Mechanistic Study Of A Cistanche-Based Mongolian Herbal Prescription in The Treatment Of Acute Kidney Injury: A Network Pharmacology, Molecular Docking, And In Vivo Validation Approach
Oct 24, 2025
2. Results
2.1 Network Pharmacology Analysis
2.1.1 Screening of Active Components and Targets of Cistanche-Based Mongolian Prescription and Construction of Component-Target Network
According to the screening criteria, a total of 75 active components were collected, including 15 from Piper longum, 8 from Areca nut, 13 from Alpinia officinarum, 32 from Cinnamon, 7 from Pomegranate, and 4 from Amomum villosum. Among them, Pomegranate and Alpinia officinarum shared 3 components, and Amomum villosum and Alpinia officinarum shared 1 component. The corresponding targets included 97 for Piper longum, 23 for Areca nut, 188 for Alpinia officinarum, 440 for Cinnamon, 181 for Pomegranate, and 78 for Amomum villosum. After removing duplicates, a total of 682 unique targets were obtained. These components and targets were imported into Cytoscape to generate a visual network (Figure 1), where larger nodes indicate higher degree centrality.
2.1.2 Collection of AKI Targets
AKI-related targets were collected from the following databases:
GeneCards: 11,304 targets
OMIM: 638 targets
TTD: 4 targets
DrugBank: 46 targets
PharmGKB: 91 targets
After removing duplicates, a total of 11,792 unique AKI-related targets were identified. A Venn diagram was created to visualize the overlap (Figure 2).
2.1.3 Screening of Core Components and Targets and Construction of PPI Network
From the component-target network in section 2.1.1, 7 core active components of the Cistanche-based Mongolian prescription were identified (see Table 2). A Venn diagram showed 613 overlapping targets between the prescription and AKI-related targets (Figure 3). These were input into the STRING database to construct a PPI network, identifying 54 potential targets and 6 core targets (Figure 4).
Cistanche-Based Mongolian Prescription for Kidney Health Improvement
2.1.4 GO and KEGG Enrichment Analysis
GO enrichment analysis yielded 3,943 annotations, including 3,454 biological processes (BP), 194 cellular components (CC), and 295 molecular functions (MF). The top 10 entries by –log10(P-value) were visualized.
BP: DNA-binding transcription factor activity, steroid hormone regulation, myeloid cell differentiation
CC: transcription regulator complex, membrane raft, vesicles
MF: transcription factor binding, phosphatase binding, cytokine binding (Figure 5)
KEGG pathway analysis identified 212 enriched pathways, with the top 20 visualized. These included:
PI3K/Akt signaling pathway
IL-17 signaling pathway
TNF signaling pathway
HIF-1 signaling pathway (Figure 6)
A compound-target-disease-pathway network was constructed to illustrate the multi-target, multi-pathway mechanism of the Cistanche-based Mongolian medicine in treating AKI (Figure 7).
2.1.5 Molecular Docking Results
The core components Quercetin, T-Muurolol, Hexadecanoic Acid, Alpha-Caryophyllene, Linoleic Acid, Cis-Cinnamaldehyde, and Phenethyl Acetate were docked with the core targets AKT1 (PDB: 1unq), TNF (PDB: 5uui), IL6 (PDB: 1alu), TP53 (PDB: 3d06), IL1B (PDB: 5r8q), and EGFR (PDB: 8a27).
A binding energy below 0 kJ/mol indicates potential binding, while < –7 kJ/mol suggests strong binding. All docking energies were below –7 kJ/mol, showing strong binding interactions. The top 6 interactions were visualized (Figure 9):
IL1B–T-Muurolol
TP53–Cis-Cinnamaldehyde
TP53–Alpha-Caryophyllene
IL1B–Alpha-Caryophyllene
TNF–T-Muurolol
TP53–T-Muurolol
T-Muurolol and Alpha-Caryophyllene had the strongest interactions with targets, making them likely key components in Cistanche-based treatment for AKI.
2.2 In Vivo Experimental Results
2.2.1 General Condition, Body Weight, and Kidney Index of Mice
Mice in the model group showed reduced food intake and urine output, lethargy, and increased respiratory and heart rates. These symptoms were improved in the treatment groups. Compared with the control, model group mice had significantly reduced body weight (P < 0.01, F = 6.979), while both low and high-dose groups showed significant weight recovery (P < 0.01, Figure 11). The kidney index was significantly elevated in the model group (P < 0.01, F = 8.56) and decreased dose-dependently after treatment (P < 0.05, Figure 12).
2.2.2 Effect of Cistanche-Based Prescription on BUN, Scr, and UA
Model group mice had significantly elevated blood urea nitrogen (BUN), serum creatinine (Scr), and uric acid (UA) levels compared to control (P < 0.01). Both treatment groups reduced BUN (P < 0.01, F = 12.46), and the high-dose group significantly reduced Scr and UA levels (P < 0.01, F = 12.8 and 13.77, respectively). UA was significantly lower in the high-dose group than in the low-dose group (P < 0.05, Figure 13).
2.2.3 Histopathological Changes in Kidney Tissue (HE Staining)
HE staining showed normal kidney morphology in the control group. The model group exhibited tubular epithelial cell shedding, casts, glomerular swelling, and inflammatory infiltration. Both treatment groups showed attenuated pathological damage (Figure 14).
2.2.4 Effect on TNF-α, IL-6, and IL-1β
These inflammatory cytokines are core targets of Cistanche-based treatment. Levels of TNF-α, IL-6, and IL-1β were significantly elevated in the model group (P < 0.01, Figure 15). Both treatment groups significantly reduced these levels, with the high-dose group showing more pronounced effects.
2.2.5 Effect on PI3K/Akt Pathway Protein Expression
Compared with the control, PI3K and p-Akt/Akt protein expression levels were significantly decreased in the model group (P < 0.05). Treatment significantly restored these levels in a dose-dependent manner (P < 0.01, Figure 16).
3. Discussion
AKI remains a major global health issue, with over 2 million deaths annually. Its complex etiology, high incidence, and poor prognosis place a heavy burden on patients and healthcare systems. Currently, no effective drugs are available; treatment is largely supportive and expensive.
According to traditional Mongolian medicine, the kidney is governed by the Badagan principle and belongs to the "water" element, regulating body fluids. An excess of Badagan leads to internal dampness and kidney dysfunction. The Cistanche-based Mongolian formula, composed of Cistanche, Pomegranate, Cinnamon, Piper longum, Alpinia officinarum, and Amomum villosum, is designed to warm the kidney and restore balance among the three roots (Badagan, Heyi, and Shira).
Each herb contributes specific functions:
Cistanche: tonifies kidney yang, improves blood circulation
Piper longum: dispels cold, strengthens constitution
Alpinia officinarum: warms the stomach, relieves pain
Cinnamon: warms kidney yang, boosts energy
Pomegranate: quenches thirst, relieves pain
Amomum villosum: astringes and warms the spleen
Together, they expel kidney cold and restore balance, thus treating kidney disorders.
This study used network pharmacology to systematically explore the active compounds and mechanisms of Cistanche-based Mongolian medicine. The core components identified-Quercetin, T-Muurolol, Hexadecanoic Acid, Alpha-Caryophyllene, Linoleic Acid, Cis-Cinnamaldehyde, and Phenethyl Acetate-are supported by literature:
Quercetin reduces apoptosis and inflammation in AKI
T-Muurolol, from essential oils, has anti-inflammatory properties
Hexadecanoic Acid inhibits cell proliferation
Alpha-Caryophyllene has anti-inflammatory, antimicrobial, and anticancer effects
Linoleic Acid modulates macrophage-mediated inflammation
Cis-Cinnamaldehyde exhibits antioxidant and antibacterial activity
Phenethyl Acetate regulates the nervous system via aromatherapy
PPI network analysis revealed major targets: AKT1, TNF, IL6, TP53, IL1B, EGFR. These are involved in inflammation, cell survival, apoptosis, and immune regulation.
GO and KEGG enrichment suggested complex biological processes and key pathways, especially PI3K/Akt, IL-17, TNF, and HIF-1. The PI3K/Akt pathway was most enriched and plays a critical role in metabolism, growth, and inflammation. Modulating this pathway can mitigate AKI by reducing apoptosis and inflammation.
Molecular docking confirmed strong binding between core compounds and targets, supporting their relevance in AKI treatment.
In vivo experiments validated the network pharmacology findings:
Improved body weight and kidney index
Reduced BUN, Scr, and UA
Alleviated histopathological damage
Decreased TNF-α, IL-6, IL-1β levels
Upregulated PI3K and p-Akt/Akt expression
Mechanistically, PI3K converts PIP2 into PIP3, which activates Akt, suppressing inflammatory cytokine release. Reducing inflammation promotes tubular epithelial repair and restores renal function.
Conclusion
This study reveals that Cistanche-based Mongolian medicine treats AKI through multiple active compounds, targets, and pathways-particularly via the PI3K/Akt signaling pathway, which modulates inflammation and protects renal function. While the network pharmacology findings were validated in animal models, future in vitro and large-scale clinical studies are needed to support clinical use and development of traditional Mongolian medicine for AKI.
