Therapeutic Benefits of Spleen Tyrosine Kinase Inhibitor Administration on Binge Drinking-Induced Alcoholic Liver Injury, Steatosis, and Inflammation in Mice

Abstract

Background: Binge drinking is increasingly recognized as an important cause of liver disease with limited therapeutic options for patients. Binge alcohol use, similar to chronic alcohol consumption, induces numerous deregulated signaling events that drive liver damage, steatosis, and inflammation. In this article, we evaluated the role of spleen tyrosine kinase (SYK), which modulates numerous signaling events previously identified linked in the development alcohol-induced liver pathology.

Methods: A 3-day alcohol binge was administered to C57BL/6 female mice, and features of alcoholic liver disease were assessed. Some mice were treated daily with intraperitoneal injections of a SYK inhibitor (R406; 5 to 10 mg/kg body weight) or drug vehicle control. Liver and serum samples were collected and were assessed by Western blotting, biochemical, ELISA, electrophoretic mobility shift assays, real-time quantitative polymerase chain reaction, and histopathological analysis.

Results: We found that binge drinking induced significant SYK activation (SYK(Y525/526) ) with no change in total SYK expression in the liver. Functional inhibition of SYK activation using a potent SYK inhibitor, R406, was associated with a significant decrease in alcohol-induced hepatic inflammation as demonstrated by decreased phospho-nuclear factor kappa beta (NF-κB) p65, NF-κB nuclear binding, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1 mRNA in the liver. Compared to vehicle controls, SYK inhibitor treatment decreased alcohol binge-induced hepatocyte injury indicated by histology and serum alanine aminotransferase. Strikingly, SYK inhibitor treatment also resulted in a significant reduction in alcohol-induced liver steatosis.

Conclusions: Our novel observations demonstrate the role of SYK, activation in the pathomechanism of binge drinking-induced liver disease highlighting SYK a potential multifaceted therapeutic target.

Keywords: Alcoholic Hepatitis; Binge Drinking; Nonreceptor Tyrosine Kinase.

Figure 1. Acute/Binge alcohol induced liver injury in mice induces SYK activation

Female C57BL/6 mice received sugar or ethanol gavage daily for three days. Liver samples were stained by H&E (A) or Oil-red-O (B), and liver injury and steatosis was quantified by measuring serum ALT (A) and liver triglycerides (B), respectively. Total liver expression of TNF-α, MCP-1 and pro–IL-1β in the liver was analyzed by qPCR (C). Total liver protein was analysed for total SYK (D) and activated SYK^Y525/526 (E) expression by western blotting using β-actin as loading control. *p<0.05 compared to baseline sugar control was considered statistically significant by student t-test for 7–10 mice per experimental group.

Figure 2. SYK inhibitor treatment decreased acute/binge alcohol induced activated SYK and ERK1/2 expression in the liver

C57BL6 mice received alcohol or isocaloric sugar gavage daily for three days with and without SYK inhibitor or vehicle treatment (5mg/kg) as indicated. Total liver protein was analysed for total SYK (A), activated SYK^Y525/526 (B) by western blotting using β-Actin as normalization control, phospho ERK1/2 [pThr202/Tyr204] by ELISA (C). *^/#p<0.05 compared to baseline sugar gavage control or alcohol gavage test group was considered statistically significant by ANOVA for 7–10 mice per experimental group.

Figure 3. SYK inhibitor treatment reduces acute/binge alcohol induced phospho-NF-κB p65, NF-κB nuclear binding, hepatic inflammatory cytokine production and hepatocyte injury

Female C7BL/6 mice received daily alcohol or isocaloric sugar gavage over three days with or without SYK inhibitor treatment (5mg/kg) as indicated. Total liver protein was analysed for phospho- NF-κB p65 by western blotting using β-Actin as protein loading control (A). Nuclear liver extracts were analysed for NF-κB promoter binding by EMSA (B). Total liver RNA or protein was analysed for TNF-α (C) and MCP-1 (D) by quantitative RT-PCR using 18s RNA as normalization control and ELISA. Hepatocyte injury was analysed by H&E histology and serum ALT (E). A representative slide of 6–8 mice per experimental group is presented. *^/#p<0.05 compared to baseline or EtOH vehicle group was considered statistically significant by ANOVA for 6–8 mice per experimental group.

Figure 4. Acute/binge alcohol induced hepatic steatosis is ameliorated with SYK inhibitor treatment

C57BL/6 mice received daily alcohol or isocaloric sugar gavage for three days with or without SYK inhibitor treatment (5mg/kg) as indicated. Liver steatosis was evaluated by Oil-red-O staining (A) and liver triglyceride assay (B). Liver RNA was analysed for ADRP (C), FASN (D), PRDM16 (E) and UCP1 (F). *^/#p<0.05 compared to baseline or EtOH vehicle group was considered statistically significant by ANOVA for 6–8 mice per experimental group.