Xuebijing Protects Against Septic Acute Liver Injury Based on Regulation of GSK-3β Pathway

Abstract

Xuebijing (XBJ), the only drug approved for the sepsis and multiple organ dysfunction, and its protective effects against acute liver injury (ALI) and its mechanism. The aim of this study was to evaluate the protective effect of XBJ on cecal ligation and perforation (CLP)-induced mouse ALI model and LPS-induced RAW264.7 cell ALI model. Mice were pretreated with XBJ before the CLP model was established, and serum and liver tissues were collected at the end of the experiment to assess the levels of inflammatory factors and liver injury. Results showed that XBJ pretreatment reduced liver/body weight, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in serum, and inhibited levels of pro-inflammatory factors in serum. Cells were treatment with XBJ and modeled by LPS modeling increased cell viability in the XBJ-treated group compared to the model group and XBJ also decreased serum pro-inflammatory factors in a dose-dependent manner. Western blot detected that XBJ also up-regulated the phosphorylated levels of glycogen synthase kinase-3β (p-GSK-3β) and cAMP-response element-binding protein (p-CREB) and down-regulated the phosphorylated level of nuclear factor kappa-B (p-NF-κB) in liver and cell. After overexpression of GSK-3β in cells, the mechanism was further investigated using CO-IP analysis. The binding of p-NF-κB and p-CREB to CREB-binding protein (CBP) was increased and decreased, respectively, indicating that GSK-3β regulated inflammation by regulating the binding of p-NF-κB and p-CREB to CBP. The present studies suggested that the hepatoprotective effect of XBJ may be through up-regulation of GSK-3β (Ser9) and increasing the binding of p-CREB to CBP, thereby alleviating the inflammatory response.

Keywords: CREB; GSK-3β; NF-κB; Xuebijing; acute hepatic injury.

FIGURE 1

XBJ alleviates acute liver injury (ALI) in C57BL/6 mice with cecal ligation perforation (CLP) sepsis (A) XBJ improved survival in mice with ALI from sepsis. (B) XBJ reduced liver/body weight in mice with ALI from sepsis (C) Phenotype and H and E staining of livers from each group of ALI mice. (D) Representative images of GSK-3β immunofluorescence in vivo (E) The expression of GSK-3β in liver of mice with ALI. Con, control group that underwent sham surgery; CLP, model group with CLP surgery; L-XBJ: group with CLP surgery + XBJ low dose group (4 ml/kg/day); H-XBJ: group with CLP surgery + XBJ high dose group (8 ml/kg/day); DXM: group with CLP surgery + Dexamethasone (5 mg/kg/day). Compared with the control group, #p < 0.05, ##p < 0.01 were considered to be significant; Compared with the CLP group, *p < 0.05, **p < 0.01 were considered to be significant. Data are expressed as mean ± SD.

FIGURE 2

XBJ improves liver function (A) XBJ reduced AST in serum of mice with ALI induced by CLP. (B) XBJ reduced ALT in serum of mice with ALI induced by CLP (C) XBJ reduces MPO in the liver of mice with CLP-induced ALI. Con: control group that underwent sham surgery; CLP: model group with CLP surgery; L-XBJ: group with CLP surgery + XBJ low dose group (4 ml/kg/day); H-XBJ: group with CLP surgery + XBJ high dose group (8 ml/kg/day); DXM: group with CLP surgery + Dexamethasone (5 mg/kg/day). Compared with the control group, #p < 0.05, ##p < 0.01 were considered to be significant; Compared with the CLP group, *p < 0.05,**p < 0.01 were considered to be significant. Data are expressed as mean ± SD.

FIGURE 3

XBJ modulates the serum levels of inflammatory factors in mice with CLP-induced ALI (A) XBJ increased IL-10 in serum of mice with CLP-induced ALI. (B) XBJ decreased TNF-α in serum of mice with CLP-induced ALI (C) XBJ decreased IL-6 in serum of mice with CLP-induced ALI. (D) XBJ decreased IL-12 in serum of mice with CLP-induced ALI (E) XBJ decreased IL-1β in serum of mice with CLP-induced ALI. Con: control group that underwent sham surgery; CLP: model group with CLP surgery; L-XBJ: group with CLP surgery + XBJ low dose group (4 ml/kg/day); H-XBJ: group with CLP surgery + XBJ high dose group (8 ml/kg/day); DXM: group with CLP surgery + Dexamethasone (5 mg/kg/day). Compared with the control group, #p < 0.05, ##p < 0.01 were considered to be significant; Compared with the CLP group, *p < 0.05, **p < 0.01 were considered to be significant. Data are expressed as mean ± SD.

FIGURE 4

XBJ regulates the activities of GSK-3β, NF-κB and CREB in the liver of CLP-induced ALI mice (A) Representative immunoblots of GSK-3β, NF-κB and CREB protein expression in liver lysates. (B-D) Administration of XBJ increased GSK-3β and CREB activity, while NF-κB activity was reduced in vivo. Con: control group that underwent sham surgery; CLP: model group with CLP surgery; L-XBJ: group with CLP surgery + XBJ low dose group (4 ml/kg/day); H-XBJ: group with CLP surgery + XBJ high dose group (8 ml/kg/day); DXM: group with CLP surgery + Dexamethasone (5 mg/kg/day). Compared with the control group, #p < 0.05, ##p < 0.01 were considered to be significant; Compared with the CLP group, *p < 0.05, **p < 0.01 were considered to be significant. Data are expressed as mean ± SD.

FIGURE 5

XBJ treatment increases the viability and GSK-3β (Ser9) expression of RAW264.7 cells (A) Concentration screening for XBJ cell experiments. (B) XBJ improved the viability of LPS-induced sepsis ALI cells (C) Representative images of GSK-3β immunofluorescence in vitro. (D) The expression of GSK-3β in cell with ALI. Con: control group without LPS treatment; LPS: model group with LPS treatment; 100-XBJ: group with LPS treatment + XBJ injection diluted 100 times; 50-XBJ: group with LPS treatment + XBJ injection diluted 50 times; 25-XBJ: group with LPS treatment + XBJ injection diluted 25 times. Compared with the control group, #p < 0.05, ##p < 0.01 were considered to be significant; Compared with the CLP group, *p < 0.05, **p < 0.01 were considered to be significant. Data are expressed as mean ± SD.

FIGURE 6

XBJ modulates the levels of inflammatory factors in LPS-induced ALI cells (A) XBJ increased IL-10 in RAW264.7 cells with LPS-induced ALI. (B) XBJ decreased TNF-α in RAW264.7 cells with LPS-induced ALI (C) XBJ decreased IL-6 in RAW264.7 cells with LPS-induced ALI. (D) XBJ decreased IL-12 in RAW264.7 cells with LPS-induced ALI. (E) XBJ decreased IL-1β in RAW264.7 cells with LPS-induced ALI. Con: control group without LPS treatment; LPS: model group with LPS treatment; 100-XBJ: group with LPS treatment + XBJ injection diluted 100 times; 50-XBJ: group with LPS treatment + XBJ injection diluted 50 times; 25-XBJ: group with LPS treatment + XBJ injection diluted 25 times. Compared with the control group, #p < 0.05, ##p < 0.01 were considered to be significant; Compared with the CLP group, *p < 0.05, **p < 0.01 were considered to be significant. Data are expressed as mean ± SD.

FIGURE 7

XBJ regulates the activities of GSK-3β, NF-κB and CREB in LPS-induced ALI cells (A) Representative immunoblots of GSK-3β, NF-κB and CREB protein expression in cell lysates. (B-D) Administration of XBJ increased GSK-3β and CREB activity, while NF-κB activity was reduced in vitro. Con: control group without LPS treatment; LPS: model group with LPS treatment; 100-XBJ: group with LPS treatment + XBJ injection diluted 100 times; 50-XBJ: group with LPS treatment + XBJ injection diluted 50 times; 25-XBJ: group with LPS treatment + XBJ injection diluted 25 times. Compared with the control group, #p < 0.05, ##p < 0.01 were considered to be significant; Compared with the CLP group, *p < 0.05, **p < 0.01 were considered to be significant. Data are expressed as mean ± SD.

FIGURE 8

Overxepression of GSK-3β regulates the expression pNF-κB and CREB (Ser133) (A) GSK-3β was successfully overexpressed in cells. (B) Representative immunoblots of p-NF-κB and p-CREB (C) GSK-3β-OE increased the expression of pNF-κB in cells. (D) GSK-3β-OE decreased the expression of CREB in cells. Con: control group; GSK-3β-OE: GSK-3β overexpression group. *p < 0.05, **p < 0.01 were considered to be significant. Data are expressed as mean ± SD.