| Objective: Pulmonary vascular endothelial cells dysfunction is an important pathophysiology feature of pulmonary disorders,such as acute respiratory distress syndrome(ARDS),chronic obstructive pulmonary disease and pulmonary arterial hypertension.Research shows that Nod-like receptor pyrin domain-containing protein 3(NLRP3)inflammsome,interleukin-1 beta(IL-1β)and interleukin-18(IL-18)paly an important role in airway diseases,such as pneumonia,asthma,and ARDS.This study is aimed to investigate whether lipopolysaccharide(LPS)combined with adenosine triphosphate(ATP)could activate NLRP3 inflammasome in human pulmonary artery endothelial cells(HPAECs)as well as the mechanism underlying.N-acetylcysteine(NAC)is employed to prove the effect of reactive oxygen species(ROS)on inflammasome activation.Blocking inflammsome activation via caspase-1 specific inhibitor z-YVAD-FMK is designed to observe the influence of inflammsome activation on cell apoptosis.Methods: Cultured HAPECs were divided into eight different groups: control group,LPS(1,5,10 μg/ml,5.5 h or 11.5 h)+ATP(5 m M,0.5h)group,LPS(1,5,10 μg/ml,6 h)group,ATP(5 m M,0.5 h)group,NAC(10 m M,0.5h)+ LPS(1 μg/ml,5.5 h)+ATP(5 m M,0.5h)group,NAC(10 m M,0.5 h)group,z(2 μM,0.5 h)+ LPS(1 μg/ml,11.5 h)+ATP(5 m M,0.5 h)group,z(2 μM,0.5 h)group.Cell vitality was assessed by cell counting kit-8.The levels of Il-1β and Il-18 in supernatant were analyzed by enzyme-linked immunosorbent assay(ELISA).The expression of cysteine aspartic specific protease(caspase)-1,caspase-3,B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),phosphorylation of nuclear factor-kappa B(NF-κB)and p38 mitogen activated protein kinase(MAPK)was determined by western blot.Intracellular and extracellular reactive oxygen species(ROS)level was detected by DCFH-DA fluorescent probe.Cell apoptosis was evaluated by Hoechst 333432 staining assay as well as annexin ⅴ and propidium iodide(PI)labeling flow cytometric analysis.Results: 1.Different concentration of LPS(1,5 and 10 μg/ml,6 h)alone had no effects on HPAECs.Cell viability of LPS+ATP group was decreased to 71.08%±0.88%,which is remarkably lower than that of control group(P<0.05).2.The levels of IL-1β and IL-18 in supernatant of LPS+ATP group were 17.25±2.96 pg/ml and 19.81±5.27 pg/ml,which were higher than that of control group(7.05±1.13 pg/ml and 10.78±2.47 pg/ml),LPS group(9.35±2.05 pg/ml and 11.57±3.34 pg/ml)and ATP group(9.75±1.62 pg/ml and 11.62±3.45 pg/ml)(P<0.05).The expression of caspase-1 in LPS+ATP group was significantly higher than that of control group,LPS group and ATP group(P<0.05).3.The concentrations of intracellular and extracellular ROS in LPS+ATP group were 39.19±3.9 and 118.74±26.28,which were both significantly higher than that of control group(9.8±1.24 pg/ml and 28.5±9.32 pg/ml),NAC+LPS+ATP group(15.39±4.78 and 65.26±13.76)and NAC group(15.29±3.4 and 45.76±10.37)(P<0.05).The levels of IL-1β and IL-18 in supernatant of NAC+LPS+ATP group were 10.49±1.26 pg/ml and 14.85±1.88 pg/ml,which were achievably lower than that of LPS+ATP group(16.96±5.45 pg/ml and 21.24±2.83 pg/ml)(P<0.05).NAC could remarkably inhibit the expression of caspase-1 as well as phosphorylation of NF-κB(p65)and p38 MAPK in LPS+ATP group(P<0.05).The apoptosis rate of LPS+ATP group was 19.27%±5.29%,which was significantly higher than that of control group(7.54%±0.45%)(P<0.05).The apoptosis rate of NAC+LPS+ATP group was 12.55%±1.29%,which was achievably lower than that ofLPS+ATP group(P<0.05).4.The apoptosis rate of LPS(1 μg/ml,11.5 h)+ATP(5 m M)group was 31.74%±7.75%,which was significantly higher than that of control group(8.12%±0.75%)(P<0.05).The apoptosis rate of z+LPS+ATP group was 18.36%±4.67%,which was achievably lower than that of LPS(1 μg/ml,11.5 h)+ATP(5 m M)group(P<0.05).The Hoechst 3342 staining positive rate of LPS(1 μg/ml,11.5 h)+ATP(5 m M)group was 21.43%±3.35%,which was significantly higher than that of control group(2.93%±0.75%)(P<0.05).The Hoechst 3342 staining positive rate of z+LPS+ATP group was 14.03%±2.55%,which was achievably lower than that of LPS(1 μg/ml,11.5 h)+ATP(5 m M)group(P<0.05).Caspase-1 inhibitor could alleviate the up-regulation of caspase-3 and Bax/Bcl-2 as well as the apoptosis of HPAECs activated by LPS with ATP.Conclusion: High level of ROS played an important role in LPS combing with ATP-induced NLRP3 inflammasome activation as well as apoptosis of HPAECs. |
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