PD is an advanced neurodegenerative syndrome frequently perceived among the elderly, nevertheless, there are no effective cures. The Pathological representative of PD is the loss of dopaminergic neurons or dopamine deficiency (Agafonov et al.,2024 Werner and Olanow,2022). Furthermore, the dopaminergic neuron loss generates motor symptoms of PD due to either putamen dopamine loss or dopaminergic neuron loss (Raza et al.,2019, Dionisio etal.,2021, Chakrabarti and Bisaglia,2023). The generation of ROS causes OS and mitochondrial dysfunction the common pathogenic mechanisms associated with progressive neurodegenerative diseases (Hassanzadeh and Rahimmi,2018, Hu et al.,2018, Desouky et al.,2023). Dopaminergic neurodegeneration consequences from OS transformed the morphology of mitochondria, which directs mitochondrial dysfunction (Hu et al.,2018, Desouky et al.,2023). Thus, the discovery of innovative beneficial ingredients against PD by aiming ROS formation and OS is clinically important. In the current report, we presented a neuroprotective effect of SL (5 and 10 µM) against PD using rotenone-stimulated SK-N-SH cells, an in vitro model of PD. SL decreased rotenone-induced ROS generation, OS, and apoptosis in neuroblastoma cells in a concentration-related way. As well, SL enhances rotenone-reduced SK-N-SH cell proliferation, MMP, and induced apoptotic protein expression. We then further explored the protective molecular mechanism of SL on rotenone-stimulated apoptotic cell death of neuroblastoma cells.
Rotenone is a strong lipophilic pesticide targeting the mitochondrial complex consequently harming neuronal development and triggering neurochemical pathological and behavioral changes (Erro et al.,2021, Ibarra et al,2023). Recently, rotenone weakened the behavioral parameters with a steady elevation in the akinesia and catalepsy and a drop in locomotor activities (Ibarra et al.,2023, El-Shamarka et al.,2023). Dopaminergic injury may be linked with behavioral parameters (Ibarra et al.,2023, El-Shamarka et al.,2023a). Rotenone is allied to mitochondrial dysfunction, α-synuclein accretion, oxidative injury, and cell apoptosis. In this study, SL (5 and 10 µM) could attenuate rotenone-stimulated mitochondrial impairment, OS, and apoptosis in neuroblastoma cells and showed a neuroprotective effect. Previously, it has been demonstrated that SL exerts neuroprotective activity (Jung et al.,2015).
This research established that rotenone is toxic to neuroblastoma cells, as documented in earlier investigations (Tamilselvam et al.,2013). Here in, SL (5 and 10 µM) could reduce dose-related way of the toxic sequence of rotenone-stimulated cells. The MTT experiment is extensively utilized to determine cell viability by reducing the MTT tetrazolium salt to formazan. This reaction is primarily catalyzed by proliferating cells containing mitochondrial dehydrogenases (Tamilselvam et al.,2013a). Cell viability is determined as per the mitochondrial optimal function, therefore it plays a crucial part in controlling cell death signaling by the contribution of ROS production, regulating cellular energy metabolism, and the discharge of apoptotic mediators into the cytosol (Wang et al.,2022, Zhang et al.,2021). Findings acquired from the MTT test in this current work propose a conventional neuroprotective effect of SL on rotenone-facilitated mitochondrial dysfunction.
Mitochondria show a vital part in the progression and apoptosis of cells, which are the major sources of intracellular ROS and also the key targets of OS (Moors et al.,2017). ROS formed due to oxidative injury caused by the PD pathogenesis (Grootveld,2022). Mitochondrial dysfunction originates from ROS formation which destructively affects cellular structures such as DNA, lipids, and proteins (Grootveld,2022, Grootveld et al.,2019). High-level production of ROS and cell death has been established in the former PD models (Moors et al.,2017, Goyal et al.,2023). Our findings also exhibit that rotenone induction can trigger the overproduction of ROS in neuroblastoma cells, as substantiated by prior investigations (Tamilselvam et al.,2013, Grootveld, 2022, Juan et al.,2021). Herein, rotenone treatment exposed high contents of LDH and MDA, however, decreasing the activities of GPx and SOD in the neuroblastoma cells. Hence, it has been established that rotenone is directed to anabatic apoptosis, hypergeneration of ROS, and higher OS in the PD model. Moreover, it is demonstrated that the ROS creation and apoptotic capability of rotenone-administered neuroblastoma cells were attenuated by SL (5 and 10 µM) in a concentration-related mode, meanwhile, we also observed that SL decreased the LDH and MDA and ameliorated the GPx and SOD actions in this in vitro model of PD. To the greatest of our understanding, we were the first to prove that SL has the role of averting apoptotic cell death, diminishing ROS creation, inhibiting OS, and elevating MMP in rotenone-stimulated neuroblastoma cells.
Furthermore, we explored the apoptotic mechanisms in an in vitro PD model treatment with SL. Bcl-2, as an anti-apoptotic protein reduces cell death activated via numerous latent mechanisms including OS, elevated caspases and Bax will trigger neuronal cell death in PD (Li et al.,2020, Li et al.,2023). In this study, rotenone declined the Bcl-2 protein expression, while elevating the protein level of Bax and caspases. As a result of rotenone induction, increasing the discharge of Cyt-c from the mitochondria, which activates caspases-12, 9, and 3 terminating with apoptosis (41–43). SL treatment elevates the mitochondrial permeability, and averts the cyt c discharge from the mitochondria, thereby preventing caspases12, 3, and 9, thus restoring the imbalance in the expression profiles of Bax and Bcl-2, and preventing cell death. Furthermore, over expression of Bcl-2 interrupts the pro-apoptotic proteins of Bax and averts the mitochondrial release of cyt c, thus suppressing the stimulation of caspases, and apoptosis (Li et al.,2020, Li et al.,2023, Wu et al.,2018). It has been documented that complex I anticipation by rotenone may be the consequence of the mitochondrial permeability transition pores (PTP) opening, which makes a precise conformational transformation of complex I and a huge generation of ROS (Li et al.,2023, Wu et al.,2018). Rise in ROS within the mitochondria are recognized to root auxiliary mitochondrial membrane depolarization and discharge of ROS. In the existing work, we demonstrated that SL expressively suppressed Cyt-c, Bax, and Caspase-12pathways, while enhancing MMP and Bcl-2 protein expression in this PD model. Therefore, we established that SL repressed the apoptosis by regulating the apoptosis-related protein expressions.
Earlier reports have exposed that the dysregulation of the PI3K/Akt/mTOR network has been linked to the injury of dopaminergic neurons in PD (Yao et al 2022, Li et al.,2023). Our data established that rotenone alleviated PI3K, Akt, and mTOR phosphorylation in neuroblastoma cells, which is related to the preceding documents (Yao et al 2022, Li et al.,2023, Wang et al.,2022). Administration of neuroblastoma cells with SL (5 and 10 µM), the p-PI3K, p-Akt, and p-mTOR protein expression were markedly upregulated dose-dependably than the in vitro model of PD. These findings specified that SL activated the PI3K, Akt, and mTOR phosphorylation, and augmented the PI3K/Akt/mTOR pathway in this PD cellular model. Activated PI3K/AKT/mTOR signaling subdues cell apoptosis and autophagy, thus defending the cells from OS damage (Wang et al.,2022, Zhang et al.,2021, Peng et al.,2019). Agreeing with the above results, we rationally accomplish that SL protects the SK-N-SH cells by triggering the PI3K/ Akt/mTOR signaling.
In conclusion, SL could increase cell proliferation of rotenone-induced SK-N-SH neuroblastoma cells, MMP, while reducing neuronal injury, mitochondrial dysfunction, ROS generation, and apoptosis through regulating apoptotic proteins. PI3K/AKT/mTOR signaling pathway suppresses cell apoptosis and promotes the stimulation of autophagy, thus protecting the cells from OS damage. Here, we found that rotenone down-regulated the phosphorylation level of PI3K, Akt, and mTOR in SK-N-SH cells. Furthermore, SL (5 and 10 µM/ml) ameliorates the PI3K/Akt/ mTOR pathway in the in vitro PD model in a dose-related way. These results emphasize that SL might be a possible remedial component for PD, which could promote further research on animal models and clinical trials as a new therapeutic agent.