| Background:Lung ischemia-reperfusion injury(LIRI),as a significant clinical issue,is also a intractable problem that has plagued the clinicians for many years,which usually occurs in lung transplantation,heart and lung transplantation,revascularization therapy of pulmonary embolism,cardio-pulmonary resuscitation,pulmonary arterioplasty,shock and various cardiac surgery under cardiopulmonary bypass.In recent years,with the continuous progress and innovation of medical technology,lung transplantation has gradually become the effective and ultimate method for the treatment of end-stage lung diseases.Lungs are extremely susceptible to injury,in spite of advances in surgical management and immunosuppression,outcomes for lung transplantation are the worst of any solid organ transplantation.It is reported that the patient one-year survival rate following lung transplantation is only about 70%,and patients tend to succumb to post-operative mortality within 30 days of surgery.The success of lung transplantation is limited by high rates of primary graft dysfunction(PGD)because of ischemia-reperfusion injury characterized by robust inflammation,alveolar damage,and vascular permeability.Because PGD is a complication that leads to severe early and long-term consequences for lung transplant recipients,transplant teams tend to be very conservative in the selection of donor lungs.PGD,largely because of ischemia-reperfusion injury,is associated with significant early and late mortality after transplant,and severe(Grade 3)PGD has an incidence of approximately 28.8%within 72h of transplant.In addition,PGD is also a risk factor for late graft rejection,the major cause of mortality in recipients beyond one year of transplant.As we all know,ischemia-reperfusion injury is ine-vitable after lung transplantation which can cause lung damnification,such as pulmonary vascular endothelial injury,microcirculation dysfunction,and changes in hemorheology,and even can lead to graft failure.Methods for prevention of PGD after lung transplant are urgently needed,and understanding mechanisms of ischemia-reperfusion injury is critical for the development of novel and effective therapeutic approaches.Therefore,from the actual situation of ischemia-reperfusion injury after lung transplantation,how to intervene or protect the lung before ischemia-reperfusion is the key to improve both short-and long-term outcomes after lung transplantation.In lung transplantation,organ ischemia and subsequent reperfusion is unavoidable and commonly leads to acute,sterile inflammation after transplantation called ischemia-reperfusion injury(IRI).Currently,there are no therapeutic agents clinically utilized to specifically prevent ischemia-reperfusion injury,and treatment strategies are limited to supportive care.The annual number of lung transplantations keeps growing,and major research efforts have been aimed toward:better preserving donor lungs,expanding the donor lung pool,reconditioning of marginal donor lungs via ex-vitro lung perfusion,and developing some kind of methods to prevent or treat ischemia-reperfusion injury.In recent years,a large number of studies have described strategies to limit lung ischemia-reperfusion injury in experimental settings,which often reveal mechanistic insight.Many of these strategies involved the use of various antioxidants,anti-inflammatory agents,mesenchymal stem cells,and ventilation with gaseous molecules.Further advancements have been achieved in understanding mechanisms of innate immune cell activation,neutrophil infiltration,endothelial barrier dysfunction and oxidative stress reaction.Recent studies have identified a variety of strategies including:antioxidant strategies using free radical scavengers or inhibitors of oxidant-producing enzymes(e.g.,methylene blue or N-acetylcysteine);anti-inflammatory strategies using inhibitors of proinflammatory transcription factors(e.g.,inhibitor of Egr-1)or inflammatory mediators(e.g.,?1-antitrypsin,anticytokine or anti-HMGB1 agents);ventilation with gaseous molecules(e.g.,carbon monoxide)or inhaled anesthetic sevoflurane that display anti-inflammatory properties;growth factors(e.g.,keratinocyte growth factor-2);dietary supplements such as creatine;and cell-based therapies such as application of mesenchymal stem cells.In addition,a recent study showed that treatment with diannexin(a homodimer of annexin V,an anionic phospholipid-binding protein with potent anticoagulant activity)significantly reduced ischemia-reperfusion injury in lung transplantation model by reducing cell death and tissue inflammation.Although many experimental studies have described various strategies to prevent or improve ischemia-reperfusion injury,these have not yet been translated into clinical applications.Therefore,the thrust of future research should aim to better understand mechanisms of lung ischemia-reperfusion injury to identify more effective therapeutic targets and also aim to push the most promising therapies toward clinical trails.Silymarin is an active element extracted from milk thistle Silybum marianum.Silymarin and its major active ingredient,with the function of anti-oxidative stress,anti-inflammatory,maintaining cell membrane fluidity,protecting liver cell membranes,promoting liver cell repair and regeneration,have been used for the treatment of various liver diseases traditionally.Along with the deepening of research,silymarin has shown that it also has the important role of regulating cell cycle,inhibiting apoptosis,immune regulation,cardiovascular and neural protection,and anti-ischemia-reperfusion injury,with these new discoveries,its clinical applications are also more and more widely.Although many studies have shown that silymarin can reduce the ischemia-reperfusion injury of various organs through anti-inflammatory,anti-oxidation and inhibition of apoptosis,but the role of silymarin in pulmonary vascular dysfunction induced by lung ischemia-reperfusion injury and its mechanisms have not been reported.In our study,we establish the model of lung ischemia-reperfusion injury in rats in vivo and to observe the effect of silymarin preconditioning on lung inflammatory responses,oxidative stress,caspase-3/9 activities,HIF-la and iNOS protein expressions,and pulmonary diastolic function after lung ischemia-reperfusion injury,and to clarify the role of silymarin on pulmonary artery diastolic dysfunction induced by lung ischemia-reperfusion injury,and to explore its possible mechanism.Part 1:Effect of lung ischemia-reperfusion injury on pulmonary vascular functionObjective:To establish the model of lung ischemia-reperfusion injury in rats in vivo and to observe the effect of lung ischemia-reperfusion injury on diastolic function of isolated pulmonary artery rings.Methods:Male Sprague-Dawley rats were randomly divided into two groups(n=10):sham group(S group)and ischemia-reperfusion group(IR group).Animals in IR group were underwent IR procedure while the rats in sham group were submitted to the same surgical protocol as described but without ligation.After 60 minutes of ischemia,the left pulmonary artery occlusion was cleared and the artery was reperfused with blood.Following 24 hours of reperfusion,the rats were scarificed by decollation following anesthetization.The pulmonary arteries were dissected out from the lung tissue and the connective tissue was removed under a microscope.The pulmonary arteries were cut into pulmonary artery rings with a length of about 2.5 mm.According to the experiment,the endothelium of some rings was removed by gently rubbing the lumen with human hair.The isolated pulmonary artery rings were divided into endothelium intact group and endothelium-denued group.The diastolic responses of newly isolated pulmonary artery rings to acetylcholine and nitroglycerin were measured by in vitro microvascular tension measurement system.Results:1.Acetylcholine-induced relaxant response of isolated pulmonary artery rings1.1 In case of endothelium-intact,the relaxant response curve of pulmonary artery rings induced by acetylcholine in group S rised slowly in the concentration range from 1×10-9 to 1×10-7,and the curve increased rapidly,when the cumulative concentration reached up to 3×10-7,subsequently,the rise tendency of relaxant response curve became flat;showing that the diastolic function of pulmonary artery rings was good;on the whole,the relaxant response curve of pulrmonary artery rings induced by acetylcholine in group IR rised gently,indicating that the diastolic function of pulmonary artery rings declined.Comparing with group S,the diastolic function of pulmonary artery rings induced by acetylcholine decreased significantly in group IR(P<0.01).1.2 After endothelial denudation,the relaxant response curves of pulmonary artery rings induced by acetylcholine in group S and group IR presented on slowly increasing,but they were generally more straight,suggesting that diastolic function of pulmonary artery rings in both groups decreased obviously;there was no significant difference between S group and IR group(P>0.05).2.Nitroglycerin-induced relaxant responses of isolated pulmonary artery rings2.1 In case of endothelium-intact,relaxant response curves of pulmonary artery rings induced by nitroglycerin in group S and group IR showed gradually rise during concentration range from 1×10-9 to 1×10-7,and both curves increased rapidly,when the cumulative concentration reached up to 3×10-7;while,since the concentration attains 1×10-5,the rise tendency of relaxant responses curves became flat,hinting that diastolic function of pulmonary artery ring in both groups were good;there was no significant difference between two groups(P>0.05).2.2 After endothelial denudation,variation tendency of relaxant responses curve of pulmonary artery ring induced by nitroglycerin in group S and group IR was basically consistent with that in case of endothelium-intact,indicating that diastolic function of pulmonary artery ring in both groups were good;there was no significant difference between two groups(P>0.05).Conclusions:Endothelium-dependent pulmonary arterial dilatation but not endothelium-independent relaxation was significantly impaired by lung ischemia-reperfusion injury.Part 2:The role of silymarin on pulmonary vascular dysfunction induced by lung ischemia-reperfusion injuryObjective:To establish the model of lung ischemia-reperfusion injury in rats in vivo and to observe the effect of silymarin preconditioning on lung inflammatory responses,oxidative stress,caspase-3/9 activities,HIF-1? and iNOS protein expressions after ischemia-reperfusion injury and pulmonary diastolic function,and to clarify the role of silymarin on pulmonary artery diastolic dysfunction induced by lung ischemia-reperfusion injury,and to explore its possible mechanism.Methods:Male Sprague-Dawley rats were randomly divided into three groups(n=10):sham group(S group),ischemia-reperfusion group(IR group)and silymarin pretreatment group(SM group),IR group and SM group were underwent ischemia-reperfusion procedure as described in the first part.The rats in SM group were administered with silymarin 250mg/kg/day intragastrically(diluted with normal saline to 2ml)for 8 consecutive days before operation.The other two groups were given equal volume of normal saline daily for 8 consecutive days before surgery.Following 24 hours of reperfusion,the rats were scarificed by decollation following anesthetization and the left lung tissue were collected.The pulmonary artery rings were obtained as previously described.The levels of NF-?B,TNF-?,IL-6 and IL-1?in lung tissue were measured by enzyme-linked immunosorbent assay(ELISA).The content of MDA in lung tissue was detected by thiobarbituric acid color chromatography.The activity of SOD in lung tissue was detected by spectrophotometry.The expression of caspase-9 and Caspase-3 in lung tissue was detected by Spectrophotometry.The expression of HIF-1? and iNOS protein in lung tissue was detected by Western blotting.The diastolic responses of newly isolated pulmonary artery rings to acetylcholine and nitroglycerin were measured by in vitro microvascular tension measurement system.The lung wet/dry weight ratio(W/D)was calculated.Results:1.Comparison of W/D in three groups:Compared with the S group,the W/D of the lung tissue in IR group and SM group were significantly increased(P<0.01);Compared with the IR group,the W/D of the lung tissue in SM group were significantly decreased(P<0.01).2.Comparison of inflammatory cytokine levels in three groups:Compared with S group,the levels of NF-?B,TNF-?,IL-6 and IL-1? in IR group and SM group were significantly increased(P<0.01);Compared with IR group,the levels of NF-?B,TNF-a,IL-6 and IL-1? in IR group and SM group were significantly decreased(P<0.01).3.Comparison of oxidative stress in three groups:Compared with S group,the levels of MDA in IR group and SM group were significantly increased and the activities of SOD were significantly decreased(P<0.01).Compared with IR group,the level of MDA in SM group was significantly decreased and the activity of SOD was significantly increased(P<0.01).4.Comparison of caspase-3/9 in three groups:Compared with S group,the activities of Caspase-3 and Caspase-9 in IR group and SM group were significantly increased(P<0.01).Compared with IR group,the activities of Caspase-3 and Caspase-9 in SM group were significantly decreased(P<0.01).5.Comparison of HIF-1? and iNOS protein expression in three groups:Compared with S group,the protein expression of HIF-la and iNOS in IR group and SM group were significantly increased(P<0.01).Compared with S group,the protein expression of HIF-l? and iNOS in SM group were significantly decreased(P<0.01).6.Comparison of diastolic function of pulmonary artery in three groups:Relaxant response curves of pulmonary artery ring induced by acetylcholine in these three groups showed gradually rise during concentration range from 1×10-9 to 1×10-7 and the three curves increased rapidly,when the cumulative concentration reached up to 3×10-7,with the highest increase rate in S group,followed by SM group,and smallest in IR group,and then the rising trend of diastolic response curves appeared to be gentle,indicating that pulmonary artery diastolic function in S group was good,then SM group and worst in IR group.Compared with S group,the diastolic function of the isolated pulmonary artery induced by acetylcholine in the IR group and the SM group was significantly decreased(P<0.01).Compared with IR group,the diastolic function of pulmonary artery induced by acetylcholine was significantly improved(P<0.01).Conclusion:Silymarin can significantly improve endothelium-dependent pulmonary artery diastolic function induced by lung ischemia-reperfusion injury,and its mechanism may be related to its anti-inflammatory,anti-oxidative stress,inhibition of apoptosis and regulation of HIF-1?-iNOS signaling pathway. |
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