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Objective To observe the apeptosis in liver injury following limbs ischemia-reperfusion(IR) in rats and the protective effects of taurine.Methods The model of limbs ischemia-reperfusion injury was established.30 Wistar rats were randomized into 3 groups: control group,IR group and tanrine + reperfusion group (TR group) (n = 10 for each group).The levels of malondialdehyde (MDA) and xanthineoxidas (XOD), calcium and myeloper-oxidase (MPO) in the liver tissue were measured.DNA fragmentation was observed and analyzed by agarose gel e-lectrophoresis.Apoptosis was detected by TUNEL methods.The morphologic changes were observed with HE stai-ning.Results Compared with control group,the values of MDA, XOD, MPO, calcium in liver tissue were increased significantly in IR group (P<0.01), but the values of those in TR group were lower than in IR group (P<0.01).The percentage of apeptosis cell was higher in IR group than in control group(P<0.01).Compared with IR group, the percentage of apoptosis cell was lower in TR group (P<0.01).IR group presented DNA ladder pattern, while TR group showed no specific DNA ladder pattern in agarose gel electrophoresis.Conclusion Apoptosis participates in the liver injury after limb ischemia-reperfusion.Taurine can mitigate the liver injury and apoptosis after limb is-chemia-reperfusion injury in rats.
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BACKGROUND: Rabbit serum samples are widely used in basic researches, and two-dimensional gelelectrophoresis (2-DE) is the most classic technique for protein separation. Therefore, it is of great significance to establish a stable technique system of 2-DE for rabbit serum.OBJECTIVE: To establish a 2-DE technique system for rabbit serum protein separation. DESIGN, TIME AND SETTING: A single sample observational experiment was performed at Tianjin Key Laboratory of Biomarkers for Occupational and Environmental Hazard of Chinese People's Armed Police Forces Medical College from June to July in 2008. MATERIALS: Six healthy rabbits were provided by the Animal Experimental Center of Tangshan Vocational Technical College METHODS: Health rabbit serum was dissolved in rehydration sample loading buffer before and after eliminating high abundance proteins to make proteins in it schizolysis adequately. After reductive alkylation, the samples were loaded into the rehydration tray to undergo passive rehydration for 14 hours. Isoelectric focusing (IEF) electrophoresis was followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After sliver staining, the gel was analyzed by PDQuest7.4. MAIN OUTCOME MEASURES: ①Efficiency of eliminating high abundance proteins. ②Two-dimensional eleotrophoregrams (2-D electrophoregrams)RESULTS: Distinct 2-D eleotrophoregrems were obtained with high resolution and good reproducibility. The removal of high abundance proteins in serum failed to result in better 2-D electrophoregrams.CONCLUSION: We have successfully established a 2-DE technique for rabbit serum proteome, which can lay the foundation for the further study of serum proteomics of diseases.
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BACKGROUND: Limb ischemia reperfusion (LIR) as a stressor leads to gastric mucosal injury, and then results in the occurrence of stress ulcer.OBJECTIVE: To observe the effects of LIR on gastric mucosal injury, investigate part of the mechanism, and the role of several transient limb ischemia in the occurrence of gastric mucosal injury.DESIGN: A randomized grouping design and controlled animal experiment.SETTING: Department of Pathophysiology of North China Coal Medical College.MATERIALS: The experiment was carried out in the pathophysiological laboratory of North China Coal Medical College from January to June 2002. Fifty-four healthy adult male Wistar rats were randomly divided into three groups with 18 rats in each group. Ischemia reperfusion (I/R) group: The rats were duplicated into models according to the Rosenthal method that under superficial anesthesia with ether, the roots of both hindlimbs were ligated by wrapping with rubber strap, blood flow was blocked for 4 hours and then recovered to perfusion for 4 hours, and finally killed by bleeding from abdominal aorta. Ischemic preconditioning group: Before model establishment, blood flow of both hindlimbs was blocked for 5 minutes, and then recovered to perfusion for 5 minutes, which was repeated for four times, and the following operations were the same as those in the I/R group. Control group: The operations were the same as those in the I/R group,but both hindlimbs were ligated at relaxation without blocking the blood flow.METHODS: Sections of gastric mucosa were prepared, and then observed under light microscope and electron microscope, and the index of gastric mucosal injury was determined according to the Guth standard. The colorimetric assay was performed with 721 spectrophotometer at 650 nm, and the amount of gastric combining mucus was calculated.Meanwhile, the blood flow of gastric mucosa, contents of phospholipid and hexosamine in gastric mucus, content of nitric oxide in plasma and gastric tissue and activity of nitric oxide synthase (NOS) in gastric mucosa were determined.MAIN OUTCOME MEASURES: Index of gastric mucosal injury, amount of gastric combining mucus, blood flow of gastric mucosa, contents of phospholipid and hexosamine in gastric mucus, contents of nitric oxide in plasma and gastric tissue and NOS activity in gastric mucosa.RESULTS: All the 54 rats were involved in the analysis of results. ① In the I/R group, gastric mucosal injury was serious, edema, hyperemia, erosion and disintegration of gland of mucosal glands were observed, infiltration of inflammatory cells (formation of ulcer) was observed in basal and inferior mucosa. In the ischemic preconditioning group, the gastric mucosa was complete, and the damaged severity was milder than that in the I/R group; Under electron microscope, the organell structures of gastric parietal and chief cells were incomplete and destroyed. The cell injuries in the ischemic preconditioning group were milder than those in the I/R group (index of injury: 18.00±10.71, 34.00±15.01, P＜ 0.01). ② The blood flow and combining mucosal amount of gastric mucosa, contents of phospholipid and hexosamine in gastric mucus in the ischemic preconditioning group and I/R group were all obviously lower than those in the control group [(2.12±0.56), (10.84±2.56), (25.52±2.97) mL/(kg·h); (2.01±0.91), (2.79±0.73), (3.99±0.87) mg;(7.68±1.95), (9.74±1.04), (11.98±1.98) mg/g; (3.83±1.18), (5.42±0.47), (5.76±1.21) mg/g, P ＜ 0.05, 0.01], those the above indexes were all higher in the ischemic preconditioning group than in the I/R group. ③ The contents of nitric oxide in plasma and gastric tissue and NOS activity in gastric mucosa in the ischemic preconditioning group and I/R group were significantly lower than those in the control group [(250.0±5.6), (270.0±11.3), (210.0±7.4) μmol/L; (9.34±0.67), (11.34±1.00), (7.50±0.67) μ kat/g, P ＜ 0.01], those were also signficantly higher in the ischemic preconditioning group than in the I/R group.CONCLUSION: As a stressor, LIR can lead to gastric mucosal injury, and cause stress ulcer.Ischemic preconditioning can alleviate the gastric mucosal injury following LIR
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AIM To evaluate the possible role of endogenous nitric oxide (NO) in the development of lung injury after hind limbs ischemia reperfusion (LIR), and the effect of taurine on endogenous NO on the model of lung injury following LIR. METHODS Wistar rats were divided into three groups: control group, ischemia reperfusion group (IR) and taurine+IR (Tau+IR). The changes of PaO 2 ,PaCO 2,lung index (LI) and lung permeability index (LPI) were observed. The content of malondialdehyde (MDA), NO and endothelin (ET) in both plasma and the lung as well as the content of nitric oxide synthase (NOS) and taurine in the lung were detected. The immunohistochemical method was used to detect level in the expression of NOS protein. RESULTS Taurine improved the pulmonary respiratory following LIR significantly, lightened lung edema, decreased the content of ET in both plasma and lung tissue, increased the content of NO in both plasma and lung tissue and promoted the level in the expression of NOS protein. CONCLUSION Using taurine may lighten lung injury following LIR and this protective effect may due to increasing the content of endogeneous NO and decreasing the level of ET.
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Objective To investigate cell injury and apoptosis of L-6TG cells induced by simulated ischemia-reperfusion,and to explore its regulating mechanism.Methods Cultured L-6TG cells were randomly divided into control group(C)and simulated ischemia-reperfusion group(IR).The L-6TG cells of C group were cultivated under normal condition in mimicking reperfusion solution,while the L-6TG cells of IR group were cultivated in mimicking ischemic solution for 4 hours,and then cultivated in mimicking reperfusion solution for 4 hours.Superoxide dismutase(SOD),xanthine oxidase(XOD)and free calcium in L-6TG cells were determined in each group;mitochondrial respiration was measured by MTT method.Apoptosis was identified by flow cytometer with propidium iodide(PI)staining method and agarose gel electrophoresis;the immunohistochemical method was used to determine the expression of bcl-2 and caspase-3,and their profiles were half-quantitatively analyzed by automatism image analytical system.Results Compared with the L-6TG cells of C group,after suffering from ischemia and reperfusion each for 4 hours,XOD,free calcium in L-6TG cells and apoptotic percentage were all increased significantly(287.88?8.47 vs 42?3.94 U/g prot,1.547?0.017 vs 7.23?0.28 lnMCF,7.23%?0.28% vs 0.69%?0.07%,P
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Objective To observe the injury of myocardium and to investigate the role of oxidative stress after limbs ischemia reperfusion in rats. Methods 20 male healthy Wistar rats were randomly divided into 2 groups: control group(C) and ischemia-reperfusion group (IR). The biochemical methods were used to measure the contents of malondialdehyde (MDA), myeloperoxidase (MPO), ATP, lactic acid (LD) and ATPase of myocardium. The levels of creatine kinase (CK) and creatine kinase MB isoenzyme (CK-MB) in plasma were also observed. The morphologic changes of myocardium were observed with microscope. Results It was found that after rats’ limbs suffering from ischemia-reperfusion, the level of ATP decreased, the activities of three kinds of ATPase (Na~+-K~+-ATPase, Ca~ 2+ ATPase and Mg~ 2+ ATPase) decreased in different degree (P
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AIM: To observe the degree of gastric mucosal injury following limb ischemia/reperfusion(LIR),and to investigate the mechanism of gastric mucosal injury and the influence of the series of brief ischemia/reperfusion of hind limbs of rats on gastric mucosal injury. METHODS: Referring to Rosenthal's method, the model rats which underwent 4 hours of ischemia and 4 hours of reperfusion in hind limbs were made. The indexes of gastric mucosal injury after LIR and ischemic preconditioning (IPC) + LIR were determined. The morphologic changes were observed with light microscope and transmission electron microscop respectively. The GMBF,histologic lesion score, gastric barrier mucus, phospholipids, hexosamine, nitric oxide and nitric oxide synthase in mucus were measured in different groups. RESULTS: Serious damage in gastric mucosa was observed under microscope and EM after LIR. But less serious damage was observed in the IPC group. After LIR, compared to the control group, the GMBF and the content of gastric barrier mucus, phospholipids and hexosamine in mucus decreased significantly. There was significant difference in most indexes between the control group and the IPC group, but compared to LIR group, the histologic lesion score decreased significantly and the GMBF and the content of gastric barrier mucus, phospholipids, hexosamine ,nitric oxide and nitric oxide synthase in mucus increased significantly. CONCLUSION: LIR caused the gastric mucosa injury. IPC alleviated the damage of gastric mucosa following ischemia/reperfusion in hind limbs.
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In order to explore the effects of printing and dyeing wastewater untreated and treated by bacterial biotechnological method on the specific immune response of Carassius auratus, the tested fish were preimmunized intraperitoneally with Aeromonas hydrophila vaccine then were separately placed into control clean water,untreated printing and dyeing wastewater (dilution ratio 13%,pH=7) and treated water 5 fish of every group were killed on the day 2,4,6,8,10,14,18,25,35 th to detect their immune indexes The results indicated that total leucocytic numbers of blood and serum lysozyme level in fish of clean water and treated water increased rapidly and reached their peaks on the day 8th and 6th respectively after vaccination,then reduced to baseline and maintatined for a period of time Leucocytic numbers in fish in untreated wastewater group decreased on day 2nd then increased gradully to normal,serum lysozyme level in this group had decreasing tendency and was lower than the other 2 groups from beginning to end However titer of anti A hydrophila antibody in serum of untreated water group was only lower than that of treated water group on the day 6th when it appeared After this antibody titers of these 3 groups had no significant defference It indicated that printing and dyeing wastewater inhibited immune response of Carassius auratus ,but water treated by bacterial biotechnological method had no this kind of toxic effect
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Aim To investigate the effect of L-arginine(L-Arg) on myocardium in rat which suffered from limb ischemia-reperfusion.Methods Models of limb ischemia reperfusion were made by tourniquet methods.L-Arg(150 mg?kg-1) were intravascularly injected before reperfusion.Contents of malondialdehyde(MDA),myeloperoxidase(MPO) and tumour necrosis factor(TNF-?) in myocardium were measured.Levels of creatine kinase(CK),creatine kinase MB isoenzyme(CK-MB) and nitric oxide(NO) in plasma were deter-mined.The mean arterial pressure(MAP,left venteicular systolic pressure(LVSP),maximal rise rate of left venteicular pressure(dp/dtmax)and the maximal fall rate of left venteicular pressure(-dp/dtmax)were monitored.Morphologic changes of myocardium were evaluated after reperfusion.Results After rats' limbs suffering from ischemia-reperfusion,levels of MDA,MPO and TNF-? in myocardium,CK,CK-MB and NO in plasma increased differently(P
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AIM: To investingate the potential role of apoptosis in the development of acute lung injury(ALI) following limbs ischemia-reperfusion(LIR) in rats and the effects of raurine. METHODS: The model of LIB injury in rats was made. By using TUNEL, electrophoresis, reverse-transcription polymerase chain reaction(RT-PCR) and immunohistochemistry techniques, pulmonary apoptosis, Fas/FasL expressions and Ca~(2+), reactive oxygen species (ROS),the ratio of DNA double chain in tissue were detected in different groups.RESULTS: The rapid appearance of apoptosis in alveolar epithelial cells and pulmonary vascular endothelial cells were observed after LIR in rats. The ratio of DNA double chain decreased and tissue Ca~(2+) increased. The expression of Fas/FasL mRNA and protein was up-regulated in lung tissue of rats after LIR, which was related to the elevation of alveolar epithelial cells and pulmonary vascular endothelial cell apoptosis. Taurine decreased alveolar epithelial cell and pulmonary vascular endothelial cell apoptosis not by down-regulating expression of Fas/FasL system. CONCLUSIONS: These results suggest that the excessive apoptosis and Fas/FasL system expression may play a role in the pathogensis of ALI following LIB in rats, and taurine decreases alveolar epithelial cell and pulmonary vascular endothelial cell apoptosis.
