Therapeutic Potential And Molecular Mechanisms Of Cistanche Deserticola in Asthma: Targeting PI3K-AKT Pathway And INOS-Mediated Inflammation
May 19, 2025
Identification of Asthma-Associated Hub Genes via WGCNA Analysis
To investigate key genes associated with asthma, we utilized the GSE67472 dataset from the Gene Expression Omnibus (GEO) database. This dataset includes samples from 62 individuals diagnosed with asthma who had not received inhaled corticosteroid treatment, and a control group of 43 healthy individuals.
Differentially expressed genes (DEGs) were analyzed and visualized using volcano plots and heatmaps (Figures 7A and 7B). Through Weighted Gene Co-expression Network Analysis (WGCNA), we identified 27 gene modules significantly associated with asthma development when compared to healthy controls (Figure 7C).
Among these, the "skyblue" module showed the strongest positive correlation with asthma
(correlation coefficient: r = 0.62, p = 2e-12, Figure 7D).
Figure 7E illustrates the adjacency heatmap depicting relationships between the trait-related modules and the skyblue module.
We further analyzed the correlation between module membership (MM) and gene significance (GS) for asthma within the skyblue module (Figure 7F), revealing a strong positive correlation (correlation = 0.84, p = 1.1e-41), emphasizing the robust association between the skyblue module and asthma.
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Subsequent functional enrichment analysis of genes within the skyblue module confirmed their involvement in asthma-related biological processes and pathways, including IgE binding, asthma, and the IL-17 signaling pathway (Figures 7G–J).
Figure 7. Identification of Core Asthma-Related Genes Using WGCNA
A–B. Visualization of DEGs using volcano plot and heatmap.
C. Clustering dendrogram of co-expressed genes associated with asthma.
D. Module–trait relationships in asthma. Each cell shows the correlation coefficient and p-value.
E. Heatmap showing adjacency relationships of module eigengenes.
F. Correlation between module membership and gene significance for asthma in the skyblue module.
G–J. Functional enrichment analysis of genes in the skyblue module.
iNOS as a Therapeutic Indicator of Cistanche Treatment in Asthma
To explore key asthma-related genes, we compiled 259 genes from multiple disease databases, including GeneCards, Therapeutic Target Database, DisGeNET, and OMIM. By intersecting these genes with those from the skyblue module and PG-targeted genes, we identified iNOS (inducible nitric oxide synthase) as a core gene (Figure 8A).
Further analysis of RNA-seq data revealed that iNOS expression was decreased following Cistanche treatment (Figure 8B). Additionally, molecular docking was performed to validate the binding affinity between PG and the iNOS protein (Figure 8C). Notably, favorable interactions were observed involving amino acid residues such as arginine, glutamate, and tryptophan. The measured binding energy of -8.97 kcal/mol indicated a strong binding affinity between PG and iNOS (Supplementary Table 2).
To confirm the functional effect of PG on iNOS, in vitro assays were conducted (Figure 8D). The results demonstrated that PG significantly attenuated HDM-induced iNOS activity, supporting the molecular docking findings.
We then examined the effect of PG on iNOS protein expression. Western blot analysis showed that HDM treatment increased iNOS expression in MLE12 cells, which was reduced upon PG treatment (Figure 8E). Similarly, qPCR analysis of RNA extracted from mouse lung tissue revealed a significant increase in iNOS expression in HDM mice, which was reduced following Cistanche treatment (Figure 8F).
Immunofluorescence assays further validated these observations, showing elevated iNOS expression in HDM-exposed mice and reduction after Cistanche treatment (Figure 8G and Supplementary Figure 3). In parallel, we assessed the expression level of PI3K (Supplementary Figure 3). Compared to the control group, the HDM group exhibited upregulation of PI3K, suggesting its involvement in the asthmatic response.
Notably, Cistanche at a low dose (20 mg/kg) did not significantly improve PI3K expression altered by HDM. However, higher concentrations of Cistanche effectively downregulated PI3K expression. This regulatory effect on PI3K expression correlated with improvements in airway hyperresponsiveness (AHR) and reduced pathological changes in asthmatic mice, indicating the therapeutic potential of Cistanche via modulation of the PI3K pathway.
Figure 8. iNOS as a Therapeutic Target of Cistanche in Asthma
A. Identification of iNOS as a potential target through intersection of gene sets.
B. TPM values of iNOS from RNA-seq data.
C. Molecular docking confirms interaction between PG and iNOS.
D. Fluorescence intensity indicating iNOS activity.
E. Western blot analysis of iNOS protein levels in different experimental groups.
F. qPCR analysis of iNOS transcript levels across groups.
G. Immunofluorescence analysis showing iNOS expression patterns in various treatment groups.
All experimental data represent at least three independent replicates and are expressed as mean ± standard deviation (SD).
Conclusion, Highlights, and Prospective Applications
Cistanche deserticola is a treasured traditional Chinese herb, long acclaimed for its efficacy in addressing various health conditions. Modern scientific investigations have revealed its broad-spectrum bioactivities, including anti-inflammatory, antioxidant, anti-aging, and immune-enhancing effects (Li et al., 2016). Notably, studies have explored its therapeutic potential in diseases such as diabetes, where it has shown promise in regulating postprandial blood glucose levels and improving insulin resistance (Xiong et al., 2013). Additionally, in neurological contexts, Cistanche has demonstrated potential in alleviating blood-brain barrier damage after ischemic stroke by modulating neuroinflammation (Liao et al., 2023). In oncology, phenylethanoid glycosides from Cistanche tubulosa have exhibited antitumor effects, suggesting therapeutic potential in hepatocellular carcinoma (Peng et al., 2021).
Although research into the respiratory benefits of Cistanche is still in its early stages, preliminary findings suggest its potential advantages. For instance, echinacoside, a bioactive compound found in Cistanche, has shown promise in attenuating oxidative stress-induced endothelial cell death, thereby alleviating lung injury symptoms (Xie et al., 2023).
In the context of asthma, our study marks the first attempt to explore the therapeutic potential and underlying mechanisms of Cistanche. Using a house dust mite (HDM)-induced asthma mouse model, Cistanche was administered as a therapeutic intervention. Encouragingly, our results demonstrate its significant effects in controlling asthma, including reduced airway hyperresponsiveness (AHR), diminished inflammatory cell infiltration, and alleviation of pulmonary inflammation and mucus production in allergic asthma.
Cytokines such as IL-4 and IL-5 play pivotal roles in asthma pathogenesis (Michael, 2013; Giovanni, 2017; Patrick & Peter, 2007). IL-4 is crucial for stimulating B cells to produce IgE antibodies, leading to elevated IgE levels-a hallmark of allergic asthma and predictor of exacerbation risk. IL-5, on the other hand, is essential for the growth and activation of eosinophils, which release pro-inflammatory mediators that exacerbate airway inflammation and mucus secretion.
Our transcriptomic analysis revealed that Cistanche treatment disrupted pathways associated with IL-4, IL-5, and eosinophils-a finding further supported by GSVA results. GSEA further highlighted the association between Cistanche treatment and type 2 immune cytokine responses, underscoring its therapeutic relevance in asthma. Experimental results also demonstrated that Cistanche reduced serum IgE, IL-5, and IL-13 levels in asthmatic mice, further confirming its efficacy in alleviating asthma symptoms.
Notably, our GSEA results suggested that Cistanche participates in glucocorticoid-related biological processes. Given the adverse effects and limited sensitivity observed in some patients during long-term corticosteroid therapy (Ip et al., 2018), this finding opens avenues for exploring alternative glucocorticoid therapies. Recent studies have revealed that active compounds in Cistanche, such as acteoside, exert significant effects on glucocorticoid-induced osteoporosis (Li et al., 2023), further supporting the feasibility of Cistanche as an alternative or adjunctive therapy for asthma.
The PI3K-AKT signaling pathway plays a crucial role in asthma pathophysiology. Recent studies have shown that miR-200a promotes asthma development via the PI3K-AKT-FOXC1 axis (Lin et al., 2014). This pathway also regulates cyclin D1, which is critical in airway smooth muscle remodeling (Feng et al., 2014), and modulates expression of asthma-related molecules such as IL-7, TNF-α, and IgE (Winer et al., 2022; Jing et al., 2019; Dera et al., 2020).
In our study, the major bioactive compound of Cistanche was identified via LC-HRMS as pinoresinol 4-O-β-D-glucopyranoside (PG), with molecular formula C₂₆H₃₂O₁₁. PG belongs to lignans-plant-derived compounds known for their anti-inflammatory, antioxidant, and immunosuppressive properties (Osmakov et al., 2022)-and emerged as a promising candidate for asthma treatment. Target enrichment analysis for PG demonstrated strong associations with the PI3K-AKT pathway, suggesting that its therapeutic effects may be mediated through this mechanism.
Nitric oxide (NO), a gaseous free radical (N=O), is abundantly present in the human respiratory and cardiovascular systems (Antosova et al., 2017). In asthma, NO serves as an inflammatory biomarker, frequently measured as fractional exhaled nitric oxide (FeNO) to assess disease severity and guide steroid therapy (Smith et al., 2005; Chiara et al., 2012). Elevated FeNO levels, closely linked to IL-13-mediated type 2 airway inflammation, were also observed in our asthma model. NO is primarily synthesized by nitric oxide synthases (NOS), particularly the inducible isoform iNOS (NOS2), which is upregulated under pro-inflammatory conditions (Anavi & Tirosh, 2020). Overexpression of iNOS leads to increased NO production, promoting mucus secretion and eosinophil recruitment, thereby exacerbating asthma symptoms (Meurs et al., 2003). iNOS inhibitors have shown potential in managing FeNO levels in asthma patients (Islam et al., 2010; Hansel et al., 2003).
Through integrated analysis of asthma-related genes, WGCNA modules, and PG-targeted genes, we identified iNOS as a core gene. Cistanche treatment downregulated iNOS protein levels in asthma-model mice, suggesting suppression of inflammation-associated NO production. Molecular docking confirmed strong binding affinity between PG and iNOS, and PG treatment reduced both NOS activity and iNOS protein levels.
Existing research also indicates that the PI3K-AKT pathway regulates iNOS expression in other physiological systems (Zafirovic et al., 2019; Lu et al., 2022). In our mouse model, Cistanche treatment reduced PI3K expression in lung epithelial cells. While its role in asthma requires further exploration, our findings suggest a novel regulatory link between the PI3K-AKT pathway and iNOS in the context of Cistanche treatment. Additional mechanistic studies are needed to validate these pathways.
Schematic Summary: Mechanisms of Cistanche in Asthma Treatment
HDM exposure initiates a cascade of airway pathological changes characterized by heightened reactivity and inflammatory cell infiltration. Cistanche, a traditional herbal medicine, demonstrates the ability to counteract these HDM-induced effects via dual mechanisms:
Modulation of the PI3K pathway, influencing cellular responses and reducing asthma-related inflammation;
Direct targeting of iNOS by its bioactive component PG, resulting in suppression of iNOS expression and activity. PG-mediated downregulation of iNOS offers a potential molecular intervention for alleviating key asthma symptoms.
Citation
Liu, J., Gao, J., Xiong, A., Li, G., Zhong, K., Bai, L., Li, X., Ran, Q., Xiong, Y., & He, X. (2025). Exploring Cistanche's therapeutic potential and molecular mechanisms in asthma treatment. Phytomedicine: International Journal of Phytotherapy and Phytopharmacology, 136, 156265.
