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\begin{document}

\title{Verification of key genes and pathways explores the anti-inflammation
mechanism of NR4a1on macrophage through bioinformatics analysis}



\author[1]{Juntao Tan}%
\author[1]{Teng Teng}%
\author[1]{Su Chen}%
\author[1]{Tiegeng Huang}%
\author[1]{Zhenni Xiao}%
\author[1]{Xiaotian Luo}%
\author[1]{Yixin Tang}%
\affil[1]{University of South China Affiliated Nanhua Hospital}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
NR4a1 has been demonstrated to exert a protective role in various
chronic inflammatory disease. The underlying mechanism has yet to be
clarified. This research aimed to explore the anti-inflammatory
mechanism of NR4a1on macrophage through bioinformatics analysis and
further verified by a series of experiments. In this study, The GSMs
(GEO samples) that LPS(lipopolysaccharide) treated macrophage derived
from wild type and NR4a1 knockout mice were extracted from GSE68167 gene
expression profiles in Gene Expression Omnibus (GEO) database. DEGs
(Differentially expressed genes) screened out. The gene ontology (GO)
and KEGG (Kyoto Encyclopedia of Genes and Genomes pathway) enrichment
were conducted. PPI (Protein-protein interaction) network that comprised
49 nodes was mapped. The enrichment of biological process (BP) was
principally and significantly revealed in signal transduction,
inflammatory response and positive regulation of chemokine production.
In terms of KEGG pathway, DEGs were primarily enriched in
Cytokine-cytokine receptor interaction, JAK-STAT signaling pathway and
cAMP signaling pathway. In the PPI network, IL10, IFNG, Fos, IL19, PDE4B
NPY, Cnr1, MMP13, Rtn1, UCHL1 were selected as Hub gene. PDE4B and cAMP
signaling pathway may associate with anti-inflammatory function of
NR4a1. Our data showed that NR4a1 could promotes the protein level of
cAMP, p-PKA and decreased p-p65, p-I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α \selectlanguage{english}through down-regulating PDE4B.
NR4a1-downregulated PDE4B expression notably decreased the LPS induced
mRNA level of IL-6 and IL1\selectlanguage{greek}β. \selectlanguage{english}This study also pointed the potential
physiological processes regulated by NR4a1.Our findings supports that
NR4a1 may be a considerable therapeutic target in inflammatory disease.%
\end{abstract}%



\sloppy


\textbf{Introduction:}

Inflammatory diseases are being considered a serious issue, for which
inhibiting the prolonged and excessive macrophage-regulated inflammatory
processes can serve as a considerable therapy~{[}1,2{]}. The orphan
nuclear receptor NR4a1, also known as Nurr7, NGFI, NAK-1, has been
recognized as a key regulator in inflammation~{[}2,3{]}. NR4a1 in all
kinds of inflammatory diseases has been investigated, such as
atherosclerosis, inflammatory bowel diseases, and rheumatoid arthritis,
exerting a protective role against inflammation~{[}4{]}. Inflammatory
stimulation could promote the NR4a1 expression via NF-\selectlanguage{greek}κ\selectlanguage{english}B in
macrophage~{[}5{]}. However, NR4a1 exerts the anti-inflammatory effects
depending on the negative regulator for NF\selectlanguage{greek}κ\selectlanguage{english}B~{[}6-8{]}. NR4a1-KO
macrophage displayed the increased productive in pro-inflammatory
cytokines and this up-regulation was decreased by NF\selectlanguage{greek}κ\selectlanguage{english}B
inhibitor~{[}9,10{]}. Over-expressed NR4a1 exhibits increased IKK
expression,which is a inhibitory role in NF\selectlanguage{greek}κ\selectlanguage{english}B signaling{[}9,10{]}. NR4a1
can directly have effect on p65 to suppress
neuroinflammation~{[}9,10{]}. NR4a1 is also considered a regulator in
metabolism during inflammatory responses of immune cells~{[}11,12{]}.
Despite the several previous study, the understanding of downstream
genes of NR4a1 remains lacked~{[}13{]}.

To investigate the functioning mechanism of NR4a1 in macrophage, the
GSE681767 were employed for analysis. The GO and KEGG enrichment were
conducted in DAVID online database. Protein-protein interaction (PPI)
network was mapped by Cytoscape. The results showed that PDE4B and cAMP
signaling pathway may play key roles in anti-inflammatory function of
NR4a1. PDE4B serves as a selectively hydrolytic enzyme of second signal
messenger cyclic adenosine monophosphate(cAMP)~{[}14{]}, of which the
deficiency represses activation of nuclear factor kappa-B (NF-\selectlanguage{greek}κ\selectlanguage{english}B) in
various cell stimulated with lipopolysaccharide(LPS)~{[}15{]}. cAMP has
been proved to play an anti-inflammatory function, employed as a
solution of inflammation~{[}14,16{]}. cAMP-activated PKA can inhibit the
NF\selectlanguage{greek}κ\selectlanguage{english}B signaling through prohibiting the degradation of I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{english}\selectlanguage{greek}α{[}14,17{]}.
Previous studies have demonstrated the close relation of NR4a1's
anti-inflammatory function with the inhibited NF\selectlanguage{greek}κ\selectlanguage{english}B
signaling~{[}7,18,19{]}. Thus, we assumed that NR4a1 may regulates PDE4B
expression to exert anti-inflammatory function.

\textbf{Method}

\textbf{Microarray date and DEGs screen}

In this research, GSE68167, a gene expression profile based on GPL6887
Illumina MouseWG-6 v2.0 expression beadchip composed of 18 samples was
extracted from GEO database(www.ncbi.nlm.nih.gov), which contains gene
expression profiles~{[}20{]}. From these samples, the macrophage
extracted from NR4a1 deficit or wild type mice treated with LPS 8 hours
were chosen for analysis by GEO2R. LogFc\textgreater{}1 and
P\textless{}0.05 were set as threshold to screen out the significant
DEGs.

\textbf{Functional enrichment analysis}

The DEGs were analyzed by DAVID database(https://david.ncfcrf.gov), an
online tool providing a functional annotation of genes and discover
enriched functional-related gene group~{[}21{]}. The Gene Ontology (GO)
constructs the annotation of genes, gene products and sequence~{[}22{]}.
KEGG pathway analysis was carried out to explore the gene linking
functional and genomic information~{[}23{]}. P\textless{}0.05 were
considered significant.

\textbf{Integration of protein-protein interaction network and Hubgenes}

The relationships between DEGs were assessed using STRING online
database(https://cn.string-db.org) PPI network mapped by
Cytoscape(https://cystoscape)~{[}24{]}. All nodes were ranked by Degree
in cytoHubba.

\textbf{Cell culture and Transfection}

RAW264.7 cells obtained from Zhong Qiao Xin Zhou Biotechnology were
cultured in DMEM supplemented with 10\% fetal bovine serum, 100 units/mL
penicillin and 100 g/mL streptomycin at 37 C in 5\% CO2. siNR4a1 and
plasmid were Designed and produced by IGE biotechnology. The sequence of
siNR4a1: Forward:GCCAUUAGAUGAGACCUAUdTdT Reverse:AUAGGGUCUCA

UCUAAUGGGCdTdT. siRNA and plasmid Transfection was conducted by
lipofectamine 3000 (Thermo Fisher Scientific). After 40 hours
transfection, cells were stimulated by LPS for another 8 hours for
further testing.

\textbf{Quantitative reverse transcription polymerase chain reaction
(qRT-PCR) assay}

According to the manufacturer, total RNA was isolated from RAW264.7 cell
by TRIzol (Cwbio). RNA was translated into cDNA using PrimeScript RT
Master Mix(Takara Bio). The qRT-PCR was run on the LightCycler\selectlanguage{ngerman}® 480
Software using TB Green® Premix Ex Taq\selectlanguage{english} II (TaKaRa, Bio) with gene
specific primers. Gene expression level was calculated employing the
2\selectlanguage{english}\textsuperscript{-ΔΔCt} method. The primer sequences were listed
in\emph{Table S1} .

\textbf{Western Blot}

RAW264.7 cell were transfected by siRNA or plasmid, hydrolyzed in RIPA
lysis buffer for 30 minutes after stimulated with LPS for 8 hours. The
BCA protein were employed to detect the protein concentration, then
separated by 10\% SDS-PAGE. Membranes were blocked in 5\% milk for 1
hour and then treated with antibodies overnight. The primary antibodies
were: anti-PED4B antibody, anti-cAMP antibody, anti-NR4a1 antibody were
bought from Abcam Technology, USA. Anti-PKA antibody,
anti-phosphorylated-PKA antibody, anti-p65 antibody,
anti-phosphorylated-65 antibody, anti-I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α \selectlanguage{english}antibody, anti-phosphorylated
antibody, anti-GAPDH antibody were purchased from Cell Signaling
Technology, USA. After washed and incubated with mouse or rabbits
secondary antibodies for 1 hour, the protein bands were detected using
the enhanced chemiluminescence reagents.

Statistical analysis

All data were expressed as mean \selectlanguage{ngerman}±SD. One-way analysis of variance
(ANOVA) was employed to compare the multiple groups. Statistic package
for social science (SPSS) version. 13.0 was employed for statistical
analysis. When \emph{P} value \textless{} 0.05, the differences were
considered significant.

\textbf{Results}

\textbf{DEG identification, KEGG and GO Enrichment}

As displayed in heat map \emph{Fig S1} , there are 108 DEGs in the heat
map. KEGG and GO enrichment of DEGs were fabricated using DAVID online
database. As \emph{Fig1 (A,B,C)}shows, GO consisted of biological
process (BP), cellular components (CC), molecular functions (MF). BP
analysis of DEGs mainly shows signal transduction, positive regulation
of inflammatory response and chemokine production. For CC terms, DEGs
were enriched mainly in cytoplasm, membrane, and cytosol; while in
protein binding, protein kinase binding, Calcium ion binding and
collagen binding for MF. KEGG pathway shows the enrichment of DEGs
mainly in cytokine-cytokine receptor, JAK-STAT signaling pathway and
cAMP signaling pathway (\emph{Fig1 D} ).

\textbf{PPI network and Hubgenes}

All DEGs were analyzed by STRING online database. Using the Cytoscape
software, 49 DEGs were mapped to the PPI network (\emph{Fig 2} ). The
top 10 Hub genes referred to IL10, IFNG, Fos, IL19, NPY, Cnr1, PDE4B,
MMP13, Rtn1, UCHL1, which were assessed and ranked by the plugin
cytoHubba in Cytoscape (Table2)\textbf{.} Phosphodiesterase 4B (PDE4B),
a member of phosphodiesterase (PDEs) family, serves as a selectively
hydrolytic enzyme of second signal messenger cAMP(cyclic adenosine
monophosphate){[}15,25{]}, playing a vital role in signal transduction
of inflammation{[}15{]}. The inhibitor of PDE4 has demonstrated a
prominent validity in treating inflammatory disease such as asthma,
chronic obstructive pulmonary disease, inflammatory bowel disease,
atopic dermatitis, rheumatoid arthritis, and other disease{[}15,26{]}.
cAMP-activated PKA exerts an effect to inhibit NF\selectlanguage{greek}κ\selectlanguage{english}B signaling
pathway~{[}10,27,28{]}. PDE4B deficit macrophage exhibit the decreased
NF\selectlanguage{greek}κ\selectlanguage{english}B activation and reactive oxygen species level induced by LPS, and
the suppressed inflammatory response~{[}14,29,30{]}. The
anti-inflammatory ability of NR4a1 has association with NF\selectlanguage{greek}κ\selectlanguage{english}B signaling
pathway. Therefore, we speculate that NR4a1 may regulate the PED4B
expression with anti-inflammatory function and a close relation of
NR4a1-regulated PDE4B to cAMP-PKA signaling and NF\selectlanguage{greek}κ\selectlanguage{english}B signaling pathway.

\textbf{NR4a1 promotes the activation of cAMP-PKA signaling through
down-regulating PED4B expression.}

Our previous results implied a key role of PED4B in NR4a1-regulated
inflammatory macrophage. First, we established the NR4a1 silenced or
over-expressed RAW264.7 macrophage by transfecting pcDNA3.1-NR4a1 or
siNR4a1 (\emph{Fig S1} ). The expression of PDE4B was appraised by
western blot and RT-PCR. As shown in \emph{Fig3 (A,B),} It can be seen
that LPS prominently elevated the protein and mRNA level of PED4B.
Compared to the LPS group, pcDNA3.1-NR4a1 group exhibited the decreased
PED4B expression and the NR4a1 silenced group showed the increased. We
further evaluated the components of cAMP signaling pathway by western
blot(\emph{Fig2 C,D} ). LPS stimulation could significantly decrease the
cAMP, p-PKA protein level. pcDNA3.1-NR4a1group exhibited higher cAMP,
p-PKA protein level and the siNR4a1 group showed lower. However,
cotransfection of pcDNA3.1-PDE4B and pcDNA3.1-NR4a1 group exhibited the
decreased cAMP, p-PKA protein level compared to pcDNA3.1- NR4a1
group(\emph{Fig2 E,F} ) .

\textbf{NR4a1} \selectlanguage{greek}\textbf{supresses the LPS induced activation of NFκB
signaling pathway and the IL-6 and IL-1β mRNA level by down-regulating
PED4B expression}\selectlanguage{english}

Our previous discussion has been illustrated that the activation of NF\selectlanguage{greek}κ\selectlanguage{english}B
signaling pathway regulated by NR4a1 and cAMP activated PKA. NF-\selectlanguage{greek}κ\selectlanguage{english}B,
acting as a transcription factor, could manipulate a number of
proinflammatory cytokines expression such as IL-6 IL1\selectlanguage{greek}β\selectlanguage{english}~{[}31,32{]}. We
further detected the effects of NR4a1 and PDE4B on LPS induced
activation of NF\selectlanguage{greek}κ\selectlanguage{english}B and the expression of IL-6 and IL1\selectlanguage{greek}β. \selectlanguage{english}As depicted
in\emph{Fig4(A,B,E),} pcDNA3.1NR4a1 group displayed the decreased
protein level of p-p65 and p-I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α \selectlanguage{english}and mRNA level of IL-6 and IL-1\selectlanguage{greek}β \selectlanguage{english}and
the siNR4a1 group showed higher compared to LPS treated group. However,
the protein level of p-p65 and p-I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α \selectlanguage{english}and mRNA level of IL-6 and IL-1\selectlanguage{greek}β
\selectlanguage{english}of co-transfected group were higher than the pcDNA3.1-NR4a1
group(\emph{Fig4 C,D,F} ).

\textbf{Discussion}

Previous studies has shown the noticeable anti-inflammatory effects of
NR4a1. In this study, we explored the mechanism of NR4a1 through
bioinformatics analysis. We employed the GSE68167 dataset and selected
108 DEGs to be analyzed using DAVID and KEGG pathway and GO term
analysis. BP analysis mainly shows signal transduction, positive
regulation of chemokine production and inflammatory response. CC terms
displayed enrichment of DEGs mainly in cytoplasm, membrane, and cytosol;
while MF indicated the protein binding, calcium ion binding and callagen
binding. KEGG showed the enrichment of DEGs mainly in cytokine-cytokine
receptor, JAK-STAT signaling pathway and cAMP signaling pathway. We used
STRING and Cytoscape to construct the gene network, where 49 DEGs were
mapped and top Hub gene

IFNG, IL10, Fos, IL19, PDE4B, Cnr1, MMP13, NPY, Rtn1, UCHL1 were ranked
by CytoHubba. Among these Hub genes, IFNG encodes IFN\selectlanguage{greek}γ, \selectlanguage{english}IL10 and IFN\selectlanguage{greek}γ
\selectlanguage{english}are considered the important inflammatory cytokine and NR4a1 defects
macrophage showed the decreased IL10 while the increased IFN\selectlanguage{greek}γ
\selectlanguage{english}production~{[}10,33,34{]}. NR4a1 regulates the expression of early gene,
including Fos in cancer~{[}35{]}. Fos together with c-jun forms AP-1
plays a key role in inflammatory type macrophage~{[}36{]}. UCHL1 has
been reported to exert the anti-inflammatory effects via MAPK and NF\selectlanguage{greek}κ\selectlanguage{english}B
signaling pathway in LPS stimulated macrophage~{[}37{]}.
Phosphodiesterase 4B(PDE4B), a member of the phosphodiesterase (PDEs)
family, serving as a selectively hydrolytic enzyme of the second signal
messenger cyclic adenosine monophosphate (cAMP) to play a crucial role
in cAMP signaling pathway~{[}15,25{]}. PDE4B, NPY and FOS were found to
be enriched in cAMP signaling pathway, where the former is critical in
signal transduction of inflammation~{[}15,25,30{]}. The inhibitor of
PDE4B has been shown exert an effective validity in treating various
inflammatory diseases~{[}15{]}. PED4B defected macrophage shows the
decreased LPS induced NF\selectlanguage{greek}κ\selectlanguage{english}B activation and reactive oxygen species
level~{[}30,38{]}.

Our data demonstrated that NR4a1 showed the effects of increasing cAMP
and p-PKA levels through down-regulating PED4B expression. We also found
that NR4a1 can inhibit LPS induced NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling activation and the
mRNA level of IL-6 and IL-1\selectlanguage{greek}β \selectlanguage{english}by down-regulating PED4B expression. The
anti-inflammatory function of NR4a1 has been proved to be related to
restrict NF\selectlanguage{greek}κ\selectlanguage{english}B signaling pathway~{[}33,39{]} Interestingly, cAMP
activated PKA could effectively inhibit the NF\selectlanguage{greek}κ\selectlanguage{english}B regulated gene
transcription~{[}10,27{]}.Thus, we hypothesized that NR4a1 may regulates
the NF\selectlanguage{greek}κ\selectlanguage{english}B signaling pathway through PDE4B-cAMP-PKA signaling, which is
also worthy to further detecting. In addition, bioinformatics analysis
showed that the effect of NR4a1 in macrophage may be related to Fos,
UCHL1, MMP13. JAK-STAT signaling pathway and cytokine-cytokine
interaction may be regulated by NR4a1.

\textbf{Conclusion}

Among the NR4a1 mediated several pathways, NF\selectlanguage{greek}κ\selectlanguage{english}B signaling lies one of
the most crucial pathway. NR4a1 also manipulates the metabolism of
inflammatory response of immune cells. In our study, we found that NR4a1
can promotes the cAMP signaling pathway and inhibits the LPS induced the
activation of NF\selectlanguage{greek}κ\selectlanguage{english}B signaling and the mRNA level of IL-6 and IL-1\selectlanguage{greek}β \selectlanguage{english}by
down-regulating the PDE4B expression. Our study implied that NR4a1 plays
a protective role with the potential to serve as an excellent therapy
for inflammatory diseases.

\textbf{Funding}

The present study was supported by the Natural Sciences Foundation of
Hunan Province \emph{(g} \emph{rant no. 2019JJ50538, 2019JJ40268),}
Scientific Research Fund Project of Hunan Provincial Health Commission
B2019123.

\textbf{Author contributions}

Juntao Tan : Validation, visualization and completed manuscripts. Teng
Teng : Visualization and revised manuscripts. Chen Su, Tiegen Huang and
Zhenni Xiao: Data curation. Xiaotian Luo: Supervision. Yixin Tang:
Conceptualized projects and supervision.

\textbf{Declaration of Competing Interest}

None.

\textbf{Acknowledgements}

This work were supported by the Natural Sciences Foundation of Hunan
Province (2019JJ50538, 2019JJ40268) and Scientific Research Fund Project
of Hunan Provincial Health Commission (B2019123).

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