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\begin{document}

\title{Trilobatin attenuates blood-brain barrier dysfunction induced by
cerebral ischemia reperfusion by targeting MMP9: Involvement of
APOE4/CypA/NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling\selectlanguage{english}}



\author[1]{Linying Feng}%
\author[1]{Yeli Li}%
\author[1]{Mu Lin }%
\author[1]{Dianyou Xie}%
\author[1]{Yunmei Luo}%
\author[2]{Nana Chen}%
\author[2]{Zhi-Xu He}%
\author[2]{Qihai Gong}%
\author[1]{zhu yizhun}%
\author[2]{Jianmei Gao}%
\affil[1]{Macau University of Science and Technology}%
\affil[2]{Zunyi Medical University}%


\vspace{-1em}



  
  \date{March 13, 2025}


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\begin{abstract}
Background and Purpose: Blood-brain barrier (BBB) breakdown is one of
the most crucial pathological changes of cerebral ischemia-reperfusion
(I/R) injury. Trilobatin (TLB), a naturally occurring food additive,
exerts neuroprotective effect against cerebral I/R injury as
demonstrated in our previous study. This study was designed to
investigate the effect of TLB on disruption of BBB after cerebral I/R
injury. Experimental Approach: Rats with focal cerebral ischemia caused
by transient middle cerebral artery occlusion (MCAO) and brain
microvascular endothelial cells along with human astrocytes to mimic
blood brain barrier (BBB) injury caused by oxygen and glucose
deprivation (OGD) followed by reoxygenation (OGD/R). Key results: The
results showed that TLB effectively maintained the integrity of BBB and
inhibited neuronal loss following cerebral I/R challenge. Furthermore,
TLB dramatically increased tight junction proteins including ZO-1,
occludin and claudin 5, as well as decreased the levels of
apolipoprotein E (APOE) 4, cyclophilin A (CypA), and phosphorylated
nuclear factor kappa B (NF-\selectlanguage{greek}κ\selectlanguage{english}B), thereby reduced proinflammatory
cytokines. In addition, TLB also decreased Bax/Bcl-2 ratio and
cleaved-caspase 3 level along with reduced the number of apoptotic
neurons. Intriguingly, molecular docking and transcriptomics predicted
MMP9 was a prominent gene evoked by TLB treatment. Furthermore, the
protective effect of TLB on OGD/R-induced the loss of BBB integrity in
human brain microvascular endothelial cell and astrocyte co-cultures in
vitro was markedly reinforced by knockdown of MMP9. Conclusions and
implications: Our findings reveal a novel property of TLB: saving BBB
disruption following cerebral I/R via targeting MMP9 and inhibiting
APOE4/CypA/NF-\selectlanguage{greek}κ\selectlanguage{english}B axis.%
\end{abstract}%



\sloppy


Gao Jianmei (Orcid ID: 0000-0003-3683-7956)

\selectlanguage{greek}\textbf{Trilobatin attenuatesblood-brain barrier dysfunction induced by
cerebral ischemia reperfusion by targeting MMP9: Involvement of
APOE4/CypA/NF-κB signaling}\selectlanguage{english}

Linying Feng\textsuperscript{1,2}, Yeli Li\textsuperscript{1,2}, Mu
Lin\textsuperscript{1,2}, Dianyou Xie\textsuperscript{1,2}, Yunmei
Luo\textsuperscript{1,2}, Nana Chen\textsuperscript{2}, Zhixu
He\textsuperscript{3}, Qihai Gong\textsuperscript{1,2}, Yizhun
Zhu\textsuperscript{1}, Jianmei Gao\textsuperscript{1,2*}

\textsuperscript{1}Department of Pharmacy, Macau University of Science
and Technology, Macau, China

\textsuperscript{2}Key Laboratory of Basic Pharmacology of Ministry of
Education and Joint International Research Laboratory of Ethnomedicine
of Ministry of Education, Zunyi Medical University, Zunyi, China

\textsuperscript{3}The Collaborative Innovation Center of Tissue Damage
Repair and Regeneration Medicine of Zunyi medical University

Corresponding author: Prof. Jianmei Gao, Ph.D.

6 Xuefu West Road, Zunyi City, Guizhou Province, 563000, P. R. China

Tel: +86-851-286-423-03

mail: gaojianmei@zmu.edu.cn

\textbf{Acknowledgements}

This work was supported by Natural Science Foundation of China
(No.82160756), Science and Technology Support Plan of Guizhou Province
(No. {[}2020{]}1Y010), The hundred level of high-level innovative
talents in Guizhou Province (No. QKHRCPT 20165684), Collaborative
Innovation Center of Chinese Ministry of Education (No. 2020-39),
Science and technology planning project of Zunyi (No. {[}HZ2021{]}187),
The Natural Science Foundation of Guizhou, China (No.2019-1329).

\textbf{Author contribution}

LYF, YLL, ML, and NNC performed the experiments. DYX and YML helped with
bioinformatics analysis and molecular docking analysis. All authors were
involved in analysis of data. LYF wrote the manuscript. JMG, QHG ,YZZ,
and HZX design the experiments and revised the manuscript.

\textbf{Confict of interest}

The authors declare no conflicts of interest.

\textbf{Declaration of transparency and scientific rigour}

This Declaration acknowledges that this paper adheres to the principles
for transparent reporting and scientific rigour of preclinical research
as stated in the BJP guidelines for Design \& Analysis, Immunoblotting
and Immunochemistry and Animal Experimentation and as recommended by
funding agencies, publishers and other organizations engaged with
supporting research.

\textbf{Data availability statement}

The authors declare that data supporting the findings of this study are
available within the article and it's Supplementary information
(Supplementary SFig.1, 2). Uncropped Western blots are shown in
Supplementary material. All relevant datasets used and/or analyzed in
this study are available from the corresponding author upon reasonable
request.

\textbf{Hosted file}

\verb`Manuscript.docx`  available at \url{https://authorea.com/users/510398/articles/587532-trilobatin-attenuates-blood-brain-barrier-dysfunction-induced-by-cerebral-ischemia-reperfusion-by-targeting-mmp9-involvement-of-apoe4-cypa-nf-%CE%BAb-signaling}

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