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Berberine Facilitates Angiogenesis Against Ischemic Stroke Through Modulating Microglial Polarization via AMPK Signaling

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Abstract

Evidence suggests that microglia/macrophages can change their phenotype to M1 or M2 and participate in tissue damage or repair. Berberine (BBR) has shown promise in experimental stroke models, but its effects on microglial polarization and long-term recovery after stroke are elusive. Here, we investigated the effects of BBR on angiogenesis and microglial polarization through AMPK signaling after stroke. In the present study, C57BL/6 mice were subjected to transient middle cerebral artery occlusion (tMCAO), intragastrically administrated with BBR at 50 mg/kg/day. Neo-angiogenesis was observed by 68Ga-NODAGA-RGD micro-PET/CT and immunohistochemistry. Immunofluorescent staining further exhibited an increase of M2 microglia and a reduction of M1 microglia at 14 days after stroke. In vitro studies, the lipopolysaccharide (LPS)-induced BV2 microglial cells were used to confirm the AMPK activation effect of BBR. RT-PCR, Flow cytometry, and ELISA all demonstrated that BBR could inhibit M1 polarization and promote M2 polarization. Furthermore, treatment of human umbilical vein endothelial cells (HUVEC) with conditioned media collected from BBR-treated BV2 cells promoted angiogenesis. All effects stated above were reversed by AMPK inhibitor (Compound C) and AMPK siRNA. In conclusion, BBR treatment improves functional recovery and promotes angiogenesis following tMCAO via AMPK-dependent microglial M2 polarization.

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Abbreviations

BBR:

Berberine

tMCAO:

Transient middle cerebral artery occlusion

HUVEC:

Human umbilical vein endothelial cell

LPS:

Lipopolysaccharide

CI/RI:

Cerebral ischemia and reperfusion injury

CNS:

Central nervous system

AMPK:

Adenosine 5’-monophosphate-activated protein kinase

CCA:

Common carotid artery

ECA:

External carotid artery

ICA:

Internal carotid artery

MCA:

Middle cerebral artery

rCBF:

Regional cerebral blood flow

TTC:

2,3,5-Triphenyltetrazolium chloride

HE:

Hematoxylin eosin

OSEM3D:

3-Dimensional ordered subset expectation maximization algorithm

DMSO:

Dimethyl sulfoxide

PBS:

Phosphate-buffered saline

PVDF:

Polyvinylidene difluoride

TBST:

TBS-Tween20

DM:

Conditioned medium

LKB1:

Liver kinase B1

CaMKKβ:

Calcium/calmodulin‐dependent protein kinase kinase beta

PPARγ:

Peroxisome proliferator-activated receptor γ

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Acknowledgements

This work was supported by the National Nature Science Foundation of China (Grant No. 81773987 to YZ), Program of Nanjing Health and Family Planning Commission (YKK188107), and Hospital Pharmaceutical Research Program of Nanjing Pharmaceutical Commission (2017YX001). We would like to thank Prof. Hongguang Xie (Nanjing First Hospital) and Dr. Bangshun He (Nanjing First Hospital) for their informative advice.

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Author notes

  1. Junrong Zhu, Dingwen Cao, and Chao Guo have contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacy, Nanjing First Hospital, Nanjing Medical University, Nanjing, 210006, People’s Republic of China

    Junrong Zhu, Dingwen Cao, Manman Liu, Yifu Tao & Yanli Zhao

  2. School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing, 211198, People’s Republic of China

    Junrong Zhu, Dingwen Cao, Manman Liu, Yanli Zhao, Yingdong Zhang, Weirong Fang & Yunman Li

  3. Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Xi’an, 710000, People’s Republic of China

    Chao Guo

  4. Department of Neurology, Nanjing First Hospital, Nanjing Medical University, Nanjing, 210006, People’s Republic of China

    Junshan Zhou & Yingdong Zhang

  5. Department of Nuclear Medicine, Nanjing First Hospital, Nanjing Medical University, Nanjing, 210006, People’s Republic of China

    Feng Wang

  6. Department of Endocrinology, Nanjing First Hospital, Nanjing Medical University, Nanjing, 210006, People’s Republic of China

    Jing Wei

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  1. Junrong Zhu
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  12. Yunman Li
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Contributions

All authors listed contributed immensely to this study. YZ, YL, and JZ conceived and designed the study. DC, CG, and ML performed the experiments. JW, JZ, FW, and YZ analyzed the data. JZ and DC wrote the paper. YZ, WF, and YL reviewed and edited the manuscript. All authors read and approved the final version of the manuscript.

Corresponding authors

Correspondence to Yingdong Zhang, Weirong Fang or Yunman Li.

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Zhu, J., Cao, D., Guo, C. et al. Berberine Facilitates Angiogenesis Against Ischemic Stroke Through Modulating Microglial Polarization via AMPK Signaling. Cell Mol Neurobiol 39, 751–768 (2019). https://doi.org/10.1007/s10571-019-00675-7

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