Protective Effects of Agastache rugose Against Cyclophosphamide-Induced Cognitive Impairment in Mice: Experimental Validation and Compound-Target Prediction

INTRODUCTION

Cyclophosphamide (CYP) has been extensively used as an anticancer and immunosuppressive agent (de Jonge et al., 2005). Although the chemotherapeutic agent was initially thought not to cross the blood-brain barrier when administered at standard clinically relevant doses, 17 - 75% of patients, who received the chemotherapy, have reported several adverse effects related to learning and memory such as concentration difficulties and cognitive impairment (Silberfarb, 1983; Hodgson et al., 2013; Lange et al., 2014; Matsos and Johnston, 2019). Furthermore, the extent of cognitive dysfunction was proportional to the dose of chemotherapy (Ahles and Saykin, 2001). Several molecular mechanisms underlying the CYP-induced deficits in learning and memory function have been studied, which include oxidative stress, inflammation, neuronal cell death, and interference with the function of neurotransmitters and neurotrophic factors (Bagnall-Moreau et al., 2019).

Agastache rugosa (AR), also known as Korean mint, is a member of the Lamiaceae family (Rinik et al., 2024), and it is widely distributed across East Asia, including Korea, China, Japan, and Russia. AR, a major component of the traditional formula Gwakhyangjeonggi-san, has been historically used for digestive disorders, including diarrhea, abdominal discomfort, and vomiting (Ko et al., 2013). However, recent accumulating pharmacological evidence suggests that AR may broaden the therapeutic potential beyond classical indications by its antioxidant, anti-inflammatory, anti-aging, anti-bacterial, and anti-tumor activities. These effects are largely attributed to its bioactive constituents, including flavonoids (e.g., acacetin, tilianin), phenylpropanoids (e.g., rosmarinic acid), lignans, and terpenoids (Nam et al., 2020; Song et al., 2024). In this study, we aimed to broaden the indications of AR treatment by investigating its ameliorative effects in a mouse model of neurodegeneration and memory impairment induced by CYP administration. Moreover, we examined the possible target genes under the protective effects of AR against CYP-induced neurotoxicity by using network pharmacology analysis, which has been widely applied to explain the multi-component, multi-target, and multi-pathway behavior of medicinal herbs (Song et al., 2024). To this end, we performed the analysis using encephalitis (a clinical diagnosis broadly regarded as reflecting neuroinflammatory conditions) as a keyword, allowing us to identify the predicted target genes potentially related to neuroinflammatory processes.

MATERIALS AND METHODS

RESULTS

1. The antioxidative activities of AR extract in DPPH- and ABTS-scavenging assays

To determine the antioxidant activity of AR extract, we measured free radical scavenging rates in DPPH and ABTS assays at different concentrations of the extracts (0, 5, 10, 20, 100, and 500 mg/mL; Fig. 1). AR extracts at concentrations ≥ 20 mg/mL exerted significantly higher antioxidative activities in DPPH (p = 0.610 and p < 0.001 for the other comparisons) and ABTS (p = 0.009 and p < 0.001 for remaining comparisons) assays compared with AR 5 mg/mL-treated group. At concentrations below 100 mg/mL, the AR extract exhibited a steeply increasing antioxidant effect in a dose-dependent manner, whereas the slope of scavenging rates became gentler above 100 mg/mL.

Fig. 1.

The percentage of DPPH (A) and ABTS (B) radical scavenging activity showed the antioxidant benefits of AR.All data are presented as mean ± standard error (SE). **p < 0.01 and ***p < 0.001 indicates a significant difference compared to AR 5 mg/mL-treated group. DPPH, 1,1-diphenyl-2-picrylhydrazyl; ABTS, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); AR, Agastache rugosa extract.

2. Protective effects of AR extract in the CYP-induced cognitive impairment

We evaluated memory and cognitive performance by measuring the freezing time during the contextual fear conditioning test (Fig. 2). There were no differences between groups in the training phase, but in the testing phase, the CYP group showed a significantly lower freezing time than that of the control group (p = 0.015). However, AR pretreatment before CYP administration tended to increase the freezing time, although the significance was observed in only CYP+AR200 group (p = 0.040).

Fig. 2.

Freezing time (%) during training (A) and testing (B) phases of the contextual fear conditioning test.All data are presented as mean ± standard error (SE). *p < 0.05 indicates a significant difference compared to CYP group. CYP, cyclophosphamide treated group; CYP+AR100, cyclophosphamide and Agastache rugosa extract at a dose of 100 mg/kg treated group; CYP+AR200, cyclophosphamide and Agastache rugosa extract at a dose of 200 mg/kg treated group.

3. Ameliorative effect of AR extract against CYP-induced suppression of hippocampal neurogenesis

Proliferating progenitor cells and immature neurons in the dentate gyrus (DG) of adult mouse hippocampus were quantified based on the number of Ki67- and DCX-positive cells, respectively (Fig. 3). Representative microscopic images are presented in Fig. 3A. CYP administration significantly decreased the positive cells of Ki67- (Fig. 3B) and DCX-positive cells (Fig. 3C) compared with the control group (p = 0.016 and p = 0.033, respectively). However, pretreatment with AR at 100 and 200 mg/kg ameliorated the reduction of Ki67-positive cells (both p = 0.016), and 100 mg/kg of AR significantly increased the expression of DCX-positive cells (p = 0.033).

Fig. 3.

Pretreatment of AR extract improved the decrease in the number of Ki67- and DCX-positive cells in the subgranular zone of the hippocampus after CYP treatment.(A) Representative microscopic images of Ki67- (arrows) and DCX-positive cells in the hippocampal DG. Quantitative analysis of Ki67-positive cells (B) and DCX-positive cells (C). Scale bar is 100 μm. All data were expressed as mean ± standard error (SE). *p < 0.05 indicates a significant difference compared to CYP group. CYP, cyclophosphamide treated group; CYP+AR100, cyclophosphamide and Agastache rugosa extract at a dose of 100 mg/kg treated group; CYP+AR200, cyclophosphamide and Agastache rugosa extract at a dose of 200 mg/kg treated group. DCX: Doublecortin.

4. Active small molecules and target genes

The literature search was conducted for 11 active components identified through liquid and gas chromatographic analyses in a 70% ethanol extract of AR (Nam et al., 2020; Song et al., 2024). These components were rosmarinic acid, tilianin, acacetin 7-O-(6"-O-malonyl)-beta-D-glucopyranoside, isoagastachoside, acacetin 7-O-(2"-O-acetyl-6"-malony)-beta-D-glucopyranoside, acacetin, limonene, menthone, estragole, pulegone, and methyl eugenol. Rosmarinic acid and tilianin did not pass the ADME criteria according to public databases. Acacetin 7-O-(6"-O-malonyl)-beta-D-glucopyranoside, isoagastachoside, and acacetin 7-O-(2"-O-acetyl-6"-malony)-beta-D-glucopyranoside were excluded because of lack of related ADME data from public databases. Finally, six components including acacetin, limonene, menthone, estragole, pulegone, and methyl eugenol were selected. Their OB was ≥ 30 % and -0.3 < BBB according to TCMSP. DL values were ‘yes’ according to SwissADME databases. Structurally, these components were classified as one flavonoid (acacetin) and five monoterpenes (limonene, menthone, estragole, pulegone, and methyl eugenol). The monoterpenes had higher OB, DL, and BBB values than the flavonoids (Table 1).

Table 1.

Information related to active components was collected from public databases.

5. Potential target genes and PPI

Based on STITCH and SwissTargetPrediction databases, 659 genes associated with the six active components of AR were identified, and 2,290 encephalitis-related proteins from the GeneCards database were retrieved. The overlap between the two datasets resulted in 161 common proteins (Fig. 4A). The network between 161 potential target proteins and six active components is shown in Fig. 4B. This network consisted of 167 nodes and 285 edges, of which ACHE, KDR, and PARP1 were the core proteins associated with five active components. ALOX5, CYP1A2, HMOX1, MAOB, MPO, NOS2, and SRD5A1 were related to four active components. CCR5, which had a high relevance score with encephalitis in the GeneCards database, had links to limonene and pulegone.

Fig. 4.

Network of six active components–161 potential target proteins related to encephalitis.(A) Venn diagram showing the intersection of 659 proteins associated with AR and 2,290 proteins involved in encephalitis. (B) The network comprised 167 nodes and 285 edges. Pink rectangles are active components, and light blue ovals represent potential target proteins. AR, Agastache rugosa extract.

We conducted the PPI analysis for analyzing the interaction of 161 target proteins using the frequently used STRING database (Wei et al., 2021) and visualized the PPI network using Cytoscape 3.10.1. The topological characteristics are calculated by the “network analyzer” function of this software (Chen et al., 2021; Gao et al., 2021). The genes with a high confidence score were TP53, BCL2, CCL2, CD4, EGFR, IL2, JUN, MAPK3, MMP9, PPARG, PTGS2, and SRC (Fig. 5). A high confidence score indicates strong evidence for functional or physical relationship between target proteins. The top 10 proteins of edge count were TP53, EGFR, PTGS2, BCL2, MAPK3, SRC, PPARG, JUN, CD4, and CCL2. The top 10 proteins of betweenness centrality were EGFR, MAPK3, IL2, PPARG, GPT, MMP9, JUN, BCL2, RPL19, and DRD2. The betweenness centrality indicates the frequency with which the shortest paths between any pair of nodes pass through that node; a larger value in the network indicates that the node has greater importance (Lee et al., 2020).

Fig. 5.

PPIs analysis of potential target proteins.Large red circles mean high confidence score, and the number of arrows indicates primary related proteins.

6. Pathway analysis related to encephalitis

The signaling pathways were analyzed using the DAVID and KEGG databases with the p-value (p < 0.05) correction algorithm (Fig. 6) (Xu et al., 2022). A total of 128 pathways were searched in the DAVID database, 48% of which were pathways classified into the category of human diseases (Fig. 6A), and the top 10 pathways are shown in Fig. 6B. The 12 pathways related to encephalitis were serotonergic synapse, estrogen signaling pathway, neuroactive ligand-receptor interaction, chemical carcinogenesis-reactive oxygen species cAMP signaling pathway, arachidonic acid metabolism, apoptosis, PI3K-Akt signaling pathway, NOD-like receptor signaling pathway, neurotrophin signaling pathway, Alzheimer’s disease, and glutathione metabolism. A total of 86 potential target proteins were involved in the encephalitis-related pathways (Fig. 6C). The six proteins BCL2, JUN, MAP2K1, MAPK1, MAPK3, and PIK3CB were associated with more than five pathways.

Fig. 6.

Categories of KEGG pathways of AR (A), top 10 of KEGG pathway enrichment analysis of potential target genes of AR (B), and the network consisting of encephalitis-related proteins (mint oval) and KEGG pathways (orange triangles) with 112 nodes and 189 edges.

DISCUSSION

In this study, we demonstrated the neuroprotective effects of AR extract in a mouse model of CYP-induced neurodegeneration. We confirmed the anti-oxidant activity of AR extract. Additionally, pretreatment of AR extract significantly improved the cognitive impairment in contextual fear conditioning test and restored the reduced immunoreactivities of DCX and Ki67 in the hippocampus of CYP-treated mouse. Furthermore, by analyzing active ingredients of AR extract based on the published literature and conducting a network pharmacology analysis of AR-related target genes in CYP-induced neuroinflammation, we predicted several potential target genes that may be involved in the protective effects of AR extract against CYP-induced neurodegeneration.

Although chemotherapy is known to cause cognitive impairment in cancer patients, the underlying mechanisms remain poorly understood. Previous studies have shown a link between chemotherapy-induced hippocampal-dependent memory deficits and impairment in hippocampal neurogenesis (Sekeres et al., 2021). Similarly, in the present study, we identified cognitive impairment and decrease of hippocampal neurogenesis in CYP-administered mice. Among several mechanisms underlying CYP-induced neurogenesis impairment, neuroinflammation and oxidative stress appear to play critical roles. Intraperitoneal injection of CYP induces inflammatory responses in the central nervous system, leading to the activation of glial cells (Hirshman et al., 2020). Activated glial cells secrete inflammatory cytokines, which could inhibit the proliferation and differentiation of neural progenitor cells in the hippocampal DG and damage the neurogenic microenvironment (Monje et al., 2003; Sato, 2015). In parallel, CYP metabolism generates reactive oxygen species and toxic aldehyde byproducts, which induce oxidative stress in neural stem cells, subsequently leading to the decline in neurogenesis (Hussein et al., 2024). CYP-induced impairment in neurogenesis and synaptic plasticity, likely driven by neuroinflammation and oxidative stress, could ultimately be associated with cognitive impairment (Ibrahim et al., 2024). Therefore, targeting neuroinflammation and oxidative stress may offer a promising strategy to ameliorate the chemotherapy-induced cognitive deficits.

AR has been traditionally used to treat digestive disorders, including diarrhea, abdominal discomfort, and vomiting (Ko et al., 2013). Several studies have shown that AR extract and its compounds exhibit various therapeutic effects including anti-oxidant (Desta et al., 2016), anti-microbial (Shin, 2004), anti-inflammatory (Zielińska and Matkowski, 2014), and cardiovascular effects (Cao et al., 2017). To explore the target genes potentially involved in the therapeutic effects of AR against CYP-induced neuroinflammation, we conducted a network pharmacology analysis based on AR compounds identified in our previous study (Nam et al., 2020; Song et al., 2024). 11 constituents of AR were classified as five flavonoids (acacetin, tilianin, acacetin 7-O-(6"-O-malonyl)-beta-D-glucopyranoside, isoagastachoside, acacetin 7-O-(2"-O-acetyl-6"-malony)-beta-D-glucopyranoside), one phenylpropanoid (rosmarinic acid) and five monoterpenes (limonene, menthone, estragole, pulegone, and methyl eugenol). Among the 11 components, five were excluded due to lack of information in the databases or failure to meet the ADME criteria. Subsequent network analysis showed that five small molecules including limonene, menthone, estragole, pulegone, and methyl eugenol had high values of OB, DL, and BBB permeability. These five compounds exhibit significant potential in improving cognitive function through various mechanisms including anti-inflammation, anti-oxidation and neuroprotection (de Sousa et al., 2024; Eddin et al., 2021; Gogoi et al., 2020; Park et al., 2022; Roy et al., 2018; Wang and Heinbockel, 2018). These characteristics collectively indicate that the AR compounds could be promising candidates for the treatment of neurodegenerative diseases.

Next, to identify potential genes associated with the therapeutic effects of AR compounds in CYP-induced neuroinflammation, we performed a target prediction analysis followed by PPI network and functional enrichment analyses. The 659 proteins associated with AR and the 2,290 encephalitis-related proteins formed an intersection of 161 proteins. Analysis of the interactions between 161 proteins revealed that TP53, BCL2, CCL2, CD4, EGFR, IL2, JUN, MAPK3, MMP9, PPARG, PTGS2, and SRC were key nodes exerting significant influence within the PPI network. Among the proteins, BCL2, EGFR, JUN, MAPK3, and PPARG were identified as overlapping proteins with both the top 10 proteins ranked by edge count and the top 10 proteins ranked by betweenness centrality. Of the five proteins, BCL2, JUN, and MAPK3 were found to be involved in more than five of 12 KEGG pathways related with encephalitis. The KEGG pathways that included all three proteins were estrogen signaling pathway, apoptosis, and neurotrophin signaling pathway. Meanwhile, there are various studies about that BCL2, JUN, and MAPK3 are associated with neuroinflammation, neurodegeneration and cognitive deficits. BCL2, generally recognized as anti-apoptotic protein, could mediate neuronal differentiation by regulating lipopolysaccharide-induced inflammation in cortical neural stem cells, suggesting a protective effect of BCL2 in neuroinflammation (Park and Han, 2022). c-Jun, a product of JUN gene, plays a complex role by regulating neurodegeneration, neuroinflammation, neuronal regeneration, and neuroplasticity (Raivich, 2008). Similarly, MAPK3, also known as ERK1, is essential for normal physiological brain function such as learning and memory, but it also involved in neuroinflammation and neurodegeneration (Impey et al., 1999; Peng et al., 2010; Sun and Nan, 2017). Therefore, dysregulating the three key genes, BCL2, JUN, and MAPK3, may induce alternation in neuronal microenvironment, ultimately leading to cognitive deficits.

In conclusion, we confirmed the protective effects of AR extract, which attenuated neuronal damage in a CYP-induced cognitive impairment mouse model. Furthermore, based on the network pharmacology analysis, AR compounds may exert their neuroprotective effects against CYP-induced neuroinflammation through key candidate target genes, BCL2, JUN, and MAPK3, although further validation is required to confirm these targets and their roles.

Acknowledgments

This work was supported by a grant from the Jeju Institute of Korean Medicine (JIKOM-C-230004), Korea Institute of Oriental Medicine (ERT2011180 and KSN2511030), Convergence Research Group Project of the National Research Council of Science (CRC21021) and Regional Innovation System & Education (RISE) through the Gwangju RISE Center, funded by the Ministry of Education (MOE) and the Gwangju Metropolitan Government (20250527000003426624).

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