Effects of matrine on HepG2 cell proliferation and expression of tumor relevant proteins in vitro

Figures & data

Table 1.  L₁₈ (2¹×3⁷) scheme of the combinatorial orthogonal test.

Groups	Medium	Celldensity	Serumcontent	Antibiotics concentration	pH	Washing times	Trypsin concentration	Digestion times	Score
1	DMEM	8 × 10^4/mL	10%	50 IU	7	1 min	0.25%	1 min	6.3
2	DMEM	8 × 10^4/mL	15%	80 IU	7.2	2 min	0.15%	2 min	7
3	DMEM	8 × 10^4/mL	20%	20 IU	7.4	3 min	0.05% + EDTA	3 min	5
4	DMEM	4 × 10^4/mL	10%	20 IU	7	2 min	0.15%	3 min	8.3
5	DMEM	4 × 10^4/mL	15%	50 IU	7.2	3 min	0.05% + EDTA	1 min	9.7
6	DMEM	4 × 10^4/mL	20%	80 IU	7.4	1 min	0.25%	2 min	7.3
7	DMEM	1 × 10^4/mL	10%	20 IU	7.2	1 min	0.05% + EDTA	3 min	5.7
8	DMEM	1 × 10^4/mL	15%	50 IU	7.4	2 min	0.25%	1 min	4.7
9	DMEM	1 × 10^4/mL	20%	80 IU	7	3 min	0.15%	2 min	6.
10	RPMI-1640	8 × 10^4/mL	10%	50 IU	7.4	3 min	0.15%	1 min	6.3
11	RPMI-1640	8 × 10^4/mL	15%	80 IU	7	1 min	0.05% + EDTA	2 min	6
12	RPMI-1640	8 × 10^4/mL	20%	20 IU	7.2	2 min	0.25%	3 min	5.3
13	RPMI-1640	4 × 10^4/mL	10%	80 IU	7.2	3 min	0.25%	2 min	3.3
14	RPMI-1640	4 × 10^4/mL	15%	20 IU	7.4	1 min	0.15%	3 min	5
15	RPMI-1640	4 × 10^4/mL	20%	50 IU	7	2 min	0.05% + EDTA	1 min	7.3
16	RPMI-1640	1 × 10^4/mL	10%	80 IU	7.4	2 min	0.05% + EDTA	2 min	5
17	RPMI-1640	1 × 10^4/mL	15%	20 IU	7	3 min	0.25%	3 min	4.3
18	RPMI-1640	1 × 10^4/mL	20%	50 IU	7.2	1 min	0.15%	1 min	5.7

To determine the optimal cultivation condition of HepG2 cells, eight impact factors shown above were selected according to the combinatorial orthogonal test design (L₁₈ (2¹×3⁷)). Cell auxodrome and the divisional index of the HepG2 cells were obtained, and the average stage of HepG2 growth was scored from 0 to 10 corresponding to every parallel treatment. The results indicate that Group 5 reached the highest score 9.7 and was determined as the optimal cultivation condition.

Table 2.  Groups and concentration of matrine.

Groups	Treatment	Groups	Treatment
Control	10 mM PBS	Control	10 mM PBS
1	0.3 mg/mL matrine	1	0.1 mg/mL matrine
2	0.5 mg/mL matrine	2	0.15 mg/mL matrine
3	0.8 mg/mL matrine	3	0.2 mg/mL matrine
4	1 mg/mL matrine	4	0.25 mg/mL matrine
5	1.5 mg/mL matrine	5	0.3 mg/mL matrine
6	2 mg/mL matrine	6	0.35 mg/mL matrine
7	2.5 mg/mL matrine	7	0.4 mg/mL matrine
8	3 mg/mL matrine	8	0.45 mg/mL matrine
9	3.5 mg/mL matrine	9	0.5 mg/mL matrine

Figure 1.  Matrine changed the morphology of HepG2 and induced part cells to death. HepG2 cell incubated with different concentration of matrine for 4 days were harvested for H&E staining (×400magnification) to view cell morphology under multifunctional microscope. Untreated cells served as control (A), and treated cells were shown in 0.2 mg/mL (B); 1.5 mg/mL (C); 3.0 mg/mL (D) and the green arrow indicated the plasmatorrhexis.

Figure 2.  Matrine inhibited the proliferation of HepG2 cells in vitro compared with the control. HepG2 cells were incubated in presence of matrine at different concentrations (from 0.1 to 0.5 mg/mL, and from0.3 to 3.5 mg/mL) for 4 days, and cell proliferation was determined by MTT assay to calculate the proliferation inhibition rate (%). Negative-inhibition effects were found at the concentration of matrine below 0.2 mg/mL as shown in left section, whereas positive dose-dependent inhibition of HepG2 cell growth by matrine as described in right section.

Figure 3.  Matrine down-regulated the expression of AFP and PCNA proteins. HepG2 cells incubated with absence (E, G) or presence of 0.8 mg/mL matrine for 2 days were determined with immunohistochemistry analysis. HepG2 cells were viewed under multifunctional microscope (×400magnification). Immunostaining intensity of AFP (F) and PCNA (H) were weaker than control. The decrease demonstrated the down-regulation of protein expression.

Figure 4.  Matrine down-regulated the expression of C-myc, Bcl-2, and up-regulated the expression of Bax. HepG2 cells cultured in DMEM (upper row, I, J, K) or DMEM containing 0.8 mg/mL matrine (lower row, L, M, N) for 2 days were detected with immunostaining. HepG2 cells were viewed under multifunctional microscope (×400magnification). Expression of C-myc (first column) and Bcl-2 (second column) were seen in the cell nucleolus and cytoplasm of matrine-treated cells and the staining intensity was weaker than that of untreated cells. Bax (third column) expressed stronger than untreated cells. The mean grade of staining intensity was higher compared with control cells.