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
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% N 2 and 5% CO 2 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 Ca 2+ 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