ABSTRACT
Objective To investigate the effect of intratracheal injection of c-Jun N-terminal kinase ( JNK) siRNA plasmid on ischemia-reperfusion ( I∕R) injury in a rat model of lung transplantation. Meth-ods ExperimentⅠ Thirty-two male Wistar rats, weighing 250-280 g, were divided into 2 groups ( n=16 each) using a random number table method: control group ( group C) and JNK siRNA group. JNK siR-NA plasmid 2 mg∕kg ( diluted to 0. 2 ml in sterile phosphate buffer solution) was intratracheally injected in JNK siRNA group. Scrambled siRNA plasmid 2 mg∕kg ( diluted to 0. 2 ml in sterile phosphate buffer solu-tion) was intratracheally injected in group C. Six rats in each group were sacrificed at 48 h of administra-tion, and left lung tissues were removed for determination of the expression of JNK and JNK mRNA ( by Western blot and real-time polymerase chain reaction, respectively) . The other 10 rats left in each group were used for left lung transplantation. Experiment Ⅱ Thirty male Wistar rats, weighing 250-280 g, were divided into 3 groups ( n=10 each ) using a random number table method: sham operation group ( group S) , transplanted lung I∕R group ( group I∕R) and transplanted lung I∕R+JNK siRNA group ( group I∕R+JNK siRNA) . In group I∕R and group I∕R+JNK siRNA, the left lung transplantation was performed, and the donor lungs were obtained from the living donors in group C and group JNK siRNA, respectively. At 15 min of mechanical ventilation and 30, 60, 90 and 120 min of reperfusion, arterial blood samples were obtained for blood gas analysis, PaO2 was recorded, and the oxygenation index ( PaO2 ÷ FiO2 ) was calculated. Arterial blood samples were obtained at 120 min of reperfusion in transplanted lungs for determi-nation of concentrations of interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α) and interferon-γ( IFN-γ) in serum ( using enzyme-linked immunosorbent assay) , and the rats were sacrificed and left lung tissues were removed for microscopic examination of the pathological changes which were scored and for de-termination of wet∕dry lung weight ratio ( W∕D ratio) , and nuclear factor kappa B ( NF-κB) and activator protein-1 ( AP-1) activities ( using enzyme-linked immunosorbent assay) . Results ExperimentⅠ Com-pared with group C, the expression of JNK and JNK mRNA was significantly down-regulated in group JNK siRNA (P<0. 05). ExperimentⅡ Compared with group S, the oxygenation index was significantly de-creased, and the concentrations of serum IL-8, TNF-α and IFN-γ, W∕D ratio, lung injury score and ac-tivities of NF-κB and AP-1 were increased in I∕R and I∕R+JNK siRNA groups ( P<0. 05) . Compared with group I∕R, the oxygenation index of receptors were significantly increased, and the concentrations of serum IL-8, TNF-α and IFN-γ, W∕D ratio, lung injury score and activities of NF-κB and AP-1 were decreased in group I∕R+JNK siRNA ( P<0. 05) . Conclusion Intratracheal injection of JNK siRNA can reduce trans-planted lung I∕R injury, and the mechanism may be related to inhibiting inflammatory responses of rats.
ABSTRACT
Objective Using small interfering RNA (siRNA) against p38 and simulated lung transplantation model,we discussed the effect of p38 siRNA on hypoxia/reoxygenation injury of pulmonary microvascular endothelial cells (PMVECs) after lung transplantation.Methods We transfected the PMVECs with p38 siRNA or non-targeting (NT) siRNA.After 48 h,these cells were exposed to simulated ischemia-reperfusion.At 2 h and 4 h of reperfusion,we detected lactate dehydrofenase (LDH) leakage rate,malondialdehyde (MDA) levels,superoxide dismutase (SOD) activity,cell apoptosis,and the serum levels of interleukin (IL)-1,IL-6 and tumor necrosis factor (TNF)-α.Protein levels of p38,NF-κB and AP-1 were detected.Untreated PMVECs served as the negative control.Results As compared with NT siRNA,p38 siRNA reduced LDH leakage rate (22.3 ± 5.7 vs.45.1 ± 6.2 and 46.3 ± 7.3 vs.75.6 ± 12.4),decreased MDA levels (4.1 ± 2.2 vs.7.1 ± 2.1 and 3.9 ± 0.5 vs.6.1 ± 1.2),increased SOD activity (12.8 ± 3.2 vs.9.4 ± 1.1 and 10.8 ± 1.2 vs.7.0 ± 1.1),and inhibited apoptosis (2.8 ± 0.6 vs.4.1 ± 1.4 and 3.1 ± 1.1 vs.5.8 ± 1.3).p38 siRNA reduced the levels of IL-1 (288 ± 89 vs.592 ± 95 and 380 ± 94 vs.775 ± 175) and IL-6 (38 ± 5 vs.70 ± 12 and 80 ± 20 vs.118-± 17),however,had no influence on TNF-α level.Silencing p38 gene decreased phosphorylation of p65 and inhibitor of nuclear factor kappa-B kinase β,and increased inhibitor of nuclear factor kappa-B expression.However,p38 siRNA had no effect on the phosphorylation of c-Jun and c-Fos.Conclusion Through inhibiting the NF-κB classic activation pathway,p38 siRNA reduced oxidative stress,inflammation and apoptosis of rat PMVECs,protected membrane integrity,and reduced hypoxia/reoxygenation injury.
ABSTRACT
Objective To investigate the effect of hypercapnia on hypoxic-ischemic brain injury in rats.Methods Forty-eight adult male SD rats were randomly divided into three groups: sham group (group S), hypoxic ischemic group (group HI) and hypercapnia group (group HP), n=16 in each group.Levine`s model was used to cause hypoxic-ischemic brain injury.In group S, the left common carotid artery was separated without ligation for 1 h, then ventilation with air maintaining the normal levels of PaO2 and PaCO2 for 3 h.In group HI, the left common carotid artery was separated and ligated for 1 h, PaO2 was maintained at 30-49 mm Hg by ventilating with low concentration (11%-13%) O2 for 3 hours.Based on group HI, PaCO2 in group HP was maintain at 60-80 mm Hg by inhalation of mixture gas containing (11%-13%) O2-8%CO2-N2 for 3 hours.FITC-dextran was used to measure the permeability of blood-brain barrier, TUNEL staining were used to observe the changes in the structure of the cerebral cortex.The expressions of aquaporin AQP4 and RECA-1 in cerebral cortex were detected by immunofluorescence and western blot.Results The level of brain water content, permeability of blood brain barrier and AQP4 expression were significantly increased in group HP as compared with group S and group HI (P<0.05).The histopathologic damage,as well as neuronal apoptotic index were aggravated in group HP as compared with group HI (P<0.05).Conclusion Hypercapnia may aggravate the brain damage during severe hypoxic-ischemic brain injury.This may associate with the increased expression AQP4 and the damage of blood-brain barrier.