Our study analyzed the active components, targets, and related signaling pathways of DR in improving memory impairment by using network pharmacology. Because of the advantages of network pharmacology research strategy, from “one target, one drug” to “network target, multicomponent therapy”, we also provided an overview of the central role of Akt1 in the network. In addition, we confirmed xanthogalenol was the interactive object with Akt1 in DR.
Memory impairment is one of the most urgent public health concerns due to its high prevalence, chronicity, and disabling features. Timely and accurate diagnosis of memory impairment is critical. However, there are no specific drugs for the treatment of memory impairment at present. In search of clues to resolve this question, we were focused on the reciprocal interactions between bone and brain. On one hand, brain regulates bone homeostasis via multiple pathways. For example, leptin is a powerful inhibitor of bone remodeling through a central relay . On the other hand, there is growing evidence demonstrating bone as an endocrine organ. Therefore, bone returns the favor to brain in the process of acute stress response , and age-related memory loss . Osteocalcin plays an important role in these aspects. It has been reported that DR increased the expression of osteocalcin in rat with steroid‑induced avascular necrosis of the femoral head . In view of this, DR may ameliorate memory impairment indirectly through osteocalcin. However, we explored the possibility of their direct interaction. Therefore, BBB score was used for screening of brain-penetrating compounds from DR. 7 active components, including xanthogalenol, could cross BBB, and were considered as prime candidates.
Xanthogalenol exerts antiproliferative activity against on mammalian cancer cells through unidentified mechanisms . Using network pharmacological analysis, we identified NGF-TRKA signaling pathway may mediate the effect of xanthogalenol on memory impairment. Since its discovery, NGF has been proved to play a critical role in a variety of developmental processes related to nerve cells. Hippocampus is one of the most NGF-rich sites in brain . There is a strong connection between NGF and cholinergic system in hippocampus, both of which are required for supporting spatial memory . Therefore, NGF-based therapy has been under continual development of treatment for memory impairment caused by Alzheimer’s Disease , and diabetes . There are generally considered to be two types of NGF receptors: the low-affinity NGF receptor p75 neurotrophin receptor (p75NTR) and the high affinity NGF receptor TRKA . When TRKA is activated by NGF, the downstream cAMP response element-binding protein promotes regeneration, survival, and proliferation of neurons . On the other hand, binding of NGF and p75NTR triggers neuronal apoptosis by activating C-Jun N-terminal kinase signaling pathway . Thus, imbalance of TRKA/p75NTR signaling pathway mediates impairment of spatial learning and memory in offspring caused by maternal subclinical hypothyroidism . The same phenomenon has also been reported in Alzheimer’s Disease . Hence, one treatment strategy for memory impairment is to develop small molecule agonists for TRKA, or antagonists against p75NTR. In our study, it is more likely that xanthogalenol plays a protective role in memory impairment through activating NGF-TRKA signaling pathway.
According to the Neurotrophin signaling pathway from KEGG, upstream of Akt cascade is phosphatidylinositol-3-kinase (PI3K), while downstream of Akt cascade is related to axonal outgrowth, synapse formation, and cell survival. Thus, there is no doubt that some of nervous system drugs act through their interaction with Akt in the central nervous system. Three Akt isoforms are expressed in human brain, Akt1, Akt2, and Akt3. Despite high overall homology across species, the isoforms have distinct biological functions in neurological disorders. Accumulating evidence suggests that Akt1 has role in schizophrenia , Akt2 in gliomas  and Akt3 in brain growth . More specifically, Akt1 is more important than Akt2 and Akt3 for synaptic plasticity mediated by gene expression change and new proteins synthesis . Our result also showed that Akt1 was implicated in memory impairments associated with synaptic plasticity. Oxiracetam, a common drug for improving memory impairments, ameliorates ischemic stroke induced neuronal apoptosis through up-regulating PI3K/Akt signaling pathway in rats . Oxiracetam also alleviates learning and memory impairments in vascular dementia by activating Akt/mTOR signaling pathway in neurons . In molecular docking analysis, Lys30, Thr34 and Tyr38 were the predicted binding site in Akt1 for Oxiracetam. It also predicted that xanthogalenol was able to bind to Akt1at the binding sites of Val4 and Glu49. Although these two binding sites was not identical to each other, they were structurally very close. This would mean that xanthogalenol may play neuroprotective roles by a similar mechanism as oxiracetam. Akt1 itself is phosphorylated and activated by Thr308 and Ser473 , which are located relatively far from the binding domain of xanthogalenol. However, a recent study reveals an activity-induced Akt1 SUMOylation at Lys64 and Lys276 is required for long-term potentiation of synaptic transmission . Since neuronal activity-regulated protein SUMOylation plays a vital role in memory, this mechanism may mediate the effect of xanthogalenol on memory impairment because of spatial proximity between the amino acid residue, especially between Glu49 and Lys64. Therefore, these sites are potential targets for future drug design.