Integration of Network Pharmacology and Experimental Validation to Explore the Pharmacological Mechanisms of Zhuanggu Busui Formula Against Osteoporosis

Abstract

Osteoporosis (OP) is a common skeletal disease, characterized by decreased bone formation and increased bone resorption. As a novel Chinese medicine formula, Zhuanggu Busui formula (ZGBSF) has been proved to be an effective prescription for treating OP in clinic, however, the pharmacological mechanisms underlying the beneficial effects remain obscure. In this study, we explored the pharmacological mechanisms of ZGBSF against OP via network pharmacology analysis coupled with in vivo experimental validation. The results of the network pharmacology analysis showed that a total of 86 active ingredients and 164 targets of ZGBSF associated with OP were retrieved from the corresponding databases, forming an ingredient-target-disease network. The protein-protein interaction (PPI) network manifested that 22 core targets, including Caspase-3, BCL2L1, TP53, Akt1, etc, were hub targets. Moreover, functional enrichment analyses revealed that PI3K-Akt and apoptosis signalings were significantly enriched by multiple targets and served as the targets for in vivo experimental study validation. The results of animal experiments revealed that ZGBSF not only reversed the high expression of Caspase-3, Bax, Prap, and low expression of Bcl-2 in osteoblasts of the OP mouse model but also contributed to the phosphorylation of Akt1 and expression of PI3K, thereby promoting osteogenesis and ameliorating the progression of OP. In conclusion, this study systematically and intuitively illustrated that the possible pharmacological mechanisms of ZGBSF against OP through multiple ingredients, targets, and signalings, and especially the inhibition of the apoptosis and the activation of PI3K-Akt signaling.

Keywords: PI3K-Akt signalling; Zhuanggu Busui formula; apoptosis; network pharmacology; osteoporosis; pharmacological mechanisms.

Figure 1

A flowchart for explaining the mechanism of ZGBSF against OP through targets identification, network construction, enrichment analysis, and experimental validation.

Figure 2

The drug-target-disease network and PPI network construction. (A) The collection of predictive targets for OP. (B) The intersection targets of ZGBSF-related targets and OP-related targets. (C) The network of drug-target-disease includes 10 kinds of herbs, 86 active components, and 164 target genes. Circle represents active components and rectangle represents related targets. (D) PPI network of predicted targets of ZGBSF against OP. (E) The significant proteins of the PPI network were extracted from (D). (F) Screening 22 key proteins of ZGBSF in the treatment of OP were extracted from (E).

Figure 3

GO and KEGG functional analysis. (A) The top 10 of GO enrichment analysis in Biological processes (BP), cellular components (CC) and molecular function (MF). (B) The top 30 signaling pathways were analyzed by KEGG. (C, D) The PI3K-Akt signaling pathway (C) and apoptosis (D) were described in detail. The larger the nodes, the darker the color, and the more genes were enriched.

Figure 4

ZGBSF ameliorates bone loss in OVX mice. (A) Representative μCT images of distal femoral. Quantification of bone mineral density (BMD) (B), bone volume fraction (BV/TV) (C), trabecular thickness (Tb.Th) (D), trabecular number (Tb.N) (E) and trabecular separation (Tb.Sp) (F). (G) Hematoxylin-eosin staining of the distal femur. (H) The area of trabecular bone (%). (I) The area of lipid droplets (%). Data were presented as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, NS indicated no significant difference.

Figure 5

ZGBSF promotes osteogenesis and fails to inhibit osteoclastogenesis. (A) Immunohistochemistry of Runx2, Alp, and Osx of the distal femur. Red arrows indicated the high expression of Runx2, Alp, and Osx. (B–D) The quantification of Runx2, Alp, and Osx expression. (E) TRAP staining of the distal femur. (F) The number of osteoclasts (OC.N/mm²). The arrows represented the positive expression. Data were presented as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, NS indicated no significant difference.

Figure 6

ZGBSF suppresses apoptosis of osteoblasts to ameliorate OP in OVX mice. (A) Immunofluorescence of Bcl-2, Bax, Caspase-3 and Parp in distal femur. (B–E) The quantification of Bcl-2, Bax, Caspase-3 and Parp positive cells. (F) Tunel staining was performed to examine the apoptosis status of osteoblasts. (G) The quantification of TUNEL positive rates in the distal femur. White arrows indicated the positive expression. Data were presented as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.

Figure 7

ZGBSF activates the PI3K-Akt signaling pathway of femoral tissue. (A) Immunohistochemistry of t-Akt1, Akt1(S473), and PI3K of distal femur. (B, C) The quantification of Akt1(S473)/t-Akt1, and PI3K expression. Red arrows indicated the positive expression. Data were presented as mean ± SEM of three independent experiments. **P < 0.01.