Herbs were purchased from Tongchun Pharmaceutical Co., Ltd (Fuzhou, Fujian, China). Their quality met the criteria described in the Pharmacopoeia of the People’s Republic of China. To prepare water extract, Astragalus membranaceus (60 g), Ligustrum lucidum (60 g), Ganoderma lucidum (30 g), Rhizoma dioscorea (30 g), Prunellae vulgaris (30 g) and Hedyotis diffusa Willd (60 g) were pulverized into extremely fine powders with a mortar and pestle and immersed in distilled water, respectively. The mixture was simmered for 2 h and filtered. The solution was concentrated so that it contained 2.66 g of dry herbs per 1 ml and was stored at 4°C until use.

Human HepG2 hepatoma cells were purchased from the Shanghai Institute of Life Science, Chinese Academy of Sciences (Shanghai, China). Cells were maintained in RPMI-1640 culture medium (Gibco-BRL, Carlsbad, CA, USA) with 100 ml/l of fetal calf serum, 1×10⁵ U/l of penicillin and 100 mg/l of streptomycin (Gibco-BRL) in an incubator (Thermo Fisher Scientific, Rockford, IL, USA) at 37°C with 5% CO₂. Cell morphology was observed using an inverted microscope (Olympus, Tokyo, Japan).

Cells were cultured in the absence or presence of water extract of FZQJ at the final concentrations of 0.5, 1, 1.5 and 2 mg/ml for 24, 48 and 72 h respectively. Then, 20 μl of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Invitrogen Life Technologies, Carlsbad, CA, USA) was added to each well and incubated for another 4 h prior to discarding the medium. The purple-blue formazan precipitate was dissolved in 100 μl of dimethyl sulfoxide (DMSO). The absorbance (OD) was measured at 490 nm with a microplate reader (BioTek Instruments Inc., Winooski, VT, USA). The cell viability ratio was calculated according to the following formula: Cell viability ratio (%) = average OD_{treatment group}/average OD_{vehicle group} × 100%.

In brief, following treatment with different concentrations of water extract of FZQJ for 24 h, the cells were fixed with 4% paraformaldehyde and then incubated with 1 μg/ml of 4,6-diamidino-2-phenylindole (DAPI) staining solution (Beyotime Inc., Shanghai, China) at room temperature for 5 min and washed with PBS 3 times. The morphology of nuclei in HepG2 cells was observed under a fluorescence microscope (Olympus) at an excitation wavelength of 350 nm.

Cells were incubated in 6-well plates for 24 h in the absence or presence of different concentrations of FZQJ water extract. Then cells were digested by trypsinase and washed twice with cold PBS. A final concentration of 1×10⁶ cells/ml of single-cell suspension was prepared. Apoptosis and mitochondrial membrane potential (Δψ) were measured by a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) with Annexin-V fluorescein isothiocyanate (FITC) or 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1; BD Biosciences). Cells (10,000) were acquired and analyzed using CellQuest software (BD Biosciences). Each determination was performed as three parallel assays.

Total cellular RNA was isolated using the TRIzol one-step method according to the manufacturer’s instructions (Invitrogen Life Technologies). Single-stranded cDNA was synthesized using oligo (dT) primer (Takara, Dalian, China) in a 20 μl reaction mixture. Bcl-2 and Bax mRNA were evaluated using PCR. The primer pairs of Bcl-2, Bax and GAPDH were as follows: Bcl-2, forward 5′-CAG CTG CAC CTG ACG CCC TT-3′ and reverse 5′-GCC TCC GTT ATC CTG GAT CC-3′; Bax, forward 5′-TGC TTC AGG GTT TCA TCC AGG-3′ and reverse 5′-TGG CAA AGT AGA AAA GGG CGA-3′; GAPDH, forward 5′-AGA AGG CTG GGG CTC ATT TG-3′ and reverse 5′-AGG GGC CAT CCA CAG TCT TC-3′. DNA amplification was performed for 40 cycles following an initial denaturation step at 94°C for 5 min in a thermo cycler by using the following program: denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 30 sec and a final extension at 72°C for 10 min. PCR reagent kit was obtained from Takara. Finally, the amplified products were separated on a 1.2% agarose gel and examined using a Gel Doc 2000 Imaging System (Bio-Rad, Hercules, CA, USA).

HepG2 cells were collected and lysed in lysis buffer (Beyotime Inc.) for 10 min following treatment with different concentrations of FZQJ water extract for 24 h. Following centrifugation at 12,000 × g at 4°C for 20 min, the supernatant was collected and the protein concentration determined using the Bradford assay. Equal amounts of denatured protein were separated on SDS-PAGE gels and transferred onto PVDF membranes. These membranes were then put into blocking solution for 1 h and incubated in solution with either monoclonal anti-human Bax or Bcl-2 primary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4°C with agitation and then in horseradish peroxidase (HRP)-conjugated secondary antibody (Beyotime Inc.) for at least 1 h. Protein was detected with ECL solution (Beyotime Inc.) using a chemiluminescence imaging system (Bio-Rad).

The activities of caspase-3 and -9 were examined using caspase-3 and -9 colorimetric assay kits (KeyGen Biotech, Nanjing, Jiangsu, China). Briefly, HepG2 cells were collected and lysed on ice with 100 μl of lysis buffer (containing 1% dithiothreitol) following treatment with different concentrations of FZQJ water extract for 24 h. Following centrifugation at 11,000 × g for 1 min, the protein concentration of the supernatant was determined using the Bradford assay. Equal amounts of protein were incubated with specific substrates of caspase-3 or -9 at 37°C in the dark for 4 h. Finally the samples were determined at 405 nm by a microplate reader (BioTek Instruments Inc.). The activities of caspase -3 or -9 were calculated according to the formula: average OD_treatment/average OD_vehicle.

Phosphorylated P38 MAPK (p-P38 MAPK) was evaluated using flow cytometry. The cells were digested and rinsed with PBS following treatment with different concentrations of FZQJ water extract for 48 h. Then cells were suspended, fixed with fixation buffer for 10 min at 37°C, permeabilized on ice for 30 min and stained with Alexa Fluor^® 647 mouse anti-p-P38 MAPK antibody (BD Biosciences). The cells were analyzed with the flow cytometer.

Data were analyzed using the SPSS 16.0 statistical package. All results are expressed as the mean ± standard deviation (SD) of at least three experiments. The data for multiple comparisons were performed by one-way ANOVA. P<0.05 was considered to indicate a statistically significant difference.

FZQJ made according to the patent (13). The growth of HepG2 cells was evaluated using an MTT assay. As shown in Fig. 1, in the presence of indicated concentrations of water extracts, cell proliferation was inhibited in a dose- and time-dependent manner. The inhibitory rate of HepG2 cells was able to reach as high as 92.57% at the highest concentration for 72 h. The results demonstrated that water extract of FZQJ markedly inhibited the proliferation of HepG2 cells.

We first evaluated apoptosis of HepG2 cells treated with indicated doses of FZQJ for 24 h through observing the morphological changes using an inverted fluorescence microscope. As shown in Fig. 2A, a decreased cell number was associated with morphological changes of cells following the addition of FZQJ to the medium; polygon- or spindle-shaped cells became round, shrunk and collapsed. Following staining of the nuclei with DAPI (a fluorescent DNA-binding agent), more brightened nuclei were observed in the FZQJ-treated cells, with chromatin pyknosis and fragmentation (Fig. 2B). Clearly, FZQJ water extract induced marked apoptotic morphological alterations.

Next, cells were evaluated flow cytometrically by Annexin-V staining. Annexin-V is a cellular protein used as a probe to detect cells that have expressed phosphatidylserine on the cell surface (an event identified in apoptosis). Therefore, Annexin-V positive cells are considered to be apoptotic cells. As displayed in Fig. 2C, the percentage of apoptotic cells stained with Annexin V markedly increased in FZQJ-treated cells in a dose-dependent manner. Taken together, these data revealed that FZQJ extract induced apoptosis of HepG2 cells.

Apoptosis is often accompanied by a decrease of Δψ. Loss of Δψ is important in the early apoptotic process (14). JC-1 is a lipophilic fluorochrome that is found to be sensitive to Δψ and is used as an indicator of Δψ during apoptosis. JC-1 has two different formations, aggregates and monomers. In normal cells with a polarized Δψ, JC-1 aggregates stay in the mitochondria and emit red fluorescence (red channel) and monomers stay in the cytoplasm and exhibit green fluorescence (green channel). Therefore, JC-1-treated normal cells demonstrate green and red channels on flow cytometers. When the mitochondria Δψ depolarizes, JC-1 does not form aggregates in the mitochondria but remains as monomers in the cytoplasm, resulting in increased numbers of cells with reduced JC-1 fluorescence in the red channel. As shown in Fig. 3, JC-1 fluorescence was observed in red and green channels (R2 region) in the vehicle-treated cells, indicating that the majority of cells were alive. In the presence of increasing concentrations of FZQJ, there was a significant increase in the number of cells with lowered red fluorescence (R3 region), indicative of a depolarized Δψ. Thus, the data indicate that FZQJ-induced apoptosis was associated with the depolarization of Δψ.

The activities of caspase-3 and -9 are closely associated with the mitochondria-dependent apoptosis pathway. We next examined whether caspase-3 and -9 were involved in FZQJ-induced depolarization of Δψ in HepG2 cells. The activities of caspase-3 and -9 were examined by colorimetric assay. The results demonstrated that the activities of caspase-3 and -9 were markedly increased in a dose-dependent manner. Following treatment with 2 mg/ml of FZQJ extract for 24 h, 2.77-fold and 2.99-fold increases were observed for caspase-3 and -9 activity compared with the vehicle control, respectively (Fig. 4). The data suggested that water extract of FZQJ could decease Δψ in HepG2 cells via the activation of caspase-3 and -9.

Antiapoptotic Bcl-2 and proapoptotic Bax are members of the Bcl-2 family and are critical in apoptosis controlled by mitochondria. To further investigate how FZQJ induces mitochondria-dependent apoptosis, the expression of Bcl-2 and Bax were evaluated in mRNA and protein levels by RT-PCR and western blot analysis, respectively. As shown in Fig. 5A, compared with the vehicle control, FZQJ decreased Bcl-2 mRNA and increased Bax mRNA expression in a dose-dependent manner. Similar results were observed in western blot analyses (Fig. 5B), indicating that FZQJ induced depolarization of Δψ in HepG2 cells through regulating the expression of members of the Bcl-2 family.

Activation of P38 MAPK has been demonstrated to lead to cell death via stimulating mitochondrial Bax translocation and activating caspase -9 and -3 (15). To investigate whether FZQJ induces mitochondrial apoptosis via activation of P38 MAPK, phosphorylation of P38 MAPK was evaluated. As shown in Fig. 6, FZQJ treatment caused a dose-dependent increase of p-P38 MAPK level in HepG2 cells, indicating that FZQJ could activate P38 MAPK.