Herbal Compound "Jiedu Huayu" Reduces Liver Injury in Rats via Regulation of IL-2, TLR4, and PCNA Expression Levels

Abstract

Aim of the Study. To investigate the preventative effects of Jiedu Huayu (JDHY) on D-galactosamine (D-GalN) and lipopolysaccharide-induced acute liver failure (ALF) and to evaluate the possible mechanisms of action. Materials and Methods. ALF was induced in Wistar rats by administrating D-GalN (900 mg/kg) and lipopolysaccharide (10 μg/kg). After treatment with JDHY granules, the levels of blood alanine aminotransferase, aspartate aminotransferase, total bilirubin, and prothrombin time were determined. Proliferating cell nuclear antigen was detected by immunohistochemistry staining. The expression of interleukin-2 (IL-2) and toll-like receptor 4 (TLR4) was examined by fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. Results. JDHY treatment dramatically improved liver function and increased survival rates in an ALF model in rats. We observed a decrease in IL-2 and TLR4 expression following treatment with JDHY in liver cells from ALF rats using qRT-PCR and Western blot analysis. Conclusion. We hypothesize that the therapeutic potential of JDHY for treating ALF is due to its modulatory effect on the suppression of inflammation and by promoting hepatocyte regeneration. Our results contribute towards validation of the traditional use of JDHY in the treatment of liver disease.

Figure 1

Paeoniflorin was used as a quality control and analyzed by HPLC (B1–B3).

Figure 2

Serum levels of ALT, AST, TBIL, and PT after treatment. (a) ALT; (b) AST; (c) TBIL; (d) PT. Control: negative control, rats were given with distilled water via gavage; model: rats injected with LPS + D-GalN; low dose: rats injected with LPS + D-GalN and treated with a low dose of JDHY; high dose: rats injected with LPS + D-GalN and treated with a high dose of JDHY. For control, model, low dose, and high dose groups, the ALT levels were 37.45 ± 7.01 U/L, 571.47 ± 95.18 U/L, 383.46 ± 82.41 U/L, and 182.64 ± 71.34 U/L, respectively. AST levels were 88.93 ± 10.23 U/L, 1097.43 ± 124.66 U/L, 673.16 ± 102.13, and 569.32 ± 100.57, respectively. TBIL levels were 2.36 ± 0.78 μmol/L, 47.11 ± 5.74 μmol/L, 37.55 ± 4.23 μmol/L, and 28.36 ± 3.85 μmol/L, respectively. PT was 11.53 ± 1.24 s, 33.78 ± 2.41 s, 25.36 ± 2.04 s, and 19.32 ± 1.74 s, respectively. For (a), (b), (c) and (d), ^#P < 0.05 compared to the control group, ^▲P < 0.05 compared to the model group, and ^◆P < 0.05 compared to the low dose group.

Figure 3

Survival studies. Control: negative control given distilled water via gavage; model: rats injected with LPS + D-GalN; low dose: rats injected with LPS + D-GalN and treated with a low dose of JDHY; high dose: rats injected with LPS + D-GalN and treated with a high dose of JDHY. ^#P < 0.05 compared to the model group.

Figure 4

PCNA immunohistochemistry. (a) PCNA immunohistochemistry; (b) PCNA positive rates per group. Control: negative control given distilled water via gavage; model: rats injected with LPS + D-GalN; low dose: rats injected with LPS + D-GalN and treated with a low dose of JDHY; high dose: rats injected with LPS + D-GalN and treated with a high dose of JDHY. The PCNA positive (light density of positive cumulative, IOD) rates were 3394.51 ± 614.63, 502.81 ± 102.67, 1576.84 ± 288.27, and 2172.43 ± 363.60 in control, model, low dose, and high dose groups, respectively. ^#P < 0.05 compared to the control group, ^▲P < 0.05 compared to the model group, and ^◆P < 0.05 compared to the low dose group.

Figure 5

mRNA and protein expression levels of IL-2. (a) IL-2 mRNA expression, (b) IL-2 protein expression, (c) amplification curves, and (d) Western blot analysis. Control: negative control given distilled water via gavage; model: rats injected with LPS + D-GalN; low dose: rats injected with LPS + D-GalN and treated with a low dose of JDHY; high dose: rats injected with LPS + D-GalN and treated with a high dose of JDHY. For control, model, low dose, and high dose groups, the IL-2 mRNA expression levels were 1.83 ± 0.24, 23.12 ± 3.07, 15.33 ± 2.21, and 10.25 ± 2.06 and the protein expression levels were 0.21 ± 0.08, 0.76 ± 0.13, 0.52 ± 0.09, and 0.25 ± 0.05, respectively. ^#P < 0.05 compared to control group, ^▲P < 0.05 compared to model group, and ^◆P < 0.05 compared to the low dose group.

Figure 6

mRNA and protein expression levels of TLR4. (a) TLR4 mRNA expression levels, (b) TLR4 protein expression levels, (c) amplification curves, and (d) Western blot analysis. Control: negative control given distilled water via gavage; model: rats injected with LPS + D-GalN; low dose: rats injected with LPS + D-GalN and treated with a low dose of JDHY; high dose: rats injected with LPS + D-GalN and treated with a high dose of JDHY. For control, model, low dose, and high dose groups, the TLR4 mRNA expression levels were 1.21 ± 0.02, 10.02 ± 2.23, 8.51 ± 1.32, and 6.22 ± 1.02. Protein expression levels were 0.19 ± 0.04, 0.55 ± 0.11, 0.47 ± 0.07, and 0.27 ± 0.06, respectively. ^#P < 0.05 compared to control group, ^▲P < 0.05 compared to model group, and ^◆P < 0.05 compared to the low dose group.