\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[greek,ngerman,english]{babel}



\usepackage{float}








\begin{document}

\title{Empagliflozin ameliorated chondrocytes inflammation, catabolism and
senescence and osteoarthritis via suppressing the nuclear factor kappa-B
signal pathway.}



\author[1]{Bin Ru}%
\author[2]{Zhonggai Chen}%
\author[1]{Langhai Xu}%
\author[1]{Shun Li}%
\author[1]{Wenjun Cai}%
\affil[1]{Zhejiang Provincial People's Hospital}%
\affil[2]{Yuying Children's Hospital of Wenzhou Medical College}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Background Extracellular matrix (ECM) degradation, chondrocyte
inflammation, and cellular senescence contribute to the pathology of
osteoarthritis (OA). Empagliflozin, a selective inhibitor of
sodium-glucose cotransporter-2 (SGLT2), has been reported to show the
anti-inflammatory properties in several conditions. However, whether
empagliflozin can be used to improve OA is still unknown. Methods The
protective effects and underlying mechanism of empagliflozin in OA were
investigated in vitro and in vivo. Cell viability, catabolic markers,
inflammatory mediators, cellular senescence level, cartilage
degeneration were evaluated by CCK-8 assay, safranine O staining, ELISA,
real-time PCR, western blot, \selectlanguage{greek}β-\selectlanguage{english}galactosidase Staining, and histological
analysis. Results We found that empagliflozin significantly
downregulated the expression of catabolic enzymes (MMP9 and MMP13), and
decreased the expression of inflammatory mediators (NO, PGE2, IL-6,
COX2, and INOS), and reduced the cellular senescence level in
IL-1\selectlanguage{greek}β-\selectlanguage{english}treated chondrocytes by inhibiting the nuclear factor kappa-B
(NF-\selectlanguage{greek}κ\selectlanguage{english}B) signaling pathway. What's more, empagliflozin prevented
cartilage degeneration in DMM-induced OA mice model with significant
lower OARSI grade. Conclusion Our findings reveal that empagliflozin
inhibited chondrocytes ECM degradation, inflammation and cellular
senescence in vitro and prevented cartilage degeneration in vivo by
suppressing NF-\selectlanguage{greek}κ\selectlanguage{english}B signal pathway, indicating a therapeutic potential in
OA treatment.%
\end{abstract}%



\sloppy


\textbf{Empagliflozin ameliorated chondrocytes inflammation, catabolism
and senescence and osteoarthritis via suppressing the nuclear factor
kappa-B signal pathway.}

\textbf{Bin Ru\textsuperscript{1\#}, Zhonggai Chen\textsuperscript{2\#},
Langhai Xu\textsuperscript{1\#}, Shun Li\textsuperscript{1*}, Wenjun
Cai\textsuperscript{1*}}

\textsuperscript{1}Department of Pain, Zhejiang Provincial People's
Hospital, People's Hospital of Hangzhou Medical College, Hangzhou,
China.

\textsuperscript{2}The Second Affiliated Hospital and Yuying Children's
Hospital of Wenzhou Medical University, Wenzhou, China.

\textsuperscript{\#} Bin Ru, Zhonggai Chen, Langhai Xu contributed
equally to this work

\textsuperscript{*} Correspondence to:

Wenjun Cai (caiwj0302@163.com) and Shun Li (1148449287@qq.com),
Department of Pain, Zhejiang Provincial People's Hospital, People's
Hospital of Hangzhou Medical College, Hangzhou, China.

\textbf{Contributions:} Wenjun Cai \& Shun Li: Conceptualization,
Methodology. Bin Ru: Project administration, Investigation. Zhonggai
Chen: Writing Original Draft, Data Curation. Langhai Xu: Acquisition,
Analysis and interpretation of data.

\textbf{ACKNOWLEDGMENTS:} This study was supported by grants from the
Department of Education of Zhejiang Province (NO. Y202044592), the
Natural Science Foundation of Zhejiang Province (NO. LGF20H310004) and
the National Natural Science Foundation of China (NO. 81901122).

\textbf{CONFLICT OF INTERESTS:} The authors declare that there is no
conflict of interests.

\textbf{DATA AVAILABILITY STATEMENT:} The raw data of this study are
available from the corresponding author upon reasonable request.

\textbf{Abstract}

\textbf{Background}

Extracellular matrix (ECM) degradation, chondrocyte inflammation, and
cellular senescence contribute to the pathology of osteoarthritis (OA).
Empagliflozin, a selective inhibitor of sodium-glucose cotransporter-2
(SGLT2), has been reported to show the anti-inflammatory properties in
several conditions. However, whether empagliflozin can be used to
improve OA is still unknown.

\textbf{Methods}

The protective effects and underlying mechanism of empagliflozin in OA
were investigated in vitro and in vivo. Cell viability, catabolic
markers, inflammatory mediators, cellular senescence level, cartilage
degeneration were evaluated by CCK-8 assay, safranine O staining, ELISA,
real-time PCR, western blot, \selectlanguage{greek}β-\selectlanguage{english}galactosidase Staining, and histological
analysis.

\textbf{Results}

We found that empagliflozin significantly downregulated the expression
of catabolic enzymes (MMP9 and MMP13), and decreased the expression of
inflammatory mediators (NO, PGE2, IL-6, COX2, and INOS), and reduced the
cellular senescence level in IL-1\selectlanguage{greek}β-\selectlanguage{english}treated chondrocytes by inhibiting
the nuclear factor kappa-B (NF-\selectlanguage{greek}κ\selectlanguage{english}B) signaling pathway. What's more,
empagliflozin prevented cartilage degeneration in DMM-induced OA mice
model with significant lower OARSI grade.

\textbf{Conclusion}

Our findings reveal that empagliflozin inhibited chondrocytes ECM
degradation, inflammation and cellular senescence in vitro and prevented
cartilage degeneration in vivo by suppressing NF-\selectlanguage{greek}κ\selectlanguage{english}B signal pathway,
indicating a therapeutic potential in OA treatment.

\textbf{Keywords:} empagliflozin; chondrocyte; inflammation; senescence;
NF-\selectlanguage{greek}κ\selectlanguage{english}B.

\textbf{1 Introduction}

Osteoarthritis (OA) is a common joint disease with high rate of
incidence and wide coverage in the elderly population, which causes a
severe economic burden on families and society \textsuperscript{1,2}.
Current research has shown that OA is characterized by cartilage
degradation, underlying bone remodeling, and synovial
inflammation\textsuperscript{3}. Several risk factors, such as age,
gender, obesity and genetics, may affect the development of OA
\textsuperscript{2,4}. Nevertheless, the underlying molecular mechanism
among OA remains obscure.

Increasing evidence suggests that low-grade inflammation is a key
mediator of the pathogenesis of OA \textsuperscript{5}. Chondrocytes, as
the only cellular component of cartilage, play a central role in the
synthesis of cartilage matrix. OA is defined as the accumulation of
degenerative factors leading to the decease of chondrocytes and the
degradation of extracellular matrix (ECM). During OA, pro-inflammatory
factors, such as IL-1\selectlanguage{greek}β, \selectlanguage{english}TNF-\selectlanguage{greek}α, \selectlanguage{english}trigger a series of pathogenic reactions
of chondrocytes, promoting inflammation, senescence, and
apoptosis\textsuperscript{6}. Current treatment is aimed at relieving
the symptoms of OA but cannot slow down disease progression. Therefore,
it is necessary to further facilitate identification of emerging
pharmaceutical therapies.

Empagliflozin (Empa), a selective inhibitor of sodium-glucose
cotransporter-2 (SGLT2), lowers blood sugar by reducing glucose
reabsorption in the renal tubules. Various studies showed favorable
effects of Empa on glycaemic improvement, body weight, blood pressure,
arterial stiffness, and endothelial function \textsuperscript{7-9}. In
recent years, Empa has been reported to regulate macrophage polarity and
exert anti-inflammatory effects \textsuperscript{10}. Also, Empa has
hepatic protective effect against cholestatic liver injury via its
anti-oxidant and anti-inflammatory properties \textsuperscript{11}.
However, the effect of Empa on inflammation and senescence in OA has not
been reported yet

In our study, we hypothesized that Empa might inhibit inflammation in
chondrocytes during the process of OA. We concentrated on the
anti-inflammatory effect of Empa on chondrocytes and the underlying
mechanism. We hope that our study will provide a new therapeutic
approach for the treatment of OA in the future.

\textbf{2 Materials and methods}

\textbf{Reagents}

Recombinant mouse IL-1\selectlanguage{greek}β \selectlanguage{english}was purchased from R\&D Systems, UK.
Empagliflozin was purchased from MedChemExpress, China. Dulbecco's
modified Eagle's medium (DMEM), penicillin/streptomycin, fetal bovine
serum (FBS), and 0.5\% trypsin were obtained from Gibco BRL, Grand
Island, NY, USA. Collagenase type II was purchased from Sigma-Aldrich,
St, Louis, MO, USA.

\textbf{Chondrocyte isolation and culture}

Primary chondrocytes were obtained from 5-day-old C57BL/6 mice as
previously described \textsuperscript{12}. Briefly, the articular
cartilage from the knee joint of mice was cut into 1
mm\textsuperscript{3} particles and then was digested with DMEM
containing 1\% penicillin/streptomycin, 0.5\% trypsin, and 1\%
Collagenase type II for 2 h. After filtered through a 40 \selectlanguage{greek}μ\selectlanguage{english}m cell
strainer, the digestive suspensions were placed in a centrifuge at 1500
rpm. The centrifuged cell deposits were resuspended and grown in DMEM
supplemented with 10\% FBS and 1\% penicillin/streptomycin at 37 \selectlanguage{ngerman}°C with
5\% CO\textsubscript{2}. The cells were passaged to passage 3 and then
used for the following experiments.

\textbf{Cell viability assay}

5 x 10\textsuperscript{3} cells were seeded in 96-well plates and
treated with different concentrations (0, 0.5, 1, and 5 \selectlanguage{greek}μ\selectlanguage{english}M) of
empagliflozin with or without IL-1\selectlanguage{greek}β \selectlanguage{english}(10 ng/ml) for 24 h. The equal
volume of vehicle was used in the control group. To analyze cytotoxicity
of empagliflozin on chondrocytes, CCK-8 assays (MCE, HY-K0301) were
conducted according to the manufacturer's instruction. The optical
density (OD) value was read at a wavelength of 450 nm.

\textbf{Safranine O staining}

Chondrocytes were incubated with different concentrations (0, 1, and 5
\selectlanguage{greek}μ\selectlanguage{english}M) of empagliflozin with or without IL-1\selectlanguage{greek}β \selectlanguage{english}(10 ng/ml) for 24 h. Then the
cells were fixed with 4\% paraformaldehyde (PFA) solution for 20 min.
After washing with PBS, 0.1\% Safranine O dye solution were added to
stain the cells for 5 min. After washing with PBS twice, cells were
imaged by gross camera.

\textbf{Western blot}

After treatment, equal amounts of cells were lysed with RIPA for 60min.
The lysates were quantified by BCA kit (Beyotime, China). The samples
were then separated with SDS-polyacrylamide gels and transferred into
nitrocellulose membranes. After being blocked and cut into proper
sections, membranes were incubated with different primary antibodies
against GAPDH (Abcam, \#ab181602), MMP9 (Abcam, \#ab76003), MMP13
(Abcam, \#ab39012), COX2 (CST, \#12282), INOS (Abcam, \#ab3523), P53
(Abcam, \#ab90363), P21 (Abcam, \#ab215971), NF-\selectlanguage{greek}κ\selectlanguage{english}B P65 (CST, \#8242),
P-NF-\selectlanguage{greek}κ\selectlanguage{english}B P65 (Ser536) (CST, \#3033), I\selectlanguage{greek}κ\selectlanguage{english}B (CST, \#4812), and P-I\selectlanguage{greek}κ\selectlanguage{english}B (Ser32)
(CST, \#2859) at 4 \selectlanguage{ngerman}°C overnight. The membranes were then incubated with
luminesced secondary antibodies and detected with the BioRad System.
GAPDH was used as control.

\textbf{Real-time PCR (RT-PCR)}

Chondrocytes were pretreated with empagliflozin (0, 1, and 5 \selectlanguage{greek}μ\selectlanguage{english}M) for 2
h, and followed by incubating with IL-1\selectlanguage{greek}β \selectlanguage{english}(10 ng/ml) for 24 h. Afterward,
RNAiso reagent (Takara, China) was used to extract total RNA from
chondrocytes. The total RNA was reversely transcribed into cDNA with
cDNA synthesis kit (Takara). SYBR Green PCR Master Mix (Takara) was used
for RT-PCR with an ABI StepOnePlus System (Applied Biosystems, UK). The
primers used for the RT-PCR were shown in Table 1.

\textbf{Assessment of nitric oxide (NO)}

The NO concentration in cell suspensions was measured with Griess
reagent (Sigma, G4410). Chondrocytes were pretreated with empagliflozin
at various concentrations (0, 1, and 5 \selectlanguage{greek}μ\selectlanguage{english}M) for 2 h and incubated with
IL-1\selectlanguage{greek}β \selectlanguage{english}(10 ng/ml) for 24 h, the NO levels in the cell culture medium were
assessed with Griess reagent according to the manufacturer. The relative
levels of NO were normalized to cell count.

\textbf{ELISA}

The levels of MMP9, MMP13, PGE2 and IL-6 were measured using ELISA kits
(AdipoGen LIFE SCIENCES, Germany). Chondrocytes were incubated with
IL-1\selectlanguage{greek}β \selectlanguage{english}(10 ng/ml) for 24 h before pretreatment for 2 h with empagliflozin
at various concentrations (0, 1, and 5 \selectlanguage{greek}μ\selectlanguage{english}M). The levels of MMP9, MMP13,
PGE2 and IL-6 were detected by collecting the cell culture supernatants
from each sample with ELISA kits according to the manufacturer. The
relative levels were normalized to cell count.

\textbf{Senescence analysis}

\selectlanguage{greek}β-\selectlanguage{english}galactosidase Staining assay was performed according to the
manufacture's instruction. After treatment, chondrocytes were fixed for
20 min and then incubated with \selectlanguage{greek}β-\selectlanguage{english}galactosidase staining buffer
(Beyotime, China) at 37\selectlanguage{ngerman}°C for 24h and subsequently observed under a
microscope.

\textbf{Animal experiments}

DMM-induced OA mouse model was used in the animal experiments as
previously described \textsuperscript{13}. Thirty 12-week-old C57BL/6
mice were randomly divided into 3 groups. Mice in the OA group and the
OA+Empa group were anesthetized and the medial meniscus in their knee
joints was surgically removed while mice in the Sham group were
anesthetized and performed a sham operation. A week after surgery,
intra-articular injection of 5 \selectlanguage{greek}μ\selectlanguage{english}M empagliflozin was conducted once a
week in the OA+Empa group, and equal volume of vehicle was conducted in
the OA group and the Sham group. After 4 weeks of treatment, the mice
were sacrificed and the knee samples were harvested for histological
analysis. This study was performed in line with the principles of the
Declaration of Helsinki. Approval was granted by the Ethics Committee of
Zhejiang Provincial People's Hospital, People's Hospital of Hangzhou
Medical College, Hangzhou, China.

\textbf{Histological analysis}

The mouse knee samples were fixed with 4\% paraformaldehyde solution for
48 h and then decalcified with 0.5 M ethylenediaminetetraacetic acid
(EDTA) until the tissues became soft. The samples were embedded in
paraffin and cut into 5 mm sections by a microtome. The tissue sections
were subsequently stained with safranin O and then observed by optical
microscopy. The OARSI grading system (0--6) was used to evaluate the
sections.

\textbf{Statistical analysis}

All data are presented as means \selectlanguage{ngerman}± SDs. One-way ANOVA with a subsequent
post hoc Tukey's test was used for comparisons. The value of p
\textless{} 0.05 was considered to indicate significant differences.

\textbf{3 Results}

\selectlanguage{greek}\emph{3.1 Effects of empagliflozin and IL-1β on cell viability and
chondrocyte phenotype maintenance}\selectlanguage{english}

The effects of empagliflozin and IL-1\selectlanguage{greek}β \selectlanguage{english}on cell viability and chondrocyte
phenotype maintenance were evaluated by CCK-8 assay and the safranine O
staining. The results demonstrated that empagliflozin had no
cytotoxicity on mouse chondrocyte viability with concentration [?]5 \selectlanguage{greek}μ\selectlanguage{english}M at
24 h. Besides, chondrocytes treated with 1 and 5 \selectlanguage{greek}μ\selectlanguage{english}M concentrations of
empagliflozin could significantly improve the down-regulated cell
viability caused by IL-1\selectlanguage{greek}β \selectlanguage{english}(Figure 1A). Similarly, the results of
safranine O staining revealed that chondrocytes were apparently less
stained after 24 h IL-1\selectlanguage{greek}β \selectlanguage{english}treatment, but the mouse chondrocytes treated
with 1 and 5 \selectlanguage{greek}μ\selectlanguage{english}M of empagliflozin were observably reverse the IL-1\selectlanguage{greek}β
\selectlanguage{english}caused safranine O stain loss (Figure 1B). Thus, 1 and 5 \selectlanguage{greek}μ\selectlanguage{english}M
concentrations of empagliflozin were used for the following in vitro
tests, and a concentration of 5 \selectlanguage{greek}μ\selectlanguage{english}M was used for the following in vivo
tests.

\emph{3.2} \selectlanguage{greek}\emph{Effects of empagliflozin on IL-1β-induced ECM
degradation in mouse chondrocytes}\selectlanguage{english}

Here the expression levels of catabolic markers of ECM were detected by
ELISA, RT-PCR and western blot to evaluate the effects of empagliflozin
on IL-1\selectlanguage{greek}β-\selectlanguage{english}induced ECM degradation in mouse chondrocytes. As shown in
Figure 2, administration of empagliflozin could counteract the
increments of MMP9 and MMP13 secretion level in the cell medium caused
by IL-1\selectlanguage{greek}β. \selectlanguage{english}In addition, pretreatment with empagliflozin significantly
decreased IL-1\selectlanguage{greek}β-\selectlanguage{english}induced upregulation of matrix-degrading genes (MMP9,
MMP13) in a dose-dependent manner at both mRNA level and protein level.
Based on the results above, empagliflozin could protect mouse
chondrocytes from ECM degradation by inhibiting the MMPs.

\selectlanguage{greek}\emph{3.3 Effects ofempagliflozin on IL-1β-induced inflammation in mouse
chondrocytes}\selectlanguage{english}

To discover the anti-inflammation effects of empagliflozin on mouse
chondrocytes, the expression of several inflammatory mediators in mouse
chondrocytes was assessed by ELISA, RT-PCR and western blot. As
illustrated in Figure 3A, the production and secretion of
proinflammatory factors (NO, PGE2, and IL-6) was increased under IL-1\selectlanguage{greek}β
\selectlanguage{english}treatment, and empagliflozin pretreatment could dose-dependently offset
these increments. Moreover, the results also showed that empagliflozin
significantly decreased IL-1\selectlanguage{greek}β-\selectlanguage{english}induced upregulation of other inflammatory
genes (COX2 and INOS) in both mRNA and protein expression levels (Figure
3B-C). These data demonstrated that empagliflozin could significantly
inhibited the production of IL-1\selectlanguage{greek}β-\selectlanguage{english}induced inflammatory mediators in
mouse chondrocytes.

\selectlanguage{greek}\emph{3.4 Effects of empagliflozin on IL-1β-induced senescence in mouse
chondrocytes}\selectlanguage{english}

Senescence analysis was conducted to evaluate the effects of
empagliflozin on the IL-1\selectlanguage{greek}β-\selectlanguage{english}induced senescence in mouse chondrocytes by
western blot and \selectlanguage{greek}β-\selectlanguage{english}galactosidase Staining Assay. It was found that the
levels of senescence markers (P21 and P53) were upregulated by IL-1\selectlanguage{greek}β
\selectlanguage{english}treatment, but this effect was largely weakened by empagliflozin (Figure
4A). Besides, \selectlanguage{greek}β-\selectlanguage{english}galactosidase Staining results showed that the
proportion of SA-\selectlanguage{greek}β-\selectlanguage{english}Gal-positive cells obviously increased after IL-1\selectlanguage{greek}β
\selectlanguage{english}treatment but decreased in the presence of empagliflozin (Figure 4B).
These data collectively suggested that empagliflozin exerted the
protective role in IL-1\selectlanguage{greek}β-\selectlanguage{english}induced cellular senescence of mouse
chondrocytes.

\selectlanguage{greek}\emph{3.5 Effects of empagliflozin on IL-1β-induced NF-κB pathway
activation in mouse chondrocytes}\selectlanguage{english}

To explore the underlying mechanism, the effects of empagliflozin on
NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway activation were investigated. As illustrated in Figure 5,
empagliflozin activated the NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway in mouse chondrocytes
represented as higher levels of the activated status of P65
(phosphor-P65, p-P65) and I\selectlanguage{greek}κ\selectlanguage{english}B (phosphor-I\selectlanguage{greek}κ\selectlanguage{english}B, p-I\selectlanguage{greek}κ\selectlanguage{english}B). The above
IL-1\selectlanguage{greek}β-\selectlanguage{english}induced NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway activation was inhibited by empagliflozin
pretreatment. The results indicated that empagliflozin could inhibit
IL-1\selectlanguage{greek}β-\selectlanguage{english}induced NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway activation in mouse chondrocytes.

\emph{3.6 Effects of empagliflozin on cartilage degeneration in mouse OA
model}

To investigate the anti-osteoarthritic effects of empagliflozin in vivo,
DMM-induced OA mouse model was used. The articular cartilage tissue
samples from mice in the OA group exhibited typical OA features with
reduced abundance of chondrocytes and loss of ECM. However, 4-week
intra-articular injection of 5 \selectlanguage{greek}μ\selectlanguage{english}M empagliflozin could improve these
changes (Figure 6A). The OARSI grade of the OA+Empa group was lower than
the OA group with significant difference. The data demonstrated that
empagliflozin showed anti-osteoarthritic effect in mouse model.

\textbf{4 Discussion}

OA is a common clinical degenerative joint disease in elderly
population, which is one of the main causes of chronic pain and joint
disability. OA can severely reduce the quality of life of affected
individuals. Current therapeutic goal of OA in clinical practice is to
relieve the symptoms but cannot slow down the disease progression.
Non-steroidal anti-inflammatory drugs (NSAIDs) are generally considered
to be the first-line pharmacological treatments for OA. However, NSAIDs
cannot achieve the long-term pain relief for OA patients, and they are
associated with significant side efforts such as gastric ulceration,
renal impairment, and cardiovascular accidents \textsuperscript{14}. At
the end stage of OA, a replacement surgery remains to be the only
treatment option. Thus, identification of emerging pharmaceutical
therapies is urgently needed in the treatment of OA. Empagliflozin, a
selective inhibitor of sodium-glucose cotransporter-2 (SGLT2), has been
reported to show the anti-inflammatory properties in several conditions
such as cardiovascular diseases, Alzheimer's disease, liver injury, and
chronic kidney disease \textsuperscript{15-19}. In this study, we
discovered the protective effect of empagliflozin on cartilage
degeneration via inhibition of NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway.

Previously thought to simply be damaged from ''wear and tear'', OA is
now understood as a complex cell-mediated process. Current research has
shown that OA is characterized by progressive cartilage degradation, but
also underlying bone remodeling, osteophyte formation, and synovial
inflammation \textsuperscript{3,20}. A series of pathological factors
such as ECM degradation, chondrocyte inflammation, oxidative stress,
mitochondrial dysfunction, abnormal mechanical load, and senescent
molecules contribute to the degeneration of chondrocytes, ultimately
causing cartilage damage and OA development \textsuperscript{21,22}.
Chondrocytes, as the only cellular component of cartilage, play a
central role in the balance of ECM metabolism through the synthesis of
cartilage matrix. However, the inflammatory factors like IL-1\selectlanguage{greek}β \selectlanguage{english}can
disrupt the balance of ECM metabolism by increasing the expression of
catabolic enzymes including MMPs, causing the gradual loss of ECM. MMPs
inhibitors could be promising agents for the treatment of OA
\textsuperscript{23}. Our data revealed that IL-1\selectlanguage{greek}β \selectlanguage{english}upregulated the
expression of matrix-degrading enzyme (MMP9 and MMP13) and activated the
inflammatory cascade reaction by the secretion of inflammatory cytokines
(NO, PGE2, IL-6, COX2, and INOS) in mouse chondrocytes, whereas these
effects could be offset by the administration of empagliflozin. These
founds indicated that empagliflozin could protect mouse chondrocytes
from IL-1\selectlanguage{greek}β-\selectlanguage{english}induced ECM degradation and inflammatory reaction. What's
more, chondrocyte senescence is also involved in the pathological
process of OA. Jeon et al found that senescent cells accumulated in the
articular cartilage and synovium after anterior cruciate ligament
transection, and local clearance of these senescent cells attenuated the
development of post-traumatic OA \textsuperscript{24}. Therefore, the
senescence analysis of chondrocytes was conducted in this study.
Cellular senescence is generally regarded as a cell state characterized
by an irreversible cell-cycle arrest \textsuperscript{25}. P21 and P53
are the key factors for promoting senescence. We found that
empagliflozin could counteract the elevated expression of senescence
markers (P21 and P53) induced by IL-1\selectlanguage{greek}β. \selectlanguage{english}Empagliflozin could also
decrease the SA-\selectlanguage{greek}β-\selectlanguage{english}Gal-positive cells. Thus, empagliflozin could
alleviate IL-1\selectlanguage{greek}β-\selectlanguage{english}induced senescence in mouse chondrocytes in vitro. All
these results displayed the antidegradation role of empagliflozin on
chondrocytes in vitro. The effect of empagliflozin on cartilage damage
in vivo was also observed in DMM-induced OA mice model. We found that
empagliflozin could also protect mouse knee cartilage from wear and
matrix degeneration.

Multiple signaling pathways are involved in OA such as NF-\selectlanguage{greek}κ\selectlanguage{english}B,
Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin, HIFs, TGF\selectlanguage{greek}β/ΒΜ\selectlanguage{english}P and so on \textsuperscript{26}. NF-\selectlanguage{greek}κ\selectlanguage{english}B
signaling participates in many OA-associated events, including
chondrocyte catabolism, survival, and inflammation \textsuperscript{27}.
NF-\selectlanguage{greek}κ\selectlanguage{english}B transcription factor has been considered as a disease-contributing
factor of OA for a long time \textsuperscript{27}. When NF-\selectlanguage{greek}κ\selectlanguage{english}B exists in
the cytoplasm, it is in an inactive state and cannot enter the nucleus
to play its function because it binds to the inhibitory protein I\selectlanguage{greek}κ\selectlanguage{english}B.
Once cells are stimulated by certain inductive factors, I\selectlanguage{greek}κ\selectlanguage{english}B is
phosphorylated and the molecular conformation of I\selectlanguage{greek}κ\selectlanguage{english}B changes, resulting
in the activation and nuclear translocation of
NF-\selectlanguage{greek}κ\selectlanguage{english}B\textsuperscript{28,29}. Activation of NF-\selectlanguage{greek}κ\selectlanguage{english}B ,on the one hand, can
directly bind to the promoters of MMPs genes to promote the expression
of matrix-degrading enzyme \textsuperscript{30}. On the other hand,
activation of NF-\selectlanguage{greek}κ\selectlanguage{english}B regulates the transcription of many nuclear genes
related to inflammation, including cyclooxygenase (COX2) and inducible
nitric oxide synthase (iNOS) genes \textsuperscript{31,32}. COX2 and
iNOS can promote the production and secretion of induce prostaglandin
(PGE2) and nitric oxide (NO). Increased PGE2 and NO levels can
upregulate the expression of MMPs, ultimately causing cartilage
degeneration. NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling pathway was focused in this study. Our
results showed that empagliflozin inhibited the phosphorylation of I\selectlanguage{greek}κ\selectlanguage{english}B
and P65, displaying the suppressive role on NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling. Thus, the
underlying mechanism of empagliflozin in mouse cartilage protective
roles is related to the inhibition of NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway.

However, there are still some limitations in our study. First at all, we
confirmed the protective effect of empagliflozin on preventing cartilage
degeneration via inhibiting the activation of NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway. But whether
NF-\selectlanguage{greek}κ\selectlanguage{english}B is the direct target of empagliflozin needs to be further
identified. Secondly, we have only demonstrated the effects of
empagliflozin on cellular inflammation and senescence in chondrocytes.
The effects of empagliflozin on other aspects involving the onset and
progression of OA deserve further investigation.

\textbf{5 Conclusions}

We demonstrated that empagliflozin inhibited chondrocytes ECM
degradation, inflammation and cellular senescence in vitro and prevented
cartilage degeneration in vivo by suppressing the NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway
activation. Our findings indicate a therapeutic potential of
empagliflozin in OA treatment.

\textbf{References}

1 Roos EM,Arden NK. Strategies for the prevention of knee
osteoarthritis. Nat Rev Rheumatol 2016; 12: 92-101.

2 O'Neill TW, McCabe PS,McBeth J. Update on the epidemiology, risk
factors and disease outcomes of osteoarthritis. Best Pract Res Clin
Rheumatol 2018; 32: 312-326.

3 Loeser RF, Goldring SR, Scanzello CR,Goldring MB. Osteoarthritis: A
disease of the joint as an organ. Arthritis \& Rheumatism 2012; 64:
1697-1707.

4 Palazzo C, Nguyen C, Lefevre-Colau MM, Rannou F,Poiraudeau S. Risk
factors and burden of osteoarthritis. Ann Phys Rehabil Med 2016; 59:
134-138.

5 Robinson WH, Lepus CM, Wang Q, Raghu H, Mao R, Lindstrom TM,Sokolove
J. Low-grade inflammation as a key mediator of the pathogenesis of
osteoarthritis. Nat Rev Rheumatol 2016; 12: 580-592.

6 Liu-Bryan R,Terkeltaub R. Emerging regulators of the inflammatory
process in osteoarthritis. Nat Rev Rheumatol 2015; 11: 35-44.

7 Frampton JE. Empagliflozin: A Review in Type 2 Diabetes. Drugs 2018;
78: 1037-1048.

8 Santos-Gallego CG, Requena-Ibanez JA, San Antonio R, Garcia-Ropero A,
Ishikawa K, Watanabe S, Picatoste B, Vargas-Delgado AP, Flores-Umanzor
EJ, Sanz J, Fuster V,Badimon JJ. Empagliflozin Ameliorates Diastolic
Dysfunction and Left Ventricular Fibrosis/Stiffness in Nondiabetic
Heart~Failure: A Multimodality Study. JACC Cardiovasc Imaging 2021; 14:
393-407.

9 Kolijn D, Pabel S, Tian Y, L\selectlanguage{ngerman}ódi M, Herwig M, Carrizzo A, Zhazykbayeva
S, Kovács Á, Fülöp G, Falcão-Pires I, Reusch PH, Linthout SV, Papp Z,
van Heerebeek L, Vecchione C, Maier LS, Ciccarelli M, Tschöpe C, Mügge
A, Bagi Z, Sossalla S,Hamdani N. Empagliflozin improves endothelial and
cardiomyocyte function~in human heart failure with preserved ejection
fraction via reduced pro-inflammatory-oxidative pathways and protein
kinase G\selectlanguage{greek}α \selectlanguage{english}oxidation. Cardiovasc Res 2021; 117: 495-507.

10 Xu L, Nagata N, Nagashimada M, Zhuge F, Ni Y, Chen G, Mayoux E,
Kaneko S,Ota T. SGLT2 Inhibition by Empagliflozin Promotes Fat
Utilization and Browning and Attenuates Inflammation and Insulin
Resistance by Polarizing M2 Macrophages in Diet-induced Obese Mice.
EBioMedicine 2017; 20: 137-149.

11 Shakerinasab N, Azizi M, Mansourian M, Sadeghi H, Salaminia S, Abbasi
R, Shahaboddin ME,Doustimotlagh AH. Empagliflozin Exhibits
Hepatoprotective Effects Against Bile Duct Ligation-induced Liver Injury
in Rats: A Combined Molecular Docking Approach to In Vivo Studies. Curr
Pharm Des 2022; 28: 3313-3323.

12 Cho C, Kang LJ, Jang D, Jeon J, Lee H, Choi S, Han SJ, Oh E, Nam J,
Kim CS, Park E, Jeong SY, Park CH, Shin YS, Eyun SI,Yang S. Cirsium
japonicum var. maackii and apigenin block Hif-2\selectlanguage{greek}α-\selectlanguage{english}induced osteoarthritic
cartilage destruction. J Cell Mol Med 2019; 23: 5369-5379.

13 Park E, Lee CG, Han SJ, Yun SH, Hwang S, Jeon H, Kim J, Choi CW, Yang
S,Jeong SY. Antiosteoarthritic Effect of Morroniside in Chondrocyte
Inflammation and Destabilization of Medial Meniscus-Induced Mouse Model.
Int J Mol Sci 2021; 22.

14 Gutthann SP, Garc\selectlanguage{ngerman}ía Rodríguez LA,Raiford DS. Individual nonsteroidal
antiinflammatory drugs and other risk factors for upper gastrointestinal
bleeding and perforation. Epidemiology 1997; 8: 18-24.

15 Fu J, Xu H, Wu F, Tu Q, Dong X, Xie H,Cao Z. Empagliflozin inhibits
macrophage inflammation through AMPK signaling pathway and plays an
anti-atherosclerosis role. Int J Cardiol 2022; 367: 56-62.

16 Gohari S, Reshadmanesh T, Khodabandehloo H, Karbalaee-Hasani A,
Ahangar H, Arsang-Jang S, Ismail-Beigi F, Dadashi M, Ghanbari S, Taheri
H, Fathi M, Muhammadi MJ, Mahmoodian R, Asgari A, Tayaranian M,
Moharrami M, Mahjani M, Ghobadian B, Chiti H,Gohari S. The effect of
EMPAgliflozin on markers of inflammation in patients with concomitant
type 2 diabetes mellitus and Coronary ARtery Disease: the EMPA-CARD
randomized controlled trial. Diabetol Metab Syndr 2022; 14: 170.

17 La Grotta R, de Candia P, Olivieri F, Matacchione G, Giuliani A,
Rippo MR, Tagliabue E, Mancino M, Rispoli F, Ferroni S, Berra CC,
Ceriello A,Prattichizzo F. Anti-inflammatory effect of SGLT-2 inhibitors
via uric acid and insulin. Cell Mol Life Sci 2022; 79: 273.

18 Li N,Zhou H. SGLT2 Inhibitors: A Novel Player in the Treatment and
Prevention of Diabetic Cardiomyopathy. Drug Des Devel Ther 2020; 14:
4775-4788.

19 Maayah ZH, Ferdaoussi M, Takahara S, Soni S,Dyck JRB. Empagliflozin
suppresses inflammation and protects against acute septic renal injury.
Inflammopharmacology 2021; 29: 269-279.

20 Charlier E, Deroyer C, Ciregia F, Malaise O, Neuville S, Plener Z,
Malaise M,de Seny D. Chondrocyte dedifferentiation and osteoarthritis
(OA). Biochem Pharmacol 2019; 165: 49-65.

21 Motta F, Barone E, Sica A,Selmi C. Inflammaging and Osteoarthritis.
Clin Rev Allergy Immunol 2023; 64: 222-238.

22 Abramoff B,Caldera FE. Osteoarthritis: Pathology, Diagnosis, and
Treatment Options. Med Clin North Am 2020; 104: 293-311.

23 Hu Q,Ecker M. Overview of MMP-13 as a Promising Target for the
Treatment of Osteoarthritis. Int J Mol Sci 2021; 22.

24 Jeon OH, Kim C, Laberge RM, Demaria M, Rathod S, Vasserot AP, Chung
JW, Kim DH, Poon Y, David N, Baker DJ, van Deursen JM, Campisi
J,Elisseeff JH. Local clearance of senescent cells attenuates the
development of post-traumatic osteoarthritis and creates a
pro-regenerative environment. Nat Med 2017; 23: 775-781.

25 Gorgoulis V, Adams PD, Alimonti A, Bennett DC, Bischof O, Bishop C,
Campisi J, Collado M, Evangelou K, Ferbeyre G, Gil J, Hara E,
Krizhanovsky V, Jurk D, Maier AB, Narita M, Niedernhofer L, Passos JF,
Robbins PD, Schmitt CA, Sedivy J, Vougas K, von Zglinicki T, Zhou D,
Serrano M,Demaria M. Cellular Senescence: Defining a Path Forward. Cell
2019; 179: 813-827.

26 Yao Q, Wu X, Tao C, Gong W, Chen M, Qu M, Zhong Y, He T, Chen S,Xiao
G. Osteoarthritis: pathogenic signaling pathways and therapeutic
targets. Signal Transduct Target Ther 2023; 8: 56.

27 Choi MC, Jo J, Park J, Kang HK,Park Y. NF-\selectlanguage{greek}κ\selectlanguage{english}B Signaling Pathways in
Osteoarthritic Cartilage Destruction. Cells 2019; 8.

28 Wang XF, Zhang AP, Sun ZY, Liu C, Kuang LH,Tian JW. {[}Expression of
NF-\selectlanguage{greek}κ\selectlanguage{english}B in a degenerative human intervertebral disc model{]}. Zhonghua Yi
Xue Za Zhi 2017; 97: 1324-1329.

29 Mortezaee K, Najafi M, Farhood B, Ahmadi A, Shabeeb D,Musa AE. NF-\selectlanguage{greek}κ\selectlanguage{english}B
targeting for overcoming tumor resistance and normal tissues toxicity. J
Cell Physiol 2019; 234: 17187-17204.

30 Zheng Y, Lin Y, Chen Z, Jiao Y, Yuan Y, Li C, Xu X,Cao P.
Propionibacterium acnes induces intervertebral discs degeneration by
increasing MMP-1 and inhibiting TIMP-1 expression via the NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway.
Int J Clin Exp Pathol 2018; 11: 3445-3453.

31 Lin Y, Tang G, Jiao Y, Yuan Y, Zheng Y, Chen Y, Xiao J, Li C, Chen
Z,Cao P. Propionibacterium acnes Induces Intervertebral Disc
Degeneration by Promoting iNOS/NO and COX-2/PGE(2) Activation via the
ROS-Dependent NF-\selectlanguage{greek}κ\selectlanguage{english}B Pathway. Oxid Med Cell Longev 2018; 2018: 3692752.

32 Wang J, Hu J, Chen X, Huang C, Lin J, Shao Z, Gu M, Wu Y, Tian N, Gao
W, Zhou Y, Wang X,Zhang X. BRD4 inhibition regulates MAPK, NF-\selectlanguage{greek}κ\selectlanguage{english}B
signals, and autophagy to suppress MMP-13 expression in diabetic
intervertebral disc degeneration. Faseb j 2019; 33: 11555-11566.

\textbf{Tables}

Table 1 Primers used for RT-PCR in this study\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Gene & Forward & Reverse\tabularnewline
\midrule
\endhead
MMP9 & CTTCACCGGCTAAACCACCT & TGTCCCTAACGCCCAGTAGA\tabularnewline
MMP13 & TGATGGACCTTCTGGTCTGGC & CATCCACATGGTTGGGAAGTTCTG\tabularnewline
COX2 & GGTCTGGTGCCTGGTCTGATGAT & GTCCTTTCAAGGAGAATGGTGC\tabularnewline
INOS & GCAAACCCAAGGTCTACGTTCA & GAGCACGCTGAGTACCTCATTG\tabularnewline
GAPDH & TCACTGCCACCCAGAC & TGTAGGCCATGAGGTCCAC\tabularnewline
\bottomrule
\end{longtable}



\textbf{Figure legends}

Figure 1. Effects of empagliflozin and IL-1\selectlanguage{greek}β \selectlanguage{english}on cell viability and
chondrocyte phenotype maintenance of mouse chondrocytes. (A) CCK-8
analysis, *p\textless{}0.05 versus the IL-1\selectlanguage{greek}β \selectlanguage{english}group. (C) Gross view of
safranine O stained mouse chondrocytes that treated with empagliflozin
at various concentrations (0, 1, and 5 \selectlanguage{greek}μ\selectlanguage{english}M) with or without IL-1\selectlanguage{greek}β \selectlanguage{english}(10
ng/ml) for 24 h.

Figure 2. Effects of empagliflozin on IL-1\selectlanguage{greek}β-\selectlanguage{english}induced ECM degradation in
mouse chondrocytes. Chondrocytes were pretreated with empagliflozin at
various concentrations (0, 1, and 5 \selectlanguage{greek}μ\selectlanguage{english}M) for 2 h and then incubated with
IL-1\selectlanguage{greek}β \selectlanguage{english}(10 ng/ml) for 24 h. (A) ELISA analysis of MMP9 and MMP13 in
treated cells. (B) The mRNA expression levels of MMP9 and MMP13 in
treated cells. (C) The protein expression levels of MMP9 and MMP13 in
treated cells. *p\textless{}0.05 versus control group,
\#p\textless{}0.05 versus model group.

Figure 3. Effects of empagliflozin on IL-1\selectlanguage{greek}β-\selectlanguage{english}induced inflammation in
mouse chondrocytes. Chondrocytes were pretreated with empagliflozin at
various concentrations (0, 1, and 5 \selectlanguage{greek}μ\selectlanguage{english}M) for 2 h and then incubated with
IL-1\selectlanguage{greek}β \selectlanguage{english}(10 ng/ml) for 24 h. (A) Nitrite production, PGE2 secretion, and
IL-6 secretion in treated cells. (B) The mRNA expression levels of COX2
and INOS in treated cells. (C) The protein expression levels of COX2 and
INOS in treated cells. GAPDH as an internal control. *p\textless{}0.05
versus control group, \#p\textless{}0.05 versus model group.

Figure 4. Effects of empagliflozin on IL-1\selectlanguage{greek}β-\selectlanguage{english}induced senescence in mouse
chondrocytes. Chondrocytes were pretreated with empagliflozin at various
concentrations (0, 1, and 5 \selectlanguage{greek}μ\selectlanguage{english}M) for 2 h and then incubated with IL-1\selectlanguage{greek}β
\selectlanguage{english}(10 ng/ml) for 24 h. (A) The protein expression levels of P21 and P53 in
treated cells. GAPDH as an internal control. (B) \selectlanguage{greek}β-\selectlanguage{english}galactosidase
staining results of treated cells. *p\textless{}0.05 versus control
group, \#p\textless{}0.05 versus model group. Bar = 50 \selectlanguage{greek}μ\selectlanguage{english}m

Figure 5. Effects of empagliflozin on the IL-1\selectlanguage{greek}β-\selectlanguage{english}induced NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway
activation in mouse chondrocytes. Chondrocytes were pretreated with
empagliflozin at various concentrations (0, 1, and 5 \selectlanguage{greek}μ\selectlanguage{english}M) for 2 h and
then incubated with IL-1\selectlanguage{greek}β \selectlanguage{english}(10 ng/ml) for 15 min. The protein expression
levels of P-P65, P65, p-I\selectlanguage{greek}κ\selectlanguage{english}B, and I\selectlanguage{greek}κ\selectlanguage{english}B in treated cells. The ratio of
p-P65 to P65 as well as the radio of p-I\selectlanguage{greek}κ\selectlanguage{english}B to I\selectlanguage{greek}κ\selectlanguage{english}B was calculated. GAPDH
as an internal control. *p\textless{}0.05 versus control group,
\#p\textless{}0.05 versus model group.

Figure 6. Effects of empagliflozin on cartilage degeneration in mouse OA
model. (A) Microscopic images of safranin O stained mouse knee joint
sections. Bar = 500 \selectlanguage{greek}μ\selectlanguage{english}m. (B) The OARSI grades. *p\textless{}0.05 versus
control group, \#p\textless{}0.05 versus model group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/fig1/fig1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/fig2/fig2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/fig3/fig3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/fig4/fig4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/fig5/fig5}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/fig6/fig6}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

