Effect Of Heme Oxygenase-1 On Golgi Stress In Endotoxin-Induced Acute Lung Injury

Objective:Oxidative stress is essential pathological process in endotoxin-induced acute lung injury.Heme oxygenase-1（HO-1）has an important role in sepsis-induced acute lung injury.Golgi apparatus stress（GA stress）is a kind of adaptive response of Golgi apparatus in response to oxidative stress-related disease.However,the function of GA stress in endotoxin-induced acute lung injury has not been reported.Therefore,we plan to explore the function of GA stress,and the regulation of HO-1 on GA stress in endotoxin-induced acute lung injury,which would provide some therapeutic targets for acute lung injury.Method:In order to explore the role of Golgi stress in acute lung injury and the function of HO-1 on Golgi stress,we conducted the experiments both in vivo and in vitro,which could be divided into the following four parts:1.In order to evaluate the role of Golgi stress in acute lung injury,the mice were injected with LPS（10mg/kg）by tail vein and sacrificed 12 hours later to establish acute lung injury models.Then we detected the levels of albumin and IL-6 in BALF by ELISA.HE staining and TUNEL staining was used to see lung injury levels and apoptosis,respectively.Immunofluorescence staining was used to observe the structure of Golgi apparatus.We also detected the expression of Golgi structure-related proteins（Golgin 97,GM130,MannosidaseⅡ）,Golgi stress-related protein（GOLPH3）by RT-qPCR,as well as the levels of ROS in lung tissue.2.In order to explore the function of HO-1 on Golgi stress,HO-1^fl/fl/CAG-Cre and CAG-Cre mice were treated with LPS（10mg/kg）for 12 hours before sacrificing.And same methods were used to detect above biomarkers.3.In order to find out the function of HO-1 on Golgi stress in vitro,the MLE-12cells were transfected with HO-1si RNA to knockdown the expression of HO-1 before the stimulation of LPS（5μg/ml）.Cells viability,ROS levels,SOD activities and Golgi stress levels were evaluated after the stimulation of LPS for 24 hours.4.To investigate the mechanism of HO-1 on Golgi stress both in vivo and in vitro,DMOG was used to stimulate HIF-1α/HO-1 signaling pathway.Then we used same methods to evaluate lung injury and Golgi stress both in vivo and in vitro.Results:1.The treatment of LPS induced severe lung injury,higher cytokines,apoptosis and ROS levels in mice.Simultaneously,LPS induced Golgi fragmentation by decreasing the expression levels of Golgi structure related proteins（GM130,MannosidaseⅡ,Golgin 97）,and increasing the expression levels of Golgi stress related protein GOLPH3.Therefore,LPS stimulation resulted in Golgi stress in acute lung injury.2.The knockout of HO-1 in vivo and in vitro aggravated oxidative stress,inflammation,apoptosis and reduced the expression of Golgi structure related proteins（GM130,MannosidaseⅡ,Golgin 97）,and increased the expression of Golgi stress related protein GOLPH3,which caused the fragmentation of Golgi.Therefore,HO-1 is related to Golgi stress in endotoxin-induced acute lung injury.3.The activation of HIF-1α/HO-1 signaling pathway after the pretreatment of DMOG,attenuates Golgi stress and oxidative stress by increasing the levels of GM130,MannosidaseⅡ,Golgin 97,and decreasing the expression of GOLPH3 in LPS-induced acute lung injury in mice and in endotoxin-activated alveolar epithelial cells.Therefore,the activation of HIF-1α/HO-1 signaling pathway reduce acute lung injury by improving Golgi stress.Conclusion:LPS stimulation induced Golgi stress by promoting Golgi fragmentation and decreasing the expression of Golgi structure-related proteins（Golgin97,GM130,MannosidaseⅡ）and increasing the levels of Golgi stress related protein GOLPH3 in endotoxin-induced acute lung injury both in vivo and in vitro.The down-regulation of HO-1 exacerbated septic lung injury by promoting Golgi stress,and increasing oxidative stress,apoptosis and inflammation.Furtherly,the protective effect of HO-1 on Golgi stress was enhanced by the activation of HIF-1α/HO-1 signaling pathway.In conclusion,HO-1 plays a crucial role in alleviating endotoxin-induced acute lung injury by regulating Golgi stress,and its mechanism is related to HIF-1α/HO-1 signaling pathway,which may provide a therapeutic basis for the treatment of acute lung injury.