| Objective: This study aims to establish pulmonary fibrosis model in rats and collagen deposition model in human lung adenocarcinoma epithelial cells(A549 cells)induced by nickel oxide nanoparticles(NiO NPs).Then we further explore the roles of long noncoding RNA(lnc RNA)MEG3,transforming growth factor-β1(TGF-β1),phosphatidylinositol-3-kinase(PI3K)/AKT signaling pathway and epithelial mesenchymal transition(EMT)in NiO NPs-induced pulmonary fibrosis.Methods: 1.In vivo experiment: 32 healthy adult male Wistar rats were randomly divided into four groups.The rats in each group were respectively exposed to 0,0.015,0.06 and 0.24 mg/kg NiO NPs by intratracheal instillation,twice a week for 9 weeks.After the last exposure for 24 hours,the lungs of rats were collected for the follow-up experiment.2.In vitro experiment:(1)A549 cells were treated with different concentrations of NiO NPs suspension(0,25,50 and 100 μg/m L)for 24 h.(2)A549 cells were pretreated with 10 μM TGF-βRI inhibitor SB431542 or 10 μM PI3 K inhibitor LY294002 for 1 h,and then treated with 100 μg/m L NiO NPs suspension for 24 h.(3)Normal A549 cells and MEG3 overexpressed A549 cells(LV-MEG3 A549 cells)were treated with 100 μg/m L NiO NPs for 24 h.Following the different treatments,the cells were harvested for the subsequent experiments.3.Index detection:(1)Hematoxylin-eosin(HE)staining was used to observe the pathological changes of rat lung tissues,and picro-sirius red staining was performed to assess collagen deposition of rat lung tissues.The content of hydroxyproline in lung tissue was measured by biochemical method.(2)The relative expression level of lncRNA MEG3 was detected by reverse transcription-quantitative polymerase chain reaction(RT-q PCR).(3)Western blot was employed to detect the protein levels of collagen type I alpha 1 chain(Col-I),TGF-β1,Vimentin,α-smooth muscle actin(α-SMA),Fibronectin,E-cadherin,the total protein levels of PI3 K,AKT,mechanistic target of rapamycin kinase(m TOR)and their phosphorylated levels(p-PI3 K,p-AKT and p-m TOR).(4)The protein contents and subcellular location of Col-I,Vimentin,α-SMA,Fibronectin and E-cadherin were detected by immunofluorescence staining.Results: 1.In vivo experiments:(1)HE staining showed that the alveolar septum was wider,and the alveolar wall was thicker in 0.06 and 0.24 mg/kg NiO NPs groups than those in control group.Picro-sirius red staining showed that NiO NPs could cause excessive collagen deposition in rat lung tissues.The contents of hydroxyproline and Col-I in rat lung tissues were significantly increased in 0.06 and0.24 mg/kg NiO NPs groups(P < 0.05).(2)Compared with control group,the protein levels of TGF-β1,p-PI3 K,p-AKT,p-m TOR,Vimentin,α-SMA and Fibronectin in rat lung tissues were increased(P < 0.05),while the protein level of E-cadherin was decreased in 0.06 and 0.24 mg/kg NiO NPs groups(P < 0.05).(3)Compared with control group,the relative expression level of lncRNA MEG3 was down-regulated with the increased dosage of NiO NPs in rat lung tissues(P < 0.05).2.In vitro experiment:(1)Compared with control group,the protein levels of TGF-β1,p-PI3 K,p-AKT,p-m TOR,Vimentin,α-SMA and Fibronectin in A549 cells were up-regulated(P < 0.05),while the protein level of E-cadherin was decreased in 100 μg/m L NiO NPs group(P < 0.05).(2)Compared with control group,the relative expression level of lncRNA MEG3 was decreased in A549 cells treated with 50 and 100 μg/m L NiO NPs(P < 0.05).(3)Compared with 100 μg/m L NiO NPs group,the protein levels of Col-I,p-PI3 K,p-AKT,p-m TOR,Vimentin,α-SMA and Fibronectin in A549 cells were reduced(P < 0.05),while the protein expression levels of E-cadherin was elevated in 100 μg/m L NiO NPs + SB431542 group(P < 0.05).Immunofluorescence staining showed that NiO NPs could cause E-cadherin to be transported from membrane protein to cytoplasm,while Vimentin was released from nucleus to cell surface,which could be partially inhibited by SB431542.(4)Compared with 100μg/m L NiO NPs group,the protein levels of Col-I,p-PI3 K,p-AKT,p-m TOR,Vimentin,α-SMA and Fibronectin were decreased in A549 cells(P < 0.05),while the protein level of E-cadherin was increased in NiO NPs + LY294002 group(P < 0.05).(5)Compared with normal A549 cells treated with 100 μg/m L NiO NPs,the protein levels of TGF-β1,Col-I,p-PI3 K,p-AKT,p-m TOR,Vimentin,α-SMA and Fibronectin were down-regulated(P < 0.05),while the protein level of E-cadherin was up-regulated in LV-MEG3 A549 cells treated with 100 μg/m L NiO NPs(P < 0.05).Conclusion: NiO NPs can induce pulmonary fibrosis in rats,and its mechanism is related to the down-regulated lncRNA MEG3 induced by NiO NPs,which lead to the over-expression of TGF-β1 protein,activation of PI3 K / AKT signaling pathway and EMT occurrance. |
