Abstract
Recent advances indicate a possible role of phytochemicals as modulatory factors in signaling pathways. We have previously demonstrated PHLPP2-mediated suppression of Nrf2 responses during oxidant attack. The present study was designed to explore Nrf2-potentiating mechanism of morin, a flavonol, via its possible role in intervening PHLPP2-regulated Akt/GSK3β/Fyn kinase axis. Efficacy of morin was evaluated against oxidative stress-mediated damage to primary hepatocytes by tert-butyl hydroperoxide (tBHP) and acetaminophen. The anti-cytotoxic effects of morin were found to be a consequence of fortification of Nrf2-regulated antioxidant defenses since morin failed to sustain activities of redox enzyme in Nrf2 silenced hepatocytes. Morin promoted Nrf2 stability and its nuclear retention by possibly modulating PHLPP2 activity which subdues cellular Nrf2 responses by activating Fyn kinase. Pull-down assay using morin-conjugated beads indicated the binding affinity of morin towards PHLPP2. Molecular docking also revealed the propensity of morin to occupy the active site of PHLPP2 enzyme. Thus, dietary phytochemical morin was observed to counteract oxidant-induced hepatocellular damage by promoting Nrf2-regulated transcriptional induction. The findings support the novel role of morin in potentiating Nrf2 responses by limiting PHLPP2 and hence Fyn kinase activation. Therefore, morin may be exploited in developing novel therapeutic strategy aimed at enhancing Nrf2 responses.
Highlights
Such an occurrence is prevented through a coordinated defense network, such as the Antioxidant Response Element (ARE)/Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) pathway, that guards the cellular makeup from a possible oxidative insult
To explore the key protective mechanism behind its role as an antioxidant, we investigated whether morin could protect isolated hepatocytes from toxicity generated due to oxidative overload
For this we employed tert-butyl hydroperoxide (tBHP), a pro-oxidant, wherein GSH depletion is believed to be a prime causative factor in the cytotoxicity ensued by oxidative stress [21]. 250 mM concentration of tBHP at 90 min exposure was selected for studies involving cytoprotection by morin since this concentration was observed to establish significant cell death due to oxidative stress [7] which is indicative of collapse in cellular defensive mechanisms
Summary
Capacity to act as signaling moieties involved in maintenance of cellular homeostasis [1]. Their accumulation within the cell may lead to oxidative imbalances, altered homeostasis and subsequent cell death. Such an occurrence is prevented through a coordinated defense network, such as the Antioxidant Response Element (ARE)/Nrf pathway, that guards the cellular makeup from a possible oxidative insult. When confronted with oxidant challenge, Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), translocates to the nucleus, where, in association with other proteins, it keeps a panel of cytoprotective genes under its tight regulation [2]. Any irregularity in its mechanism potentiates free radical-induced stress, thereby disturbing normal cellular physiology
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