Role Of Pulmonary Fibrosis Related Factors In The Development Of Silicosis

Objective To detect the toxicity of formaldehyde on human bronchial epithelial cells, 16 HBE cells were treated with different concentrations of formaldehyde. The influence of formaldehyde on asthma associated Th1 / Th2 / Th17 type cytokines TNF-α expression was tested as well as NF-κB, COX-2, JNK and ERK expression in order to explore formaldehyde-induced asthma mechanisms and determine the biomarker for early diagnosis of occupational asthma.Methods 1. Formaldehyde exposure 16 HBE human bronchial epithelial cell proliferation activity test Take the number of cells grown on 96-well plates were seeded in the period, culture to confluent cell monolayer bottom of the hole at 37 °, 5% CO2 conditions, divided into six groups according to the experimental requirements 24 hours exposed to formaldehyde(formaldehyde exposure concentrations To 0.04,0.08,0.16,0.32,0.64 mmol / l, with serum-free medium with formaldehyde 1640 AR configuration), each concentration located six parallel holes, using MTT assay in human bronchial epithelial cell proliferative activity and detecthalf 16 HBE lethal dose LC50. 2. Studied cell supernatant Th1 type cytokine changes in epithelial cells after acute exposure to formaldehyde, 2,4 hours bronchial Logarithmic growth period of 16 HBE cells were seeded onto 48-well plates using serum-free medium, different concentrations of formaldehyde solution(with serum-free medium to a concentration of 0.04,0.16,0.32,0.64,1.2 mmol / l) staining After drug and incubated in a 37 °, 5% CO2 incubator for 2,4 hours sterile supernatant taken after high speed centrifugation cells(IL-2, IL-10,IL-12, IFN-γ) measured with an ELISA kit Th1 type cells factor levels in human bronchial epithelial cells after acute exposure to formaldehyde changing role of Th1 cytokines. 3. Cell supernatants were Th2 cytokines changes of epithelial cells exposed to formaldehyde 2,4 hour acute bronchial Logarithmic growth period 16 HBE cells were seeded in 48-well plates, serum-free medium with different concentrations of formaldehyde solution(with serum-free medium to a concentration of 0.04,0.16,0.32,0.64,1.2 mmol / l) exposure After cultured at 37 °, 5% CO2 incubator sterile 2,4 hours to take the high-speed supernatant after centrifugation Th2 cytokines measured by ELISA(IL-4, IL-5, IL-6, IL-13) Content of human bronchial epithelial cells after acute exposure to formaldehyde changing role of Th2 cytokines. 4. After the acute human bronchial epithelial cells exposed to formaldehyde 2,4 hour class Th17 cell supernatants change research and TNF-α cytokine Logarithmic growth period 16 HBE cells were seeded in 48-well plates, serum-free medium with different concentrations of formaldehyde solution(with serum-free medium to a concentration of 0.04,0.16,0.32,0.64,1.2 mmol / l) exposure After the after-cultured in 37 °, 5% CO2 incubator sterile 2,4 hours to take the high-speed centrifugal supernatant measured Th17(IL-8, IL-17) and TNF-α cytokine levels by ELISA kit Research in human bronchial epithelial cells after acute exposure to formaldehyde class and Th17 cytokines TNF-α change. 5. WESTERN BLOT assay with formaldehyde exposure 16 HBE cell signal transductionprotein NF-κB, COX-2, ERK expression Take on 16 HBE cells grown on 6-well plates were inoculated period, serum-free medium, different concentrations of formaldehyde solution(with serum-free medium to a concentration of 0.04,0.16,0.64,1.2 mmol / l) after exposure, WESTERN BLOT measured by NF-κB, COX-2, ERK expression after culture content in 37 °, 5% CO2 incubator sterile 2,4 hours total protein was extracted.Results 1. Human bronchial epithelial cells(16HBE) cell viability and inversely related to formaldehyde exposure dose. Compared with the control group(0mmol / l), cell survival 0.32,0.64 mmol / l dose group decreased significantly, the difference was statistically significant(P <0.05). Formaldehyde on human bronchial epithelial cells(16HBE) median lethal dose concentration 0.4658 mmol / l. 2. TH1 cytokines test results show: 2 hours when exposed to formaldehyde, and the control group(0.00 mmol / l) compared to, 0.04,0.16 mmol / l cell supernatant reduce formaldehyde exposure dose IL-2 group content, differences were statistically significant(P<0.05), IFN-γ, IL-12 between groups was not significantly change, the difference was not statistically significant(P> 0.05); 4 hours when exposed to formaldehyde, and the control group(0.00 mmol / l) compared to formaldehyde exposure dose group within the IL-2, IL-12 decreased, the difference was statistically significant(P <0.05); compared with the control group between(0.00 mmol / l), IFN-γ group no significant difference did not change significantly(P> 0.05). 3. TH2 cytokines test results show: 2 hours when exposed to formaldehyde, and the control group(0.00 mmol / l) compared to cell supernatant IL-5 content increased exposure dose group, IL-6 content decreased The differences were statistically significant(P <0.05), IL-4, IL-10, IL-13 between groups did not change significantly the difference was not statistically significant(P> 0.05); 4 hours when exposed to formaldehyde, and the control group(0.00 mmol / l) compared to the 1.2mmol / l dose of IL-4 levels increased dose within 0.04,0.16,0.64 content increased IL-5, IL-5 content of 1.2mmol / l dose group within reduce,IL-6 group difference was statistically significant between content(P <0.05), IL-10, IL-13 inter-group difference was not statistically significant change significantly(P> 0.05). 4. TH17 cytokines TNF-α and test results show: 2 hours when exposed to formaldehyde, and the control group(0.00 mmol / l) compared to cells exposed to supernatant dose IL-8 content decreased, the difference There was statistically significant(P <0.05), IL-17, TNF-α between groups did not change significantly the difference was not statistically significant(P> 0.05); 4 hours when exposed to formaldehyde, multivariate analysis of variance, with the control group( 0.00 mmol / l) compared within 0.04,0.16 mmol / l dose of IL-8 levels increased and continue to increase the concentration of reducing formaldehyde content of IL-8, TNF-α in each group levels were increased, the difference statistics significance(P<0.05), IL-17 content did not change significantly between the groups, the difference was not statistically significant(P> 0.05). 5. Formaldehyde increased nuclear transcription factor(NF-κB) and COX-2 expression levels in a dose-dependent way, the effect on the expression of ERK is not obvious.Conclusions 1. Formaldehyde inhibited human bronchial epithelial cell proliferation at low dose and caused cell death at higher dose. 2. Acute exposure to formaldehyde reduced the concentration of Th1 type cytokines, including IL-2 and IL-12, increased the concentration of Th2 type cytokines including IL-4 and IL-5, increased the concentration of IL-8 which is blong to Th17 type cytokines and TNF-α level, resulted in increasing airway inflammation to induce the development of asthma. 3. Upregulation of nuclear transcription factor(NF-κB), COX-2, JNK, ERK protein levels by acute exposure to formaldehyde promoted the secretion of inflammatory cells, deteriorated airway inflammation as well.