Green Tea Extract Protects Against Diethylnitrosamine‐Mediated Liver Injury And Cell Proliferation By Attenuating STAT3 And iNOS Expression In High Fat‐Induced Obese Mice With Nonalcoholic Steatohepatitis

Abstract

Nonalcoholic steatohepatitis (NASH) increases hepatocellular carcinoma (HCC) risk by increasing inflammation and oncogenesis. Antiinflammatory activities of green tea extract (GTE) protect against dietary high-fat (HF)-induced NASH. I hypothesized that antiinflammatory and anti-oncogenic activities of GTE during NASH would also prevent diethylnitrosamine (DEN)-induced development towards HCC. Male C57BL/6J mice (4 wk old) were fed a HF diet devoid of, or supplemented with, GTE at 2% (w/w) and received once weekly intraperitoneal injections of saline vehicle or DEN (60 mg/kg; 5 and 7 wk old) until 25 wk old. Gross pathological observation indicated no tumors, as expected. GTE protected against obesity-associated parameters, histological and biochemical evidence of NASH, and hepatic TNFα and MCP-1 expression in both saline-and DENinjected mice (P<0.05). GTE attenuated serum alanine aminotransferase (ALT) activity, hepatic malondialdehyde (MDA), hepatic iNOS and survivin mRNA expression, signal transducer and activator of transcription 3 (STAT3), and hepatocyte proliferating cell nuclear antigen (PCNA) otherwise exacerbated by DEN. Hepatic GSTP protein expression increased in mice fed GTE. Serum ALT was correlated (r = 0.71-0.83; P<0.0001) with MDA, iNOS, STAT3, survivin and PCNA. iNOS correlated with PCNA, STAT3 (r = 0.64-0.65; P<0.05) and survivin iii (r = 0.66; P<0.0001), suggesting that iNOS-induced inflammation regulates liver injury and oncogenic cell proliferation. GTE lowers NASH-and DEN-mediated HCC risk by increasing GSTP and attenuating iNOS-mediated liver injury and survivin-mediated cell proliferation. I would like to express my sincerest gratitude towards my advisor Dr. Richard Bruno. He allowed me to enter his lab with very limited research experience and provided me many opportunities to learn not only research skills, but many life skills as well. His passion for research and passion for his students' growth has affected me in the best of ways. He challenged me academically and always encouraged the best in me by providing mentorship and friendship. He aided me in many abstracts, posters and oral presentations during my years as a master's student. This research could not have been done without our collaborators Dr. Jennifer Thomas-Ahner and Dr. Steven Clinton and I would like to thank them for sharing their time and extensive knowledge and expertise. I would also like to sincerely thank my other committee members, Dr. Amanda Bird and Dr. Ouliana Ziouzenkova for the time, care and advice they had given me. Dr. Bird's passion for research, expertise and kindness was greatly encouraging especially during experiments and data analysis. During times of uncertainty, Dr. Ziouzenkova provided insight and outlook on my research and my potential future career. Both committee members truly care about students and it was a privilege to learn under their guidance. v I would like to thank my colleagues Dr.