Study on the mechanism of action of Er Zhi Wan in combating Alzheimer’s disease based on serum metabolomics and network pharmacology
Er Zhi Wan comes from the classic Ming Dynasty prescription “Yi Bian” Volume 1, and has the effects of nourishing kidney essence, stopping bleeding, and strengthening the body and bones. Er Zhi Wan is composed of holly seeds and drought lotus grass. Pharmacological studies have shown that Er Zhi Wan has anti-aging, anti-inflammatory, and anti-tumor effects. Related studies have found that Er Zhi Wan can improve the learning and memory abilities of D-galactose-induced aging rats, has anti-aging functions, clears brain free radicals, and protects neurons. The active ingredients of Er Zhi Wan, such as quercetin, geraniol, β – sitosterol, naringenin, and icarium, may improve Alzheimer’s disease (AD), and the mechanism of action of these compounds is closely related to the PI3K/Akt signaling pathway. At present, research on Er Zhi Wan has only been conducted at the overall therapeutic level, and its anti AD mechanism has not been elucidated, greatly hindering its clinical application.
AD is a progressive neurodegenerative disease that currently affects over 50 million people worldwide and is expected to become a major issue for an aging population, imposing a huge burden on patients, families, and healthcare systems. AD is the most common age-related dementia, accounting for approximately 70% of all dementia cases. Although the final positive diagnosis of AD still requires post-mortem analysis of brain tissue, an increasing number of biomarkers for cerebrospinal fluid, blood, and positron emission tomography imaging have been proposed, and it is expected to provide early diagnosis, sensitivity, and specific diagnosis for living patients by combining clinical standards and evaluations. The characteristics of AD are extracellular amyloid beta (A β) plaques, neuronal inclusions composed of truncated and phosphorylated forms of microtubule stabilizing protein tau (neurofibrillary tangles), malnourished neural processes, loss of synapses and neurons, and a prominent gliosis involving changes in the morphology and function of microglia and astrocytes. In recent years, blood metabolomics technology has played a huge role in the diagnosis and mechanism research of AD, and can even be used as a specific detection tool for screening AD. The metabolic basis of AD is poorly understood, and the relationship between metabolic system abnormalities and the pathogenesis of AD is still unclear. Understanding how overall metabolic disorders are associated with the severity of AD neuropathology and the ultimate expression of AD symptoms in high-risk populations is crucial for developing effective disease improvement treatments. Network pharmacology is an emerging, interdisciplinary, and cutting-edge discipline in the era of artificial intelligence and big data for the systematic study of drugs. It emphasizes the analysis of molecular relationships between drugs and treatment targets from the perspective of system level and biological networks. It is widely used in the discovery of active compounds in drugs and traditional Chinese medicine, the interpretation of overall mechanisms of action, and the analysis of drug combinations and formula compatibility rules. It provides new ideas for the study of complex systems in traditional Chinese medicine and new technological support for clinical rational drug use and new drug development. Therefore, this study used high-throughput metabolomics combined with network pharmacology techniques to investigate it, in order to discover potential components and core metabolites related to the disease, and clarify the mechanism of action of the drug.

This study discovered 15 blood biomarkers closely related to AD through metabolomics techniques, and their metabolic mechanisms and physiological significance are related to the development of AD in different stages. The following is an explanation.

The learning and memory mechanism of the body is closely related to metabolites such as 5-HT. Tyrosine is a non essential amino acid produced by hydroxylation of phenylalanine in humans and animals. It is a precursor of monoamine neurotransmitters such as adrenaline, norepinephrine, and dopamine. Dopamine regulates psychological activity, emotions, recognition, thinking, and reasoning through the midbrain margin, midbrain cortex, substantia nigra striatum, and nodule funnel pathway, indirectly affecting memory. 3-methoxy-4-hydroxyphenylethylene glycol sulfate and homovanillic acid are both metabolites of the dopamine metabolic pathway system. In this study, the levels of 3-methoxy-4-hydroxyphenylene glycol sulfate and N-acetyl5-hydroxytryptamine in the serum of the model group rats were significantly higher than those in the sham operation group, while the levels of vanillic acid and vanillyl mandelic acid in the model group were lower than those in the sham operation group. Er Zhi Wan can regulate the levels of endogenous metabolites in AD rats (P<0.05), suggesting that Er Zhi Wan may enhance the learning and memory abilities of AD rats by regulating the levels of monoamine neurotransmitters and their metabolites.

The main inhibitory amino acid neurotransmitter gamma aminobutyric acid in the nervous system is also believed to be associated with cognitive function. Gamma aminobutyric acid plays an important role in maintaining brain excitation balance and inhibiting nerve conduction. Autopsy and animal model studies have shown that there is dysfunction of the gamma aminobutyric acid system in the pathological process of AD. 4-Guanidobutyric acid is a precursor of gamma aminobutyric acid. In patients with arginase deficiency, elevated levels of 4-guanidinobutyric acid lead to various clinical syndromes such as intellectual disability, neurodevelopmental arrest, and epilepsy. In this study, Er Zhi Wan reduced the content of 4-guanidinobutyric acid in the blood of AD rats and was inferred to increase the content of gamma aminobutyric acid in the hippocampus, indicating that Er Zhi Wan can promote the synthesis of gamma aminobutyric acid, reduce the damage caused by hyperammonemia to the nervous system, and thus exert neuroprotective effects.

Glutamic acid and aspartic acid are two main excitatory amino acid neurotransmitters that play important roles in the transmission of excitatory information in the nervous system. They are related to the survival, synaptic formation, and plasticity of neurons. Glutamate and aspartate are excitatory amino acids released from the presynaptic membrane, selectively binding to G protein mediated glutamate receptors and N-methyl-D-aspartate (NMDA) receptors, leading to the opening of calcium channels. This increases the influx of Ca2+into the postsynaptic membrane and participates in the generation and maintenance of long-term potentiation (LTP) of synaptic transmission. LTP is considered an indicator of neuronal physiological activity during the process of memory formation and consolidation. The content of glycine and aspartic acid in the model group significantly increased (P<0.01), while the content of glycine and aspartic acid in the treatment group significantly decreased (P<0.01). The results indicate that Er Zhi Wan can inhibit the synthesis of excitatory amino acids in the brain, thereby exerting a protective effect on nerve cells.

Taurine is a conditionally essential amino acid, particularly distributed in the cerebral cortex, hippocampus, and cerebellum. In the central nervous system, taurine, as the second most abundant endogenous amino acid, plays a role in neural regulation, osmotic regulation, calcium homeostasis maintenance, membrane stability, antioxidant function, anti-inflammatory process, and neuroprotection. Taurine has also shown neuroprotective effects in many pathological conditions, such as AD and Huntington’s disease. During the progression of AD, A β monomers aggregate into neurotoxic soluble oligomers A β, leading to cognitive impairment. According to reports, the taurine content in the brain of AD patients is reduced, and taurine can inhibit the accumulation of A β. In addition, taurine has a significant impact on the intellectual development of rats. Injecting taurine into the body can increase the content of acetylcholine in the brain, and long-term use of taurine has no toxicity or other side effects. In this study, after administration of Er Zhi Wan, the blood taurine concentration in the model group rats decreased, while it significantly increased after treatment with Er Zhi Wan. This result indicates that Er Zhi Wan can inhibit the accumulation of A β protein by regulating taurine metabolism.

Adenosine is responsible for regulating, integrating, and fine-tuning neuronal activity, and affecting related brain functions including sleep and wakefulness, cognition and memory, as well as neuronal damage and degeneration. Uridine, as a precursor of membrane phospholipid biosynthesis, has been shown to increase the number of brain synapses and improve learning and memory in the long term by reducing early apoptotic cell death in newborns. Compared with sham surgery, the levels of adenosine and uridine in the model group were significantly increased, while the levels of adenosine and uridine in the treatment group were much lower than those in the model group. The restoration of adenosine and uridine to healthy levels may improve the learning and memory of AD rats by affecting the structure and function of neurons in Er Zhi Wan.

Tryptophan is an essential amino acid for the human body, involved in the synthesis of proteins and some important active substances (serotonin, nicotinamide adenine dinucleotide, and niacin). Imbalance in the canine urinary amino acid pathway is associated with AD. The kynurenine pathway consists of two branches, leading to the formation of xanthine and kynurenine or the production of 3-hydroxyanthranilate and 3-methoxyanthranilate. Canine uric acid, as an N-methyl-D-aspartate receptor antagonist, is considered to have neuroprotective effects, while metabolites from other branches, including 3-hydroxyanthranilic acid and 3-methoxyanthranilic acid salts, are believed to have neurotoxicity. The accumulation of 3-hydroxyanthranilic acid and 3-methoxyanthranilic acid salts may lead to apoptosis of astrocytes and certain nerve cells, thereby weakening the work of the microglial neural network, reducing the synthesis of neurotrophic factors, and causing damage to the entire nervous system. Er Zhi Wan reduces the content of 3-hydroxyanthranilate in the urine of AD rats, indicating that Er Zhi Wan can protect nerve cells by reducing neurotoxic substances.

In addition, studies have shown that tissue protease D may be an important diagnostic factor for AD. The study examined changes in selected protein levels associated with the pathogenesis of Alzheimer’s disease in plasma samples from control subjects and patients with cognitive impairment. In order to accurately classify the disease, 56 participants underwent white matter hyperintensities using clinical cognitive tests, amyloid positron emission tomography, and magnetic resonance imaging scores. The plasma levels of cathepsin D in the subjects were examined by immunoblotting and enzyme-linked immunosorbent assay (ELISA). To examine the correlation between plasma tissue protease D levels and AD related factors and clinical characteristics through statistical analysis. Through quantitative immunoblotting and ELISA analysis, we found that compared with the control group, the plasma levels of cathepsin D (a major lysosomal protease) deposited in amyloid plaques in the brain were reduced. The level of plasma cathepsin D is negatively correlated with the total score of the Clinical Dementia Rating Scale. The comprehensive multivariate logistic regression model indicates that plasma tissue protease D levels have high performance in distinguishing AD from non AD patients. These results indicate that plasma tissue protease D levels are closely related to AD and can be developed as a diagnostic biomarker phenological selector for AD.

After intervention with Er Zhi Wan in AD rats, the levels of uridine, 3-hydroxyanthranilic acid, p-cresol sulfate, p-cresol, N-acetylserotonin, 3-methoxy-4-hydroxyphenylene glycol sulfate, 4-guanidinobutyric acid, adenosine, creatinine, vanillic acid, taurine, indolyloxy sulfate, hypervanadate, citric acid, and phenylacetylglycine were restored to normal. The results of pathway enrichment analysis indicate that Er Zhi Wan may achieve synergistic intervention in AD by regulating processes such as tryptophan metabolism, taurine and low taurine metabolism, citric acid cycle (TCA cycle), glyoxylate and dicarboxylic acid metabolism. The results of network pharmacology show that multiple components in the formula work together, involving multiple targets related to AD, reflecting the synergistic effect of multiple components and targets in traditional Chinese medicine prescriptions. The most important discovery is the intersection of metabolomics and network pharmacology, namely cathepsin D, which may be the core target and key to the efficacy of Er Zhi Wan in treating AD. The directly acting component related to it is quercetin, which is present in both Ligustrum lucidum and Alternanthera philoxeroides, and has been confirmed to be one of the effective ingredients of this formula. Based on the above results, this study believes that it has significant research significance in the treatment of AD, and further validation research will gradually focus on the related work of quercetin on tissue protease D. The conclusion of this study provides a theoretical basis for the study of the anti AD mechanism of Er Zhi Wan, and further research is still needed.