Identification of kukoamine a as an anti-osteoporosis drug target using network pharmacology and experiment verification

Abstract

Background: Osteoporosis (OP) is a major and growing public health problem characterized by decreased bone mineral density and destroyed bone microarchitecture. Previous studies found that Lycium Chinense Mill (LC) has a potent role in inhibiting bone loss. Kukoamine A (KuA), a bioactive compound extract from LC was responsible for the anti-osteoporosis effect. This study aimed to investigate the anti-osteoporosis effect of KuA isolated from LC in treating OP and its potential molecular mechanism.

Method: In this study, network pharmacology and molecular docking were investigated firstly to find the active ingredients of LC such as KuA, and the target genes of OP by the TCMSP platform. The LC-OP-potential Target gene network was constructed by the STRING database and network maps were built by Cytoscape software. And then, the anti-osteoporotic effect of KuA in OVX-induced osteoporosis mice and MC3T3-E1 cell lines were investigated and the potential molecular mechanism including inflammation level, cell apoptosis, and oxidative stress was analyzed by dual-energy X-ray absorptiometry (DXA), micro-CT, ELISA, RT-PCR, and Western Blotting.

Result: A total of 22 active compounds were screened, and we found KuA was identified as the highest active ingredient. Glycogen Phosphorylase (PYGM) was the target gene associated with a maximum number of active ingredients of LC and regulated KuA. In vivo, KuA treatment significantly increased the bone mineral density and improve bone microarchitecture for example increased BV/TV, Tb.N and Tb.Th but reduced Tb.Sp in tibia and lumber 4. Furthermore, KuA increased mRNA expression of osteoblastic differentiation-related genes in OVX mice and protects against OVX-induced cell apoptosis, oxidative stress level and inflammation level. In vitro, KuA significantly improves osteogenic differentiation and mineralization in cells experiment. In addition, KuA also attenuated inflammation levels, cell apoptosis, and oxidative stress level.

Conclusion: The results suggest that KuA could protect against the development of OP in osteoblast cells and ovariectomized OP model mice and these found to provide a better understanding of the pharmacological activities of KuA again bone loss.

Keywords: Kukoamine A; Network pharmacology; Osteoblast cells; Osteoporosis; Ovariectomized mice.

Fig. 1

The experimental result show that KuA significantly increased the bone mineral density of the spine and tibia in OVX mice. A KuA spectrometry analyses and total experiment flowchart. B Representative figure of bone mineral density in mice. C BMD of L4. D BMD of the tibia. These results found that KuA improves the bone mass of the tibia and spine in ovariectomized mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. KuA: Kukoamine A; OVX: ovariotomy; L4: Lumber 4; BMD: bone mineral density

Fig. 2

KuA significantly improved the tibia bone microstructure and mechanical properties in OVX mice. A Representative figure in the tibia. B BV/TV of the tibia. C Tb.N of tibia. D Tb.Sp of the tibia. E Tb.Th of the tibia. These results found that KUA improved the bone microstructure of the tibia in ovariectomized mice. F, G The maximum force of the tibia. Mechanical results showed that KUA improve the maximum stress of the tibia in ovariectomized mice. The bar is 0.7 mm. *P < 0.05, **P < 0.01,***P < 0.001,****P < 0.0001. KuA: Kukoamine A; OVX: ovariotomy; L4: Lumber 4; BMD: bone mineral density. BV/TV: bone volume over total volume; Tb.Th: trabecular thickness; Tb.N: trabecular number, Tb.Sp: trabecular spacing

Fig. 3

KuA significantly increased the bone microstructure of the spine in OVX mice. A Representative figure in L4. B BV/TV of L4. C Tb.Sp of L 4. D Tb.N of L 4. E Tb.Th of L4. These results found that KuA improved the bone microstructure of the spine in ovariectomized mice. The bar is 0.3 mm. *P < 0.05, **P < 0.01,***P < 0.001,****P < 0.0001. KuA: Kukoamine A; OVX: ovariotomy; L4: Lumber 4; BMD: bone mineral density. BV/TV: bone volume over total volume; Tb.Th: trabecular thickness; Tb.N: trabecular number, Tb.Sp: trabecular spacing

Fig. 4

The gene expression analyses in osteoporosis samples from the GEO dataset. A Gene normalization diagram shows this dataset has a good consistency. B Gene volcano map. C Principal genetic analysis. D Uniform Manifold Approximation and Projection. D Heatmap analysis found that the top 20 genes expressed in GEO dataset. UMAP: Uniform Manifold Approximation and Projection, PCA: Principal genetic analysis

Fig. 5

Potential target genes and PPI network map of KuA for OP. A The Venn results of potential genes of KuA therapy for OP. B Counts and lists of the top genes of PPI network map. C The PPI network map of 24 target genes. 723 protein nodes and 8743 edges were obtained for intersection genes. After screening with DC > 61 and a BC range of 20–113.2, the first 20 proteins were selected in Table 5 (in descending order of degree), with a total of 322 edges. PPI: Protein–Protein Interaction; KuA: Kukoamine A; OP: osteoporosis

Fig. 6

Topological analysis of the protein–protein interaction network (A) and GO/KEGG enrichment analysis (B, C). GO: genetic selection; KEGG: biological pathways. GO patents were linked with response to stimulus, metabolic process, and biological regulation

Fig. 7

A Target genes-miRNA. B TCM compound-disease regulatory network. C The 3D map of binding of KuA. The Vina score results of KuA, Linarin, aurantiamide acetate, and acacetin increased steadily, indicating that KuA has the strongest and most stable binding affinity for PYGM. 3D images of acacetin, alexandrine, emodin, KuA, and Kulactone to PYGM. KuA: Kukoamine A; OVX: ovariotomy; TCM: Traditional Chinese Medicine

Fig. 8

KuA protects against cell apoptosis and oxidative stress level in OVX mice. A mRNA expression in the tibia. B Bax/BCL-2 level. C The original membrane of the western blotting. D cytochrome c level. E Caspase-3. F MnSOD. G CuZnSOD. H H₂O₂ level. *P < 0.05, **P < 0.01,***P < 0.001,****P < 0.0001. KuA: Kukoamine A; OVX: ovariotomy; Bax: BCL2 Associated X, Apoptosis Regulator; BCL2: BCL2 Apoptosis Regulator; SOD, Superoxide Dismutase; MDA: malondialdehyde

Fig. 9

KuA increased the osteoblastic differentiation and mineralized nodule formation of osteoblastic MC3T3-E1 cells. A MDA. B Relative ALP activity. C Alizarin Red SOD. D Relative cell viability. E mRNA level in MC3T3-E1 cells. *P < 0.05, **P < 0.01,***P < 0.001,****P < 0.0001. KuA: Kukoamine A; OVX: ovariotomy; ALP, Alkaline Phosphatase; PYGM, Glycogen Phosphorylase, Muscle Associated; OCN, osteocalcin; Osterix: Sp7 Transcription Factor