A Study On The Role And Mechanism Of Carbon Monoxide Inhibit Acute Lung Injury Induced By Lipopolysaccharide

BackgroundAlveolar epithelial cells and pulmonary vascular endothelial cells damage induced by acute lung injury(ALI)is caused by direct or indirect wounding agents,and it shows decreased lung compliance,refractory hypoxemia,hypercapnia and respiratory distress,and is easy to develop into acute respiratory distress syndrome(ARDS)finally.The pathogenesis of ALI is very complex,and it was not explained clearly until now.The previous studies regarded inflammation as the deciding factor of inducing and exacerbation ALI.When the body was stimulated by exogenous or disordered by itself,the activation of immune system caused related chemokine and inflammatory factors releasing,neutrophil assembling in quantity,which resist and eliminate the diverse factors to body.But inflammation is harmful to body else,excessive inflammatory reaction will aggravate damage of physiology.The main performance is damaging of blood gas exchange in lung.Compared with adults,the development of pulmonary alveolar in newborn is not perfect enough,and it is vulnerable to the various external stimulus because its limited self-regulation.In 1989,Faix reported 11 cases of full-term newborn ALI/ARDS firstly.After that,all ALI/ARDS cases about full-term newborn,premature and low-birth weight infants were reported successively.In recent years,the researchers found that the newborn ALI was different from adult's and children's along with more and more reports of neonatal ALI/ARDS.It is faster onset,progress more quickly,and more severe hypoxemia.In all neonatal ALI inducing factors,two most important and common causes are meconium inhalation and infection.Carbon monoxide(CO)exists in the body very little.It is mainly generated by Heme oxygenase(HO)catalysting Heme.The present study showed that CO played important roles in physiological regulation in body,including dilate blood vessels,anti-inflammation,anti-apoptosis and anti-proliferation.The researchers found that low concentration of CO significantly relieved the damage of ALI in various kinds of animal model of ALI.The mechanism of CO was mainly focus on its anti-inflammation.But its anti-apoptotic effect was not concerned by now,especially for alveolar type ? epithelial cells(AEC-?).ObjectivesTo explore the role and mechanism of carbon monoxide on apoptosis of newborn rat AEC-? in LPS induced ALI.MethodsThe role of carbon monoxide on LPS inducing newborn rat ALI1.Creating a newborn rat ALI model induced by LPS.To verify the model by observing the lung tissue pathological,Wet-to-dry ratio(W/D),leukocyte counts and protein content changes in BALF.2.iCORM3 preparation: Place CORM3 solution at room temperature for 18 hours.Make it release CO gas fully,and the remainder is i CORM3 which is made of carbonyl construction.3.To observe the role of CORM3 inhibit newborn rat ALI induced by LPS.Observation index include H-E staining,W/D radio,changes of cell counts and protein content in BALF.4.To observe the role of CORM3 inhibit newborn rat AEC-? apoptosis in ALI induced by LPS.Observing apoptosis of alveolar cells by TUNEL staining.Observing AEC-? apoptosis by TUNEL plus SP-C immunofluorescent assay.The role of carbon monoxide on A549 cells apoptosis induced by LPS1.To observe the changes of A549 cells activity by MTT assay.2.To observe the role of different CORM3 concentrations on the changes of A549 cells activity by MTT assay.3.To observe the role of CORM3 on A549 cells apoptosis induced by LPS by TUNEL staining.4.To observe the mitochondrial membrane potential change of A549 cells by JC-10 staining.5.To test the content change of Cytochrome C in mitochondrial and cytoplasm by western-blot.6.To test the changes of Caspase 3 activity in A549 cells by ELISA.ResultsThe role of carbon monoxide on LPS inducing newborn rat ALI1.Compared with the control group,the LPS group of lung tissue structure disordered obviously,lung interval significantly broadened,and numerous PMN assembled in the alveoli and interstitial.W/D ratio,leukocyte number and protein content in BALF raised significantly,and it's statistically different(P < 0.05).The CORM3 group compared with the LPS group,lung tissue pathology change markedly improved,and the counts of PMN in alveoli and interstitial decreased significantly.W/D ratio,number of BALF cell and protein content increased obviously dropped,the difference was statistically significant(P < 0.05).No obvious changes in the above observations and measurements in The ICORM3 group.All these illustrate that CORM3 inhibited the newborn rat ALI induced by LPS.2.Compared with the control group,the counts of apoptotic AEC-?significantly increased in LPS group.The CORM3 group compared with the LPS group,the number of apoptotic AEC-?decreased significantly.The ICORM3 group compared with the LPS group,that index had no obvious change.It concluded that AEC-?apoptosis existed in newborn rat ALI induced by LPS,and CORM3 inhibited it.The role of carbon monoxide on A549 cells apoptosis induced by LPS.1.With the increasement of concentration of LPS,the A549 cell activity decreased obviously,and it changed most significantly between 100-400 ug/ml.2.Compared with the LPS group,A549 cell vitality enhanced obviously after intervention with CORM3.The increased trend was positively correlated with CORM3 concentration change.No obvious change between the iCORM3 group and the LPS group.3.Compared with the control group,the TUNEL fluorescent signal markedly improved in the LPS group,which certified the counts of apoptotic A549 cell increased.The CORM3 group compared with LPS,the number of A549 cells decreased.While,it had little change in the iCORM3 group.4.In the control group,the JC-10 existed inner mitochondrial membrane in the form of the polymer.It appeared red fluorescent signal under microscope,and the mitochondrial membrane potential level was high at this time.JC-10 mainly exists in the form of monomer in the LPS group,which appears green fluorescence under microscope.This showed that the mitochondrial membrane potential decreased obviously.And after intervention with CORM3,the mitochondrial membrane potential rebounded,but iCORM3 had no effect.5.In the control group,the Cytochrome C existed mainly in mitochondrial and little in cytoplasm.After stimulated with LPS,the Cytochrome C decreased in mitochondrial,and intracytoplasmic Cytochrome C increased in contrast.Cytochrome C in mitochondria increased gradually with CORM3 treatment.In i CORM3 group,Cytochrome C content in mitochondria had no obvious change compared with LPS group.These results suggest that: mitochondrial membrane potential decreased with LPS's stimulation,then permeability enhancement,and Cytochrome C in mitochondrial transfered into the cytoplasm at last.CORM3 could suppress the Cytochrome C transfer into cytoplasm from mitochondria.6.Contrast to control group,the activity of Caspase 3 had no change whether stimulated with LPS or treated with CORM3.Conclusions1.CO relieves newborn rat ALI induced by LPS.2.CO can inhibit AEC-? apoptosis when LPS induce acute lung injury in neonate rat.3.CO can reduce A549 cells death induced by LPS,and the mechanism maybe caspase-independent.