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Published on: January 30, 2014
Ursolic Acid Ameliorated Neuronal Damage by Restoring Microglia-Activated MMP/TIMP Imbalance in vitro
Luying Qiu1, Yaxuan Wang2, Yuye Wang1,3
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.
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.
Insights
Ursolic acid (UA) protects neurons from cell death in ischemic stroke models by stabilizing the matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) balance. This study investigated UA
Area of Science:
- Neuroscience
- Cell Biology
- Pharmacology
Background:
- Ischemic stroke and cerebral ischemia-reperfusion (I/R) injury are critical conditions.
- Activated microglia release matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), disrupting the MMP/TIMP balance and causing neuronal damage.
- Ursolic acid (UA) shows neuroprotective effects in vivo, but its mechanisms in vitro remain unclear.
Purpose of the Study:
- To investigate whether UA reduces neuronal damage in an in vitro oxygen and glucose deprivation-reoxygenation (OGDR) model.
- To determine if UA reverses the MMP/TIMP imbalance caused by microglia in I/R injury.
- To elucidate the underlying mechanisms of UA's neuroprotective effects.
Main Methods:
- Established an in vitro OGDR model using SH-SY5Y cells and microglia-conditioned medium (MCM) stimulated by LPS and IFNγ.
- Assessed cell viability, lactate dehydrogenase activity, intraneuronal Ca2+ concentration, and apoptosis.
- Measured MMP9 and TIMP1 levels using ELISA, and confirmed UA's effects using recombinant MMP9 (rMMP9) and anti-TIMP1.
Main Results:
- Microglia-conditioned medium (MCM) reduced SH-SY5Y cell viability after OGDR, an effect restored by UA.
- UA treatment significantly improved cell viability, reduced lactate dehydrogenase activity, intraneuronal Ca2+ concentration, and apoptosis.
- UA corrected the MMP/TIMP imbalance by decreasing MMP9 and increasing TIMP1 expression, with effects mitigated by rMMP9 and anti-TIMP1.
Conclusions:
- UA inhibits microglia-induced neuronal cell death in an in vitro model of ischemic reperfusion injury.
- UA stabilizes the MMP9/TIMP1 imbalance, offering a neuroprotective mechanism.
- UA demonstrates potential as a therapeutic agent for ischemic stroke and related injuries.
