1.Effect of Anti-Oxidative of Ethyl Pyruvate and Taurine on the Red Blood Cell Storage at 4 ℃.
Shu-Qiang GAO ; Shu-Hui GAO ; Chen-Hui ZHU ; Xiao-Yan YUAN ; Li-Xia REN
Journal of Experimental Hematology 2022;30(3):890-896
OBJECTIVE:
To investigate the anti-oxidative effect of ethyl pyruvate (EP) and taurine (TAU) on the quality of red blood cells stored at 4±2 ℃, hemolysis, energy metabolism and lipid peroxidation of the red blood cells in the preservation solution were studied at different intervals.
METHODS:
At 4±2 ℃, the deleukocyte red blood cells were stored in the citrate-phosphate-dextrosesaline-adenine-1 (CPDA-1) preservation (control group), preservation solution with EP (EP-AS), and TAU (TAU-AS) for long-term preservation. The enzyme-linked immunoassay and automatic blood cell analyzer were used to detect hemolysis and erythrocyte parameters. Adenine nucleoside triphosphate (ATP), glycerol 2,3-diphosphate (2,3-DPG) and malondialdehyde (MDA) kits were used to test the ATP, 2,3-DPG and MDA concentration.
RESULTS:
During the preservation, the rate of red blood cell hemolysis in EP-AS and TAU-AS groups were significantly lower than that in CPDA-1 group (P<0.01). The MCV of EP-AS group was increased with the preservation time (r=0.71), while the MCV of the TAU-AS group was significantly lower than that in the other two groups (P<0.05). The concentration of ATP and MDA in EP-AS and TAU-AS groups were significantly higher than that in CPDA-1 group at the 14th day (P<0.01). The concentrations of 2,3-DPG in the EP-AS and TAU-AS groups were significantly higher than that in the CPDA-1 group from the 7th day (P<0.01).
CONCLUSION
EP and TAU can significantly reduce the red blood cell hemolysis rate, inhibit the lipid peroxidation level of red blood cells, and improve the energy metabolism of red blood cells during storage. The mechanism of EP and TAU may be related to their antioxidation and membrane protection effect, so as to improve the red blood cell quality and extend the preservation time.
2,3-Diphosphoglycerate/metabolism*
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Adenine
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Adenosine Triphosphate/metabolism*
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Blood Preservation
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Citrates/pharmacology*
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Erythrocytes/metabolism*
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Glucose/pharmacology*
;
Hemolysis
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Humans
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Pyruvates
;
Taurine/pharmacology*
2.Effect of 3-bromopyruvate on mitochondrial membrane potential and apoptosis of human breast carcinoma SK-BR-3 cells.
Yuanyuan ZHANG ; Zhe LIU ; Qianwen ZHANG ; Zhenhua CHAO ; Pei ZHANG ; Fei XIA ; Chenchen JIANG ; Hao LIU ; Zhiwen JIANG
Journal of Southern Medical University 2013;33(9):1304-1307
OBJECTIVETo study the effect of glycolysis inhibitor 3-bromopyruvate (3-BrPA) in inducing apoptosis of human breast carcinoma cells SK-BR-3 and the possible mechanism.
METHODSMTT assay was used to detect the growth inhibition induced by 3-BrPA in breast cancer cells SK-BR-3. The apoptotic cells were detected by flow cytometry with propidium iodide (PI). ATP levels in the cells were detected by ATP assay kit, and DHE fluorescent probe technique was used to determine superoxide anion levels; the mitochondrial membrane potential was assessed using JC-1 staining assay.
RESULTSMTT assay showed that the proliferation of SK-BR-3 cells was inhibited by 3-BrPA in a time- and concentration-dependent manner. Exposure to 80, 160, and 320 µmol·L(-1) 3-BrPA for 24 h resulted in cell apoptosis rates of 6.7%, 22.3%, and 79.6%, respectively, and the intracellular ATP levels of SK-BR-3 cells treated with 80, 160, 320 µmol·L(-1) 3-BrPA for 5 h were 87.7%, 60.6%, and 23.7% of the control levels. 3-BrPA at 160 µmol·L(-1) increased reactive oxygen levels and lowered mitochondrial membrane potential of SK-BR-3 cells.
CONCLUSION3-BrPA can inhibit cell proliferation, reduce the mitochondrial membrane potential and induce apoptosis in SK-BR-3 cells, the mechanism of which may involve a reduced ATP level by inhibiting glycolysis and increasing the reactive oxygen level in the cells.
Apoptosis ; drug effects ; Cell Line, Tumor ; Female ; Glycolysis ; Humans ; Membrane Potential, Mitochondrial ; drug effects ; Pyruvates ; pharmacology ; Reactive Oxygen Species ; metabolism
3.The Antitumor Effect and Hepatotoxicity of a Hexokinase II Inhibitor 3-Bromopyruvate: In Vivo Investigation of Intraarterial Administration in a Rabbit VX2 Hepatoma Model.
Hwan Jun JAE ; Jin Wook CHUNG ; Hee Sun PARK ; Min Jong LEE ; Ki Chang LEE ; Hyo Cheol KIM ; Jung Hwan YOON ; Hesson CHUNG ; Jae Hyung PARK
Korean Journal of Radiology 2009;10(6):596-603
OBJECTIVE: The purpose of this study was to compare the antitumor effect and hepatotoxicity of an intraarterial delivery of low-dose and high-dose 3-bromopyruvate (3-BrPA) and those of a conventional Lipiodol-doxorubicin emulsion in a rabbit VX2 hepatoma model. MATERIALS AND METHODS: This experiment was approved by the animal care committee at our institution. VX2 carcinoma was implanted in the livers of 36 rabbits. Transcatheter intraarterial administration was performed using low dose 3-BrPA (25 mL in a 1 mM concentration, n = 10), high dose 3-BrPA (25 mL in a 5 mM concentration, n = 10) and Lipiodol-doxorubicin emulsion (1.6 mg doxorubicin/ 0.4 mL Lipiodol, n = 10), and six rabbits were treated with normal saline alone as a control group. One week later, the proportion of tumor necrosis was calculated based on histopathologic examination. The hepatotoxicity was evaluated by biochemical analysis. The differences between these groups were statistically assessed with using Mann-Whitney U tests and Kruskal-Wallis tests. RESULTS: The tumor necrosis rate was significantly higher in the high dose group (93% +/- 7.6 [mean +/- SD]) than that in the control group (48% +/- 21.7) (p = 0.0002), but the tumor necrosis rate was not significantly higher in the low dose group (62% +/- 20.0) (p = 0.2780). However, the tumor necrosis rate of the high dose group was significantly lower than that of the Lipiodol-doxorubicin treatment group (99% +/- 2.7) (p = 0.0015). The hepatotoxicity observed in the 3-BrPA groups was comparable to that of the Lipiodol-doxorubicin group. CONCLUSION: Even though intraarterial delivery of 3-BrPA shows a dose-related antitumor effect, single session treatment seems to have limited efficacy when compared with the conventional method.
Animals
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Disease Models, Animal
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Dose-Response Relationship, Drug
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Doxorubicin/administration & dosage/pharmacology
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Infusions, Intra-Arterial
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Iodized Oil/administration & dosage/pharmacology
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Liver Neoplasms, Experimental/*drug therapy/radiography
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Pyruvates/administration & dosage/*pharmacology
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Rabbits
;
Statistics, Nonparametric
;
Tomography, X-Ray Computed
4.3-bromopyruvate enhances cisplatin sensitivity of hepatocellular carcinoma cells in vitro.
Surong ZHAO ; Yuanyuan ZHANG ; Chengzhu WU ; Hongmei LI ; Chenchen JIANG ; Zhiwen JIANG ; Hao LIU
Journal of Southern Medical University 2014;34(1):25-30
OBJECTIVETo investigate the effect of 3-bromopyruvate (3-BP) in sensitizing hepatocellular carcinoma cells to cisplatin-induced apoptosis and its possible mechanism.
METHODSThe growth inhibition of HepG2 and SMMC7721 cells following exposures to different concentrations of 3-BP and cisplatin was measured by MTT assay. The apoptosis of cells treated with 100 µmol/L 3-BP with or without 8 µmol/L cisplatin was assessed using flow cytometry with PI staining, and the activity of caspase-3 and intracellular ATP level were detected using commercial detection kits; the expression of XIAP and PARP was analyzed using Western blotting.
RESULTS3-BP produced obvious inhibitory effects on HepG2 and SMMC7721 cells at the concentrations of 50-400 µmol/L with IC50 values of 238.9∓13.9 µmol/L and 278.7∓11.7 µmol/L for a 48-h treatment, respectively. Cisplatin also inhibited the growth of HepG2 and SMMC7721 cells at the concentrations of 2-32 µmol/L, with IC50 values of 16.4∓0.9 µmol/L and 20.9∓1.8 µmol/L after a 48-h treatment, respectively. Treatment with 100 µmol/L 3-BP combined with 8 µmol/L cisplatin for 48 h resulted in a growth inhibition rate of (60.6∓2.2)% in HepG2 cells and (56.8∓2.3)% in SMMC7721 cells, which were significantly higher than those in cells treated with 3-BP or cisplatin alone. The combined treatment for 48 h induced an apoptotic rate of (51.1∓4.3)% in HepG2 cells and (46.5∓3.9)% in SMMC7721 cells, which were also markedly higher than those in cells with 3-BP or cisplatin treatment alone.
CONCLUSION3-BP can sensitize HepG2 and SMMC7721 cells to cisplatin-induced apoptosis possibly by causing intracellular ATP deficiency, down-regulating XIAP, and increasing caspase-3 activity.
Adenosine Triphosphate ; metabolism ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; pathology ; Caspase 3 ; metabolism ; Cisplatin ; pharmacology ; Hep G2 Cells ; Humans ; Liver Neoplasms ; pathology ; Pyruvates ; pharmacology ; X-Linked Inhibitor of Apoptosis Protein ; metabolism
5.Ethyl Pyruvate Has Anti-Inflammatory and Delayed Myocardial Protective Effects after Regional Ischemia/Reperfusion Injury.
In Seok JANG ; Mi Young PARK ; Il Woo SHIN ; Ju Tae SOHN ; Heon Keun LEE ; Young Kyun CHUNG
Yonsei Medical Journal 2010;51(6):838-844
PURPOSE: Ethyl pyruvate has anti-inflammatory properties and protects organs from ischemia/reperfusion (I/R)-induced tissue injury. The aim of this study was to determine whether ethyl pyruvate decreases the inflammatory response after regional I/R injury and whether ethyl pyruvate protects against delayed regional I/R injury in an in vivo rat heart model after a 24 hours reperfusion. MATERIALS AND METHODS: Rats were randomized to receive lactated Ringer's solution or ethyl pyruvate dissolved in Ringer's solution, which was given by intraperitoneal injection 1 hour prior to ischemia. Rats were subjected to 30 min of ischemia followed by reperfusion of the left coronary artery territory. After a 2 hours reperfusion, nuclear factor kappaB, myocardial myeloperoxidase activity, and inflammatory cytokine levels were determined. After the 24 hours reperfusion, the hemodynamic function and myocardial infarct size were evaluated. RESULTS: At 2 hours after I/R injury, ethyl pyruvate attenuated I/R-induced nuclear factor kappaB translocation and reduced myeloperoxidase activity in myocardium. The plasma circulating levels of inflammatory cytokines decreased significantly in the ethyl pyruvate-treated group. At 24 hours after I/R injury, ethyl pyruvate significantly improved cardiac function and reduced infarct size after regional I/R injury. CONCLUSION: Ethyl pyruvate has the ability to inhibit neutrophil activation, inflammatory cytokine release, and nuclear factor kappaB translocation. Ethyl pyruvate is associated with a delayed myocardial protective effect after regional I/R injury in an in vivo rat heart model.
Animals
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Anti-Inflammatory Agents/*pharmacology
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Cell Nucleus/metabolism
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Cytoplasm/metabolism
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Heart/physiopathology
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Inflammation
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Male
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Myocardial Infarction/prevention & control
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Myocardium/*metabolism
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NF-kappa B/metabolism
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Peroxidase/metabolism
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Pyruvates/*pharmacology
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Rats
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Rats, Sprague-Dawley
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Reperfusion Injury/*drug therapy/*metabolism
6.FDG-PET for Evaluating the Antitumor Effect of Intraarterial 3-Bromopyruvate Administration in a Rabbit VX2 Liver Tumor Model.
Hee Sun PARK ; Jin Wook CHUNG ; Hwan Jun JAE ; Young Il KIM ; Kyu Ri SON ; Min Jong LEE ; Jae Hyung PARK ; Won Jun KANG ; Jung Hwan YOON ; Hesson CHUNG ; Kichang LEE
Korean Journal of Radiology 2007;8(3):216-224
OBJECTIVE: We wanted to investigate the feasibility of using FDG-PET for evaluating the antitumor effect of intraarterial administration of a hexokinase II inhibitor, 3-bromopyruvate (3-BrPA), in a rabbit VX2 liver tumor model. MATERIALS AND METHODS: VX2 carcinoma was grown in the livers of ten rabbits. Two weeks later, liver CT was performed to confirm appropriate tumor growth for the experiment. After tumor volume-matched grouping of the rabbits, transcatheter intraarterial administration of 3-BrPA was performed (1 mM and 5 mM in five animals each, respectively). FDG-PET scan was performed the day before, immediately after and a week after 3-BrPA administration. FDG uptake was semiquantified by measuring the standardized uptake value (SUV). A week after treatment, the experimental animals were sacrificed and the necrosis rates of the tumors were calculated based on the histopathology. RESULTS: The SUV of the VX2 tumors before treatment (3.87+/-1.51[mean+/-SD]) was significantly higher than that of nontumorous liver parenchyma (1.72+/-0.34) (p < 0.0001, Mann-Whitney U test). The SUV was significantly decreased immediately after 3-BrPA administration (2.05+/-1.21) (p = 0.002, Wilcoxon signed rank test). On the one-week follow up PET scan, the FDG uptake remained significantly lower (SUV 1.41+/-0.73) than that before treatment (p = 0.002), although three out of ten animals showed a slightly increasing tendency for the FDG uptake. The tumor necrosis rate ranged from 50.00% to 99.90% (85.48%+/-15.87). There was no significant correlation between the SUV or the SUV decrease rate and the tumor necrosis rate in that range. CONCLUSION: Even though FDG-PET cannot exactly reflect the tumor necrosis rate, FDG-PET is a useful modality for the early assessment of the antitumor effect of intraarterial administration of 3-BrPA in VX2 liver tumor.
Animals
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Disease Models, Animal
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Enzyme Inhibitors/*pharmacology
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Feasibility Studies
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Fluorodeoxyglucose F18
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Infusions, Intra-Arterial
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Injections, Intra-Arterial
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Liver Neoplasms, Experimental/*drug therapy/pathology/*radionuclide imaging
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Necrosis
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*Positron-Emission Tomography
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Pyruvate Dehydrogenase Complex/antagonists & inhibitors
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Pyruvates/*pharmacology
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Rabbits
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Radiopharmaceuticals
7.Effects of ethyl pyruvate on myocardial apoptosis and expression of Bcl-2 and Bax proteins after ischemia-reperfusion in rats.
Jialong, GUO ; Kailun, ZHANG ; Yanmei, JI ; Xionggang, JIANG ; Shunqing, ZUO
Journal of Huazhong University of Science and Technology (Medical Sciences) 2008;28(3):281-3
In order to study the effects of ethyl pyruvate on cardiomyocyte apoptosis following ischemia/reperfusion (I/R) in vitro and the expression of Bcl-2 and Bax proteins, isolated rat hearts were perfused in a Langendorff model. Twenty-four rats were randomly divided into 3 groups (n=8 in each group): control group was perfused for 120 min. In the I/R group, after 30 min stabilization the injury was induced by 30 min global ischemia followed by 60 min reperfusion. Ethyl pyruvate (EP) group was set up with the same protocol as I/R group except that it was supplied with 2 mmol/L EP 15 min before ischemia and throughout reperfusion. Myocardial malonaldehyde (MDA) content was measured. Myocardial apoptotic index (AI) was tested by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. The expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax in cardiac myocytes was detected by immunohistochemistry. As compared with control group, the content of MDA, myocardial AI and the expression of Bcl-2, Bax proteins were increased significantly in I/R group, but the content of MDA, myocardial AI and the expression of Bax protein were decreased obviously and the expression of Bcl-2 protein was up-regulated in EP group (P<0.05). These results demonstrate that EP could inhibit apoptosis of cardiac myocytes possibly via alleviating oxidative stress, up-regulating Bcl-2 and down-regulating Bax proteins.
Apoptosis
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In Situ Nick-End Labeling
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Malondialdehyde/pharmacology
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Myocardium/*pathology
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Myocytes, Cardiac/cytology
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Oxidative Stress
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Proto-Oncogene Proteins c-bcl-2/*metabolism
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Pyruvates/*pharmacology
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Rats, Sprague-Dawley
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Reperfusion Injury
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Tissue Distribution
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bcl-2-Associated X Protein/*metabolism
8.Effect of Inslulin Administration on the Physical Performance of Rats and Mice.
Won Kyu LEE ; Hae Kun PARK ; Sung Il HONG ; Doo Hee KANG
Yonsei Medical Journal 1973;14(1):10-17
The correlation between muscle glycogen content and physical performance in mice was evaluated by investigating whether an increase in glycogen content in skeletal muscle with insulin administration can improve the physical performance without other effects of exercise. Albino rats(group I) were divided into two groups, i.e., insulin and saline administered group. The former experimental group was treated with protamine zinc insulin(15U/kg/day) subcutaneously for two weeks to increase the content of the muscle glycogen and the latter control group with saline. Mice (group II) were also divided into insulin treated and control groups and both groups were subjected to running exercise on an animal treadmill up to point of exhaustion once every day. After two weeks of insulin treatment, the muscle glycogen content, the maximal running time and the maximal swimming time were measured in non-exercised group I. In group II, after 12 days of insulin and saline administration, the muscle glycogen content, the maximal running time, concentrations of lactate and pyruvate in the blood were measured before and after the maximal exhaustive running. The results were summarized as follows. In group I, the muscle glycogen content, the maximal running time and the maximal swimming time of the insulin administered group were significantly greater of the control groups. In group II, the maximal running time was significantly greater(P < 0.01) in the experimental group than of the control group, while the muscle glycogen content revealed no significant difference between the two groups. On the other hand, lactate concentration and lactate/pyruvate ratios in the blood were significantly lower in the experimental group than those of the control groups. From the above results, it may be concluded that the elevation of muscle glycogen content alone by insulin treatment without any previous physical training can improve physical performance of rats. And insulin was also found to improve physical performance even in experimental animals which had been subjected to a longterm of exercise.
Anaerobiosis/drug effects*
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Animal
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Blood Glucose/analysis
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Body Weight
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Exertion/drug effects*
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Glycogen/analysis*
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Injections, Subcutaneous*
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Insulin/administration & dosage
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Insulin/pharmacology*
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Lactates/blood
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Male
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Metabolism/drug effects*
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Mice
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Muscles/analysis*
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Pyruvates/blood
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Rats
9.Effect of glycolytic inhibitor 3-BrPA on the proliferation and apoptosis of mouse splenic lymphocytes in mixed lymphocytes culture.
Ruiqing ZHOU ; Jing WANG ; Dafa QIU ; Xiaomin NIU ; Ziwen GUO ; Huiqing HE ; Xiaojun XU
Chinese Journal of Hematology 2014;35(7):637-640
OBJECTIVETo study the effect of glycolytic inhibitor 3-Bromopyruvate (3-BrPA) on the proliferation and apoptosis of mouse spleen lymphocytes and explore its mechanism.
METHODSAn one-way mixed lymphocyte culture (MLC) system was established, including BALB/c mouse spleen cells (H-2d) as stimulator and C57BL/6 mouse spleen cells (H-2b) as responder. With treatment of 3-BrPA at different concentrations (0-200 μmol/L), lymphocyte proliferation capacity was detected by the CCK-8 method, the expression of CD3, CD4, and CD8 by flow cytometry, and the concentrations of cytokine interleukin (IL)-4 and interferon (IFN)-γ in the supernatant by ELISA.
RESULTSAt a middle or high dose (over 20 μmol/L), 3-BrPA displayed a dose-dependent inhibitory effect on lymphocyte proliferation in the MLC system. The 50% inhibitory concentration (IC50) were 48.6, 41.2, and 41.9 μmol/L after 24, 36, and 48 h culture, respectively. With treatment of 50 μmol/L 3-BrPA, the IFN-γ level [(164.25 ± 20.14) ng/L] was significantly lower, compared with control [(277.61 ± 18.46) ng/L]. The IL-4 level [(31.06 ± 6.06) ng/L] was significantly higher, compared with control [(28.64 ± 3.97) ng/L]. Consequently, the IFN-γ/IL-4 ratio decreased significantly.
CONCLUSIONThese results indicate that 3-BrPA had a significant inhibitory effect on the proliferation of mouse spleen lymphocytes cultured in MLC system, accompanied with the Th2-biased secretion of cytokines.
Animals ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Lymphocyte Culture Test, Mixed ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Pyruvates ; pharmacology ; Spleen ; cytology ; metabolism
10.Influence of splenic high mobility group box-1 protein on immune function of regulatory T lymphocytes in scald rats.
Li-feng HUANG ; Feng-hua YAO ; Yong-ming YAO ; Li-tian ZHANG ; Ning DONG ; Zhi-yong SHENG
Chinese Journal of Burns 2010;26(2):104-108
OBJECTIVETo observe the influence of high mobility group box-1 protein (HMGB1) derived from spleen on the phenotype of regulatory T lymphocytes (Treg) and HMGB1-mediated immune function in severely scalded rats after delayed resuscitation.
METHODSOne hundred and four Wistar rats were divided into normal control group (NC, n = 8), sham scald group (SS, n = 32), scald group (S, n = 32), and ethyl pyruvate (EP) treatment group (EPT, n = 32) according to the random comparison table. Rats in the latter 2 groups were subjected to 30%TBSA full-thickness scald, which were intraperitoneally injected with Ringer solution or EP solution at post scald hour (PSH) 6 (delayed antishock treatment) and administered with 4 mL Ringer solution or EP solution per 12 hours after PSH 12 till PSH 48. Rats in SS group were treated the same as that of S group except for sham scald with 37 degrees C water. Injured rats were sacrificed at post scald day (PSD) 1, 3, 5, 7 (rats in NC group were also sacrificed), and CD4(+)CD25(+)Treg were isolated from spleen with magnetic-activated cell sorting method. The content of HMGB1 in spleen and IL-2 level in supernatant were determined with ELISA. The expression of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) on Treg was determined with flow cytometry, and the proliferation activity of T lymphocytes was also detected (recorded as absorbance value). Data were processed with analysis of variance among groups and independent samples t test.
RESULTS(1) Compared with that of rats in SS group and EPT group, the expression of splenic HMGB1 in S group increased significantly on PSD 1 through PSD 7 [peaked on PSD 1: (46.7 +/- 8.3) ng/mg protein]. (2) Compared with that in SS group, the expression of CTLA-4 in S group was enhanced significantly on PSD 1 through PSD 5 (with t value respectively 10.459, 12.051, 4.029, P < 0.05 or P < 0.01); while that in EPT group decreased significantly on PSD 1 through PSD 7 as compared with that from S group (with t value respectively 2.796, 9.913, 9.581, 10.022, P < 0.05 or P < 0.01). (3) Compared with that of rats in SS group, the proliferation activity of T lymphocytes in S group was markedly suppressed on PSD 1 through PSD 7 (nadir on PSD1: 0.167 +/- 0.059), and release of IL-2 was decreased significantly [nadir on PSD 5: (44 +/- 24) pg/mL]. T lymphocytes proliferation activity was restored and excretion of IL-2 increased in EPT group as compared respectively with that of S group at each time point.
CONCLUSIONSThe release of HMGB1 may stimulate splenic Treg to mature, thereby induce suppression of proliferation activity of T lymphocytes and immune function. EP can ameliorate immune dysfunction in animals with delayed resuscitation through inhibiting the synthesis and release of HMGB1.
Animals ; Antigens, CD ; metabolism ; Burns ; immunology ; CTLA-4 Antigen ; Cell Proliferation ; HMGB1 Protein ; metabolism ; Interleukin-2 ; metabolism ; Male ; Pyruvates ; pharmacology ; Rats ; Rats, Wistar ; Spleen ; cytology ; immunology ; T-Lymphocytes, Regulatory ; cytology ; immunology