The Hepatoprotective Effect of Haoqin Qingdan Decoction against Liver Injury Induced by a Chemotherapeutic Drug Cyclophosphamide

Abstract

Haoqin Qingdan decoction (HQQD), a modern Chinese formula, has been widely used in Eastern Asia. Our study focuses on the hepatoprotective effect of HQQD against cyclophosphamide-induced hepatotoxicity. S180, a kind of ascites tumor cells, was used to establish S180-bearing mice, followed by the injection of cyclophosphamide (CP, 80 mg/kg) every other day for 5 times. HQQD was used intragastrically at the dose of 80 g/kg, 40 g/kg, and 20 g/kg twice a day for 12 days. HL-7702 hepatic cell line was incubated with HQQD-medicated serum. Then we detected the effects of HQQD on (i) tumor suppression; (ii) morphological examination; (iii) SOD, MDA, GSH, ALT, and AST; (iv) cleaved caspase-3 expression and (v) cellular viability. CP caused dramatic elevations of AST, ALT, and MDA, while HQQD notably attenuated these elevations. SOD and GSH were notably increased, which were efficiently attenuated by HQQD. CP injection significantly increased apoptosis by increasing cleaved caspase-3 expression, which was obviously inhibited by HQQD, accompanied by the improvement of cells viability. Histopathological examinations supported the above findings. Therefore, HQQD may protect liver tissue through attenuating oxidative stress and the caspase-3-dependent intrinsic apoptosis induced by CP, which suggests the potentially therapeutic effect of HQQD in the use of alkylating agent for cancer chemotherapy.

Figure 1

HQQD inhibits the tumor growth. (a) The tumor weights treated by HQQD with low (20 g/kg), moderate (40 g/kg), and high dosage (80 g/kg). (b) The inhibition rate (%) produced by HQQD. Tumor inhibition rate calculated as percent compared to control. Data displayed as mean of 10 mice in each group. The experiment was independently repeated for 3 times. ^∗∗∗ p < 0.001 versus control group.

Figure 2

Representative photomicrographs of H&E staining on hepatic morphology show that HQQD reverse liver injury induced by CP from the pathological feature. All of the mice were killed at the end of experiment, and liver tissues were quickly harvested for histopathology analysis. The structure in the mice treated by HQQD was recovered to some extent, with the less necrosis, neutrophils infiltration, and cellular edema.

Figure 3

HQQD effects on oxidative stress parameters: (a) changes in SOD activity in different treated mice; (b) changes in MDA concentration in different treated mice; (c) changes in the GSH activity in different treated mice. Data expressed as mean ± SD for 7 mice in each group. ^∗ p < 0.05 versus control group, ^∗∗∗ p < 0.001 versus control group; ^# p < 0.05 versus the CP group, ^## p < 0.01 versus the CP group, and ^### p < 0.001 versus the CP group.

Figure 4

Suppressive effect of HQQD on elevated serum enzyme activities. HQQD with low and moderate dose significantly inhibited the ALT (a) and AST (b) levels. HQQD with high dose significantly inhibited AST level. Data expressed as mean ± SD for 7 mice in each group. ^∗∗ p < 0.01 versus control group, ^∗∗∗ p < 0.001 versus control group; ^# p < 0.05 versus the CP group, ^## p < 0.01 versus the CP group.

Figure 5

Effect of HQQD on cleaved caspase-3 expression. (a) The effect on cleaved caspase-3 protein formation caused by 80 mg/kg CP was examined at dose of 40 g/kg of HQQD. Data were normalized to the β-actin protein, which was used as an internal control. (b) Bar graphs were shown as means ± SD of three mice per group. Values represented the mean optical density ratio relative to the internal control. ^∗ p < 0.05 versus the control group; ^# p < 0.05 versus the CP group.

Figure 6

(a) Trendline represented the relation between cell viabilities and different dose of CP. (b) Bar graphs showed the changes of hepatic cells viability determined via CCK-8 assay. HQQD of moderate and low dose of medicated serum improved the cells viability. Data expressed as mean ± SD for 6 wells in each group. ^∗∗∗ p < 0.001 versus the control group; ^### p < 0.001 versus the CP group.