RESUMO
Objective:To investigate the role of polyomavirus enhancer activator 3 (PEA3) in hyperoxia-induced injury of type Ⅱ alveolar epithelial cells (AEC Ⅱ) and the underlying mechanism.Methods:AEC Ⅱ cells were cultured in vitro and divided into hyperoxia group and normoxia group.After 24 h, 48 h and 72 h of hyperoxia or air treatment, cells were collected and the best treatment time was selected at 48 h. AEC Ⅱ cells were divided into 3 groups: control group, negative control group (transfected with negative control) and PEA3 over expression group (transfected with PEA3 overexpression plasmid). Each group was further divided into hyperoxia subgroup and normoxia subgroup.Cells were harvested at 48 h after hyperoxia or normoxia treatment.Reactive oxygen species (ROS), Nod-like receptor domain 3 (NLRP3), monocyte chemoattractant protein-1 (MCP-1), interleukin(IL)-1β, IL-6, IL-8, IL-18, surfactant protein C (SP-C), aquaporins 5 (AQP5), PEA3 and manganese superoxide dismutase (MnSOD) levels were detected.Differences were compared by the t-test and repeated measures analysis of variance using SPSS 20.0 statistical software. Results:The interaction of grouping and treatment duration had significant effects on ROS, IL-1β, IL-6, IL-8, IL-18, SP-C and AQP5 levels in AEC Ⅱ cells ( F=19.857, 20.132, 23.133, 18.673, 28.341, 27.333 and 34.217, respectively, all P<0.05). At 24 h, 48 h and 72 h, ROS level in hyperoxia group was 1.78, 1.94 and 2.26 times higher than that in normoxia group ( t=18.649, 17.486 and 19.385, respectively all P<0.05). NLRP3 and MCP-1 levels were significantly upregulated in hyperoxia group.IL-1β level was 1.33, 1.69, and 1.65 times higher in hypoxia group at 24 h, 48 h and 72 h than that of normoxia group; IL-6 level was 1.26, 1.56 and 2.12 timers higher; IL-8 level was 1.13, 1.47 and 2.34 times higher; and IL-18 level was 1.46, 1.72 and 1.95 times higher, respectively (all P<0.05). The protein expression of SP-C was downregulated, while that of AQP5 was significantly upregulated in hypoxia group.The RNA expression of SP-C was 22%, 63% and 72% lower in hypoxia group than that in normoxia group at 24 h, 48 h and 72 h ( t=3.982, 16.328 and 20.259, P<0.05, respectively), and that of AQP5 was 1.92, 5.23 and 7.36 times higher ( t=14.631, 18.945 and 19.521, respectively, all P<0.05). There were significant differences in ROS, IL-1β, IL-6, IL-8, IL-18, SP-C and AQP5 levels at 24 h, 48 h and 72 h in hyperoxia group ( F=22.343, 20.566, 23.701, 19.222, 32.146, 40.278 and 37.107, respectively, all P<0.05). After 48 h of PEA3 overexpression, compared with the hyperoxic negative control group, ROS level in hyperoxic AEC Ⅱ cells overexpressing PEA3 decreased by 34% ( t=14.635, P<0.05). NLRP3 and MCP-1 were downregulated in hyperoxic AEC Ⅱ cells after overexpression of PEA3.IL-1β, IL-6, IL-8 and IL-18 levels decreased by 29%, 22%, 27% and 18%, respectively ( t=15.895, 17.872, 18.749 and 15.274, all P=0.000). SP-C was upre-gulated and AQP5 was downregulated by overexpression of PEA3 in hyperoxic AEC Ⅱ cells.In addition, PEA3 and MnSOD levels were significantly enhanced. Conclusions:Overexpression of PEA3 can alleviate the increase of ROS level in AEC Ⅱ cells, block the activation of various inflammatory pathways and reduce the transformation from AEC Ⅱ to AEC Ⅰ cells via enhancing MnSOD level.
RESUMO
Objective To investigate the effects of diammonium glycyrrhizinate (DG) on Toll-like receptor 4 in rat alveolar epithelial cells induced by paraquat (PQ).Methods Rats in the PR and PDR groups were induced 2 h by RU486 (100 nmol/L).Then rats in the DG and PDR groups were induced 2 h by DG (0.6 mg/mL).Finally,PQ (0.6 mg/mL) were administered and induced 24h in the PQ,DG,PR and PDR groups,while,the NE group was induced 24 h by absolute ethyl alcohol (0.33 μ mol/L),and the NS group was induced 24 h without drugs.MTT assay was used to measure the cell growth and inhibitioneffects of PQ and DG on cells.The ELISA assay was applied to measure the levels of the TLR-4,Myd88,NF-κB P65 and GR.The gene expressions ofTLR-4,Myd88,NF-κB P65 and GR were detected by RT-PCR.Results The survival rate of rat alveolar epithelial cells was decreased by PQ (200,400,600,800,1 000,1 500,2 000 μmol/L),and the IC50 value for 24 h was 927.045 μmol/L.The inhibition rates were (11.74±1.44)％,(18.76±1.30)％,(28.74±0.54)％,(40.30±0.55)％,(51.24±0.76)％,(68.19±1.10)％,(83.16±0.59)％ in the 200,400,600,800,1 000,1 500,2 000 pmol/L PQ treatment groups,respectively.And the inhibition rates were (48.01±1.37)％,(40.68±2.33)％,(32.76±4.11)％,(34.12±4.3)％,(39.22±2.23)％,(51.26±-0.39)％ in the 0.2,0.4,0.6,0.8,1,and 2 mg/mL DG treatment groups,respectively.The levels of TLR4,Myd88,NF-κB P65,and TNF-a in the PQ and PR groups were higher than those in the NS group (all P＜0.01).While,the levels of GR in the PQ and PR groups were lower than that in the NS group (all P＜0.01).And,the levels of TLR4,Myd88,NF-rκB P65,and TNF-α in the DG and PDR groups were lower than those in the PQ group (all P＜0.01).But the levels of GR in the DG and PDR groups were higher than that in the PQ group (all P＜0.01).Conclusions Diammonium Glycyrrhizinate can attenuate the injury of rat alveolar epithelial cells induced by paraquat,can decrease the levels of TLR-4,Myd88,NF-KB and TNF-α,and increase the GR level.