rCsHscB Derived from Clonorchis sinensis: A Carcinogenic Liver Fluke Ameliorates LPS-Induced Acute Hepatic Injury by Repression of Inflammation

Abstract

Sepsis-associated acute liver injury caused by spillovers of bacteria and endotoxins (lipopolysaccharide, LPS) into the liver remains a public health issue due to the lack of specific therapeutic approaches. Previous studies showed that the recombinant protein HscB (rCsHscB) of Clonorchis sinensis, a carcinogenic liver fluke, had an anti-inflammatory effect and could alleviate inflammatory diseases such as enteritis; however, whether it can prevent sepsis-associated acute liver injury induced by LPS is still unknown. In our current study, the therapeutic effects and the potential mechanisms of rCsHscB on LPS-induced acute liver injury were investigated both in vivo and in vitro. The data showed that rCsHscB prevented LPS-induced liver damage, as demonstrated by histopathological observation and hepatic damage markers (the activities of serum ALT and AST) in a murine model of sepsis-associated acute liver injury. rCsHscB also significantly reversed the high levels of serum IL-6 and MCP-1 induced by LPS. In addition, rCsHscB attenuated the production of LPS-induced proinflammatory cytokines, including IL-6 and TNF-α, in a macrophage cell line-RAW264.7, through possible mediation by the MAPK signaling pathway in vitro. In conclusion, the present study demonstrates that rCsHscB derived from a fluke C. sinensis protects against sepsis-associated acute liver injury induced by LPS, which may be attributed to the inhibition of the MAPK signaling pathway. Our present study provides a potential therapeutic strategy for sepsis-associated acute liver injury.

Keywords: Clonorchis sinensis; LPS; MAPK; rCsHscB; sepsis-associated liver injury.

Figure 1

Histologic changes of liver tissues upon rCsHscB treatment in LPS-induced acute liver injury (H&E staining). Mice were divided into four groups, including the control group (PBS), rCsHscB (1.25 mg/kg body weight/mouse, i.p.) group, LPS (5 mg/kg body weight per mouse, i.p.) group, and LPS (5 mg/kg body weight per mouse, i.p.) + rCsHscB (1.25 mg/kg body weight per mouse, i.p.) group. For the LPS + rCsHscB group, mice were treated by intraperitoneal instillation of LPS (5 mg/kg body weight per mouse) 20 min before rCsHscB (intraperitoneal injection, 1.25 mg/kg body weight per mouse) injection. Then, 12 h later, mice were anesthetized, and the liver from each mouse was collected for H&E staining. (A) Histologic changes of the liver in different groups of mice. Arrow represents the inflammatory cell infiltrations in the liver of mice; the triangle indicates the proliferated biliary epithelium cells. (B) Numbers of infiltrated inflammatory cells in the liver of these groups. *** indicates that p value is less than <0.001, compared with indicated group.

Figure 2

The effects of rCsHscB on hepatic damage markers ALT and AST in the liver of mice stimulated by LPS. Mice were divided into four groups: the control group, rCsHscB group, LPS group, and LPS + rCsHscB group. The serum of each mouse from the respective group was collected 12 h after LPS treatment. (A) The activity of AST in the sera of mice from each group. (B) The activity of ALT in the sera of mice from each group. ** indicates that p value was less than 0.01; *** indicates that p value is less than <0.001, compared with indicated group.

Figure 3

rCsHscB reduces the secretion of serum IL-6 and MCP-1 in sepsis-associated acute liver injury mice induced by LPS. Mice were grouped, including the control group, rCsHscB group, LPS group, and LPS + rCsHscB group. The sera of the mice were collected after LPS treatment for 12 h, and the concentrations of IL-6 (A) and MCP-1 (B) in the sera were determined by ELISA. *** indicates that p value is less than <0.001, compared with indicated group.

Figure 4

rCsHscB attenuates LPS-induced inflammatory response in RAW264.7 cells. RAW 264.7 cells were stimulated with medium (negative control), rCsHscB (100 ng/mL), LPS (100 ng/mL), and LPS (100 ng/mL) + rCsHscB (100 ng/mL) for 24 h, the supernatants were collected, and IL-6 (A), TNF-α (B) and IL-10 (C) in the supernatants of cultured cells were determined by ELISA. * indicates that p value was less than 0.05; ** indicates that p value was less than 0.01; *** indicates that p value is less than <0.001, compared with indicated group.

Figure 5

MAPK pathway may mediate the depression of rCsHscB in LPS-induced acute liver injury. (A–D) RAW 264.7 cells were stimulated with medium (negative control), rCsHscB (100 ng/mL), LPS (100 ng/mL), and LPS (100 ng/mL) + rCsHscB (100 ng/mL) for 24 h, the cells were collected and lysed, and phosphorylated proteins of MAPK including ERK, JNK, and p38 in RAW264.7 cells were detected by Western blot (A). (B) Analysis of phosphorylation of ERK by ImageJ software. (C) Analysis of phosphorylation of JNK by Image J. (D) Analysis of phosphorylation of p38 by Image J software. (E) Part of the liver of mice from the control group, rCsHscB group, LPS group, and LPS + rCsHscB group was lysed, and the phosphorylated ERK1/2 was detected by western blot. ** indicates that p value is less than 0.01; *** indicates that p value is less than <0.001, compared with indicated group.