Dapk1 expression in mice of infantile pneumonia
Firstly, gene chip was used to analyze the changes expression of pro-inflammation gene in mice of infantile pneumonia. As shown in Fig. 1A, Dapk1 expression maybe one pro-inflammation gene and was up-regulated in mice of infantile pneumonia as 24 h after induction model. Next, we found that Dapk1 mRNA and protein expressions in 6, 12 or 24 h after induction model, compared to sham mice (Fig. 1B-1D).
Dapk1 gene increased inflammation in vitro model
Then, we used vitro model to analyze the effects of Dapk1 gene on infantile pneumonia. Over-expression of Dapk1 increased TNF-α, IL-6, IL-1β and IL-18 levels in vitro model, compared to negative group (Fig. 2A-2D). Down-regulation of Dapk1 reduced TNF-α, IL-6, IL-1β and IL-18 levels in vitro model, compared to negative group (Fig. 2E-2H). These results showed that Dapk1 gene increased inflammation in vitro model of infantile pneumonia.
Dapk1 promotes p38MAPK-mediated NF-κB Expression
Gene chip was used to analyze inflammation signaling pathway by regulation of Dapk1 in infantile pneumonia. As shown in Fig. 3A-3B, p38MAPK/NF-κB expression signaling pathway may a target spot for the function of Dapk1 in infantile pneumonia. Over-expression of Dapk1 induced Dapk1, p-p38MAPK and NF-κB protein expression in vitro model of infantile pneumonia, compared to negative group (Fig. 3C-3F). Down-regulation of Dapk1 suppressed p-p38MAPK and NF-κB protein expression in vitro model of infantile pneumonia, compared to negative group (Fig. 3C-3F). IF showed over-expression of Dapk1 induced p-p38MAPK expression in vitro model of infantile pneumonia, compared to negative group (Fig. 3G). Done-regulation of Dapk1 suppressed Dapk1, p-p38MAPK and NF-κB protein expression in vitro model of infantile pneumonia, compared to negative group (Fig. 3F, 3H-3J).
The regulation of p38MAPK participated in the effects of Dapk1 in infantile pneumonia
The study evaluated the role of p38MAPK in the function of Dapk1 in infantile pneumonia. Dehydrocorydaline nitrate (10 nM, p38MAPK agonist, MedChemExpress) induced p-p38MAPK and NF-κB protein expressions in vitro model following with down-regulation of Dapk1 (Fig. 4A-4C). Then, the activation of p38MAPK also increased the TNF-α, IL-6, IL-1β and IL-18 levels in vitro model following with down-regulation of Dapk1 (Fig. 4D-4G).
p38 MAPK-IN-1 (20 nM, 38MAPK inhibitor, MedChemExpress) suppressed p-p38MAPK and NF-κB protein expressions in vitro model following with up-regulation of Dapk1 (Fig. 5A-5C). Then, the inhibition of p38MAPK also reduced the TNF-α, IL-6, IL-1β and IL-18 levels in vitro model following with up-regulation of Dapk1 (Fig. 5D-5G).
The regulation of NF-κB participated in the effects of Dapk1 in infantile pneumonia
Lastly, si-NF-κB suppressed the up-regulation of Dapk1 on NF-κB protein expression, and the TNF-α, IL-6, IL-1β and IL-18 levels in vitro model, compared with up-regulation of Dapk1 group (Fig. 6). NF-κB plasmid induced NF-κB protein expression, and the TNF-α, IL-6, IL-1β and IL-18 levels in vitro model by down-regulation of Dapk1, compared with down-regulation of Dapk1 group (Fig. 7).