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. 2024 Jun 15;16(6):2190-2211.
doi: 10.62347/RQHY5963. eCollection 2024.

Integrating network pharmacology with experimental validation to investigate the mechanism of Wuwei Zishen formula in improving perimenopausal syndrome

Xuewen Fu  1 , Hui Wang  2 , Meichen Gai  3 , Yuanhua Dai  3 , Jun Chang  3 , Hong Zhang  3
Affiliations

Integrating network pharmacology with experimental validation to investigate the mechanism of Wuwei Zishen formula in improving perimenopausal syndrome

Xuewen Fu et al. Am J Transl Res. .

Abstract

Objectives: To investigate the role of the Wuwei Zishen formula (WWZSF) in treating and preventing perimenopausal syndrome (PMS) and to understand its mechanism.

Methods: Network pharmacology and molecular docking was used to predict active compounds, potential targets, and pathways for PMS treatment using WWZSF. Female Sprague-Dawley (SD) rats were induced with D-galactose (D-gal) to establish a PMS model and treated with Kunbao pill (KBP) and WWZSF. Estrus cycles were observed using vaginal smears. Serum sex hormones were measured using the enzyme-linked immunosorbent assay (ELISA). Histological changes in the uterus and ovaries were evaluated using hematoxylin-eosin staining (HE). Western blot was used to assess the protein expression levels of Cleaved Caspase-3, p62, BAX/Bcl-2, p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR in the uterus and ovaries.

Results: A total of 70 active compounds and 440 potential targets were screened out. Important targets and pathways, including AKT1, Bcl-2, Caspase-3, mTOR, and the PI3K/AKT/mTOR pathways, and molecular docking verified their high affinities to key WWZSF components. In vivo experiments showed that WWZSF can ameliorate the morphological abnormalities of the uterus and ovaries, increase sex hormone levels and organ index, and restore the estrus cycles in PMS rats. Moreover, the western blot results showed decreased Cleaved Caspase-3 and BAX/Bcl-2 protein levels in the ovarian and uterine tissues after WWZSF therapy. Concurrently, there was an increase in the expression of p62 and the ratios of p-AKT/AKT, p-mTOR/mTOR, and p-PI3K/PI3K.

Conclusion: The PI3K/AKT/mTOR signaling pathway-mediated apoptosis and autophagy pathways may be how WWZSF efficiently reduces PMS.

Keywords: D-galactose; Network pharmacology; PI3K/AKT/mTOR signaling pathway; molecular docking; perimenopausal syndrome.

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Conflict of interest statement

None.

Figures

Figure 1
Figure 1
Venn diagram was visualized to analyze the common potential targets of perimenopausal syndrome (PMS) and Wuwei Zishen formula (WWZSF).
Figure 2
Figure 2
The “Herbs-Ingredients-Targets-Disease” network.
Figure 3
Figure 3
Protein-protein interaction (PPI) network diagram.
Figure 4
Figure 4
Top 15 results of Biological processes (BP), cellular components (CC) and molecular function (MF) of Gene Ontology (GO) enrichment analysis.
Figure 5
Figure 5
Top 20 results of Kyoto Encyclopedia of Genes and Genomes (KEGG).
Figure 6
Figure 6
Molecular docking. A. 3’-O-Methylorobol and Bcl-2. B. Tetramethoxyluteolin and Caspase-3. C. Pratensein and PI3K. D. 3’-O-Methylorobol and AKT1. E. Pratensein and mTOR.
Figure 7
Figure 7
The rat estrus cycle in the last week of the experiment. A. Rat vaginal exfoliated cells stained with crystal violet solution (×40). B. The estrous cycle statistics table.
Figure 8
Figure 8
WWZSF improved the appearance, weight, apparent morphology of ovarian tissue, ovarian weight, and ovarian index of PMS rats. A. Shift in the appearance of the model rats. B. Body weight. C. Apparent morphology of ovarian tissue. D. Ovarian weight. E. Ovarian index. Data are presented as mean ± SD, n = 3. #P < 0.05 vs. D-gal.
Figure 9
Figure 9
Effects of WWZSF on the sex hormone levels in PMS rats. A. Estradiol (E2). B. Follicle-stimulating hormone (FSH). C. Luteinizing hormone (LH). Data are presented as mean ± SD, n = 3. ***P < 0.001 vs. control; #P < 0.05 vs. D-gal, ##P < 0.01 vs. D-gal, ###P < 0.001 vs. D-gal.
Figure 10
Figure 10
Effects of WWZSF on the pathological morphology of ovarian tissue observed by Hematoxylin-Eosin staining. A. Control group. B. D-gal group. C. KBP group. D. WWZSF group. Blue →: Primary follicles. Green →: Secondary follicles. Red →: Mature follicles. Black →: Atretic follicles. Yellow →: Corpus luteum. ★: Ovarian granular cells. Bar = 50 μm.
Figure 11
Figure 11
Expression of apoptosis-related proteins in ovaries tissues of rats in different groups. A. The protein bands of BAX, Bcl-2, and Cleaved Caspase-3 were detected by Western Blot. B. Quantitative analysis of expression levels of BAX/Bcl-2. C. Quantitative analysis of expression levels of Cleaved Caspase-3. Data are presented as mean ± SD, n = 3. **P < 0.01 vs. control, ***P < 0.001 vs. control; #P < 0.05 vs. D-gal, ###P < 0.001 vs. D-gal.
Figure 12
Figure 12
Expression of PI3K/AKT/mTOR signaling pathway related proteins in ovaries tissues of rats in different groups. A. The protein bands of p-PI3K, PI3K, p-AKT, AKT, p-mTOR, and mTOR were detected by Western Blot. B. Quantitative analysis of expression levels of p-PI3K/PI3K. C. Quantitative analysis of expression levels of p-AKT/AKT. D. Quantitative analysis of expression levels of p-mTOR/mTOR. Data are presented as mean ± SD, n = 3. **P < 0.01 vs. control, ***P < 0.001 vs. control; #P < 0.05 vs. D-gal.
Figure 13
Figure 13
Expression of p62 protein in ovaries tissues of rats in different groups. A. The protein band of p62 was detected using Western Blot. B. Quantitative analysis of expression levels of p62. Data are presented as mean ± SD, n = 3. ***P < 0.001 vs. control; #P < 0.05 vs. D-gal, ##P < 0.01 vs. D-gal.
Figure 14
Figure 14
WWZSF improved the apparent morphology of uterine tissue, uterine weight, and uterine index of PMS rats. A. Morphology of uterus. B. Uterus weight. C. Uterus index. **P < 0.01 vs. control; #P < 0.05 vs. D-gal, ##P < 0.01 vs. D-gal.
Figure 15
Figure 15
Effects of WWZSF on the pathological morphology of uterine tissue observed by Hematoxylin-Eosin staining. A. Control group. B. D-gal group. C. KBP group. D. WWZSF group. Black →: Uterine gland. Bar = 50 μm.
Figure 16
Figure 16
Expression of key proteins in uterus tissues of rats in different groups. A. The protein bands of BAX, Bcl-2, Cleaved Caspase-3, p-PI3K, PI3K, p-AKT, AKT, p-mTOR, mTOR, and p62 were detected by Western Blot. B. Quantitative analysis of expression levels of BAX/Bcl-2. C. Quantitative analysis of expression levels of Cleaved Caspase-3. D. Quantitative analysis of expression levels of p-PI3K/PI3K. E. Quantitative analysis of expression levels of p-AKT/AKT. F. Quantitative analysis of expression levels of p-mTOR/mTOR. G. Quantitative analysis of expression levels of p62. Data are presented as mean ± SD, n = 3. *P < 0.05 vs. control, **P < 0.01 vs. control, ***P < 0.001 vs. control; #P < 0.05 vs. D-gal, ##P < 0.01 vs. D-gal.
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