FPs and CSE. This suggests that NADPH oxidase is involved in ROS generation in endothelial cells 0 co-exposed to both UFPs and CSE. UFPs (/ml) CSE (%) 0 0 0 2.5 50 0 50 2.5 The mitogen-activated protein kinase (MAPK) pathway is in- volved in a variety of biological events and is also responsible for Fig. 7. Increased expression of IL-6 in MPMVEC from wild-type mice exposed to UFPs and/or CSE was attenuated by Finibax pre-treatment of cells with p38 inhibitor. 2 10 5 cells were seeded into each well of 6-well plates. After overnight culture, cells were pre-treated with 10 l M SB203580 for 3 h, then treated with UFPs and/or CSE for 12 h.
Cells without any treatment were used as controls. Real-time PCR was performed with iQ5 Cycler (Bio-Rad). Data are shown as mean SD of three experiments with three replicates in each experiment. Signiant difference as compared with the control, p < 0.05; # Signiant difference as compared with UFPs alone or CSE alone treated group, p < 0.05; $ Signiant difference as compared with those with UFPs and/or CSE treatment but without Bosutinib SB203580 pre-treatment, p < 0.05. There are several potential ROS generation sources in pulmon- ary endothelial cells ( Cai and Harrison, 2000; Griendling et al., 2000; Lum and Roebuck, 2001 ). Our previous studies showed that cytokine production in response to various particles. MAPKs can be activated through phosphorylation. UFPs or CSE are known to induce oxidative stress and subsequently activate MAPKs ( Karoly et al., 2007 ; Kim et al., Li et al., 2003, 2006; Low et al., 2007; Orosz et al., 2007 ).
The present study showed that co-exposure of endo- thelial cells to UFPs and CSE caused a signiant increase in phos- phorylation of Erk1/2 and p38 as compared to that when cells were exposed to UFPs or CSE alone. Further, we demonstrated that acti- vation of NADPH oxidase to generate ROS was also involved in the activation of MAPKs because exposure of endothelial cells from gp91 phox knock-out mice to UFPs and/or CSE did not cause in- creased phosphorylation of Erk1/2 and p38. Our results demonstrated that UFPs or CSE signiantly up-reg- ulated Egr-1 expression at both the transcriptional and transla- Relative IL-6 mRNA (% of control) (% of control) Relative IL-6 mRNA pg IL-6/mg cell protein purchase Marbofloxacin Relative IL-6 mRNA (% of control) Page 7 302 Y. Mo et al. / Toxicology in Vitro 26 (2012) 29503 tional levels in endothelial cells. Co-exposure to UFPs and CSE re- sulted in a signiant increase in the expression of Egr-1 as com- pared to that with exposure to UFPs or CSE alone. Egr-1 is a zinc ger transcription factor, which has 802 kDa nuclear phospho- protein consisted of 533 amino acids.
The DNA-binding domain of Egr-1 consists of three zinc ger motifs located between amino order Marbofloxacin acid 332 and 416 towards the carboxyl-terminal region of the pro- tein ( Khachigian et al., 1996 ). Egr-1 expression is highly regulated by many factors including oxidative stress, cellular signaling mol- ecules such as MAPKs, shear stress, mechanical injury, and Ang II, which can further stimulate transcription of several proinmma- tory genes, including TNF- a , IL-2, MCP-1 and ICAM-1 ( Houston et al., 1999; Jeong et al., 2010; Khachigian et al., 1996; Yan et al., 1999 ). Our present studies clearly demonstrated that oxidative stress and activation of MAPKs were involved in acute up-regulation of Egr-1 expression since either UFPs or CSE failed to upregulate Egr-1 expression in endothelial cells from gp91 phox knock-out mice, and the Egr-1 expression was abolished when endothelial cells were pre-treated with SB203580, a speci p38 inhibhours generated signiant ROS ( Fig. 2 ). ROS was signiantly en- hanced when cells were co-exposed to both UFPs and CSE ( Fig. 2 ).
In MPMVEC obtained from gp91 phox KO mice, no signiantly in- creased ROS generation were observed when cells were either ex- posed to UFPs, CSE or UFPs with CSE ( Fig. 2 ). Since gp91 phox is one of key components of NADPH oxidase, our results demonstrated that NADP