**4. Discussion**

Dementia is a complex condition involving the disruption of cortical function along with adverse effects on memory, reasoning, orientation, learning capacity, and emotional stability. Progressive dementia is associated with AD, a prevalent neurodegenerative disorder, characterized by excessive accumulation of neuritic plaques and neuronal loss, including abnormal tau proteins and β-amyloid. A depletion of the neurotransmitter, ACh, occurs in patients with AD. Degeneration of the cholinergic neurons is also an crucial factor that contributes toward the development of dementia [15].

In this study, mice were tested to a passive avoidance test and an MWM task in a spatial memory trial to assess their learning abilities and memory deficits [6]. SCO treatment significantly decreased cognitive function as reported in a previous study [16]. However, Brainon treatment recovered the decreased step-through latency and reduced escape latency and swim distance in passive avoidance performance and MWM trials, respectively.

ACh establishes synaptic connections between neurons; it plays an important role in the CNS and is related to cognition and memory [17]. ACh is hydrolyzed by AChE. Increased ACh levels resulting from AChE inhibition enhance learning and memory by improving cholinergic deficiency [15,18]. Acting as a non-selective muscarinic cholinergic receptor antagonist, SCO causes cholinergic dysfunction that leads to impaired learning and memory. It is known that AChE inhibitors antagonize the effects of scopolamine on spatial memory in the radial arm maze [19] in the Morris Water maze and in passive avoidance tests [20,21]. This suggests a strong correlation between anti-AChE activity and the ability to oppose these amnestic effects of scopolamine [22]. We administered SCO to mice to induce cholinergic neurodegeneration alongside cognitive deficiency and confirmed the decline in ACh levels and increase in AChE activity [23]. We discovered that Brainon treatment significantly enhanced ACh levels and decreased AChE activity in the hippocampi of SCO-injected mice.

SCO-induced neurodegeneration results in a decrease in cholinergic activity and BDNF as well as the inhibition of phosphorylated CREB expression in the hippocampal tissue [4]. High BDNF expression in the CNS, particularly in the hippocampus, is a neurotrophic factor that modulates the growth and survival of neurons. BDNF enhances synaptic plasticity and regulates memory formation by increasing the activity of enzymes related to ACh synthesis [24]. CREB also leads to neuronal synaptic plasticity through the expression of downstream targets of Bcl-2 and neuroprotective effectiveness against reactive oxygen species (ROS)-caused cell toxicity [25,26]. Disturbance of phosphorylated CREB indicates neurodegenerative disorders, such as AD as well as Huntington's and Parkinson's disease [25,27]. In this study, we assessed the relevance of the CREB–BDNF signaling mechanism in AD. Brainon upregulated BDNF protein levels as well as phosphorylated CREB protein levels in the hippocampi comparison with those in the SCO-treated group.

ROS induces oxidative stress and is significant in the pathology of AD [9,28]. Excessive oxidative damage induces neuroinflammation and results in neurotoxicity, contributing to clinical symptoms of AD, including cognitive deficits [29,30] Antioxidant enzymes, such as SOD-1 and SOD-2, play a protective role against oxidative stress by catalyzing the conversion of superoxide anions to oxygen and hydrogen peroxide. Results from previous studies show that ACh exerts a neuroprotective effect against oxidative stress by increasing the expression of SOD, and that ACh levels are positively related with those of SOD [31,32]. In the present study, treatment with Brainon recovered the antioxidant defense system by increasing SOD and ACh levels in the hippocampal tissues of SCO-induced mice.

ROS-caused oxidative stress is considerably correlated with neuroinflammation, which exacerbates neurodegenerative disorders and contributes to the progression of AD. SCO administration upregulated IL-1β, IL-6, and TNFα levels in mouse hippocampi, and these findings were consistent with the results from previous studies [33,34]. However, Brainon suppressed the SCO-induced neuroinflammatory cytokines in the SCO-treated mouse hippocampi. ROS, the primary cause of oxidative stress, is also responsible for the initiation of apoptosis. It promotes the overexpression of the proapoptotic protein, Bax, leading to cell death. The antiapoptotic protein, Bcl-2, which has effects opposite to those of Bax, is regulated by CREB. Bax upregulation and Bcl-2 downregulation promote neuronal cell death by increasing caspase activator release in the hippocampal tissue [12,35,36]. Our results suggested that SCO activated Bax, cleaved caspase-9, and cleaved-PARP, while downregulating protein expression of Bcl-2. Moreover, we found that Brainon alleviated

the overexpression of Bax and the activation of cleaved caspase-9, cleaved-PARP, and on the other hand increased protein expression of Bcl-2 in SCO-treated mice.

To summarize, Brainon was found to have an anti-amnesic effect, which could be regulated by cholinergic activity and the CREB-BDNF signaling pathway, and by virtue of its antioxidant, anti-inflammation, and anti-apoptotic properties.
