Role of the sympathetic nervous system in carbon tetrachloride-induced hepatotoxicity and systemic inflammation

Abstract

Carbon tetrachloride (CCl4) is widely used as an animal model of hepatotoxicity and the mechanisms have been arduously studied, however, the contribution of the sympathetic nervous system (SNS) in CCl4-induced acute hepatotoxicity remains controversial. It is also known that either CCl4 or SNS can affect systemic inflammatory responses. The aim of this study was to establish the effect of chemical sympathectomy with 6-hydroxydopamine (6-OHDA) in a mouse model of CCl4-induced acute hepatotoxicity and systemic inflammatory response. Mice exposed to CCl4 or vehicle were pretreated with 6-OHDA or saline. The serum levels of aminotransferases and alkaline phosphatase in the CCl4-poisoning mice with sympathetic denervation were significantly lower than those without sympathetic denervation. With sympathetic denervation, hepatocellular necrosis and fat infiltration induced by CCl4 were greatly decreased. Sympathetic denervation significantly attenuated CCl4-induced lipid peroxidation in liver and serum. Acute CCl4 intoxication showed increased expression of inflammatory cytokines/chemokines [eotaxin-2/CCL24, Fas ligand, interleukin (IL)-1α, IL-6, IL-12p40p70, monocyte chemoattractant protein-1 (MCP-1/CCL2), and tumor necrosis factor-α (TNF-α)], as well as decreased expression of granulocyte colony-stimulating factor and keratinocyte-derived chemokine. The overexpressed levels of IL-1α, IL-6, IL-12p40p70, MCP-1/CCL2, and TNF-α were attenuated by sympathetic denervation. Pretreatment with dexamethasone significantly reduced CCl4-induced hepatic injury. Collectively, this study demonstrates that the SNS plays an important role in CCl4-induced acute hepatotoxicity and systemic inflammation and the effect may be connected with chemical- or drug-induced hepatotoxicity and circulating immune response.

Fig 1. Schematic diagram of the study protocol.

Mice were randomly assigned in a blinded manner to one of four groups: group 1, control (n = 6), group 2, CCl₄ (n = 6), group 3, OHDA (n = 6), and group 4, 6-OHDA/CCl₄ (n = 6). The animals underwent 5 days of intra-peritoneal (i.p.) administration of 6-OHDA or saline. After ablation of the sympathetic nervous system, the animals were given CCl₄ or vehicle on day 6, and sacrificed 24 hours after CCl₄ poisoning. 6-OHDA = 6-hydroxydopamine, CCl₄ = carbon tetrachloride.

Fig 2. Immunofluorescent analysis of liver tyrosine hydroxylase (TH) reactive nerve fibers.

TH positive nerve fibers (arrows) were seen around the portal area in saline treated mice but not in chemical sympathectomised animals. Magnification × 200. The results are representative of four sets of experiments. Scale bar = 100 μm.

Fig 3. Absence of the sympathetic nervous system attenuated CCl₄-induced hepatotoxicity.

(A) Serum isolated from whole blood was used to determine lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels. Bars are means ± SD, n = 6 mice per group. (***p < 0.001 vs. control, ^# p < 0.05 vs. CCl₄, and ^## p < 0.01 vs. CCl₄) (B) Liver sections after CCl₄ administration were stained with hematoxylin and eosin, 200× magnification. Scale bar = 100 μm. (C) The semi-quantitative data of hepatocellular necrosis area showed sympathetic denervation resulted in lower necrosis area (^### p < 0.001 vs. CCl₄). Images are representative of n = 6 mice per group.

Fig 4. Absence of the sympathetic nervous system attenuated CCl₄-induced steatosis.

(A) Steatosis was evaluated using fat droplets area per 400× field. Scale bar = 50 μm. (B) Semi-quantitatively sympathetic denervation reduced fat droplets deposition prominently in the injured hepatocytes around the central veins compared to control at 24 h. Bars are means ± SD, n = 6 mice per group. (^## p < 0.01 vs. CCl₄).

Fig 5. Effect of ablation of the sympathetic nervous system on serum lipid profile in mice.

(A) Serum total cholesterol, triglyceride, high-density lipoprotein (HDL), very low-density lipoprotein (VLDL), and low-density lipoprotein (LDL) in mice administered sympathetic denervation for 5 d following CCl₄ intoxication. (B) Serum atherogenic index, cardiac risk factor, and LDL/HDL ratio in mice with sympathetic denervation following CCl₄ intoxication. Bars are means ± SD, n = 6 mice per group. In all figures, significant differences are noted as *p < 0.05 vs. control, **p < 0.01 vs. control, and ***p < 0.001 vs. control.

Fig 6. Prevention by sympathetic denervation of CCl₄-induced lipid peroxidation.

Mice were treated or not treated for 24 h with CCl₄, with or without pretreatment with 6-OHDA. Lipid peroxidation was assessed by malondialdehyde (MDA) measurement in liver homogenates (A) and serum (B). Results are expressed as mmol/g protein in liver homogenates and μM in serum. Data are mean ± SD for 6 mice per group. In all figures, significant differences are noted as **p < 0.01 vs. control, ^## p < 0.01 vs. CCl₄, ***p < 0.001 vs. control, and ^### p < 0.001 vs. CCl₄.

Fig 7. The effect of sympathetic denervation on CCl₄-induced systemic inflammatory markers as measured by antibody array.

(A) Inflammation array in serum from control, CCl₄-, 6-OHDA-, and 6-OHDA/CCl₄-treated mice. Altered cytokines and chemokines are indicated by boxes. (B) Quantification of the expression of eotaxin-2/CCL24, Fas ligand, granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-1α, IL-6, IL-12p40p70, keratinocyte-derived chemokine (KC), monocyte chemoattractant protein-1 (MCP-1/CCL2), and tumor necrosis factor-α (TNF-α) in each group. Data are presented as the fold of expression. Bars are mean ± SD for two replicated spots on the membrane. In all figures, significant differences are noted as *p < 0.05 vs. control, ^# p < 0.05 vs. CCl₄, **p < 0.01 vs. control, ^## p < 0.01 vs. CCl₄, and ^### p < 0.001 vs. CCl₄.

Fig 8. The effect of dexamethasone pretreatment on CCl₄-induced hepatotoxicity and systemic inflammation.

(A) Serum isolated from whole blood was used to determine LDH, ALT, AST, and ALP levels. Bars are means ± SD, n = 4 mice per group. (*p < 0.05 vs. PBS + CCl₄) (B) Inflammation array in serum from PBS + CCl₄ and Dexamethasone + CCl₄-treated mice. (C) Quantification of the expression of eotaxin-2, FasL, G-CSF, IL-1α, IL-6, IL-12p40p70, KC, MCP-1/CCL2, and TNF-α in each group. Data are presented as the fold of expression. Bars are mean ± SD for two replicated spots on the membrane. In all figures, significant differences are noted as *p < 0.05 vs. PBS + CCl₄, **p < 0.01 vs. PBS + CCl₄, and ***p < 0.001 vs. PBS + CCl₄.