Measurement of Oxidative Stress in Huh-7 Cell Line to Determine the Effectiveness of PNPLA3 Targeted Gene Therapy for Mitigation of Lipid and Alcohol Induced Oxidative Stress in the Liver

Abstract

NAFLD is a condition of increased buildup of fat in the liver, causing lipotoxicity that can manifest to cirrhosis, steatosis and fibrosis, which can cause significant and eventually irreversible damage to the liver. A key gene associated is the PNPLA3 I148M variant, which has been shown to display lipogenesis functionality. However, the role of PNPLA3 in increased ROS formation is debated. Moreover, there are no studies determining correlation between the variant and alcohol-induced oxidative stress. This project determines the following mechanistic functions of the PNPLA I148M variant to test for the efficiency of PNPLA3 gene therapy for patients with fatty liver disease and alcohol liver disease. There is a strong correlation between lipid induced oxidative stress and PNPLA3 148M variant. DCFDA shows increased concentration of H2O2 for PNPLA3 148M overexpressed cell lines for both ethanol and FFA treatment groups. Moreover, there is a statistical correlation between PNPLA3 148M overexpressed cell lines and increased mitochondrial oxidative stress by MitoSOX cellular ROS analysis methods. This study confirmed the significant decrease in oxidative stress levels for 148I variant overexpressed cell lines and proved the efficiency of PNPLA3 targeted gene therapy to NAFLD patients and suggested the potential use of the therapeutic method to patients with ALD. To ensure gene therapeutic effectiveness for patients with NAFLD and alcoholic liver diseases, further experiments may be needed to verify molecular pathways of ROS formation by PNPLA3 with qPCR analysis.