| Objective:Multi-walled carbon nanotubes(MWCNTs)exhibit unique electrical,mechanical,thermal and optic properties,which make them the first choice for a variety of industrial and consumer product applications.MWCNTs have numerous beneficial applications in electrical engineering,computer science,biomedicine and aerospace industry.Although there is no epidemiologic study to support the conclusion that MWCNTs induce pulmonary fibrosis in humans,in vivo studies demonstrate that exposure to MWCNTs via respiratory pathways results in pulmonary fibrosis.Therefore,MWCNTs could elicit a potential risk of pulmonary fibrosis in humans due to occupational or consumer exposure.Sirtuin 6(SIRT6),a nicotinamide adenine dinucleotide(NAD~+)-dependent deacetylase,belongs to the sirtuin family.SIRT6 has been proved to prevent fibrosis in liver,renal and myocardial tissues.However,to the best of our knowledge,there was no report on the role of SIRT6 in MWCNTs-induced pulmonary fibrosis.Epithelial-mesenchymal transition(EMT)is known to be one of the key processes that promote the development of pulmonary fibrosis.In the present study,we aimed to explore the role of SIRT6 in MWCNTs-induced EMT and MWCNTs-activated TGF-?/Smads signaling pathway in human bronchial epithelial BEAS-2B cells,and provided a scientific basis for finding effective therapeutic targets for MWCNTs-induced pulmonary fibrosis.Methods:The size and morphology of MWCNTs were measured by transmission electron microscopy(TEM)and scanning electron microscopy(SEM).The specific surface area(SSA)of MWCNTs was measured by Brunauer-Emmett-Teller(BET)method.Thermal gravimetric analysis(TGA)was used to analyze the purity of MWCNTs.The air-dried tube sheets were tested by X-ray diffraction(XRD)to identify the elemental composition on the surface of MWCNTs.The dispersion status and stability of MWCNTs dispersed in culture medium was evaluated by dynamic light scattering(DLS).TEM was used to examine uptake of MWCNTs by BEAS-2B cells.CCK-8 assay was performed to determine the doses of MWCNTs(0.25,1,4 and 16?g/cm~2)used in the study.BEAS-2B cells were treated with MWCNTs at the concentrations of 0.25,1 and 4?g/cm~2 for 72 h,and Western blot analysis of endogenous SIRT6,the epithelial marker E-cadherin,and the mesenchymal markers vimentin and?-SMA was performed.BEAS-2B cells were infected with an adenoviral vector encoding SIRT6 or SIRT6 shRNA for 24 h,and subsequently treated with 4?g/cm~2 of MWCNTs for 72 h.The protein levels of E-cadherin,vimentin,?-SMA and SIRT6 were measured by Western blot.BEAS-2B cells were infected with an adenoviral vector encoding SIRT6 for 24 h,and subsequently treated with 4?g/cm~2 of MWCNTs for 72 h.The mRNA levels of MMP-2,MMP-9,COL3A1 and TGF-?1 were measured by real-time PCR.The protein levels of MMP-2and MMP-9 were measured by Western blot.Transwell assay was used to detect changes in cell migration abilities.BEAS-2B cells were infected with wild-type SIRT6 or the H133Y mutant without histone deacetylase activity for 24 h,and subsequently treated with 4?g/cm~2 of MWCNTs for 1 h.Phosphorylation of Smad2 and Smad3 were measured by Western blot.Results:1.Characterization and dispersion state of MWCNTs:The outer diameter of MWCNTs was 8-15 nm,the inner diameter was 3-5 nm,and the length was 10-50?m.The surface area was estimated to be 140.77 m~2/g.The purity of MWCNTs was>98%.Fe and Ni were two major metal contaminants with contents of 0.2%and 0.5%,respectively.Agglomerates had a highest average hydrodynamic diameter of 356 nm at72 h.Meanwhile,the polydispersion index(PDI)was 0.45 at 72 h.No significant change in hydrodynamic diameter and PDI of MWCNTs was observed from 0 to 72 h.The results suggested good stability of MWCNTs dispersed in culture medium for at least 72 h.2.Cellular uptake of MWCNTs:BEAS-2B cells were treated with 4?g/cm~2 of MWCNTs for 72 h,and photographed by TEM.MWCNTs could be internalized by BEAS-2B cells,and detected mostly as agglomerates inside cellular vesicles in the cytoplasm.3.MWCNTs induced EMT in BEAS-2B cells:BEAS-2B cells were treated with MWCNTs at the concentration of 0.25,1 and 4?g/cm~2 for 72 h.Significant loss of the epithelial marker E-cadherin after treatment with 1 and 4?g/cm~2 of MWCNTs(P<0.01),and upregulation of the mesenchymal markers vimentin and?-SMA in response to MWCNTs at 0.25,1 and 4?g/cm~2 were observed in a dose-dependent manner(P<0.05or P<0.01).4.Expression of SIRT6 in MWCNTs-treated BEAS-2B cells:Compared with that of control,the protein levels of SIRT6 were elevated as doses of MWCNTs increased,and achieved the highest level after treatment with 4?g/cm~2 of MWCNTs at 72 h(P<0.05).5.Effect of SIRT6 on MWCNTs-induced EMT:SIRT6 overexpression restored MWCNTs-induced downregulation of the epithelial marker E-cadherin and upregulation of the mesenchymal markers vimentin and?-SMA(P<0.05 or P<0.01).SIRT6 knock-down failed to influence EMT markers in MWCNTs-treated cells.6.Effects of SIRT6 on MWCNTs-induced EMT-like cell behaviors:The mRNA levels of MMP-2,MMP-9,TGF-?1 and COL3A1 and the protein levels of MMP-2 and MMP-9 were significantly reversed by overexpression of SIRT6 in response to MWCNTs(P<0.05 or P<0.01).What's more,overexpression of SIRT6 almost completely reversed cell migration induced by MWCNTs(P<0.01).7.Effect of SIRT6 on MWCNTs-activated TGF-?1/Smad2 signal pathway:Phosphorylation of Smad2 and Smad3 protein levels were all significantly upregulated after treatment with 4?g/cm~2 of MWCNTs for 1 h(P<0.01).Overexpression of SIRT6significantly reduced phosphorylation of Smad2(P<0.01),but not Smad3 in BEAS-2B cells treated with MWCNTs.The SIRT6(H133Y)mutant without histone deacetylase activity could not inhibit phosphorylation of Smad2 induced by MWCNTs.Conclusions:The protein level of SIRT6 was increased after treatment with MWCNTs for 72 h in BEAS-2B cells.Overexpression of SIRT6 prevented MWCNTs-induced EMT by inhibition of TGF-?1/Smad2 signaling pathway in BEAS-2B cells,which depended on its deacetylase activity. |
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