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. 2021 May 17:15:2059-2089.
doi: 10.2147/DDDT.S307015. eCollection 2021.

An Integrated Analysis of Network Pharmacology, Molecular Docking, and Experiment Validation to Explore the New Candidate Active Component and Mechanism of Cuscutae Semen-Mori Fructus Coupled-Herbs in Treating Oligoasthenozoospermia

Xue Bai  1 , Yibo Tang  2 , Qiang Li  1 , Dan Liu  1 , Guimin Liu  1 , Xiaolei Fan  2 , Zhejun Liu  2 , Shujun Yu  1 , Tian Tang  1 , Shuyan Wang  2 , Lingru Li  3 , Kailin Zhou  4 , Yanfei Zheng  3 , Zhenquan Liu  1   3
Affiliations

An Integrated Analysis of Network Pharmacology, Molecular Docking, and Experiment Validation to Explore the New Candidate Active Component and Mechanism of Cuscutae Semen-Mori Fructus Coupled-Herbs in Treating Oligoasthenozoospermia

Xue Bai et al. Drug Des Devel Ther. .

Abstract

Purpose: One of the most common types of male infertility is recognized as oligoasthenozoospermia (OA), characterized by low sperm count and quality in males. As a traditional Chinese medicine (TCM), Cuscutae Semen-Mori Fructus coupled-herbs (CSMFCH) has been known to act a curative effect on OA for thousands of years. Nevertheless, the substantial basis and molecular mechanism of CSMFCH in treating OA remain elusive.

Methods: Herein, an integrated approach, including network pharmacology, molecular docking, and experiment validation, was utilized to reveal the new candidate active component and mechanism of CSMFCH in treating OA.

Results: The results show that kaempferol is the most significant bioactive component of CSMFCH on OA. The mechanism and targets of CSMFCH against OA are relevant to hormone regulation, oxidant stress, and reproductive promotion. In order to validate network pharmacology results, molecular docking and experiment validation were conducted. In detail, molecular docking was employed to verify the strong binding interactions between kaempferol and the core targets. UHPLC-Q-Orbitrap-MS was used to identify kaempferol in the CSMFCH extract. In vitro and in vivo experiments further proved CSMFCH and kaempferol could enhance the mouse Leydig (TM3) and mouse Sertoli (TM4) cell viability, improve the male reproductive organ weights, sperm quality, and decrease testis tissue damage in the OA mouse model induced by CP.

Conclusion: Our results not only identify the new candidate active component of CSMFCH in treating OA but also provide new insights into the mechanisms of CSMFCH against OA.

Keywords: kaempferol; network pharmacology; oligoasthenozoospermia; traditional Chinese medicine.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
CSMFCH component-target network. (A) Venn diagram: 137 components (green section), and 18 bioactive components filtered by two models relevant to ADME (blue section stands for the components of OB ≥ 30%, red section stands for DL ≥ 0.18). (B) CS component-target network, including 316 nodes and 714 edges. (C) MF component-target network, including 165 nodes and 373 edges. (D) Construction of CSMFCH component-target visual network, including 325 nodes and 835 edges. Turquoise node and orange node stand for CS and MF, respectively. Purple nodes and pink nodes stand for bioactive components from CS and MF, respectively. Yellow node stands for the common components from CS and MF. Blue nodes stand for targets.
Figure 2
Figure 2
GO and KEGG pathway enrichment analyses of the CSMFCH component-target network (p-value ≤ 0.05). (A) The PPI network. (B) The clusters of PPI network. The colors indicate diverse clusters or binding site sets that are predicted by the MCODE algorithm. (C) The top 20 biological processes. (D) The top 20 cellular components. (E) The top 20 molecular functions. (F) The top 20 KEGG pathways. The bar plot and different colors show the enrichment scores [-log10 (P-value)] of the top 20 significant enrichments.
Figure 3
Figure 3
OA-related targets. (A) Venn diagram: OA-related targets are 495, including 473 common targets between oligospermia and asthenozoospermia, and 31 targets from the search term “oligoasthenozoospermia”. (B) Venn diagram: the number of OA-related targets from the five different databases is 415, 78, 51, 36, and 30, respectively. The common target is androgen receptor (AR).
Figure 4
Figure 4
GO and KEGG pathway enrichment analyses of the OA-related targets (p-value ≤ 0.05). (A) The PPI network. (B) The clusters of PPI network. The colors represent different clusters or sets of binding sites predicted by MCODE algorithm. (C) The top 20 biological processes. (D) The top 20 cellular components. (E) The top 20 molecular functions. (F) The top 20 KEGG pathways. The bar plot and different colors show the enrichment scores [-log10 (P-value)] of the top 20 significant enrichments.
Figure 5
Figure 5
CSMFCH-OA common-target network. (A) Intersection of Venn diagram: 64 targets are common to CSMFCH and OA. (B) Common-target network, including 80 nodes and 218 edges. The size of the circle represents the node degree of the target protein. Purple nodes and pink nodes stand for bioactive components from CS and MF, respectively. Yellow node stands for the common components from CS and MF. Blue nodes stand for targets.
Figure 6
Figure 6
The PPI network and cluster analysis. (A) The PPI information generated by the STRING database. (B) The PPI network visualized using the Cytoscape software. (C) Cluster 1 (score = 9.6). (D) Cluster 2 (score = 5.455). (E) Cluster 3 (score = 3.333). (F) Cluster 4 (score = 3). The size and color of the node represent the degree of the target protein. The width and color of the edge represent the combined score of the target protein.
Figure 7
Figure 7
GO and KEGG pathway enrichment analyses of the PPI network (p-value ≤ 0.05). (A) The top 20 biological processes. (B) The top 20 cellular components. (C) The top 20 molecular functions. (D) The top 20 KEGG pathways. The color scales indicate the different thresholds for the p-values. (E and G) The GO enrichment of the PPI network using the ClueGo plugin. Show only pathways with ***P≤0.001. (F and H) The KEGG pathway analysis of the PPI network using the ClueGo plugin. Show only pathways with *P≤0.05.
Figure 8
Figure 8
GO and KEGG pathway enrichment analyses of the clusters (p-value ≤ 0.05). (A) The top 20 biological processes for cluster 1. (B) The top 20 KEGG pathways for cluster 1. (C) The top 20 biological processes for cluster 2. (D) The top 20 KEGG pathways for cluster 2. (E) The top 20 biological processes for cluster 3. (F) The top 20 KEGG pathways for cluster 3. (G) The top 20 biological processes for cluster 4. (H) Nine KEGG pathways for cluster 4. The color scales indicate the different thresholds for the p-values.
Figure 9
Figure 9
Core component-target-pathway network. The V nodes represent the top 20 KEGG signaling pathways. The ellipse nodes represent the targets related to the pathways. The diamond nodes represent ingredients from CS, and the hexagon nodes represent ingredients from MF. The size of the circle represents the node degree of the target protein.
Figure 10
Figure 10
The molecular structure of kaempferol was shown as 2D diagrams (A) and 3D diagrams (B).
Figure 11
Figure 11
Estrogen signaling pathway. The red rectangle represents the targets related to the PPI network.
Figure 12
Figure 12
Molecular models of the binding of kaempferol from CSMFCH to the predicted targets (A and B) AKT1, (C and D) EGFR, (E and F) MAPK3, (G and H) ESR1, (I and J) CYP19A1, and (K and L) AR shown as 3D diagrams and 2D diagrams.
Figure 13
Figure 13
Mass spectrum of CSMFCH in the negative ion mode. (A) Total ion current chromatogram of CSMFCH. (B) ESI-MS/MS spectra of kaempferol.
Figure 14
Figure 14
Mass spectrum of CSMFCH in the positive ion mode. (A) Total ion current chromatogram of CSMFCH. (B) ESI-MS/MS spectra of kaempferol.
Figure 15
Figure 15
The absorbance and cell viability of TM3 and TM4 cells after CSMFCH and kaempferol treatment. (A) The absorbance of TM3 cells after CSMFCH treatment. (B) The cell viability of TM3 cells after CSMFCH treatment. (C) The absorbance of TM3 cells after kaempferol treatment. (D) The cell viability of TM3 cells after kaempferol treatment. (E) The absorbance of TM4 cells after CSMFCH treatment. (F) The cell viability of TM4 cells after CSMFCH treatment. (G) The absorbance of TM4 cells after kaempferol treatment. (H) The cell viability of TM4 cells after kaempferol treatment. Data: n = 6, mean ± SD, experiments performed in triplicate. *P < 0.05 and **P < 0.01 versus non-treatment control group.
Figure 16
Figure 16
The body, testicular, and epididymal weight of each group. (A) The body weight. (B) The testicular weight. (C) The epididymal weight. Data: n = 6, mean ± SD, experiments performed in triplicate. ##P < 0.01 versus the NC group, *P < 0.05 and **P < 0.01 versus the MC group.
Figure 17
Figure 17
The sperm quality of each group. (A) The sperm density. (B) The sperm viability. (C) The sperm motility. (D) The abnormal sperm motility. Data: n = 6, mean ± SD, experiments performed in triplicate. ##P < 0.01 versus the NC group, **P < 0.01 versus the MC group.
Figure 18
Figure 18
The sperm morphology (×400). (A) The normal control group. (B) The model control group. (C) The low-dose CSMFCH group. (D) The medium-dose CSMFCH group. (E) The high-dose CSMFCH group. (F) The kaempferol group. Data: n = 6, experiments performed in triplicate.
Figure 19
Figure 19
HE staining of testicular tissues (×400). (A) The normal control group. (B) The model control group. (C) The low-dose CSMFCH group. (D) The medium-dose CSMFCH group. (E) The high-dose CSMFCH group. (F) The kaempferol group. Data: n = 6, experiments performed in triplicate.
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