plasma nitric oxide (NO)level and atherosclerotic lesion in high cholesterol-fed (HCD ) rabbits.Methods Twenty male Japanese white rabbits were divided randomly into two groups,which were fed with normal diet (control group,n =10)or HCD (experimental group, n =10 )for 1 6 weeks.At the end of the experiment,plasma lipid and NO levels were measured.The gross atherosclerotic lesions in each group were detected by Sudan IV staining while intimal lesion area was measured by hematoxylin/eosin (HE)and elastica van gieson (EVG).Moreover,the macrophages (MΦ)and smooth muscle cells (SMC)were detected by immunohistochemical staining.The correlation analysis was made to reveal the relationship between atherosclerotic lesions and plasma NO level.Results Compared with those in control group, the total cholesterol (TC),triglyceride (TG),low-density lipoprotein cholesterol (LDL-C)and NO levels all increased significantly in experimental group.Atherosclerotic lesions appeared on the vascular wall in the latter group.The area of atherosclerotic lesions and MΦ in the plaque had a positive association with plasma NO level. Conclusion There is a relationship between plasma NO level and the size of HCD-induced atherosclerotic lesions in rabbits.Meanwhile,the MΦ positive area in the atherosclerotic plaque is also associated with plasma NO level in cholesterol-fed rabbits,suggesting that plasma NO level may be associated with the occurrence and progress of early atherosclerosis.
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Objective The aim of this study was to generate human cholesteryl ester transfer protein ( CETP) transgenic rabbits and analyze their biological properties.Methods We generated human CETP transgenic rabbits by DNA microinjection, and detected the expression of human CETP by real-time PCR and Western blot assay.The activity of CETP was measured using an activity assay kit.Results Human CETP transgenic rabbits were successfully generated by DNA microinjection.Compared with wide type rabbits, the expression of human CETP was dramatically increased in the liver of the human CETP transgenic rabbits.The plasma CETP activity was also much higher in the liver of human CETP transgenic rabbits than that of control rabbits.Conclusions The model of human CETP transgenic rabbits is successfully established by DNA microinjection.It will provide a useful tool for the studies of CETP biological function and its involvement in the mechanisms of cardiovascular diseases.
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Objective To observe the clinical efficacy of duloxetine in the treatment of malignant neuropathic pain with depression.Methods 60 patients were randomly divided into two groups as study group(30 cases) and contrdl group(30 cases) and treated for 4 weeks.The patients of study group were treated with duloxetine and oxycontin,and the patients of control group were treated with oxycontin only.Numberical rating scale (NRS) on pain,criteria of pain relief and Hamilton depression scale(HAMD,17 items) score were used to assess the therapeutic effect before and after treatment.Results By the end of the fourth week of treatment,the average usage of oxycontin of the study group was significantly less than control group((45.6±8.5) mg vs (88.2±5.2)mg,P＜0.05).The effective rate of pain relief in the study group was significantly higher than that in control group (93.3％ vs 73.3％,P＜0.05).Comparing pre-treatment,the score of HAMD of the study group had a remarkable decrease ((11.45±4.56) vs (23.07±5.47),P＜0.01).In comparison to the score of control group,study group had a significant effect ((11.45±4.56) vs (18.75±4.21),P＜0.01).Conclusion Duloxetine is one of effective agents in the treatment of malignant neuropathic pain with depression,which can alleviate depression and relieve pain.Duloxetine have mild adverse effects and good tolerance.
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Objective To study the effect of Qige San ethyl acetate extract on the proliferation of human esophageal carcinoma Eca109 cells and to explore its possible mechanism. Methods Inhibitory effects of Qige San ethyl acetate extract on the proliferation of esophageal carcinoma Eca109 cells were detected by MTT assay, cell apoptosis was detected by flow cytometer, and the expression of protein STAT3, Bcl-2 and Caspase 9 was detected by Western blotting method. Results In the range of 10~100 μg/mL, Qige San ethyl acetate extract inhibited the proliferation of Eca109 cells effectively (P<0.05) . Compared with the control group, Qige San ethyl acetate extract in the concentrations of 1.47, 33.26, 75.52 μg/mL significantly increased the apoptotic rate of Eca109 cells within 48h ( P<0.01). And Western blotting results showed that the ex pression levels of STAT3 and Bcl-2 were reduced, and Caspase 9 was increased with the increase of drug concentration. Conclusion Qige San ethyl acetate extract could significantly inhibit the proliferation of Eca109 cells and induce cell apoptosis, and its mechanism is probably associated with the inhibition of signal transducers and activators of transcription 3 (STAT3) signaling pathway.
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Objective To investigate the effect of allicin on the development of atherosclerosis in apoE-/-mice and explore its underlying mechanism from the perspective of protein S-nitrosylation.Methods Thirty male apoE-/- mice were randomly divided into 3 groups:control group (saline,ig),low-dose group (allicin,9 mg/kg·d, ig)and high-dose group (allicin,18 mg/kg·d,ig).They were fed with high cholesterol diet for 12 weeks.The levels of plasma lipids,oxidized-LDL (ox-LDL),malondialdehyde,tumor necrosis factor-alpha and nitric oxide (NO)were measured.The atherosclerotic lesions in aortic root were evaluated after hematoxylin and eosin staining and elastica van Gieson and immunohistochemical staining,respectively.Furthermore,in vitro experiments were performed using human umbilical vein endothelial cells (HUVECs).The HUVECs were treated with allicin (10μmol/L or 20 μmol/L)for 24 hours in the presence of ox-LDL (50 μg/mL).The level of NO in supernatant was measured by a nitrate/nitrite assay. The protein S-nitrosylation of the HUVECs was detected through immunofluorescence.Results The histological analysis revealed that allicin treatment not only significantly decreased the areas of the atherosclerotic lesion (all P <0.05)but also suppressed the macrophage accumulation and smooth muscle cell proliferation in the lesion.There was no significant difference in the levels of plasma lipids between control and treated groups.However,allicin exerted obvious anti-oxidative and anti-inflammatory effects. Interestingly,the allicin treatment led to marked increase of the plasma NO level (P <0.05)and aortic protein S-nitrosylation.The experiments in vitro further proved that the allicin up-regulated the levels of NO and protein S-nitrosylation in HUVECs treated with ox-LDL (P < 0.01 ).Conclusion Allicin can inhibit the development of atherosclerosis.The mechanism is associated with the up-regulation of protein S-nitrosylation in endothelial cells, which plays an important role in anti-oxidization and anti-inflammation.
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Objective:To explore the feasibility and safety of CT-guided hookwire localization of small lung nodule in video-as-sisted thoracic surgery. Methods: Preoperative localization of small lung nodule was performed using the CT-guided hookwire tech-nique, followed by video-assisted thoracic surgery in the wedge resection. The next mode of operation depends on the results of frozen biopsy. Results:Preoperative localization with CT-guided hookwire was performed in 34 patients between February 2012 and March 2014. The diameter of lung nodule ranged from 5 mm to 22 mm. CT-guided hookwire localization was successful in all patients, with a median positioning time of 23 min. Puncture needles were detached from two of the total patients during the surgery, and three other pa-tients showed pneumothorax by CT scan after localization. Conclusion:Preoperative hookwire localization of small lung nodule is an accurate and safe approach to improve the rate of wedge resection in video-assisted thoracic surgery.