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Shuxuening injection facilitates neurofunctional recovery via down-regulation of G-CSF-mediated granulocyte adhesion and diapedesis pathway in a subacute stroke mouse model - 30/05/20

Doi : 10.1016/j.biopha.2020.110213 
Zhixiong Li a, b, 1, Guangxu Xiao a, b, 1, Ming Lyu a, b, c, Yule Wang a, b, Shuang He a, b, Hongxia Du a, b, Xintong Wang a, b, Yuxin Feng a, b, Yan Zhu a, b,
a State Key Laboratory of Component-based Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Beihua South Road, JingHai District, Tianjin, 301617, China 
b Research and Development Center of TCM, Tianjin International Joint Academy of Biotechnology & Medicine, 220 Dongting Road, TEDA, Tianjin, 300457, China 
c Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China 

Corresponding author at: State Key Laboratory of Component-based Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Beihua South Road, JingHai District, Tianjin, 301617, China.State Key Laboratory of Component-based Chinese MedicineTianjin University of Traditional Chinese MedicineBeihua South RoadJingHai DistrictTianjin301617China

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Highlights

SXNI facilitated neurofunctional recovery in subacute stroke mice.
SXNI inhibited granulocyte adhesion and diapedesis pathway in subacute stroke mice.
SXNI down-regulated the expression of G-csf and its downstream E-selectin and Mac-1.

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Abstract

Post-stroke neural damage is a serious health concern which does not yet have an effective treatment. We have shown previously that Shuxuening injection (SXNI), a Ginkgo biloba extract-based natural medicine, protects brain after an acute ischemic stroke, but its efficacy for post-stroke recovery is not known. This study was to investigate whether SXNI can improve the prognosis of stroke at a subacute phase. Mice with cerebral ischemia-reperfusion injury (CIRI) were established by middle cerebral artery occlusion (MCAO), and drugs or saline were injected by the tail vein every 12 h after reperfusion. The therapeutic effect of SXNI was evaluated by survival rate, modified neurologic severity scores (mNSS), open-field test, locomotive gait patterns, cerebral infarction volume, brain edema and histopathological changes. Subsequently, a combined method of RNA-seq and Ingenuity® Pathway Analysis (IPA) was performed to identify key targets and pathways of SXNI facilitating the prognosis of stroke in mouse brain. The results of the transcriptome analysis were verified by real time reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), western blot (WB) and immunohistochemistry (IHC). The experimental results showed that in the new subacute stroke model, SXNI markedly improves the survival rate, neurological and motor functions and histopathological changes, and significantly reduces cerebral infarction and edema volume. RNA-seq analysis of subacute stroke mice with or without SXNI (3 mL/kg) indicated 963 differentially expressed genes (DEGs) with a fold change ≥ 1.5 and a P-value ≤ 0.01. IPA analysis of DEGs showed that granulocyte adhesion and diapedesis ranked first in the pathway ranking, and the most critical gene regulated by SXNI was G-csf. Simultaneously, RT-PCR, ELISA, WB and IHC results demonstrated that SXNI not only obviously reduced the mRNA expression levels of key genes G-csf, Sele and Mac-1 in this pathway, but also significantly decreased the protein expression levels of G-CSF in serum and E-selectin and MAC-1 in brain tissues. In summary, our research suggested that SXNI can exert a remarkable neurofunctional therapeutic effect on stroke mice via down-regulating G-CSF to inhibit granulocyte adhesion and diapedesis. This study provides experimental evidence that SXNI may fulfill the need for stroke medicine targeting specifically at the recovery stage.

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Abbreviations : SXNI, G-CSF, CIRI, I/R, MCAO, mNSS, IPA, RT-PCR, ELISA, WB, IHC, DEGs, GBE, TTLs, ECs, TCM, CCA, ECA, ICA, PPA, MCA, TTC, H&E, RNA-Seq, BOS, RF, RH, LF, LH, FC, PPI, UR

Keywords : Shuxuening injection, Subacute stroke, Cerebral ischemia-reperfusion injury, Neurofunctional recovery, Granulocyte adhesion and diapedesis, G-CSF


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