In this study, we utilized network pharmacology analysis to elucidate the mechanisms underlying the therapeutic effect of QFY in AD. We first identified the active ingredients and predicted targets of QFY, followed by disease correlation analysis and visualization of the C-T and C-T-AD networks. Subsequently, we performed a PPI network analysis to further explore the potential targets of QFY in AD and verified by molecular docking. Simultaneously, a target-organ localization network between the screened targets and 84 organs in the human body was established to investigate the potential organ targets of QFY. Further, GO and pathway enrichment analyses were constructed to reveal the action pathway of QFY in treating AD. Finally, an animal experiment was carried out to verify the results of bioinformatics. Based on these studies, we explored the mechanisms of QFY in treating AD.
Principal findings and comparison with other studies
Our results revealed a total of 203 potential targets among 87 active compounds in QFY, with each compound having approximately 45 targets. The results demonstrate that the treatment effect of QFY on AD is likely due to its combined effect on multiple targets, highlighting its potential as a possible therapeutic strategy for treating AD. In our network pharmacology analysis, we identified 102 potential targets related to AD from among 87 compounds in QFY. Notably, isorhamnetin, formononetin, 7-methoxy-2-methyl isoflavone, stigmasterol, and β-sitosterol were among the main active compounds in QFY, with a higher number of predicted targets. This suggests that these compounds may play a crucial role in the pharmacological action of QFY in the treatment of AD. Isorhamnetin alleviates inflammatory response and apoptosis through the Akt/SIRT1/Nrf2/HO-1 signaling pathway [35], regulates apoptosis and autophagy through the P38/PPAR-α pathway [36], and protects against Aβ-induced cytotoxicity and Aβ aggregation [37]. Formononetin has been found to have potential benefits in protecting against AD and regulating vascular inflammation in AD through the Nrf2 pathways [38]. It also has a neuroprotective effect and can improve learning and memory in a mouse model of AD [39]. Further, it has identified seven key target genes for formononetin in the treatment of patients with AD. The biological processes included apoptosis, energy pathways, metabolism, and signal transduction [40]. β-Sitosterol also has potential treatment effects on the management of AD. For example, β-sitosterol gradually improved the working memory, spontaneous alternation behavior, and motor coordination of transgenic mice [41]. β-Sitosterol is beneficial in neurodegenerative diseases such as AD by promoting inner mitochondrial membrane fluidity to enhance mitochondrial function [42] and alleviating inflammation via ERK/p38 and NF-κB pathways [43]. Stigmasterol can decrease the phosphorylation of MAPKs to protect neurons against Aβ25-35-induced injury, with anti-AD effects [44]. According to the results of molecular docking analysis, stigmasterol formed stable structures with NOS2, PPARG, MAPK14, AGER, and NOS3, while β-sitosterol showed better binding activity and stable conformations with PTGS1, PTGS2, GSK3B, ACHE, NF-κB, and other targets. These findings suggest that the active ingredients in QFY may improve cognitive function in AD by modulating inflammation-related targets.
A target-organ localization network between the screened targets and 84 organs in the human body was established to investigate the potential organ targets of QFY. The results showed that the lungs, heart, liver, kidneys, brain, and blood were the main organs associated with the potential drug targets of QFY. Interestingly, the targets of QFY are located highly not only in the brain but also in other organs, especially the liver and blood, which may be related to drug absorption, metabolism, and clearance. Therefore, it is necessary to consider the comprehensive effects of QFY on multiple organs and systems when developing therapeutic strategies for the treatment of AD.
The results of GO and pathway analyses revealed that the action pathway of QFY in treating AD was primarily related to metabolism, inflammatory response, apoptosis, aging, and memory. The alterations in energy metabolism in normal brain aging and AD, including glycolipid metabolism and mitochondrial metabolism, were connected to inflammatory responses via redox regulation [45]. Inflammatory response and apoptosis are also important processes that contribute to neuroinflammation and neuronal cell death in AD. Aging is a primary AD risk factor, and memory impairment is a hallmark symptom of AD. Similarly, NF-κB, STAT3, MAPK, PPARG, caspase 3 (CASP3), and NOS in the C-T-AD network showed higher enrichment. These findings indicated that these genes are the key part of the network and that inflammation, apoptosis, and metabolism may be the main processes through which QFY treats AD. Therefore, the regulation of these biological processes and signaling pathways may be key mechanisms underlying the therapeutic effects of QFY in the treatment of AD.
The enrichment analysis revealed that the AGE-RAGE pathway was significantly enriched. The binding of AGEs to their receptor RAGE activates a range of signaling pathways, including PI3K-AKT, JAK-STAT, MAPK, and NF-κB signaling. The accumulation of AGEs is a hallmark of connective tissue aging and is characterized by a gradual decline in regenerative capacity [46]. AGEs can upregulate the mRNA expression of MCP-1, TNF-α, IL-6, IL-1, COX-2, and iNOS and activate the RAGE/NF-κB pathway [47]. The prolonged cellular disturbance brought on by ligand-RAGE interactions is a hallmark of chronic diseases such as diabetes, inflammation, and AD. As a pattern recognition receptor, overexpression of RAGE can trigger a range of signals, including PI3K/Akt and MAPK pathways [48]. These signals ultimately lead to NF-κB activation and inflammation [49]. Besides, RAGE enables extracellular HMGB1-LPS complex transport to lysosomes to activate cytosolic caspase-11 in macrophages and endothelial cells [50]. Abnormal activation of JNK occurs in AD patients and AD transgenic mice, and inhibition of JNK activation can reduce neuroinflammation and synaptic loss [51]. JNK is a subfamily of MAPKs and plays a critical role in the regulation of insulin signaling, inflammation, apoptosis, and caspase-3 activity in diabetes and the upregulation of pro-inflammatory cytokines such as MCP-1, IL-6, IL-8, and TNF-α in AD pathology [52]. ERK and p38 MAPK, the two other main members of the MAPK family, also play a role by working together to activate NF-κB signaling, leading to neuroinflammation [53]. Moreover, the activation of the JNK and p38 MAPK pathways can promote apoptosis [54]. Apoptosis is an important mechanism leading to synaptic dysfunction and neuronal loss in AD [55]. The results of a genome-wide association study (GWAS) suggested that abnormalities in JAK-STAT signaling were associated with the pathogenesis of AD [56]. Moreover, the levels of STAT3 are reduced in the hippocampus of AD patients [57]. The activation of STAT3 has been shown to improve cognitive deficits in animal models of AD through the regulation of NMDA receptor (NMDAR) expression [58]. In summary, we predicted that QFY can alleviate neuroinflammation and apoptosis through RAGE-related pathways.
Proteins collaborate within the PPI network to carry out essential molecular processes within the cell. Anomalies in a specific protein can disrupt the function of other proteins in the network, ultimately leading to the development of a disease. Consistent with the results of enrichment analysis, the PPI network between QFY and AD displayed significant interactions with NF-κB, STAT3, MAPKs, and TLRs. The primary enrichment pathways identified in Cluster 1 were the AGE-RAGE pathway and the PI3K-AKT pathway. The NF-κB is particularly notorious for its role in mediating inflammatory responses and its canonical pathway is a key characteristic in AD development [59]. TLRs are transmembrane proteins that initiate innate and adaptive immune responses through recognized cellular damage-associated molecular patterns (DAMPs). However, dysfunctional or excessive TLR activation can contribute to various dysfunctions such as autoimmune, inflammatory, and age-associated diseases [60]. TLR4 activation leads to NF-κB activation and proinflammatory cytokine release through the activation of both myeloid differentiation primary response protein 88 (MyD88)-dependent and MyD88-independent signals [61]. Based on our analysis, we hypothesize that the mechanism underlying the therapeutic effects of QFY on AD involves the RAGE pathway and NF-κB pathway, including targets such as MAPKs, TLRs, and NF-κB. Our study suggests that QFY exerts its beneficial effects by inhibiting neuroinflammation, which is a key contributor to the development and progression of AD.
During our research, network pharmacology analysis indicated that the anti-AD mechanism of QFY is associated with inflammation, and this mechanism may be mediated by AGER and NF-κB. Minocycline, a broad-spectrum tetracycline antibiotic, is a microglial cell inhibitor that can suppress microglia activation and reduce the expression and release of inflammatory mediators [62, 63]. Through its anti-inflammatory and neuroprotective actions, minocycline has been shown to have a beneficial effect in ameliorating spatial memory decline [64]. As a result, we chose minocycline as our experimental positive control drug. Our MWM test results demonstrated that QFY can partially enhance spatial memory. Both TLR4 and AGER are capable of activating the downstream target NF-κB, thereby promoting inflammation upon binding to their respective ligands [65]. Extracellular AGEs can directly bind to myeloid differentiation 2 (MD2), which is a co-receptor of TLR4. This binding leads to the formation of an AGEs-MD2-TLR4 complex and initiates pro-inflammatory pathways [66]. Meanwhile, RAGE has been discovered in a variety of immune cells that are essential for maintaining the immune response. It has been found that many of the extracellular ligands that initiate RAGE signaling are implicated in both acute and chronic immune responses. Following RAGE activation, the proinflammatory transcription factor NF-κB and its downstream target genes are induced. Interestingly, RAGE has a functional NF-κB binding site in its proximal promoter, making it an NF-κB-regulated target gene as well [67]. Our findings are consistent with the hypothesis that QFY can ameliorate the inflammation of the brain by reducing the protein expression of TLR4 and AGER. Specifically, QFY intervention reversed the upregulation of TLR4 and AGER protein expression in AD rats. Additionally, our immunofluorescence results revealed that QFY reduced the entry of NF-κB into the nucleus, thereby inhibiting NF-κB activation. Although there were no significant differences in the NF-κB mRNA expression among the different groups, this can be attributed to the fact that mRNA represents the total expression of NF-κB. The entry of NF-κB into the nucleus represents its activation, which in turn promotes the activation of microglia. Therefore, it is normal that there is a difference between the total expression and activation of NF-κB. Furthermore, QFY reduced the mRNA expression levels of inflammatory factors such as IL-1β and TNF-α in AD rats. Additionally, QFY reduced microglia activation, as microglia are the main mediators of neuroinflammation and the natural immune cells of the central nervous system. Our results suggest that QFY can exert anti-inflammatory effects in the brain through multiple pathways, thereby enhancing learning and memory in AD rats.