The Role Of ANG?/ANG1-7 In ER Stress-induced Endothelial Apoptosis During Seawater Aspiration-induced Acute Lung Injury

BackgroundSeawater drowning is a common cause of unintentional death.And the injury caused by inspiration of seawater after drowning can also bring significant social and public health problems.Acute lung injury(ALI)and acute respiratory distress syndrome(ARDS)are the most common complications after seawater drowning.In seawater aspiration-induced ALI,lung edema and inflammatory cells infiltration are caused by the injury of alveolar epithelial-endothelial barrier.And apoptosis of alveolar cells especially pulmonary microvascular endothelial cells after seawater stimulation is the main cause of lung blood-air barrier injury.Therefore,the reduction of pulmonary microvascular endothelial cell apoptosis will play an important role in the treatment of seawater aspiration induced ALI.Endoplasmic reticulum(ER)is the major organelle responsible for protein biosynthesis and modification.ER proteostasis is altered after injury and protein folding stress reaction will be generated in damaged cells.ER stress can induce unfolded protein response(UPR).UPR is an adaptive reaction in cells.However,excessive ER stress and UPR can also trigger apoptosis.UPR is initiated by three transmembrane proteins anchoring on the ER-IRE1,PERK and ATF6.The activation of PERK and IRE1 signaling pathway participated in ER stress induced apoptosis.Activation of PERK signaling pathway can ultimately promote the expression of CHOP which can induce the apoptosis via regulating the expression of bcl-2 family.Apoptosis induced by activation of IRE1 pathway was through the phosphorylation of JNK(which is the key molecule in the mitochondrial apoptosis pathway).In our previous study,we found that PERK-CHOP pathway participated in the seawater stimulation induced apoptosis.On the other hand,activation of JNK also plays a key role in the seawater stimulation induced apoptosis.However,whether IRE1 activated by ER stress can regulate the JNK pathway in seawater induced apoptosis need to be further investigated.Besides renin-angiotensin system(RAS)in body,there is an intrinsic angiotensin system(all the factors in the system were produced by the cells)existed in the lung microvascular endothelial cells.This intrinsic system include angiotensin II(ANG?)and its counter-regulatory axis ACE-2/ANG1-7.In our previous study,we found that seawater stimulation can induce the expression of ANG? and inhibit the expression of ACE-2/ANG1-7.However,Losartan(an inhibitor of ANG? receptor AT1)can reduce the inflammation,lung edema and apoptosis in seawater aspiration induced ALI.And its inhibition effect in the apoptosis induced by seawater was related to the depression of JNK pathway.Furthermore,the activation of JNK pathway is also a key step in the ER stress induced apoptosis,but whether ANG?/ANG1-7 system can regulate apoptosis induced by ER stress in seawater aspiration induced ALI need to be investigated.In this study,we established the seawater stimulation induced injury model both in rat lung tissue and RPMVECs and investigated the role of ANG?/ANG1-7 system in the regulation of apoptosis induced by ER stress during seawater aspiration induced ALI.Moreover,we designed the tandem gene based on the mechanism study and tested its effect in the apoptosis induced by seawater.Objective:In this study,we established the seawater stimulation induced injury model both in rat lung tissue and RPMVECs and investigated the role of ANG?/ANG1-7 system in the regulation of apoptosis induced by ER stress during seawater aspiration induced ALI.Furthermore,we designed the tandem gene based on the mechanism study and tested its effect in the apoptosis induced by seawater.Methods:Part 1: The seawater stimulation induced injury models were established in SD rat and RPMVECs.The effect of ER stress inhibitor 4-PBA on the pathological change(H-E staining),W/D ratio and Evans blue permeability after seawater stimulation were investigated.The expression of ER stress related protein Grp78,CHOP,p-IRE1,XBP1 s and apoptosis related protein cleaved-caspase3,bax,bcl-2 after seawater stimulation were tested by Western blot.And the effects of 4-PBA on the expression of these proteins were also investigated.Furthermore,the changes of apoptosis rates in RPMVECs were tested by Flow cytometry.Part 2: The expression of ACE-2 protein was tested by Western blot and expression of ANG?,ANG1-7 were tested by ELISA.And the effects of 4-PBA on the expression of these proteins were also investigated at the same time.We also investigated the effects of IRE1 inhibitor 4?8C and PERK inhibitor GSK2656157 on the expression of ANG?/ANG1-7 system in RPMVECs.In addition,we used SAR(Saralasin,an ANG? receptor AT1 inhibitor)and A779(an ANG1-7 receptor Mas inhibitor)to investigate the effect of ANG?/ANG1-7 system in the regulation of apoptosis induced by ER stress during seawater aspiration induced ALI.And we also investigated the effect of ANG?/ANG1-7 system on the activation of JNK pathway.Part 3: Based on the protection effects of ACE-2/ANG1-7 on the ER stress induced apoptosis after seawater stimulation and XBP1 s expression characteristics,we designed and established a tandem gene,promotor-ACE-2 gene-ANG1-7 gene(promotor is a start-up sequence which can combined with XBP1s),via plasmid vector.Through checking in gene bank and some related detections,we finally chose start-up sequence of wfs1 gene(a downstream gene of XBP1s)as the promoter of tandem gene.The changes of apoptosis were tested after the tandem gene transfection into the RPMVECs.Results:Part 1: The rat lung tissue appears significant pathological changes,W/D ratio and Evans blue permeability increase after seawater aspiration.Seawater and ER stress agonist MG132 promoted the expression of ER stress related protein CHOP and Grp78,and activated IRE1-XBP1 s pathway which is closely related to apoptosis.In addition,seawater and MG132 both promoted RPMVECs apoptosis rates and increased the expression of cleaved-caspase3 and bax,inhibited anti-apoptotic protein bcl-2 expression.Moreover,addition of ER stress inhibitor 4-PBA restrained the expression of ER stress related proteins and inhibited apoptosis after seawater aspiration.Part 2: Seawater and MG132 significantly promoted the expression of ANG? and inhibited ACE-2/ANG1-7 in RPMVECs.At the same time,the expression of apoptosis related protein cleaved-caspase3 and bax were significantly promoted and the expression of anti-apoptotic protein bcl-2 was inhibited.On the other hand,addition of ER stress inhibitor 4-PBA restrained the apoptosis rates and the expression of ANG? and promoted ACE-2/ANG1-7 both in lung tissues and RPMVECs.We further found that addition of IRE1 inhibitor 4?8C restrained the expression of ANG? and promoted ACE-2/ANG1-7,but the effect of PERK inhibitor GSK2656157 was not significant.Moreover,pretreatment with SAR(an ANG? receptor AT1 inhibitor)before seawater and MG132 stimulation inhibited the expression of cleaved-caspase3,bax and promoted the expression of bcl-2.Pretreatment with ANG1-7 in RPMVECs also inhibited the expression of cleaved-caspase3,bax and promoted the expression of bcl-2.However,pretreatment with A779(an ANG1-7 receptor Mas inhibitor)inhibited the apoptosis in RPMVECs.In addition,seawater and MG132 stimulation both activated the phosphorylation of JNK pathway.Pretreatment with SAR and ANG1-7 in RPMVECs both inhibited the phosphorylation of JNK,but addition with A779 promoted the phosphorylation of JNK.These results showed that the effect of ANG?/ANG1-7 system in the regulation of apoptosis induced by ER stress during seawater aspiration induced ALI was through JNK pathway.Part 3: Firstly,we designed and established a tandem gene,promotor-ACE-2 gene-ANG1-7 gene(promotor is a start-up sequence which can combined with XBP1s),via plasmid vector.After checking in gene bank and specificity analysis,we chose wfs1 and atf3 gene promoter that the specificity of combining with XBP1 s were better than other genes.After PCR testing,we found that seawater and MG132 stimulation promoted the expression of wfs1 and atf3 mRNA,but the expression of wfs1 mRNA was higher than atf3.This result showed that the specificity of wfs1 gene promoter combining with XBP1 s is better than atf3 gene promoter.Therefore,we chose the start-up sequence of wfs1 as the tandem gene promoter.The content of ACE-2 mRNA was significantly promoted after the tandem gene transfected into the seawater stimulated RPMVECs and this effect was concentration dependent.In addition,with no seawater stimulation,the expression of ACE-2 and ANG1-7 were same in control group and tandem gene transfection group.After seawater stimulation,the expression of ACE-2 and ANG1-7 were significantly inhibited.However,the expression of ACE-2 and ANG1-7 were promoted in tandem gene transfected RPMVECs after seawater stimulation.This result showed that the tandem gene can be activated after seawater stimulation.Moreover,without seawater stimulation,apoptosis rates was very low and the expression of cleaved-caspase3,bax and apoptosis related JNK phosphorylation were also very low in both control group and tandem gene transfection group.After seawater stimulation,apoptosis rate and apoptosis related proteins in RPMVECs without tandem gene transfection were significantly promoted.However,apoptosis rate and apoptosis related proteins were significantly inhibited in tandem gene transfected RPMVECs after seawater stimulation.Conclusion:1.Seawater stimulation activated ER stress and ultimately induced apoptosis in lung tissue and RPMVECs.The activation of IRE1-JNK pathway plays an important role in ER stress induced apoptosis during seawater aspiration induced ALI.2.ER stress(particularly activation of IRE1 pathway)promoted the expression of ANG? and inhibited the expression of ACE-2/ANG1-7 in seawater aspiration induced ALI.3.In seawater aspiration induced ALI,the interaction of ANG? and its receptor AT1 promoted ER stress induced apoptosis.However,the interaction of ANG1-7 and its receptor Mas inhibited apoptosis induced by ER stress.And these effects were related to JNK pathway.4.The activation of IRE1 in ER stress induced by seawater stimulation led to splicing of XBP1 u and caused the rapid expression of XBP1 s.Base on this character,we designed and established a tandem gene,promotor-ACE-2 gene-ANG1-7 gene(promotor is a start-up sequence which can combined with XBP1s),via plasmid vector.After transfection with this tandem gene,apoptosis induced by ER stress was significantly inhibited in seawater aspiration induced ALI.