| Osteosarcoma is a common primary malignant bone tumor,which mostly occurs in children and adolescents.At present,the chemotherapy drugs for osteosarcoma have the disadvantages of high side effects and destruction of body immunity,and patients are prone to develop chemotherapy resistance and reduce the treatment effect.Therefore,trying new drugs to improve the therapeutic effect of osteosarcoma has become the focus of research.Traditional Chinese medicine has many advantages,such as low toxic and side effects,simultaneous regulation of multiple targets and reduction of drug resistance.Pilose antler is a precious traditional Chinese medicine in China,which can resist tumor,protect nervous system,protect bones and enhance immunity.Research shows that pilose antler has complex components,and pilose antler mainly plays a major role by velvet antler polypeptide and other active components.At present,there are few studies on the pharmacodynamic components and mechanism of action of pilose antler in the treatment of osteosarcoma.In this paper,the mechanism of velvet antler polypeptide and other active ingredients of pilose antler regulating the death,migration and invasion of osteosarcoma cells was explored through network pharmacological prediction and in vitro cell experiments.The research contents are as follows:(1)The material basis and molecular mechanism of pilose antler in the treatment of osteosarcoma were obtained by network pharmacological analysis.It was found that TNF-α、Notch1 can be used as the key target of pilose antler in the treatment of osteosarcoma;17β-estradiol and estrone can be used as the key components of pilose antler in the treatment of osteosarcoma.TNF-α Necrotic apoptosis can promote the death of tumor cells,and Notch signaling pathway mediated by Notch1 can regulate the migration and invasion of tumor cells.It is speculated that the active components of pilose antler can regulate the death,migration and invasion of osteosarcoma cells through these two pathways.(2)In vitro cell experiments were conducted to explore the mechanism of 17β-estradiol,estrone and velvet antler polypeptide regulating osteosarcoma cell death.MTT assay showed that both 17β-estradiol and estrone could effectively inhibit the proliferation of MG63 cells.Enzyme linked immunosorbent assay showed that 17β-estradiol and estrone could up regulate the expression of TNF-α,RIPK3 and MLKL proteins in MG63 cells,and TNF,RIPK3 and MLKL mediated necroptosis of osteosarcoma cells.These results suggest that 17β-estradiol and estrone can promote necroptosis in osteosarcoma cell line MG63.Similarly,the enzyme-linked immunosorbent assay showed that velvet antler polypeptide could also up regulate the expression of TNF-α,RIPK3 and MLKL in osteosarcoma cell line MG63,thereby promoting the occurrence of necroptosis.(3)In vitro cell experiment was conducted to explore the mechanism of velvet antler polypeptide regulating the migration and invasion of osteosarcoma cells.The results of Cell colony formation assay showed that 80 μg/m L pilose antler polypeptide could inhibit the proliferation of osteosarcoma cells MG63 and U2OS;The results of Wound healing(scratch)assay and Transwell assay showed that velvet antler polypeptide effectively significant inhibition the migration and invasion of osteosarcoma cells MG63 and U2OS;The results of Western blot and q PCR showed that 20 μg/m L and 80 μg/m L velvet antler polypeptide treated cells,the expression of Notch1,Hes1 protein and their m RNA decreased significantly,indicating that velvet antler polypeptide can effectively inhibit the expression of Notch signaling pathway in osteosarcoma cells MG63 and U2 OS.To sum up,velvet antler polypeptide,17β-estradiol and estrone can promote osteosarcoma cell death by promoting necroptosis.Velvet antler polypeptide can down regulate the expression of Notch signaling pathway,thus inhibiting the migration and invasion of osteosarcoma cells. |
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