Atthapan Morchang^1,2, Shilu Malakar³, Siripat Aluksanasuwan^1,2, Keerakarn Somsuan^1,2, Narongsuk Munkong⁴, Kevalin Vongthoung⁵

Abstract

Purpose: To investigate the molecular mechanism underlying the antioxidant effect of red sticky rice bran extract (RRBE; a pigmented strain of Oryza sativa L.) in human hepatocellular carcinoma cell line. Methods: Human hepatocellular carcinoma HuH-7 cells were treated with the ethanol extract of RRBE in the presence or absence of EX-537 (Sirtuin 1 inhibitor), dexamethasone (NF-κB inhibitor), brusatol (Nrf-2 inhibitor), or HO-1 inh (HO-1 inhibitor) before exposure to hydrogen peroxide-induced oxidative stress. Intracellular ROS and glutathione levels were assessed using CellROX™ green reagent and GSH-Glo™ glutathione assay, respectively. Levels of nuclear factor erythroid 2-related factor 2 (Nrf-2) and expression of heme oxygenase-1 (HO-1) were determined using immunofluorescent assay and real-time polymerase chain reaction (RT-PCR), respectively. Results: None of the tested inhibitors affected the ability of RRBE to reduce the level of ROS. Treatment with RRBE significantly decreased intracellular ROS and glutathione levels in HuH-7 cells undergoing oxidative stress (in the presence of BST and HO-1 inh; p < 0.05). Furthermore, Nrf-2 expression and HO-1 gene were significantly enhanced and upregulated respectively in HuH-7 cells pre-treated with RRBE (p < 0.05). Conversely, these effects were attenuated in the presence of brusatol. Conclusion: Antioxidant property of RRBE is mediated through the activation of Nrf-2 and HO-1 pathways. These insights pave way for the development of functional foods or supplementary medicines aimed at preventing and treating NCDs in the future.