The Role Of Toll-like Receptors Signal Pathway On The Protective Effect Of Nitric Oxide Against The Mouse Lung Ischemia-Reperfusion Injury

PartⅠ Short Term of Nitric Oxide Inhalation Protect Against the Mouse Lung Ischemia-Reperfusion InjuryObjective:To investigate the effect of inhaled nitric oxide (NO) preconditioning with light concentration and different terms on lung ischemia-reperfusion injury in mice.Background:NO can participate in the signal transduction, inflammation and other links. It has the function of diastolic blood vessels and topical anti-inflammatory. Inhaled with low concentration NO preconditioning has protective effect on lung ischemia/reperfusion injury. This study used lung ischemia/reperfusion model in mice and investigated the best length of inhaled NO pretreatment.Methods and Results:Specific pathogen-free C57BL mice (male,6-8weeks old) were divided into five groups:Sham (S) group, Ischemia/Reperfusion (IR) group, NO1-min preconditioning group (NO1-min), NO10-min preconditioning group (NO10-min), NO60-min preconditioning group (NO60-min). The changes of PaO2, wet-dry weight ratio (W/D), myeloperoxidase (MPO) in the injury lung were measured after60minutes of left lung ischemia. The changes of TLR2/4activations and plasma TNF-α were measured in this procedure. Compared with IR group, PaO2increased and W/D, MPO decreased evidently in NO10-min group (P<0.05). The changes in NO60-min group were similar with NO10-min group (P>0.05). There was no difference between NO1-min and IR group (P>0.05). In NO10-min group, the expressions of TLR2/4mRNA and TLR2/4proteins were diminished and TNF-a concentrations were decreased.Conclusion:Short term (10min) of inhalation NO protected lung IR injury in mice. But the protective effect of NO was not increased with the longer term of inhalation NO. Inhaled NO could inhibit the activations of TLR2/4in the lung after IR injury. Part Ⅱ Application of Real-Time PCR Gene Chip Technology to Detect the Role of Toll-like Protein Receptor Signaling Pathways in the Protective Effect of Nitric Oxide on Lung Ischemia-Reperfusion Injury in MiceObjective:Application of Real-Time PCR gene chip technology to detect toll-like protein receptor signaling pathways in nitric oxide (NO) on pulmonary ischemia-reperfusion injury in mice protection in the process of change, in order to understand the mechanism of NO lung protection.Background:Low concentration NO preconditioning has protective effect on pulmonary ischemia-reperfusion injury. In the first part of our studies found that lung protection during the process of the pretreatment of inhaled NO, Toll2/4expression activation was significantly inhibited; Functional Real-Time PCR, gene chip technology to Superarray companies in USA, the second generation of the gene chip, function classification has a high sensitivity, linear range wide good repeatability high resolution, high specificity. We used the technology to understand the toll-like receptor protein signaling pathways in the process of pretreatment with NO of lung protection.Methods and Results:6-8weeks male C57BL mice, divided into Sham group (S group), Ischemia-Reperfusion group (I/R group),10min NO pretreatment group (NO10-min group). Using Real-Time PCR gene chip technology to detect toll-like receptor mediated signal transduction protein gene expression patterns change, according to the results of91compared with I/R group, the NO10-min group toll-like receptor mediated signal transduction protein gene expression by80, including toll-like receptor proteins1/2/4/8/9, cohesion MyD88protein, the effect of molecular IRAK1, downstream pathway and target genes IKBKB, IRF3, NFKB2(P<0.05).Conclusion:At low concentrations of inhaled NO pretreatment in the process of lung ischemia-reperfusion injury in mice, the activations of toll-like receptor signaling pathways were significantly inhibited, NO can inhibit toll-like receptor signaling pathways to reduce lung injury. Part Ⅲ Toll-like receptors signaling pathways involved in pretreatment of nitric oxide in mice lung ischemia-reperfusion injury of protectionObjective:Application of knockout mice (MyD88-/-), lung ischemia-reperfusion in mice model was established, exploring toll-like protein receptor signaling pathways are involved in nitric oxide (NO) on pulmonary ischemia-reperfusion injury in mice.Background:Low concentration NO preconditioning has protective effect on pulmonary ischemia-reperfusion injury, this experimental study found that inhaled NO pretreatment of lung ischemia-reperfusion injury protection process, toll-like protein receptor signaling pathways of gene expression level activation was significantly inhibited, which as the core hub of MyD88signaling pathway molecules, expression inhibited significantly.Methods and Results:6-8weeks (MyD88+/+) of male C57BL mice, divided into blank group (group S), ischemia reperfusion group (I/R group),10min NO pretreatment group (NO10-min);6-8weeks male MyD88-/-knockout mice (C57BL as genetic background), divided into blank group (group S), ischemia reperfusion group (I/R group),10min NO pretreatment group (NO10-min), respectively, at6h after reperfusion, RT-PCR detection by2/4toll-like receptors, TRAF6, IRF3level of gene expression changes, and ELISA to detect TNF alpha, IL-6level of protein expression changes. Results show that the MyD88+/+and MyD88-/-within the group, NO pretreatment can inhibit toll-like receptors by2/4, TRAF6, IRF3the activation of gene expression (P<0.05), and inflammatory factor TNF alpha, IL-6protein expression significantly were reduced (P<0.05).Conclusion:Toll-like receptor signaling pathways through the MyD88-dependent pathway and MyD88-undependent pathway to participate in the NO in mice lung ischemia-reperfusion injury of protection.