Zhongyuan Li,1,* Pengfei Xu,2,* Yang Deng,3,* Rui Duan,1 Qiang Peng,1 Shiyao Wang,1 Zhaohan Xu,1 Ye Hong,1 Yingdong Zhang1 

1Department of Neurology, Nanjing First Hospital, Nanjing Medical University, Nanjing, 210000, People’s Republic of China; 2Department of Neurology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230001, People’s Republic of China; 3Department of Neurology, Nanjing First Hospital, China Pharmaceutical University, Nanjing, 210006, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Ye Hong; Yingdong Zhang, Department of Neurology, Nanjing First Hospital, Nanjing Medical University, No. 68, Changle Road, Nanjing, 210000, People’s Republic of China, Email dg1735053@smail.nju.edu.cn; zhangyingdong@aliyun.com

Background: After ischemic stroke (IS), microglia and astrocytes undergo polarization, transforming into a pro-inflammatory phenotype (M1 or A1). According to previous studies, exosomes might play an important role in the interplay between M1 microglia and A1 astrocytes after IS.
Methods: We used the microglial oxygen-glucose deprivation/reperfusion (OGD/R) model and ultracentrifugation to extract M1 microglial exosomes (M1-exos). Subsequently, we identified circSTRN3 enriched in exosomes through RNA sequencing and detected the role of circSTRN3 in astrocyte activation based on bioinformatics analysis, immunofluorescence, Western blotting, and polymerase chain reaction analysis. We validated these findings in the middle cerebral artery occlusion/reperfusion (MCAO/R) model of adult male C57BL/6J mice. Finally, we confirmed the correlation among circSTRN3, miR-331-5p, and stroke severity score in exosomes isolated from peripheral blood of IS patients.
Results: Our findings revealed that M1-exos promoted A1 astrocyte activation. CircSTRN3 was abundant in M1-exos, which could sponge miR-331-5p to affect mitochondrial antiviral signaling protein (MAVS), activate NF-κB pathway, and participate in A1 astrocyte activation. In addition, overexpressed circSTRN3 augmented the infarct size and neurological dysfunction in MCAO/R models, while miR-331-5p mimics reversed the effect. Furthermore, circSTRN3 in IS patients was positively correlated with stroke severity score (R2 = 0.83, P < 0.001), while miR-331-5p demonstrated a negative correlation with the same score (R2 = 0.81, P < 0.001).
Conclusion: Taken together, our research indicated that circSTRN3 from M1-exos could promote A1 astrocyte activation and exacerbate ischemic brain injury via miR331-5p/MAVS/NF-κB axis.

Keywords: ischemic stroke, microglia, astrocyte, exosome, circSTRN3, miR-331-5p