The shared KEGG pathways between icariin-targeted genes and osteoporosis

Abstract

Osteoporosis is a common metabolic bone disease that affects about 40% of postmenopausal women. Treatment options for osteoporosis are limited, however. Icariin is an herbal substance that has been shown to improve bone mass, but the mechanisms are largely unknown. Using bioinformatics analysis, we have identified the hub genes and KEGG pathways shared between icariin-targeted genes and osteoporosis. The top five shared KEGG pathways were the Toll-like receptor signaling pathway, adipocytokine pathway, neurotrophin signaling pathway, NOD-like receptor signaling, and B cell receptor signaling pathway; the hub genes were RELA, NFKBIA, and IKBKB, belonging to the NF-κB family. The identified icariin-targeted genes are involved in inflammation, insulin resistance, apoptosis, and immune responses, and regulate the PI3K-Akt, NF-κB, MAPK, and JNK signaling pathways. Our in vitro data show that icariin inhibits apoptosis in human mesenchymal stem cells by suppressing JNK/c-Jun signaling pathway. Together, these findings indicate that icariin exerts its anti-osteoporotic function by inhibiting JNK/c-Jun signaling pathway, and suggest that icariin may be a promising treatment option for osteoporosis.

Keywords: JNK; bioinformatics; icariin; osteogenesis; osteoporosis.

Figure 1

The interaction networks of icariin-targeted genes. (A) Interaction network constructed by Cytoscape. (B) Weighted interaction network.

Figure 2

Identification of shared KEGG pathways of icariin-targeted genes and osteoporosis. 58 icariin-targeted genes related KEGG pathways, and 110 osteoporosis related KEGG pathways were found; 26 (18.3%) shared KEGG pathways were identified.

Figure 3

Gene enrichment analysis. RELA, NFKBIA, and IKBKB were involved in all five pathways. The top three genes by degree were AKT1, JUN, and SRC.

Figure 4

Circular visualization of chromosomal positions and connectivity of icariin-targeted genes. The names of the genes are shown in the outer circle. For the outer heatmap, red represents high degree, and green represents low degree. For the middle heatmap, pink represents high betweenness, and blue represents low betweenness. For the inner heatmap, brown represents high closeness, and black represents low closeness. Lines coming from each gene point to their specific chromosomal locations on the chromosomal circle. The three hub genes are shown in red.

Figure 5

Icariin-targeted genes related to the top 5 shared KEGG pathways. (A) Icariin-targeted genes related to Toll-like receptor pro-inflammatory signaling: (1) PI3K-Akt signaling, (2) NF-kB, and (3) MAPK signaling pathway. (B) Icariin-targeted genes related to Adipocytokine signaling, and insulin resistance. (C) Icariin-targeted genes related to Neurotrophin signaling regulating cell survival and apoptosis. (4) JNK signaling pathway is involved in the process. (D) Icariin-targeted genes related to NOD-like receptor signaling pathway. (2) NF-kB, and (3) MAPK signaling pro-inflammatory pathways are involved. (E) Icariin-targeted genes related to B cell receptor signaling. (2) NF-kB pathway is involved, regulating B cell ontogeny, autoimmunity anergy, and immune responses.

Figure 6

Icariin inhibits apoptosis of hMSC cells in vitro. (A) The cellular morphology of hMSCs; (B) CCK-8 proliferation assay of icariin-treated hMSCs; (C) Bcl-2 and Bax gene expression in icariin-treated hMSCs measured by qRT-PCR; (D, E) Apoptosis assessed by flow cytometry.

Figure 7

Icariin inhibits apoptosis of hMSCs by suppressing c-Jun/JNK signaling. (A) Expression of c-Jun, p-c-Jun, JNK, and p-JNK analyzed in icariin-treated hMSCs by western blotting; (B, C) Phosphorylation levels of c-Jun and JNK in hMSCs transfected with c-Jun or JNK plasmids and treated with icariin (10 μg/ml), analyzed by western blotting; (D, E) CCK-8 proliferation assay of icariin-treated hMSCs; (F) Apoptosis analyzed by flow cytometry of icariin-treated hMSCs; (G) Apoptosis analyzed in hMSCs transfected with c-Jun and JNK plasmids, and treated with icariin; (H, I) Bcl-2 and Bax mRNA levels analyzed by qRT-PCR.