In Vitro Effects And Mechanism Of HMGB1- Induced On The Expression Of Asthma-Related Inflammatory Cytokines From Bronchial Epithelial Cells

Objective To investigate the in vitro the effect and mechanism of HMGB1-induced on the expression of asthma-related inflammatory cytokines from bronchial epithelial cells.Methods 16-HBE were coincubated with different groups of control, HMGB1(100,500,2000ng/ml). Then cells mRNA (cultured within 24h) and supernatants were isolated for qPCR and ELISA analysis of the expression of TSLP, IL-8, IL-6, EGF, VEGF, MMP-9, IL-1β and TNF-a. Moreover, total mRNA and cell protein lysates were collected for qPCR and Western blot analysis of RAGE, TLR2, and TLR4 expression. (2)、ELISA was used to measure the inflammatory cytokines secretion after RAGE (20μg/ml), TLR2 (20μg/ml) and TLR4 (20μg/ml) blocked by specific antibodies. (3)、Western blot and ELISA were used to measure the phosphorylation level of p38 MAPK、 ERK1/2、PI3K and the inflammatory cytokines secretion after these pathways inhibited by SB202190 (10μM)、U0126 (10μM) and LY294002 (10μM)Results (1).Compared with control groups, HMGB1-500g/ml significantly increased the mRNA expression and secretion of TNF-α、TSLP、MMP-9、VEGF (P<0.05). HMGB1-200ng/ml can increase more mRNA expression and secretion of TNF-α、TSLP、MMP-9 and VEGF than HMGB1-500g/ml did(P<0.05). (2).Compared with control groups, HMGB1-500g/ml significantly increased the mRNA expression and protein level of RAGE (P<0.05). Compared with HMGB1-500g/ml groups, HMGB1-200ng/ml can increase more mRNA and protein expression (P<0.05). (3). HMGB1 can both activate p38 MAPK and ERK1/2. Compared with HMGB1-2000ng/ml, p38 MAPK inhibition (increased about 45%～60%) made more reduction of TNF-α、TSLP、 MMP-9、VEGF secretion than ERKl/2 inhibition did (increased about 10%～ 30%).Conclusion 1. HMGB1can activate the inflammatory responses of 16-HBE by increasing proinflammatory factor expression (TNF-α、TSLP、 MMP-9 and VEGF) in vitro.2. This effect was mediated by activation of p38 MAPK and ERK1/2 via RAGE. In this process, p38 MAPK plays a relatively greater role than ERK1/2.