| Objectives To explore the relationship between s RAGE and cytokines in the human serum samples,and preliminarily to determine the possible role of s RAGE in the process of silicosis.After TGF-?1-induced EMT cell models were established,the key ligands in combination with RAGE involved in regulating silicosis fibrosis were identified and the possible mechanisms of RAGE-mediated silicosis were discussed.Methods 59 cases of silicosis patients(mainly engaged in pioneering,crushing,wind drilling,excavation,stone processing,etc.)have been selected from the in Beidaihe Sanatorium of Chinese coal workers.And 18 cases in the first stage,15 cases in the second stage and 21 cases in the third stage were included in the study groups.In addition,12 healthy adult males were selected as the control group.The levels of s RAGE,TNF-?,TGF-?1,IL-6 and IL-1? in serum were measured by enzyme-linked immunosorbent assay(ELISA)kit.The basic data of age,working age,blood pressure,body mass index(BMI),age of first exposure to dust and lung function were collected.Pearson product moment correlation analysis was used to analyze the correlation between s RAGE and TNF-?,TGF-?1,IL-6,IL-1?,TGF-?1 levels was analyzed by Pearson product moment correlation.Human alveolar type II epithelial cells(A549)and rat alveolar epithelial cells(RLE-6TN)were cultured routinely,and were divided into control group randomly,5ng/ml TGF-?1 1 stimulation group and 10ng/ml TGF-?1 1 stimulation group.After cells were stimulated by TGF-beta 1 for 48 or 72 hours the expression of EMT markers was detected by Western blot.Real-time quantitative PCR(q RT-PCR)was used to detect the m RNA expression level of EMT-related markers after 48 hours of TGF-?1 stimulation.After the EMT model was established successfully,the expression of RAGE was detected by Western blot and q RT-PCR,and the protein levels of RAGE ligand S100A4 and HMGB1 was detected.After determining the optimal concentration and time of TGF-?1 stimulation of RAGE with EMT,,the advanced glycation end product receptor inhibitor(FPS-ZM1)was given to RLE-6TN cell,meanwhile the cells were divided into the following six groups: control group,TGF-beta 1 stimulation group,TGF-beta 1+1ug/ml FPS-ZM1 group,TGF-beta 1+5ug/ml FPS-ZM1 group,TGF-beta 1+10ug/ml FPS-ZM1 group,10ug/ml FPS-ZM1 group and 10ug/ml FPS-ZM1 group.The expression of EMT-related markers and RAGE protein was detected by Western blot.Results 1 The levels of s RAGE in the control group was significantly lower than those in the first stage of silicosis and the second stage of silicosis(all p<0.05);the levels of TNF-? in the second stage of silicosis and the third stage of silicosis were distincly higher than those in the control group(all p<0.05);the levels of IL-6 in the second stage of silicosis and the third stage of silicosis were higher than the control group,and the levels of IL-6 in the second stage of silicosis were lower than those in the third stage of silicosis(all p< 0.05);The level of IL-1? was higher than that of the control group and the first stage of silicosis group(p<0.05).2 The results of correlation analysis showed that the levels of s RAGE were positively negatively correlated with TNF-?,IL-6 and IL-?1,and the correlation coefficients were-0.241?-0.288 and-0.413,respectively(all p<0.05).3 After A549 cells were induced by TGF-?1 for 48 h,EMT-related markers changed significantly.The protein and m RNA level of CDH1 in 5 ng/ml TGF-?1 and 10 ng/ml TGF-?1 groups were significantly lower than those in the control group,and the protein level of CDH2 was significantly higher than that in the control group(all p<0.05).The protein levels of RAGE,HMGB1 and S100A4 in 5 ng/ml TGF-?1 group and 10ng/ml TGF-?1 group weresignificantly higher than those in the control group(all p<0.05).The m RNA level of HMGB1 and S100A4 in 5ng/ml TGF-?1 group was higher than that in the control group(all p<0.05).4 After TGF-?1 induced RLE-6TN cells for 48 hours,the epithelial markers protein expression of ZO-1 and CDH1 m RNA in 5ng/ml TGF-?1 were significantly lower than those in the control group,the protein and m RNA levels of CDH2 and the m RNA expression of ?-SMA were markedly higher than that of the control group(all p<0.05).RAGE,HMGB1,S100A4 protein levels and m RNA expression were significantly higher than that of the control group.After TGF-?1 induced cells 72 hours,the ZO-1 protein levels in 10 ng/ml TGF-?1 group was significantly lower than that in the control group,and the protein levels of CDH2 were significantly higher than that in the control group(all p<0.05).5 FPS-ZM1 inhibited RAGE expression in RLE-6TN cells: The levels of ZO-1 in TGF-?1+1?g/ml FPS-ZM1 group,TGF-?1+5?g/ml FPS-ZM1 group,TGF-?1+10?g/ml FPS-ZM1 group were higher than that in the model group(all p<0.05),while the levels of Vimentin and CDH2 were lower than that in the model group(all p<0.05).The level of ?-catenin in the 5 ng/ml TGF-?1 group was increased with the increase of RAGE compared with the model group,and the ?-catenin in TGF-?1+1?g/ml FPS-ZM1,TGF-?1+10?g/ml FPS-ZM1 groups were decreased with the decrease of RAGE protein expression(all p<0.05).Conclusions 1 The expression of s RAGE was decreased in serum of silicosis patients.s RAGE was negatively correlated with the levels of TNF-?,IL-6 and IL-?1,and s RAGE may regulate the occurrence of silicosis fibrosis by affecting the levels of cytokines.2 In the cell model of alveolar epithelial-mesenchymal transition induced by TGF-?1,the activity of HMGB1/S100A4-RAGE pathway was enhanced.3 Inhibition of RAGE can alleviate the occurrence of EMT induced by TGF-?1.And RAGE could be involved in regulating the EMT process induced by TGF-beta 1 by affecting the beta-catenin signaling pathway.Figure10;Table15;Reference 122... |
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