| ?Objective?To explore the effect of autophagy in Toluene diisocyanate(TDI)-induced inflammatory response and Airway remodeling in human airway epithelial cells,so as to provide new ideas for the pathogenesis of TDI asthma,and provide direction for further prevention and treatment of occupational asthma.?Methods?The TDI-HSA conjugate was synthesized and the concentration of TDI in the TDIhuman serum albumin(HSA)conjugate was measured by the Gutmann method,and the HSA content in the TDI-HSA conjugate was determined by the BCA method.Human bronchial epithelial(16HBE)cells were stimulated with different concentrations of TDI-HSA and HSA,and the highest concentration was determined by cell viability assay(CCK-8).Flow cytometry and ELISA were used to detect the expression level of ROS and the level of inflammatory reaction in 16 HBE cells at different concentrations.The autophagy activation of 16 HBE cells exposed to different concentrations of TDI-HSA was detected by transmission electron microscopy and Western blot.After determining the optimal concentration,the effects of autophagy inhibitor 3-methyladenine(3-MA)and autophagy agonist rapamycin(Rapa)on inflammatory response and airway remodeling in 16 HBE cells were investigated.The experiment was divided into five groups,solvent control group,TDI-HSA group,Rapa group,TDI-HSA+Rapa group and TDI-HSA+3-MA group.The survival rate of each group was detected by CCK-8 method.Autophagy was detected by transmission electron microscopy and Western blot.The changes of autophagic flow were detected after transfection of 16 HBE cells with GFP-RFP-LC3 adenovirus.The inflammatory reaction of each group was detected by ELISA and immunofluorescence;The expression levels of airway remodeling related proteins in each group were detected by Western blot.?Results?1.Determination of TDI mass concentration.The mass concentration of TDI in the TDI-HSA synthesized in this study was 18.30?g/mL,the mass concentration of HSA was 1650?g/mL,and the molar ratio of TDI to HSA was 4.33.The TDI-HSA mass concentrations in this study are expressed as the mass concentration of HSA therein.The mass concentration of TDI in the 40?g/mL TDI-HSA group was 0.44?g/mL,the mass concentration of TDI in the 80?g/mL TDI-HSA group was 0.89?g/mL,and the mass of TDI in the 120?g/mL TDI-HSA group.Concentration 1.33?g/mL2.Cell survival rate experiment.The effect of different concentrations of TDI-HSA on the survival rate of 16 HBE cells was detected by CCK-8.The results showed that when the concentration was 120?g/mL or less,the cell viability of the different doses of TDI-HSA group or HSA group were compared,and the cell viability of the same dose of TDI-HSA group and HSA group were compared,the difference was not statistically significant(P>0.05).The TDI-HSA group and the HSA group did not affect cell viability at doses below 120?g/mL.3.Effect of TDI-HSA on the expression of ROS,IL-4 and IL-6 in 16 HBE cells.After treatment of 16 HBE cells with different concentrations of TDI-HSA(40,80,120?g/mL)for 24 h,the expression levels of ROS,IL-4 and IL-6 were significantly higher than those of the same concentration HSA group.The expression levels of ROS,IL-4 and IL-6 in 16 HBE cells increased significantly with the increase of TDI-HSA dose,the difference was statistically significant(P<0.05).4.Effect of TDI-HSA on the level of autophagy in 16 HBE cells.After treatment of 16 HBE cells with different concentrations of TDI-HSA(40,80,120?g/mL)for 24 h,the autophagy-related structures in the control group(0?g/mL)and the same concentration of HSA group 16 HBE were not observed by transmission electron microscopy.In the TDI-HSA group,the number of autophagic lysosomes in 16 HBE cells increased significantly,and the number of mitochondrial vacuoles increased.Western blot analysis showed that compared with the control group,the relative expression levels of Beclin1 protein and LC3?-II/I ratio in 16 HBE cells of different concentrations of TDI-HSA group were increased,and the expression of P62 protein was decreased,the expression levels of HMGB1 and RAGE protein were increased,and the differences were statistically significant(P<0.05).With the increase of TDI-HSA concentration,the expression levels of Beclin1 and HMGB1 were significantly increased,the ratio of LC3?-II/I was significantly decreased,and the expression level of P62 protein was significantly decreased,the difference was statistically significant(P<0.05).5.Effect of different test substances on the cytotoxicity of 16 HBE cells.The CCK-8 test results showed that compared with the control group,the survival rate of TDI-HSA group,TDI-HSA+Rapa group,TDI-HSA+3-MA group and Rapa group remained above 90%,indicating different test substances had no obvious cytotoxicity to 16 HBE cells.6.GFP-RFP-LC3? transfection of 16 HBE cells to observe autophagy flux.After GFP-RFP-LC3? double-labeled adenovirus was transfected into 16 HBE cells,the yellow spots and red spots were quantified by TDI-HSA after fluorescence microscopy.The results showed that a small amount of autophagosomes and autolysosomes were detected in the control group;a large number of yellow and red spots appeared in the TDI-HSA treatment group;the number of autophagosomes and autolysosomes was significantly reduced,and autophagy was reduced after inhibition of autophagy with 3-MA;Rapa actively activated autophagy,which was the same as TDI-HSA-stimulated cells.7.Effect of different test substances on autophagy of 16 HBE cells.Western blot was used to detect the changes of autophagy-related proteins Beclin1 and LC3-II/I ratio to assess the occurrence of autophagy.Compared with the control group,the LC3-II/I ratio of TDI-HSA group was significantly increased,and the expression of beclin1 protein was significantly increased(P<0.05).Compared with TDI-HSA group,the ratio of LC3-II/I in TDI-HSA+RAPA group was significantly increased,and the expression of beclin1 protein was increased(P<0.05),indicating that autophagy activity was enhanced;TDI-HSA+3-MA group LC3-II/I ratio decreased significantly,beclin1 protein expression decreased(P<0.05),indicating decreased autophagy activity.8.Effect of autophagy on TDI-HSA-induced inflammatory response in 16 HBE cells.The results of ELISA showed that the expression levels of IL-5,IL-6 and IL-8 in 16 HBE cells of TDI-HSA group were significantly higher than those in the control group(P<0.05).Compared with TDI-HSA group,the expression levels of IL-5,IL-6 and IL-8 in 16 HBE cells of TDI-HSA+Rapa group were significantly increased(P<0.05).The expression levels of IL-5,IL-6 and IL-8 in 16 HBE cells of TDI-HSA+3-MA group were significantly decreased(P<0.05).The results of immunofluorescence assay showed that the number of caspase-1 and NLRP3 in 16 HBE cells in TDI-HSA group was significantly increased compared with the control group,the difference was statistically significant(P<0.05).Compared with the TDI-HSA group,the expression of caspases-1 and NLRP3 was significantly increased in the TDI-HSA+Rapa group,and significantly decreased in the TDI-HSA+3-MA group,the difference was statistically significant(P<0.05).9.The effect of autophagy on TDI-HSA-induced airway remodeling in 16 HBE cells.Western blot analysis showed that compared with the control group,the expression of E-cadherin protein was decreased in 16 HBE cells,and the expression of vimentin,?-SMA,MMP-9 and Mucin5 AC protein was significantly increased in the TDI-HSA group(P< 0.05).Compared with the TDI-HSA group,the E-cadherin protein of 16 HBE cells in the TDI-HSA+Rapa group was significantly decreased,and the expression of vimentin,?-SMA,MMP-9 and Mucin5 AC protein was significantly increased(P<0.05).The expression of E-cadherin protein in 16 HBE cells of TDI-HSA+3-MA group was significantly increased,and the expression of vimentin,?-SMA,MMP-9 and Mucin5 AC protein was significantly decreased(P<0.05).?Conclusions?1.TDI can increase the level of ROS and activate autophagy in human bronchial epithelial cells.2.TDI can induce inflammatory response,promote EMT process and MMP-9,Mucin5 AC protein expression in 16 HBE cell,autophagy inhibitor can reduce TDI-induced airway inflammatory response and airway remodeling related protein expression. |
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