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.