The Protective Effect And Mechanism Of Exogenous Hydrogen Sulfide Donor On Formaldehyde-induced Pulmonary Epithelial Mesenchymal Transition In Vitro

Background Formaldehyde（FA）is a common environmental pollutant that can cause a series of health damages such as respiratory system damage.Epithelial-mesenchymal transition（EMT）plays a key role in lung development and the occurrence and development of various lung diseases.However,it is unknow whether formaldehyde induce the EMT process of pulmonary.Hydrogen Sulfide（H2S）,one of the recognized gas signal molecules,is widely involved in a range of cell functions and the physiological and pathological processes of various diseases,as well as plays an important therapeutic role in a variety of lung diseases.Our previous study showed that hydrogen sulfide is involved in the regulation of nickel chloride-induced pulmonary epithelial-mesenchymal transition.However,the role and mechanism of hydrogen sulfide in formaldehyde-induced pulmonary EMT remains unclear.Objective In the study,we investigate the role of exogenous hydrogen sulfide donor Na HS in formaldehyde-induced EMT in pulmonary through in vitro experiments by using the lung bronchial epithelial cell line（BEAS-2B）as a model,which is of great significance for elucidate the mechanism of formaldehyde-induced lung injury,and will also provide new ideas for the treatment of formaldehyde-induced lung injury.Methods BEAS-2B cells in good growth state,were used for the following experiments:1.To verify whether FA induces EMT by activating the TGF-β/Smads and MAPKs signaling pathway,BEAS-2B cells were treated with different concentrations of FA（0,25,50,75,100μM-24h）for different times（0,6,12,24,36h-100μM）,or pretreated cells with TGF-β/Smads and MAPKs signaling pathway inhibitors and then short-term exposure to formaldehyde（100μM,24h）;the cell morphology was observed by phase contrast microscope following by photographed and recorded.Western Blot assay detected the expression levels of epithelial and mesenchymal marker proteins and TGF-β/Smads and MAPKs signaling pathway proteins in BEAS-2B cells after treatment with formaldehyde.2.In order to investigate the effect and mechanism of hydrogen sulfide on FA-induced lung EMT,BEAS-2B cells were treated with FA at different concentrations（0,25,50,75,100μM-24h）and different times（0,6,12,24,36h-100μM）,and the H₂S content in the cells was detected by chemical fluorescence assay.Western Blot was used to detect the protein expression levels of CBS,CTH and 3-MST.To investigate whether hydrogen sulfide protects FA-induced lung EMT through regulating TGF-β/Smads and MAPKs signaling pathway,BEAS-2B cells were pretreated with sodium sulfide and treated with formaldehyde for a short period（100μM,24h）.Morphological changes of BEAS-2B cells were observed by phase contrast microscope.Western Blot was used to detect the expression levels of epithelial and interstitial marker proteins and TGF-β/Smads and MAPKs signaling pathway proteins.3.To investigate whether hydrogen sulfide protects FA-induced lung EMT by inhibiting TGF-β/Smads and MAPKs signaling pathway by regulating NRF2-ROS levels,BEAS-2B cells were pretreated with Na HS or NAC and then treated with FA（100μM,24h）.The level of total ROS in cells was detected by chemical fluorescence assay.Western Blot was used to detect the expression of NRF2 total protein,cytoplasmic and nuclear protein,as well as TGF-β/MAPKs signaling pathway marker protein in cells.Results 1.FA exposure resulted in the cell morphology changes of lung bronchial epithelial cells（BEAS-2B）（the cells changed from paving stones to fusiform,and the cell space became larger）.The expression levels of epithelial markers E-cadherin were declined,while the expression levels of interstitial markers N-cadherin,vimentin,ɑ-SMA and transcription factor zeb-1 were increased,presenting an obvious time-dose-effect relationship,and the difference was statistically significant compared with the control group（P<0.05）.2.FA exposure induces lung EMT by activating TGF-β/Smads and MAPKs signaling pathway:BEAS-2B cells were treated with FA at different concentration（0,25,50,75,100μM-24h）and different times（0,6,12,24,36h-100μM）.After formaldehyde exposure,the protein expression levels of TGF-β,P-SMad2,P-Smad3,P-ERK,P-JNK and P-P38 in BEAS-2B cells increased in a time and dose dependent manner.BEAS-2B cells were pretreated with TGF-β/Smads and MAPKs signaling pathway inhibitor before treated with short-term formaldehyde exposure（100μM,24h）.Compared with FA exposure group,TGF-β/p-ERK/p-JNK/p-p38 protein expression levels in TGF-β/Smads and MAPKs inhibitor+FA group were decreased,while the epithelial marker E-cadherin protein expression was increased and interstitial marker N-cadherin and Vimentin protein expression was declined.The difference was statistically significant（P<0.05）.3.FA reduced the level of H₂S and down-regulated the protein levels of hydrogen sulfide synthase（CBS,CTH,3-MST）in BEAS-2B cells:After BEAS-2B cells were treated with formaldehyde at different concentration（0,25,50,75,100μM-24h）and different times（0,6,12,24,36h-100μM）,the H₂S level decreased,and the protein expression levels of CBS,CTH,and 3-mst were down-regulated,which showed obvious time-dose-effect relationship.The difference was statistically significant（P<0.05）.4.Na HS can restore the reduction of H₂S and lung EMT induced by FA:Compared with FA exposure group,Na HS+FA group increased the level of H₂S and restored the morphology of lung bronchial epithelial cells（BEAS-2B）,as well as up-regulated the expression of E-cadherin protein while down-regulated the expression levels of N-cadherin,vimentin,ɑ-SMA and zeb-1 protein.The difference was statistically significant（P<0.05）.5.Na HS can inhibit FA-activated TGF-β/Smads and MAPKs signaling pathway:The expression levels of TGF-β,p-smad2,p-smad3,p-ERK,p-JNK and p-p38 protein in TGF-β/Smads and MAPKs inhibitor+FA group were down-regulated and the difference was statistically significant compared with the FA group（P<0.05）.6.Na HS can restore FA-induced down-regulation the expression levels of NRF2total protein,cytoplasmic and nuclear protein in cells:BEAS-2B cells were treated with FA at different concentration（0,25,50,75,100μM-24h）and different times（0,6,12,24,36h-100μM）,and the levels of total protein,cytoplasmic protein and nuclear protein of NRF2 in BEAS-2B cells decreased in a time and dose dependent manner.These results suggest that FA exposure can down-regulated the expression levels of NRF2 protein in BEAS-2B cells.Cells exposed to FA（100μM,24h）were pretreated with Na HS（100μM）,and the expression levels of total protein,cytoplasmic and nuclear protein of NRF2 in BEAS-2B cells were up-regulated in FA+Na HS group,compared with FA group.The differences were statistically significant（P<0.05）.7.Na HS can restore the increased of total ROS induced by FA:BEAS-2B cells were treated with FA at different concentration（0,25,50,75,100μM-24h）and different times（0,6,12,24,36h-100μM）,the level of total ROS in BEAS-2B cells increased in a time and dose dependent manner.BEAS-2B cells exposed to FA（100μM,24h）after pretreated with Na HS（100μM,1h）,and the level of total ROS in cells decreased in FA+Na HS group compared with FA group.The differences were statistically significant（P<0.05）.8.NAC can inhibit FA-induced lung EMT by inhibiting TGF-β/MAPKs signaling pathway:Compared with FA exposure group,the morphology of BEAS-2B cells in NAC+FA group was recovered,and the expression levels of E-cadherin protein were up-regulated,while the expression levels of N-cadherin and vimentin protein were declined.The protein expression levels of TGF-β,p-Smad2,p-Smad3,p-ERK,p-JNK and p-P38 were declined.The differences were statistically significant（P<0.05）.Conclusion1.Formaldehyde induces lung EMT by activating TGF-β/Smads and MAPKs pathway.2.Formaldehyde reduces the level of endogenous H₂S and inhibits the protein expression level of hydrogen sulfide synthase.Exogenous hydrogen sulfide donor Na HS inhibits FA-activated TGF-β/Smads and MAPKs signaling pathway by supplementing intracellular H₂S,which in turn reverses the epithelial-mesenchymal transition.3.Na HS may inhibit the production of ROS by up-regulating NRF2 protein expression,and then inhibit TGF-β/Smads and MAPKs signaling pathway,thereby reversing FA-induced lung epithelial-mesenchymal transform.