AIM:To investigate the role of canonical transient receptor potential channel 1 ( TRPC1 ) in the migration of human bronchial epithelial cells (16HBE) induced by transforming growth factor-β1 ( TGF-β1).METH-ODS:Silencing of TRPC1 gene expression was performed by siRNA.The cell activity and apoptosis were measured by CCK-8 assay and flow cytometry, respectively.The migration and invasion abilities of the 16HBE cells were detected by wound-healing assay and Transwell assay.The protein expression of E-cadherin and vimentin was determined by Western blot.RESULTS:TGF-β1 treatment significantly enhanced the cell migration distance compared with control groups ( P<0.01 ) .The results of CCK-8 assay and flow cytometry indicated that there were no significant difference in proliferation and apoptosis among TRPC1 siRNA group, TGF-β1 group and control group (P>0.05).The results of wound-healing and Tr-answell assays showed that migration and invasion abilities in TRPC1 siRNA +TGF-β1 group were markedly suppressed compared with TGF-β1 group (P<0.01).The protein expression of E-cadherin and vimentin induced by TGF-β1 was in-hibited by TRPC1 silencing compared with TGF-β1 group (P<0.05).CONCLUSION:TRPC1 is involved in the migra-tion of human bronchial epithelial cells induced by TGF-β1 through regulating the protein expression of E-cadherin and vim-entin.

[ ABSTRACT] AIM:To investigate the role of canonical transient receptor potential channel 1 ( TRPC1 ) in the epithelial-mesenchymal transition ( EMT) of human bronchial epithelial ( HBE) cells induced by transforming growth fac-tor-β1 (TGF-β1).METHODS:EMT of 16HBE cells induced by TGF-β1 were identified by microscopy, immunofluores-cence and Western blotting.Immunofluorescence, real-time PCR and Western blotting were applied to detect the mRNA and the protein expression of TRPC1 in the 16HBE cells.The influence of SKF96365 (a TRPC1 blocker) and siRNA-me-diated silencing of TRPC1 on the EMT of the 16HBE cells were detected by microscopy and Western blotting.RESULTS:Treatment with TGF-β1 induced significant morphological changes of the 16HBE cells.Exposure to TGF-β1 decreased the expression of E-cadherin protein (P<0.01) and increased the expression of α-SMA protein (P<0.05) in the 16HBE cells.Immunofluorescence observation indicated that TRPC1 expression in the 16HBE cells was positive.The expression of TRPC1 at mRNA and protein levels was significantly increased in the 16HBE cells after stimulation with TGF-β1 ( P<0.05).The morphological changes of the 16HBE cells induced by TGF-β1 were inhibited by SKF96365 and TRPC1 silen-cing compared with TGF-β1 group.The protein expression of E-cadherin andα-SMA induced by TGF-β1 were inhibited by SKF96365 and TRPC1 silencing compared with TGF-β1 group (P<0.05).CONCLUSION:TGF-β1 induces EMT with the mechanism of up-regulating TRPC1 in human bronchial epithelial cells.

AIM:To investigate the role of caveolin-1 on epithelial-mesenchymal transition (EMT) in human bronchial epithelial (HBE) cells induced by transforming growth factor beta 1 (TGF-β1).METHODS:Immunofluorescence, real-time PCR and Western blot were applied to detect the mRNA and the protein expression of caveolin-1 in the 16HBE cells during EMT.The influence of siRNA-mediated silencing of caveolin-1 on EMT in the 16HBE cells was detected by Western blot.RESULTS:Caveolin-1 was widely present on the cell membrane of the 16HBE cells.The expression of caveolin-1 at mRNA and protein levels was significantly decreased in a time-dependent manner in the 16HBE cells compared with control group (P<0.05) after stimulation with TGF-β1.The morphologic changes of the 16HBE cells induced by TGF-β1 were promoted by caveolin-1 silencing compared with TGF-β1 group.The protein expression of E-cadherin and α-SMA induced by TGF-β1 was promoted by caveolin-1 silencing compared with TGF-β1 group (P<0.05).The phosphorylation levels of AKT and Smad3 were the highest at 30 min and increased significantly compared with control group (P<0.05) after stimulated with TGF-β1.Treatment of the 16HBE cells with TGF-β1 for 30 min after silencing caveolin-1 gene for 24 h significantly increased the phosphorylation levels of AKT and Smad3 compared with TGF-β1 group (P<0.05).CONCLUSION:TGF-β1 down-regulates the expression of caveolin-1 in the 16HBE cells.Caveolin-1 may participate in TGF-β1/Smad pathway and PI3K-AKT pathway, which are the signal transduction pathways for TGF-β1 inducing EMT.

OBJECTIVETo establish a detection method based on gas chromatography-mass spectrometry (GC-MS) for concentrations of volatile nitrosamine compounds in urine, and apply it to the test of real samples.

METHODSTarget compounds dichloromethane in urine samples was extracted with dichloromethane through liquid-liquid extraction, then the dichloromethane extract was filtrated, evaporated with nitrogen at 40°C to dryness, and the volume was set with 0.2 ml dichloromethane. Analysis of nine volatile nitroso-compounds were performed with GC-MS under selected ion monitoring mode, external reference method was used for quantification, and the detection limit, repeatability and sensitivity were evaluated. In addition, nine volatile nitroso-compounds of 92 urine samples in a town of Anhui province were measured.

RESULTSA good linear range of 2 - 200 ng/ml (with correlation coefficient 0.9985 - 0.9999) were obtained for the above mentioned nine kinds of analyte, and the lowest examination concentration was 0.05 - 0.50 ng/ml. The addition standard recoveries were 68%-102% with the RSD of 0.4% - 5.5% (n = 3). The detection limits were 0.001 - 0.013 ng/ml urine. The detection rate of N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitrosodiethylamine (NDEA), N-nitrosodi-n-propylamine (NDPA), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR), N-nitrosopiperidine (NPIP), N-nitrosodi-n-butylamine (NDBA) and N-nitrosodiphenylamine (NDPhA) were 71% (65), 74% (68), 65% (60), 80% (73), 92% (85), 78% (72), 76% (70), 87% (80), 98% (90), respectively, with the results (0.27 ± 0.12), (0.75 ± 0.29), (0.06 ± 0.02), (0.16 ± 0.07), (23.66 ± 5.18), (1.01 ± 0.35), (0.38 ± 0.11), (2.47 ± 0.52) and (15.13 ± 3.48) nmol/g creatinine.

CONCLUSIONSA gas chromatography-mass spectrometry detect method was developed for low level volatile nitrosamines in urine samples.

Gas Chromatography-Mass Spectrometry ; Humans ; Nitrosamines ; urine ; Urinalysis ; methods ; Volatile Organic Compounds ; urine