The Effect And Mechanism Of Sambucus Sibirica Nakai Bark Extract On Rat Femoral Fracture Healing

Objective: In this study,the model of femoral fracture in SD rats was established,and the effect and mechanism of Sambucus Sibirica Nakai bark extract on fracture healing were explored,so as to provide a theoretical basis for further development and clinical utilization.Methods: The first part: 108 3-month-old male SD rats were randomly divided into 5 groups: model group,the ethanol extract group,the aqueous extract group,positive control group,sham operation group.The osteogenic effect of Sambucus Sibirica Nakai bark extract on femoral fracture healing in rats was evaluated by X-ray,Micro-CT,Biomechanical experiment and Histological methods.The second part: Blood samples were taken at 2,4 and 6 weeks after administration,and the levels of serum osteocalcin(BGP),osteoprotegerin(OPG),type I collagen carboxyl terminal peptide(PICP),alkaline phosphatase(ALP),acid phosphatase(ACP),calcium(Ca)and phosphorus(P)were determined by enzyme linked immunosorbent assay(Elisa).The third part: The effects of Sambucus Sibirica Nakai bark extract on Wnt/β-catenin signal pathway and growth factors were evaluated by RT-PCR,Western Blot and immunohistochemistry.Results: The first part: 1.The results of X-ray examination: at the second week of administration,the fracture line of the model group was obvious,while that of the ethanol extract group,the aqueous extract group and the positive control group was blurred,and the fibrous callus could be seen.At the 4th week,the fracture line in the model group became blurred and a small amount of callus formed,while in the ethanol extract group,the aqueous extract group and positive control group,a large number of callus formed and the fracture line was blurred.At the 6th week,there were a large number of bony callus formation and callus shaping in the ethanol extract group,the aqueous extract group and positive control group,the medullary cavity recanalized and the fracture line disappeared.Compared with the positive control group,the score in the ethanol extract group was significantly higher than that in the model group.2.Micro-CT detection: the experimental results showed that in the 2nd,4th and 6th week,BMD,BV/TV,Tb.Th and Tb.N in the ethanol extract group and the aqueous extract group were significantly higher than those in the model group,and the number of trabeculae in the ethanol extract group and the aqueous extract group was not only larger than that in the model group,but also the trabecular thickness was larger than that in the model group.The microstructure showed that the healing of the ethanol extract group and the aqueous extract group was better than that of model group.3.Biomechanical experiment: the maximum load and maximum deflection of the ethanol extract group and the aqueous extract group were significantly better than those of the model group.4.The results of tissue section showed that at the second week,a small number of chondrocytes were found in both the ethanol extract group and the aqueous extract group.a large number of mesenchymal cells,fibroblasts,mononuclear macrophages,osteoblasts,osteocytes,and neovascularization began to form closely arranged fibrous connective tissue,which initially linked the fracture segments,while there were a small number of mesenchymal cells,osteoblasts and osteocytes in the model group.Granulation tissue and loosely arranged fibrous connective tissue were formed by fibroblasts and inflammatory cells.At the 4th week,a large number of chondrocytes,osteoblasts and osteoclasts could be seen in the fracture site of rats in the ethanol extract group and the aqueous extract group,and many kinds of bone appeared at the broken end and under the periosteum of the fracture,while a small number of chondrocytes and osteoblasts could be seen in the model group.the fracture segments were linked by closely arranged fibrous connective tissue.At the 6th week,the osteoid increased in the ethanol extract group and the aqueous extract group,the bone trabeculae were connected to lamellar bone,and the osteogenic process was obvious,while a small amount of new bone trabeculae and less osteoid could be seen at the fracture end of the model group.The second part: At the second week of drug intervention,compared with the sham operation group,the serum levels of ALP,OPG,ACP,BGP,PICP,Ca and P in the model group were significantly higher than those in the sham operation group.Compared with the model group,the serum levels of ALP,ACP,BGP,PICP and P in the positive control group,the serum levels in the ethanol extract group and the aqueous extract group were significantly higher,and the serum Ca levels in the ethanol extract group and the aqueous extract group were significantly lower than those in the model group(P<0.01).The level of ALP,ACP,BGP and PICP decreased with the process of fracture healing,while the content of OPG increased with the process of fracture healing,the difference was statistically significant(P<0.05).The third part: 1.Using RT-PCR and Western Blot technology,we found that,compared with the model group,the protein expression of Wnt2,β-catenin and p-GSK3β in the ethanol extract group and the aqueous extract group increased significantly,and the difference was statistically significant.2.Compared with the model group,the expression of bone morphogenetic protein 2(BMP-2)and vascular endothelial growth factor(VEGF)in the ethanol extract group and the aqueous extract group increased significantly,increased gradually at the 2nd and 4th week,reached the peak at the 4th week,and decreased gradually at the 6th week when osteoblasts matured and callus formed.Conclusion: The experiment found that,by the observation of gross specimens,the extract of Sambucus Sibirica Nakai bark could promote the formation of callus,improve the quality of callus,significantly increase the density of bone structure,increase the number and thickness of trabeculae,and significantly accelerate fracture healing.The experiment confirmed that Sambucus Sibirica Nakai can increase the level of bone metabolic markers,increase the content of blood phosphorus,promote calcium deposition,increase the absorption of trace elements,and bone mineral density,accelerate bone healing.The molecular biology experiment showed that the extract of Sambucus Sibirica Nakai bark can activate Wnt/β-catenin signal pathway,secrete a variety of growth factors,such as BMP-2、VEGF,promote the deposition of calcified cartilage,and finally remodel and ossify the calcified cartilage to complete the repair process,thus promoting fracture healing.