The Research Of Human Bronchial Epithelium Injured By Black Gunpowder Smog And Protective Effect Of Puerarin

This study was main research 1, The platform establishment of human bronchial epithelial cell inflammatory injured by black gunpowder smog. 2, This study investigated the gunpowder smog induced BEAS-2B cells inflammatory injury to reveal the species of cytokine involved and the mechanism of cytokine produced. 3, According to the study of pre-treated puerarin in gunpowder smog induced cells injury to illustrate the protection of puerarin and the mechanism of puerarin protected cells. The previous research has established the stable platform of gunpowder smog induced BEAS-2B cells inflammatory injury. The change of celluar morphology were observed by inverted microscope after cell exposed gunpowder smog for 10 min and went on cultured for 24 hours in incubator. After stimulated by gunpowder smog, the cytokine produced by BEAS-2B cells were tested by MILLIPLEX○RMAP Human Cytokine/Chemokine Magnetic Bead Panel Kit. BEAS-2B cells were divided into three groups, control group(BEAS-2B cells were normally cultured for 48 hours), smog group(BEAS-2B cells were exposed gunpowder smog for 10 min), smog+puerarin group(the BEAS-2B cells were exposed smog for 10 min after pre-treated with puerarin for 2 hours, the puerarin of dosage was 100μg/ml). The components of smog were tested by gas detector. The cellular viability was determined by CCK-8 after gunpowder smog stimulated and puerarin(the dose of 25μg/ml 、 50μg/ml 、 100μg/ml) pre-treated. The level of inflammatory cytokine IL-8 in BEAS-2B cells culture supernatant after smog impacted and puerarin pretreated were detected through ELISA. The groups included control group, smog groups(the different culture time, 0h, 3h, 6h, 9h, 12 h, 24h), smog+puerarin groups (25μg/ml, 50μg/ml, 100μg/ml), smog+NAC groups(2m M, 4m M, 6m M, 8m M, 10 m M), smog+Bay11-7082 groups(1μM, 2μM, 4μM, 8μM) and smog+SB203580(10μM). After smog impacted on the BEAS-2B cells and pre-treated puerarin in smog induced the BEAS-2B cells injury, the intracellular ROS level was determined by DCFH-DA probe. This experiment detected the protein of P-P38 MAPK and P-IκBα by Western Blot, in order to investigate the mechanism of smog induced cell injury and the protective mechanism of puerarin. The results suggested 1, After the BEAS-2B cell were treated with smog, morphological changed were observed such as from irregular to round, thin between cells, compared to control group. 2, The result of cytokine panel shown that the release of GM-CSF,IL-6,IL-8,MCP-1 increased after injured by gunpowder smog compared to control group(P<0.05). The expressed of IL-8 was stable in smog effect. When intervention with puerarin, the cytokine significantly reduced compared to smog group. 3, The component of smog including O2, CO, H2 S. When the black gunpowder bumming, the percent contents of O2 decreased gradually, and H2 S was 88.2ppm remain unchanged within 10 min. 4, The cellular viability of BEAS-2B were greatly decreased after irritated with black gunpowder smog, whereas the cellular viability of puerarin groups were elevated in dose-dependent manner compared to smog group. 5, The level of intracellular ROS was increased after gunpowder smog treated, the smog+puerarin groups reduced in dose dependent. 6, The results of IL-8 tested by ELISA have suggested that the reduced level of IL-8 accompanied with the NAC, puerarin, Bay11-7082 dosage increasing. The level of IL-8 in smog+SB203580 group decreased significantly. 7, The protein of P-P38 MAPK expression was increasing after gunpowder smog stimulated for 10 min, while the P-IκBα expression highly at 2 hours after gunpowder smog stimulated for 10 min. When pretreated with NAC, puerarin, Bay11-7082, SB203580, the protein of P-P38 MAPK and P-IκBα expression was decreased. The study shown that the gunpowder smog induced BEAS-2B cells injured such as cytokine increased, ROS elevated and the protein of P-P38 MAPK and P-IκBα expressed highly. The intervention of NAC, puerarin, Bay11-7082 and SB203580 reduced the IL-8 and protein of P-P38 MAPK and P-IκBα expression. The study suggested that the black gunpowder smog induced the cytokine expression highly in BEAS-2B cells may be through the ROS, P38 MAPK and NF-κB signaling pathway. Simultaneously, the study indicated that puerarin may be through this signaling pathway to protect BEAS-2B cells from injury by black gunpowder smog.