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Journal of Chinese Integrative Medicine ›› 2013, Vol. 11 ›› Issue (5): 327-336.doi: 10.3736/jintegrmed2013047

• Research Article • Previous Articles     Next Articles

Cardioprotection of Shenfu preparata on cardiac myocytes through cytochrome P450 2J3

Yong Xiaoa,b, Zeng-chun Maa, Yu-guang Wanga, Hong-ling Tana, Xiang-ling Tanga, Qian-de Lianga, Cheng-rong XiaoaYue Gaoa,b()   

  1. a. Beijing Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China
    b. School of Pharmaceutical Sciences, Central South University, Changsha 410013, Hunan Province, China
  • Received:2013-05-24 Accepted:2013-07-18 Online:2013-09-10 Published:2013-09-15


To evaluate whether Shenfu injection (SFI) protects against cardiac myocyte injury induced by Fupian injection (FPI) in vitro.


H9c2 cells were separately treated with FPI, Renshen injection (RSI) and SFI. Cell viability, lactate dehydrogenase (LDH) release, spontaneous beating rate of primative cardical cells, caspase-3/7 activity, cell apoptosis, and cytochrome P450 2J3 (CYP2J3) mRNA expression were analyzed.


The viability of H9c2 cells treated with SFI (37 and 75 mg/mL) was significantly higher than that of H9c2 cells treated with FPI (25 and 50 mg/mL) (P<0.05, P<0.01, respectively). LDH activity of H9c2 cells treated with SFI (75 mg/mL) was significantly decreased (P<0.01) compared with that of H9c2 cells treated with FPI (50 mg/mL). SFI (150 mg/mL) significantly attenuated FPI (100 mg/mL)-induced spontaneous beating rate decrease in primary myocardial cells after 4-hour treatment. Compared with FPI (12 and 25 mg/mL), SFI (18 and 37 mg/mL) treatment could effectively reverse the change of caspase-3/7 activity (P<0.01 and P<0.01, respectively). Compared with FPI (6 and 25 mg/mL), apoptotic cells decreased signi?cantly (P<0.05, P<0.01, respectively) when H9c2 cells were incubated with SFI (9 and 37 mg/mL). The expression of CYP2J3 mRNA was down-regulated by FPI, while RSI and SFI could up-regulate the expression of CYP2J3 (P<0.01), which suggested the potential mechanism of protection of RSI against cardiac myocyte damage induced by FPI treatment.


These observations indicate that SFI has the potential to exert cardioprotective effects against FPI toxicity. The effect was possibly correlated with the activation of CYP2J3.

Key words: Shenfu decoction, Cardiotonic agents, Cytochrome P450 2J3, Plant extracts, In vitro

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. ( 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."


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."

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