益气散瘀解毒方有效成分抑制骨肉瘤的机制研究OA

Objective:To explore the mechanism by which the main active components of the Yi-Qi-San-Yu-Jie-Du formula inhibit the occurrence and development of osteosarcoma through regulating the REGγ/RNF149/BRAF/MEK/ERK signa-ling pathway.Methods:Mass spectrometry and immunohistochemical experiments were conducted on samples of adjacent normal tissues and osteosarcoma tissues from 9 osteosarcoma patients.Osteosarcoma cell lines 143B and MG63 were used as research subjects.The expression of REGγ protein was regulated by shRNA interference and transient transfection methods.Western Blot and RT-PCR were used to verify the transfection efficiency and detect the expression levels of RNF149,BRAF,MEK,and ERK.Co-immunoprecipitation experiments and in vitro degradation experiments were per-formed to verify the interaction between REGγ and RNF149 proteins.The main components of Astragalus,Coptis,and Polyphyllin in the compound were identified by high-performance liquid chromatography.After treating the cells with these components,CCK-8,cell plate cloning,Transwell,Western Blot,and RT-PCR were used to detect cell proliferation ability and the expression levels of the REGγ/RNF149/BRAF/MEK/ERK signaling pathway.REGγ overexpression was achieved by transient transfection,and CCK-8 and cell plate cloning experiments were used to detect the cytotoxic effects of berberine,polyphyllin Ⅰ,and the compound on the two osteosarcoma cell lines.Western Blot and RT-PCR were used to detect changes in gene expression levels.C16-PAF,an activator of the MEK/ERK pathway,was used to treat the cells alone or in combination with the compound,and CCK-8 was used to detect cell survival rates.Results:REGγ was highly expressed in osteosarcoma tissues.Knockdown of REGγ significantly inhibited the proliferation and invasion abilities of the two osteosarcoma cell lines.The mRNA level of RNF149 did not change significantly,but the protein level increased significantly.The protein levels of BRAF,p-MEK,and p-ERK decreased significantly.REGγ was involved in regulating the ubiquitin and ATP-independent protein degradation process of RNF149.Berberine and polyphyllin Ⅰ were the main components of the Yi-Qi-San-Yu-Jie-Du formula that exerted anti-cancer effects,and the compound more significantly inhibited the proliferation ability of the two osteosarcoma cell lines than the two effective monomer components,which was consistent with the changes in protein expression levels after REGγ knockdown.Overexpression of REGγ significantly inhibited the cytotoxic effects of berberine,polyphyllin Ⅰ,and the compound on the two osteosarcoma cell lines.Overex-pression of REGγ significantly inhibited the ability of the compound to upregulate RNF149 protein expression and down-regulate BRAF,p-MEK,and p-ERK protein expression levels.C16-PAF significantly inhibited the cytotoxic effects of berberine,polyphyllin Ⅰ,and the compound on the two osteosarcoma cell lines.Conclusion:Yi-Qi-San-Yu-Jie-Du formula participates in the regulation of osteosarcoma occurrence and development through the REGγ/RNF149/BRAF/MEK/ERK signaling pathway,and berberine and polyphyllin Ⅰ are its main active components.