Neurodegenerative diseases, including Alzheimer which results in a decrease of human cognitive function, have become the main obstacles that degrade human life span and the overall wellbeing [38].

Through aging, Aβ is a product that naturally emerges and accumulates in the brain. Accumulated Aβ gradually forms aggregates with beta-sheet structures and has the capacity to alter the intercellular redox balance of neurons cells. This is considered a major cause of the onset of cognitive dysfunction [12,13,39]. Among Aβ variances, the Aβ_25–35 fragment is the shortest segment of full-length Aβ₁–₄₂that can form a beta-sheet; this fragment was proven to have similar oxidative neurotoxicity output compared with the full-length Aβ₁–₄₂ [14]. With good solubility and stress-induced efficiency, interest in this undecapeptide Aβ_25–35 has grown over the last decade [15].

In detail, both full-length and short Aβs were proven to form ion-like channels in cell membranes that promote Ca²⁺ influx, destabilize intercellular balance, and produce cellular oxidative stress [40,41,42]. Another source of oxidative stress arises as Aβ_25–35 and Aβ₁–₄₂were also both found to cause mitochondrial abnormalities via the deactivation of mitochondrial complex IV [43,44,45,46]. In neurodegenerative progression, ROS overproduction from these important events causes damage to the cellular structure, increases MDA, LDH, and protein carbonyl levels, eventually initiates neuron cell death mechanisms, and promotes neuro-cognitive impairments [10,47,48]. Explaining this similar effect of Aβ₂₅–₃₅ and Aβ_1-42 is still a topic of debate, but the single methionine-35 located in both Aβs was mentioned as its redox states are significantly important for Aβ-correlated free radical oxidative stress and neurotoxicity causes [45,49,50,51].

In the in vitro study, our results demonstrated that Aβ_25–35 induced injury in HT-22 cells, including massive ROS release, resulting in MDA and LDH leakage and increased protein carbonyl levels, which substantially reduced HT-22 cell viability. Melittin dramatically mitigated Aβ_25–35-induced oxidative stress injury by reducing ROS, MDA, SOD, and eventually, the protein carbonyl levels. To further explain these phenomena, we examined the melittin effect on the generation of HO-1, an important component of the cellular antioxidant system. This protein expression is inducible and has been demonstrated to shield cells against oxidative damage [16]. HO-1 expression is regulated by Nrf2. In the primitive stage, Nrf2 is retained in the Nrf2-Keap1 complex, and activation of cellular protection mechanisms leads to the separation of Nrf2 from the complex and transfer to the nucleus. Nrf2 acts as a transcription factor in the antioxidant response element (ARE) gene region, which in turn upregulates the expression of the HO-1 gene [10,33]. Our results showed that under cellular stress-induced conditions, melittin markedly upregulated the nuclear translocation of Nrf2 and enhanced the overall production of antioxidant enzyme activity, suggesting that the beneficial effect of melittin on Aβ_25–35-induced HT22 cell injury was attributed to its antioxidant properties. This is in line with the findings of previous studies indicating that melittin enhances Nrf2 nuclear translocation and subsequently upregulates the expression of important antioxidant genes such as HO-1 [8,9].

One other mechanism of melittin can be related to the TrkB/CREB/BDNF pathway, which is a commonly studied direction along with antioxidant aspects [11,31,52,53]. The activation of TrkB can lead to downstream enhancement of both cellular antioxidant defensive and brain-derived neurotrophic factor neuro-proliferative shields [10]. In our experiment, Aβ_25–35 presence significantly depleted the TrkB/CREB/BDNF pathway. Remarkably, melittin induced p-TrkB activation, increased the amount of p-CREB transcription factor, and in turn upregulated the expression of BDNF [54]. Moreover, BDNF was examined to stimulate subsets of Trk receptors and can further protect neuronal cells from oxidative stress-induced cell death [36,55]. The grounds above suggested the mechanism on how melittin demonstrated the ability to protect neuron cell HT22 apoptosis induced by Aβ_25–35, an protective effect which was confirmed by the normalization of Bax/Bcl-2 ration, apoptosis-inducing factor, Calpain, CytoC, and CleaCas3.

For in vivo research, the injection of Aβ_25–35 into mice brains was a utilized as a mean to induce oxidative stress, initiating synaptic loss, suppressing neurogenesis, and resulting in cognitive impairments in animal experiments [15,20,21,22]. This model elicits a considerable degree of Alzheimer’s progression signs and neurodegeneration conditions in general [21].

In this study, melittin significantly enhanced the memory and learning abilities of cognitive impairment-induced animals, a novel finding. The neuroprocessing ability of mice is closely related to hippocampal physiology [56]. Among the many anatomical parts of the hippocampus, the dentate gyrus is commonly studied and plays a key role in the formation, recall, and discrimination of episodic memory [57]. The significant increase in neurogenesis in this region has proven the effect of melittin at an anatomical scale.

To further explore the antioxidant property of melittin, we measured ROS and MDA levels in the hippocampus and serum; the levels of these parameters were significantly increase due to Aβ_25–35 ICV., and melittin treatment clearly decreased these oxidative stress markers. Our results showed melittin reduced the amount of NO accumulation and iNOS protein expression in the hippocampus, which indicates melittin’s effect in lowering neuronal-derived nitric oxide—a key element stimulating neural diseases [58,59].

Maintenance of the balance of the cholinergic system is necessary for normal memory function [30]. Previous studies have revealed that patients with Alzheimer’s disease have downregulated expression of mAChR 1; hence, it is an important neuroreceptor to study in the cholinergic system [30]. In the synaptic cleft, ACh binds to postsynaptic mAChRs, and the synaptic signal communicates sequentially with the cyclic adenosine monophosphate/protein kinase A/CREB signaling pathway via G-coupled protein receptors. This bridge reflects the reality that the cholinergic signaling system and intercellular neurotrophic factors are two reciprocal entities. The disruption of one can cause a negative effect on the other and they both synergistically facilitate the grounds for neuronal grow and brain normal cognitive functions [60]. In our study, downregulation of mAChR 1 expression by Aβ_25–35 in hippocampal tissues was significantly normalized by melittin pretreatment. Excessive AChE activity leads to a decrease in the Ach level in hippocampal cholinergic synapses. Aβ_25–35 ICV. injection augmented AChE activity by 1.5-fold in hippocampal tissue, whereas pretreatment with melittin completely attenuated the excessive activation of AChE and increased ACh levels. Regarding intercellular neurotrophic factors, the transcription factor CREB, which plays a key role in BDNF synthesis, is essential for memory and synaptic plasticity [61]. In line with the in vitro experiments, our in vivo results showed that melittin-treated mice also had increased hippocampal p-CREB and BDNF levels.

Therefore, through both in vivo and in vitro experiments, melittin had proved itself to be a drug candidate combating neurovegetative disease.

The drug administration route of melittin is often subcutaneous, which can cause adverse effects if over-dosed [62]. Although melittin exhibited the ability to recover cognitive function in neurodegenerative-induced models, its irritation properties should be alleviated, and an optimal dosage for humans should be determined. In other disease research with melittin, up-to-date recombinant technology and computational bioinformatics modified the specific amino acid sequences and created a specialized-engineered-melittin. This produced effective augmentation and enhanced drug delivery, which enabled melittin to event be intravenously injected and to target a specific group of malarian cells [63]. Such advances can alleviate the side effects of melittin and further increase the popularity of melittin treatment.