M. L. Arffa5, M. A. Zapf5, A. N. Kothari1, V. Chang5, G. N. Gupta1, X. Ding1, M. M. Al-Gayyar2,4, W. Syn3, N. M. Elsherbiny4, P. C. Kuo1, Z. Mi1 1Loyola University Chicago Stritch School Of Medicine,Department Of Surgery,Maywood, IL, USA 2University Of Mansoura,Department Of Clinical Biochemistry,Mansoura, DAKAHLIA, Egypt 3Institute Of Hepatology,London, LONDON, United Kingdom 4University Of Tabuk,Department Of Pharmaceutical Chemistry,Tabuk, TABUK, Saudi Arabia 5Loyola University Chicago Stritch School Of Medicine,Maywood, IL, USA
Introduction: Osteopontin (OPN) expression is upregulated in liver injury and promotes fibrosis. Epidemiological studies suggest that green tea consumption is associated with lower incidence of liver disease. Epigallocatechin-3-gallate (EGCG) is a polyphenol found in green tea that abrogates hepatic fibrosis. Our previous results show that EGCG reduces OPN protein expression by increasing mRNA degradation. EGCG upregulates expression of multiple miRNAs in HepG2 cells, including miRNA 221. In Silico analysis identified the 3’UTR of OPN mRNA as a potential target for miRNA 221. Since miRNA expression varied depending on EGCG treatment protocols, we also identified miRNA 181a and 10b as candidates. We investigated whether these miRNAs mediate EGCG dependent OPN mRNA decay and their role both in vitro using HepG2 cells and in vivo using a rat model of thioacetamide (TAA) liver injury and fibrosis.
Methods: HepG2 cells were treated with EGCG [0.02-20µg/ml], and real time-PCR (RT-PCR) was used to quantify miRNA expression. Antagomirs and mimics were transfected. OPN protein was quantified by ELISA. MicroRNA was extracted from rat liver tissue and quantified by RT-PCR. Immunohistochemical (IHC) staining was performed for trichrome, p53, ki67, and OPN. IHC profiler was used to analyze OPN staining.
Results: EGCG treatment [0.02-20 µg/ml] of HepG2 cells increased expression of miRNAs 221, 181a, and 10b (p<0.05), with 221 having the highest expression. 2µg/ml of EGCG was chosen for downstream experiments because it is physiologically relevant. 2µg/ml EGCG decreased OPN protein levels by ~0.6 fold (p<0.01) while antagomir inhibition of only miRNA 221 reversed this effect (p<0.01). However, all three mimic miRNAs caused a ~0.6-fold decrease in OPN protein levels (p<0.01). Hepatic tissue from TAA rats treated with EGCG had upregulated expression of all three miRNAs (p<0.05). IHC analysis showed that TAA treated rats developed mild to moderate steatohepatitis and fibrosis. Rats simultaneously treated with EGCG showed no signs of liver damage. TAA rats had increased expression of ki67, p53, and OPN, indicating increased hepatocyte proliferation, apoptosis, and fibrosis, respectively, while EGCG+TAA rats did not have increased expression.
Conclusion: In in vitro and in vivo models of TAA induced hepatocyte injury, EGCG inhibits OPN dependent injury and fibrosis by primarily upregulating miRNA221 to accelerate OPN degradation. EGCG may have utility as a protective agent in settings of liver injury.