BRP, a polysaccharide fraction isolated from Boschniakia rossica, protects against galactosamine and lipopolysaccharide induced hepatic failure in mice

Abstract

The aim of this study was to investigate the hepatoprotective effect of BRP, a polysaccharide fraction isolated from Boschniakia rossica, against galactosamine and lipopolysaccharide induced fulminant hepatic failure. Mice were injected with a single dose of galactosamine/lipopolysaccharide with or without pretreatment of BRP. Results showed marked reduction of hepatic necrosis, serum marker enzymes and levels of tumor necrosis factor-α and interleukin-6 in BRP pretreated mice when compared with galactosamine/lipopolysaccharide-challenged mice. Mice pretreated with BRP decreased the activation of caspases-3 and caspase-8, and showed a reduced level of DNA fragmentation of liver cells. BRP also reduced hepatic lipid peroxidation, increased potential of hepatic antioxidative defense system, and reduced hepatic nitric oxide level which was elevated by galactosamine/lipopolysaccharide injection. Immunoblot analysis showed down-regulation of inducible nitric oxide synthase and cyclooxygenase-2 proteins of liver tissues in BRP pretreated group when compared with galactosamine/lipopolysaccharide-challenged group. Furthermore, treatment with galactosamine/lipopolysaccharide markedly increased toll-like receptor 4, nuclear level of nuclear factor-κB, and phosphorylation of both extracellular signal-regulated kinase and c-Jun N-terminal kinase in liver tissues. However, these increases were attenuated by pretreatment with BRP. The results suggest that BRP alleviates galactosamine/lipopolysaccharide-induced liver injury by enhancing antioxidative defense system, suppressing inflammatory responses and reducing apoptotic signaling.

Keywords: Boschniakia rossica; hepatic failure; mice; polysaccharide.

Fig. 1

BRP reduced serum ALT and AST of mice with GalN/LPS-induced liver injury. Mice were orally administered with BRP, silymarin or a vehicle for 3 days prior to administration with GalN/LPS. Blood samples were collected at 8 h after GalN/LPS exposure. Data are presented as mean ± SD, n = 12. ^#p<0.05 as compared with the control group; *p<0.05 as compared with the model group; ^†p<0.05, significantly different from 120 mg/kg BRP group. Control: normal control; Model: GalN/LPS alone; BRP: GalN/LPS plus BRP (120 and 240 mg/kg); SIL: GalN/LPS plus silymarin (50 mg/kg).

Fig. 2

BRP improved liver histological alterations in GalN/LPS-challenged mice. Liver sections were stained with hematoxylin-eosin for morphological evaluation (200×). Mice were orally administered with BRP, silymarin or a vehicle for 3 days prior to administration with GalN/LPS. (A) Control; (B) Model; (C) BRP 120 mg/kg; (D) BRP 240 mg/kg; (E) Silymarin 50 mg/kg.

Fig. 3

BRP reduced serum TNF-α and IL-6 contents in GalN/LPS-challenged mice. Mice were orally administered with BRP, silymarin or a vehicle for 3 days prior to administration with GalN/LPS. Blood samples were collected at 2 h after GalN/LPS exposure for measurement of circulating serum TNF-α (A) and IL-6 (B). Data are presented as mean ± SD, n = 12. ^#p<0.05 as compared with the control group; *p<0.05 as compared with the model group; ^†p<0.05, significantly different from 120 mg/kg BRP group. Control: normal control; Model: GalN/LPS alone; BRP: GalN/LPS plus BRP (120 and 240 mg/kg); SIL: GalN/LPS plus silymarin (50 mg/kg).

Fig. 4

BRP suppressed hepatic DNA fragmentation in GalN/LPS-challenged mice. Mice were orally administered with 240 mg/kg BRP, 50 mg/kg silymarin or a vehicle for 3 days prior to administration with GalN/LPS. The animals were sacrificed 8 h after GalN/LPS exposure. Lane M: marker; Lane 1: control; Lane 2: model; Lane 3: BRP 240 mg/kg; Lane 4: silymarin 50 mg/kg.

Fig. 5

BRP suppressed hepatic caspase-3 and caspase-8 cleavages in GalN/LPS-challenged mice. Mice were orally administered with 240 mg/kg BRP, 50 mg/kg silymarin or a vehicle for 3 days prior to administration with GalN/LPS. Liver samples were collected at 8 h after GalN/LPS exposure for measurement of cleaved caspase-3 (A) and caspase-8 (B). GAPDH was used as an internal control and the relative level of protein expression was normalized to the control. Data are presented as mean ± SD, n = 6. ^#p<0.05 as compared with the control group; *p<0.05 as compared with the model group. Control: normal control; Model: GalN/LPS alone; BRP 240: GalN/LPS plus BRP (240 mg/kg); SIL: GalN/LPS plus silymarin (50 mg/kg).

Fig. 6

BRP reduced hepatic caspase-3 and caspase-8 activities in GalN/LPS-challenged mice. Mice were orally administered with BRP, silymarin or a vehicle for 3 days prior to administration with GalN/LPS. Liver samples were collected at 8 h after GalN/LPS exposure. Data are presented as mean ± SD, n = 12. ^#p<0.05 as compared with the control group; *p<0.05 as compared with the model group; ^†p<0.05, significantly different from 120 mg/kg BRP group. Control: normal control; Model: GalN/LPS alone; BRP: GalN/LPS plus BRP (120 and 240 mg/kg); SIL: GalN/LPS plus silymarin (50 mg/kg).

Fig. 7

BRP reduced hepatic LOOH and TBARS contents in GalN/LPS-challenged mice. Mice were orally administered with BRP, silymarin or a vehicle for 3 days prior to administration with GalN/LPS. Liver samples were collected at 8 h after GalN/LPS exposure. Data are presented as mean ± SD, n = 12. ^#p<0.05 as compared with the control group; *p<0.05 as compared with the model group. Control: normal control; Model: GalN/LPS alone; BRP: GalN/LPS plus BRP (120 and 240 mg/kg); SIL: GalN/LPS plus silymarin (50 mg/kg).

Fig. 8

BRP up-regulated hepatic HO-1 protein expression in GalN/LPS-challenged mice. Mice were orally administered with 240 mg/kg BRP, 50 mg/kg silymarin or a vehicle for 3 days prior to administration with GalN/LPS. Liver samples were collected at 8 h after GalN/LPS exposure. GAPDH was used as an internal control and the relative level of protein expression was normalized to the control. Data are presented as mean ± SD, n = 6. ^#p<0.05 as compared with the control group; *p<0.05 as compared with the model group; ^†p<0.05, significantly different from 240 mg/kg BRP group. Control: normal control; Model: GalN/LPS alone; BRP 240: GalN/LPS plus BRP (240 mg/kg); SIL: GalN/LPS plus silymarin (50 mg/kg).

Fig. 9

BRP reduced hepatic NO content in GalN/LPS-challenged mice. Mice were orally administered with BRP, silymarin or a vehicle for 3 days prior to administration with GalN/LPS. Liver samples were collected at 8 h after GalN/LPS exposure. Data are presented as mean ± SD, n = 12. ^#p<0.05 as compared with the control group; *p<0.05 as compared with the model group. Control: normal control; Model: GalN/LPS alone; BRP: GalN/LPS plus BRP (120 and 240 mg/kg); SIL: GalN/LPS plus silymarin (50 mg/kg).

Fig. 10

BRP down-regulated hepatic iNOS and COX-2 protein expression in GalN/LPS-challenged mice. Mice were orally administered with 240 mg/kg BRP, 50 mg/kg silymarin or a vehicle for 3 days prior to administration with GalN/LPS. Liver samples were collected at 8 h after GalN/LPS exposure for measurement of iNOS (A) and COX-2 (B). GAPDH was used as an internal control and the relative level of protein expression was normalized to the control. Data are presented as mean ± SD, n = 6. ^#p<0.05 as compared with the control group; *p<0.05 as compared with the model group; ^†p<0.05, significantly different from 240 mg/kg BRP group. Control: normal control; Model: GalN/LPS alone; BRP 240: GalN/LPS plus BRP (240 mg/kg); SIL: GalN/LPS plus silymarin (50 mg/kg).

Fig. 11

BRP down-regulated nuclear NF-κB p65 protein expression in livers of GalN/LPS-challenged mice. Mice were orally administered with 240 mg/kg BRP, 50 mg/kg silymarin or a vehicle for 3 days prior to administration with GalN/LPS. Liver samples were collected at 2 h after GalN/LPS exposure. Lamin B1 was used as an internal control and the relative level of protein expression was normalized to the control. Data are presented as mean ± SD, n = 6. ^#p<0.05 as compared with the control group; *p<0.05 as compared with the model group. Control: normal control; Model: GalN/LPS alone; BRP 240: GalN/LPS plus BRP (240 mg/kg); SIL: GalN/LPS plus silymarin (50 mg/kg).

Fig. 12

BRP suppressed hepatic JNK and ERK activation in GalN/LPS-challenged mice. Mice were orally administered with 240 mg/kg BRP, 50 mg/kg silymarin or a vehicle for 3 days prior to administration with GalN/LPS. Liver samples were collected at 2 h after GalN/LPS exposure for measurement of JNK (A) and ERK (B). GAPDH was used as an internal control and the relative level of protein expression was normalized to the control. Data are presented as mean ± SD, n = 6. ^#p<0.05 as compared with the control group; *p<0.05 as compared with the model group; ^†p<0.05, significantly different from 240 mg/kg BRP group. Control: normal control; Model: GalN/LPS alone; BRP 240: GalN/LPS plus BRP (240 mg/kg); SIL: GalN/LPS plus silymarin (50 mg/kg).

Fig. 13

BRP down-regulated TLR4 protein expression in livers of GalN/LPS-challenged mice. Mice were orally administered with 240 mg/kg BRP, 50 mg/kg silymarin or a vehicle for 3 days prior to administration with GalN/LPS. Liver samples were collected at 2 h after GalN/LPS exposure. GAPDH was used as an internal control and the relative level of protein expression was normalized to the control. Data are presented as mean ± SD, n = 6. ^#p<0.05 as compared with the control group; *p<0.05 as compared with the model group; ^†p<0.05, significantly different from 240 mg/kg BRP group. Control: normal control; Model: GalN/LPS alone; BRP 240: GalN/LPS plus BRP (240 mg/kg); SIL: GalN/LPS plus silymarin (50 mg/kg).