Drug Design, Development and Therapy (Aug 2023)
Ursolic Acid Ameliorated Neuronal Damage by Restoring Microglia-Activated MMP/TIMP Imbalance in vitro
Abstract
Luying Qiu,1 Yaxuan Wang,2 Yuye Wang,1,3 Fang Liu,1 Shumin Deng,1 Weishuang Xue,1 Yanzhe Wang1 1Department 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; 2Department of Anesthesiology, The First Affiliated Hospital of China Medical University, Shenyang, People’s Republic of China; 3Department of Neurology, China-Japan Friendship Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, People’s Republic of ChinaCorrespondence: Yanzhe Wang, Department of Neurology, Key Laboratory for Neurological Big Data of Liaoning Province, The First Affiliated Hospital of China Medical University, No. 155, Nanjing North Street, Heping District, Shenyang, Liaoning Province, 110001, People’s Republic of China, Tel +86-15040289417, Email [email protected]: 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
