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\begin{document}

\title{Anti-inflammatory effect of BMP326, a novel benzothiazole derivative:
Possible involvement of the NF-\selectlanguage{greek}κ\selectlanguage{english}B and MAPKs Signaling Pathways in
LPS-induced RAW264.7 macrophages\selectlanguage{english}}



\author[1]{huijie Guo}%
\author[1]{xingyan Luo}%
\author[1]{shuxia Yang}%
\author[1]{yang Liu}%
\author[1]{chunfen Mo}%
\affil[1]{Chengdu Medical College}%


\vspace{-1em}



  
  \date{June 08, 2024}


\begingroup
\let\center\flushleft
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\endgroup





\selectlanguage{english}
\begin{abstract}
Background: Benzothiazole and its derivatives have been extensive
studied due to their versatile biological properties and pharmaceutical
applications. We recently found that the BMP326
(1-(1,3-benzothiazol-2-yl)-3-(2-methoxyphenyl)-1H-pyrazol-5-ol), a novel
benzothiazole derivative, have anti-inflammatory properties in
lipopolysaccharide(LPS)-induced RAW264.7 macrophages and there were no
relevant reports previously. Methods and Results: Treatment with
BMP326(5, 10 and 20 \selectlanguage{greek}μ\selectlanguage{english}M) can significantly inhibit nitric oxide
production and down-regulate mRNA expression of nitric oxide synthase
(iNOS) and cyclooxygenase-2 (COX-2) but did not cause significant
cytotoxicity on RAW264.7 macrophages. It is also observed BMP326 can
inhibit LPS-stimulated interleukin (IL)-6, interleukin(IL)-1\selectlanguage{greek}β, \selectlanguage{english}tumor
necrosis factor (TNF)-\selectlanguage{greek}α \selectlanguage{english}production depending on its dosage. The gene
transcription levels of IL-6, IL-1\selectlanguage{greek}β \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}were reduced under BMP326
exposure in LPS-treated RAW264.7 cells. In addition, we explored the
inhibitory mechanisms of BMP326 on the production of pro-inflammatory
mediators. The results showed that BMP326 inhibited nuclear factor kappa
B (NF-\selectlanguage{greek}κ\selectlanguage{english}B) activation by reducing the phosphorylation of p65 and I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α.
\selectlanguage{english}Moreover, the phosphorylation of p38 MAPK (p38), extracellular
signal-regulated kinases 1/2 (ERK1/2), and c-Jun N-terminal kinase (JNK)
in RAW264.7 cells which are stimulated with LPS were suppressed in a
dose-dependent manner. Conclusions: In summary, these results suggest
that BMP326 exerts anti-inflammatory properties via suppression of the
NF-\selectlanguage{greek}κ\selectlanguage{english}B and MAPKs signaling pathways.%
\end{abstract}%



\sloppy


\selectlanguage{greek}\textbf{Anti-inflammatory effect of BMP326, a novel benzothiazole
derivative: Possible involvement of the NF-κB and MAPKs Signaling
Pathways in LPS-induced RAW264.7 macrophages}\selectlanguage{english}

Hui-Jie Guo\textsuperscript{a\#}, Xing-Yan Luo\textsuperscript{a\#},
Shu-Xia Yang\textsuperscript{a\#}, Yang Liu\textsuperscript{a*} and
Chun-Fen Mo\textsuperscript{a*}

\textsuperscript{a}Center of Science and Research, Chengdu Medical
College, Chengdu, Sichuan, China

\textsuperscript{\#}Hui-Jie Guo, Xing-Yan Luo and Shu-Xia Yang
contributed equally to this work.

\emph{*Correspondence to} : Chun-Fen Mo, Center of Science and Research,
Chengdu Medical College, Xindu Avenue 783, Chengdu, Sichuan 610513,
China. E-mail: mochunfen@cmc.edu.cn. Yang Liu, Center of Science and
Research, Chengdu Medical College, Xindu Avenue 783, Chengdu, Sichuan
610513, China. E-mail:\emph{liuyang@cmc.edu.cn}

The authors' email address are as follows: Hui-Jie
Guo:\emph{guohuijie@cmc.edu.cn};Xing-Yan
Luo:\emph{luoxingyan@cmc.edu.cn},Shu-Xia Yang:
\emph{shuxiay@126.com},Yang Liu:\emph{liuyang@cmc.edu.cn},Chun-Fen Mo:
\emph{mochunfen@cmc.edu.cn} .

\textbf{Abstract}

\textbf{Background:} Benzothiazole and its derivatives have been
extensive studied due to their versatile biological properties and
pharmaceutical applications. We recently found that the BMP326
(1-(1,3-benzothiazol-2-yl)-3-(2-methoxyphenyl)-1H-pyrazol-5-ol), a novel
benzothiazole derivative, have anti-inflammatory properties in
lipopolysaccharide(LPS)-induced RAW264.7 macrophages and there were no
relevant reports previously.

\textbf{Results:} Treatment with BMP326(5, 10 and 20 \selectlanguage{greek}μ\selectlanguage{english}M) can
significantly inhibit nitric oxide production and down-regulate mRNA
expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)
but did not cause significant cytotoxicity on RAW264.7 macrophages. It
is also observed BMP326 can inhibit LPS-stimulated interleukin (IL)-6,
interleukin(IL)-1\selectlanguage{greek}β, \selectlanguage{english}tumor necrosis factor (TNF)-\selectlanguage{greek}α \selectlanguage{english}production depending
on its dosage. The gene transcription levels of IL-6, IL-1\selectlanguage{greek}β \selectlanguage{english}and TNF-\selectlanguage{greek}α
\selectlanguage{english}were reduced under BMP326 exposure in LPS-treated RAW264.7 cells. In
addition, we explored the inhibitory mechanisms of BMP326 on the
production of pro-inflammatory mediators. The results showed that BMP326
inhibited nuclear factor kappa B (NF-\selectlanguage{greek}κ\selectlanguage{english}B) activation by reducing the
phosphorylation of p65 and I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α. \selectlanguage{english}Moreover, the phosphorylation of p38
MAPK (p38), extracellular signal-regulated kinases 1/2 (ERK1/2), and
c-Jun N-terminal kinase (JNK) in RAW264.7 cells which are stimulated
with LPS were suppressed in a dose-dependent manner.

\textbf{Conclusions:} In summary, these results suggest that BMP326
exerts anti-inflammatory properties via suppression of the NF-\selectlanguage{greek}κ\selectlanguage{english}B and
MAPKs signaling pathways.

\textbf{Keywords:} BMP326; LPS-induced RAW264.7 macrophages; NF-\selectlanguage{greek}κ\selectlanguage{english}B ;
MAPKs;

\textbf{Background}

Inflammation is an essential biological protective mechanism against
various foreign infection or physical damage. The key features are
redness, heat, swelling, pain and impaired function {[}1{]}.Macrophages
as the vital immune cells play an important role in host defense through
producing inflammatory mediators {[}2{]}. However, the chronic
inflammation characterized by prolonged overproduction of inflammatory
mediators may aggravate several system disorder and diseases such as
rheumatoid arthritis{[}3{]}, cancer{[}4{]}, bacterial sepsis{[}5{]},type
2 diabetes{[}6{]}, hearing loss{[}7{]}, heart failure{[}8{]} and
autoimmune diseases{[}9{]}. Anti-inflammatory drugs are widely used in
the clinic, but many drugs are notoriously known for their side effects
or individual differences among patients. Therefore, it is imperative to
explore a novel low toxicity and effective anti-inflammation drug to
relieve patient pain.

Lipopolysaccharide (LPS), the structural component of Gram-negative
bacteria's outer membrane, can initiate the inflammatory cascades via
Toll-like receptors 4 (TLR4) in macrophages. LPS stimulated
RAW264.7macrophages that producing inflammatory cytokines as an
inflammatory model that is extensively used {[}10{]}. Nuclear factor-\selectlanguage{greek}κ\selectlanguage{english}B
(NF-\selectlanguage{greek}κ\selectlanguage{english}B) activation and Mitogen-activated protein kinases (MAPKs) pathway
has been recognized as the classical signaling pathway that involved in
the inflammatory response. MAPKs (Mitogen-activated protein kinases)
contain three families (extracellular signal-regulated kinase
{[}ERK{]}1/2, p38, and c-Jun N-terminal kinase {[}JNK{]}).Some or even
all of them are involved in regulating of the pro-inflammatory cytokine
production{[}11{]}.Furthermore, activated NF-\selectlanguage{greek}κ\selectlanguage{english}B as one of the most
important regulators reacts to the extra-and intra-cellular stimuli
through up regulating the expression of various pro-inflammatory
mediators{[}12{]}. There have been many reports about these two
prototypical pro-inflammatory signaling pathways {[}13, 14{]}.

Benzothiazole is an aromatic heterocyclic compound which has been
researched wildly due to its extensive pharmaceutical applications.
Benzothiazole and its derivatives show a wide range of biological
activities, such as anticancer{[}15-17{]}, anti-inflammatory{[}18-20{]},
immunosuppressive{[}21,22{]}, antitubercular{[}23{]},
antidiabetic{[}24{]}, antimicrobial{[}25{]}, analgesic{[}26{]},
antiviral{[}27{]}, antimalarial {[}28{]}, anticonvulsant{[}29{]}and
other activities{[}30{]}.Some drugs derived from benzothiazole have been
used in clinical treatment of various diseases, e.g ethoxzolomide serves
as diuretic. Frentizole is used as an antiviral as well as
immunosuppressive agent. Tiaramide is an Anti-inflammatory agent.
Zopolrestat is an important drug with anti diabetic effect{[}31{]}.
Meanwhile, some Benzothiazole derivatives like Pramipexole and
Riluzoleare still in clinical trials{[}32{]}. To discover new
derivatives of benzothiazole with anti-inflammatory activity, a series
of benzothiazole compounds were synthesized and screened. We found that
BMP326 is one of the most effective and no related preceding research is
observed.

In this study, we indicate the anti-inflammatory effects and investigate
the possible mechanism underlying the action of BMP326in LPS-induced
RAW264.7 macrophages. It inhibited the production of inflammation medium
(NO, IL-6, IL-1\selectlanguage{greek}β, \selectlanguage{english}TNF\selectlanguage{greek}α\selectlanguage{english}) and decreased the gene transcription levels of
iNOS, IL-6, IL-1\selectlanguage{greek}β \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}in LPS-treated RAW264.7 cells. The mechanism
of its anti-inflammatory effects of BMP326 is involved in the NF-\selectlanguage{greek}κ\selectlanguage{english}B and
MAPKs signaling pathways. Potentially, BMP326 may be used as a lead
compound for design and development of new anti-inflammatory drugs for
therapeutic use in inflammatory diseases.

\textbf{Materials and methods}

\textbf{Reagents and Antibody}

BMP326 (Fig.1a) was purchased from ChemBridge Corp (San Diego, CA, USA).
We dissolved it in dimethyl sulfoxide (DMSO) and stored in -80 at a
concentration of 40mM. Lipopolysaccharide (LPS, E.coli O55:B5) was
purchased from Sigma--Aldrich and which was dissolved in PBS as a 1\selectlanguage{greek}μ\selectlanguage{english}g/ml
stock solution. The following rabbit monoclonal antibodies for western
blot assay were purchased from Cell Signaling Technology:
anti-phospho-p65, anti-p65, anti-phospho-I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α, \selectlanguage{english}anti- I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α,
\selectlanguage{english}anti-phospho-p38MAPK, anti-p38, anti-phospho-Erk1/2, anti-Erk1/2,
anti-phospho-JNK, anti-JNK, anti-GAPDH and HRP-goat anti-Rabbit IgG. The
antibodies for ELISA assays were purchased from eBioscience, include
mouse TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6 and HRP-linked antibody.

\textbf{Cell culture}

The murine macrophage cell line RAW264.7 was obtained from Key
Laboratory of Biotherapy (Chengdu Sichuan University, China) and
cultured in RPMI1640 (SH30809.01B, Hyclone) supplemented with 10\% fetal
bovine serum (FBS) (SFBS, BOVOGEN) at 37 in a 5\% CO2 incubator. Cells
were routine sub-cultured after reaching 80\%--90\% confluence.

\textbf{Cell viability assay}

To evaluate the cytotoxicity of BMP326 on RAW264.7 macrophages, the cell
viability was determined by using the Cell Counting Kit (Dojingdo,
Kumamoto, Japan). Briefly, Raw264.7 macrophages were seeded in 96-well
plates at a density of 1\selectlanguage{ngerman}×10\textsuperscript{5}cells/well and incubated
at 37\selectlanguage{english}overnight. Then the cells were treated with or without BMP326
(0-40\selectlanguage{greek}μ\selectlanguage{english}M) at different concentrations for 24 h. For additional
incubation, CCK-8(10 \selectlanguage{greek}μ\selectlanguage{english}l/well) solution was added to the wells and
incubated for another 4 h. The absorbance was measured at a wavelength
of 450 nm using SpectraMax M5 microplate reader (Sunnyvale, CA, USA).

\textbf{Measurement of NO production}

TheGriess Reagent Kit (Beyotime, Shanghai, China) was used to detect the
NO production according to the manufacturer's instructions. Briefly,
Raw264.7 macrophages were seeded in 96-well plates at a density of
1\selectlanguage{ngerman}×10\textsuperscript{5} cells/well and incubated at 37\selectlanguage{english} overnight. Cells
were treated with different concentrations of BMP326 (0-20\selectlanguage{greek}μ\selectlanguage{english}M) for 3 h
then added LPS (100 ng/ml) for another 24 h. Subsequently culture
supernatant was collected and mixed with Griess reagent. The absorbance
was measured at a wavelength of 540 nm by using microplate reader.
Obtain the output of NO produced through the standard curve.

\textbf{Quantitative Real-Time polymerase chain reaction}

RAW264.7 macrophages were seeded in 6-well plates at a density of
1\selectlanguage{ngerman}×10\textsuperscript{6} cells/well and incubated at 37\selectlanguage{english} overnight. Cells
were pretreated with BMP326(0-20\selectlanguage{greek}μ\selectlanguage{english}M) for 3 h prior to adding 100 ng/ml
LPS for another 24 h. Total RNA were extracted by Trizol Reagent
(TaKaRa, Japan) according to the manufacturer's protocol. Concentration
and purity of total RNA were measured with NanoDrop 2000
Spectrophotometer (Thermo scientific, Wilmington , DE, USA).1\selectlanguage{greek}μ\selectlanguage{english}g total
RNA was reverse transcribed to cDNA following the PrimeScript RT
reagent kit (TaKaRa). Universal Probe Library and FastStart Universal
Probe Master (ROX) were purchased from Roche for RT-PCR analyses. The
relative genes expressions level were quantitated by the CFX Connect
Real-Time System (Bio-Rad Laboratories, USA). All the sequences of
primers used in this study were synthesized by Sangon Biotech (Shanghai,
China), the sequences information are listed in Supplemental Table 1.

\textbf{Enzyme-linked immunosorbent assay (ELISA)}

RAW264.7 macrophages were seeded in 96-well plates at a density of
1\selectlanguage{ngerman}×10\textsuperscript{5} cells/well and incubated at 37\selectlanguage{english} overnight. Cells
were pretreated with BMP326 at different concentrations (0-20\selectlanguage{greek}μ\selectlanguage{english}M) for 3 h
prior to adding 100 ng/ml LPS for another 24 h. IL-6, IL-1\selectlanguage{greek}β \selectlanguage{english}and TNF-\selectlanguage{greek}α
\selectlanguage{english}secretion levels were detected in culture supernatant by commercial
ELISA kits (Multi Sciences, China) according to the manufacturer's
instructions. Ultimately, the absorbance was determined with a
microplate reader at 492 nm. Three replicates were conducted for each
sample.

\textbf{Western Blot Analysis}

RAW264.7 macrophages were seeded in 6-well plates at a density of
2\selectlanguage{ngerman}×10\textsuperscript{6} cells/well and incubated in low serum medium
(1640+1\%FBS) at 37\selectlanguage{english} overnight. The cells were pretreated in the absence
or presence of BMP326 (0-20\selectlanguage{greek}μ\selectlanguage{english}M) for 2hr and then stimulated with or
without LPS (100ng/ml) for another 30min. Discarded the medium and
rinsed cells with the ice-cold PBS before added Lysis buffer with
phosphatase and protease inhibitors (Thermo Scientific, USA). Whole cell
lysates were centrifuged at 13,000 g for 15 min at 4\selectlanguage{ngerman}°C. Protein
concentrations were measured by the BCA Protein Assay kit (BioTeke,
China). Equal amounts of protein(30\selectlanguage{greek}μ\selectlanguage{english}g) were denatured in 5\selectlanguage{ngerman}×SDS loading
buffer at 100\selectlanguage{english}@C for 10 min. Different proteins were separated on 30\%
SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride
(PVDF) membrane (Millipore, USA). After blocking for 1 h in 5\% skim
milk, the membranes were incubated with specific primary antibodies
overnight at 4. The dilution ratio was 1:000 except rabbit anti-GAPDH
for 1:3000. After washing 3 times with TBST buffer, the membranes were
incubated with the secondary antibodies (1:3000) for 1 h at room
temperature following washing 3 times with TBST buffer again. The
protein bands were revealed by chemiluminescence (Millipore, USA) and
detected by ChemiDoc XRS+ (Bio-Rad, USA).

\textbf{Statistical analysis}

Data are expressed as means \selectlanguage{ngerman}± SD, Statistical analyses was performed
using GraphPad Prism 6 (San Diego, USA).Student's t-tests were performed
to determine differences between the BMP326 treatment group and control
groups. P \textless{}0.05 was considered as statistically significant.

\textbf{Results}

\textbf{The cytotoxic effects of BMP326 on RAW264.7 cell viability}

To confirm the optimal condition of LPS on RAW264.7 macrophages, NO
production were detected at 24h after treated with different
concentrations of LPS, and 100ng/ml is the optimal stimulate condition
(Fig 1b). The CCK-8 assay was performed at 24 h after treatment with the
indicated concentrations of BMP326 (0 to 40\selectlanguage{greek}μ\selectlanguage{english}M) and 100ng/ml LPS to
determine the effect of BMP326 on RAW264.7 cell viability. BMP326 at
40\selectlanguage{greek}μ\selectlanguage{english}M showed obvious cytotoxic effects but BMP326 (0-20\selectlanguage{greek}μ\selectlanguage{english}M) did not cause
significant cytotoxicity on RAW264.7 macrophages (Fig 1c). Therefore,
LPS (100ng/ml) and BMP326 (0-20\selectlanguage{greek}μ\selectlanguage{english}M) were conducted for the following
experiments in this study.

\textbf{Effect of BMP326 onNO production and the mRNA expression level
of iNOS and COX-2 in LPS-stimulated RAW264.7 cell lines}

As an initial indicator of the anti-inflammatory effects of BMP326 on
LPS stimulated RAW264.7 cells, iNOS and its mediated pro-inflammatory
mediator COX-2 induction and NO production were determined using the
qRT-PCR and Griess reagent separately{[}33, 34{]}.Compared with LPS
stimulation only,BMP326 markedly inhibited iNOS mRNA expression, NO
production and COX-2 mRNA expression level also reduced in concentration
dependent manner (Fig. 2a, 2b and 2c).These results showed that BMP326
has a preliminary anti-inflammatory effect in this inflammatory model.

\selectlanguage{greek}\textbf{Effect of BMP326 onpro-inflammatory mediator TNF-α, IL-6, and
IL-1β production in LPS-stimulated RAW264.7 cells}\selectlanguage{english}

To further determine whether BMP326 has anti-inflammatory activity, we
detected several critical pro-inflammatory cytokines including IL-6,
IL-1\selectlanguage{greek}β \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}which are involved in multiple inflammatory
pathways{[}35{]}. Both the mRNA and protein expression levels of these
pro-inflammatory mediator were significantly increased in LPS-stimulated
RAW264.7 cells. But pre-treatment of the BMP326 inhibited the mRNA
expression levels of IL-6, IL-1\selectlanguage{greek}β \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}(Fig. 3a, 3b and 3c). As our
prediction, similar results showed that the secretion of TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-6and
IL-1\selectlanguage{greek}β \selectlanguage{english}in culture supernatant were considerably decreased by BMP326 in a
dose-dependent manner (Fig. 3d, 3e and 3f). These results confirm that
BMP326 has effective anti-inflammatory properties in LPS-induced
RAW264.7 cells.

\selectlanguage{greek}\textbf{BMP326 might have anti-inflammatory effects through the NF-κB
pathway}\selectlanguage{english}

Previous studies have showed that NF-\selectlanguage{greek}κ\selectlanguage{english}B as a well-known classical
transcription factor involved in regulating the expression of
inflammatory mediators{[}36{]}. NF-\selectlanguage{greek}κ\selectlanguage{english}B as an inactive complex exists
within the cytoplasm of unstimulated normal cells. p65 subunit is an
important component of NF-\selectlanguage{greek}κ\selectlanguage{english}B which generally bound to the I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α \selectlanguage{english}family.
The I\selectlanguage{greek}κ\selectlanguage{english}B kinase complex (IKK), as an important upstream kinase to
phosphorylate I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α \selectlanguage{english}and then I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α \selectlanguage{english}degradation from p65 when cells are
stimulated by LPS. Activated NF-\selectlanguage{greek}κ\selectlanguage{english}B p65 translocate to nuclei and trigger
the transcription of downstream proinflammatory signaling{[}37{]}. As
shown in the previous results, we detected the highest level of
phospho-NF-\selectlanguage{greek}κ\selectlanguage{english}B p65 protein occurred at 30min after LPS stimulating.
However, BMP326 markedly attenuated the LPS-induced phospho-NF-\selectlanguage{greek}κ\selectlanguage{english}B p65
and phospho-I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α \selectlanguage{english}expression in a concentration-dependent manner compared
with LPS-stimulated along (Fig.4a and 4b). These results suggest that
the anti-inflammatory effect of BMP326 in LPS stimulated RAW264.7 cells
might related to the NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway.

\textbf{BMP326 might have anti-inflammatory effects through the MAPK
pathway}

Multiple studies have found that MAPK signaling pathway plays critical
role in LPS-stimulated RAW264.7 macrophages{[}38{]}. So we proceeded to
analyze whether BMP326 inhibited inflammatory cytokines via the MAPK
signaling pathways. Compared to the blank group, the highest level of
phosphorylation p38MAPK, JNK, ERK1/2 were detected at 30min after LPS
stimulating (Previous research results showed). However, pretreatment
with BMP326 in different concentration can reversed the phosphorylation
levels of p38 MAPK, JNK and ERK and resulted in 61.7\%, 57.9\% and
80.6\% of inhibition respectively (Fig.4d, 4e and 4f). These results
indicate that BMP326 inhibits inflammatory cytokines expression through
MAPK signaling pathways in LPS-induced RAW264.7 macrophages.

\textbf{Discussion}

Chronic inflammation threatens human health which is characterized by
tissue infiltration by macrophages and lymphocytes {[}39{]}. Among the
efforts to identify novel candidates for the treatment of inflammatory
diseases, we investigated the anti-inflammatory effects of BMP326 - a
novel benzothiazole derivative in LPS-induced RAW264.7 macrophages. Our
results demonstrated that BMP326 with the high anti-inflammatory
activity could effectively suppress the inflammatory cytokines
production. And preliminary molecular mechanisms underlying these
effects involved in NF-\selectlanguage{greek}κ\selectlanguage{english}B and MAPK signaling pathways in RAW264.7
macrophage cells.

NO over production during a variety of inflammatory diseases as well as
the macrophages stimulated by LPS. iNOS as one isoform of nitric oxide
synthase (NOS) family plays a crucial part in production of NO{[}40{]}.
Therefore, a good strategy to control the inflammation is to reduce the
NO production through suppression of iNOS gene expression. In our
results, BMP326 significantly inhibited iNOS mRNA expression and NO
production but without cytotoxicity in 20\selectlanguage{greek}μ\selectlanguage{english}M (Fig1, Fig2A,2B).
Cyclooxygenase (COX) involved in the prostaglandin biosynthesis and
plays an important role in the development of inflammation. This enzyme
exists as COX-1 and COX-2 isomers.COX-1 constitutively expressed in most
tissues and organs and responsible for maintaining homeostasis of
physiology include gastric and renal integrity whereas COX-2 induces
inflammatory conditions. Many traditional Nonsteroidal anti-inflammatory
drugs (NSAIDs) are non-selective inhibitors for both COX-1 and COX-2,
thereby accompanied by side effects such as gastrointestinal erosion and
renal damage {[}41, 42{]}.So an ideal strategy for the reduction of
inflammation is to screen selective COX-2 inhibitors. Our results
displayed that BMP326 can effectively reduce the COX-2 mRNA expression
in a dose-dependent manner but it has no effect on the expression of
COX-1(Fig2C, 2D). Pro-inflammatory cytokines released is the typical
manifestation in the early stages of inflammation, and played important
roles in development of the inflammatory process. As macrophages express
IL-6 when they are induced by microbes or LPS and then regulate
production of TNF-\selectlanguage{greek}α \selectlanguage{english}and IL-1\selectlanguage{english}\selectlanguage{greek}β{[}43{]}. TNF-\selectlanguage{greek}α \selectlanguage{english}is another early responsive
pro-inflammatory cytokines which leads to the activation of the
downstream inflammation-related signaling pathways{[}44{]}.IL-1\selectlanguage{greek}β \selectlanguage{english}as a
key proinflammatory cytokines produced by the inflammasome, which
involved in a number of inflammatory diseases{[}45{]}. Increasing
evidences suggest that inhibited the production of inflammatory
cytokines is becoming a therapeutic strategy. As shown by our results,
LPS significantly induced the mRNA expressions and cytokine secretions
of IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, and TNF-\selectlanguage{greek}α\selectlanguage{english}compared with the control group, but BMP326
inhibited the expression of pro-inflammatory cytokines in a
dose-dependent manner (Fig3A-F). Consequently, BMP326 can be used as a
relatively safe and effective anti-inflammation drug.

Then we explored the potential signaling pathway. Our research utilized
Raw264.7 macrophages which are stimulated with LPS as the inflammatory
model to explore the anti-inflammatory effect of BMP326. LPS as the
outer membrane structural component of Gram-negative bacteria can be
recognized by TLR4 in macrophages and then activating the downstream
signaling pathways{[}46{]}. Many studies have shown that nuclear
factor-\selectlanguage{greek}κ-\selectlanguage{english}gene binding (NF-\selectlanguage{greek}κ\selectlanguage{english}B) and mitogen activated protein kinase
(MAPK) signaling pathways play a crucial part in inflammation
regulation. Activated NF-\selectlanguage{greek}κ\selectlanguage{english}B translocated to the nucleus and bind to
specific DNA binding site then promotes the transcription of
inflammatory genes including iNOS, COX-2, IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, TNF\selectlanguage{english}\selectlanguage{greek}α{[}38{]}.The
MAPK family includes three members (ERK, p38, and JNK). LPS-induced
phosphorylation of these MAPKs increased the expression of iNOS, COX-2,
and inflammatory cytokines in RAW264.7 cells{[}47{]}. Available results
indicated that LPS induced phosphorylation of I\selectlanguage{greek}κ\selectlanguage{english}B-\selectlanguage{greek}α, \selectlanguage{english}P65, JNK, P38 and
ERK are significantly reduced after pretreatment with BMP326.
Admittedly, it is unclear whether there are other regulatory mechanisms
for BMP326 to exert anti-inflammatory effects. Further experiments are
needed to verify this.

\textbf{Conclusions}

Until now, the anti-inflammation effect of BMP326 has not been explored
. We firstly demonstrated that BMP326 exerts anti-inflammatory
properties and clarified the potential mechanism(Fig5). Therefore, these
results suggest that BMP326 may be used as a lead compound for design
and development of new anti-inflammatory drugs for therapeutic use in
inflammatory diseases.

\textbf{Declarations}

\textbf{Ethics approval and consent to participate:} Not applicable

\textbf{Consent for publication:}Not applicable

\textbf{Availability of data and materials:} All data generated or
analysed during this study are included in this published article {[}and
its supplementary information files{]}.

\textbf{Funding:} This work was supported by the Project of Clinical
Science Research Fund of Chengdu Medical College(Grant no.
2022LHZYYB-06)and the Project of Chengdu Health and Family Planning
Commission(Grant no. 2021076).

\textbf{Competing Interests:} The authors declare that they have no
competing interests.

\textbf{Acknowledgements:} Not applicable

\textbf{Figures}

Fig.1 Effects of BMP326 on the viability of RAW264.7 macrophages. (a)
Chemical structure of BMP326. (b)RAW264.7 macrophages were treated with
different concentrations of LPS(0-1000ng/ml) for 24 h, Griess Reagent
Kit detected that 100ng/ml LPS is the optimal stimulate condition for
following researches. (c) RAW264.7 macrophages were pretreated with
BMP326 (0-20 \selectlanguage{greek}μ\selectlanguage{english}M) for 3 h and were stimulated with or without LPS (100
ng/ml) for another 24 h. The cells viability was determined by CCK-8
assays. \#P \textless{} 0.05 vs. Control group; **P \textless{} 0.01 vs.
LPS alone.

Fig.2 BMP326 inhibited LPS-induced iNOS gene expression and NO synthesis
in RAW264.7 macrophages. RAW264.7 Macrophages were pretreated with or
without various concentrations of BMP326(0-20 \selectlanguage{greek}μ\selectlanguage{english}M) for 3 h before LPS(100
ng/ml) stimulation for another 24 h.(a) The culture medium was used for
measuring the level of NO by Griess reagents.(b,c,d) The mRNA levels of
iNOS, COX-1 and COX-2 were measured by qRT-PCR.Values are referred as
mean \selectlanguage{ngerman}± SD,\#P \textless{} 0.05 vs. Control group,*P \textless{} 0.05 vs.
LPS alone, **P \textless{} 0.01 vs. LPS alone.

Fig.3 BMP326 inhibited the inflammatory cytokines secretion and
expression of mRNA in LPS-induced RAW264.7 macrophages. RAW264.7
macrophages were treated as described before (a,b,c). The mRNA levels of
IL-6 , TNF-\selectlanguage{greek}α \selectlanguage{english}and IL-1\selectlanguage{greek}β \selectlanguage{english}were measured by qRT-PCR. (b) The effects of
BMP326 on secretion of IL-6 , TNF-\selectlanguage{greek}α \selectlanguage{english}and IL-1\selectlanguage{greek}β\selectlanguage{english}were measured by ELISA.
Values are referred as mean \selectlanguage{ngerman}± SD, \#\#P \textless{} 0.01 vs. Control
group,*P \textless{} 0.05 vs. LPS alone, **P \textless{} 0.01 vs. LPS
alone.

Fig.4 Effects of BMP326 on the NF-\selectlanguage{greek}κ\selectlanguage{english}B and the MAPK signaling pathways in
LPS-induced RAW264.7 macrophages. Cells were pretreated with the
indicated concentrations(0-20 \selectlanguage{greek}μ\selectlanguage{english}M) of BMP326 for 3 h and then stimulated
with LPS (100ng/mL) for another 30 min. Protein lysate were harvested
and analyzed by western blot with specific antibodies.(a) Protein levels
of phospho-p65 were measured.(b)Protein levels of phospho-I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α \selectlanguage{english}were
measured. Protein levels of phospho-ERK(c),phospho-JNK(d) and
phospho-p38(e) were measured. GAPDH was used as an internal standard
control and blot bands densitometry were analyzed with Image J software.
Values are referred as mean \selectlanguage{ngerman}± SD, \#\#P \textless{} 0.01 vs. Control
group,*P \textless{} 0.05 vs. LPS alone, **P \textless{} 0.01 vs. LPS
alone.

Fig.5 The possible molecular mechanism underlying the anti-inflammatory
effect of BMP326. BMP326 suppressed the production of inflammatory
mediators and pro-inflammatory cytokines through inhibition of NF-\selectlanguage{greek}κ\selectlanguage{english}B and
MAPKs signaling pathway in LPS-stimulated RAW264.7 cells.

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