10-Oxo-trans-11-octadecenoic acid generated from linoleic acid by a gut lactic acid bacterium Lactobacillus plantarum is cytoprotective against oxidative stress

Highlights

• We evaluated the effect of modified fatty acids generated by Lactobacillus plantarum.

• 10-Oxo-trans-11-ocatadecenoic acid (KetoC) protected cells from oxidative stress.

• KetoC activated the Nrf2-ARE pathway to promote antioxidative gene expression.

• KetoC promoted the expression of antioxidative enzymes in mice organs.

• The cytoprotective effect of KetoC was because of α,β-unsaturated carbonyl moiety.

Abstract

Oxidative stress is a well-known cause of multiple diseases. The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway plays a central role in cellular antioxidative responses. In this study, we investigated the effects of novel fatty acid metabolite derivatives of linoleic acid generated by the gut lactic acid bacteria Lactobacillus plantarum on the Nrf2-ARE pathway. 10-Oxo-trans-11-octadecenoic acid (KetoC) protected HepG2 cells from cytotoxicity induced by hydrogen peroxide. KetoC also significantly increased cellular Nrf2 protein levels, ARE-dependent transcription, and the gene expression of antioxidative enzymes such as heme oxygenase-1 (HO-1), glutamate-cysteine ligase modifier subunit (GCLM), and NAD(P)H:quinone oxidoreductase 1 (NQO1) in HepG2 cells. Additionally, a single oral dose administration of KetoC also increased antioxidative gene expression and protein levels of Nrf2 and HO-1 in mouse organs. Since other fatty acid metabolites and linoleic acid did not affect cellular antioxidative responses, the cytoprotective effect of KetoC may be because of its α,β-unsaturated carbonyl moiety. Collectively, our data suggested that KetoC activated the Nrf2-ARE pathway to enhance cellular antioxidative responses in vitro and in vivo, which further suggests that KetoC may prevent multiple diseases induced by oxidative stress.

Introduction

Oxidative stress is due to an imbalance between production of reactive oxygen species (ROS) and antioxidative mechanisms (Halliwell, 2009). Since excessive ROS and oxidation products damage biomolecules such as proteins, nucleic acids, and lipids, impairing their biological functions, oxidative stress has been implicated in multiple diseases. Particularly, many ROS are produced in liver during energy metabolism and detoxification, which can cause various liver diseases including non-alcoholic steatohepatitis (NASH) (Polimeni et al., 2015, Rolo et al., 2012).

Cellular defense mechanisms against oxidative stress are naturally present in the human body. One such mechanism is the nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway, which plays a central role in cellular antioxidative responses (Taguchi et al., 2011, Rushmore and Pickett, 1990, Alam et al., 1999). Under normal conditions, protein expression of the transcription factor Nrf2 is suppressed by binding to the Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm, which leads to Nrf2 ubiquitin-dependent proteasomal degradation. Under oxidative stress, electrophilic xenobiotics or oxidation products cause conformational changes in Keap1 through modification of cysteine residues, leading to the release of Nrf2 and thus preventing it from degradation. Nrf2 is then transported into the nucleus where it binds to the ARE in the promoter region of antioxidative and phase II detoxification enzyme genes to induce their expression. Since these enzymes quench ROS and eliminate electrophilic substances, Nrf2 activation in liver is expected to ameliorate diseases associated with oxidative stress, such as NASH (Chartoumpekis and Kensler, 2013, Shimozono et al., 2013, Tanaka et al., 2008). Interestingly, many natural dietary ingredients have been previously identified as Nrf2 activators, including sulforaphane in broccoli sprouts and curcumin in turmeric (Magesh et al., 2012, Kwak et al., 2007, Balogun et al., 2003, Izumi et al., 2012).

The oxidation products (or metabolites) of polyunsaturated fatty acids (PUFAs) such as nitro linoleic acids (Villacorta et al., 2007), 4-hydroxynonenal (Chen et al., 2005) and 15-deoxy-Δ^{12, 14}-prostaglandin J₂ (Yu et al., 2006) have been reported to be endogenous inducers of Nrf2 activation (Kansanen et al., 2012, Wang et al., 2009). Additionally, 9-oxo-trans-7-octadecenoic acid derived from green alga, Ulva lactuca, has been reported to activate the Nrf2-ARE pathway in human neuroblastoma IMR-32 cells (Wang et al., 2013). These reports indicate that derivatives of long chain fatty acids can activate the Nrf2-ARE pathway.

Here, we focused on the metabolic intermediates derived from PUFAs generated by gut microorganisms as potential Nrf2 activators. We previously revealed in detail the saturation pathway of linoleic acid (LA) by Lactobacillus plantarum AKU1009a (Fig. 1), which is the representative lactic acid bacterium utilized in fermented foods and is one of the gut microorganisms (Kishino et al., 2013). L. plantarum converts LA to conjugated LA and oleic acid through multi-enzymatic reactions in the host gastrointestinal tract. Additionally, the efficient production of intermediates such as hydroxy fatty acids, oxo fatty acids, conjugated fatty acids, and partially saturated fatty acids from PUFAs was achieved by the enzymes in the metabolic pathway in L. plantarum. These fatty acid intermediates can be detected in host organs, and therefore could be involved in host health conditions. For instance, the fatty acid metabolites generated by L. plantarum have been reported to improve intestinal epithelial barrier impairment (Miyamoto et al., 2015), promote adipogenesis (Goto et al., 2015), control immune system (Bergamo et al., 2014) and suppress lipogenesis (Nanthirudjanar et al., 2015).

In the present study, we investigated the effect of hydroxy and oxo fatty acids derived from LA generated by L. plantarum on cellular antioxidative responses regulated by the Nrf2-ARE pathway using human hepatocellular carcinoma cell line HepG2 as a hepatocyte model since the liver is constantly exposed against oxidative stress. As a result, we found that among the metabolites derived from LA, 10-oxo-trans-11-octadecenoic acid (KetoC) provided the most potent cytoprotective effects against oxidative stress through activation of the Nrf2-ARE pathway. We also confirmed that KetoC induced the expression of antioxidative enzymes in vivo.