Abstract
Peroxynitrite-mediated nitrosative stress in the brain has been associated with various neurodegenerative disorders. Recent evidence highlights peroxisome proliferator-activated receptor γ (PPARγ) as a critical neuroprotective factor in neurodegenerative diseases. Here, we observed the effect of the herb hydroxysafflor yellow A (HSYA) during nitrosative stress in neurons and investigated the mechanism based on PPARγ protection. We found that a single exposure of primary neurons to peroxynitrite donor SIN-1 caused neuronal injury, which was accompanied by the increase of PPARγ nitration status and lack of activation of the receptor, as measured by PPARγ DNA-binding activity, by agonist (15d-PGJ2 or rosiglitazone) stimulation. The crucial role of PPARγ in neuronal defense against nitrosative stress was verified by showing that pretreatment with 15d-PGJ2 or rosiglitazone attenuated SIN-1-induced neuronal injury but pretreatment with GW9662, a PPARγ antagonist, aggravated SIN-1-induced neuronal injury. The addition of HSYA not only inhibited SIN-1-induced neuronal damage but prevented PPARγ nitrative modification and resumed PPARγ activity stimulated by either 15d-PGJ2 or rosiglitazone. Furthermore, HSYA also showed the ability to rescue the neuroprotective effect of 15d-PGJ2 or rosiglitazone when the agonists were coincubated with SIN-1. Finally, in vivo experiments demonstrated that the administration of HSYA also efficiently blocked PPARγ nitration and loss of activity in the SIN-1-injected hippocampus and reversed the increased neuronal susceptibility which was supported by the inhibition of Bcl-2 protein downregulation induced by SIN-1. The results suggest that HSYA protects neurons from nitrosative stress through keeping PPARγ as a functional receptor, allowing a more effective activation of this neuroprotective factor by the endogenous or exogenous agonist. Our findings provide new clues in understanding the role of the neuroprotective potential of the herbal HSYA.
Highlights
Produced nitric oxide (NO) and superoxide lead to the generation of peroxynitrite (ONOO−)
lactate dehydrogenase (LDH) release assay for cytotoxicity and MTT assay for cell viability (b), Hoechst staining for apoptotic cells (c), and Western blotting for 3-nitrotyrosine (3-NT) expression (d) were carried out after 24 h incubation
Neurons treated with SIN-1 exhibited cytotoxic damage, as determined by LDH released into the media and MTT assay for cell viability (Figure 1(b)), the Hoechst 33258 staining assay for apoptotic nuclei (Figure 1(c)), or protein nitrative modification based on 3-NT formation (Figure 1(d))
Summary
Produced nitric oxide (NO) and superoxide lead to the generation of peroxynitrite (ONOO−). Peroxynitritemediated nitrosative stress causes severe damage to proteins, lipids, and DNA, resulting in cell apoptosis or death. The concentration of 3-NT has been reported to increase in a wide range of neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease, and traumatic or ischemic brain injury [2,3,4,5]. The formation of 3-NT was elevated markedly and the significantly elevated 3-NT was positively correlated with infarct volume in ischemic animals [2]. The inhibition of 3-NT formation protects against brain injury in these disorders [2,3,4,5]. Peroxynitrite-mediated nitrosative stress represents an important pathogenic mechanism of neurodegenerative diseases
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