The pathophysiological molecular mechanism of VCI is complex. The use of herbal compounds containing AM, AM and its extracts to treat VCI has been confirmed by experimental research and clinical trial research results (Li et al. 2017; Mei et al. 2017), but the mechanism of AM and its main effective ingredients in treating VCI have not been fully clarified.
In this study, 87 active ingredients of AM were screened through TCMSP. The results of molecular docking displayed that among the 20 effective ingredients, calycosin and quercetin may be the core active compounds of AM for VCI treatment. Calycosin whose mechanism may be associated with anti-autophagy, anti-apoptosis and anti-inflammatory is a phytoestrogen isolated from AM (Wang et al. 2018). It has neuroprotective effects on cerebral ischemia-reperfusion injury. The experimental studies revealed that calycosin can alleviate the inflammatory reaction by suppressing MAPK and TLR/NF-κB signaling pathways, and improve sensory and movement disorders in degenerative diseases of the nervous system (Yang et al. 2019). Studies have also indicated that quercetin is a flavonol compound with all kinds of biological activities that can inhibit the MAPK signaling pathway and its downstream targets p-38 MAPK, p-ERK, and p-JNK. Meanwhile, quercetin can also reduce the levels of TNF-α, IL-6 and IL-1β, relieving neuroinflammatory reaction and neuralgia. Additionally, the molecular docking results of this study showed that AM has the strongest binding energy of calycosin and MAPK3, which may regulate the growth, differentiation, and neuroinflammation of nerve cells through the MAPK signaling pathway. In this study, the results are presented in accordance with the findings of Ye et al (Ye et al. 2021).
By intersecting the key targets of AM active ingredients and VCI disease targets, 158 key targets of AM interfering with VCI were obtained, so as to construct the PPI network diagram. Topology parameter calculation showed that the important potential active ingredients may be AKT1, TNF, IL6, SRC, EGFR, MAPK3, CASP3, HSP90AA1, JUN, MMP9, showing multi-target intervention in VCI. Further analysis of the combined module showed that the key targets are MAPK3 and MMP9. MMP9 can degrade the extracellular matrix, damage cell structure, cause apoptosis, increase cerebral vascular permeability, and destroy blood-brain barrier (Chakraborty et al. 2018). In parallel, it can also play the role of neuro-inflammatory mediators and reduce the stability of atherosclerotic plaques. Overall, MMP9 is a hazard factor for cerebral infarction and concurrent cognitive impairment (Yang et al. 2017). After cerebral ischemic injury, the MAPK family is highly phosphorylated, activating the MAPK signaling pathway, and up-regulating TNF-α, IL-6 ,IL-1β and other neuroinflammatory factors (Lee et al. 2015).
The KEGG enrichment analysis of this study indicated that the important pathways of AM in the treatment of VCI include Lipid and atherosclerosis, PI3K/Akt, MAPK, Rap1, and Ras signaling pathway, etc. Among them, PI3K/Akt signaling transduction pathway is a classic signaling transduction pathway, which can resist apoptosis, promote the survival of neuron cells and interfere with autophagy of neuron cells. In addition, the PI3K/Akt signaling transduction pathway, which can protect nerve cells and regulate neuronal cell apoptosis caused by cerebral ischemia and hypoxia, is related to cognitive dysfunction caused by cerebrovascular diseases. Akt, a key regulator of the PI3K/Akt signaling pathway, binds to 14-3-3 by phosphorylation the Ser136 site of Bad, preventing it from inhibiting the anti-apoptotic protein Bcl-2 and decreasing neuronal apoptosis (Pang et al. 2020). Akt's subtype AKT1 is a serine/threonine kinase encoding protein that affects the regulation of nerve cells. Its oxidative modification can lead to a decline in synaptic function and cause cognitive dysfunction (Ahmad et al. 2017). Experimental studies have also confirmed that activating of Gi protein/c-Src/Pyk2/EGFR/PI3K/Akt/p42/p44 MAPK cascade can lead the activation of the pro-inflammatory factor activator protein-1 (AP-1) containing JUN, inhibit neuroinflammation and reduce neuronal apoptosis (Yang et al. 2017; Liou et al. 2019).
MAPK signaling pathway regulates cell growth, differentiation, proliferation, cytoskeleton remodeling, and cell migration. It has extensive distribution and expression in the central nervous system. Furthermore, the MAPK signaling pathway is closely associated with cerebral ischemia injury and repair, which affects the long-term potentiation (LTP) of learning and memory formation in the hippocampus by regulating inflammatory factors and oxidative stress (Revest et al. 2014). The PI3K/Akt and MAPK signaling pathways are important downstream pathways of brain-derived neurotrophic factor (BDNF), and their common mediator is epithelial growth factor receptor (EGFR). EGFR can induce the small G protein Ras to activate the serine/threoninase Raf after activating SOS1. Ser338 site on Raf-1 can activate the downstream MAPK/ERK signaling, while Ser259 site can reversely inhibit MAPK/ERK pathway. The PI3K/Akt signaling pathway can activate Ser259 site to inhibit MAPK/ERK pathway activity (Park et al. 2011; Horn et al. 2015). It is worth noting that there is crosstalk among PI3K/Akt, MAPK and Ras signaling pathways, which are strongly correlated with the pathogenesis and treatment of VCI. The important pathway of AM in the treatment of VCI is closely related to neurotrophic, neuroinflammation, autophagy, and apoptosis.