RESUMO
Salvianolic acid B (SB) is a natural compound with protective effect against ischemia-reperfusion heart injury. However, the signal network of SB including both direct target proteins and downstream signal-related proteins has not been clarified. In the present study, epidermal growth factor receptor (EGFR) was predicted to be the most possible direct protein target of SB by INVDOCK, a ligand-protein inverse-docking algorithm. Possible signal-related proteins of SB in H9C2 cells, including both under normal condition and under ischemia-reperfusion injury, were searched using 2-DE analysis. Totally, 14 signal-related proteins were found. Finally, signal network from EGFR to the signal-related proteins was established using bioinformatic analysis. Interestingly, 9 of the 14 signal-related proteins could be included in a network together with EGFR through direct interaction or only one intermediate partner. The signal cascade from EGFR to heat shock protein 27 (HSP27) and mitofilin (IMMT, inner membrane mitochondrial protein) might be the most important cascade. The signal network was certified by measuring the binding affinity of SB to EGFR in vitro, the effect of SB on internalization and phosphorylation of EGFR, the effect of SB on viability and proliferation of H9C2 cells, and the expression of inner membrane mitochondrial protein in the presence of EGFR inhibitor AG 1478.
Assuntos
Benzofuranos/análise , Transdução de Sinais , Animais , Benzofuranos/metabolismo , Western Blotting , Linhagem Celular , Biologia Computacional , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas Mitocondriais/análise , Proteínas Mitocondriais/metabolismo , Proteínas Musculares/análise , Proteínas Musculares/metabolismo , Ligação Proteica , Proteômica , Ratos , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
This paper is aimed to report the development of a method for the determination of the binding rate of plasma protein with salvianolic acid B. In vitro, equilibrium dialysis method was used to imitate the binding process between salvianolic acid B and plasma protein, in vivo, ultrafiltration method was used and the binding rate with HPLC was determined. Plasma samples were treated with methanol to precipitate the protein, and the buffer solution was directly determined after filtering. The calibration curve of the buffer solution was linear in the range of 0.5-20 microg mL(-1). The calibration curve of the plasma was linear in the range of 2-200 microg mL(-1). The extract recovery was 68.6%-81.9%. RSDs of intra- and inter-day precisions were all less than 8.5%. The binding rates of plasma protein with salvianolic acid B in vitro was 75.2% and in vivo was 92.1%. This paper shows the high binding power of salvianolic acid B to plasma protein with high sensitivity, good reproduction, simple management and fulfilling the requirement.