Neuromodulatory effect of 4-(methylthio)butyl isothiocyanate against 3-nitropropionic acid induced oxidative impairments in human dopaminergic SH-SY5Y cells via BDNF/CREB/TrkB pathway
Prabhjot Kaur, Shivani Attri, Davinder Singh, Farhana Rashid, Sharabjit Singh, Avinash Kumar, H. Kaur, Neena Bedi, Saroj Arora
Abstract
Abstract Mitochondrial impairment, energetic crisis and elevated oxidative stress have been demonstrated to play a pivotal role in the pathological processes of Huntington’s disease (HD). 3-Nitropropionic acid (3-NPA) is a natural neurotoxin that mimics the neurological dysfunctions, mitochondrial impairments and oxidative imbalance of HD. The current investigation was undertaken to demonstrate the neuroprotective effect of 4-(methylthio)butyl isothiocyanate (4-MTBITC) against the 3-NPA induced neurotoxicity in human dopaminergic SH-SY5Y cells. The experimental evidence of oxidative DNA damage by 3-NPA was elucidated by pBR322 DNA nicking assay. In contrast, the 4-MTBITC considerably attenuated the DNA damage, suggesting its free radical scavenging action against 3-NPA and Fenton's reagent. The dose and time-dependent increase of 3-NPA revealed its neurotoxic dose as 0.5 mM after 24 h of treatment of SH-SY5Y cells in MTT assay. In order to determine the optimal dose at which 4-MTBITC protects cell death, the 3-NPA (IC 50 ) induced cells were pretreated with different concentrations of 4-MTBITC for 1 h. The neuroprotective dose of 4-MTBITC against 3-NPA was found to be 0.25 μM. Additionally, the elevated GSH levels in cells treated with 4-MTBITC indicate its propensity to eliminate reactive species generated as a result of 3-NPA-induced mitochondrial dysfunction. Likewise, it was determined through microscopic and flow cytometric experiments that 3-NPA's induced overproduction of reactive species and a decline in mitochondrial membrane potential (MMP) could be efficiently prevented by pre-treating cells with 4-MTBITC. To elucidate the underlying molecular mechanism, the RT-qPCR analysis revealed that the pre-treatment of 4-MTBITC effectively protected neuronal cells against 3-NPA-induced cell death by preventing Caspase-3 activation, Brain-derived neurotrophic factor (BDNF) upregulation, activation of cAMP response element-binding protein (CREB) and Nrf2 induction. Together, our findings lend credence to the idea that pre-treatment with 4-MTBITC reduced 3-NPA-induced neurotoxicity by lowering redox impairment, apoptotic state, and mitochondrial dysfunction. The present work, in conclusion, presented the first proof that the phytoconstituent 4-MTBITC supports the antioxidant system, BDNF/TrkB/CREB signaling, and neuronal survival in dopaminergic SH-SY5Y cells against 3-NPA-induced oxidative deficits.