Tubiechong patching promotes tibia fracture healing in rats by regulating angiogenesis through the VEGF/ERK1/2 signaling pathway

Ethnopharmacological relevance: The external use of traditional Chinese medicine (TCM) to treat fractures has a long history of clinical application and theoretical basis, and is also one of the characteristic treatment methods of TCM with significant efficacy and many advantages. Among the commonly used external Chinese medicines, Tubiechong is noteworthy.Aim of the study: To elucidate whether local patching of Tubiechong can promote fracture healing and explore its mechanism of action. Materials and methods: A rat tibia fracture model was constructed by the modified Einhorn modeling method. Xray films were taken to evaluate the progress of fracture healing. Serum bone alkaline phosphatase (BALP), osteocalcin (BGP) and the C-terminal content of collagen type I (CTX-I) were analyzed by ELISA. CD31 immunohistochemistry was used to evaluate angiogenesis in the tibia segment. The effects of Tubiechong decoction (TD) on HUVEC proliferation, migration and invasion were detected by MTT assay, wound healing assay and Transwell migration assay, respectively. RNA-seq was performed to identify differentially expressed genes (DEGs). Enrichment of functions and signaling pathway analysis were performed based on the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Quantitative real time polymerase chain reaction (qRT-PCR) was used to study gene expression levels. Western blotting (WB) was used to detect the expression of relevant regulatory proteins.Results: The healing time of rat tibia fractures in the three TD dose groups was shortened. The serum levels of BALP, BGP and CTX- I in the TD-treated group were higher than those in the NC group. The X-ray results showed that on the 7th day after surgery, the fracture healing degree of the high-dose TD group was significantly better than that of the NC group, and the fracture healing degrees of each TD treatment group were significantly higher than those of the NC group on the 14th, 17th, and 21st days after the operation. The CD31 immunohistochemistry results showed that the number of blood vessels and the vascular area in the TD treatment group were higher than those in the NC group. In vitro, TD promoted the proliferation, wound healing and migration of HUVECs. GO analysis of transcriptome sequencing results showed that TD significantly altered the expression of genes related to cell growth, metabolism, and motility. According to KEGG annotations, VEGFA was upregulated. Eight DEGs were enriched in the VEGFA-VEGFR2 signaling pathway, of which six were upregulated. KEGG signaling pathway analysis showed that the most abundant DEGs were in mitogen-activated protein kinase (MAPK) signaling pathway. qRT-PCR showed that VEGFA gene expression in HUVECs was 7.8 times that of the control group after 1 mg/mL TD treatment for 24 h, and WB experiments showed that its protein expression was 3 times that of the control group. WB results showed that the phosphorylated ERK gene was highly expressed, while the expression levels of phosphorylated P38 and phosphorylated JNK protein remained unchanged.Conclusion: Tubechong patching therapy promotes tibia fracture healing in rats by regulating angiogenesis through the VEGF/ERK1/2 signaling pathway.