Dapk1 promoted inflammation of infantile pneumonia by p38MAPK/NF-κB signaling pathway

The present study was designed to investigate the function of death-associated protein kinase 1 (DAPK1) in infantile pneumonia and explore the potential mechanism of the actions. Male C57BL/6 mice were injected with 2 mg/kg of LPS for the mice model of infantile pneumonia. A549 cells were treated with 100 ng/ml of LPS for vitro model of infantile pneumonia. Dapk1 mRNA and protein expressions in 6, 12 or 24 h after induction model of mice. Dapk1 gene increased inflammation in vitro model through activation of p38MAPK-mediated NF-κB expression. The inhibition of p38MAPK or NF-κB reduced the pro-inflammation effects of DAPK1 in infantile pneumonia. Our study demonstrates that Dapk1 promoted inflammation of infantile pneumonia by p38MAPK/NF-κB signaling pathway, may be achieved inflammation by activation of p38MAPK/NF-κB signaling pathway.


Introduction
Mycoplasmal pneumonia is a respiratory infection caused by mycoplasma pneumoniae, which is clinically manifested by fever, cough, sputum expectoration, anorexia, headache, sore throat, substernal pain and chills (Bai et al. 2018). The more atypical clinical symptoms are generally detected in younger children, often accompanied with multi-system and multi-organ functional damage (Guo and Cheng 2018). Infantile mycoplasmal pneumonia is one of the main causes of respiratory infection in children (Guo and Cheng 2018). If not treated in time, it may cause bronchiectasis, atelectasis and pulmonary interstitial fibrosis, and even death in severe cases . At present, the etiology of infantile pneumonia is not completely clear, which might be associated with the humoral and cellular immunity of the children . Therefore, anti-infection and symptomatic treatment are the main therapeutic approaches.
NF-kappaB (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signaling pathways play important roles in the inflammatory process (Bai et al. 2017;Liu et al. 2019). Both of them can promote the occurrence and development of inflammatory response in lung tissue by regulating multiple inflammatory factors, such as interleukin (IL)-12, IL-13 and tumor necrosis factor (TNF)-α and promoting the infiltration of inflammatory cells (Bai et al. 2017;Liu et al. 2019). Many studies have shown that NF-κB and p38 MAPK signaling pathways are involved in pneumonia caused by Streptococcus pneumonia (Lee et al. 2009;Quan et al. 2019). However, there are relatively rare studies on the role of NF-κB and p38 MAPK signaling pathways on MP-infected pneumonia (Kang et al. 2018).
Death-associated protein kinase 1 (DAPK1) is a serine/ threonine protein kinase regulated by calmodulin (CaM), involved in a variety of pathophysiological processes in the body (Arko-Boham et al. 2020). Previous studies have shown that DAPK1 is one of the positive regulators of apoptosis, which is widely involved in apoptosis induced by multiple pathways (Daniunaite et al. 2020;Shin and Chung 2020;Wang et al. 2020). Recent studies have also found that in addition to regulating apoptosis and autophagy, DAPK1 also plays a vital role in a series of inflammation regulation (Daniunaite et al. 2020;Shin and Chung 2020;Wang et al. 1 3 2020). The present study was designed to investigate the function of DAPK1 in infantile pneumonia and explore the potential mechanism of the actions.

Histological examination
Lung tissue samples after mice sacrificed were collected and fixed with 4% paraformaldehyde for 24 h at room temperature. All experiments have been approved by the Ethics Committee of The Affiliated Changzhou NO.2 People's Hospital of Nanjing Medical University. Lung tissue samples fixed with paraformaldehyde were paraffin-embedded. Lung tissue samples were cut into 5 μm sections using a paraffin-slicing machine and stained with hematoxylin and eosin. Lung tissues were observed under light microscopy (magnification, 100 ×; BH3-MJL; Olympus Corporation, Tokyo, Japan).

Quantitative real-time PCR detection
Total RNA was extracted from tissue sample or cells samples by using Trizol reagent (Life Technologies Corporation, Carlsbad, CA, USA). Taqman MicroRNA Reverse Transcription Kit and Taqman Universal Master Mix II with TaqMan MRNA Assay (Applied Biosystems, Foster City, CA, USA) were used for testing the gene expression level. The primer sequences of Dapk1: forward primer 5′-ACA GTG ATT TCC TCA AGA CTC TTG G-3′ and reverse primer 5′-CAG CAG AGT CCC CAC TCA CCT CCT -3′; Actin: forward, 5′-TCA CTA TCG GCA ATG AGC GG-3′; reverse, 5′-TTG GCT ACA ACT ACA-3′.

Immunofluorescent staining
A549 cell was fixed with 4% paraformaldehyde for 15 min at room temperature and then incubated with using 0.25% Triton X-100 for 15 min. A549 cell was incubated with p-p38 MAPK (1:100) at 4 °C overnight after blocking with 5% BSA for 1 h. A549 cell was incubated with goat anti-rabbit IgG-cFL 555 antibody (1:100) for 2 h at room temperature and stained with DAPI for 15 min and washed with PBS for 15 min. The images of A549 cell obtained using a Zeiss Axioplan 2 fluorescent microscope (carl Zeiss AG, Oberkochen, Germany).

Statistical analysis
All experiments were repeated at least three times. p values were calculated using two-tailed Student's t test between two groups, or one-way analysis of variance for more than two groups. Statistical analyses were performed by using SPSS 19.0 statistical software. p value of < 0.05 was considered to indicate a statistically significant result.

Dapk1 expression in mice of infantile pneumonia
First, 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 proinflammation 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-d).
These results showed that Dapk1 mRNA and protein expressions in model of infantile pneumonia.

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-d). Down-regulation of Dapk1 reduced TNF-α, IL-6, IL-1β and IL-18 levels in vitro model, compared to negative group (Fig. 2e-h). These results showed that Dapk1 gene increased inflammation in vitro model of infantile pneumonia. These results demonstrated Dapk1 promoted inflammation reactions in model of infantile pneumonia.

Dapk1 promotes p38MAPK-mediated NF-κB expression
We used Dapk1 in vitro model and then cell samples were used to analyze the regulation gene expression using gene microarray hybridization. Gene microarray hybridization was used to analyze inflammation signaling pathway by regulation of Dapk1 in infantile pneumonia. As shown in Fig. 3a and b, NF-κB expression was markedly up-regulated and p38 MAPKNF-κB expression signaling pathway, and online bioinformatics tools (TargetScan, the regulation genes of Dapk1, inflammation genes and the regulation genes of infantile pneumonia) found that p38 MAPK/NF-κBmay 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-f). Down-regulation of Dapk1 suppressed p-p38MAPK and NF-κB protein expression in vitro model of infantile pneumonia, compared to negative group (Fig. 3c-f). IF showed over-expression of Dapk1 induced Dapk1 and 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, h-j). These results confirmed that Dapk1 promotes p38MAPK-mediated NF-κB expression to promote inflammation reactions in model of infantile pneumonia.

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-c). 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-g).

The regulation of NF-κB participated in the effects of Dapk1 in infantile pneumonia
Finally, 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). These results reported that p38MAPK/NF-κB is an important signal pathway of Dapk1 in infantile pneumonia.

Discussion
Infantile pneumonia is a common pediatric disorder in lung tissues, which occurs at all ages of children, especially in infants and young children (Hyvärinen et al. 2005). The common pathogens mainly bacteria, viruses and mycoplasma. Anti-infection and symptomatic treatment are used by Western Medicine. And most children can be cured within approximately 1 week (Li et al. 2019). However, a considerable number of children, especially infants under 6 months of age, often have prolonged illness and delayed healing, such as persistent rales, phlegm in the throat, constant sputum and coughing, excessive sweating, poor appetite and loose stools (Tomari et al. 2018). According to Western Medicine, only anti-infection and symptomatic treatment can be continued, which generally fail to significantly relieve the condition. In addition, some children have worsening diarrhea, causing microbiota imbalance, and even nosocomial cross infection. The present study demonstrated that Dapk1 mRNA and protein expressions in 6, 12 or 24 h after induction model. Li et al. showed that Dapk1 increased inflammation in LPS-induced acute lung injury via p38MAPK/NF-κB signaling pathway (Li et al. 2020). At this stage, this paper first reported that Dapk1 may regulated inflammation in infantile pneumonia, which was an important innovation point for this experiment. Heat map and microarray data (a), interpretation of result (b), Dapk1, p-p38MAPK and NF-κB protein expression in vitro model by over-expression of Dapk1 (c-f); p-p38MAPK protein expression (immunofluorescence, g), Dapk1, p-p38MAPK and NF-κB protein expression in vitro model by down-regulation of Dapk1 (f, g-j). Negative negative mimic group, Dapk1 over-expression of Dapk1 group, siDapk1 down-regulation of Dapk1 group. ## p < 0.01 compared with negative mimic group p38, a member of the MAPK family, is mainly involved in apoptosis and differentiation, cell inflammation and oxidative stress (Liu et al. 2019). p38 is a key factor of the MAPK signaling pathway (Liu et al. 2019;Acchioni et al. 2019). The activation of p38 MAPK can affect the activity of transcription factors, thereby regulating the inflammatory response (Liu et al. 2019;Acchioni et al. 2019). Studies have reported that the p38 MAPK signaling pathway can promote the phosphorylation and degradation of I-κBα, thereby activating the NF-κB pathway (Guo et al. 2019;Zuo et al. 2017). Furthermore, this study demonstrated that Dapk1 promotes p38MAPK-mediated NF-κB expression to increase inflammation in vitro model of infantile pneumonia. Wu et al. reveal that tumor suppressor Dapk1 inhibits necroptosis by p38 MAPK activation .
NF-κB is an important nuclear transcription factor that regulates the immune response and inflammatory pathways. Under the normal state of the body, NF-κB binds to I-κBα in an inactive state in the plasma (Li et al. 2019;Liu et al. 2018;Xie et al. 2017). Under the stimulation of pathogenic factors, I-κBα is phosphorylated and degraded, subsequently separated from the NF-κB heterodimer to expose Fig. 4 The induction of p38MAPK participated in the effects of Dapk1 in infantile pneumonia. p-p38MAPK and NF-κB protein expression (a-c), TNF-α (d), IL-6 (e), IL-1β (f) and IL-18 (g) levels.
Negative negative mimic group, siDapk1 down-regulation of Dapk1 group, siDapk1 + p38 a down-regulation of Dapk1 and p38 agonist group. ## p < 0.01 compared with negative mimic group; **p < 0.01 compared with down-regulation of Dapk1 group Fig. 5 The inhibition of p38MAPK participated in the effects of Dapk1 in infantile pneumonia. p-p38MAPK and NF-κB protein expression (a-c), TNF-α (d), IL-6 (e), IL-1β (f) and IL-18 (g) levels. Negative negative mimic group, Dapk1 over-expression of Dapk1 group, Dapk1 + p38 i over-expression of Dapk1 and p38 inhibitor group. ## p < 0.01 compared with negative mimic group; **p < 0.01 compared with over-expression of Dapk1 group the nuclear localization signal of p50 protein to activate NF-κB . Activated NF-κB transfers to the nucleus and combines with the target gene κB to induce the expression of inflammatory factors, which is an important transcription factor to cause inflammatory response caused in infantile pneumonia (Xie et al. 2017). The present study demonstrated that the inhibition of p38 MAPK or NF-κB reduced the effects of Dapk1 on inflammation of infantile pneumonia. Wu et al. suggest that DAPK1 modulates a curcumin-induced G2/M arrest and by NF-κB activation (Wu et al. 2013). These results showed that Dapk1 regulated p38 MAPK/ NF-κB pathway to promote inflammation in infantile pneumonia. This experiment only used vitro model to validate our conclusion, which was an insufficient for this experiment, and we will verify Dapk1 is the key gene of infantile pneumonia in mice model.
In conclusion, the present study demonstrates that Dapk1 gene and protein expression was up-regulated in infantile pneumonia. Dapk1 induced p38MAPK/NF-κB signaling pathway to promote inflammation. These results reveal a novel pro-inflammation of Dapk1 in infantile pneumonia, suggesting that it may be a promising therapeutic target for the treatment of IP.

Declarations
Conflict of interest There are no potential conflicts of interest to disclose. Fig. 6 The inhibition of NF-κB participated in the effects of Dapk1 in infantile pneumonia. NF-κB protein expression (a, b), TNF-α (c), IL-6 (d), IL-1β (e) and IL-18 (f) levels. Negative negative mimic group, Dapk1 over-expression of Dapk1 group, Dapk1 + siNF-κB over-expression of Dapk1 and down-regulation of NF-κB group. ## p < 0.01 compared with negative mimic group; **p < 0.01 compared with over-expression of Dapk1 group Fig. 7 The induction of NF-κB participated in the effects of Dapk1 in infantile pneumonia. NF-κB protein expression (a, b), TNF-α (c), IL-6 (d), IL-1β (e) and IL-18 (f) levels. Negative negative mimic group, siDapk1 down-regulation of Dapk1 group, siDapk1 + NF-κB down-regulation of Dapk1 and over-expression of NF-κB group. ## p < 0.01 compared with negative mimic group; **p < 0.01 compared with down-regulation of Dapk1 group Ethical approval This study was approved by the Ethics committee of the Affiliated Changzhou NO.2 People's Hospital of Nanjing Medical University.
Informed consent All patients have given their written informed consent.