背景:中药(TCM)外用治疗骨折具有悠久的临床应用历史和理论基础,是中医特色治疗方法之一,疗效显著,具有诸多优点。在常用的外用中药中,Tubiechong值得注意。
目的:阐明土必冲局部修补是否能促进骨折愈合,并探讨其作用机制。
方法:采用改良的Einhorn造模方法建立大鼠胫骨骨折模型。拍摄X线片以评估骨折愈合的进展。血清骨碱性磷酸酶(BALP),通过ELISA分析骨钙蛋白(BGP)和I型胶原蛋白(CTX-I)的C端含量。CD31免疫组织化学用于评估胫骨段中的血管生成。痰必冲汤(TD)对HUVEC增殖的影响,通过MTT法检测迁移和侵袭,伤口愈合试验和Transwell迁移试验,分别。进行RNA-seq以鉴定差异表达的基因(DEG)。基于基因本体论(GO)和京都基因和基因组百科全书(KEGG)数据库进行功能的富集和信号通路分析。定量实时聚合酶链反应(qRT-PCR)用于研究基因表达水平。蛋白质印迹法(WB)检测相关调节蛋白的表达。
结果:三个TD剂量组大鼠胫骨骨折愈合时间缩短。血清BALP水平,TD治疗组的BGP和CTX-I高于NC组。X光检查结果显示,手术后第7天,高剂量TD组骨折愈合程度明显优于NC组,14日各TD治疗组骨折愈合程度明显高于NC组,17日,手术后第21天.CD31免疫组化成果显示,TD医治组的血管数目和血管面积均高于NC组。体外,TD促进了扩散,创伤愈合和HUVECs迁移。GO转录组测序分析结果显示,TD显著改变了细胞生长相关基因的表达,新陈代谢,和运动性。根据KEGG注释,VEGFA上调。8个DEGs在VEGFA-VEGFR2信号通路中富集,其中六个被上调。KEGG信号通路分析表明,DEGs最丰富的是丝裂原活化蛋白激酶(MAPK)信号通路。qRT-PCR显示,1mg/mLTD处理24h后,HUVECs中VEGFA基因表达量为对照组的7.8倍,WB实验表明其蛋白表达量是对照组的3倍。WB结果显示磷酸化ERK基因高表达,而磷酸化P38和磷酸化JNK蛋白的表达水平保持不变。
结论:土贝红修补疗法通过VEGF/ERK1/2信号通路调节血管新生,促进大鼠胫骨骨折愈合。
BACKGROUND: 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.
OBJECTIVE: To elucidate whether local patching of Tubiechong can promote fracture healing and explore its mechanism of action.
METHODS: A rat tibia fracture model was constructed by the modified Einhorn modeling method. X-ray 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.
CONCLUSIONS: Tubechong patching therapy promotes tibia fracture healing in rats by regulating angiogenesis through the VEGF/ERK1/2 signaling pathway.