Figure 1

The plant of Renshen and Fuzi and the chemical structures of its major extracts Renshen and Fuzi (A) are crude herbal drugs isolated from the dried root of rhizome of Panax ginseng C. A. Mey and Aconitum carmichaeli Debx., respectively. The Shenfu injection (B) manufactured by Ya’an Sanjiu Pharmaceutical Co., Ltd. (http://www.999.com/) has completed a phase II clinical trial in the USA (No. NCT00797953). Both Renshen injection and Fupian injection are the intermediate products of Shenfu injection, which is composed of Renshen injection and Fupian injection with a ratio of 1:2. One millimeter of Shenfu injection contains extracts of 0.1 g Renshen and 0.2 g Fupian. The chemical structures of the major components (ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Rg2) from Renshen and the major Fuzi extracts (benzoylhypaconitine and benzoyldeoxyaconitine) are shown in C and D, respectively."

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Figure 2

The effects of SFI on H9c2 cell viability (Inverted fluorescence microscopy, ×100)The cells were treated with FPI, RSI and SFI at varied concentrations for 24 h, then the media were removed and the cells were incubated for 48 h in complete media before analyzing the media for stain with Zeiss Aviovert 200. A1: RSI 25 mg/mL; A2: RSI 50 mg/mL; A3: RSI 100 mg/mL; B1: FPI 25 mg/mL; B2: FPI 50 mg/mL; B3: FPI 100 mg/mL; C1: SFI 25 mg/mL; C2: SFI 50 mg/mL; C3: SFI 100 mg/mL. SFI: Shenfu injection; RSI: Renshen injection; FPI: Fupian injection."

Figure 3

The effects of SFI on cell viability in H9c2 cellsThe cells were treated with FPI, RSI and SFI at varied concentrations for 24 h, then the media were removed and cells were incubated for 48 h in complete media before analyzing the media cells viability. Data are expressed as mean ± standard deviation of three separate experiments. *P < 0.05, **P < 0.01, vs control; △P < 0.05, △△P < 0.01, vs FPI-treated cells. SFI: Shenfu injection; RSI: Renshen injection; FPI: Fupian injection."

Figure 4

The effects of SFI on LDH release in H9c2 cells The cells were treated with FPI, RSI and SFI at varied concentrations for 24 h, then the media were removed and cells were incubated for 48 h in complete media before analyzing the media for LDH release. Data are expressed as mean ± standard deviation of three separate experiments. *P < 0.05, **P < 0.01, vs control; △△P < 0.01, vs FPI-treated cells.SFI: Shenfu injection; RSI: Renshen injection; FPI: Fupian injection; LDH: lactate dehydrogenase."

Figure 5

The effects of SFI on the spontaneous beating rate in primary myocardial cellsData are expressed as mean ± standard deviation of three separate experiments. *P < 0.05, **P < 0.01, vs control; △P<0.05, vs FPI-treated cells. RSI: Renshen injection; FPI: Fupian injection; SFI: Shenfu injection."

Figure 6

The effects of SFI on level of caspase3/7 activityH9c2 cells were plated at the density of 5×104 /mL in 96-well white plates. Caspase-3/7 activity was measured with Acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-CHO) as the substrate. A total of 100 μL of caspase-3/7 reagent was added to each well and further incubated in the dark for 1 h at room temperature, finally read with a ?uorescence spectrophotometer. Data are expressed as mean ± standard deviation of three separate experiments. *P < 0.05, **P < 0.01, vs control; △△P < 0.01, vs FPI-treated cells.SFI: Shenfu injection; RSI: Renshen injection; FPI: Fupian injection."

Figure 7

Apoptosis analysis of SFI on H9c2 cells by Annexin V-propidium iodide with flow cytometry methodH9c2 cells were pretreated with different concentrations of FPI, RSI and SFI for 24 h. Cells were harvested and apoptosis was confirmed by flow cytometry method (staining with both Annexin V-?uorescein isothiocyanatepropidium iodide) 6 h later. Data are expressed as mean ± standard deviation of three separate experiments. **P < 0.01, vs control; △P < 0.05, △△P <0.01, vs FPI-treated cells.SFI: Shenfu injection; RSI: Renshen injection; FPI: Fupian injection."

Figure 8

The effects of RSI (A), FPI (B), and SFI (C) on CYP2J3 mRNA expression in H9c2 cellsNormalization relative to GAPDH was performed. Levels of mRNA are expressed relative to control cardiomyoblast in the mean ± standard deviation values derived from three independent experiments. *P < 0.05, **P < 0.01, vs control.SFI: Shenfu injection; RSI: Renshen injection; FPI: Fupian injection; GAPDH: glyceraldehydes-3-phosphate dehydrogenase."