Scutellarin Loaded on Ultradeformable Nanoliposome Scutellarin EDTMP (S-UNL-E) Promotes Osteogenesis in Osteoporotic Rats

Abstract

Scutellarin is known as a safe, effective, and low-cost traditional Chinese medicine and has a variety of biological activities. Studies reported that the scutellarin loaded on ultradeformable nanoliposome scutellarin EDTMP (S-UNL-E) could promote osteoblast differentiation and bone formation in vitro. However, its effect on promoting osteogenesis in vivo is still unclear. In this study, pharmacology network and transcriptome sequencing were used to screen the potential targets and pathways of scutellarin in treating osteoporosis. The female Sprague-Dawley (SD) rats were operated on with bilateral oophorectomy and femoral defect to establish an osteoporosis model and then treated separately with bone dust, single scutellarin, 40 mg/kg ultradeformable nanoliposome scutellarin (S-UNL), and the optimal concentration of 40 mg/kg S-UNL-E for a total of 56 d to detect the parameters of trabecular bones. And qRT-PCR and western blot were performed to determine the expression of prostaglandin-endoperoxide synthase 2 (PTGS2), alkaline phosphatase (ALP), transcription factor 4 (TCF4), and β-catenin. Results of microscopic computed tomography (Micro-CT) of trabecular bones showed that single scutellarin, S-UNL, and S-UNL-E all promoted the bone formation of osteoporotic rats, in which S-UNL-E manifested the most remarkable therapeutic effect. And it is found that 40 mg/kg of S-UNL-E increased the expression of PTGS2, ALP, TCF4, and β-catenin, which indicated that S-UNL-E stimulated the secretion of ALP in bone defect areas to promote bone healing, and increased PTGS2 expression thereby enhancing the transcription and translation of key gene β-catenin and TCF4 in the Wnt/β-catenin signaling pathway to treat osteoporotic rats.

Figure 1

Target genes of scutellarin for the treatment of osteoporosis based on network pharmacology analysis. (a) Venn diagram of overlapped genes. (b) PPI network of overlapped genes.

Figure 2

Bioinformatics analysis of DEGs. (a) Heat map. The horizontal coordinates are the samples, and the vertical coordinates are the normalized values of the differential gene FPKM. The redder the color, the higher the expression, and the greener the color, the lower the expression. (b) Volcano map. The horizontal coordinates are the log2FoldChange values, the vertical coordinates are −log10padj values, and the blue dashed line indicates the threshold line for the DEG screening criteria. SE: S-UNL-E group; NC: sham group.

Figure 3

GO and KEGG enrichment analysis of DEGs. (a) GO enrichment analysis of down-DEGs. (b) GO enrichment analysis of reversed down-DEGs. (c) KEGG enrichment analysis of down-DEGs. (d) KEGG enrichment analysis of reversed down-DEGs. The horizontal coordinates are the ratio of the number of DEGs to the total number of DEGs that are annotated to GO/KEGG terms, and the vertical coordinates are the GO/KEGG terms.

Figure 4

Relative mRNA and protein expressions of PTGS2, β-catenin, TCF4, and ALP after osteoblasts were treated with 2 × 10⁷, 2 × 10⁸, and 2 × 10⁹ S-UNL-E. (a–d) The mRNA levels were analyzed by qRT-PCR. (e) The expression levels were analyzed by western blot. (f–i) The western blot results were qualified. ^∗∗∗p < 0.001vs. control.

Figure 5

SUN-L-E promoted the bone healing in osteoporotic rats. (a) BV/TV (%). (b) Tb.Th (mm). (c) Tb.N (mm⁻¹). (d) Conn.D (mm⁻³). (e) Tb.Sp (mm). ^∗∗p < 0.01 and ^∗∗∗p < 0.001vs. sham. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001vs. model.

Figure 6

Relative mRNA and protein expressions of PTGS2, β-catenin, TCF4, and ALP of rats after being treated with 40 mg/kg S-UNL-E. (a–d) The mRNA levels were analyzed by qRT-PCR. (e) The expression levels were analyzed by western blot. (f–i) The western blot results were qualified. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001vs. sham. ^#p < 0.05, ^##p < 0.01, and ^##p < 0.001vs. model.