4. Discussion
Rosemary, an herb of economic and gustatory repute, is employed in traditional medicines in many countries[31]. Rosemary contains CA, CS and abietane-type phenolic diterpenes[32, 33], which account for most of its biological and pharmacological actions[34, 35]. In our previous study, we identified DACA as an abietane type diterpene with antioxidant effects. However, no investigations have explored the role of DACA in PD. The present study demonstrates that: (1) DACA ameliorates MPTP-induced motor dysfunction and decrease of TH level, and induces the upregulation of Nrf2 and its downstream antioxidant enzymes in the midbrain and striatum; (2) DACA alleviate MPP+-induced oxidative stress, mitochondrial damage and autophagy abnormalities. (3) The neuroprotective mechanism of DACA operates via the activation of Nrf2.
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by resting tremor, bradykinesia, myotonia, postural and gait disturbances as the primary motor manifestations[36]. The behavioral disorders associated with Parkinson’s disease are attributed to the degenerative demise of dopaminergic neurons, and alterations in TH expression exhibit a close correlation with dopaminergic neuronal death[37, 38]. In this study, DACA treatment effectively mitigated MPTP-induced motor dysfunction and significantly elevated TH levels in the midbrain and striatum. Nrf2 is the master regulator of cellular redox status[39]. Overexpression of its downstream proteins under neurotoxic conditions prevents hydrogen peroxide accumulation, lipid peroxidation, and, consequently, neuronal loss[40]. The findings of our study demonstrate that DACA has the potential to enhance the expression levels of Nrf2, HO-1, GCLC, and GCLM in both the midbrain and striatum.This demonstrates that Nrf2 activation using DACA might be able to help brain tissue repair and promote functional recovery.
Accumulating evidence from human studies and various experimental models of PD suggests that oxidative stress plays a pivotal role in both the initiation and progression[41]. The data obtained from our study demonstrated that DACA exhibited a protective effect against MPP+-induced oxidative stress in cells, thereby suggesting its potential as a potent antioxidant compound. The generation of ROS is intricately associated with oxidative stress-induced damage and the pathophysiology underlying neurological dysfunction and neuronal cell death processes. SOD serves as the primary line of defense against free radicals, exhibiting robust and efficient scavenging activity by utilizing free radicals as substrates. GSH, an indispensable antioxidant, effectively mitigates oxidative stress instigated by ROS. Notably, DACA significantly attenuated MPP+-induced ROS production and concurrently enhanced the levels of SOD and GSH. Simultaneously, DACA facilitated nuclear translocation of Nrf2 and upregulated the expression of HO-1, GCLC, and GCLM.
Excessive oxidative stress selectively targets and impairs mitochondria, resulting in their functional impairment. The preservation of mitochondrial integrity and bioenergetic functions is crucial for cellular homeostasis[42]. To maintain mitochondrial and cellular homeostasis while preventing the detrimental effects of damaged mitochondria on cells, a process known as mitophagy occurs wherein cells selectively sequester and degrade dysfunctional or impaired mitochondria[43, 44]. The results demonstrated that DACA effectively reversed the decline in mitochondrial membrane potential and the elevation of mitochondrial reactive oxygen species induced by MPP+. Additionally, DACA exerted regulatory effects on intracellular apoptosis-related proteins Bax and Bcl-2, leading to a significant increase in the Bcl-2/Bax ratio, thereby inhibiting MPP+-induced apoptosis. Interestingly, DACA modulated mitophagy through modulation of p62, PINK1, and LC3 expression.
A plethora of studies have demonstrated that Nrf2 not only governs oxidative stress, but also exerts a pivotal role in preserving mitochondrial function and regulating mitophagy[41] [45]. In order to elucidate the localization of Nrf2 in mediating the neuroprotective effects of DACA, we employed a selective inhibitor targeting Nrf2. Intriguingly, treatment with this specific Nrf2 inhibitor abrogated the protective efficacy of DACA against MPP+-induced cellular damage. However, with the presence of a specific p62-Nrf2 inhibitor, substantial levels of Nrf2 were still observed within the cellular milieu. This suggests that the neuroprotective effect of DACA is through Nrf2. And unlike most natural small molecules of Nrf2 activators, Nrf2 activation by DACA is not via p62.