AD is a complicated neurodegenerative disease characterized by a progressive loss of cognitive function resulted from many etiological factors that causes and pathogenesis are not clear. So, combination treatments rather than monotherapy are warranted for the development of effective approaches in treating AD. The unique advantages of TCM such as naturally sourced, multitargeted, individual mode and a holistic concept are obvious in treating complicated diseases especially those with a poor efficacy to western medicine alone. In the theory of TCM, AD is mainly caused by old and weak with deficiency of healthy Qi as a result of endogenesis of turbid phlegm, blinding the clear orifices. The basic pathogenesis is deficiency of origin and excess of sign. XXD is a classical formula which is used to resolve phlegm, induce resuscitation and nourish the heart to calm the mind. XXD has been used to treat dementia clinically for a long history from hundreds of years ago in China. But it is undeniable that the complexity of compounds and compatibility in the use of TCM, as well as the dynamic process of the development of chronic and complex diseases, bring great challenges to the research of TCM. With the continuous development of bioinformatics, TCM network pharmacology has become a topic on hot spots to reveal above mentioned complex biological processes from the perspective of integrated multi-component networks[22].There are few studies on the mechanism of XXD in the prevention and treatment of AD. To reveal the beneficial effects of XXD on AD, we used network pharmacology approach to explore the putative active compounds and potential mechanisms.
In our study, we constructed the following network to reveal the potential targets and pathways of XXD in AD treatment: compound-target network, PPI network of shared targets-corresponding compounds and compound-hub target -pathway network. After integrating and consolidating information from diverse sources of available databases, 10 active compounds of XXD acted on 208 different targets associated with AD. According to the shared targets-corresponding compounds network,10 compounds and 208 shared targets are highly connected and they can be defined as vital compounds in XXD. In XXD, according to the TCM theory in formula prescribing, the Jun or “monarch herb” is Pinellia ternate (Banxia), exerting the effects of resolving phlegm. The Chen or “minister herbs” consist of Panax ginseng (Renshen) and Aconitum carmichaeli Debx(Fuzi), with the function of tonifying Qi and Warming and activating Yang Qi, respectively. In addition, Glycyrrhiza uralensis Fisch (Gancao), playing the role as Zuoshi or “adjuvant and guide herb”, helps tonifying Qi and harmonizing various herbs. In our study, three of main active compounds including Gomisin B, baicalein and Karanjin, were identified from the “monarch herbs”, the “minister herbs” and the“adjuvant and guide herb”, respectively. This prescription feature might provide the pharmacological evidences for XXD to be used as multi-target regulation in treating AD.
Moreover, according to the pathway- target-compound network, components in XXD such as licochalcone a, Gomisin B,7-Methoxy-2-methyl isoflavone, baicalein, Glypallichalcone, zizyphusine,7-Acetoxy-2-methylisoflavone, Karanjin, Girinimbin and 6-prenylated eriodictyol interacted with a large number of targets, indicating the important roles in the anti-AD system. Some of them have been reported to show biological activities against AD in the past. For example, schisantherin B(Gomisin B) identified in Renshen was able to protect against learning and memory impairments and remit the toxicity caused by excessive activation of hyperphosphorylated tau in the dementia mouse model induced by Aβ1−42 through regulating glial glutamate transporter type 1 (GLT-1), which has an effect on the level of p-tau protein[23]. Baicalein in Banxia was proved to show therapeutic potential for AD through reducing oxidative stress, anti-inflammatory properties, inhibiting aggregation of amyloid proteins, stimulating neurogenesis and differentiation action and anti-apoptosis effects[24].A research showed that karanjin in Fuzi possesses learning and memory improvement [25].
According to GO enrichment analysis, BP terms enriched by target genes were mainly concentrated in neurodegeneration-related progressions such as positive regulation of kinase activity, positive regulation of cell death and synaptic signaling. Through KEGG enrichment analysis, we found that the mechanisms of XXD in treatment of AD mainly include Alzheimer's disease pathway, neuroactive ligand-receptor interaction pathway, serotonergic synapse and MAPK signaling pathway.
The Alzheimer's disease pathway with a high degree, containing insulin signaling pathway with hub targets such as PIK3CA and AKT, is thought to be tightly associated with the effect of XXD on AD. Previous research indicated that insulin signaling pathway depicts a strong correlation of energy utilization, mitochondrial function, oxidative stress, synaptic plasticity and cognitive function and the occurrence and development of AD is partially attributed to the impairment of insulin signaling pathway[26].
Some receptor genes control the neuroactive ligand-receptor interaction pathway, which regulates learning and memory ability through enhancing cholinergic function[27], and may participate in cognitive and emotional regulation[28]. The abnormal function of GABAA (γ-aminobutyric acid A)receptor, one of the important receptors of neuroactive ligand-receptor interaction pathway, is closely associated with AD[29].
Serotonergic synapse is one of the primary pathological factors in neuropsychiatric symptoms[30, 31]. Previous researches provided sufficient evidence that serotonergic synapse pathway are likely to play a role in memory dysfunction or AD[32, 33].
Mitogen-Activated Protein Kinase (MAPK) pathways, including the Extracellular Signal-Regulated Kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 pathways, play the critical roles in regulating cellular functions such as cell apoptosis, synapse plasticity, neural cell survival and neuroinflammation. All the MAPKs are activated in related neurons in patients with AD, suggesting that MAPK pathways are involved in AD pathophysiology and pathogenesis[34]. Furthermore, inhibition of these pathways depressed multiple pathological features including Aβ production, tau hyperphosphorylation and synaptic loss, and also made the degeneration of cognitive function in AD animal models[35, 36]. Among the potential active ingredients, baicalien was demonstrated to alleviate neurotoxicity in Aβ25−35-induced PC12 cells via the increased expression of MAPK pathway, suggesting its neuroprotective effect [37].
We performed molecular docking simulation between 8 hub target genes and 9 main active compounds to verify the importance of hub targets. The results showed that all of the target-compound pairs possess high scores of docking affinity, especially, TP53, PIK3CA, AKT1. Previous studies have shown that these genes regulate multiple functions contributing to AD progression. As a downstream effector of DAPK-1 and a Ca2+/calmodulin (CaM)-dependent serine/threonine protein kinase pathways ,TP53 plays an important role in cell apoptosis involving transcriptional induction of proapoptotic genes which consequently trigger mitochondrial pathways.TP53 has been suggested as potential therapeutic targets of AD because of its correlation with AD-related neurodegeneration in the brain[38]. Akt1, a cell survival kinase, mediate Akt1 signaling through its oxidative modification, occurring in the APP/PS1 transgenic mouse model of AD. The loss of Akt1 kinase lead to potential synaptic dysfunction in neurodegeneration including AD[39]. PIK3CA, a catalytic subunit of PI3K, plays a critical role in PI3K/AKT signaling pathways and affects the regulation of the pathogenesis of AD via inhibition of tyrosine phosphatases to protective against Aβ toxicity[40].