Sympathetic Nervous System Control of Carbon Tetrachloride-Induced Oxidative Stress in Liver through α-Adrenergic Signaling

Abstract

In addition to being the primary organ involved in redox cycling, the liver is one of the most highly innervated tissues in mammals. The interaction between hepatocytes and sympathetic, parasympathetic, and peptidergic nerve fibers through a variety of neurotransmitters and signaling pathways is recognized as being important in the regulation of hepatocyte function, liver regeneration, and hepatic fibrosis. However, less is known regarding the role of the sympathetic nervous system (SNS) in modulating the hepatic response to oxidative stress. Our aim was to investigate the role of the SNS in healthy and oxidatively stressed liver parenchyma. Mice treated with 6-hydroxydopamine hydrobromide were used to realize chemical sympathectomy. Carbon tetrachloride (CCl4) injection was used to induce oxidative liver injury. Sympathectomized animals were protected from CCl4 induced hepatic lipid peroxidation-mediated cytotoxicity and genotoxicity as assessed by 4-hydroxy-2-nonenal levels, morphological features of cell damage, and DNA oxidative damage. Furthermore, sympathectomy modulated hepatic inflammatory response induced by CCl4-mediated lipid peroxidation. CCl4 induced lipid peroxidation and hepatotoxicity were suppressed by administration of an α-adrenergic antagonist. We conclude that the SNS provides a permissive microenvironment for hepatic oxidative stress indicating the possibility that targeting the hepatic α-adrenergic signaling could be a viable strategy for improving outcomes in patients with acute hepatic injury.

Figure 1

Effect of 6-OHDA on CCl₄ induced changes on hepatocyte morphology. Representative micrographs of transmission electron microscopy (magnification 6,000x or 12,000x) in the liver tissues. (a and e) The saline + olive oil group; (b and f) the 6-OHDA + olive oil group; (c and g) the saline + CCl₄ group; and (d and h) the 6-OHDA + CCl₄ group. Arrows denote rough endoplasmic reticulum. Arrowheads denote perinuclear space. C, chromatin; G, glycogen deposits; L, lipid drops; Vac, vacuolization. Scale bar denotes 100 nm in (a)–(d) and 500 nm in (e)–(h).

Figure 2

Effect of 6-OHDA on CCl₄ induced changes in hepatocyte nuclei. Representative micrographs of DAPI stained nuclei (magnification 200x or 400x) in the liver tissues. Typical images were selected from each experimental group (original magnification 200x or 400x). Scale bar = 100 μm in 200x and 50 μm in 400x.

Figure 3

Effect of 6-OHDA on CCl₄ induced oxidative DNA damage. Oxidized DNA in liver was measured 24 hours after exposure to olive oil or CCl₄ following pretreatment with saline of 6-OHDA. Data plotted are mean and SD (n = 6 animals in each group).  ∗∗ denotes significant differences (p < 0.01) compared with the saline + olive oil group.  # denotes significant differences (p < 0.05) compared with the saline + CCl₄ group.

Figure 4

Effect of 6-OHDA on levels of hepatic cytokines and chemokines in the absence and following CCl₄ treatment. (a) A cytokine array assay in the liver of the saline + olive oil group, the 6-OHDA + olive oil group, the saline + CCl₄ group, and the 6-OHDA + CCl₄ group was measured. Altered cytokines (twofold or more), including IL-1α, IL-10, leptin, TIMP-2, sTNFR I, GM-CSF, CCL3, CCL5, CCL9, CCL11, and CXCL11, are indicated by boxes. (b) The relative density of cytokines and chemokines was normalized with the internal control and expressed as a ratio of the expression level of cytokines and chemokines in each group divided by the expression level in the saline + olive oil group. Each value represents the average of two replicated spots on the membrane. In all figures,  ∗ denotes significant differences compared with the saline + olive oil group (p < 0.05). ∗∗ denotes significant differences compared with the saline + olive oil group (p < 0.01).  # denotes significant differences compared with the saline + CCl₄ group (p < 0.05).  ## denotes significant differences compared with the saline + CCl₄ group (p < 0.01).

Figure 5

Effect of α- or β-adrenergic blocker on CCl₄ induced hepatic injury and oxidative DNA damage. Histological features (a) and area of necrosis (b) of liver sections stained with hematoxylin and eosin 24 h after CCl₄ treatment. Typical images were selected from each experimental group (original magnification 200x). The saline + CCl₄ group showing hepatocellular necrosis; the phentolamine + CCl₄ group showing mild hepatocellular necrosis. (c) Oxidized DNA in liver was measured 24 hours after exposure to CCl₄ following pretreatment with saline, phentolamine, or nadolol. Data plotted are mean and SD (n = 4 animals in each group).  ∗ denotes significant differences (p < 0.05) compared with the saline + CCl₄ group.  ∗∗∗ denotes significant differences (p < 0.001) compared with the saline + CCl₄ group.