Experimental Study Of Attenuated Mechanism Of Shenfu Injection And Ginsenoside Re

Abstract:Shenfu praeparata, a classical prescription commonly used in clinical sides, is derived from the dried root or rhizome of Ginseng Radix Et Rhizoma Rubra and Aconiti Lateralis Radix. Ginseng can exert pharmacological activity, such as anti tumor, anti-aging and anti-fatigue, improving cardiovascular function and immunity, while aconiti with blood pressure improvement, antishock, antithrombotic, anti hypoxia, anti myocardial ischemia and anti slow arrhythmia. Shenfu injection has therapeutic effect on cardiovascular disease and shock. Ancient physicians confirm its efficacy in clinical practice. However, the rearch on mechanisms of Shenfu injection attenuation and synergy are few. The study take ginseng and aconite as the research object, and investigate the compatibility of the two drugs attenuated mechanism through in vivo and vitro experiments, and supplying theoretic basis for clinical and new medicine in using drugs.In vivo, The arrhythmia model was prepared by intravenous injections of aconitine20μg/kg in rats, ECG changes were observed by BioPAC MP-100. amiodarone group15mg/kg, Renshen injection group0.5g/kg, Fupian injection group0.5g/kg, Shenfu injection group0.75g/kg. In vitro, evaluating whether Renshen injection protects against Fupian injection-induced H9c2cell injury from the level of myocardial cells, H9c2cell were separately treated with Fupian injection (FPI), Renshen injection (RSI) and Shenfu injection (SFI). Cell viability, LDH release, spontaneous beating rate of primative cardical cells, Caspase3/7activity and cell apoptosis was analyzed. Based on PXR-CYP3A (Pregnane X Receptor-Cytochrome P4503A) stable translation cell lines, active components were screened to clarify the basis of drug interaction of Shenfu injection, screening chemical composition of Shenfu injection with induction or inhibition of PXR-CYP3A pathway, and confirm it from the activity level; Finally, it investigate the effect of ginsenosides Re on CYP enzymes on the mRNA and protein level, simultaneously detecte its effect on downstream apoptotic gene mRNA levels.The results showed that1) the heart rate of Shenfu group and amiodarone group was accelerated significantly. While, Q-T interval and P-R interval all appeared significant decline. The heart rate of acontine group was accelerated and then prolonged. Shenfu group, ginseng group and amiodarone group could delay the appearing time of VPB;2) the treatment of SFI (37,75mg/mL) on H9c2cell viability was significantly higher than FPI (25,50mg/mL)(p<0.05, p＜0.001, respectively). The treatment of SFI (75mg/mL) on LDH activity was significantly decreased (p＜0.001) compared with FPI (50mg/mL). SFI (150mg/mL) significantly attenuates FPI (100mg/mL)-induced spontaneous beating rate decrease in primary myocardial cells at4h treatment. Compared with FPI (12,25mg/mL); SFI (18,37mg/mL) treatment could effectively reduce elevated caspase-3/7 activity (p<0.01, p＜0.01, respectively). Apoptotic nuclear characteristics decreased significantly (p<0.05, p＜0.00l, respectively) when the H9c2cells were incubated with SFI (9,37mg/mL) compared with FPI (6,25mg/mL).3) Reporter gene screening study results showed that comparied with DMSO group, the fold induction of ginsenoside Rc, Rf, Rb2, Rg2, F2, F1had a statistical significance (P＜0.05) with treatment concentration exceeded50μmol/L for36、48and60h, while cells were treated with drug (200umol·L-1) for48and60h, the fold induction of ginsenoside, Rb2, Rg2, F1were significantly higher than RIF group (P<0.05). Enzyme activity results showed that ginsenoside Rc, Rf, Rb2, F2, F1could increase the enzyme activity of CYP3A4(P＜0.05) at48h.4) With the increase of concentration and time, the effect of ginsenoside Re on CYP2C11, CYP2J3mRNA levels were induced to a good effect, with a good inhibition on CYP4A1mRNA levels. The effect of ginsenoside Re on CYP4A1protein levels also has a good inhibition, and with a good induction on the downstream apoptotic gene Bcl-2and inhibition on Bax.In summary, this study take Shenfu injection as the research object, preliminarily discuss attenuated mechanisms from the perspective of CYP450enzymes. Compatibility with other prescription could provide new ideas for medicine studies.