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. 2024 Mar 9;10(6):e27646.
doi: 10.1016/j.heliyon.2024.e27646. eCollection 2024 Mar 30.

Mechanism of Bazi Bushen capsule in delaying the senescence of mesenchymal stem cells based on network pharmacology and experimental validation

Yaping Zhang  1 , Tongxing Wang  2   3   4 , Yanfei Song  2   3   5 , Meng Chen  2   3   5 , Bin Hou  2   3 , Bing Yao  2   3   5 , Kun Ma  2   3   6 , Yahui Song  2   3 , Siwei Wang  1   2   3 , Dan Zhang  1   2   3 , Junqing Liang  2   3 , Cong Wei  1   2   3
Affiliations

Mechanism of Bazi Bushen capsule in delaying the senescence of mesenchymal stem cells based on network pharmacology and experimental validation

Yaping Zhang et al. Heliyon. .

Abstract

Ageing is becoming an increasingly serious problem; therefore, there is an urgent need to find safe and effective anti-ageing drugs.

Aims: To investigate the effects of Bazi Bushen capsule (BZBS) on the senescence of mesenchymal stem cells (MSCs) and explore its mechanism of action.

Methods: Network pharmacology was used to predict the targets of BZBS in delaying senescence in MSCs. For in vitro studies, MSCs were treated with D-gal, BZBS, and NMN, and cell viability, cell senescence, stemness-related genes, and cell cycle were studied using cell counting kit-8 (CCK-8) assay, SA-β-galactosidase (SA-β-gal) staining, Quantitative Real-Time PCR (qPCR) and flow cytometry (FCM), respectively. Alkaline phosphatase (ALP), alizarin red, and oil red staining were used to determine the osteogenic and lipid differentiation abilities of MSCs. Finally, the expression of senescence-related genes and cyclin-related factors was detected by qPCR and western blotting.

Results: Network pharmacological analysis suggested that BZBS delayed cell senescence by interfering in the cell cycle. Our in vitro studies suggested that BZBS could significantly increase cell viability (P < 0.01), decrease the quantity of β-galactosidase+ cells (P < 0.01), downregulate p16 and p21 (P < 0.05, P < 0.01), improve adipogenic and osteogenic differentiation, and upregulate Nanog, OCT4 and SOX2 genes (P < 0.05, P < 0.01) in senescent MSCs. Moreover, BZBS significantly reduced the proportion of senescent MSCs in the G0/G1 phase (P < 0.01) and enhanced the expression of CDK4, Cyclin D1, and E2F1 (P < 0.05, P < 0.01, respectively). Upon treatment with HY-50767A, a CDK4 inhibitor, the upregulation of E2F1 was no longer observed in the BZBS group.

Conclusions: BZBS can protect MSCs against D-gal-induced senescence, which may be associated with cell cycle regulation via the Cyclin D1/CDK4/E2F1 signalling pathway.

Keywords: Bazi bushen capsule; Cell cycle; Cell senescence; Mesenchymal stem cells; Network pharmacology.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Graphical abstract of anti-ageing validation of BZBS based on network pharmacology and in vitro experiments (Created with BioRender.com).
Fig. 2
Fig. 2
The network mechanism of anti-ageing effects exerted by BZBS. (A) Enrichment analysis of KEGG pathway for targets regulated by BZBS exerting anti-ageing effects. (B) Key targets for the anti-ageing effects of BZBS.
Fig. 3
Fig. 3
Effects of BZBS on cell viability, p21 and p16 expression, and SA-β-gal staining in D-gal-induced senescent MSCs. (A) MSCs samples with no less than 1 × 106 cells/well were collected from each group. Cell viability was measured using the CCK-8 kit, as described in the Methods section. Effects of BZBS on cell viability of D-gal-induced MSCs (n = 6); (B) and (C) MSCs samples with no less than 1 × 106 cells/well were collected from each group. Total RNA was collected, and qPCR was performed according to the method described in the Methods section. Effect of BZBS on the expressions of p21 and p16 in senescent MSCs (n = 3, n = 6); (D) and (E) According to the method described in the Methods section, the MSCs were fixed at room temperature, then stained with SA-β-gal, and finally recorded using an inverted microscope. Effect of BZBS on SA-β-gal-positive ratio in senescent MSCs and quantitative analysis of SA-β-gal-positive cells. (n = 4; Magnification: 200x). The results represent the mean ± SD. vs Control, *p < 0.05, **p < 0.01; vs Model, #p < 0.05, ##p < 0.01.
Fig. 4
Fig. 4
Effect of BZBS on the differentiation potential of senescent MSCs. Each group of MSCs was fixed at room temperature and stained according to the method described in the Methods section. (A) Effect of BZBS on adipogenic potential in senescent MSCs. (Oil red O staining, Magnification: 200x). (B), (C), and (D) Effect of BZBS on osteogenic potential in senescent MSCs. (ALP staining, Magnification: 100×; Alizarin red staining, Magnification: 50x and 100x). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 5
Fig. 5
Effect of BZBS on the expression of stemness-associated genes in senescent MSCs. MSCs samples with no less than 1 × 106 cells/well were collected from each group. Total RNA was collected and qPCR was performed according to the method described in the Methods section. (A), (B), and (C) Effect of BZBS on the expression of Nanog, OCT4, and SOX2 in senescent MSCs (n = 6, n = 6, n = 5). The results represent the mean ± SD. vs Control, *p < 0.05, **p < 0.01; vs Model, #p < 0.05, ##p < 0.01.
Fig. 6
Fig. 6
Effect of BZBS on the cell cycle of senescent MSCs. (A) FCM was used to detect the cell cycle. MSCs samples with no less than 2 × 106 cells/well were collected for PI staining in each group, and the cell cycle was detected according to the method described in the Methods section. Effect of BZBS on the distribution of cell cycle in senescent MSCs (n = 6); (B) and (E) Effect of BZBS on the expression of Cyclin D1 in senescent MSCs (n = 6, n = 3); (C) and (F) Effect of BZBS on the expression of CDK4 in senescent MSCs (n = 6, n = 3); (D) Effect of BZBS on the expression of E2F1 in senescent MSCs (n = 5); (G) Effect of BZBS on the expression of E2F1 in senescent MSCs after the treatment with CDK4 inhibitor (HY-50767A) (n = 6). The results represent the mean ± SD. vs Control, *p < 0.05, **p < 0.01; vs Model, #p < 0.05, ##p < 0.01.
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