Study on mechanism of Yiqi Huayu Formula promoting fracture healing in ovariectomized mice based on type H angiogenesis
Objective:To explore the mechanism of Yiqi Huayu Formula on fracture healing and type H angiogenesis in ovariectomized mice.Methods:96 3-month-old C57BL/6 female mice were randomly divided into sham operation group with 33 mice,model group with 33 mice and Yiqi Huayu Formula group with 30 mice.Bilateral ovariectomized models were established in the model group and the Yiqi Huayu Formula group,while part of the adipose tissue around the ovary was removed in the mice of the sham operation group.Eight weeks after the operation,left mid-tibial fracture operation was performed in all mice.After fracture,mice in Yiqi Huayu Formula group were intragastrically administrated with Yiqi Huayu Formula once a day for 4 consecutive weeks,while mice in the sham operation group and model group were treated with equal volume of saline.The tibia tissue of mice in each group was collected,and the fracture healing was observed by Micro-CT scanning,and the histomorphological changes of callus tissue were observed by HE staining.The maximum loading force of tibia of mice was detected by three-point bending test,and the proportion of type H vascular endothelial cells in total endothelial cells of callus tissue of mice in each group at different time was analyzed by flow cytometry,and the distribution of type H vessels in callus tissue was observed by immunofluorescence double staining.Results:On the 28th day after fracture,Micro-CT analysis results showed that the callus tissue of mice in the sham operation group and Yiqi Huayu Formula group was basically completed shaping,while the callus in the model group was still in the shaping stage.The results of HE staining showed that the bone cortex of the fracture part in the sham operation group was relatively flat,while the callus tissue was still obvious in the model group,and small callus tissue was also observed in Yiqi Huayu Formula group.The maximum loading force of the healed tibia in Yiqi Huayu Formula group was significantly higher than that in the model group.The results of flow cytometry and immunofluorescence staining showed that the proportion of type H vascular endothelial cells in total endothelial cells in the fracture tissue of mice in the sham operation group was increased on the 3rd day after fracture,and reached the peak on the 7th day.The proportion of type H vascular endothelial cells in the model group was increased on the 5th day after fracture,and the proportion was significantly lower than that in the sham operation group at each time point.The cell proportion in Yiqi Huayu Formula group was increased on the 3rd day after fracture,earlier than that in the model group.Compared with the model group,the proportion of type H vascular endothelial cells and type H angiogenesis in the fracture part of mice in Yiqi Huayu Formula group were significantly increased on the 3rd and 5th day after fracture.Conclusion:Yiqi Huayu Formula can increase type H angiogenesis at the early stage of fracture healing in ovariectomized mice,which may be one of the mechanisms to promote fracture healing in ovariectomized mice.
osteoporotic fractureovariectomyYiqi Huayu Formulatype H vessel
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