A Study On The Role Of Src Kinase In Liver Injury And Lung Injury In Endotoxemic Mice Via Regulating Nrf2 Nuclear Export

Backgroud and objective:Endotoxemia is a systemic inflammatory response caused by infection. It is one of the main reasons for the death of infection and Intensive Care Unit(ICU) patients. In spite of the rapid development of medicine in recent years, the mortality of endotoxemia remains high(30%-60%). Liver and lung are easily injured in endotoxemia, while the pathogenesis of liver and pulmonary injury is complex and there is no effective therapeutic strategies for it at present. Nuclear factor-erythroid-2-related factor 2(Nrf2) is a key transcription factor in endogenous anti-injury system. It plays a pivotal role in the anti-injury of liver and lung. Src tyrosine kinase is an upstream kinase in Nrf2 signaling pathway. It also plays a pivotal role in liver injury and lung injury in endotoxemia via regulating Nrf2 nuclear export. The previous study of our team found that high dose tumor necrosis factor-alpha(TNF-α) and hydrogen peroxide(H2O2) decrease the Nrf2 transcriptional activity in pulmonary microvascular endothelial cells(PMVECs). Therefore, we speculate that Src kinase catalyze Nrf2Y568 phosphorylation which leads to Nrf2 nuclear export and then degradation and endogenous anti-injury activity decline is another important mechanism on liver injury and lung injury in endotoxemia. The aim of this study was to explore the role of Src kinase in liver injury and lung injury in endotoxemia via regulating Nrf2 nuclear export by using the model of LPS-induced liver injury and lung injury, and in vitro experiment, providing new insight and therapeutic targets to intervene in liver injury and lung injury in endotoxemia through endogenous anti-injury pathway.Methods:1 The effects of Src kinase inhibition on liver injury and lung injury in endotoxemic mice1.1 Established the model of liver injury and lung injury in endotoxemic mice via LPS intraperitoneal injection.1.2 The effects of Src kinase inhibition on liver injury and lung injury were evaluated by HE staining1.3 The levels of SOD、MDA、MPO in liver and lung of endotoxemia mice were detected by kits.1.4 Serum levels of ALP in endotoxemia mice were detected by kits.1.5 The levels of Nrf2 expression in liver and lung of endotoxemia mice were detected by western blots.2 The role of Nrf2 nuclear export in LPS-induced A549 cell injury.2.1 The distribution change of Nrf2 in LPS treated cell were detected by immunofluorescence.2.2 The distribution change of Nrf2 after Src kinase inhibitor PP2 and nuclear transport protein CRM1 inhibitor LMB administration in LPS treated cell were detected by immunofluorescence.2.3 The effects of Nrf2+/+ and Nrf2Y568 A adenovirus transfection on distribution change of Nrf2 were detected by immunofluorescence.2.4 Following LPS treatment, the viability change of normal cells, PP2 and LMB administration cells, Nrf2+/+ and Nrf2Y568 A adenovirus transfection cells were detected by cell counting kit-8(CCK-8).Results:1. Src kinase inhibitor PP2 significantly increased the levels of SOD and Nrf2, while decreased the levels of MPO and MDA of liver and lung in endotoxemic mice. PP2 also decrease the serum levels of ALP and apparently attenuated the liver injury and lung injury.2. LPS-induced Nrf2 nuclear export could be blocked by PP2 and LMB. Nrf2 couldn’t export out of the nuclear when the 568 th tyrosine was mutated.3. PP2 and LMB administration, Nrf2Y568 A adenovirus transfection could increase cell viability after LPS stimulation.Conclusions:1. We established a model of liver injury and lung injury in endotoxemic mice successfully via LPS intraperitoneal injection.2. Blocking Nrf2 nuclear export by Src kinase inhibitor has important effects on liver injury and lung injury in endotoxemic mice.3. Blocking Nrf2 nuclear export play a pivotal role in cytoprotection. Administration of Src kinase inhibitor may be an effective method to enhance cell anti-injury ability via blocking Nrf2 nuclear export.