| Background Ischemia-reperfusion(IR)is a major cause of acute lung injury(ALI)during lung transplantation,cardiopulmonary bypass,pulmonary sleeve resection,trauma and cardiopulmonary resuscitation.Even if the patients with acute lung injury were supported by advanced intensive care,the mortality is still above30-40%.The pathological manifestations of lung ischemia-reperfusion injury(LIRI)are severe inflammatory reactions in the lungs,infiltration of inflammatory cells,the lung epithelium and endothelial cells damaged,and the air-blood barrier dysregulated.Inflammatory reaction is one of the factors that cause and aggravate secondary injury after IR.During inflammatory process,variety of inflammatory factors and inflammatory mediators participate in the IR injury.Cholinergic anti-inflammatory pathway(CAP)is a regulatory pathway that links nerves and the immune system.It is an important part in inflammatory reflex.It has become an important research target between nervous system and immune system that activating or inhibiting CAP interventiontoregulatesystemicinflammationandimmuneresponse.Ultrasound-based therapy stimulates the spleen's intrinsic alpha7 nicotinic acetylcholine receptor(?7nAChR)dependent CAP to suppress inflammatory responses,then reduce tissue inflammatory and oxidative stress responses.This provides a new insight and method for LIRI lung protection.Part?Effects of LIPUS pretreatment on the inflammatory response of lung ischemia-reperfusion injury in ratsOBJECTIVE This research was aimed to build lung ischemia-reperfusion injury model of rats in vivo,and observe the effects of low-intensity pulsed ultrasound(LIPUS)pretreatment on the inflammatory response in lung tissue after IR injury.Methods Thirty male Sprague-Dawley rats,aged 8-12 weeks and weighed200-250 g,were randomly divided into three groups.After anesthesia was induced by intraperitoneal injection of 10%chloral hydrate 5ml/kg,Tracheal intubation followed by small animal ventilator,breathing frequency 70bpm,breathing ratio of 1:2,tidal volume:10-12ml/kg.Sham group S,left chest thoracic free left hilar,not blocked;ischemia-reperfusiongroupIR,theestablishmentofratlung ischemia-reperfusion lung injury experimental model.Thoracotomy was performed on the 5th intercostal space of the left anterior chest and free left hilar.50 U heparin was diluted with saline to 500?L and injected into the caudal vein.The left hilar was closed with a noninvasive microvascular clamp at 10 min after filling,blocked 45 min and reperfused 180 min.The group L was treated with low intensity pulsed ultrasound,the diameter of the probe was 1.5 cm and the frequency was 1.02 MHz.The ultrasound power of the probe was 0.8w/cm~2with pulse wave.The rats were anesthetized and placed on the test bed and irradiated with ultrasound for 30min,and then treated with IR.At the end of the experiment,animals were sacrificed by left atrial exsanguination and the chest was dissected to remove the entire left lung tissue.Haematoxylin and eosin stain was used for lung tissue biopsy,lung wet/dry weight ratio was measured.The contents of IL-1 and IL-6 in lung homogenate were determined by enzyme-linked immunosorbent assay(ELISA),Immunohistochemistry was used to detect the protein expression of TNF-?.Results Compared with the group S,the lung tissue showed obvious damage after ischemia-reperfusion.The alveolar wall thickening and edema degree after IR were higher,the pathological score of lung tissue was significantly higher(P<0.05),the wet/dry weight ratio and MDA content increased,the activity of SOD was inhibited,the IL-1,IL-6 and TNF-?increased.After low-intensity pulsed ultrasound pretreatment,the lung histopathological score of L group was significantly lower than that of IR group(P<0.05).The wet/dry weight ratio of lung tissue decreased,the content of MDA decreased,the inhibition degree of SOD activity decreased,and the inflammatory indexes of IL-1,IL-6 and TNF-?decreased significantly(P<0.05).Conclusion In LIRI model of rat,LIPUS pretreatment can improve inflammatory exudation of lung tissue after LIRI,and decrease the lung tissue W/D and the content of MDA,and reduce the inhibition of SOD activity,and decrease the levels of IL-1,IL-6 and TNF-?.LIPUS pretreatment have protective effect on LIRI by inhibit inflammatory response in rats.Part?Mechanism of LIPUS pretreatment protective on lung ischemia-reperfusion injury in ratsObjective This research was aimed to build lung ischemia-reperfusion injury model of rats in vivo,and investigate the role of CAP in LIPUS pretreatment on LIRI and explore the mechanism of LIPUS pretreatment.Methods Forty male Sprague-Dawley rats,aged 8-12 weeks and weighed200-250 g,were randomly divided into four groups.After anesthesia was induced by intraperitoneal injection of 10%chloral hydrate 5ml/kg,Tracheal intubation followed by small animal ventilator,breathing frequency 70bpm,breathing ratio of 1:2,tidal volume:10-12ml/kg.Sham group S,left chest thoracic free left hilar,not blocked;ischemia-reperfusion group IR,the establishment of rat lung ischemia-reperfusion lung injury experimental model.Thoracotomy was performed on the 5th intercostal space of the left anterior chest and free left hilar.50 U heparin was diluted with saline to 500?L and injected into the caudal vein.The left hilar was closed with a noninvasive microvascular clamp at 10 min after filling,blocked 45 min and reperfused 180 min.The group L was treated with low intensity pulsed ultrasound,the diameter of the probe was 1.5 cm and the frequency was 1.02 MHz.The ultrasound power of the probe was 0.8w/cm~2 with pulse wave.The rats were anesthetized and placed on the test bed and irradiated with ultrasound for 30min,and then treated with IR.The rats of group LA were injected intraperitoneally with?7nAchR antagonist methyl Nippon Buping 2mg/Kg,and then treated with the pretreatment group after 30 min.At the end of the experiment,animals were sacrificed by left atrial exsanguination and the chest was dissected to remove the entire left lung tissue.The expression of?7 nAchR and NF-?B p65 protein was detected by Western Blot.The nuclear translocation of NF-?B p65 was detected by immunofluorescence.The relative quantitative expression of TNF-?mRNA in lung tissue was detected by q-PCR.Results NF-?B p65 was mainly located in the cytoplasm,and only a small amount of NF-?B p65 and TNF-?mRNA were expressed in the absence of IR treatment.The expression of?7nAchR protein was decreased after IR treatment.NF-?B p65 translocation from the cytoplasm to the nucleus,NF-?B p65 protein expression increased significantly,TNF-?mRNA expression increased(P<0.05);while the expression of?7nAChR protein in LIPUS preconditioning group was significantly increased(P<0.05).The expression of NF-?B p65 protein was significantly decreased and the expression of TNF-?mRNA was decreased.However,after intraperitoneal injection of?7 nAChR antagonist methyl Nippon Lipin 2mg/Kg,LIPUS preconditioning and IR after treatment,the number of NF-?B p65 positive cells translocated from the cytoplasm to the nucleus significantly increased,the expression of NF-?B p65 protein increased,and the expression of TNF-?mRNA increased(P<0.05).Conclusion In LIRI model of rat,LIPUS pretreatment have protective effect by activating the?7nAchR-dependent CAP,and then inhibit NF-?B translocation and activation,and decrease the expression level of TNF-?m RNA in lung tissue of LIRI. |
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