Ursolic Acid Ameliorated Neuronal Damage by Restoring Microglia-Activated MMP/TIMP Imbalance in vitro

Luying Qiu; Yaxuan Wang; Yuye Wang; Fang Liu; Shumin Deng; Weishuang Xue; Yanzhe Wang

doi:10.2147/DDDT.S411408

Ursolic Acid Ameliorated Neuronal Damage by Restoring Microglia-Activated MMP/TIMP Imbalance in vitro

Drug Des Devel Ther. 2023 Aug 22:17:2481-2493. doi: 10.2147/DDDT.S411408. eCollection 2023.

Authors

Luying Qiu¹, Yaxuan Wang², Yuye Wang^{1

3}, Fang Liu¹, Shumin Deng¹, Weishuang Xue¹, Yanzhe Wang¹

Affiliations

¹ Department of Neurology, Key Laboratory for Neurological Big Data of Liaoning Province, The First Affiliated Hospital of China Medical University, Shenyang, People's Republic of China.
² Department of Anesthesiology, The First Affiliated Hospital of China Medical University, Shenyang, People's Republic of China.
³ Department of Neurology, China-Japan Friendship Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, People's Republic of China.

Abstract

Purpose: The oxygen and glucose deprivation-reoxygenation (OGDR) model is widely used to evaluate ischemic stroke and cerebral ischemia-reperfusion (I/R) injury in vitro. Excessively activated microglia produce pro-inflammatory mediators such as matrix metalloproteinases [MMPs] and their specific inhibitors, tissue inhibitors of metalloproteinases [TIMPs], causing neuronal damage. Ursolic acid (UA) acts as a neuroprotective agent in the rat middle cerebral artery occlusion/reperfusion (MCAO/R) model keeping the MMP/TIMP balance with underlying mechanisms unclear. Our study used OGDR model to determine whether and how UA reduces neuronal damage by reversing MMP/TIMP imbalance caused by microglia in I/R injury in vitro.

Methods: SH-SY5Y cells were first cultured with 95% N₂ and 5% CO₂ and then cultivated regularly for OGDR model. Cell viability was tested for a proper UA dose. We established a co-culture system with SH-SY5Y cells and microglia-conditioned medium (MCM) stimulated by lipopolysaccharide (LPS) and interferon-gamma (IFNγ). MMP9 and TIMP1 levels were measured with ELISA assay to confirm the UA effect. We added recombinant MMP9 (rMMP9) and TIMP1 neutralizing antibody (anti-TIMP1) for reconfirmation. Transmission electron microscopy was used to observe cell morphology, and flow cytometry and Annexin V-FITC and PI labeling for apoptotic conditions. We further measured the calcium fluorescence intensity in SH-SY5Y cells.

Results: The MCM significantly reduced cell viability of SH-SY5Y cells after OGDR (p<0.01), which was restored by UA (0.25 µM) (p<0.05), whereas lactate dehydrogenase activity, intraneuronal Ca²⁺ concentration, and apoptosis-related indexes were showed significant improvement after UA treatment (p<0.01). UA corrected the MMP/TIMP imbalance by decreasing MMP9 expression and increasing TIMP1 expression in the co-culture system (p<0.01) and the effects of UA on SH-SY5Y cells were mitigated by the administration of rMMP9 and anti-TIMP1 (p<0.01).

Conclusion: We demonstrated that UA inhibited microglia-induced neuronal cell death in an OGDR model of ischemic reperfusion injury by stabilizing the MMP9/TIMP1 imbalance.

Keywords: glucose deprivation-reoxygenation; matrix metalloproteinase; microglia; neuroinflammation; oxygen; ursolic acid.

MeSH terms

Glucose
Humans
Macrophages
Matrix Metalloproteinase 9
Microglia*
Neuroblastoma*
Ursolic Acid

Substances

Glucose
Matrix Metalloproteinase 9

Grants and funding

This study was supported by the National Natural Science Foundation of China, No. 81901189 (belongs to YZW) and the National Natural Science Foundation of China, No. 82201480 (belongs to WSX). The funding sources had no role in study conception and design, data analysis or interpretation, paper writing or deciding to submit this paper for publication.