Iranian crack induces hepatic injury through mitogen-activated protein kinase pathway in the liver of Wistar rat

Abstract

Objectives: Iranian crack (IC) is a heroin-based substance manifesting various pathologic side effects. Herein, we aimed to investigate the mechanism of IC-induced liver injuries in Wistar rats.

Materials and methods: Twenty male Wistar rats were randomly divided into two groups: control, and IC (0.9 mg/kg/day/IP, for 30 days). Mitochondrial reactive oxygen species (ROS) production was measured by DCF fluorescence staining. The expression of tumor necrosis factor-alpha (TNF-α), interleukin 1β (IL-1β), and phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (c-JNK) were assessed by immunoblotting assay. The intensity of collagen fiber in the liver was also determined by Trichrome-Masson staining. Furthermore, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) activities were measured using colorimetric methods.

Results: Our results showed that ROS production, p38 MAPK, c-JNK phosphorylation levels, and expression of TNF-α and IL-1β were significantly elevated in the liver tissue of IC group as compared to the control group. Moreover, collagen fiber and ALT activity were increased in the liver tissue of IC group compared to the control group. However, there was no statistically significant difference in the levels of ALP between two groups. In addition, there was a positive correlation between the intensity of collagen fiber and the ALT activity, and the levels of TNF-α and IL-1β and liver enzymes activities including ALP, ALT, and AST.

Conclusion: Our findings revealed that IC-induced liver cells injury is partially mediated by MAPK stress kinases. Therefore, regular liver examination in substance abuse is strongly recommended.

Keywords: Cytokines; Iranian Crack; Liver fibrosis; Transaminase; c-JNK; p38 MAPK.

Figure 1

Effects of Iranian crack (IC) on the hepatic mitochondrial reactive oxygen species (ROS) production. (A) Fluorescent microscopic images of DCF-stained mitochondria in the liver tissue. (Aa) normal saline control group; (Ab) IC-received (0.9 mg/kg/day) group. (B) Quantified DCF fluorescence by Fluorimeter (Biotek), which was normalized against mg protein in mitochondrial extract and presented as fold of control per group. Values are represented as mean ± SEM (n=5); ***P<0.001 vs. control group

Figure 2

Effects of Iranian crack (IC) on the phosphorylation of p38 MAPK and JNK in the liver tissue. (A) Immunoblotting images of phosphor-p38 MAPK, total p38 MAPK, phosphor-JNK and total JNK proteins in normal saline (NS)-received rat and IC-treated rats. (B) Densitometry analysis of phosphor-p38 MAPK normalized against total p38 MAPK and presented as fold change of control. (C) Densitometry analysis of phosphor-JNK normalized against total JNK and presented as fold change of control. Values are represented as mean±SEM; **P<0.01, ***P<0.001 vs. control group

Figure 3

Effects of Iranian crack (IC) on the expression of tumor necrosis factor-alpha (TNF-α) and interleukin 1β (IL-1β) in the liver tissue. (A) Immunoblotting images of TNF-α, IL-1β, β-actin proteins (as the internal control) in the normal saline (NS)-received rat and IC-treated rats. (B) Densitometry analysis of TNF-α normalized against β-actin and presented as fold change of control (C) Densitometry analysis of IL-1β normalized against β-actin and presented as fold change of control. Values are represented as mean ± SEM; **P<0.01 vs. control group

Figure 4

Effect of Iranian crack (IC) on the liver tissue inflammation and fibrosis. A) 400× hematoxylin and eosin (H&E)- stained sections and B) Trichrome Masson (MT)-stained sections. (Aa) Photomicrographs of liver sections of normal control group showed well-structured and arrangement of the normal liver architecture and few inflammatory cell infiltration; (Ab) Photomicrographs of liver sections of IC-administered group showed inflammation and mononuclear cell infiltration in the liver tissue (black arrow); (Ba) the rate of collagen as a marker of fibrosis in normal saline control group (blue color) (Bb) representative of the collagen and other fibrotic agents accumulation in the liver tissue of IC-treated rats (blue color). (C) The score of liver fibrosis in different groups. Values are presented as mean ± SEM (n=5); *P<0.05 vs. control group