Severe steatosis increases hepatocellular injury and impairs liver regeneration in a rat model of partial hepatectomy

Abstract

Objective: The aim of this study was to assess the influence of severe steatosis with inflammation on hepatocellular recovery after 70% hepatectomy in a rat model of diet-induced steatosis.

Background: Patients with steatosis have an increased risk of inflammatory complications after liver resection. This might be attributable to Kupffer cell-mediated inflammation in steatotic livers causing progressive injury.

Methods: Male Wistar rats were fed a standard methionine- and choline-deficient diet for 1 or 5 weeks. A 70% partial hepatectomy (PH) was performed, after which rats were killed at 24, 48, or 72 hours. The extent of steatosis and inflammation was determined by assessment of hepatic triglycerides, cytokine content, and histopathology. Outcome parameters were: liver regeneration (MIB-5 proliferation rate, mitotic index, and regenerating liver mass), hepatocellular injury (plasma aminotransferases, lipid peroxidation, histopathology, and apoptosis), Kupffer cell-mediated proinflammatory response (TNF-alpha, IL-1beta, IL-6, IL-10 in plasma and liver) and antioxidant content (total glutathione).

Results: Methionine- and choline-deficient diet induced uncomplicated steatosis after 1 week (<30% hepatocytes affected without inflammation) and severe steatosis after 5 weeks (>60% hepatocytes affected, including prominent inflammation) as confirmed by histopathology. After PH, liver regeneration was impaired at all time points in the severe steatosis group as compared with the mild and control groups (P < 0.05). Hepatocellular injury was significantly increased in the severe steatosis group at all time points (P < 0.05). Kupffer cell-mediated inflammatory responses were aggravated in the severe steatosis group along with decreased antioxidant content (P < 0.05). Necrosis was the main type of cell death in severe steatotic livers compared with mainly apoptotic cell death in mild steatotic and normal livers.

Conclusion: Steatosis with prominent inflammation impaired liver regeneration probably because of increased hepatocellular lipid peroxidation and damage in concert with Kupffer cell-mediated proinflammatory responses. These results suggest an increased risk of performing extensive liver resection in the presence of severe steatosis.

FIGURE 1. Micrograph of liver tissue of control rat (A) showing no pathologic changes. After a 1-week MCD diet (B), mainly microvesicular steatosis with occasional macrovesicular foci and no inflammatory cells were observed. After 5 weeks of MCD diet (C), mainly macrovesicular steatosis and prominent inflammation were observed. All slides stained with hematoxylin and eosin (original magnification ×20). The histopathologic changes corresponded with increased hepatic triglycerides content (D). Values are mean ± SEM. *P < 0.05 versus controls. **P < 0.05 versus the mild steatosis group.

FIGURE 2. Liver regeneration after PH was evaluated by measuring increase in postoperative liver mass (A) and hepatocyte proliferation index (B). A decreased response was seen in the severe steatotic rats with respect to both parameters in contrast to no differences between the mild steatotic and control rats. Values are mean ± SEM. *P < 0.05 versus controls. **P < 0.05 versus the mild steatosis group.

FIGURE 3. Hepatocellular damage after PH assessed by plasma ALT (A) and AST (B) was most increased in the severe steatotic rats. Values are mean ± SEM. *P < 0.05 versus controls. **P < 0.05 versus the mild steatosis group.

FIGURE 4. Histopathology scores in hematoxylin and eosin-stained liver tissue (A) after PH was significantly increased in severe steatotic rats compared with no changes between the control and mild steatotic rats (P < 0.01). The rate of apoptotic cells (B) remained increased in the severe steatotic rats compared with decreasing rates in the control and mild steatotic rats after PH. Values are mean ± SEM. *P < 0.05 versus controls. **P < 0.05 versus the mild steatosis group.

FIGURE 5. Effect of severe and mild steatosis on hepatic lipid peroxidation after PH as measured by free TBARS (A) and hepatic total GSH response (B). Hepatic TBARS was increased in the severe steatotic rats after PH and hepatic GSH was increased in the mild steatotic rats compared with severe steatotic and control rats. Values are mean ± SEM. *P < 0.05 versus controls. **P < 0.05 versus the mild steatotic group.

FIGURE 6. Kupffer cell-mediated inflammatory response was increased in severe steatotic rats as assessed by plasma (A) and hepatic cytokines (B) and by the amount of hepatic resident and circulatory macrophages (C–E, ED-1-positive macrophages in the control, mild steatosis, and severe steatosis groups, respectively). Values are mean ± SEM. *P < 0.05 versus controls. **P < 0.05 versus the mild steatotic group.