The Effect And Molecular Mechanism Of Poldip2 In Acute Lung Injury Induced By Lipopolysaccharide

1.Background and objectivesAcute lung injury(ALI)or acute respiratory distress syndrome(ARDS)is a critical illness caused by various pathogenic factors other than cardiogenic factors such as severe infection and trauma.The clinical manifestations are acute respiratory distress,refractory hypoxemia and non-cardiogenic pulmonary edema.The pathogenesis of ALI/ARDS is complicated,a variety of pulmonary(including pneumonia,gastroeso-phageal reflux,and drowning,etc.)or non-pulmonary(including severe sepsis,trauma,and massive blood transfusion,etc.)diseases can cause the disease.The incidence of ALI/ARDS is high,and the disease progresses rapidly with high mortality rate.Although great progresses have been made in airway management and protective mechanical ventilation in recent years,there is still a lack of effective drug treatments.Searching for key drug treatment targets in the pathogenesis of ALI/ARDS is still the focus of scientific research in respiratory and critical care medicine.The pathogenesis of ALI/ARDS has not yet been fully elucidated.Studies have shown that ALI/ARDS is an uncontrolled inflammatory response,and its remarkable pathological feature is diffuse damage to alveolar epithelial cells and pulmonary capillary endothelial cells.Oxidative stress and inflammation play an important role in its pathogenesis.A large number of reactive oxygen species(ROS)directly damage lung tissues,causing oxidative damage to lung parenchymal cells and lung interstitial tissue edema.Nicotinamide adenine dinucleotide phosphate(NADPH)oxidase is the main enzyme that produces ROS in alveolar epithelial cells.Intracellular ROS has both cellular defense and a second messenger-mediated signaling pathway,which plays a key role in the pathophysiological process of lung inflammation and ischemia/reperfusion injury.Studies have confirmed that oxidative stress induced by lipopolysaccharide(LPS)via ROS from activated NADPH oxidase is an important event in the progression of ARDS lung inflammation.Therefore,in-depth study of the specific pathogenesis of LPS-induced inflammatory injury is important for treatment of ARDS.Polymerase delta-interacting protein 2(Poldip2)is a multifunctional protein.Previous studies have shown that as an important cofactor,it can interact with human DNA polymerase?p50 subunit and proliferating cell nuclear antigen proliferating cell nuclear antigen(PCNA).Poldip2 is ubiquitously expressed in various human tissues,including lung,kidney,and brain.Which mainly involved in DNA replication and damage repair,mitochondrial function and extension,and regulation of extracellular matrix.In addition,studies have shown that Poldip2 acts as a chaperone protein of NADPH oxidase 4(Nox4)in vascular smooth muscle cells(VSMCs),and participates in the regulation of Nox4 activity.Preliminary experiments of our group showed that intratracheal atomization of LPS significantly up-regulated protein level Poldip2 in lungs of C57BL/6 mice.However,whether Poldip2 is involved in LPS-induced ARDS and the specific molecular mechanism are currently unclear.Therefore,our study aimed to construct an adeno-associated virus mediated short-hairpin RNA targeted to the mouse Poldip2 gene and down-regulate the expression of Poldip2 in the lung of mice,and to explore the role of Poldip2 in LPS-induced ARDS.Meanwhile,recombined lentiviral vector containing Poldip2 or empty vector were used to up-regulate or down-regulate the expression of Poldip2 via transfected A549 cells were used to clarify the mechanism of Poldip2 regulating LPS-induced oxidative stress and inflammation and to provide a new target for clinical treatment of ARDS.2.Research methods Part 1:Study 1:Construction of intratracheal atomization of LPS-inducedARDS mouse model.Methods1.C57BL/6 mice were selected to construct LPS-induced ARDS model.Twenty-four healthy male C57BL/6 mice were randomly divided into 4 groups,including intratracheal atomization normal group,intratracheal atomization group,intraperitoneal injection normal group,and intraperitoneal injection group(n=6 for each group).Mice in the experimental group were sprayed intratracheally or injected intraperitoneally with LPS 5 mg/Kg to establish a mouse model of ARDS,while mice in the control group were given intratracheal nebulized or intraperitoneal injected equal volume of sterile saline.Lung inflammatory injury in mice were observed with different administration methods 24 hours later,then the mouse model closest to the pathological characteristics of ARDS was selected.2.Thirty-six healthy male C57BL/6 mice were selected and randomly divided into 6groups:intratracheal atomization of LPS at different time points(0,3,6,12,24,and 48h)groups(n=6 for each group).Mice in the intratracheal atomization groups were intratracheally nebulized with LPS 5 mg/Kg,and were sacrificed at corresponding time points after LPS treatment,the lung tissues were extracted to detect the pathological score,inflammatory factors and oxidative stress indicators.Results1.Intratracheal atomization of LPS group has the most severe lung inflammatory injury,which is the closest to the pathological characteristics of ARDS.2.After intratracheal atomization of LPS,the pathological score,MDA content and concentrations of TNF-a and IL-1?in BALF reached the peak at 24 h.While SOD activity in lung tissues reached its lowest level at 24 h.Conclusions1.A mouse model of ARDS induced by intratracheal atomization of LPS was successfully established.2.The optimal dose and time point of intratracheal atomization of LPS-induced mouse ARDS are 5 mg/kg and 24 h,respectively.Study 2:The effect and mechanism of Poldip2 knockdown in reducing oxidative stress and inflammation in LPS-induced mice ARDSMethods1.In order to clarify the correlation between Poldip2 and ARDS,Thirty-six healthy male C57BL/6 mice were selected and randomly divided into 6 groups:intratracheal atomization of LPS at different time points(0,3,6,12,24,and 48 h)groups(n=6 for each group).Mice in LPS-treated group were sacrificed at the corresponding time points after LPS stimulation,lung tissues were extracted to detect the protein level of Poldip2.2.Twelve healthy male C57BL/6 mice were selected and randomly divided into short-hairpin RNA targeted to the mouse Poldip2 gene(Poldip2sh RNA)and a negative control group(NCsh RNA).Recombinant adeno-associated virus type 6(AAV-6)vector were intratracheal injected to mice.The mice were sacrificed 5 weeks later,and lung tissues were extracted to verify the expression of Poldip2.3.In order to clarify the role of Poldip2 in ARDS,Twenty-four healthy male C57BL/6mice that intratracheally injected with 6.5 x10¹⁰vg/mouse(in 50?l)of recombinant AAV6 vector expressing Poldip2sh RNA or NCsh RNA were selected and randomly divided into 4 groups(n=6 for each group):a negative control(NCsh RNA)group,a negative control plus LPS-treated group(NCsh RNA+LPS),a Poldip2 knockdown(Poldip2sh RNA)group and a Poldip2 knockdown plus LPS-treated group(Poldip2sh RNA+LPS).We aimed to detect the effect of Poldip2 knockdown on oxidative stress and inflammation in lung tissues and explore its mechanism during ARDS.Results1.Compared with the Control group,the Poldip2 level in lung tissues of mice in the intratracheal atomization of LPS group increased in a time-dependent manner,reaching the highest peak at 24 h.2.Transfection of AAV-6 Poldip2sh RNA significantly down-regulate the expression of Poldip2 in mouse lungs.3.Poldip2 knockdown significantly alleviated the inflammation in lungs of ARDS mice.4.Poldip2 knockdown markedly reduced the total protein levels of BALF in ARDS mice.5.Poldip2 knockdown obviously inhibited oxidative stress in the lungs of ARDS mice.6.Poldip2 knockdown significantly down-regulated the expression of COX-2 and up-regulated the expression of HO-1 in lungs of ARDS mice.Conclusions1.A mouse model with Poldip2 knockdown in lungs was successfully established.2.Poldip2 knockdown significantly reduced oxidative stress and inflammation in lung tissues under LPS challenge,indicating that down-regulation of Poldip2 expression protects against LPS-induced ARDS.Part 2:The mechanism of Poldip2 in human alveolar epithelial cells under LPS challenge by regulating the Nox4 signaling pathway.Methods1.Oxidative stress and inflammation are the critical pathological changes in LPS-induced ARDS in alveolar epithelial cells.The human lung adenocarcinoma cell line A549 cells injury model was constructed under LPS stimulation,the serum-free media containing different concentrations of LPS(0,2.5,5,10,15,20,and 40?g/m L)were used to stimulate A549 cells for 24 h,cell viability was detected by MTT assay.2.To clarify that Poldip2 plays an important role during LPS-stimulated A549 cells,cells were randomly divided into a saline group(Control)and a LPS-treated group,the Poldip2 levels at different time points(0,3,6,12,and 24 h)after LPS stimulation were determined.We also detected the interaction between Poldip2 and Nox4,which specialized in producing of ROS.3.To explore the role of Poldip2 in LPS-stimulated A549 cells.Recombined lentiviral vector containing short-hairpin RNA targeted to human Poldip2 gene(sh Poldip2)and a negative control lentiviral(sh Cont)were used to transfect A549 cells,the treatment groups were incubated with 10?g/m L LPS after culture for 72 h,and the no-treatment groups were stimulated with the same amount of PBS.A549 cells were devided into 4groups:a negative control(sh Cont)group,a negative control plus LPS-treated(sh Cont+LPS)group,a Poldip2 knockdown(sh Poldip2)group,and a Poldip2knockdown plus LPS-treated(sh Poldip2+LPS)group.We aimed to explore the effect of Poldip2 knockdown on the oxidative stress and inflammation under LPS stimulation in A549 cells.4.To investigate the role of Poldip2 in LPS-stimulated A549 cells.Recombined lentiviral vector containing Poldip2(Lv Poldip2)and empty vector(Lv Cont)were used to transfect A549 cells.The treatment groups were incubated with 10?g/m L LPS after culture for 72 h,and the no-treatment groups were stimulated with the same amount of PBS.A549 cells were devided into 4 groups:a empty vector(Lv Cont)group,a empty vector plus LPS-treated(Lv Cont+LPS)group,a Poldip2 overexpression(Lv Poldip2)group,and a Poldip2 overexpression plus LPS-treated(Lv Poldip2+LPS)group.We aimed to explore the effect of Poldip2 overexpression on the oxidative stress and inflammation in LPS stimulated A549 cells.5.To further clarify the downstream pathway of Poldip2 is mediated by Nox4 during ARDS.The si RNA targeting Nox4(si Nox4)and negative control si RNA(si Cont)were used to transfect A549 cells.The treatment groups were incubated with 10?g/m L LPS after culture for 72 h,and the no-treatment groups were stimulated with the same amount of PBS.A549 cells were devided into 4 groups:a negative control(si Cont)group,a negative control plus LPS-treated(si Cont+LPS)group,a Nox4 knockdown(si Nox4)group,and a Nox4 knockdown plus LPS-treated(si Nox4+LPS)group,We aimed to explore the effect of Nox4 knockdown on the oxidative stress and inflammation in LPS stimulates A549 cells.Results1.The ARDS model can be successfully induced by LPS stimulation for 12h in A549cells.2.The Poldip2 level increased at a dose/time-dependent manner under LPS stimulation in A549 cells.3.Downexpression of Poldip2 significantly attenuated the oxidative stress and inflammation under LPS stimulation.4.Overexpression of Poldip2 significantly enhanced the oxidative stress and inflammation under LPS stimulation.5.Poldip2 interacted with Nox4 under LPS stimulation in A549 cells.6.Nox4 knockdown significantly reduced the oxidative stress and inflammation under LPS stimulation,but had no effect on Poldip2 expression.Conclusions1.Poldip2 acted as an upstream regulatory protein and interacted with Nox4.2.Poldip2 knockdown or overexpression involved in the regulation of LPS-induced oxidative stress and inflammation in A549 cells by down-regulating or up-regulating Nox4 protein level,NADPH oxidase activity,and downstream signaling pathways,respectively.