The Mechanism Of LncRNA Gm16410 Regulating TGF-?1/smad2/3 Pathway In Lung EndMT Induced By PM_2.5 Exposure And The Protective Effect Of CEOs

Purpose:To investigate the molecular mechanism of lung EndMT production induced by PM_2.5 exposure and the protective effect of compound essential oil.To explain the role of lncRNA Gm16410 regulating TGF-?1/Smad2/3 in lung EndMT induced by PM_2.5 exposure.These findings provide new potential therapeutic strategies for PM_2.5-induced lung EndMT.Methods:Mouse model was established using Balb/c mice.Primary mouse pulmonary microvascular endothelial cells?MHC?and human umbilical vein endothelial cells?HUVEC?were exposed to PM_2.5 in vitro.?1?PM_2.5 sample collection and treatment:PM_2.5 was collected from October2015 to March 2016 in Langfang City,Hebei Province.After ultrasonic dissolution,70%ethanol extraction,rotary evaporation and lyophilization,the PM_2.5particles was prepared and placed in a refrigerator at-20°C for later use.A 10 mg/m L suspension with PM_2.5 particles and normal saline was ultrasonically shaken before use.?2?In vivo experiment:Balb/c mice were exposed to PM_2.5?300?g/m3?and inhaled 1%compound essential oil?normal saline configuration?.The pathological change of lung tissue was observed by HE staining.Masson staining and immunohistochemistry were used to observe the change of collagen and EndMT in mice.The expression of CD31,CDH5,Acta2 and COL1?1 in lung tissue was detected by q PCR.The expression of CD31,CD34,CDH5,Vimentin,Col1,Acta2 and S100A4in lung tissue was detected by Western blot.Microarray and q PCR analysis were used to detect differentially expressed lncRNA in mice lung tissue exposed to PM_2.5.?3?In vitro experiment:Two kinds of cells were exposed to PM_2.5for 0 h,6 h,12h,24 h,36 h and 48 h respectively.Two kinds of cells were exposed to compound essential oil?CEOs?for 6 h,12 h,24 h,36 h and 48 h respectively.MTT assay was used to analyze the change of cell viability.Cell scratch assay was applied to detect the change of cell migration ability,reflecting the influence of PM_2.5on cell functions.The expression of CD31,CDH5,Acta2 and COL1?1 was detected by q PCR under different action time in the PM_2.5group and the CEOs group.The expression of CD31,CDH5,Acta2,Col1 and S100A4 was detected by Western blot under different action time in the PM_2.5group and the CEOs group.The change of fluorescence intensity of CD31,Acta2 and S100A4 in two kinds of cells was detected by immunofluorescence.These experiments were used to explore the protective effect of CEOs in lung EndMT induced by PM_2.5 exposure combined with the vivo experiments.TGF-?pathway inhibitor SB431542 was used to investigate the role of TGF-?1/Smad2/3 pathway in EndMT induced by PM_2.5exposure.Microarray and q PCR were used to screen and detect the expression of lncRNA Gm16410 to explore the mechanism of lncRNA Gm16410 on EndMT.Cell scratch assay was applied to detect the change of cell migration ability after overexpression lncRNA Gm16410 in the PM_2.5group.Western blot was used to detect the change of relevant factors in TGF-?1/Smad2/3 pathway and EndMT after overexpression lncRNA Gm16410 to investigate the mechanism of lncRNA Gm16410 regulating TGF-?1/Smad2/3 pathway in lung EndMT induced by PM_2.5 exposure.Results:?1?In vivo experiment:HE staining showed that there was focal hyperemia,hemorrhage and inflammatory cell infiltration in lung tissue of the PM_2.5 group.After intervention of CEOs,pulmonary hemorrhage and inflammatory cells were decreased in mice.Masson staining showed that the change of collagen around the pulmonary vessels in the PM_2.5 group increased slightly.Compared with the PM_2.5 group,the change of collagen in the CEOs group had not quite different.Immunohistochemical results showed that the expression of Acta2 in the PM_2.5 group was significantly higher than that in the control group.Compared with the PM_2.5 group,the expression of Acta2in the CEOs group decreased remarkably.q PCR results showed that the expression of CDH5 and CD31 decreased in the PM_2.5group,and the expression of Acta2 and COL1?1 increased significantly.Compared with the PM_2.5 group,the expression of CDH5 and CD31 increased significantly and the expression of Acta2 and Col1decreased in the CEOs group.Western blot showed that the expression of CD31,CD34and CDH5 decreased slightly,the expression of Col1 and Acta2 increased in the PM_2.5group,but Vimentin had not quite different.In the CEOs group,the expression of CD31,CD34 and CDH5 was up-regulated significantly,the expression of Col1,Acta2and Vimentin decreased obviously.?2?In vitro experiments:PM_2.5 exposure began to increase cell viability after 24 h by MTT assay.Moreover,cell migration began to increase after 36 h,indicating that functional changes were associated with the changes in the endothelial and mesenchymal markers.The cell viability and cell migration decreased slightly in the CEOs group.q PCR results showed that the expression of Acta2 and COL1?1 increased significantly with the prolongation of action time,the expression of CDH5 and CD31decreased significantly.At the protein level,the expression of CDH5 and CD31decreased gradually,while the expression of Col1,Vimentin,Acta2 and S100A4increased gradually.CEOs had a beneficial intervention effect on EndMT induced by PM_2.5 exposure in vitro,especially at the protein level.Immunofluorescence showed that the change of fluorescence intensity was consistent with the above experiments.In conclusion,CEOs could alleviate the lung EndMT induced by PM_2.5 exposure in vivo and vitro.Western blot showed that the expression of TGF-?1,TGF-?R1 and p-Smad2/3increased in MHC exposed by PM_2.5 after 48 h,but the expression of TGF-?1,TGF-?R1and p-Smad2/3 decreased significantly after treatment with TGF-?pathway inhibitor SB431542 in MHC exposed by PM_2.5,and the expression of EndMT related factors was reversed.Immunofluorescence showed that the change of fluorescence intensity was consistent with the above experiments.The results showed that PM_2.5 exposure would induce EndMT in lung tissue by activating TGF-?1/Smad2/3 pathway.q PCR results showed that the expression of lncRNA Gm16410 decreased gradually in MHC by PM_2.5exposure,which was consistent with the result of microarray in vivo.Cell scratch showed that lncRNA Gm16410 could alleviate the cell migration in MHC exposed by PM_2.5 exposure after 48 h.Western blot showed that the expression of TGF-?1,TGF-?R1 and p-Smad2/3 decreased significantly,and the expression of EndMT related factors were reversed obviously with lncRNA Gm16410 in MHC by PM_2.5exposure after 48 h.PM_2.5 exposure could activate TGF-?1/Smad2/3 pathway by down regulating the expression of lncRNA Gm16410 to induce EndMT in the lung.Conclusion:?1?CEOs could reduce lung EndMT caused by PM_2.5 exposure.?2?PM_2.5 exposure would activate TGF-?1/Smad2/3 pathway by down regulating the level of lncRNA Gm16410 to induce lung EndMT.